Coffee and caffeine potentiate the antiamyloidogenic activity of melatonin via inhibition of Aβ oligomerization and modulation of the Tau-mediated pathway in N2a/APP cells

PubMed Central

Zhang, Li-Fang; Zhou, Zhi-Wei; Wang, Zhen-Hai; Du, Yan-Hui; He, Zhi-Xu; Cao, Chuanhai; Zhou, Shu-Feng

2015-01-01

There is an increasing prevalence of Alzheimer’s disease (AD), which has become a public health issue. However, the underlying mechanisms for the pathogenesis of AD are not fully understood, and the current therapeutic drugs cannot produce acceptable efficacy in AD patients. Previous animal studies have shown that coffee (Coff), caffeine (Caff), and melatonin (Mel) have beneficial effects on AD. Disturbed circadian rhythms are observed in AD, and chronotherapy has shown promising effects on AD. In this study, we examined whether a combination of Coff or Caff plus Mel produced a synergistic/additive effect on amyloid-β (Aβ) generation in Neuro-2a (N2a)/amyloid precursor protein (APP) cells and the possible mechanisms involved. Cells were treated with Coff or Caff, with or without combined Mel, with three different chronological regimens. In regimen 1, cells were treated with Coff or Caff for 12 hours in the day, followed by Mel for 12 hours in the night. For regimen 2, cells were treated with Coff or Caff plus Mel for 24 hours, from 7 am to 7 am the next day. In regimen 3, cells were treated with Coff or Caff plus Mel with regimen 1 or 2 for 5 consecutive days. The extracellular Aβ40/42 and Aβ oligomer levels were determined using enzyme-linked immunosorbent assay (ELISA) kits. The expression and/or phosphorylation levels of glycogen synthase kinase 3β (GSK3β), Erk1/2, PI3K, Akt, Tau, Wnt3α, β-catenin, and Nrf2 were detected by Western blot assay. The results showed that regimen 1 produced an additive antiamyloidogenic effect with significantly reduced extracellular levels of Aβ40/42 and Aβ42 oligomers. Regimen 2 did not result in remarkable effects, and regimen 3 showed a less antiamyloidogenic effect compared to regimen 1. Coff or Caff, plus Mel reduced oxidative stress in N2a/APP cells via the Nrf2 pathway. Coff or Caff, plus Mel inhibited GSK3β, Akt, PI3K p55, and Tau phosphorylation but enhanced PI3K p85 and Erk1/2 phosphorylation in N2a/APP cells. Coff or Caff, plus Mel downregulated Wnt3α expression but upregulated β-catenin. However, Coff or Caff plus Mel did not significantly alter the production of T helper cell (Th)1-related interleukin (IL)-12 and interferon (IFN)-γ and Th2-related IL-4 and IL-10 in N2a/APP cells. The autophagy of cells was not affected by the combinations. Taken together, combination of Caff or Coff, before treatment with Mel elicits an additive antiamyloidogenic effects in N2a/APP cells, probably through inhibition of Aβ oligomerization and modulation of the Akt/GSK3β/Tau signaling pathway. PMID:25565776

Coffee and caffeine potentiate the antiamyloidogenic activity of melatonin via inhibition of Aβ oligomerization and modulation of the Tau-mediated pathway in N2a/APP cells.

PubMed

Zhang, Li-Fang; Zhou, Zhi-Wei; Wang, Zhen-Hai; Du, Yan-Hui; He, Zhi-Xu; Cao, Chuanhai; Zhou, Shu-Feng

2015-01-01

There is an increasing prevalence of Alzheimer's disease (AD), which has become a public health issue. However, the underlying mechanisms for the pathogenesis of AD are not fully understood, and the current therapeutic drugs cannot produce acceptable efficacy in AD patients. Previous animal studies have shown that coffee (Coff), caffeine (Caff), and melatonin (Mel) have beneficial effects on AD. Disturbed circadian rhythms are observed in AD, and chronotherapy has shown promising effects on AD. In this study, we examined whether a combination of Coff or Caff plus Mel produced a synergistic/additive effect on amyloid-β (Aβ) generation in Neuro-2a (N2a)/amyloid precursor protein (APP) cells and the possible mechanisms involved. Cells were treated with Coff or Caff, with or without combined Mel, with three different chronological regimens. In regimen 1, cells were treated with Coff or Caff for 12 hours in the day, followed by Mel for 12 hours in the night. For regimen 2, cells were treated with Coff or Caff plus Mel for 24 hours, from 7 am to 7 am the next day. In regimen 3, cells were treated with Coff or Caff plus Mel with regimen 1 or 2 for 5 consecutive days. The extracellular Aβ40/42 and Aβ oligomer levels were determined using enzyme-linked immunosorbent assay (ELISA) kits. The expression and/or phosphorylation levels of glycogen synthase kinase 3β (GSK3β), Erk1/2, PI3K, Akt, Tau, Wnt3α, β-catenin, and Nrf2 were detected by Western blot assay. The results showed that regimen 1 produced an additive antiamyloidogenic effect with significantly reduced extracellular levels of Aβ40/42 and Aβ42 oligomers. Regimen 2 did not result in remarkable effects, and regimen 3 showed a less antiamyloidogenic effect compared to regimen 1. Coff or Caff, plus Mel reduced oxidative stress in N2a/APP cells via the Nrf2 pathway. Coff or Caff, plus Mel inhibited GSK3β, Akt, PI3K p55, and Tau phosphorylation but enhanced PI3K p85 and Erk1/2 phosphorylation in N2a/APP cells. Coff or Caff, plus Mel downregulated Wnt3α expression but upregulated β-catenin. However, Coff or Caff plus Mel did not significantly alter the production of T helper cell (Th)1-related interleukin (IL)-12 and interferon (IFN)-γ and Th2-related IL-4 and IL-10 in N2a/APP cells. The autophagy of cells was not affected by the combinations. Taken together, combination of Caff or Coff, before treatment with Mel elicits an additive antiamyloidogenic effects in N2a/APP cells, probably through inhibition of Aβ oligomerization and modulation of the Akt/GSK3β/Tau signaling pathway.

Immunophenotype of infiltrating cells in protocol renal allograft biopsies from tacrolimus-versus cyclosporine-treated patients.

PubMed

Serón, Daniel; O'Valle, Francisco; Moreso, Francesc; Gomà, Montse; Hueso, Miguel; Grinyó, Josep M; Garcia del Moral, Raimundo

2007-03-15

The prevalence of subclinical rejection is lower in patients receiving tacrolimus than in patients treated with cyclosporine. However, it is not known whether this difference is related to the modulation of a specific cell immunophenotype. We perform a two case-one control study in patients treated with tacrolimus (n=44) or cyclosporine (n=22) with a protocol biopsy performed at 4 to 6 months. Immunophenotype of infiltrating cells was evaluated with monoclonal antibodies directed against CD45 (all leukocytes), CD3 (T lymphocytes), CD68 (monocytes/macrophages), and CD20 (B lymphocytes) and expressed as interstitial positive cells/mm(2). The number of interstitial CD45 (290+/-209 vs. 696+/-560; P<0.01), CD3 (121+/-84 vs. 208+/-104; P<0.01), and CD68 (155+/-232 vs. 242+/-280; P<0.05) but not CD20 (137+/-119 vs. 197+/-154) positive cells was lower in tacrolimus-treated patients. T lymphocytes and macrophages interstitial infiltration was reduced in tacrolimus treated patients evaluated with protocol biopsies in comparison to cyclosporine-treated patients.

Assessment of apoptosis in oesophageal carcinoma preoperatively treated by chemotherapy and radiotherapy.

PubMed

Moreira, L F; Naomoto, Y; Hamada, M; Kamikawa, Y; Orita, K

1995-01-01

Apoptosis, programmed cell death, was immunohistochemically determined in 55 samples of oesophageal squamous cell carcinoma using the BM1 Mab. Sections from patients not treated (group 1, n = 12) or preoperatively treated by chemotherapy (group 2, n = 11), radiation (group 3, n = 13) or both (group 4, n = 8), and 11 additional cases of high-grade dysplasia or early cancer were examined. Most of the apoptotic cells were BM1-positive and checked by TUNEL proved to be nick end positive. They accounted for 7 (11%), 19 (29%), 21 (32%) and 26 (38%) cells per field in those 4 groups respectively. Chemotherapy and/or radiation significantly increased the number of apoptotic cells as compared to controls (p = 0.029 and p = 0.029, respectively). To assess the implications of the oncogene expression in the apoptotic pathway, additional section stained with bcl2 and p53 were negative for bcl2 and were positive for p53 in 16 samples (37%). Overall, positive cases for p53 mutation showed a significantly decreased incidence of apoptotic cells (p = 0.03). These results suggest that in situ assessment of apoptotic response better correlates to the apoptosis induced by radiation than that by chemotherapy, that abnormalities of the p53 protein decrease the apoptotic response in oesophageal carcinoma, and that immunohistochemical analysis of p53 protein helps to determine the sensitivity to these anticancer agents.

The aminobisphosphonate pamidronate controls influenza pathogenesis by expanding a γδ T cell population in humanized mice

PubMed Central

Zheng, Jian; Liu, Yinping; Sia, Sin Fun; Liu, Ming; Qin, Gang; Xiang, Zheng; Lam, Kwok-Tai

2011-01-01

There are few antiviral drugs for treating influenza, and the emergence of antiviral resistance has further limited the available therapeutic options. Furthermore, antivirals are not invariably effective in severe influenza, such as that caused by H5N1 viruses. Thus, there is an urgent need to develop alternative therapeutic strategies. Here, we show that human Vγ9Vδ2 T cells expanded by the aminobisphosphonate pamidronate (PAM) kill influenza virus–infected cells and inhibit viral replication in vitro. In Rag2−/−γc−/− immunodeficient mice reconstituted with human peripheral mononuclear cells (huPBMCs), PAM reduces disease severity and mortality caused by human seasonal H1N1 and avian H5N1 influenza virus, and controls the lung inflammation and viral replication. PAM has no such effects in influenza virus–infected Rag2−/−γc−/− mice reconstituted with Vγ9Vδ2 T cell–depleted huPBMCs. Our study provides proof-of-concept of a novel therapeutic strategy for treating influenza by targeting the host rather than the virus, thereby reducing the opportunity for the emergence of drug-resistant viruses. As PAM has been commonly used to treat osteoporosis and Paget’s disease, this new application of an old drug potentially offers a safe and readily available option for treating influenza. PMID:21708931

Effects of membrane properties on the binding activities of the HN and HC heavy-chain domains of botulinum neurotoxin A.

PubMed

Ayyar, B Vijayalakshmi; Atassi, M Zouhair

2016-12-01

Binding behaviors of the H N and the H C domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A H N and H C regions were prepared (H N 519-845 and H C 967-1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of H N 519-845 to Neuro 2a cells without affecting H C 967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both H C 967-1296 and H N 519-845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both H C 967-1296 and H N 519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K + ) depletion buffer lowered the binding of both H C 967-1296 and H N 519-845 to the cells, but seemed to exert a more pronounced effect on the binding of H C 967-1296 than on the binding of H N 519-845. Results indicate that while both the H N and H C domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells. Copyright © 2016 Elsevier B.V. All rights reserved.

[Induction of uridine 5'-diphosphate-glucuronosyltransferase gene expression by sulforaphane and its mechanism: experimental study in human colon cancel cells].

PubMed

Wang, Min; Li, Yan-Qing; Zhong, Ning; Chen, Jian; Xu, Xiao-Qun; Yuan, Meng-Biao

2005-03-30

To study the induction of expression of uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) 1A in colon cancer cells by sulforaphane (SFN) and its possible mechanism. Human colon cancer cells of the line Caco-2 were cultured and added with SFN of different terminal concentrations, all below the concentration of IC(50). RT-PCR was used to examine the expression of UGT1A mRNA induced by SFN. Western blotting was used to detect the expression of UGT1A protein. The glucuronidation rate of N-hydroxy-PhIP was measured by high performance liquid chromatography (HPLC). The nuclear localization of transcription factor Nrf2 was observed by confocal laser microscopy. (1) Expression of UGT1A mRNA was observed in the Cac0-2 cells induced by SFN of the concentrations of 10 micromol/L approximately 35 micromol/L in a dose-independent manner (P < 0.05). Sulforaphane of the concentration of 25 micromol/L induced the UGT1A mRNA expression time-dependently. The levels of UGT1A1, UGT1A8, and UGT1A10 mRNA expression were significantly increased in the cells treated with 25 micromol/L sulforaphane compared to that in the controls (P = 0.006, P = 0.017, and P = 0.008 respectively). (2) The UGT1A protein band intensity increased significantly in the Coco-2 cells treated with sulforaphane of the concentrations 10 micromol/L approximately 30 micromol/L for 24 h in comparison with the control cells. (3) When the microsomes from the untreated Caco-2 cells were incubated with N-hydroxy-PhIP there was a minor HPLC peak at the expected retention time for N-hydroxy-PhIP-N2-glucuronide. This peak was dramatically increased in the sulforaphane-treated cells, suggesting higher activities of glucuronidation of N-hydroxy-PhIP. (4) Cytoplasmic labeling of NF-E2-related factor 2 (Nrf2), a transcription factor, with no nuclear staining was observed in the non-stimulated cells, whereas an intense nuclear labeling was observed in the sulforaphane-treated cells, indicating the induction of nuclear translocation of Nrf2 by sulforaphane. (1) Low dose sulforaphane induces the expression of UGT1A, UGT1A1, UGT1A A8, and UGT1A A10 mRNA significantly. These changes are accompanied by an increase in UGT1A1 protein and increase in heterocyclic aromatic amine glucuronidation. (2) The induction of the phase II enzyme activity by SFN occurs at the transcriptional level and is regulated by Nrf2.

Further studies on the effect of adenosine cyclic monophosphate derivatives on cell proliferation in the jejunal crypts of rat.

PubMed

Tutton, P J; Barkla, D H

1982-01-01

1. Cell proliferation in the jejunal crypt epithelium of rat was measured using a stathmokinetic technique. 2. Sodium butyrate was found to promote jejunal crypt cell proliferation. 3. N6, O2'-Dibutyryl cyclic adenosine monophosphate (cAMP), N6-monobutyryl-cAMP and N6-monobutyryl-8-bromo-cAMP were found to inhibit cell proliferation when compared to sodium butyrate treated tissues. 4. 8-Chlorophenylthio-cAMP was found to inhibit cell division when compared to untreated animals. 5. O2'-Monobutyryl cAMP and 8-bromo-cAMP were not found to inhibit cell proliferation.

Long-term Fate Mapping to Assess the Impact of Postnatal Isoflurane Exposure on Hippocampal Progenitor Cell Productivity.

PubMed

Jiang, Yifei; Tong, Dongyi; Hofacer, Rylon D; Loepke, Andreas W; Lian, Qingquan; Danzer, Steve C

2016-12-01

Exposure to isoflurane increases apoptosis among postnatally generated hippocampal dentate granule cells. These neurons play important roles in cognition and behavior, so their permanent loss could explain deficits after surgical procedures. To determine whether developmental anesthesia exposure leads to persistent deficits in granule cell numbers, a genetic fate-mapping approach to label a cohort of postnatally generated granule cells in Gli1-CreER::GFP bitransgenic mice was utilized. Green fluorescent protein (GFP) expression was induced on postnatal day 7 (P7) to fate map progenitor cells, and mice were exposed to 6 h of 1.5% isoflurane or room air 2 weeks later (P21). Brain structure was assessed immediately after anesthesia exposure (n = 7 controls and 8 anesthesia-treated mice) or after a 60-day recovery (n = 8 controls and 8 anesthesia-treated mice). A final group of C57BL/6 mice was exposed to isoflurane at P21 and examined using neurogenesis and cell death markers after a 14-day recovery (n = 10 controls and 16 anesthesia-treated mice). Isoflurane significantly increased apoptosis immediately after exposure, leading to cell death among 11% of GFP-labeled cells. Sixty days after isoflurane exposure, the number of GFP-expressing granule cells in treated animals was indistinguishable from control animals. Rates of neurogenesis were equivalent among groups at both 2 weeks and 2 months after treatment. These findings suggest that the dentate gyrus can restore normal neuron numbers after a single, developmental exposure to isoflurane. The authors' results do not preclude the possibility that the affected population may exhibit more subtle structural or functional deficits. Nonetheless, the dentate appears to exhibit greater resiliency relative to nonneurogenic brain regions, which exhibit permanent neuron loss after isoflurane exposure.

Attenuation of drug-stimulated topoisomerase II-DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIalpha.

PubMed Central

Aoyama, M; Grabowski, D R; Dubyak, G R; Constantinou, A I; Rybicki, L A; Bukowski, R M; Ganapathi, M K; Hickson, I D; Ganapathi, R

1998-01-01

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIalpha phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II-DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II-DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N', N'-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II-DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIalpha. Tryptic phosphopeptide mapping of topo IIalpha protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746-29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIalpha are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons. PMID:9841887

The protective effect of aqueous extracts of roselle (Hibiscus sabdariffa L. UKMR-2) against red blood cell membrane oxidative stress in rats with streptozotocin-induced diabetes.

PubMed

Mohamed, Jamaludin; Shing, Saw Wuan; Idris, Muhd Hanis Md; Budin, Siti Balkis; Zainalabidin, Satirah

2013-10-01

The aim of this study was to investigate the protective effects of aqueous extracts of roselle (Hibiscus sabdariffa L. UKMR-2) against red blood cell (RBC) membrane oxidative stress in rats with streptozotocin-induced diabetes. Forty male Sprague-Dawley rats weighing 230-250 g were randomly divided into four groups (n = 10 rats each): control group (N), roselle-treated control group, diabetic group, and roselle-treated diabetic group. Roselle was administered by force-feeding with aqueous extracts of roselle (100 mg/kg body weight) for 28 days. The results demonstrated that the malondialdehyde levels of the red blood cell membranes in the diabetic group were significantly higher than the levels in the roselle-treated control and roselle-treated diabetic groups. The protein carbonyl level was significantly higher in the roselle-treated diabetic group than in the roselle-treated control group but lower than that in the diabetic group. A significant increase in the red blood cell membrane superoxide dismutase enzyme was found in roselle-treated diabetic rats compared with roselle-treated control rats and diabetic rats. The total protein level of the red blood cell membrane, osmotic fragility, and red blood cell morphology were maintained. The present study demonstrates that aqueous extracts of roselle possess a protective effect against red blood cell membrane oxidative stress in rats with streptozotocin-induced diabetes. These data suggest that roselle can be used as a natural antioxidative supplement in the prevention of oxidative damage in diabetic patients.

Treatment of stroke with (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate and bone marrow stromal cells upregulates angiopoietin-1/Tie2 and enhances neovascularization.

PubMed

Cui, X; Chen, J; Zacharek, A; Roberts, C; Savant-Bhonsale, S; Chopp, M

2008-09-22

Neovascularization may contribute to functional recovery after neural injury. Combination treatment of stroke with a nitric oxide donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETA-NONOate) and bone marrow stromal cells promotes functional recovery. However, the mechanisms underlying functional improvement have not been elucidated. In this study, we tested the hypothesis that combination treatment upregulates angiopoietin-1 and its receptor Tie2 in the ischemic brain and bone marrow stromal cells, thereby enhancing cerebral neovascularization after stroke. Adult wild type male C57BL/6 mice were i.v. administered PBS, bone marrow stromal cells 5x10(5), DETA-NONOate 0.4 mg/kg or combination DETA-NONOate with bone marrow stromal cells (n=12/group) after middle cerebral artery occlusion. Combination treatment significantly upregulated angiopoietin-1/Tie2 and tight junction protein (occludin) expression, and increased the number, diameter and perimeter of blood vessels in the ischemic brain compared with vehicle control (mean+ or -S.E., P<0.05). In vitro, DETA-NONOate significantly increased angiopoietin-1/Tie2 protein (n=6/group) and Tie2 mRNA (n=3/group) expression in bone marrow stromal cells. DETA-NONOate also significantly increased angiopoietin-1 protein (n=6/group) and mRNA (n=3/group) expression in mouse brain endothelial cells (P<0.05). Angiopoietin-1 mRNA (n=3/group) was significantly increased in mouse brain endothelial cells treated with DETA-NONOate in combination with bone marrow stromal cell-conditioned medium compared with cells treated with bone marrow stromal cell-conditioned medium or DETA-NONOate alone. Mouse brain endothelial cell capillary tube-like formation assays (n=6/group) showed that angiopoietin-1 peptide, the supernatant of bone marrow stromal cells and DETA-NONOate significantly increased capillary tube formation compared with vehicle control. Combination treatment significantly increased capillary tube formation compared with DETA-NONOate treatment alone. Inhibition of angiopoietin-1 significantly attenuated combination treatment-induced tube formation. Our data indicated that combination treatment of stroke with DETA-NONOate and bone marrow stromal cells promotes neovascularization, which is at least partially mediated by upregulation of the angiopoietin-1/Tie2 axis.

Interferon action: two (2'-5')(A)n synthetases specified by distinct mRNAs in Ehrlich ascites tumor cells treated with interferon.

PubMed

St Laurent, G; Yoshie, O; Floyd-Smith, G; Samanta, H; Sehgal, P B; Lengyel, P

1983-05-01

(2'-5')(A)n synthetase and RNAase L (a latent endoribonuclease) are among the mediators of interferon action. The product of (2'-5')(A)n synthetase (i.e., (2'-5')(A)n) binds, and thereby activates RNAase L. Interferons induce in Ehrlich ascites tumor (EAT) cells two mRNAs (sizes 1.5 kb and 3.8 kb), which can be translated in Xenopus oocytes into (2'-5')(A)n synthetases of 20,000 to 30,000 daltons and 85,000 to 100,000 daltons, respectively. (2'-5')(A)n synthetases of corresponding sizes are induced by interferons in EAT cells. In the cell extract the bulk of the larger enzyme is in the cytoplasmic fraction, and the bulk of the smaller one in the nuclear fraction. The only known function of (2'-5')(A)n is the activation of RNAase L, and RNAase L can be selectively crosslinked to a (2'-5')(A)n derivative in a cytoplasmic extract from EAT cells. The same (2'-5')(A)n derivative can be crosslinked to several proteins in the nuclear extract of EAT cells, and some of these proteins are induced by interferon.

Carbon nanodots prepared from o-phenylenediamine for sensing of Cu2+ ions in cells

NASA Astrophysics Data System (ADS)

Vedamalai, Mani; Periasamy, Arun Prakash; Wang, Chia-Wei; Tseng, Yu-Ting; Ho, Lin-Chen; Shih, Chung-Chien; Chang, Huan-Tsung

2014-10-01

A simple hydrothermal method was applied to prepare carbon nanodots (C dots) from o-phenylenediamine (OPD). The C dots exhibit photoluminescence at 567 nm when excited at 420 nm. In the presence of Cu2+ ions, the colour of C dots changes from yellow to orange, with an increased PL intensity as a result of the formation of Cu(OPD)2 complexes on the surfaces of C dots. The D-band to G-band ratios of C dots in the absence and presence of 80 nM Cu2+ ions are 1.31 and 4.75, respectively. The C dots allow the detection of Cu2+ ions with linearity over a concentration range of 2-80 nM, with a limit of detection of 1.8 nM at a signal-to-noise ratio of 3. The cell viability values of A549, MCF-10A, and MDA-MB-231 cells treated with 3 μg mL-1 of C dots are all greater than 99%, showing their great biocompatibility. Having great water dispersibility, photostability, chemical stability (against NaCl up to 0.5 M), great selectivity, and biocompatibility, the C dots have been employed for the localization of Cu2+ ions in the cancer cells (A549 cells) treated with 10 μM Cu2+ ions.A simple hydrothermal method was applied to prepare carbon nanodots (C dots) from o-phenylenediamine (OPD). The C dots exhibit photoluminescence at 567 nm when excited at 420 nm. In the presence of Cu2+ ions, the colour of C dots changes from yellow to orange, with an increased PL intensity as a result of the formation of Cu(OPD)2 complexes on the surfaces of C dots. The D-band to G-band ratios of C dots in the absence and presence of 80 nM Cu2+ ions are 1.31 and 4.75, respectively. The C dots allow the detection of Cu2+ ions with linearity over a concentration range of 2-80 nM, with a limit of detection of 1.8 nM at a signal-to-noise ratio of 3. The cell viability values of A549, MCF-10A, and MDA-MB-231 cells treated with 3 μg mL-1 of C dots are all greater than 99%, showing their great biocompatibility. Having great water dispersibility, photostability, chemical stability (against NaCl up to 0.5 M), great selectivity, and biocompatibility, the C dots have been employed for the localization of Cu2+ ions in the cancer cells (A549 cells) treated with 10 μM Cu2+ ions. Electronic supplementary information (ESI) available: XRD, FTIR, absorption, Raman scattering, fluorescence spectra, the effect of pH, photostability and cell viability results. See DOI: 10.1039/c4nr03213f

Grape seed procyanidin B2 ameliorates mitochondrial dysfunction and inhibits apoptosis via the AMP-activated protein kinase-silent mating type information regulation 2 homologue 1-PPARγ co-activator-1α axis in rat mesangial cells under high-dose glucosamine.

PubMed

Bao, Lei; Cai, Xiaxia; Zhang, Zhaofeng; Li, Yong

2015-01-14

Grape seed procyanidin B2 (GSPB2), an antioxidative and anti-inflammatory polyphenol in grape seed, has been found to have protective effects on diabetic nephropathy. Based on its favourable biological activities, in the present study, we aimed to investigate whether GSPB2 could inhibit apoptosis in rat mesangial cells treated with glucosamine (GlcN) under high-dose conditions. The results showed that the administration of GSPB2 (10 μg/ml) significantly increased the viability of mesangial cells treated with GlcN at a dose of 15 mM. We found that GSPB2 inhibited apoptosis in mesangial cells using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphates (dUTP) nick-end labelling staining and flow cytometry technique (P< 0·05 for both). GSPB2 treatment also suppressed oxidative stress by elevating the activity of glutathione peroxidase (P< 0·05) and superoxide dismutase (P< 0·01), as well as prevented cellular damage. GSPB2 enhanced the mRNA expression of nuclear respiratory factor 1, mitochondrial transcription factor A and mitochondrial DNA copy number in mesangial cells as determined by real-time PCR (P< 0·05 for each). Finally, GSPB2 treatment activated the protein expression of PPARγ co-activator-1α (PGC-1α), silent mating type information regulation 2 homologue 1 (SIRT1) and AMP-activated protein kinase (AMPK) in mesangial cells. These findings suggest that GSPB2 markedly ameliorates mitochondrial dysfunction and inhibits apoptosis in rat mesangial cells treated with high-dose GlcN. This protective effect could be, at least in part, due to the activation of the AMPK-SIRT1-PGC-1α axis.

Actin cytoskeleton depolymerization with clostridium spiroforme toxin enhances the secretory activity of rat melanotrophs.

PubMed

Chowdhury, H H; Popoff, M R; Zorec, R

1999-12-01

1. We measured membrane capacitance (Cm) in cultured rat melanotrophs pretreated with Clostridium spiroforme toxin (CST), which specifically depolymerizes cortical filamentous actin (F-actin). Phalloidin staining confirmed that CST treatment depolymerised the F-actin. 2. In control cells, cytosol dialysis with 1 microM Ca2+i increased Cm by 23 +/- 4 % (n = 11) relative to the resting Cm 400 s after the start of patch rupture. In CST-treated cells the increase in Cm was 32 +/- 5 % (n = 15), not significantly different from controls. The rate of Cm increase was affected transiently by CST treatment, peaking at 1 min after patch rupture. The maximal rate of Cm increase was 4.27 +/- 0.85 fF s-1 (n = 12; measured 200 s after the start of patch rupture) in controls and 8.0 +/- 1.35 fF s-1 (n = 23; measured 75 s after the start of patch rupture) in CST-treated cells (P < 0.01). 3. In control cells cytosol dialysis with 0 microM Ca2+i decreased Cm by 9 +/- 3 % (n = 7), in CST-treated cells Cm increased by 11 +/- 3 % (n = 7) relative to resting Cm 400 s after the start of cytosol dialysis. The rate of change in Cm remained constant (controls: -1 to -2 fF s-1; CST treatment: 1-2 fF s-1). 4. Transient and sustained effects of CST treatment on changes in Cm at high or low [Ca2+]i, respectively, suggest a distinct role of cytoskeleton in Ca2+-dependent and Ca2+-independent changes in Cm. Transient enhancement of the rate of Cm by CST is consistent with a barrier role of cytoskeleton in regulated exocytosis. The sustained effect of CST on Ca2+-independent changes in Cm suggests cytoskeletal involvement in endocytosis.

Advanced glycation end products promote ChREBP expression and cell proliferation in liver cancer cells by increasing reactive oxygen species.

PubMed

Chen, Hanbei; Li, Yakui; Zhu, Yemin; Wu, Lifang; Meng, Jian; Lin, Ning; Yang, Dianqiang; Li, Minle; Ding, WenJin; Tong, Xuemei; Su, Qing

2017-08-01

The aim of the study was to elucidate the mechanism by which advanced glycation end products (AGEs) promote cell proliferation in liver cancer cells.We treated liver cancer HepG2 cells with 200 mg/L AGEs or bovine serum albumin (BSA) and assayed for cell viability, cell cycle, and apoptosis. We performed real-time PCR and Western blot analysis for RNA and protein levels of carbohydrate responsive element-binding protein (ChREBP) in AGEs- or BSA-treated HepG2 cells. We analyzed the level of reactive oxygen species (ROS) in HepG2 cells treated with AGEs or BSA.We found that increased S-phase cell percentage and decreased apoptosis contributed to AGEs-induced liver cancer cell proliferation. Real-time PCR and Western blot analysis showed that AGEs stimulated RNA and protein levels of ChREBP, a transcription factor promoting glycolysis and maintaining cell proliferation in liver cancer cells. Intriguingly, the level of ROS was higher in AGEs-treated liver cancer cells. Treating liver cancer cells with antioxidant N-acetyl cystein (NAC) partly blocked AGEs-induced ChREBP expression and cell proliferation.Our results suggest that the AGEs-ROS-ChREBP pathway plays a critical role in promoting ChREBP expression and liver cancer cell proliferation.

Antifungal mechanism of an antimicrobial peptide, HP (2--20), derived from N-terminus of Helicobacter pylori ribosomal protein L1 against Candida albicans.

PubMed

Lee, Dong Gun; Park, Yoonkyung; Kim, Hee Nam; Kim, Hyung Keun; Kim, Pyoung Il; Choi, Bo Hwa; Hahm, Kyung-Soo

2002-03-08

The antifungal activity and mechanism of HP (2-20), a peptide derived from the N-terminus sequence of Helicobacter pylori Ribosomal Protein L1 were investigated. HP (2--20) displayed a strong antifungal activity against various fungi, and the antifungal activity was inhibited by Ca(2+) and Mg(2+) ions. In order to investigate the antifungal mechanism(s) of HP (2-20), fluorescence activated flow cytometry was performed. As determined by propidium iodide staining, Candida albicans treated with HP (2-20) showed a higher fluorescence intensity than untreated cells and was similar to melittin-treated cells. The effect on fungal cell membranes was examined by investigating the change in membrane dynamics of C. albicans using 1,6-diphenyl-1,3,5-hexatriene as a membrane probe and by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w) and by treating protoplasts of C. albicans with the peptide. The action of peptide against fungal cell membrane was further examined by the potassium-release test, and HP (2-20) was able to increase the amount of K(+) released from the cells. The result suggests that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membrane via pore formation or directly interacts with the lipid bilayers in a salt-dependent manner.

Protection of LLC-PK1 cells against hydrogen peroxide-induced cell death by modulation of ceramide level.

PubMed

Yoo, Jae-Myung; Lee, Youn-Sun; Choi, Heon-Kyo; Lee, Yong-Moon; Hong, Jin-Tae; Yun, Yeo-Pyo; Oh, Seikwan; Yoo, Hwan-Soo

2005-03-01

Oxidative stress has been reported to elevate ceramide level during cell death. The purpose of the present study was to modulate cell death in relation to cellular glutathione (GSH) level and GST (glutathione S-transferase) expression by regulating the sphingolipid metabolism. LLC-PK1 cells were treated with H2O2 in the absence of serum to induce cell death. Subsequent to exposure to H2O2, LLC-PK1 cells were treated with desipramine, sphingomyelinase inhibitor, and N-acetylcysteine (NAC), GSH substrate. Based on comparative visual observation with H2O2-treated control cells, it was observed that 0.5 microM of desipramine and 25 mM of NAC exhibited about 90 and 95% of cytoprotection, respectively, against H2O2-induced cell death. Desipramine and NAC lowered the release of LDH activity by 36 and 3%, respectively, when compared to 71% in H2O2-exposed cells. Cellular glutathione level in 500 microM H2O2-treated cells was reduced to 890 pmol as compared to control level of 1198 pmol per mg protein. GST P1-1 expression was decreased in H2O2-treated cells compared to healthy normal cells. In conclusion, it has been inferred that H2O2-induced cell death is closely related to cellular GSH level and GST P1-1 expression in LLC-PK1 cells and occurs via ceramide elevation by sphingomyelinase activation.

The effect of SiO2/Au core-shell nanoparticles on breast cancer cell's radiotherapy.

PubMed

Darfarin, Ghazal; Salehi, Roya; Alizadeh, Effat; Nasiri Motlagh, Behnam; Akbarzadeh, Abolfazl; Farajollahi, Alireza

2018-05-09

Recently it has been shown that radiation dose enhancement could be achievable in radiotherapy using nanoparticles (NPs). In this study, evaluation was made to determine efficiency of gold-silica shell-core NP in megavoltage irradiation of MCF7 breath cancer cells. Gold-silicon oxide shell-core NPs were obtained by conjugation of gold NP with amine or thiol functionalized silica NPs (AuN@SiO 2 and AuS@SiO 2 ). Cellular uptake and cytotoxicity of NPs were examined by fluorescent microscopy and MTT assay, respectively. MCF-7 breast cancer cells were treated with both NPs and irradiation was made with X-ray energies of 6 and 18 MV to the absorbed dose of 2, 4 and 8 Gy using Simense linear accelerator. The efficiency of radiation therapy was then evaluated by MTT and Brdu assay, DAPI staining and cell cycle analysis. TEM images indicated that synthesized NPs had average diameter of 25 nm. Cellular uptake demonstrated that the internalization of AuS@SiO 2 and AuN@SiO 2 NPs amounted to 18% and 34%, 3 h post treatment, respectively. Nontoxicity of prepared NPs on MCF-7 cells was proved by MTT and Brdu assays as well as DAPI staining and cell cycle studies. The highest enhancement in radiation dose was observed in the cells that irradiated with radiation energy of 18 MV and absorbed of 8 Gy at NPs concentration of 200 ppm. The Brdu findings revealed that the cytotoxicity and apoptosis on MCF-7 cells are dose dependent with a significantly more death in AuN@SiO 2 (amine) exposed cells (p < .05). Analysis also revealed interruption in cell cycle by demonstrating lack of cells, in S phase in amine treated cells (AuN@SiO 2 ) at given dose of 8 Gy using 18 MV X-ray in comparison to thiol treated cells. Based on the results of the study it can be concluded that the gold-silicon oxide shell-core NPs could play an effective role in radiotherapy of MCF-7 breast cancer cells.

Actin cytoskeleton depolymerization with Clostridium spiroforme toxin enhances the secretory activity of rat melanotrophs

PubMed Central

Chowdhury, Helena H; Popoff, Michel R; Zorec, Robert

1999-01-01

We measured membrane capacitance (Cm) in cultured rat melanotrophs pretreated with Clostridium spiroforme toxin (CST), which specifically depolymerises cortical filamentous actin (F-actin). Phalloidin staining confirmed that CST treatment depolymerised the F-actin. In control cells, cytosol dialysis with 1 μm Ci2+ increased Cm by 23 ± 4% (n = 11) relative to the resting Cm 400 s after the start of patch rupture. In CST-treated cells the increase in Cm was 32 ± 5% (n = 15), not significantly different from controls. The rate of Cm increase was affected transiently by CST treatment, peaking at 1 min after patch rupture. The maximal rate of Cm increase was 4.27 ± 0.85 fF s−1 (n = 12; measured 200 s after the start of patch rupture) in controls and 8.0 ± 1.35 fF s−1 (n = 23; measured 75 s after the start of patch rupture) in CST-treated cells (P < 0.01). In control cells cytosol dialysis with 0 μm Ci2+ decreased Cm by 9 ± 3% (n = 7), in CST-treated cells Cm increased by 11 ± 3% (n = 7) relative to resting Cm 400 s after the start of cytosol dialysis. The rate of change in Cm remained constant (controls: -1 to -2 fF s−1; CST treatment: 1-2 fF s−1). Transient and sustained effects of CST treatment on changes in Cm at high or low [Ca2+]i, respectively, suggest a distinct role of cytoskeleton in Ca2+-dependent and Ca2+-independent changes in Cm. Transient enhancement of the rate of Cm by CST is consistent with a barrier role of cytoskeleton in regulated exocytosis. The sustained effect of CST on Ca2+-independent changes in Cm suggests cytoskeletal involvement in endocytosis. PMID:10581310

High rate of long-term survival for high-risk lymphoma patients treated with hematopoietic stem cell transplantation as consolidation or salvage therapy.

PubMed

Espigado, I; Ríos, E; Marín-Niebla, A; Carmona, M; Parody, R; Pérez-Hurtado, J M; Márquez, F J; Urbano-Ispizua, A

2008-11-01

Patients with high-relapse-risk lymphomas or those relapsing after initial therapy have a limited probability of cure with conventional treatment. There is recent inconclusive evidence that, in such cases, intensification or salvage treatment with high-dose chemotherapy followed by hematopoietic stem cell transplantation (HSCT) increases the response rate and may improve survival. Nevertheless, published data on long-term follow-up of high-risk lymphoma patients treated with HSCT are scarce. We analyzed 101 consecutive patients receiving high-dose chemotherapy followed by HSCT after induction with standard chemotherapy. The median age was 38 years (range, 12-63 years). The diagnoses were Hodgkin's disease (n = 32), follicular lymphoma (n = 33), diffuse large B-cell lymphoma (n = 12), mantle cell lymphoma (n = 7), T-cell lymphoma (n = 14), and others (n = 3). Patients received either an autologous graft (n = 72) in first complete remission (1CR; n = 23) or in advanced stages (AS; n = 49), or an allogeneic graft (n = 29) in 1CR (n = 7) or in AS (n = 22). We concluded that transplant-related mortality was 2.7% for patients receiving an autologous HSCT and 27% for patients receiving an allogeneic HSCT. The main etiologies were graft-versus-host disease and infection in the allogeneic setting, and infection in the autologous setting. The probability of long-term (12-year) overall survival was 71%, higher than that described for high-relapse-risk lymphoma patients treated without HSCT and significantly better (P < .05) for patients who received the transplant in 1CR (89%) than in AS (65%). Finally, the probability of long-term survival was significantly better for patients treated with HSCT during the period from 2000-2007 (85%) compared with the period from 1989-1999 (72%).

Enhancing the biocompatibility of the polyurethane methacrylate and off-stoichiometry thiol-ene polymers by argon and nitrogen plasma treatment.

PubMed

Chen, Tiam Foo; Siow, Kim Shyong; Ng, Pei Yuen; Majlis, Burhanuddin Yeop

2017-10-01

Our studies focused on improving the biocompatibility properties of two microfluidic prototyping substrates i.e. polyurethane methacrylate (PUMA) and off-stoichiometry thiol-ene (OSTE-80) polymer by Ar and N 2 plasma treatment. The contact angle (CA) measurement showed that both plasma treatments inserted oxygen and nitrogen moieties increased the surface energy and hydrophilicity of PUMA and OSTE-80 polymer which corresponded to an increase of nitrogen to carbon ratios (N/C), as measured by XPS, to provide a conducive environment for cell attachments and proliferation. Under the SEM observation, the surface topography of PUMA and OSTE-80 polymer showed minimal changes after the plasma treatments. Furthermore, ageing studies showed that plasma-treated PUMA and OSTE-80 polymer had stable hydrophilicity and nitrogen composition during storage in ambient air for 15days. After in vitro cell culture of human umbilical vein endothelial cells (HUVECs) on these surfaces for 24h and 72h, both trypan blue and alamar blue assays indicated that PUMA and OSTE-80 polymer treated with N 2 plasma had the highest viability and proliferation. The polar nitrogen moieties, specifically amide groups, encouraged the HUVECs adhesion on the plasma-treated PUMA and OSTE-80 surfaces. Interestingly, PUMA polymer treated with Ar and N 2 plasma showed different HUVECs morphology which was spindle and cobblestone-shaped respectively after 72h of incubation. On the contrary, a monolayer of well-spread HUVECs formed on the Ar and N 2 plasma-treated OSTE-80 polymers. These variable morphologies observed can be ascribed to the adherence HUVECs on the different elastic moduli of these surfaces whereby further investigation might be needed. Overall, Ar and N 2 plasma treatment had successfully altered the surface properties of PUMA and OSTE-80 polymer by increasing its surface energy, hydrophilicity and chemical functionalities to create a biocompatible surface for HUVECs adhesion and proliferation. Copyright © 2017 Elsevier B.V. All rights reserved.

Ethylene is required for elicitin-induced oxidative burst but not for cell death induction in tobacco cell suspension cultures.

PubMed

Koehl, Julia; Djulic, Alma; Kirner, Veronika; Nguyen, Tach Thao; Heiser, Ingrid

2007-12-01

The signal compound ethylene and its relationships with oxidative burst and cell death were analyzed in cultured tobacco cells treated with the proteinaceous elicitor quercinin. Quercinin belongs to the protein family of elicitins and was isolated from the soil-born oak pathogen Phytophthora quercina. It was shown to induce a dose-dependent oxidative burst in tobacco cell culture in concentrations from 0.05 to 0.5 nM, and subsequently, cell death. The characteristics of quercinin-induced cell death included both membrane damage and DNA fragmentation in tobacco cell culture. At higher quercinin concentrations (2 nM), H(2)O(2) formation and ethylene biosynthesis were inhibited. Ethylene at low concentrations proved to be necessary for induction and maintenance of H(2)O(2) production in tobacco cells treated with quercinin. It was demonstrated that external addition of inhibitors of ethylene biosynthesis such as alpha-amino-oxy-acetic acid (AOA) and CoCl(2) also decreased or even inhibited the quercinin-induced oxidative burst, but did not influence cell death induction. These results demonstrate evidence for a requirement of the plant hormone ethylene for the onset of the quercinin-induced oxidative burst.

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid

PubMed Central

2013-01-01

Background Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. Results We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. Conclusion Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity. PMID:23597229

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid.

PubMed

Andres, Devon; Keyser, Brian M; Petrali, John; Benton, Betty; Hubbard, Kyle S; McNutt, Patrick M; Ray, Radharaman

2013-04-18

Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity.

Characterization of lymphopenia in patients with MS treated with dimethyl fumarate and fingolimod.

PubMed

Nakhaei-Nejad, Maryam; Barilla, David; Lee, Chieh-Hsin; Blevins, Gregg; Giuliani, Fabrizio

2018-03-01

Lymphopenia is a common occurrence of disease-modifying therapies (DMTs) for relapsing-remitting MS (RRMS). The aim of this study was to dissect the prevalence of various lymphocyte subsets in patients with RRMS treated with 2 DMTs commonly associated with lymphopenia, dimethyl fumarate (DMF), and fingolimod (FTY). Multicolor flow cytometry and multiplex assays were used to identify up to 50 lymphocyte subpopulations and to examine the expression of multiple cytokines in selected patients. We compared patients untreated (NT) or treated with FTY or DMF who did (DMF-L) or did not (DMF-N) develop lymphopenia. All FTY patients developed lymphopenia in both T-cell and B-cell compartments. CD41 T cells were more affected by this treatment than CD81 cells. In the B-cell compartment, the CD271IgD2 subpopulation was reduced. T cells but not B cells were significantly reduced in DMF-L. However, within the B cells, CD271 cells were significantly lower. Both CD41 and CD81 subpopulations were reduced in DMF-L. Within the remaining CD41 and CD81 compartments, there was an expansion of the naive subpopulation and a reduction of the effector memory subpopulation. Unactivated lymphocyte from DMF-L patients had significantly higher levels of interferon-γ, interleukin (IL)-12, IL-2, IL-4, IL-6, and IL-1β compared with DMF-N. In plasma, TNFβ was significantly higher in DMF-N and DMF-L compared with NT, whereas CCL17 was significantly higher in DMF-L compared with NT and DMF-N. This study shows that different treatments can target different lymphocyte compartments and suggests that lymphopenia can induce compensatory mechanisms to maintain immune homeostasis.

Characterization of lymphopenia in patients with MS treated with dimethyl fumarate and fingolimod

PubMed Central

Nakhaei-Nejad, Maryam; Barilla, David; Lee, Chieh-Hsin; Blevins, Gregg

2017-01-01

Objective: Lymphopenia is a common occurrence of disease-modifying therapies (DMTs) for relapsing-remitting MS (RRMS). The aim of this study was to dissect the prevalence of various lymphocyte subsets in patients with RRMS treated with 2 DMTs commonly associated with lymphopenia, dimethyl fumarate (DMF), and fingolimod (FTY). Methods: Multicolor flow cytometry and multiplex assays were used to identify up to 50 lymphocyte subpopulations and to examine the expression of multiple cytokines in selected patients. We compared patients untreated (NT) or treated with FTY or DMF who did (DMF-L) or did not (DMF-N) develop lymphopenia. Results: All FTY patients developed lymphopenia in both T-cell and B-cell compartments. CD41 T cells were more affected by this treatment than CD81 cells. In the B-cell compartment, the CD271IgD2 subpopulation was reduced. T cells but not B cells were significantly reduced in DMF-L. However, within the B cells, CD271 cells were significantly lower. Both CD41 and CD81 subpopulations were reduced in DMF-L. Within the remaining CD41 and CD81 compartments, there was an expansion of the naive subpopulation and a reduction of the effector memory subpopulation. Unactivated lymphocyte from DMF-L patients had significantly higher levels of interferon-γ, interleukin (IL)-12, IL-2, IL-4, IL-6, and IL-1β compared with DMF-N. In plasma, TNFβ was significantly higher in DMF-N and DMF-L compared with NT, whereas CCL17 was significantly higher in DMF-L compared with NT and DMF-N. Conclusions: This study shows that different treatments can target different lymphocyte compartments and suggests that lymphopenia can induce compensatory mechanisms to maintain immune homeostasis. PMID:29296636

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-beta, and H2O2.

PubMed

Lehmann, Petra; Rank, Petra; Hallfeldt, Klaus L J; Krebs, Bjarne; Gärtner, Roland

2006-08-01

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), as well as H2O2 and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-beta (5 ng/mL), or H2O2 (100 muM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. Asignificant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 microM), thyroidstimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 microM iodine, EGF (5 ng/mL) and TGF-beta (5 ng/mL), or H2O2 (100 microM) (p < 0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H2O2-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4 +/- 0.2 to 2.25 +/- 0.4 mU/microg DNA with 10 nMselenite and 2.6 + 0.4 mU/microg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-beta, EGF, iodide, and even H2O2.

Methamphetamine toxicity-induced calcineurin activation, nuclear translocation of nuclear factor of activated T-cells and elevation of cyclooxygenase 2 levels are averted by calpastatin overexpression in neuroblastoma SH-SY5Y cells.

PubMed

Chetsawang, Jirapa; Nudmamud-Thanoi, Sutisa; Phonchai, Ruchee; Abubakar, Zuroida; Govitrapong, Piyarat; Chetsawang, Banthit

2018-06-23

Methamphetamine (METH) is an addictive stimulant drug that has many negative consequences, including toxic effects to the brain. Recently, the induction of inflammatory processes has been identified as a potential contributing factor to induce neuronal cell degeneration. It has been demonstrated that the expression of inflammatory agents, such as cyclooxygenase 2 (COX-2), depends on the activation of calcineurin (CaN) and nuclear factor of activated T-cells (NFAT). Moreover, the excessive elevation in cytosolic Ca 2+ levels activates the cell death process, including calpain activation in neurons, which was diminished by the overexpression of the calpain inhibitor protein, calpastatin. However, it is unclear whether calpain mediates CaN-NFAT activation in the neurotoxic process. In the present study, we observed that the toxic high dose of METH-treated neuroblastoma SH-SY5Y cells significantly decreased cell viability but increased apoptotic cell death, the active cleaved form of calcineurin, the nuclear translocation of NFAT, and COX-2 levels. Nevertheless, these toxic effects were diminished in METH-treated calpastatin-overexpressing SH-SY5Y cells. These findings might emphasize the role of calpastatin against METH-induced toxicity by a mechanism related to calpain-dependent CaN-NFAT activation-induced COX-2 expression. Copyright © 2018. Published by Elsevier B.V.

Zinc affects miR-548n, SMAD4, SMAD5 expression in HepG2 hepatocyte and HEp-2 lung cell lines.

PubMed

Grider, Arthur; Lewis, Richard D; Laing, Emma M; Bakre, Abhijeet A; Tripp, Ralph A

2015-12-01

MicroRNAs affect disease progression and nutrient status. miR-548n increased 57 % in Zn supplemented plasma from adolescent females (ages 9 to 13 years). The purpose of this study was to determine the effects of Zn concentration in cell culture on the expression of miR-548n, SMAD4 and SMAD5 in hepatocyte (HepG2) and lung epithelium (HEp-2) cell lines. Cells were incubated for 48 h in media containing 10 % Chelex 100-treated FBS (0 μM Zn), or with 15 or 50 μM Zn, before isolation of total RNA and cDNA. Expression of miR-548n, SMAD4 and SMAD5 was measured by qPCR. The ΔΔCT method was used to calculate the fold-change, and 15 µM expression levels were used as reference values. HepG2 miR-548n expression decreased 5-fold, and SMAD4 expression increased 4-fold in the absence of Zn, while HEp-2 miR-548n expression increased 10.5-fold, and SMAD5 expression increased 20-fold in the absence of Zn. HEp-2 miR-548n expression increased 23-fold, while SMAD4 expression decreased twofold, in 50 μM Zn-treated cells. However, SMAD4 and SMAD5 expression was not correlated. These data indicate that miR-548n expression is in part regulated by Zn in a cell-specific manner. SMAD4 and SMAD5 are genes in the TGF-β/BMP signaling pathway, and SMAD5 is a putative target for miR-548n; Zn participates in regulating this pathway through controlling SMAD4 and SMAD5 expression. However, SMAD5 expression may be more sensitive to Zn than to miR-548n since SMAD5 expression was not inversely correlated with miR-548n expression.

Paraoxonase 2 modulates a proapoptotic function in LS174T cells in response to quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone

PubMed Central

Tao, Shiyu; Luo, Yanwen; Bin He; Liu, Jie; Qian, Xi; Ni, Yingdong; Zhao, Ruqian

2016-01-01

A mucus layer coats the gastrointestinal tract and serves as the first line of intestinal defense against infection. N-acyl-homoserine lactone (AHL) quorum-sensing molecules produced by gram-negative bacteria in the gut can influence the homeostasis of intestinal epithelium. In this study, we investigated the effects of two representative long- and short-chain AHLs, N-3-(oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl homoserine lactone (C4-HSL), on cell viability and mucus secretion in LS174T cells. C12-HSL but not C4-HSL significantly decreased cell viability by inducing mitochondrial dysfunction and activating cell apoptosis which led to a decrease in mucin expression. Pretreatment with lipid raft disruptor (Methyl-β-cyclodextrin, MβCD) and oxidative stress inhibitor (N-acetyl-L-cysteine, NAC) slightly rescued the viability of cells damaged by C12-HSL exposure, while the paraoxonase 2 (PON2) inhibitor (Triazolo[4,3-a]quinolone, TQ416) significantly affected recovering cells viability and mucin secretion. When LS174T cells were treated with C12-HSL and TQ416 simultaneously, TQ416 showed the maximal positive effect on cells viability. However, if cells were first treated with C12-HSL for 40 mins, and then TQ46 was added, the TQ416 had no effect on cell viability. These results suggest that the C12-HSL-acid process acts at an early step to activate apoptosis as part of C12-HSL’s effect on intestinal mucus barrier function. PMID:27364593

Cellular Encapsulation Enhances Cardiac Repair

PubMed Central

Levit, Rebecca D.; Landázuri, Natalia; Phelps, Edward A.; Brown, Milton E.; García, Andrés J.; Davis, Michael E.; Joseph, Giji; Long, Robert; Safley, Susan A.; Suever, Jonathan D.; Lyle, Alicia N.; Weber, Collin J.; Taylor, W. Robert

2013-01-01

Background Stem cells for cardiac repair have shown promise in preclinical trials, but lower than expected retention, viability, and efficacy. Encapsulation is one potential strategy to increase viable cell retention while facilitating paracrine effects. Methods and Results Human mesenchymal stem cells (hMSC) were encapsulated in alginate and attached to the heart with a hydrogel patch in a rat myocardial infarction (MI) model. Cells were tracked using bioluminescence (BLI) and cardiac function measured by transthoracic echocardiography (TTE) and cardiac magnetic resonance imaging (CMR). Microvasculature was quantified using von Willebrand factor staining and scar measured by Masson's Trichrome. Post‐MI ejection fraction by CMR was greatly improved in encapsulated hMSC‐treated animals (MI: 34±3%, MI+Gel: 35±3%, MI+Gel+hMSC: 39±2%, MI+Gel+encapsulated hMSC: 56±1%; n=4 per group; P<0.01). Data represent mean±SEM. By TTE, encapsulated hMSC‐treated animals had improved fractional shortening. Longitudinal BLI showed greatest hMSC retention when the cells were encapsulated (P<0.05). Scar size at 28 days was significantly reduced in encapsulated hMSC‐treated animals (MI: 12±1%, n=8; MI+Gel: 14±2%, n=7; MI+Gel+hMSC: 14±1%, n=7; MI+Gel+encapsulated hMSC: 7±1%, n=6; P<0.05). There was a large increase in microvascular density in the peri‐infarct area (MI: 121±10, n=7; MI+Gel: 153±26, n=5; MI+Gel+hMSC: 198±18, n=7; MI+Gel+encapsulated hMSC: 828±56 vessels/mm2, n=6; P<0.01). Conclusions Alginate encapsulation improved retention of hMSCs and facilitated paracrine effects such as increased peri‐infarct microvasculature and decreased scar. Encapsulation of MSCs improved cardiac function post‐MI and represents a new, translatable strategy for optimization of regenerative therapies for cardiovascular diseases. PMID:24113327

Peroxiredoxin Expression of Human Osteosarcoma Cells Is Influenced by Cold Atmospheric Plasma Treatment.

PubMed

Gümbel, Denis; Gelbrich, Nadine; Napp, Matthias; Daeschlein, Georg; Kramer, Axel; Sckell, Axel; Burchardt, Martin; Ekkernkamp, Axel; Stope, Matthias B

2017-03-01

To evaluate the potential involvement of redox-specific signalling pathways in cold atmospheric plasma (CAP)-induced apoptosis on human osteosarcoma cells. Osteosarcoma cell lines were treated with CAP with or without antioxidative agents and seeded in cell culture plates. Cell proliferation was determined by counting viable cells. Carrier gas-treated cells served as control. Peroxiredoxin (PRX) 1-3 expression and secretion were assessed. CAP treatment exhibited strongly attenuated proliferation rates. This effect was significantly attenuated by the addition of N-acetylcysteine (NAC). CAP-treated cells exhibited an increase of PRX 1 and 2 10 sec after treatment. The ratio of oxidized to reduced PRX1 and PRX2 was significantly altered with increasing cellular concentration of the oxidized dimer. Antioxidant supplementation with NAC increases proliferation of CAP-treated osteosarcoma cells, implicating an involvement of redox signalling. Activation of PRX1 and -2 indicate CAP affects redox homeostasis. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Comparison of the IN VITRO Cytotoxicities of Nitrogen Doped (p-TYPE) and n-TYPE Zinc Oxide Nanoparticles

NASA Astrophysics Data System (ADS)

Fujihara, Junko; Hashimoto, Hideki; Nishimoto, Naoki; Tongu, Miki; Fujita, Yasuhisa

The use of NPs in the health care field is increasing. Before their biological application, investigating the toxicities of both n-type ZnO nanoparticles (NPs) and nitrogen-doped (“p-type”) NPs is important. Using L929 cells, the cell viability, oxidative stress, apoptosis induction, inflammatory responses, and cellular uptake were assayed 24h after the addition of n-type ZnO NPs and nitrogen-doped NPs (which act as p-type) (25μg/mL). The ZnO NPs were fabricated using a gas evaporation method. Increased H2O2 generation and decreased levels of glutathione were more evident in with n-type than in those treated with nitrogen-doped (“p-type”) ZnO NPs. Caspase-3/-7 activity was higher in cells treated with n-type ZnO NPs than in those treated with nitrogen-doped (“p-type”) NPs. Elevated levels of TNF-α and IL-1β were observed in cell culture supernatants: IL-1β levels were higher in n-type ZnO NPs than nitrogen-doped (“p-type”) NPs. The cellular Zn uptake of n-type ZnO NPs was higher than nitrogen-doped (“p-type”) NPs. These findings show that n-type ZnO NPs have higher cytotoxicity than nitrogen-doped (“p-type”) ZnO NPs. This may be due to a reductive effect of n-type ZnO NPs that induces higher free radical production, reactive oxygen species (ROS) generation, and cellular uptake of this type of ZnO NPs.

Effect of N-CAM on in vitro invasion of human adenoid cystic carcinoma cells.

PubMed

França, C M; Jaeger, R G; Freitas, V M; Araújo, N S; Jaeger, M M

2001-12-01

Adenoid cystic carcinoma of salivary glands is characterised by aggressive behaviour, high rate of local recurrences, neurotropism and late metastasis. In a previous work we demonstrated that adenoid cystic carcinoma cultured cells (CAC2 cells) expressed N-CAM. It was suggested that this expression, modulated by extracellular matrix, would be correlated to cell movement. The aim of our study was to verify whether CAC2 cells presented invasion capacity. Moreover, we tested whether the neural adhesion molecule (N-CAM) would participate in this process. CAC2 cells were either previously treated, or not (control), with a monoclonal antibody against N-CAM. Invasion assays were carried out using a modified Boyden chamber (Transwell chamber). CAC2 cells (10(5)) were dispensed into Transwell upper chamber on the top of Matrigel coated filter. The cells that invaded the filters in the first 8 h were counted under light microscopy, yielding data for the invasion rates (%). Control CAC2 cells presented an invasion rate of 5.28+/-0.04%. The invasion rate raised to 6.53+/-0.2% when N-CAM was blocked with monoclonal antibody. N-CAM impaired the adenoid cystic carcinoma cell invasion in vitro. Therefore, we suggest an anti-invasive role for N-CAM in adenoid cystic carcinoma.

Prognostic role of lymphatic vessel density and lymphovascular invasion in chemotherapy-naive and chemotherapy-treated patients with invasive breast cancer

PubMed Central

Niemiec, Joanna A; Adamczyk, Agnieszka; Ambicka, Aleksandra; Mucha-Małecka, Anna; Wysocki, Wojciech M; Biesaga, Beata; Ziobro, Marek; Cedrych, Ida; Grela-Wojewoda, Aleksandra; Domagała-Haduch, Małgorzata; Wysocka, Joanna; Ryś, Janusz; Sas-Korczyńska, Beata

2017-01-01

It is assumed that the spread of breast cancer cells via the lymphatic system might be influenced by inflammatory reactions and/or the application of chemotherapy or molecularly targeted therapy. Therefore, we analysed survival according to lymphatic vessel density (LVD), lymphovascular invasion (LVI) (both assessed using podoplanin as immunohistochemical marker of lymphatic endothelium) and well-established clinico-pathological features in a group of 358 patients with invasive ductal breast cancer: 139 chemotherapy-naïve (pT1-2/pN0/M0) and 219 treated with chemotherapy (pT1-4/pN1-3/M0). Univariate analysis revealed that high LVD was related to unfavourable disease-free survival (DFS) in pN0/chemotherapy/trastuzumab-naïve patients (P = 0.028). Conversely, in pN+/chemotherapy-treated individuals high LVD was related to favourable DFS (P = 0.019). LVI was a significant indicator of survival (P = 0.005) only in pN0/chemotherapy/trastuzumab-naïve patients. The following parameters were significant independent adverse prognostic factors for DFS: (i) in pN0/chemotherapy/trastuzumab-naïve patients: high LVD (LVD > 7 vessels/mm2; RR = 2.7, P = 0.039), LVI (RR = 3.3, P = 0.046) and high tumor grade (G3 vs. G1 + G2; RR = 2.6, P = 0.030); (ii) in pN+/chemotherapy/trastuzumab-treated patients: low LVD (RR = 1.8, P = 0.042), the number of involved lymph nodes (pN3 vs. pN1-2; RR = 2.3, P = 0.012) and the breast cancer subtype (expression of steroid receptors together with HER2 immunonegativity and high proliferation index vs. other breast cancer immunophenotypes; RR = 3.0, P < 0.001). High LVD may identify high progression risk in pN0/chemotherapy/trastuzumab-naïve patients, and low progression risk in pN+/chemotherapy-treated patients. This phenomenon might be explained by potential involvement of lymphangiogenesis in two processes related to cancer eradication: a chemotherapy-stimulated activity of the immune system against cancer cells, or increased tumour drainage influencing the efficacy of cytotoxic drugs. PMID:28386369

Interfering With Lipid Raft Association: A Mechanism to Control Influenza Virus Infection By Sambucus Nigra

PubMed Central

Shahsavandi, Shahla; Ebrahimi, Mohammad Majid; Hasaninejad Farahani, Ameneh

2017-01-01

Sambucus nigra (elder) are broadly used species to treat microbial infections. The potential antiviral activity and mechanism action of elder fruit (EF) in human epithelium cell (A549) cultures infected with H9N2 influenza virus were determined. The effect of various concentrations of EF on influenza virus replication was examined by using virus titration, quantitative real time RT-PCR, fusion and lipid raft assays following two treatment procedures: A) pre-treated H9N2 virus with each concentration of EF extract and transfection of A549 cell cultures, and B) each concentrations of EF was added to H9N2 virus infected-cell cultures following virus adsorption. In both treatments with lower doses of EF increased viral titer as well as synthesized viral nucleoprotein as indicating the herb had no inhibitory effects on virus replication. In (B) trial with higher doses, 40 and 80 μg/mL of EF, a significant decrease in virus titer and viral protein synthesis were shown in EF treated cells indicating the herb affect either entry of viruses or inhibition virus particle release. The results suggest that EF treatment of the influenza virus infected-human epithelial cells may involve in lipid raft association which function as platform for formation of viral membrane fusion and budding. Differencesin treatment time and dose of EF extract in infected cells with influenza virus have a marked effect on the efficacy of the herb. PMID:29201101

Stable N-CuInSe.sub.2 /iodide-iodine photoelectrochemical cell

DOEpatents

Cahen, David; Chen, Yih W.

1985-01-01

In a photoelectrochemical solar cell, stable output and solar efficiency in excess of 10% are achieved with a photoanode of n-CuInSe.sub.2 electrode material and an iodine/iodide redox couple used in a liquid electrolyte. The photoanode is prepared by treating the electrode material by chemical etching, for example in Br.sub.2 /MeOH; heating the etched electrode material in air or oxygen; depositing a surface film coating of indium on the electrode material after the initial heating; and thereafter again heating the electrode material in air or oxygen to oxidize the indium. The electrolyte is treated by the addition of Cu.sup.+ or Cu.sup.2+ salts and In.sup.3+ salts.

Stable n-CuInSe/sub 2/iodide-iodine photoelectrochemical cell

DOEpatents

Cahen, D.; Chen, Y.W.

1984-09-20

In a photoelectrochemical solar cell, stable output and solar efficiency in excess of 10% are achieved with a photoanode of n-CuInSe/sub 2/ electrode material and an iodine/iodide redox couple used in a liquid electrolyte. The photoanode is prepared by treating the electrode material by chemical etching, for example in Br/sub 2//MeOH; heating the etched electrode material in air or oxygen; depositing a surface film coating of indium on the electrode material after the initial heating; and thereafter again heating the electrode material in air or oxygen to oxidize the indium. The electrolyte is treated by the addition of Cu/sup +/ or Cu/sup 2 +/ salts and in In/sup 3 +/ salts.

Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kyotani, Yoji, E-mail: cd147@naramed-u.ac.jp; Department of Pharmacy, Nara Medical University Hospital, Kashihara 634-8522; Ota, Hiroyo

Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned mediummore » significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation. - Highlights: ●In vitro system for intermittent hypoxia (IH) and sustained hypoxia (SH). ●IH, but not SH, induces the proliferation of rat vascular smooth muscle cell. ●Epiregulin mRNA and protein are up-regulated in IH, but not in SH. ●IH-induced erbB2 phosphorylation is attenuated by erbB2 inhibitor. ●EGF family and erbB2 receptor may be involved in IH-induced cell proliferation.« less

Comparison of the killing effects between nitrogen-doped and pure TiO2 on HeLa cells with visible light irradiation

PubMed Central

2013-01-01

The killing effect of nitrogen-doped titanium dioxide (N-TiO2) nanoparticles on human cervical carcinoma (HeLa) cells by visible light photodynamic therapy (PDT) was higher than that of TiO2 nanoparticles. To study the mechanism of the killing effect, the reactive oxygen species produced by the visible-light-activated N-TiO2 and pure-TiO2 were evaluated and compared. The changes of the cellular parameters, such as the mitochondrial membrane potential (MMP), intracellular Ca2+, and nitrogen monoxide (NO) concentrations after PDT were measured and compared for N-TiO2- and TiO2-treated HeLa cells. The N-TiO2 resulted in more loss of MMP and higher increase of Ca2+ and NO in HeLa cells than pure TiO2. The cell morphology changes with time were also examined by a confocal microscope. The cells incubated with N-TiO2 exhibited serious distortion and membrane breakage at 60 min after the PDT. PMID:23433090

TREATMENT OF STROKE WITH DETA-NONOATE AND BONE MARROW STROMAL CELLS UPREGULATES ANGIOPOIETIN-1/TIE2 AND ENHANCES NEOVASCULARIZATION

PubMed Central

CUI, X.; CHEN, J.; ZACHAREK, A.; ROBERTS, C.; SAVANT-BHONSALE, S.; CHOPP, M.

2008-01-01

Neovascularization may contribute to functional recovery after neural injury. Combination treatment of stroke with a nitric oxide donor, DETA-NONOate and bone marrow stromal cells promote functional recovery. However, the mechanisms underlying functional improvement have not been elucidated. In this study, we tested the hypothesis that combination treatment upregulates Angiopoietin1 and its receptor Tie2 in the ischemic brain and bone marrow stromal cells, thereby enhances cerebral neovascularization after stroke. Adult wild type male C57BL/6 mice were intravenously administered PBS, bone marrow stromal cells 5×105, DETA-NONOate 0.4 mg/kg or combination DETA-NONOate with bone marrow stromal cells (n=12/group) after middle cerebral artery occlusion. Combination treatment significantly upregulated Angiopoietin-1/Tie2 and tight junction protein (occludin) expression, and increased the number, diameter and perimeter of blood vessels in the ischemic brain compared with vehicle control (mean ± SE, p<0.05). In vitro, DETA-NONOate significantly increased Angiopoietin-1/Tie2 protein (n=6/group) and Tie2 mRNA (n=3/group) expression in bone marrow stromal cells. DETA-NONOate also significantly increased Angiopoietin-1 protein (n=6/group) and mRNA (n=3/group) expression in mouse brain endothelial cells (p<0.05). Angiopoietin-1 mRNA (n=3/group) was significantly increased in mouse brain endothelial cells treated with DETA-NONOate in combination with bone marrow stromal cells conditioned medium compared with cells treated with bone marrow stromal cells conditioned medium or DETA-NONOate alone. Mouse brain endothelial cell capillary tube-like formation assays (n=6/group) showed that Angiopoietin-1 peptide, the supernatant of bone marrow stromal cells and DETA-NONOate significantly increased capillary tube formation compared to vehicle control. Combination treatment significantly increased capillary tube formation compared with DETA-NONOate treatment alone. Inhibition of Angiopoietin-1 significantly attenuated combination treatment-induced tube formation. Our data indicated that combination treatment of stroke with DETA-NONOate and bone marrow stromal cells promotes neovascularization, which is at least partially mediated by upregulation of the Angiopoietin-1/Tie2 axis. PMID:18691637

Role of enzyme-treated cells in RBC antibody screening using the gel test: a study of anti-RH1, -RH2, and -RH3 antibodies.

PubMed

Conne, Jocelyne; Schneider, Philippe; Tissot, Jean-Daniel

2007-01-01

The role of enzyme-treated cells (ETCs) in red blood cell (RBC) antibody screening has been the subject of controversy, and its place in the clinical routine remains to be determined. In this work, plasma samples containing anti-RH1 (anti-D; N = 10), anti-RH2 (anti-C; N = 10), or anti-RH3 (anti-E; N = 10) antibodies were studied. The samples were diluted in nonbuffered or buffered normal saline, as well as in a pool of AB plasma samples. Titers and scores were determined by means of the gel test, using the indirect antiglobulin test (IAT) as well as ETCs, with R(0)r, r'r, or r''r test cells. Our results showed that compared to the IAT, ETCs allowed a clearer detection of anti-RH2 and anti-RH3, but not of anti-RH1 antibodies. Based on our study, it is not clear whether the ETC phase of the gel test should be maintained for RBC antibody screening. 2007 Wiley-Liss, Inc.

Novel allergic asthma model demonstrates ST2-dependent dendritic cell targeting by cypress pollen.

PubMed

Gabriele, Lucia; Schiavoni, Giovanna; Mattei, Fabrizio; Sanchez, Massimo; Sestili, Paola; Butteroni, Cinzia; Businaro, Rita; Mirchandani, Ananda; Niedbala, Wanda; Liew, Foo Y; Afferni, Claudia

2013-09-01

Cypress pollen causes respiratory syndromes with different grades of severity, including asthma. IL-33, its receptor ST2, and dendritic cells (DCs) have been implicated in human respiratory allergy. We sought to define a new mouse model of allergy to cypress pollen that recapitulates clinical parameters in allergic patients and to evaluate the implications of DCs and the IL-33/ST2 pathway in this pathology. BALB/c mice, either wild-type or ST2 deficient (ST2(-/-)), were sensitized and challenged with the Cupressus arizonica major allergen nCup a 1. Local and systemic allergic responses were evaluated. Pulmonary cells were characterized by means of flow cytometry. DCs were stimulated with nCup a 1 and tested for their biological response to IL-33 in coculture assays. nCup a 1 causes a respiratory syndrome closely resembling human pollinosis in BALB/c mice. nCup a 1-treated mice exhibit the hallmarks of allergic pathology associated with pulmonary infiltration of eosinophils, T cells, and DCs and a dominant TH2-type immune response. IL-33 levels were increased in lungs and sera of nCup a 1-treated mice and in subjects with cypress allergy. The allergen-specific reaction was markedly reduced in ST2(-/-) mice, which showed fewer infiltrating eosinophils, T cells, and DCs in the lungs. Finally, stimulation of DCs with nCup a 1 resulted in ST2 upregulation that endowed DCs with increased ability to respond to IL-33-mediated differentiation of IL-5- and IL-13-producing CD4 T cells. Our findings define a novel preclinical model of allergy to cypress pollen and provide the first evidence of a functionally relevant linkage between pollen allergens and TH2-polarizing activity by DCs through IL-33/ST2. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Immune-stimulatory effects of a bacteria-based probiotic on peripheral leukocyte subpopulations and cytokine mRNA expression levels in scouring holstein calves.

PubMed

Qadis, Abdul Qadir; Goya, Satoru; Yatsu, Minoru; Kimura, Atsushi; Ichijo, Toshihiro; Sato, Shigeru

2014-05-01

Subpopulations of peripheral leukocytes and cytokine mRNA expression levels were evaluated in scouring and healthy Holstein calves (age 10 ± 5 days; n=42) treated with a probiotic consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum. The calves were assigned to the scouring or healthy group and then subdivided into pathogen-positive treated (n=8), pathogen-positive control (n=8), pathogen-negative treated (n=6), pathogen-negative control (n=6), healthy treated (n=6) and healthy control (n=8) groups. A single dose of the probiotic (3.0 g/100 kg body weight) was given to each calf in the treatment groups for 5 days. Blood samples were collected on the first day of scour occurrence (day 0) and on day 7. In the scouring calves, smaller peripheral leukocyte subpopulations and cytokine mRNA expression levels were noted on day 0. The numbers of CD3(+) T cells; CD4(+), CD8(+) and WC1(+) γδ T cell subsets; and CD14(+), CD21(+) and CD282(+) (TLR2) cells were significantly increased in the scouring and healthy treated calves on day 7. Furthermore, interleukin-6, tumor necrosis factor-alpha and interferon-gamma mRNA expression was elevated in the peripheral leukocytes of the scouring and healthy treated calves on day 7. The scouring calves given the probiotic recovered on day 7. A significantly smaller number of peripheral leukocytes and lower cytokine mRNA expression level might be induced by scouring in calves. Repeated probiotic administration might stimulate cellular immunity and encourage recovery from scouring in pre-weaning Holstein calves.

Immune-Stimulatory Effects of a Bacteria-Based Probiotic on Peripheral Leukocyte Subpopulations and Cytokine mRNA Expression Levels in Scouring Holstein Calves

PubMed Central

QADIS, Abdul Qadir; GOYA, Satoru; YATSU, Minoru; KIMURA, Atsushi; ICHIJO, Toshihiro; SATO, Shigeru

2014-01-01

ABSTRACT Subpopulations of peripheral leukocytes and cytokine mRNA expression levels were evaluated in scouring and healthy Holstein calves (age 10 ± 5 days; n=42) treated with a probiotic consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum. The calves were assigned to the scouring or healthy group and then subdivided into pathogen-positive treated (n=8), pathogen-positive control (n=8), pathogen-negative treated (n=6), pathogen-negative control (n=6), healthy treated (n=6) and healthy control (n=8) groups. A single dose of the probiotic (3.0 g/100 kg body weight) was given to each calf in the treatment groups for 5 days. Blood samples were collected on the first day of scour occurrence (day 0) and on day 7. In the scouring calves, smaller peripheral leukocyte subpopulations and cytokine mRNA expression levels were noted on day 0. The numbers of CD3+ T cells; CD4+, CD8+ and WC1+ γδ T cell subsets; and CD14+, CD21+ and CD282+ (TLR2) cells were significantly increased in the scouring and healthy treated calves on day 7. Furthermore, interleukin-6, tumor necrosis factor-alpha and interferon-gamma mRNA expression was elevated in the peripheral leukocytes of the scouring and healthy treated calves on day 7. The scouring calves given the probiotic recovered on day 7. A significantly smaller number of peripheral leukocytes and lower cytokine mRNA expression level might be induced by scouring in calves. Repeated probiotic administration might stimulate cellular immunity and encourage recovery from scouring in pre-weaning Holstein calves. PMID:24451928

Vorinostat positively regulates synaptic plasticity genes expression and spine density in HIV infected neurons: role of nicotine in progression of HIV-associated neurocognitive disorder.

PubMed

Atluri, Venkata Subba Rao; Pilakka-Kanthikeel, Sudheesh; Samikkannu, Thangavel; Sagar, Vidya; Kurapati, Kesava Rao Venkata; Saxena, Shailendra K; Yndart, Adriana; Raymond, Andrea; Ding, Hong; Hernandez, Oscar; Nair, Madhavan P N

2014-05-15

HIV-associated neurocognitive disorder (HAND) is characterized by development of cognitive, behavioral and motor abnormalities, and occurs in approximately 50% of HIV infected individuals. In the United States, the prevalence of cigarette smoking ranges from 35-70% in HIV-infected individuals compared to 20% in general population. Cognitive impairment in heavy cigarette smokers has been well reported. However, the synergistic effects of nicotine and HIV infection and the underlying mechanisms in the development of HAND are unknown. In this study, we explored the role of nicotine in the progression of HAND using SK-N-MC, a neuronal cell line. SK-N-MC cells were infected with HIV-1 in the presence or absence of nicotine for 7 days. We observed significant increase in HIV infectivity in SK-N-MC treated with nicotine compared to untreated HIV-infected neuronal cells. HIV and nicotine synergize to significantly dysregulate the expression of synaptic plasticity genes and spine density; with a concomitant increase of HDAC2 levels in SK-N-MC cells. In addition, inhibition of HDAC2 up-regulation with the use of vorinostat resulted in HIV latency breakdown and recovery of synaptic plasticity genes expression and spine density in nicotine/HIV alone and in co-treated SK-N-MC cells. Furthermore, increased eIF2 alpha phosphorylation, which negatively regulates eukaryotic translational process, was observed in HIV alone and in co-treatment with nicotine compared to untreated control and nicotine alone treated SK-N-MC cells. These results suggest that nicotine and HIV synergize to negatively regulate the synaptic plasticity gene expression and spine density and this may contribute to the increased risk of HAND in HIV infected smokers. Apart from disrupting latency, vorinostat may be a useful therapeutic to inhibit the negative regulatory effects on synaptic plasticity in HIV infected nicotine abusers.

Aβ-Induced Insulin Resistance and the Effects of Insulin on the Cholesterol Synthesis Pathway and Aβ Secretion in Neural Cells.

PubMed

Najem, Dema; Bamji-Mirza, Michelle; Yang, Ze; Zhang, Wandong

2016-06-01

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) toxicity, tau pathology, insulin resistance, neuroinflammation, and dysregulation of cholesterol homeostasis, all of which play roles in neurodegeneration. Insulin has polytrophic effects on neurons and may be at the center of these pathophysiological changes. In this study, we investigated possible relationships among insulin signaling and cholesterol biosynthesis, along with the effects of Aβ42 on these pathways in vitro. We found that neuroblastoma 2a (N2a) cells transfected with the human gene encoding amyloid-β protein precursor (AβPP) (N2a-AβPP) produced Aβ and exhibited insulin resistance by reduced p-Akt and a suppressed cholesterol-synthesis pathway following insulin treatment, and by increased phosphorylation of insulin receptor subunit-1 at serine 612 (p-IRS-S612) as compared to parental N2a cells. Treatment of human neuroblastoma SH-SY5Y cells with Aβ42 also increased p-IRS-S612, suggesting that Aβ42 is responsible for insulin resistance. The insulin resistance was alleviated when N2a-AβPP cells were treated with higher insulin concentrations. Insulin increased Aβ release from N2a-AβPP cells, by which it may promote Aβ clearance. Insulin increased cholesterol-synthesis gene expression in SH-SY5Y and N2a cells, including 24-dehydrocholesterol reductase (DHCR24) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) through sterol-regulatory element-binding protein-2 (SREBP2). While Aβ42-treated SH-SY5Y cells exhibited increased HMGCR expression and c-Jun phosphorylation as pro-inflammatory responses, they also showed down-regulation of neuro-protective/anti-inflammatory DHCR24. These results suggest that Aβ42 may cause insulin resistance, activate JNK for c-Jun phosphorylation, and lead to dysregulation of cholesterol homeostasis, and that enhancing insulin signaling may relieve the insulin-resistant phenotype and the dysregulated cholesterol-synthesis pathway to promote Aβ release for clearance from neural cells.

Effects of calcitriol (1, 25-dihydroxy-vitamin D3) on the inflammatory response induced by H9N2 influenza virus infection in human lung A549 epithelial cells and in mice.

PubMed

Gui, Boxiang; Chen, Qin; Hu, Chuanxia; Zhu, Caihui; He, Guimei

2017-01-23

H9N2 influenza viruses circulate globally and are considered to have pandemic potential. The hyper-inflammatory response elicited by these viruses is thought to contribute to disease severity. Calcitriol plays an important role in modulating the immune response to viral infections. However, its unknown whether calcitriol can attenuate the inflammatory response elicited by H9N2 influenza virus infection. Human lung A549 epithelial cells were treated with calcitriol (100 nM) and then infected with an H9N2 influenza virus, or infected and then treated with calcitriol (30 nM). Culture supernatants were collected every 24 h post infection and the viral growth kinetics and inflammatory response were evaluated. Calcitriol (5 mg/kg) was administered daily by intraperitoneal injection to BABL/c mice for 15 days following H9N2 influenza virus infection. Mice were monitored for clinical signs of disease, lung pathology and inflammatory responses. Calcitriol treatment prior to and post infection with H9N2 influenza significantly decreased expression of the influenza M gene, IL-6, and IFN-β in A549 cells, but did not affect virus replication. In vivo, we found that calcitriol treatment significantly downregulated pulmonary inflammation in mice 2 days post-infection, but increased the inflammatory response 4 to 6 days post-infection. In contrast, the antiviral cytokine IFN-β was significantly higher in calcitriol-treated mice than in the untreated infected mice at 2 days post-infection, but lower than in untreated infected mice on days 4 and 8 post-infection. The elevated levels of pro-inflammatory cytokines and the decreased levels of antiviral cytokine are consistent with the period of maximum body weight loss and the lung damage in calcitriol-treated mice. These results suggest that calcitriol treatment might have a negative impact on the innate immune response elicited by H9N2 infection in mice, especially at the later stage of influenza virus infection. This study will provide some novel insights into the use of calcitriol to modulate the inflammatory response elicited by influenza virus infection in humans.

Increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo.

PubMed

Valbuena, D; Martin, J; de Pablo, J L; Remohí, J; Pellicer, A; Simón, C

2001-11-01

To investigate whether the deleterious effect of E(2) on embryonic implantation is due to a direct effect on the endometrium, on the embryo, or both. Prospective, controlled in vitro study. Tertiary infertility center. Fertile patients in the luteal phase with histologically normal endometrium who were attending the infertility clinic as oocyte donors (n = 14). E(2) dose-response (0, 10(-8), 10(-7), 10(-6), 10(-5), and 10(-4) M) and time course (day 2 vs. day 5) experiments were performed in an in vitro embryo adhesion assay composed of human polarized endometrial epithelial cells obtained from fertile patients and mouse embryos. Blastocyst formation rate and embryo adhesion rate. Monolayers of polarized endometrial epithelial cells expressed ERalpha at the mRNA level. The E(2) dose response of blastocysts with polarized endometrial epithelial cells (n = 235) demonstrated a progressive reduction in embryonic adhesion that was statistically significant at 10(-6) M. When polarized endometrial epithelial cells were treated alone with increasing doses of E(2) for 3 days and E(2) was then removed and blastocysts added (n = 410), embryonic adhesion was not significantly reduced, except at 10(-4) M. When 2-day mouse embryos (n = 609) were treated with increasing E(2) concentrations until day 5, the rate of blastocyst formation significantly decreased at a concentration >or= 10(-6) M, and embryonic adhesion decreased when blastocysts (n = 400) were obtained at a concentration >or= 10(-7) M. Time course experiments of embryos cultured for 2 days with polarized endometrial epithelial cells (n = 426) showed that the adhesion rate was higher at E(2) levels of 10(-7), 10(-6) and 10(-5) M compared with embryos cultured for 5 days (n = 495). High E(2) levels are deleterious to embryo adhesion in vitro, mainly because they have a direct toxic effect on the embryo that may occur at the cleavage stage.

Parkin Overexpression Ameliorates PrP106-126-Induced Neurotoxicity via Enhanced Autophagy in N2a Cells.

PubMed

Khan, Sher Hayat; Zhao, Deming; Shah, Syed Zahid Ali; Hassan, Mohammad Farooque; Zhu, Ting; Song, Zhiqi; Zhou, Xiangmei; Yang, Lifeng

2017-05-01

Transmissible spongiform encephalopathies (TSEs) are caused by the accumulation of the abnormal prion protein scrapie (PrP Sc ). Prion protein aggregation, misfolding, and cytotoxicity in the brain are the major causes of neuronal dysfunction and ultimate neurodegeneration in all TSEs. Parkin, an E3 ubiquitin ligase, has been studied extensively in all major protein misfolding aggregating diseases, especially Parkinson's disease and Alzheimer's disease, but the role of parkin in TSEs remains unknown. Here we investigated the role of parkin in a prion disease cell model in which neuroblastoma2a (N2a) cells were treated with prion peptide PrP106-126. We observed a gradual decrease in the soluble parkin level upon treatment with PrP106-126 in a time-dependent manner. Furthermore, endogenous parkin colocalized with FITC-tagged prion fragment106-126. Overexpression of parkin in N2a cells via transfection repressed apoptosis by enhancing autophagy. Parkin-overexpressing cells also showed reductions in apoptotic BAX translocation to the mitochondria and cytochrome c release to the cytosol, which ultimately inhibited activation of proapoptotic caspases. Taken together, our findings reveal a parkin-mediated cytoprotective mechanism against PrP106-126 toxicity, which is a novel potential therapeutic target for treating prion diseases.

Impact of Sustained Exposure to β-Amyloid on Calcium Homeostasis and Neuronal Integrity in Model Nerve Cell System Expressing α4β2 Nicotinic Acetylcholine Receptors*

PubMed Central

Arora, Komal; Alfulaij, Naghum; Higa, Jason K.; Panee, Jun; Nichols, Robert A.

2013-01-01

Although the interaction between β-amyloid (Aβ) and nicotinic acetylcholine receptors has been widely studied, the impact of prolonged exposure to Aβ on nAChR expression and signaling is not known. In this study, we employed a neuronal culture model to better understand the impact of sustained exposure of Aβ on the regulation of cellular and synaptic function. The differentiated rodent neuroblastoma cell line NG108-15 expressing exogenous high-affinity α4β2 nAChRs was exposed to soluble oligomeric Aβ for several days. Ca2+ responses, expression levels of α4β2 nAChRs, rate of mitochondrial movement, mitochondrial fission, levels of reactive oxygen species, and nuclear integrity were compared between Aβ-treated and untreated cells, transfected or not (mock-transfected) with α4β2 nAChRs. Sustained exposure of Aβ1–42 to α4β2 nAChR-transfected cells for several days led to increased Ca2+ responses on subsequent acute stimulation with Aβ1–42 or nicotine, paralleled by increased expression levels of α4β2 nAChRs, likely the result of enhanced receptor recycling. The rate of mitochondrial movement was sharply reduced, whereas the mitochondrial fission protein pDrp-1 was increased in α4β2 nAChR-transfected cells treated with Aβ1–42. In addition, the presence of α4β2 nAChRs dramatically enhanced Aβ1–42-mediated increases in reactive oxygen species and nuclear fragmentation, eventually leading to apoptosis. Our data thus show disturbed calcium homeostasis coupled with mitochondrial dysfunction and loss of neuronal integrity on prolonged exposure of Aβ in cells transfected with α4β2 nAChRs. Together, the results suggest that the presence of nAChRs sensitizes neurons to the toxic actions of soluble oligomeric Aβ, perhaps contributing to the cholinergic deficit in Alzheimer disease. PMID:23479730

Docosahexaenoyl ethanolamide improves glucose uptake and alters endocannabinoid system gene expression in proliferating and differentiating C2C12 myoblasts

PubMed Central

Kim, Jeffrey; Carlson, Morgan E.; Watkins, Bruce A.

2014-01-01

Skeletal muscle is a major storage site for glycogen and a focus for understanding insulin resistance and type-2-diabetes. New evidence indicates that overactivation of the peripheral endocannabinoid system (ECS) in skeletal muscle diminishes insulin sensitivity. Specific n-6 and n-3 polyunsaturated fatty acids (PUFA) are precursors for the biosynthesis of ligands that bind to and activate the cannabinoid receptors. The function of the ECS and action of PUFA in skeletal muscle glucose uptake was investigated in proliferating and differentiated C2C12 myoblasts treated with either 25 μM of arachidonate (AA) or docosahexaenoate (DHA), 25 μM of EC [anandamide (AEA), 2-arachidonoylglycerol (2-AG), docosahexaenoylethanolamide (DHEA)], 1 μM of CB1 antagonist NESS0327, and CB2 inverse agonist AM630. Compared to the BSA vehicle control cell cultures in both proliferating and differentiated myoblasts those treated with DHEA, the EC derived from the n-3 PUFA DHA, had higher 24 h glucose uptake, while AEA and 2-AG, the EC derived from the n-6 PUFA AA, had lower basal glucose uptake. Adenylyl cyclase mRNA was higher in myoblasts treated with DHA in both proliferating and differentiated states while those treated with AEA or 2-AG were lower compared to the control cell cultures. Western blot and qPCR analysis showed higher expression of the cannabinoid receptors in differentiated myoblasts treated with DHA while the opposite was observed with AA. These findings indicate a compensatory effect of DHA and DHEA compared to AA-derived ligands on the ECS and associated ECS gene expression and higher glucose uptake in myoblasts. PMID:24711795

[Inhibitory effect of Mig-7 silencing by retrovirus-mediated shRNA on vasculogenic mimicry, invasion and metastasis of human hepatocellular carcinoma cells in vitro].

PubMed

Qu, Bo; Sheng, Guan-Nan; Yu, Fei; Chen, Guan-Nan; Lv, Qi; Mao, Zhong-Peng; Guo, Long; Lv, Yi

2016-11-20

To explore the inhibitory effect of migration-inducing gene 7 (Mig-7) gene silencing induced by retroviral-mediated small hairpin RNA (shRNA) on vasculogenic mimicry (VM), invasion and metastasis of human hepatocellular carcinoma (HCC) cells in vitro. Two target sequences (Mig-7 shRNA-1 and Mig-7 shRNA-2) and one negative control sequence (Mig-7 shRNA-N) were synthesized. The recombinant retroviral vectors carrying Mig-7 shRNA were constructed, and HCC cell line MHCC-97H were transfected with Mig-7 shRNA-1, Mig-7 shRNA-2, Mig-7 shRNA-N, or the empty vector, or treated with 125 µg/mL recombinant human endostatin (ES). Mig-7 expression in the treated cells was detected using semi-quantitative PCR and Western blotting. The inhibitory effect of Mig-7 silencing on VM formation was investigated in a 3-dimensional cell culture system; the changes in cell adhesion, invasion and migration were assessed with intercellular adhesion assay, Transwell invasion assay and Transwell migration assay, respectively. The expression of Mig-7 at both mRNA and protein levels decreased significantly, VM formation, invasion and metastasis were suppressed, while intercellular adhesion increased significantly in MHCC-97H cells in Mig-7 shRNA-1 and Mig-7 shRNA-2 groups (P<0.05); such changes were not observed in cells transfected with Mig-7 shRNA-N or the empty vector, nor in cells treated with ES. Mig-7 silencing by retroviral-mediated shRNA significantly inhibits VM formation, invasion and metastasis and increases the intercellular adhesion of the HCC cells, while ES does not have such inhibitory effects.

Measurement of OH, O, and NO densities and their correlations with mouse melanoma cell death rate treated by a nanosecond pulsed streamer discharge

NASA Astrophysics Data System (ADS)

Yagi, Ippei; Shirakawa, Yuki; Hirakata, Kenta; Akiyama, Taketoshi; Yonemori, Seiya; Mizuno, Kazue; Ono, Ryo; Oda, Tetsuji

2015-10-01

Mouse melanoma cells in a culture medium are treated using a nanosecond pulsed streamer discharge plasma and the correlations between the rate of cell death and the densities of reactive species (OH, O, and NO) in the plasma are measured. The plasma is irradiated onto the culture medium surface with a vertical gas flow of an O2/N2 mixture from a glass tube at various gas flow rates and O2 concentrations. The densities of the reactive species are measured very close to the culture medium surface, where the reactive species interact with the culture medium, using laser-induced fluorescence. In the case of the N2 discharge (O2 = 0%), an increase in gas flow rate decreases OH density because it lowers the water vapor concentration by diluting the vapor, which is required for OH production. The increase in gas flow rate also leads to a decreased cell death rate. In the case of the O2/N2 discharge, on the other hand, an increase in O2 concentration at a fixed flow rate does not affect the rate of cell death, although it considerably changes the O and NO densities. These findings indicate that some reactive species derived from water vapor such as OH are responsible for the melanoma cell death, whereas those from O2, such as O and NO, are less likely responsible. They also indicate the importance of water evaporation from the culture medium surface in cell treatment.

Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell

PubMed Central

Wang, Qianli; Zhang, Junping; Guo, Zhongwu

2007-01-01

To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine. PMID:17892942

Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell.

PubMed

Wang, Qianli; Zhang, Junping; Guo, Zhongwu

2007-12-15

To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine.

Naegleria fowleri Lysate Induces Strong Cytopathic Effects and Pro-inflammatory Cytokine Release in Rat Microglial Cells

PubMed Central

Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul

2011-01-01

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A 51Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response. PMID:22072830

Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells.

PubMed

Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul; Shin, Ho-Joon

2011-09-01

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

Chronic HIV-1 Infection Induces B-Cell Dysfunction That Is Incompletely Resolved by Long-Term Antiretroviral Therapy.

PubMed

Abudulai, Laila N; Fernandez, Sonia; Corscadden, Karli; Hunter, Michael; Kirkham, Lea-Ann S; Post, Jeffrey J; French, Martyn A

2016-04-01

To determine the effect of long-term antiretroviral therapy (ART) on HIV-1-induced B-cell dysfunction. Comparative study of ART-naive and ART-treated HIV-infected patients with non-HIV controls. B-cell dysfunction was examined in patients with HIV-1 infection (n = 30) who had received ART for a median time of 9.25 years (range: 1.3-21.7) by assessing proportions of CD21 B cells (a marker of B-cell exhaustion) and proportions of tumor necrosis factor-related apoptosis-inducing ligand or B and T lymphocyte attenuator B cells, and serum levels of immunoglobulin free light chains (markers of B-cell hyperactivation). The association of these markers with serum levels of IgG1 and IgG2, and production of IgG antibodies after vaccination with pneumococcal polysaccharides were also examined. ART-naive patients with HIV (n = 20) and controls (n = 20) were also assessed for comparison. ART-treated patients had increased proportions of CD21 and tumor necrosis factor-related apoptosis-inducing ligand B cells and, furthermore, although proportions of B and T lymphocyte attenuator B cells were not significantly different from controls, they correlated negatively with CD21 B cells. Proportions of CD21 B cells also correlated negatively with current CD4 T-cell counts. In ART-naive patients with HIV, free light chains correlated with CD21 B cells and IgG1, but not IgG2. Serum IgG2:IgG1 ratios were substantially lower than normal in patients with HIV and did not resolve on ART. In ART-treated patients, IgG antibody responses to pneumococcal polysaccharides after vaccination were not associated with markers of B-cell dysfunction. B-cell dysfunction persists in patients with HIV receiving long-term ART. The causes and consequences of this require further investigation.

Non-thermal Plasma Induces Apoptosis in Melanoma Cells via Production of Intracellular Reactive Oxygen Species

PubMed Central

Sensenig, Rachel; Kalghatgi, Sameer; Cerchar, Ekaterina; Fridman, Gregory; Shereshevsky, Alexey; Torabi, Behzad; Arjunan, Krishna Priya; Podolsky, Erica; Fridman, Alexander; Friedman, Gary; Azizkhan-Clifford, Jane; Brooks, Ari D.

2012-01-01

Non-thermal atmospheric pressure dielectric barrier discharge (DBD) plasma may provide a novel approach to treat malignancies via induction of apoptosis. The purpose of this study was to evaluate the potential of DBD plasma to induce apoptosis in melanoma cells. Melanoma cells were exposed to plasma at doses that did not induce necrosis, and cell viability and apoptotic activity were evaluated by Trypan blue exclusion test, Annexin-V/PI staining, caspase-3 cleavage, and TUNEL® analysis. Trypan blue staining revealed that non-thermal plasma treatment significantly decreased the viability of cells in a dose-dependent manner 3 and 24 h after plasma treatment. Annexin-V/PI staining revealed a significant increase in apoptosis in plasma-treated cells at 24, 48, and 72 h post-treatment (p<0.001). Caspase-3 cleavage was observed 48 h post-plasma treatment at a dose of 15 J/cm2. TUNEL® analysis of plasma-treated cells demonstrated an increase in apoptosis at 48 and 72 h post-treatment (p<0.001) at a dose of 15 J/cm2. Pre-treatment with N-acetyl-L-cysteine (NAC), an intracellular reactive oxygen species (ROS) scavenger, significantly decreased apoptosis in plasma-treated cells at 5 and 15 J/cm2. Plasma treatment induces apoptosis in melanoma cells through a pathway that appears to be dependent on production of intracellular ROS. DBD plasma production of intracellular ROS leads to dose-dependent DNA damage in melanoma cells, detected by γ-H2AX, which was completely abrogated by pre-treating cells with ROS scavenger, NAC. Plasma-induced DNA damage in turn may lead to the observed plasma-induced apoptosis. Since plasma is non-thermal, it may be used to selectively treat malignancies. PMID:21046465

Anti-inflammatory activity of fisetin in human gingival fibroblasts treated with lipopolysaccharide.

PubMed

Gutiérrez-Venegas, Gloria; Contreras-Sánchez, Anabel; Ventura-Arroyo, Jairo Agustín

2014-10-01

Fisetin is an anti-inflammatory flavonoid; however, its anti-inflammatory mechanism is not yet understood. In this study, we evaluated the anti-inflammatory effect of fisetin and its association with mitogen-activated protein kinase (MAPK) and nuclear factor kappa-beta pathways in human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) obtained from Porphyromonas gingivalis. The cell signaling, cell viability, and cyclooxygenase-2 (COX-2) expression of HGFs treated with various concentrations (0, 1, 5, 10, and 15 μM) of fisetin were measured by cell viability assay (MTT), Western blotting, and reverse transcriptase polymerase chain reaction analysis on COX-2. We found that fisetin significantly reduced the synthesis and expression of prostaglandin E2 in HGFs treated with LPS. Activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK was suppressed consistently by fisetin in HGFs treated with LPS. The data indicate that fisetin inhibits MAPK activation and COX-2 expression without affecting cell viability. These findings may be valuable for understanding the mechanism of the effect of fisetin on periodontal disease.

The efficacy of facial skin cancer treatment with high-energy pulsed neodymium and Nd:YAG lasers.

PubMed

Moskalik, Konstantin; Kozlov, Alexander; Demin, Eugeny; Boiko, Ernest

2009-04-01

The aim of this study was to assess the curative and cosmetic efficacy of treatment for facial skin cancer using neodymium laser irradiation. Due to the complex anatomy of the area, therapy for facial skin cancer is difficult. Laser irradiation was used for the treatment of 3461 patients with 3624 facial skin cancer lesions of stages T(1-2)N(0)M(0:) 3346 basal cell skin cancers, 188 limited basal cell skin cancer recurrences, and 90 squamous cell skin cancers. Pulsed neodymium (Nd) and Nd:YAG lasers were used as the energy sources. The patients were followed-up from 3 mo to 5 y or more. Patients with basal cell skin cancer treated by irradiation with the Nd laser developed recurrences in 1.8% of cases, and patients treated with the Nd:YAG laser had a recurrence rate of 2.5%. Recurrences following treatment for basal cell skin cancer, and those of squamous cell skin cancer, after irradiation with the Nd laser appeared in 3.7% and 4.4% of patients, respectively. Overall, the frequency of facial skin cancer recurrences after treatment with laser irradiation was 2.1% of all the irradiated tumors. Neodymium laser irradiation is an effective method to treat facial skin cancer of stages T(1-2)N(0)M(0), and results in acceptable cosmetic results.

Unilateral Radiotherapy for the Treatment of Tonsil Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chronowski, Gregory M., E-mail: gchronowski@mdanderson.org; Garden, Adam S.; Morrison, William H.

2012-05-01

Purpose: To assess, through a retrospective review, clinical outcomes of patients with squamous cell carcinoma of the tonsil treated at the M. D. Anderson Cancer Center with unilateral radiotherapy techniques that irradiate the involved tonsil region and ipsilateral neck only. Methods and Materials: Of 901 patients with newly diagnosed squamous cell carcinoma of the tonsil treated with radiotherapy at our institution, we identified 102 that were treated using unilateral radiotherapy techniques. All patients had their primary site of disease restricted to the tonsillar fossa or anterior pillar, with <1 cm involvement of the soft palate. Patients had TX (n =more » 17 patients), T1 (n = 52), or T2 (n = 33) disease, with Nx (n = 3), N0 (n = 33), N1 (n = 23), N2a (n = 21), or N2b (n = 22) neck disease. Results: Sixty-one patients (60%) underwent diagnostic tonsillectomy before radiotherapy. Twenty-seven patients (26%) underwent excision of a cervical lymph node or neck dissection before radiotherapy. Median follow-up for surviving patients was 38 months. Locoregional control at the primary site and ipsilateral neck was 100%. Two patients experienced contralateral nodal recurrence (2%). The 5-year overall survival and disease-free survival rates were 95% and 96%, respectively. The 5-year freedom from contralateral nodal recurrence rate was 96%. Nine patients required feeding tubes during therapy. Of the 2 patients with contralateral recurrence, 1 experienced an isolated neck recurrence and was salvaged with contralateral neck dissection only and remains alive and free of disease. The other patient presented with a contralateral base of tongue tumor and involved cervical lymph node, which may have represented a second primary tumor, and died of disease. Conclusions: Unilateral radiotherapy for patients with TX-T2, N0-N2b primary tonsil carcinoma results in high rates of disease control, with low rates of contralateral nodal failure and a low incidence of acute toxicity requiring gastrostomy.« less

Arsenic trioxide induces cell cycle arrest and affects Trk receptor expression in human neuroblastoma SK-N-SH cells.

PubMed

Xiong, Xilin; Li, Yang; Liu, Ling; Qi, Kai; Zhang, Chi; Chen, Yueqin; Fang, Jianpei

2018-06-13

Arsenic trioxide (As 2 O 3 ), a drug that has been used in China for approximately two thousand years, induces cell death in a variety of cancer cell types, including neuroblastoma (NB). The tyrosine kinase receptor (Trk) family comprises three members, namely TrkA, TrkB and TrkC. Various studies have confirmed that TrkA and TrkC expression is associated with a good prognosis in NB, while TrkB overexpression can lead to tumor cell growth and invasive metastasis. Previous studies have shown that As 2 O 3 can inhibit the growth and proliferation of a human NB cell line and can also affect the N-Myc mRNA expression. It remains unclear whether As 2 O 3 regulates Trks for the purposes of treating NB. The aim of the present study was to investigate the effect of As 2 O 3 on Trk expression in NB cell lines and its potential therapeutic efficacy. SK-N-SH cells were grown with increasing doses of As 2 O 3 at different time points. We cultured SK-N-SH cells, which were treated with increasing doses of As 2 O 3 at different time points. Trk expression in the NB samples was quantified by immunohistochemistry, and the cell cycle was analyzed by flow cytometry. TrkA, TrkB and TrkC mRNA expression was evaluated by real-time PCR analysis. Immunohistochemical and real-time PCR analyses indicated that TrkA and TrkC were over-expressed in NB, and specifically during stages 1, 2 and 4S of the disease progression. TrkB expression was increased in stage 3 and 4 NB. As 2 O 3 significantly arrested SK-N-SH cells in the G2/M phase. In addition, TrkA, TrkB and TrkC expression levels were significantly upregulated by higher concentrations of As 2 O 3 treatment, notably in the 48-h treatment period. Our findings suggested that to achieve the maximum effect and appropriate regulation of Trk expression in NB stages 1, 2 and 4S, As 2 O 3 treatment should be at relatively higher concentrations for longer delivery times;however, for NB stages 3 and 4, an appropriate concentration and infusion time for As 2 O 3 must be carefully determined. The present findings suggested that As 2 O 3 induced Trk expression in SK-N-SH cells to varying degrees and may be a promising adjuvant to current treatments for NB due to its apoptotic effects.

The Study of Non-Viral Nanoscale Delivery Systems for Islet Transplantation

NASA Astrophysics Data System (ADS)

Gutierrez, Diana

Due to safety concerns associated with using viral systems clinically to expand islet cells and make them available to many more patients, significant emphasis has been placed on producing a safe and effective non-viral delivery system for biological research and gene therapy. To obtain this goal, we propose the use of an innovative technology that utilizes gold nanoparticles (AuNPs) as a non-viral method of delivery. Our laboratory was one of the first to describe the use of AuNPs in human islets and observe AuNPs can penetrate into the core of islets to deliver a gene to the vast majority of the cells, without damaging the cell. Gold nanoparticles proved to be a biocompatible delivery system both in vitro and in vivo. Thus far, gene therapy and molecular biology have focused primarily on delivering DNA of a specific gene into cells. The risk of this approach is that the DNA can be permanently incorporated into the genome and lead to damages in the cell that could result in overexpression of cancerous tumor cells. This risk does not exist with the use of mRNA. Many researchers believe mRNA is too unstable to be used as a molecular tool to overexpress specific proteins. With advances in nanotechnology, and better understanding of the translation process, methods have been developed that allow for expression of specific proteins by intracellular delivery of protein-encoding mRNA. We used AuNPs conjugated to mCherry mRNA to establish a proof of concept of the feasibility of using AuNP-mRNA to achieve increased expression of a specific protein within cells. To do this, we conjugated mCherry mRNA to AuNPs and tested the feasibility for increasing delivery efficacy and preserve functionality of human pancreatic islets. We believe that with this novel technology we can create AuNPs that allow specific mRNA to enter islets and lead to the production of a specific protein within the cell, with the aim to induce beta cell proliferation. In a previous experiment with single cells, the highest amount of protein expression was observed after 24 hours incubation with mCherry conjugated AuNPs. Based on this, human islets were treated with 12 nm, 7 nm and 2 nm mCherry AuNPs for 24 hours. The expression of mCherry protein in human islets was analyzed by 3D image reconstruction of z-stack images acquired by confocal microscopy. A minimal amount of mCherry protein was expressed in human islets when treated with mCherry mRNA coupled to the 12 nm size AuNP. Decreasing the size of the AuNPs to 7 nm or 2 nm resulted in substantial increase in mCherry protein expression throughout human pancreatic islets when treated at concentrations of 20 nM and 50 nM with mCherry mRNA AuNPs for 24 hours. We used measurements of calcium influx, KCL and mitochondrial potential to determine the effect of AuNP-mCherry mRNA treatment on islet cell function. The area under the curve was computed for intracellular calcium influx of three different islet preparations. There was no statistically significance difference between (2 nm) 20 nM versus (7 nm) 20 nM, (2 nm) 20 nM versus (7 nm) 50 nM, (2 nm) 50 nM versus (7 nm) 20 nM, (2 nm) 50 nM versus (7 nm) 50 nM. For the area under the curve for the KCL there was no significant statistical difference between the groups. In addition, mitochondrial potential indices demonstrated similarity between the control group and mCherry mRNA AuNPs treated human pancreatic islets, there was no statistical difference between the three different sizes and concentrations when compared to the non-treated group. Taken together, AuNP did not impair islet function when concentration was increased. Although, the optimal size of AuNP that was easily seen to express mCherry protein was 7 nm, when human islet cells were treated with AuNP coupled to mRNA for E2F3 (the beta-cell proliferation inducing protein), to observe whether there was any sign of enhanced beta-cell proliferation, the 12 nm sized AuNP seemed to give a slight increase in beta-cell proliferation. Transmission electron microscopy (TEM) was used to determine where within the islets the AuNPs were localized. This validated that both the 12 nm and 7 nm size AuNPs crossed the cell membrane and were found within vesicles, mitochondria and in one case the insulin granules of the islets. A notable difference that was detected under TEM for the two size of AuNPs was that the 12nm appeared predominantly in clusters where as the 7nm AuNP was more evenly distributed within the cell. Further analysis with TEM may provide insight on how the size, concentration and kinetics of the AuNPs will influence protein expression and beta-cell expansion within human pancreatic islets. (Abstract shortened by UMI.).

An Arg-Gly-Asp peptide stimulates Ca2+ efflux from osteoclast precursors through a novel mechanism

NASA Technical Reports Server (NTRS)

Yamakawa, K.; Duncan, R.; Hruska, K. A.

1994-01-01

We examined the effect of a peptide containing the Arg-Gly-Asp (RGD) sequence on 45Ca2+ efflux from osteoclast precursors. 45Ca(2+)-loaded osteoclast precursors were treated with GRGDSP (170 microM) for 10 min after 30 min of basal perfusion with a bicarbonate-containing buffer. GRGDSP significantly increased fractional efflux of Ca2+ from treated cells compared with vehicle-treated cells (P < 0.01) or cells treated with up to 200 micrograms/ml of a control peptide containing GRGESP. The effect of RGD was sustained for 15 min after the peptide was removed from the perfusate, but control levels of Ca2+ efflux returned by 1 h. The Ca2+ efflux effect of GRGDSP was most likely due to activation of the plasma membrane Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) pump, as indicated by its inhibition with vanadate and a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and the absence of an effect of Na+/Ca2+ exchange inhibition. An inhibitor of cyclic nucleotide-dependent protein kinases, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (0.1 mM), failed to inhibit GRGDSP-stimulated Ca2+ efflux. However, genistein and herbimycin A, inhibitors of protein-tyrosine kinases, blocked Ca2+ efflux stimulated by GRGDSP. The results indicate that RGD sequences of matrix proteins may stimulate Ca2+ efflux from osteoclasts through activation of protein-tyrosine kinases and suggest that GRGDSP-stimulated Ca2+ efflux is mediated via the plasma membrane Ca(2+)-ATPase.

Biocompatibility assessment of titanium dioxide nanoparticles in mice fetoplacental unit.

PubMed

Naserzadeh, Parvaneh; Ghanbary, Fatemeh; Ashtari, Parviz; Seydi, Enayatollah; Ashtari, Khadijeh; Akbari, Mohsen

2018-02-01

As the applications of titanium dioxide nanomaterials (nTiO 2 ) are growing with an ever-increasing speed, the hazardous risks of this material have become a major concern. Several recent studies have reported that nTiO 2 can cross the placental barrier in pregnant mice and cause neurotoxicity in their offspring. However, the influence of these nanoparticles on the fetoplacental unit during the pregnancy is yet to be studied. The present study reports on the effects of nTiO 2 on the anatomical structure of fetal brain and liver in a pregnant mice model. Moreover, changes in the size and weight of the fetus and placenta are investigated as markers of fetal growth. Lastly, the toxicity of nTiO 2 in primary brain and liver is quantified. Animals treated with nTiO 2 showed a disrupted anatomical structure of the fetal brain and liver. Furthermore, the fetus and placental unit in the mice treated with these nanoparticles were smaller compared to untreated controls. Toxicity analyses revealed that nTiO 2 was toxic to the brain and liver cells and the mechanism of cell death was mostly necrosis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 580-589, 2018. © 2017 Wiley Periodicals, Inc.

Oncologic outcomes of surgically treated early-stage oropharyngeal squamous cell carcinoma.

PubMed

Kass, Jason I; Giraldez, Laureano; Gooding, William; Choby, Garret; Kim, Seungwon; Miles, Brett; Teng, Marita; Sikora, Andrew G; Johnson, Jonas T; Myers, Eugene N; Duvvuri, Umamaheswar; Genden, Eric M; Ferris, Robert L

2016-10-01

The purpose of this study was to characterize oncologic outcomes in early (T1-T2, N0) and intermediate (T1-T2, N1) oropharyngeal squamous cell carcinoma (SCC) after surgery. Patients with oropharyngeal SCC treated with surgery were identified from 2 academic institutions. Of 188 patients, 143 met the inclusion criteria. Eighty-six (60%) had T1 to T2 N0 and 57 (40%) had T1 to T2 N1 disease. Sixty-five patients (45%) underwent a robotic-assisted resection, whereas the remaining had transoral (n = 60; 42%), mandible-splitting (n = 11; 8%), or transhyoid approaches (n = 7; 5%). Human papillomavirus (HPV) status was known for 97 patients (68%), and 54 (55%) were HPV positive. Three-year recurrence-free survival (RFS) was 82% (95% confidence interval [CI] = 0.75-0.89). Since 2008, HPV infection was protective of recurrence (log-rank p = .0334). A single node did not increase the risk of recurrence (p = .467) or chance of a second primary (p = .175). Complete surgical resection is effective therapy for early and intermediate oropharyngeal SCC. HPV-negative patients were at increased risk for locoregional recurrence or second primary disease. © 2016 Wiley Periodicals, Inc. Head Neck 38: First-1471, 2016. © 2016 Wiley Periodicals, Inc.

Histological, morphometric, protein and gene expression analyses of rat retinas with ischaemia-reperfusion injury model treated with sildenafil citrate.

PubMed

Zanoni, Diogo S; Da Silva, Germana A; Ezra-Elia, Raaya; Carvalho, Márcio; Quitzan, Juliany G; Ofri, Ron; Laus, José L; Laufer-Amorim, Renee

2017-06-01

The aim of this study was to better understand the role of apoptosis in a retinal ischaemia-reperfusion injury model and to determine whether sildenafil citrate treatment can prevent retinal cell apoptosis. Thirty-six rats were divided into a control group (n = 6) and two experimentally induced ischaemia-reperfusion groups (7 and 21 days; n = 15 per group). The induced ischaemia-reperfusion groups were treated with sildenafil for 7 and 21 days (n = 10 per group), and 10 animals were treated with a placebo for the same period (n = 5 per group). Paracentesis of the anterior chamber was performed with a 30-G needle attached to a saline solution (0.9%) bag positioned at a height of 150 cm above the eye for 60 min. Intraocular pressure was measured by rebound tonometer (TonoVet ® ). The eyes were analysed by histology and morphometry, and by immunohistochemistry and qRT-PCR for expression of Caspase-7, Caspase-6, Caspase-9, Tnf-r2, Fas-l, Bcl-2 and Bax. Sildenafil-treated animals showed lower levels of histopathological changes (inflammatory, cellular and tissue) than their placebo-treated counterparts at both 7 and 21 days. The retinal ganglion cell layer (RGC) was preserved in the sildenafil groups (SG), with a cell count closer to control than in the placebo groups (PG). Caspase-7 expression was significantly higher in both treated groups at 7 days compared to controls. Gene expression levels in both treatment groups differed from the controls, but in SG Bax and Caspase-6 expression levels were similar to control animals. These results suggest that the main mechanism of retinal cell death in this model is apoptosis, as there is an increase in pro-apoptotic factors and decrease in the anti-apoptotic ones. Also, sildenafil seems to protect the retinal ganglion cell layer from apoptosis. Cell survival was evident in the histological and morphometric analyses, and sildenafil treatment had a protective effect in the apoptosis pathways, with gene expression levels in SG similar to the controls. © 2017 The Authors. International Journal of Experimental Pathology © 2017 International Journal of Experimental Pathology.

Remission induction using alemtuzumab can permit chemotherapy-refractory chronic lymphocytic leukemia (CLL) patients to undergo allogeneic stem cell transplantation.

PubMed

Knauf, Wolfgang; Rieger, Kathrin; Blau, Wolfgang; Hegenbart, Ute; Von Gruenhagen, Ulrich; Niederwieser, Dietger; Thiel, Eckhard

2004-12-01

The outcome of allogeneic stem cell transplantation depends upon the disease status before transplantation. Patients with refractory disease are at high risk for relapse. To improve the curative potential of the transplant procedure, we treated 3 chemotherapy-refractory CLL patients with alemtuzumab before allogeneic stem cell transplantation. Prior to therapy, all patients suffered from B-symptoms, and had massive adenopathy, splenomegaly, thrombocytopenia, and anemia; two patients had hepatomegaly. Alemtuzumab greatly reduced tumor mass in blood and bone marrow, B-symptoms resolved, and organomegaly improved. Two patients became blood product independent. All patients proceeded to transplantation after conditioning with TBI 2 Gy (n=1) or Treosulfan (n=2) in combination with Fludarabine either from an HLA-matched sibling (n=2) or from an HLA-matched unrelated donor (n=1). All patients engrafted, and are alive and well. Two patients reached complete remission (CR); one patient attained stable partial remission (PR). These heavily pre-treated refractory patients gained substantial clinical benefit from alemtuzumab, and received successful allografts.

Off-the-Shelf Virus-Specific T Cells to Treat BK Virus, Human Herpesvirus 6, Cytomegalovirus, Epstein-Barr Virus, and Adenovirus Infections After Allogeneic Hematopoietic Stem-Cell Transplantation.

PubMed

Tzannou, Ifigeneia; Papadopoulou, Anastasia; Naik, Swati; Leung, Kathryn; Martinez, Caridad A; Ramos, Carlos A; Carrum, George; Sasa, Ghadir; Lulla, Premal; Watanabe, Ayumi; Kuvalekar, Manik; Gee, Adrian P; Wu, Meng-Fen; Liu, Hao; Grilley, Bambi J; Krance, Robert A; Gottschalk, Stephen; Brenner, Malcolm K; Rooney, Cliona M; Heslop, Helen E; Leen, Ann M; Omer, Bilal

2017-11-01

Purpose Improvement of cure rates for patients treated with allogeneic hematopoietic stem-cell transplantation (HSCT) will require efforts to decrease treatment-related mortality from severe viral infections. Adoptively transferred virus-specific T cells (VSTs) generated from eligible, third-party donors could provide broad antiviral protection to recipients of HSCT as an immediately available off-the-shelf product. Patient and Methods We generated a bank of VSTs that recognized five common viral pathogens: Epstein-Barr virus (EBV), adenovirus (AdV), cytomegalovirus (CMV), BK virus (BKV), and human herpesvirus 6 (HHV-6). The VSTs were administered to 38 patients with 45 infections in a phase II clinical trial. Results A single infusion produced a cumulative complete or partial response rate of 92% (95% CI, 78.1% to 98.3%) overall and the following rates by virus: 100% for BKV (n = 16), 94% for CMV (n = 17), 71% for AdV (n = 7), 100% for EBV (n = 2), and 67% for HHV-6 (n = 3). Clinical benefit was achieved in 31 patients treated for one infection and in seven patients treated for multiple coincident infections. Thirteen of 14 patients treated for BKV-associated hemorrhagic cystitis experienced complete resolution of gross hematuria by week 6. Infusions were safe, and only two occurrences of de novo graft-versus host disease (grade 1) were observed. VST tracking by epitope profiling revealed persistence of functional VSTs of third-party origin for up to 12 weeks. Conclusion The use of banked VSTs is a feasible, safe, and effective approach to treat severe and drug-refractory infections after HSCT, including infections from two viruses (BKV and HHV-6) that had never been targeted previously with an off-the-shelf product. Furthermore, the multispecificity of the VSTs ensures extensive antiviral coverage, which facilitates the treatment of patients with multiple infections.

Tuberin-deficiency downregulates N-cadherin and upregulates vimentin in kidney tumor of TSC patients

PubMed Central

Liang, Sitai; Salas, Tiffanie; Gencaslan, Emre; Li, Baojie; Habib, Samy L.

2014-01-01

Angiomyolipomas (AMLs) are associated with cell fibrosis in kidney of Tuberous Sclerosis Complex patients. The mechanism by which the fibrotic proteins accumulated in AMLs has not been explored. In the present study, we investigated the role of Akt/tuberin/mTOR pathway in the regulation cell fibrosis proteins. AML cells that expressed low levels of tuberin showed less expression of N-cadherin and higher of vimentin proteins compared to HEK293 cells. AML cells infected with Ad-tuberin showed a significant decrease in vimentin and an increase in N-cadherin protein expression. In addition, cells treated with rapamycin showed a significant increase in p-Akt and a decrease in p-p70S6K that was associated with a decrease expression of vimentin and a slight increase expression in N-cadherin. On the other hand, cells treated with Akt inhibitor revealed a significant decrease in p-Akt and p-p70S6K that was associated with a significant decrease in vimentin and an increase in N-cadherin expression. In addition, cells transfected with DN-Akt or DN-S6K show significant increase expression in N-cadherin and a decrease in vimentin. Moreover, cells transfected with siRNA against rictor or siRNA against raptor resulted in a decrease in vimentin and an increase N-cadherin expression. Kidney tumors from TSC patients showed significant decrease in N-cadherin and significant increased in vimentin protein expression compared to control kidney tissues. These data comprise the first report to provide the role of Akt/tuberin/mTORC1/2 in the regulation of N-cadherin and vimentin that are involved in the progression of fibrosis in kidney tumor of TSC patients. PMID:25149531

5-thiomannosides block the biosynthesis of dolichol-linked oligosaccharides and mimic class I congenital disorders of glycosylation.

PubMed

Zandberg, Wesley F; Gao, Ningguo; Kumarasamy, Jayakanthan; Lehrman, Mark A; Seidah, Nabil G; Pinto, B Mario

2012-02-13

In a cell-based assay for novel inhibitors, we have discovered that two glycosides of 5-thiomannose, each containing an interglycosidic nitrogen atom, prevented the correct zymogen processing of the prohormone proopiomelanocortinin (POMC) and the transcription factor sterol-regulatory element-binding protein-2 (SREBP-2) in mouse pituitary cells and Chinese hamster ovary (CHO) cells, respectively. In the case of SREBP-2, these effects were correlated with the altered N-linked glycosylation of subtilisin/kexin-like isozyme-1 (SKI-1), the protease responsible for SREBP-2 processing under sterol-limiting conditions. Further examination of the effects of these compounds in CHO cells showed that they cause extensive protein hypoglycosylation in a manner similar to type I congenital disorders of glycosylation (CDGs) since the remaining N-glycans in treated cells were complete (normal) structures. The under-glycosylation of glycoproteins in 5-thiomannoside-treated cells is now shown to be caused by the compromised biosynthesis of the dolichol-linked oligosaccharide (DLO) N-glycosylation donor, although the nucleotide sugars required for the synthesis of DLOs were neither reduced under these conditions, nor were their effects reversed upon the addition of exogenous mannose. Analysis of DLO intermediates by fluorophore-assisted carbohydrate electrophoresis demonstrated that 5-thiomannose-containing glycosides block DLO biosynthesis most likely at a stage prior to the GlcNAc(2) Man(3) intermediate, on the cytosolic face of the endoplasmic reticulum. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of Withania somnifera Root Extract on Spontaneous Estrogen Receptor-Negative Mammary Cancer in MMTV/Neu Mice

PubMed Central

KHAZAL, KAMEL F.; HILL, DONALD L.; GRUBBS, CLINTON J.

2015-01-01

The cancer-preventive activity of an extract of Withania somnifera (WS) roots was examined in female transgenic (MMTV/Neu) mice that received a diet containing the extract (750 mg/kg of diet) for 10 months. Mice in the treated group (N=35) had an average of 1.66 mammary carcinomas, and mice in the control group (N=33) had 2.48, a reduction of 33%. The average weights of the carcinomas were 2.36 g for mice in the treated group and 2.63 g for the controls, a difference of 10%. Labeling indices for Ki67 and proliferating cell nuclear antigen marker in mammary carcinomas of the treated group were 35% and 30% lower, respectively, than those of the corresponding control group. Expression of the chemokine was reduced by 50%. These results indicate that the root extract reduced the number of mammary carcinomas that developed and reduced the rate of cell division in the carcinomas. PMID:25368231

Protective effect of zinc on N-methyl-N-nitrosourea and testosterone-induced prostatic intraepithelial neoplasia in the dorsolateral prostate of Sprague Dawley rats.

PubMed

Banudevi, Sivanantham; Elumalai, Perumal; Sharmila, Govindaraj; Arunkumar, Ramachandran; Senthilkumar, Kalimuthu; Arunakaran, Jagadeesan

2011-09-01

Previous studies have suggested that zinc exerts anticarcinogenic and antiproliferative effects against prostate cancer both in vitro and in rat ventral prostate. Zinc accumulation diminishes early in the course of prostate malignancy and it inhibits the growth of several carcinoma cells through induction of cell cycle arrest and apoptosis. In this study, we have investigated the influence of zinc on N-methyl-N-nitrosourea (MNU) and testosterone (T)-induced prostatic intraepithelial neoplasia in the dorsolateral prostate of Sprague Dawley (SD) rats. The results indicate that zinc plays an important role in prostate carcinogenesis. Increased tumor incidence was accompanied by a decrease in prostatic acid phosphatase activity, citrate, zinc, glutathione-S-transferase, reduced glutathione, p53, B-cell lymphoma protein (Bcl-2)-associated X protein and caspase-3 levels in MNU + T-treated rats. On the contrary, significantly increased phase I drug metabolizing enzyme activities, lipid peroxide, hydrogen peroxide, proliferating cell nuclear antigen, Bcl-2 and Bcl-X(L) protein levels were observed in the dorsolateral prostate of MNU + T-treated rats. Simultaneous zinc supplementation significantly reversed these effects in MNU + T-treated rats. Signs of dysplasia, a characteristic of prostatic intraepithelial neoplasia, were evident in the dorsolateral prostatic tissue sections by MNU + T administration. However, zinc supplementation has reversed these effects in the dorsolateral prostatic histoarchitecture. These results suggest that zinc may act as an essential trace element against MNU and testosterone-induced prostatic preneoplastic progression in SD rats.

Estrogen attenuates glutamate-induced cell death by inhibiting Ca2+ influx through L-type voltage-gated Ca2+ channels

PubMed Central

Sribnick, Eric A.; Del Re, Angelo M.; Ray, Swapan K.; Woodward, John J.; Banik, Naren L.

2009-01-01

Estrogen-mediated neuroprotection is observed in neurodegenerative disease and neurotrauama models; however, determining a mechanism for these effects has been difficult. We propose that estrogen may limit cell death in the nervous system tissue by inhibiting increases in intracellular free Ca2+. Here, we present data using VSC 4.1 cell line, a ventral spinal motoneuron and neuroblastoma hybrid cell line. Treatment with 1 mM glutamate for 24 h induced apoptosis. When cells were pre-treated with 100 nM 17β-estradiol (estrogen) for 1 h and then co-treated with glutamate, apoptotic death was significantly attenuated. Estrogen also prevented glutamate-mediated changes in resting membrane potential and membrane capacitance. Treatment with either 17α-estradiol or cell impermeable estrogen did not mimic the findings seen with estrogen. Glutamate treatment significantly increased both intracellular free Ca2+ and the activities of downstream proteases such as calpain and caspase-3. Estrogen attenuated both the increases in intracellular free Ca2+ and protease activities. In order to determine the pathway responsible for estrogen-mediated inhibition of these increases in intracellular free Ca2+, cells were treated with several Ca2+ entry inhibitors, but only the L-type Ca2+ channel blocker nifedipine demonstrated cytoprotective effects comparable to estrogen. To expand these findings, cells were treated with the L-type Ca2+ channel agonist FPL 64176, which increased both cell death and intracellular free Ca2+, and estrogen inhibited both effects. From these observations, we conclude that estrogen limits glutamate-induced cell death in VSC 4.1 cells through effects on L-type Ca2+ channels, inhibiting Ca2+ influx as well as activation of the pro-apoptotic proteases calpain and caspase-3. PMID:19389388

Differential effects of retinoic acid (RA) and N-(4-hydroxyphenyl) retinamide (4-HPR) on cell growth, induction of differentiation, and changes in p34cdc2, Bcl-2, and actin expression in the human promyelocytic HL-60 leukemic cells.

PubMed

Dipietrantonio, A; Hsieh, T C; Wu, J M

1996-07-25

Incubation of the HL-60 cells with 3 microM of RA and 4-HPR resulted in suppression of cell growth and decrease in cell viability. A significant percentage of the RA-treated cells also displayed differentiation towards neutrophils, as assayed by changes in nitroblue tetrazolium reduction (NBT) and alpha-naphthyl-acetate esterase (ANAE) activities, whereas the 4-HPR treated cells remained essentially undifferentiated. Flow cytometric analysis showed 4-HPR to cause partial cell arrest in the G2/M phase after a 3-day treatment and an additional G1 phase arrest after a 7-day treatment. With RA-treated cells, a reduction in the percentage of cells in the G1 phase was observed after 7 days of treatment. In 4-HPR-treated cells an extra peak, characteristic of cells undergoing apoptosis, was found in the cell cycle phase distribution analysis. Determination of specific protein expression changes by Western blot analysis showed that the p34cdc2 was down-regulated by both chemicals. Furthermore, RA induced bcl-2 but prevented the processing of actin, whereas 4-HPR had little effect on bcl-2 but increased the specific processing of actin. These results suggest that RA promotes neutrophil differentiation and the establishment of a semi apoptosis-resistant state, possibly through the overexpression of the bcl-2 gene. By contrast, 4-HPR may trigger apoptosis by inducing overall cyto-architectural changes and specific DNA fragmentation subsequent to increased turnover of the protein actin.

Aurora B inhibitor barasertib and cytarabine exert a greater-than-additive cytotoxicity in acute myeloid leukemia cells.

PubMed

Yamauchi, Takahiro; Uzui, Kanako; Shigemi, Hiroko; Negoro, Eiju; Yoshida, Akira; Ueda, Takanori

2013-07-01

Barasertib, an aurora B inhibitor, terminates cell division, introduces polyploidy, and consequently causes apoptosis. In the present study, we evaluated the effect of the combination of barasertib and cytarabine (ara-C), a key agent for leukemia chemotherapy, on leukemic cells in vitro. Human leukemia HL-60 cells and HL-60/ara-C20 cells, a 20-fold ara-C-resistant variant, were used. The 50% growth inhibitory concentrations of an active metabolite of barasertib, barasertib-hydroxyquinazoline-pyrazol-aniline (Barasertib-HQPA), and ara-C were 51 nM and 300 nM for HL-60 cells and 70 nM and 5300 nM for HL-60/ara-C20 cells, respectively. Barasertib-HQPA induced polyploidy with a subsequent induction of sub-G1 phase apoptosis, indicating the M-phase specific cytotoxicity. Cells treated with the S-phase specific ara-C accumulated in S phase and subsequently died through apoptosis. When HL-60 cells were treated with barasertib-HQPA and ara-C in combination, a greater-than-additive apoptosis was induced. This enhancement was obtained when the cells were treated with barasertib-HQPA prior to ara-C (37.9% sub-G1) or with both concurrently (31.2% sub-G1), but not with ara-C prior to barasertib-HQPA (17.8% sub-G1). The combination effects were similarly obtained in HL-60/ara-C20 cells with 19.7% sub-G1 for barasertib-HQPA→ara-C, 18.4% sub-G1 for both concurrently, and 13.8% sub-G1 for ara-C→barasertib-HQPA, and another leukemic U937 cells with 25.4% sub-G1 for barasertib-HQPA→ara-C, 28.2% sub-G1 for both concurrently, and 16.0% sub-G1 for ara-C→barasertib-HQPA. Barasertib-HQPA inhibited aurora B autophosphorylation and histone H3 phosphorylation in all the cell lines. Barasertib-HQPA did not inhibit DNA synthesis, allowing ara-C incorporation into DNA for its cytotoxicity. Thus, barasertib-HQPA and ara-C provided a greater-than-additive cytotoxicity in leukemic cells in vitro. © 2013 Japanese Cancer Association.

N-acetylcysteine attenuates TNF-alpha-induced human vascular endothelial cell apoptosis and restores eNOS expression.

PubMed

Xia, Zhengyuan; Liu, Min; Wu, Yong; Sharma, Vijay; Luo, Tao; Ouyang, Jingping; McNeill, John H

2006-11-21

The circulatory inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in pathological conditions, such as diabetes, which initiate or exacerbate vascular endothelial injury. Both nitric oxide (NO) and reactive oxygen species may play a dual role (i.e., inhibiting or promoting) in TNF-alpha-induced endothelial cell apoptosis. We investigated the effects of the antioxidant N-acetylcysteine on TNF-alpha-induced apoptosis in human vascular endothelial cell (cell line ECV304) apoptosis, NO production and lipid peroxidation. Cultured vascular endothelial cell (ECV304) were either not treated (control), or treated with TNF-alpha (40 ng/ml) alone or TNF-alpha in the presence of N-acetylcysteine at 30 mmol/l or 1 mmol/l, respectively, for 24 h. Cell viability was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was assessed by flow cytometry. TNF-alpha-induced endothelial cell apoptosis was associated with increased inducible NO synthase but reduced endothelial NO synthase (eNOS) protein expression. NO production and the levels of the lipid peroxidation product malondialdehyde were concomitantly increased. Treatment with NAC at 30 mmol/l restored eNOS expression and further increased NO production as compared to TNF-alpha alone, resulting in improved cell viability and reduced apoptosis. This was accompanied by increased superoxide dismutase activity, increased glutathione peroxidase production and reduced malondialdehyde levels. N-acetylcysteine at 1 mmol/l, however, did not have significant effects on TNF-alpha-induced endothelial cell apoptosis and cell viability despite it slightly enhanced glutathione peroxidase production. N-acetylcysteine attenuation of TNF-alpha-induced human vascular endothelial cell apoptosis is associated with the restoration of eNOS expression.

Comparative studies of saxitoxin (STX) -induced cytotoxicity in Neuro-2a and RTG-2 cell lines: An explanation with respect to changes in ROS.

PubMed

Zhou, Zhongyuan; Tang, Xuexi; Chen, Hongmei; Wang, You

2018-02-01

Saxitoxin (STX), a paralytic shellfish toxin (PST) produced from toxic bloom-forming dinoflagellates, was selected to comparatively investigate the induction of cytotoxicity and apoptosis and a possible mechanism based on changes in the antioxidant defence system of two cellular strains: the mouse neuroblastoma cell line Neuro-2a and the rainbow trout fish cell line RTG-2. Increasing concentrations of STX (0-256 nM) presented little cytotoxic or apoptotic effects on the two cell lines. Measurements of cellular viability, lethal ratio and LDH leakage showed slight changes in Neuro-2a and RTG-2 cells (p > 0.05), and similar results were observed for cellular morphology and apoptotic rates. The contents of the main reactive oxygen species (ROS) components, superoxide anion (O 2 - ) and hydrogen peroxide (H 2 O 2 ), were markedly increased in Neuro-2a cell with STX exposure at middle (15 nM) and high (150 nM) concentrations (p < 0.05), and the simultaneous increase of the ratio of reduced/oxidized glutathione (GSH/GSSG) (p < 0.05) inferred the occurrence of oxidative stress. However, little difference was observed in all treated groups of RTG-2 cells. The activities of three antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), were significantly enhanced in Neuro-2a cells in the middle and high concentration groups (p < 0.05), while glutathione peroxidase (GPX) obviously decreased (p < 0.05) in all treated groups. Little change was found in RTG-2 cells with the same exposures. These results provided evidence that STX exposure altered the redox status of Neuro-2a cells and resulted in oxidative stress, but the same exposure exerted little effect on RTG-2 cells. Therefore, Neuro-2a cells are more sensitive than reproductive cells to STX exposure, and the antioxidant systems appears to be partly responsible for this differentiation response. Copyright © 2017 Elsevier Ltd. All rights reserved.

Minocycline blocks asthma-associated inflammation in part by interfering with the T cell receptor-nuclear factor κB-GATA-3-IL-4 axis without a prominent effect on poly(ADP-ribose) polymerase.

PubMed

Naura, Amarjit S; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C; Jordan, Joaquin; Catling, Andrew D; Rezk, Bashir M; Abd Elmageed, Zakaria Y; Pyakurel, Kusma; Tarhuni, Abdelmetalab F; Abughazleh, Mohammad Q; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C; Boulares, A Hamid

2013-01-18

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

Nitric oxide synthase inhibition reduces muscle inflammation and necrosis in modified muscle use

NASA Technical Reports Server (NTRS)

Pizza, F. X.; Hernandez, I. J.; Tidball, J. G.

1998-01-01

The objective of this study was to determine the role of nitric oxide in muscle inflammation, fiber necrosis, and apoptosis of inflammatory cells in vivo. The effects of nitric oxide synthase (NOS) inhibition on the concentrations of neutrophils, ED1+ and ED2+ macrophages, apoptotic inflammatory cells, and necrotic muscle fibers in rats subjected to 10 days of hindlimb unloading and 2 days of reloading were determined. Administration of NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the concentrations of neutrophils, ED1+ and ED2+ macrophages, and necrotic fibers in soleus muscle relative to water-treated controls. The concentration of apoptotic inflammatory cells was also significantly lower for L-NAME-treated animals compared with water-treated controls. However, the proportion of the inflammatory cell population that was apoptotic did not differ between L-NAME-treated and control animals, suggesting that L-NAME treatment did not decrease inflammatory cell populations by increasing the frequency of apoptosis. Thus, nitric oxide or one of its intermediates promotes muscle inflammation and fiber necrosis during modified muscle use and plays no more than a minor role in the resolution of muscle inflammation by inducing apoptosis of inflammatory cells.

[Elevated expression of endothelin 2 in lung tissues of asthmatic rats after exposed to cigarette smoke and its mechanism].

PubMed

Han, Fangfang; Zhu, Shuyang; Chen, Bi; Li, Jingjing

2017-08-01

Objective To study the effect of cigarette smoke exposure on the expression of endothelin 2 (ET-2) in bronchial epithelium of asthmatic rats. Methods Asthma models were established through intraperitoneal injection of 1 mL chicken ovalbumin (OVA)/Al(OH) 3 mixture (asthma model group, n=6); based on the asthma models, exposure to smoking gas lasted four weeks with 10 cigarettes per day (smoke-exposed asthma group, n=6); based on the smoke-exposed asthma models, the rats were treated with intraperitoneal injection of dexamethasone 2 mg/(kg.d), intragastric administration of ET receptor inhibitor bosentan 100 mg/(kg.d) and combined use, respectively named dexamethasone treated group, bosentan treated group, and dexamethasone-bosentan treated group, 6 rats in every group. What's more, other 6 rats were only subjected to intraperitoneal injection of 1 mL normal saline as normal controls; in addition to the injection of saline, cigarette smoke control group (n=6) was set up by the exposure to smoking gas for four weeks with 10 cigarettes per day. Bronchoalveolar lavage fluid (BALF) was collected from the upper lobe of the left lung for cell counting and classification. Pathological changes of the right upper lung lobe tissues were observed by HE staining. In other lung tissues, the expression of JNK1/2 was detected by Western blotting; ET-2 was tested by Western blotting and immunohistochemistry; thiobarbituric acid reactive substances (TBARS) assay and trace enzyme standard method were used to measure malondialdehyde (MDA) and glutathione (GSH), respectively. Results Compared with normal control group, the number of airway inflammation cells increased in the BALF, and the expressions of ET-2, JNK1/2, MDA and GSH increased in the lung tissues of cigarette smoke control group, asthma model group and cigarette smoke-exposed asthma group. Compared with cigarette smoke-exposed asthma group, the number of airway inflammation cells decreased in the BALF, and the expressions of ET-2, JNK1/2, MDA and GSH decreased in the lung tissues of the dexamethasone treated group, bosentan treated group, and dexamethasone-bosentan treated group. Airway inflammation was attenuated and the staining intensity of ET-2 in the lung tissue was reduced in the dexamethasone treated group, bosentan treated group, and dexamethasone-bosentan treated group, which were more obvious in the dexamethasone-bosentan treated group. Conclusion Cigarette smoke exposure obviously aggravates airway inflammation in asthmatic rats, and bosentan can effectively alleviate the airway inflammation. The mechanism of the inflammation may be related to ET-2 and JNK1/2 signaling pathway.

Bone marrow-derived mesenchymal stem cells ameliorate sodium nitrite-induced hypoxic brain injury in a rat model

PubMed Central

Ali, Elham H.A.; Ahmed-Farid, Omar A.; Osman, Amany A. E.

2017-01-01

Sodium nitrite (NaNO2) is an inorganic salt used broadly in chemical industry. NaNO2 is highly reactive with hemoglobin causing hypoxia. Mesenchymal stem cells (MSCs) are capable of differentiating into a variety of tissue specific cells and MSC therapy is a potential method for improving brain functions. This work aims to investigate the possible therapeutic role of bone marrow-derived MSCs against NaNO2 induced hypoxic brain injury. Rats were divided into control group (treated for 3 or 6 weeks), hypoxic (HP) group (subcutaneous injection of 35 mg/kg NaNO2 for 3 weeks to induce hypoxic brain injury), HP recovery groups N-2wR and N-3wR (treated with the same dose of NaNO2 for 2 and 3 weeks respectively, followed by 4-week or 3-week self-recovery respectively), and MSCs treated groups N-2wSC and N-3wSC (treated with the same dose of NaNO2 for 2 and 3 weeks respectively, followed by one injection of 2 × 106 MSCs via the tail vein in combination with 4 week self-recovery or intravenous injection of NaNO2 for 1 week in combination with 3 week self-recovery). The levels of neurotransmitters (norepinephrine, dopamine, serotonin), energy substances (adenosine monophosphate, adenosine diphosphate, adenosine triphosphate), and oxidative stress markers (malondialdehyde, nitric oxide, 8-hydroxy-2′-deoxyguanosine, glutathione reduced form, and oxidized glutathione) in the frontal cortex and midbrain were measured using high performance liquid chromatography. At the same time, hematoxylin-eosin staining was performed to observe the pathological change of the injured brain tissue. Compared with HP group, pathological change of brain tissue was milder, the levels of malondialdehyde, nitric oxide, oxidized glutathione, 8-hydroxy-2′-deoxyguanosine, norepinephrine, serotonin, glutathione reduced form, and adenosine triphosphate in the frontal cortex and midbrain were significantly decreased, and glutathione reduced form/oxidized glutathione and adenosine monophosphate/adenosine triphosphate ratio were significantly increased in the MSCs treated groups. These findings suggest that bone marrow-derived MSCs exhibit neuroprotective effects against NaNO2-induced hypoxic brain injury through exerting anti-oxidative effects and providing energy to the brain. PMID:29323037

Progesterone-Based Contraceptives Reduce Adaptive Immune Responses and Protection against Sequential Influenza A Virus Infections.

PubMed

Hall, Olivia J; Nachbagauer, Raffael; Vermillion, Meghan S; Fink, Ashley L; Phuong, Vanessa; Krammer, Florian; Klein, Sabra L

2017-04-15

In addition to their intended use, progesterone (P4)-based contraceptives promote anti-inflammatory immune responses, yet their effects on the outcome of infectious diseases, including influenza A virus (IAV) infection, are rarely evaluated. To evaluate their impact on immune responses to sequential IAV infections, adult female mice were treated with placebo or one of two progestins, P4 or levonorgestrel (LNG), and infected with a mouse-adapted H1N1 (maH1N1) virus. Treatment with P4 or LNG reduced morbidity but had no effect on pulmonary virus titers during primary H1N1 infection compared to placebo treatment. In serum and bronchoalveolar lavage fluid, total anti-IAV IgG and IgA titers and virus-neutralizing antibody titers but not hemagglutinin stalk antibody titers were lower in progestin-treated mice than placebo-treated mice. Females were challenged 6 weeks later with either an maH1N1 drift variant (maH1N1dv) or maH3N2 IAV. The level of protection following infection with the maH1N1dv was similar among all groups. In contrast, following challenge with maH3N2, progestin treatment reduced survival as well as the numbers and activity of H1N1- and H3N2-specific memory CD8 + T cells, including tissue-resident cells, compared with placebo treatment. In contrast to primary IAV infection, progestin treatment increased the titers of neutralizing and IgG antibodies against both challenge viruses compared with those achieved with placebo treatment. While the immunomodulatory properties of progestins protected immunologically naive female mice from the severe outcomes from IAV infection, it made them more susceptible to secondary challenge with a heterologous IAV, despite improving their antibody responses against a secondary IAV infection. Taken together, the immunomodulatory effects of progestins differentially regulate the outcome of infection depending on exposure history. IMPORTANCE The impact of hormone-based contraceptives on the outcome of infectious diseases outside the reproductive tract is rarely considered. Using a mouse model, we have made the novel observation that treatment with either progesterone or a synthetic analog found in hormonal contraceptives, levonorgestrel, impacts sequential influenza A virus infection by modulating antibody responses and decreasing the numbers and activity of memory CD8 + T cells. Progestins reduced the antibody responses during primary H1N1 virus infection but increased antibody titers following a sequential infection with either an H1N1 drift variant or an H3N2 virus. Following challenge with an H3N2 virus, female mice treated with progestins experienced greater mortality with increased pulmonary inflammation and reduced numbers and activity of CD8 + T cells. This study suggests that progestins significantly affect adaptive immune responses to influenza A virus infection, with their effect on the outcome of infection depending on exposure history. Copyright © 2017 American Society for Microbiology.

Chemopreventive effect of different ratios of fish oil and corn oil on prognostic markers, DNA damage and cell cycle in colon carcinogenesis.

PubMed

Sarotra, Pooja; Kansal, Shevali; Sandhir, Rajat; Agnihotri, Navneet

2012-03-01

Fish oil (FO) rich in n-3 polyunsaturated fatty acids (PUFAs) have a protective role in autoimmune disorders, type 2 diabetes, rheumatoid arthritis, and cancer, whereas corn oil (CO) rich in n-6 PUFAs has a proinflammatory and procarcinogenic effect. A balanced n-3/n-6 PUFA ratio in diet rather than absolute intake of either may be responsible for decreasing cancer incidence. This study was designed to evaluate the chemopreventive effect of different ratios of FO and CO on prognostic markers, DNA damage, and cell cycle distribution in colon carcinogenesis. Male Wistar rats were divided into control, N,N'-dimethylhydrazine dihydrochloride (DMH) treated, FO+CO(1 : 1)+DMH, and FO+CO(2.5 : 1)+DMH. All the groups, except control, received a weekly injection of DMH for 4 weeks. The animals were given modified AIN-76A diets and killed either 48 h later (initiation phase) or kept for 16 weeks (postinitiation phase). The animals treated with DMH in both the phases showed an increase in multiple plaque lesions, total sialic acid, lipid associated sialic acid, DNA damage and cell proliferation. However, levels of p53 in the postinitiation and cyclin D1 in both the phases were significantly elevated. FO+CO(2.5 : 1)+DMH treatment in both the phases led to a decrease in multiple plaque lesions, DNA damage, total sialic acid, lipid associated sialic acid as compared with the DMH treated group. There was a G1 arrest with a decrease in p53 and cyclin D1 levels in FO+CO(2.5 : 1) in both the phases whereas treatment with FO+CO(1 : 1)+DMH led to same results in the postinitiation phase only. This study suggests that FO+CO(2.5 : 1) is more effective in chemoprevention of experimental colon carcinogenesis.

Mugil cephalus roe oil obtained by supercritical fluid extraction affects the lipid profile and viability in cancer HeLa and B16F10 cells.

PubMed

Rosa, A; Piras, A; Nieddu, M; Putzu, D; Cesare Marincola, F; Falchi, A M

2016-09-14

We explored the changes in viability and lipid profile occurring in cancer cells, murine melanoma cells (B16F10 cells) and human cervical carcinoma cells (HeLa cells), when exposed to 24 h-treatments with an n-3 PUFA-rich oil obtained by supercritical extraction with CO2 from Mugil cephalus processed roe (bottarga). The composition of the major lipid classes of bottarga oil was determined by the (13)C NMR technique. Reversed-phase HPLC with DAD/ELSD detection was performed to analyze cells' total fatty acid profile and the levels of phospholipids, total/free cholesterol, triacylglycerols, and cholesteryl esters. Cell-based fluorescent measurements of intracellular membranes and lipid droplets were performed on bottarga oil-treated cells using the Nile red staining technique. The treatments of cancer cells with bottarga oil reduced the viability and affected the fatty acid profile, with a significant n-3 PUFA increase in treated cells. Mullet roe oil uptake modulated the cancer cell lipid composition, inducing a remarkable incorporation of health beneficial n-3 PUFA in the polar and neutral lipid fractions. Bottarga oil treatment influenced the synthesis of intracellular membranes and accumulation of cytoplasmic lipid droplets in cancer cells.

Characteristics of adverse drug reactions in a vemurafenib early post-marketing phase vigilance study in Japan.

PubMed

Uhara, H; Kiyohara, Y; Tsuda, A; Takata, M; Yamazaki, N

2018-02-01

Post-approval research or monitoring is important to determine real-world safety of new products; however, evidence is scant for vemurafenib in Japanese patients. In Japan, a unique system is officially obligated to investigate post-approval safety. Here we report the first adverse drug reaction (ADR) data from vemurafenib-treated Japanese patients with metastatic melanoma. Data were collected in an early post-marketing phase vigilance (EPPV) study. ADRs were events for which a causal relationship with vemurafenib could not be ruled out or was unknown. ADR data were collected for patients treated with vemurafenib (960 mg bid) between 26 February and 25 August 2015. Among 95 patients, 46 patients had 118 ADRs (24 serious ADRs in 13 patients). The most common serious ADRs were hypersensitivity (n = 1; 3 events), arthralgia (n = 2; 2 events), pyrexia (n = 2; 2 events) and drug eruption (n = 2; 2 events). Seven patients had serious skin disorders or hypersensitivity, six of whom had prior anti-programmed cell death-1 (PD-1) antibodies 5-35 days before starting vemurafenib. ADR reports of serious skin disorders appeared to be collected more rapidly than previously reported. Cutaneous squamous cell carcinoma developed in only one patient. EPPV in Japanese vemurafenib-treated patients identified no new safety signals. The most serious skin and hypersensitivity ADRs occurred in patients with prior anti-PD-1 exposure. Cutaneous squamous cell carcinoma appeared to be rare in Japanese patients. Further research is needed to clarify whether prior treatment with anti-PD-1 agents or racial differences affect the characteristic profile of cutaneous ADRs in Japanese patients.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukiyama, Fuyo; Nakai, Yumi; Yoshida, Masataka

Catechins have recently been reported to increase the cellular content of the hypoxia-inducible factor (HIF)-1{alpha} within mammalian cells. These catechins have a gallate moiety as a common structure. We now report that n-propyl gallate (nPG) also increases the HIF-1{alpha} protein in the rat heart-derived H9c2 cells. The increase was dose-dependent and reached a maximum at 2-4 h after the addition of nPG to the cells. nPG did not change the HIF-1{alpha} mRNA level, showing that the increase is a posttranscriptional event. Although nPG did not inhibit the HIF prolyl hydroxylase, gallate, the hydrolysis product of nPG, inhibited the enzyme completelymore » at submillimolar concentrations. Model building studies on the human HIF prolyl hydroxylase 2 showed that the two phenolate oxygen atoms of gallate form a chelate with the active site Fe{sup 2+}, while the carboxyl group of gallate forms a strong ionic/hydrogen bonding interaction with Arg383, explaining why nPG, which has an esterified carboxyl group, is unable to inhibit the hydroxylase. Together with the observation that gallate was detected in the H9c2 cells treated with nPG, these results suggest that nPG incorporated into the cells is hydrolyzed and the released gallate inhibits the HIF prolyl hydroxylase, thereby reducing the HIF degradation rate and increasing the HIF-1{alpha} content.« less

FGF-23 dysregulates calcium homeostasis and electrophysiological properties in HL-1 atrial cells.

PubMed

Kao, Yu-Hsun; Chen, Yao-Chang; Lin, Yung-Kuo; Shiu, Rong-Jie; Chao, Tze-Fan; Chen, Shih-Ann; Chen, Yi-Jen

2014-08-01

Fibroblast growth factor (FGF)-23 is a key regulator of phosphate homeostasis. Higher FGF-23 levels are correlated with poor outcomes in cardiovascular diseases. FGF-23 can produce cardiac hypertrophy and increase intracellular calcium, which can change cardiac electrical activity. However, it is not clear whether FGF-23 possesses arrhythmogenic potential through calcium dysregulation. Therefore, the purposes of this study were to evaluate the electrophysiological effects of FGF-23 and identify the underlying mechanisms. Patch clamp, confocal microscope with Fluo-4 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, calcium homeostasis and calcium regulatory proteins in HL-1 atrial myocytes with and without FGF-23 (10 and 25 ng/mL) incubation for 24 h. FGF-23 (25 ng/mL) increased L-type calcium currents, calcium transient and sarcoplasmic reticulum Ca(2+) contents in HL-1 cells. FGF-23 (25 ng/mL)-treated cells (n = 14) had greater incidences (57%, 17% and 15%, P < 0·05) of delayed afterdepolarizations than control (n = 12) and FGF-23 (10 ng/mL)-treated cells (n = 13). Compared with control cells, FGF-23 (25 ng/mL)-treated cells (n = 14) exhibited increased phosphorylation of calcium/calmodulin-dependent protein kinase IIδ and phospholamban (PLB) at threonine 17 but had similar phosphorylation extents of PLB at serine 16, total PLB and sarcoplasmic reticulum Ca(2+) -ATPase protein. Moreover, the FGF receptor inhibitor (PD173074, 10 nM), calmodulin inhibitor (W7, 5 μM) and phospholipase C inhibitor (U73122, 1 μM) attenuated the effects of FGF-23 on calcium/calmodulin-dependent protein kinase II phosphorylation. FGF-23 increases HL-1 cells arrhythmogenesis with calcium dysregulation through modulating calcium-handling proteins. © 2014 Stichting European Society for Clinical Investigation Journal Foundation.

Influence of Fractionation Scheme and Tumor Location on Toxicities After Stereotactic Body Radiation Therapy for Large (≥5 cm) Non-Small Cell Lung Cancer: A Multi-institutional Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Verma, Vivek; Shostrom, Valerie K.; Zhen, Weining

Purpose: To describe the impact of fractionation scheme and tumor location on toxicities in stereotactic body radiation therapy (SBRT) for ≥5-cm non-small cell lung cancer (NSCLC), as part of a multi-institutional analysis. Methods: Patients with primary ≥5-cm N0 M0 NSCLC who underwent ≤5-fraction SBRT were examined across multiple high-volume SBRT centers. Collected data included clinical/treatment parameters; toxicities were prospectively assessed at each institution according to the Common Terminology Criteria for Adverse Events. Patients treated daily were compared with those treated every other day (QOD)/other nondaily regimens. Stratification between central and peripheral tumors was also performed. Results: Ninety-two patients from 12 institutionsmore » were evaluated (2004-2016), with median follow-up of 12 months. In total there were 23 (25%) and 6 (7%) grade ≥2 and grade ≥3 toxicities, respectively. Grades 2 and 3 pulmonary toxicities occurred in 9% and 4%, respectively; 1 patient treated daily experienced grade 5 radiation pneumonitis. Of the entire cohort, 46 patients underwent daily SBRT, and 46 received QOD (n=40)/other nondaily (n=6) regimens. Clinical/treatment parameters were similar between groups; the QOD/other group was more likely to receive 3-/4-fraction schemas. Patients treated QOD/other experienced significantly fewer grade ≥2 toxicities as compared with daily treatment (7% vs 43%, P<.001). Patients treated daily also had higher rates of grade ≥2 pulmonary toxicities (P=.014). Patients with peripheral tumors (n=66) were more likely to receive 3-/4-fraction regimens than those with central tumors (n=26). No significant differences in grade ≥2 toxicities were identified according to tumor location (P>.05). Conclusions: From this multi-institutional study, toxicity of SBRT for ≥5-cm lesions is acceptable, and daily treatment was associated with a higher rate of toxicities.« less

Influence of Fractionation Scheme and Tumor Location on Toxicities After Stereotactic Body Radiation Therapy for Large (≥5 cm) Non-Small Cell Lung Cancer: A Multi-institutional Analysis.

PubMed

Verma, Vivek; Shostrom, Valerie K; Zhen, Weining; Zhang, Mutian; Braunstein, Steve E; Holland, John; Hallemeier, Christopher L; Harkenrider, Matthew M; Iskhanian, Adrian; Jabbour, Salma K; Attia, Albert; Lee, Percy; Wang, Kyle; Decker, Roy H; McGarry, Ronald C; Simone, Charles B

2017-03-15

To describe the impact of fractionation scheme and tumor location on toxicities in stereotactic body radiation therapy (SBRT) for ≥5-cm non-small cell lung cancer (NSCLC), as part of a multi-institutional analysis. Patients with primary ≥5-cm N0 M0 NSCLC who underwent ≤5-fraction SBRT were examined across multiple high-volume SBRT centers. Collected data included clinical/treatment parameters; toxicities were prospectively assessed at each institution according to the Common Terminology Criteria for Adverse Events. Patients treated daily were compared with those treated every other day (QOD)/other nondaily regimens. Stratification between central and peripheral tumors was also performed. Ninety-two patients from 12 institutions were evaluated (2004-2016), with median follow-up of 12 months. In total there were 23 (25%) and 6 (7%) grade ≥2 and grade ≥3 toxicities, respectively. Grades 2 and 3 pulmonary toxicities occurred in 9% and 4%, respectively; 1 patient treated daily experienced grade 5 radiation pneumonitis. Of the entire cohort, 46 patients underwent daily SBRT, and 46 received QOD (n=40)/other nondaily (n=6) regimens. Clinical/treatment parameters were similar between groups; the QOD/other group was more likely to receive 3-/4-fraction schemas. Patients treated QOD/other experienced significantly fewer grade ≥2 toxicities as compared with daily treatment (7% vs 43%, P<.001). Patients treated daily also had higher rates of grade ≥2 pulmonary toxicities (P=.014). Patients with peripheral tumors (n=66) were more likely to receive 3-/4-fraction regimens than those with central tumors (n=26). No significant differences in grade ≥2 toxicities were identified according to tumor location (P>.05). From this multi-institutional study, toxicity of SBRT for ≥5-cm lesions is acceptable, and daily treatment was associated with a higher rate of toxicities. Copyright © 2016 Elsevier Inc. All rights reserved.

Cyclin-dependent kinase 9 is a novel specific molecular target in adult T-cell leukemia/lymphoma.

PubMed

Narita, Tomoko; Ishida, Takashi; Ito, Asahi; Masaki, Ayako; Kinoshita, Shiori; Suzuki, Susumu; Takino, Hisashi; Yoshida, Takashi; Ri, Masaki; Kusumoto, Shigeru; Komatsu, Hirokazu; Imada, Kazunori; Tanaka, Yuetsu; Takaori-Kondo, Akifumi; Inagaki, Hiroshi; Scholz, Arne; Lienau, Philip; Kuroda, Taruho; Ueda, Ryuzo; Iida, Shinsuke

2017-08-31

Cyclin-dependent kinase 9 (CDK9), a subunit of the positive transcription elongation factor b (P-TEFb) complex, regulates gene transcription elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). The deregulation of CDK9/P-TEFb has important implications for many cancer types. BAY 1143572 is a novel and highly selective CDK9/P-TEFb inhibitor currently being investigated in phase 1 studies. We evaluated the therapeutic potential of BAY 1143572 in adult T-cell leukemia/lymphoma (ATL). As a result of CDK9 inhibition and subsequent inhibition of phosphorylation at serine 2 of the RNAPII CTD, BAY 1143572 decreased c-Myc and Mcl-1 levels in ATL-derived or human T-cell lymphotropic virus type-1 (HTLV-1)-transformed lines and primary ATL cells tested, leading to their growth inhibition and apoptosis. Median inhibitory concentrations for BAY 1143572 in ATL-derived or HTLV-1-transformed lines (n = 8), primary ATL cells (n = 11), and CD4 + cells from healthy volunteers (n = 5) were 0.535, 0.30, and 0.36 μM, respectively. Next, NOG mice were used as recipients of tumor cells from an ATL patient. BAY 1143572-treated ATL-bearing mice (once daily 12.5 mg/kg oral application) demonstrated significantly decreased ATL cell infiltration of the liver and bone marrow, as well as decreased human soluble interleukin-2 receptor levels in serum (reflecting the ATL tumor burden), compared with untreated mice (n = 8 for both). BAY 1143572-treated ATL-bearing mice demonstrated significantly prolonged survival compared with untreated ATL-bearing mice (n = 7 for both). Collectively, this study indicates that BAY 1143572 showed strong potential as a novel treatment of ATL. © 2017 by The American Society of Hematology.

Radiotherapy alone or combined with chemotherapy as definitive treatment for squamous cell carcinoma of the tonsil.

PubMed

Kennedy, William R; Herman, Michael P; Deraniyagala, Rohan L; Amdur, Robert J; Werning, John W; Dziegielewski, Peter; Kirwan, Jessica; Morris, Christopher G; Mendenhall, William M

2016-08-01

This study is aimed at updating our institution's experience with definitive radiotherapy (RT) for squamous cell carcinoma of the tonsil. We reviewed 531 patients treated between 1983 and 2012 with definitive RT for squamous cell carcinoma of the tonsil. Of these, 179 patients were treated with either induction (n = 19) or concomitant (n = 160) chemotherapy. Planned neck dissection was performed on 217 patients: unilaterally in 199 and bilaterally in 18 patients. Median follow-up was 5.2 years for all patients (range 0.1-31.6 years) and 8.2 years for living patients (range 1.9-31.6 years). The 5-year local control rates by T stage were as follows: T1, 94 %; T2, 87 %; T3 79 %; T4, 70 %; and overall, 83 %. Multivariate analysis revealed that local control was significantly influenced by T stage and neck dissection. The 5-year cause-specific survival rates by overall stage were as follows: I, 94 %; II, 88 %; III, 87 %; IVA, 75 %; IVB, 52 %; and overall, 78 %. Multivariate analysis revealed that cause-specific survival was significantly influenced by T stage, N stage, overall stage, fractionation, neck dissection, sex, and ethnicity. Of 77 patients treated with ipsilateral fields only, contralateral neck failure occurred in 1 %. The rate of severe complications was 12 %. Definitive RT for patients with tonsillar squamous cell carcinoma provides control rates equivalent to other modalities with a comparatively low incidence of late complications. Patients with anterior tonsillar pillar or tonsillar fossa primaries that are well lateralized with no base of tongue or soft palate extension may be treated with ipsilateral fields.

Antiapoptotic effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant in H9c2 rat cardiomyocytes.

PubMed

Chung, Hee Kyoung; Ko, Eun Mi; Kim, Sung Woo; Byun, Sung-June; Chung, Hak-Jae; Kwon, Moosik; Lee, Hwi-Cheul; Yang, Byoung-Chul; Han, Deug-Woo; Park, Jin-Ki; Hong, Sung-Gu; Chang, Won-Kyong; Kim, Kyung-Woon

2012-12-01

Granulocyte colony-stimulating factor (G-CSF) is used for heart failure therapy and promotes myocardial regeneration by inducing mobilization of bone marrow stem cells to the injured heart after myocardial infarction; however, this treatment has one weakness in that its biological effect is transient. In our previous report, we generated 5 mutants harboring N-linked glycosylation to improve its antiapoptotic activities. Among them, one mutant (Phe140Asn) had higher cell viability than wild-type hG-CSF in rat cardiomyocytes, even after treatment with an apoptotic agent (H2O2). Cells treated with this mutant significantly upregulated the antiapoptotic proteins, and experienced reductions in caspase 3 activity and PARP cleavage. Moreover, the total number of apoptotic cells was dramatically lower in cultures treated with mutant hG-CSF. Taken together, these results suggest that the addition of an N-linked glycosylation was successful in improving the antiapoptotic activity of hG-CSF, and that this mutated product will be a feasible therapy for patients who have experienced heart failure.

The protective effect of aqueous extracts of roselle (Hibiscus sabdariffa L. UKMR-2) against red blood cell membrane oxidative stress in rats with streptozotocin-induced diabetes

PubMed Central

Mohamed, Jamaludin; Shing, Saw Wuan; Md Idris, Muhd Hanis; Budin, Siti Balkis; Zainalabidin, Satirah

2013-01-01

OBJECTIVES: The aim of this study was to investigate the protective effects of aqueous extracts of roselle (Hibiscus sabdariffa L. UKMR-2) against red blood cell (RBC) membrane oxidative stress in rats with streptozotocin-induced diabetes. METHODS: Forty male Sprague-Dawley rats weighing 230-250 g were randomly divided into four groups (n = 10 rats each): control group (N), roselle-treated control group, diabetic group, and roselle-treated diabetic group. Roselle was administered by force-feeding with aqueous extracts of roselle (100 mg/kg body weight) for 28 days. RESULTS: The results demonstrated that the malondialdehyde levels of the red blood cell membranes in the diabetic group were significantly higher than the levels in the roselle-treated control and roselle-treated diabetic groups. The protein carbonyl level was significantly higher in the roselle-treated diabetic group than in the roselle-treated control group but lower than that in the diabetic group. A significant increase in the red blood cell membrane superoxide dismutase enzyme was found in roselle-treated diabetic rats compared with roselle-treated control rats and diabetic rats. The total protein level of the red blood cell membrane, osmotic fragility, and red blood cell morphology were maintained. CONCLUSION: The present study demonstrates that aqueous extracts of roselle possess a protective effect against red blood cell membrane oxidative stress in rats with streptozotocin-induced diabetes. These data suggest that roselle can be used as a natural antioxidative supplement in the prevention of oxidative damage in diabetic patients. PMID:24212844

Cellular and Hormonal Disruption of Fetal Testis Development in Sheep Reared on Pasture Treated with Sewage Sludge

PubMed Central

Paul, Catriona; Rhind, Stewart M.; Kyle, Carol E.; Scott, Hayley; McKinnell, Chris; Sharpe, Richard M.

2005-01-01

The purpose of this study was to evaluate whether experimental exposure of pregnant sheep to a mixture of environmental chemicals added to pasture as sewage sludge (n = 9 treated animals) exerted effects on fetal testis development or function; application of sewage sludge was undertaken so as to maximize exposure of the ewes to its contents. Control ewes (n = 9) were reared on pasture treated with an equivalent amount of inorganic nitrogenous fertilizer. Treatment had no effect on body weight of ewes, but it reduced body weight by 12–15% in male (n = 12) and female (n = 8) fetuses on gestation day 110. In treated male fetuses (n = 11), testis weight was significantly reduced (32%), as were the numbers of Sertoli cells (34% reduction), Leydig cells (37% reduction), and gonocytes (44% reduction), compared with control fetuses (n = 8). Fetal blood levels of testosterone and inhibin A were also reduced (36% and 38%, respectively) in treated compared with control fetuses, whereas blood levels of luteinizing hormone and follicle-stimulating hormone were unchanged. Based on immunoexpression of anti-Müllerian hormone, cytochrome P450 side chain cleavage enzyme, and Leydig cell cytoplasmic volume, we conclude that the hormone changes in treated male fetuses probably result from the reduction in somatic cell numbers. This reduction could result from fetal growth restriction in male fetuses and/or from the lowered testosterone action; reduced immunoexpression of α-smooth muscle actin in peritubular cells and of androgen receptor in testes of treated animals supports the latter possibility. These findings indicate that exposure of the developing male sheep fetus to real-world mixtures of environmental chemicals can result in major attenuation of testicular development and hormonal function, which may have consequences in adulthood. PMID:16263515

Association between radiation dose and pathological complete response after preoperative radiochemotherapy in esophageal squamous cell cancer.

PubMed

Ordu, Arif Deniz; Nieder, Carsten; Geinitz, Hans; Scherer, Vera; Kup, Philipp Günther; Schuster, Tibor; Combs, Stephanie E; Fakhrian, Khashayar

2014-12-01

This study was undertaken to examine the impact of radiation dose on pathological complete response (pCR) rates following neoadjuvant radiochemotherapy (N-RCT) for squamous cell esophageal cancer (ESCC). From 1988 to 2011, 218 patients were treated with 30-30.6 Gy (1.8-2 Gy per fraction), 39.6-40 Gy (1.8-2 Gy per fraction) or 44-45 Gy (1.8-2 Gy per fraction) and concomitant cisplatin ± 5-fluorouracil (5-FU), oxaliplatin + 5-FU or 5-FU alone. The most commonly used concomitant chemotherapy was continuous infusion of 5-FU-alone with a dose of 300 mg/m(2)/day during the whole course of treatment (n=111). To eliminate the dispersing effect of potentially different efficacy levels of these drug regimens on pCR, we excluded patients with regimens other than 5-FU-alone. Histomorphological regression grade 1a (0% residual tumor), 1b (<10% residual tumor), 2 (10-50% residual tumor) and 3 (>50% residual tumor) was observed in 26 (23%), 24 (22%), 36 (32%) and 25 (23%) patients, respectively. pCR was observed in 9 out of 71 (13%) patients treated with 30 Gy-30.6 Gy, 13 of 34 (38%) patients treated with 39.6-40 Gy and 4 of 6 (67%) patients treated with 44-45 Gy (p=0.001). Median follow-up time from the start of N-RCT was 191 months (range=2-262 months). The estimated 5-year overall survival (OS) was 33% for the whole cohort. OS at 5 years was 58% for patients with pCR compared to 25% for patients with less favorable response to N-RCT (p=0.009), respectively. The dose of radiation correlates significantly with the likelihood of achieving a pCR in stage II/III squamous cell esophageal cancer patients. Prospective randomized trials are required to definitively evaluate the impact of application of higher radiation doses on efficacy and safety/tolerability in the context of N-RCT on the clinical outcomes. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

New metabolic pathway for N,N-dimethyltryptamine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hryhorczuk, L.M.; Rainey, J.M. Jr.; Frohman, C.E.

1986-01-01

N,N-Dimethyltryptamine (DMT) undergoes a major structural alteration when added to whole human blood or its red blood cells in vitro. A new high-pressure liquid chromatography (HPLC) peak is present in extracts of these treated tissues. The compound responsible for this peak has been identified by ultraviolet spectrophotometry and by mass spectrometry as dimethylkynuramine (DMK). The enzyme responsible for this appears to be different from tryptophan 2,3-dioxygenase and also from indoleamine 2,3-dioxygenase.

Protective effects of Semiaquilegia adoxoides n-butanol extract against hydrogen peroxide-induced oxidative stress in human lens epithelial cells.

PubMed

Liang, Bing; Wei, Wei; Wang, Jianta; Zhang, Mingming; Xu, Ran; Wu, Fei; Xiao, Haitao; Tang, Lei

2016-09-01

Context Hydrogen peroxide (H2O2)-induced damage in the lens epithelium leads to cell death and cataract. Semiaquilegia adoxoides (DC.) Makino (Ranunculaceae), a folk medicine of Hmong (an ethnic group of China), has been traditionally used to treat cataract; however, the underlying molecular mechanism is yet to be uncovered. Objective This study aimed to investigate whether the n-butanol extract of S. adoxoides (nSA) is effective against the H2O2-induced oxidative stress in human lens epithelial (HLE) cells. Materials and methods Human lens epithelial (SRA 01/04) cells were stimulated by H2O2 (250 μM) in the presence or absence of nSA. The antioxidant effects of nSA were determined in terms of cell viability (MTT assay), apoptosis (AnnexinV/PI staining), radical scavenging capability (various enzymatic assays), loss of mitochondrial membrane potential (Rhodamine 123 staining), expression of apoptotic markers including caspase-3 and caspase-9 and the change of Bcl-2/Bax ratio (western blot) in the HLE cells. Results The results showed that pretreatment of nSA (250, 500 and 1000 μg/mL) markedly reduced H2O2-induced cellular apoptosis and malondialdehyde accumulation, but elevated the activities of total superoxide dismutase, catalase, glutathione peroxidase. Thus, the total antioxidative capability was enhanced upon the nSA treatment meanwhile the loss of mitochondrial membrane potential was prevented. Moreover, nSA at concentrations of 250, 500 and 1000 μg/mL also significantly suppressed the activation of caspase-3 and -9, and increased the Bcl-2/Bax ratio in the HLE cells. Discussion and conclusion Our findings suggested that nSA is a potential prophylactic agent in the prevention of cataractogeneis.

Serum From Advanced Heart Failure Patients Promotes Angiogenic Sprouting and Affects the Notch Pathway in Human Endothelial Cells

PubMed Central

Pannella, Micaela; Caliceti, Cristiana; Fortini, Francesca; Aquila, Giorgio; Sega, Francesco Vieceli Dalla; Pannuti, Antonio; Fortini, Cinzia; Morelli, Marco Bruno; Fucili, Alessandro; Francolini, Gloria; Voltan, Rebecca; Secchiero, Paola; Dinelli, GiovannI; Leoncini, Emanuela; Ferracin, Manuela; Hrelia, Silvana; Miele, Lucio; Rizzo, Paola

2017-01-01

It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta-like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT-proBNP (N-terminal of the pro-hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera-treated cells (r = −0.526 and r = −0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. PMID:26987674

The effect of pentoxifylline on spontaneous and experimental metastasis of the mouse Neuro2a neuroblastoma.

PubMed

Amirkhosravi, A; Warnes, G; Biggerstaff, J; Malik, Z; May, K; Francis, J L

1997-07-01

Pentoxifylline (PTX) has been reported to have both direct and indirect anti-tumor effects in experimental tumor models. We studied the effect of PTX on (1) the proliferation of Neuro2a mouse neuroblastoma cells in vitro and in vivo, (2) spontaneous and experimental metastasis, (3) tumor cell membrane fluidity and (4) adhesion to a fibronectin-coated surface. PTX significantly reduced the proliferation of Neuro2a cells in vitro as determined by DNA measurement (P < 0.01) and total cell count (P < 0.02). In vivo, PTX reduced the growth of subcutaneously transplanted primary tumors in syngeneic A/J mice (P < 0.01; n = 15). All seven animals (100%) receiving intravenous tumor cells developed extensive liver metastasis. In contrast, only 1/11 (9%) of animals pre-treated with oral PTX and injected with PTX-treated cells developed liver metastases. Of five mice receiving PTX-treated cells without oral pretreatment of PTX, two out of five (40%) developed liver metastases. There was a slight, but not significant (P = 0.08) increase in both experimental and spontaneous lung metastases formation in PTX-treated animals. However, tumor nodule formation on the lung surface was inefficient. PTX also increased membrane fluidity of the Neuro2a cells and significantly decreased tumor cell adhesion to fibronectin-coated microtiter wells (P < 0.01). We conclude that PTX has a cytostatic effect on the Neuro2a mouse neuroblastoma and exerts an anti-tumor effect on liver metastases following intravenous administration of neuroblastoma cells. Whether these results are directly related to the changes in membrane properties caused by pentoxifylline remains to be established.

Cooptimization of Adhesion and Power Conversion Efficiency of Organic Solar Cells by Controlling Surface Energy of Buffer Layers.

PubMed

Lee, Inhwa; Noh, Jonghyeon; Lee, Jung-Yong; Kim, Taek-Soo

2017-10-25

Here, we demonstrate the cooptimization of the interfacial fracture energy and power conversion efficiency (PCE) of poly[N-9'-heptadecanyl-2,7-carbazole-alt-5,5-(4',7'-di-2-thienyl-2',1',3'-benzothiadiazole)] (PCDTBT)-based organic solar cells (OSCs) by surface treatments of the buffer layer. The investigated surface treatments of the buffer layer simultaneously changed the crack path and interfacial fracture energy of OSCs under mechanical stress and the work function of the buffer layer. To investigate the effects of surface treatments, the work of adhesion values were calculated and matched with the experimental results based on the Owens-Wendt model. Subsequently, we fabricated OSCs on surface-treated buffer layers. In particular, ZnO layers treated with poly[(9,9-bis(3'-(N,N-dimethylamino)propyl)-2,7-fluorene)-alt-2,7-(9,9-dioctylfluorene)] (PFN) simultaneously satisfied the high mechanical reliability and PCE of OSCs by achieving high work of adhesion and optimized work function.

A microdosimetric study of {sup 10}B(n,{alpha}){sup 7}Li and {sup 157}Gd(n,{gamma}) reactions for neutron capture therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, C.K.C.; Sutton, M.; Evans, T.M.

1999-01-01

This paper presents the microdosimetric analysis for the most interesting cell survival experiment recently performed at the Brookhaven National Laboratory (BNL). In this experiment, the cells were first treated with a gadolinium (Gd) labeled tumor-seeking boronated porphyrin (Gd-BOPP) or with BOPP alone, and then irradiated with thermal neutrons. The resulting cell-survival curves indicate that the {sup 157}Gd(n,{gamma}) reactions are very effective in cell killing. The death of a cell treated with Gd-BOPP was attributed to either the {sup 10}B(n,{alpha}){sup 7}Li reactions or the {sup 157}Gd(n,{gamma}) reactions (or both). However, the quantitative relationship between the two types of reaction and themore » cell-survival fraction was not clear. This paper presents the microdosimetric analysis for the BNL experiment based on the measured experimental parameters, and the results clearly suggest a quantitative relationship between the two types of reaction and the cell survival fraction. The results also suggest new research in gadolinium neutron capture therapy (GdNCT) which may lead to a more practical modality than the boron neutron capture therapy (BNCT) for treating cancers.« less

Ketamine Alters Hippocampal Cell Proliferation and Improves Learning in Mice after Traumatic Brain Injury.

PubMed

Peters, Austin J; Villasana, Laura E; Schnell, Eric

2018-04-30

Traumatic brain injury induces cellular proliferation in the hippocampus, which generates new neurons and glial cells during recovery. This process is regulated by N-methyl-D-aspartate-type glutamate receptors, which are inhibited by ketamine. The authors hypothesized that ketamine treatment after traumatic brain injury would reduce hippocampal cell proliferation, leading to worse behavioral outcomes in mice. Traumatic brain injury was induced in mice using a controlled cortical impact injury, after which mice (N = 118) received either ketamine or vehicle systemically for 1 week. The authors utilized immunohistochemical assays to evaluate neuronal, astroglial, and microglial cell proliferation and survival 3 days, 2 weeks, and 6 weeks postintervention. The Morris water maze reversal task was used to assess cognitive recovery. Ketamine dramatically increased microglial proliferation in the granule cell layer of the hippocampus 3 days after injury (injury + vehicle, 2,800 ± 2,700 cells/mm, n = 4; injury + ketamine, 11,200 ± 6,600 cells/mm, n = 6; P = 0.012). Ketamine treatment also prevented the production of astrocytes 2 weeks after injury (sham + vehicle, 2,400 ± 3,200 cells/mm, n = 13; injury + vehicle, 10,500 ± 11,300 cells/mm, n = 12; P = 0.013 vs. sham + vehicle; sham + ketamine, 3,500 ± 4,900 cells/mm, n = 14; injury + ketamine, 4,800 ± 3,000 cells/mm, n = 13; P = 0.955 vs. sham + ketamine). Independent of injury, ketamine temporarily reduced neurogenesis (vehicle-exposed, 105,100 ± 66,700, cells/mm, n = 25; ketamine-exposed, 74,300 ± 29,200 cells/mm, n = 27; P = 0.031). Ketamine administration improved performance in the Morris water maze reversal test after injury, but had no effect on performance in sham-treated mice. Ketamine alters hippocampal cell proliferation after traumatic brain injury. Surprisingly, these changes were associated with improvement in a neurogenesis-related behavioral recall task, suggesting a possible benefit from ketamine administration after traumatic brain injury in mice. Future studies are needed to determine generalizability and mechanism.

c-fms mRNA is regulated posttranscriptionally by 1,25(OH)2D3 in HL-60 cells.

PubMed

Biskobing, D M; Fan, D; Rubin, J

1997-09-01

Macrophage colony-stimulating factor (MCSF) is required for normal osteoclast and macrophage development. The receptor for MCSF (c-fms) is expressed on the pluripotent precursor and mature osteoclasts and macrophages. We have previously shown in myelomonocytic HL-60 cells that phorbol myristate acetate (PMA) upregulates c-fms mRNA expression. This induction of c-fms is inhibited by 1,25(OH)2D3. The major regulatory control of c-fms mRNA levels by PMA has been identified as posttranscriptional. However, a role of transcript elongation in controlling levels of c-fms mRNA has also been suggested. To better understand the 1,25(OH)2D3 regulation of c-fms mRNA expression we studied nuclear run on, mRNA stability, and transcript elongation in HL-60 cells treated with 10 ng/ml phorbol myristate acetate, 10 nM 1,25(OH)2D3 alone or combined. We demonstrated by nuclear run on that c-fms was constitutively transcribed in 1,25(OH)2D3 as well as control and PMA-treated cells. Transcript elongation was evaluated by RT-PCR for exon 2 or exon 3. Both exons were minimally expressed in control and 1,25(OH)2D3-treated cells, and increased in PMA-treated cells; this increased expression was inhibited by the addition of 1,25(OH)2D3. These results fail to show differential transcript elongation. Measurement of mRNA stability demonstrated decreased mRNA half-life to 5 hours in cells treated with PMA and 1,25(OH)2D3 compared with a half-life of 8 hours in cells treated with PMA alone. Our findings demonstrate that c-fms is regulated by 1,25(OH)2D3 at the posttranscriptional level by changes in mRNA stability. This gives the cell the ability to respond to local signals with rapid changes in c-fms levels altering the ability of the cell to respond to MCSF.

Effects of inorganic nanoparticles on viability and catabolic activities of Agrobacterium sp. PH-08 during biodegradation of dibenzofuran.

PubMed

Le, Thao Thanh; Murugesan, Kumarasamy; Kim, Eun-Ju; Chang, Yoon-Seok

2014-09-01

This study investigated the cytotoxicity, genotoxicity, and growth inhibition effects of four different inorganic nanoparticles (NPs) such as aluminum (nAl), iron (nFe), nickel (nNi), and zinc (nZn) on a dibenzofuran (DF) degrading bacterium Agrobacterium sp. PH-08. NP (0-1,000 mg L(-1)) -treated bacterial cells were assessed for cytotoxicity, genotoxicity, growth and biodegradation activities at biochemical and molecular levels. In an aqueous system, the bacterial cells treated with nAl, nZn and nNi at 500 mg L(-1) showed significant reduction in cell viability (30-93.6 %, p < 0.05), while nFe had no significant inhibition on bacterial cell viability. In the presence of nAl, nZn and nNi, the cells exhibited elevated levels of reactive oxygen species (ROS), DNA damage and cell death. Furthermore, NP exposure showed significant (p < 0.05) impairment in DF and catechol biodegradation activities. The reduction in DF biodegradation was ranged about 71.7-91.6 % with single NPs treatments while reached up to 96.3 % with a mixture of NPs. Molecular and biochemical investigations also clearly revealed that NP exposure drastically affected the catechol-2,3-dioxygenase activities and its gene (c23o) expression. However, no significant inhibition was observed in nFe treatment. The bacterial extracellular polymeric materials and by-products from DF degradation can be assumed as key factors in diminishing the toxic effects of NPs, especially for nFe. This study clearly demonstrates the impact of single and mixed NPs on the microbial catabolism of xenobiotic-degrading bacteria at biochemical and molecular levels. This is the first study on estimating the impact of mixed NPs on microbial biodegradation.

Lactobacillus johnsonii N6.2 stimulates the innate immune response through Toll-like receptor 9 in Caco-2 cells and increases intestinal crypt Paneth cell number in biobreeding diabetes-prone rats.

PubMed

Kingma, Sandra D K; Li, Nan; Sun, Frank; Valladares, Ricardo B; Neu, Joe; Lorca, Graciela L

2011-06-01

Lactobacillus johnsonii (Ljo) N6.2 has been shown to mitigate the development of type 1 diabetes when administered to diabetes-prone rats. The specific mechanisms underlying this observed response remain under investigation. The objective of this study was to assess the effect of Ljo N6.2 on mucosal inflammatory response using differentiated Caco-2 monolayers. The mRNA expression levels of CCL20, CXCL8, and CXCL10 chemokines were determined by qRT-PCR. Ljo at 10(11) CFU/L induced a strong response in all chemokines examined. To assess the specific host-signaling pathways involved, we performed RT-PCR amplification of Toll-like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors. TLR7 and TLR9 expression levels were induced 4.2- and 9-fold, respectively, whereas other TLR and nucleotide-binding oligomerization domain receptors were not modified. A similar effect was observed in Caco-2 monolayers treated with Ljo cell-free extract or purified nucleic acids (NA). Increased levels of IFN type 1 and IFN regulators Stat1 and IRF7 followed the upregulation of TLR9. Activation of TLR9 was also evidenced by increased Frizzled 5 expression in Ljo-treated Caco-2 cells and an increase in the number of Paneth cells in Ljo-fed, diabetes-prone rats. These results are in agreement with the polarizing-tolerizing mechanism recently described in which the apical stimulation of TLR9 in intestinal epithelial cells leads to a higher state of immunologic alertness. Furthermore, these results suggest that live probiotics could be, in the future, replaced with select cellular components.

Cellular effect of high doses of silica-coated quantum dot profiled with high throughput gene expression analysis and high content cellomics measurements.

PubMed

Zhang, Tingting; Stilwell, Jackie L; Gerion, Daniele; Ding, Lianghao; Elboudwarej, Omeed; Cooke, Patrick A; Gray, Joe W; Alivisatos, A Paul; Chen, Fanqing Frank

2006-04-01

Quantum dots (Qdots) are now used extensively for labeling in biomedical research, and this use is predicted to grow because of their many advantages over alternative labeling methods. Uncoated Qdots made of core/shell CdSe/ZnS are toxic to cells because of the release of Cd2+ ions into the cellular environment. This problem has been partially overcome by coating Qdots with polymers, poly(ethylene glycol) (PEG), or other inert molecules. The most promising coating to date, for reducing toxicity, appears to be PEG. When PEG-coated silanized Qdots (PEG-silane-Qdots) are used to treat cells, toxicity is not observed, even at dosages above 10-20 nM, a concentration inducing death when cells are treated with polymer or mercaptoacid coated Qdots. Because of the importance of Qdots in current and future biomedical and clinical applications, we believe it is essential to more completely understand and verify this negative global response from cells treated with PEG-silane-Qdots. Consequently, we examined the molecular and cellular response of cells treated with two different dosages of PEG-silane-Qdots. Human fibroblasts were exposed to 8 and 80 nM of these Qdots, and both phenotypic as well as whole genome expression measurements were made. PEG-silane-Qdots did not induce any statistically significant cell cycle changes and minimal apoptosis/necrosis in lung fibroblasts (IMR-90) as measured by high content image analysis, regardless of the treatment dosage. A slight increase in apoptosis/necrosis was observed in treated human skin fibroblasts (HSF-42) at both the low and the high dosages. We performed genome-wide expression array analysis of HSF-42 exposed to doses 8 and 80 nM to link the global cell response to a molecular and genetic phenotype. We used a gene array containing approximately 22,000 total probe sets, containing 18,400 probe sets from known genes. Only approximately 50 genes (approximately 0.2% of all the genes tested) exhibited a statistically significant change in expression level of greater than 2-fold. Genes activated in treated cells included those involved in carbohydrate binding, intracellular vesicle formation, and cellular response to stress. Conversely, PEG-silane-Qdots induce a down-regulation of genes involved in controlling the M-phase progression of mitosis, spindle formation, and cytokinesis. Promoter analysis of these results reveals that expression changes may be attributed to the down-regulation of FOXM and BHLB2 transcription factors. Remarkably, PEG-silane-Qdots, unlike carbon nanotubes, do not activate genes indicative of a strong immune and inflammatory response or heavy-metal-related toxicity. The experimental evidence shows that CdSe/ZnS Qdots, if appropriately protected, induce negligible toxicity to the model cell system studied here, even when exposed to high dosages. This study indicates that PEG-coated silanized Qdots pose minimal impact to cells and are a very promising alternative to uncoated Qdots.

Immune response to pneumococcus and tetanus toxoid in patients with moderate-to-severe psoriasis following long-term ustekinumab use.

PubMed

Brodmerkel, Carrie; Wadman, Eric; Langley, Richard G; Papp, Kim A A; Bourcier, Marc; Poulin, Yves; Ho, Vincent; Guenther, Lyn; Kunynetz, Rod; Nigen, Simon; Vender, Ronald; Wasel, Norman; Hsu, Ming-Chun; Szapary, Philippe

2013-10-01

Little is known about the impact of long-term use of immunosuppressive agents on immune response. Assess the impact of continuous maintenance ustekinumab treatment on patients' ability to mount immune responses to pneumococcal (T-cell-independent) and tetanus toxoid (T-cell-dependent) vaccines. Ustekinumab-treated patients with moderate-to-severe psoriasis treated in the long-term extension of the Phase 3 PHOENIX 2 trial (n=60) were compared with control psoriasis patients not receiving systemic therapy (n=56). Patients were vaccinated with both 23-valent pneumococcal and tetanus toxoid vaccines. Serum samples collected pre-vaccination and 4 weeks post-vaccination were assessed for antibody responses. No differences in the ability of ustekinumab-treated patients to respond to pneumococcal or tetanus toxoid vaccinations were observed compared with controls. A ≥2-fold increase in antibody levels in ≥7 of 14 serotypes of the pneumococcal vaccine was observed in ustekinumab-treated (96.6%) and untreated control (92.6%) patients following vaccination. Ustekinumab-treated patients achieved a ≥4-fold increase (84.7%) in anti-tetanus antibody vs. 77.8% in the control group. No differences were detected in ex-vivo responses to anti-CD3/CD28 or tetanus toxoid between ustekinumab-treated and control groups. Long-term treatment (≥3 years) with ustekinumab does not compromise the immune response to T-cell-dependent/-independent vaccines in patients with moderate-to-severe psoriasis.

N-WASP and WAVE2 acting downstream of phosphatidylinositol 3-kinase are required for myogenic cell migration induced by hepatocyte growth factor.

PubMed

Kawamura, Kazuhiro; Takano, Kazunori; Suetsugu, Shiro; Kurisu, Shusaku; Yamazaki, Daisuke; Miki, Hiroaki; Takenawa, Tadaomi; Endo, Takeshi

2004-12-24

During skeletal muscle regeneration caused by injury, muscle satellite cells proliferate and migrate toward the site of muscle injury. This migration is mainly induced by hepatocyte growth factor (HGF) secreted by intact myofibers and also released from injured muscle. However, the intracellular machinery for the satellite cell migration has not been elucidated. To examine the mechanisms of satellite cell migration, we utilized satellite cell-derived mouse C2C12 skeletal muscle cells. HGF induced reorganization of actin cytoskeleton to form lamellipodia in C2C12 myoblasts. HGF treatment facilitated both nondirectional migration of the myoblasts in phagokinetic track assay and directional chemotactic migration toward HGF in a three-dimensional migration chamber assay. Endogenous N-WASP and WAVE2 were concentrated in the lamellipodia at the leading edge of the migrating cells. Moreover, exogenous expression of wild-type N-WASP or WAVE2 promoted lamellipodial formation and migration. By contrast, expression of the dominant-negative mutant of N-WASP or WAVE2 and knockdown of N-WASP or WAVE2 expression by the RNA interference prevented the HGF-induced lamellipodial formation and migration. When the cells were treated with LY294002, an inhibitor of phosphatidylinositol 3-kinase, the HGF-induced lamellipodial formation and migration were abrogated. These results imply that both N-WASP and WAVE2, which are activated downstream of phosphati-dylinositol 3-kinase, are required for the migration through the lamellipodial formation of C2C12 cells induced by HGF.

Knockdown endogenous CypA with siRNA in U2OS cells results in disruption of F-actin structure and alters tumor phenotype.

PubMed

Calhoun, Colonya C; Lu, Ying-Chun; Song, Jun; Chiu, Robert

2009-01-01

Cyclophilin A (CypA) was originally identified as a cytosolic protein possessing peptidyl-prolyl isomerase activity. CypA has been shown to play a pivotal role in the immune response, but little is known about other molecular mechanisms of CypA-mediated biologic events. In our present study, we demonstrate that knockdown CypA expression using RNAi in U2OS cells resulted in disruption of the F-actin structure, as well as decreased anchorage-independent growth, proliferation, and migration. Wild-type U2OS cells treated with cyclosporine A (CsA), a peptidyl-prolyl isomerase inhibitor, displayed the same phenotype as knockdown CypA cells, suggesting that the isomerase activity of CypA is required to maintain a normal phenotype. In vitro and in vivo binding assays revealed that CypA binds to N-WASP, which functions in the nucleation of actin via the Arp2/3 complex. Pulse-chase labeling study indicated an enhanced degradation of N-WASP in cell lacking CypA, suggesting that CypA is required for stabilizing N-WASP to form a N-WASP/Arp2/3 complex for the nucleation/initiation of F-actin polymerization.

Physically disconnected non-diffusible cell-to-cell communication between neuroblastoma SH-SY5Y and DRG primary sensory neurons.

PubMed

Chaban, Victor V; Cho, Taehoon; Reid, Christopher B; Norris, Keith C

2013-01-01

Cell-cell communication occurs via a variety of mechanisms, including long distances (hormonal), short distances (paracrine and synaptic) or direct coupling via gap junctions, antigen presentation, or ligand-receptor interactions. We evaluated the possibility of neuro-hormonal independent, non-diffusible, physically disconnected pathways for cell-cell communication using dorsal root ganglion (DRG) neurons. We assessed intracellular calcium ([Ca(2+)]) in primary culture DRG neurons that express ATP-sensitive P2X3, capsaicinsensitive TRPV1 receptors modulated by estradiol. Physically disconnected (dish-in-dish system; inner chamber enclosed) mouse DRG were cultured for 12 hours near: a) media alone (control 1), b) mouse DRG (control 2), c) human neuroblastoma SHSY-5Y cells (cancer intervention), or d) mouse DRG treated with KCl (apoptosis intervention). Chemosensitive receptors [Ca(2+)](i) signaling did not differ between control 1 and 2. ATP (10 μM) and capsaicin (100nM) increased [Ca(2+)](i) transients to 425.86 + 49.5 nM, and 399.21 ± 44.5 nM, respectively. 17β-estradiol (100 nM) exposure reduced ATP (171.17 ± 48.9 nM) and capsaicin (175.01±34.8 nM) [Ca(2+)](i) transients. The presence of cancer cells reduced ATP- and capsaicin-induced [Ca(2+)](i) by >50% (p<0.05) and abolished the 17β-estradiol effect. By contrast, apoptotic DRG cells increased initial ATP-induced [Ca(2+)](i), flux four fold and abolished subsequent [Ca(2+)](i), responses to ATP stimulation (p<0.001). Capsaicin (100nM) induced [Ca(2+)](i) responses were totally abolished. The local presence of apoptotic DRG or human neuroblastoma cells induced differing abnormal ATP and capsaicin-mediated [Ca(2+)](i) fluxes in normal DRG. These findings support physically disconnected, non-diffusible cell-to-cell signaling. Further studies are needed to delineate the mechanism(s) of and model(s) of communication.

Linagliptin reduces effects of ET-1 and TLR2-mediated cerebrovascular hyperreactivity in diabetes.

PubMed

Hardigan, Trevor; Abdul, Yasir; Ergul, Adviye

2016-08-15

The anti-hyperglycemic agent linagliptin, a dipeptidyl peptidase-4 inhibitor, has been shown to reduce inflammation and improve endothelial cell function. In this study, we hypothesized that DPP-IV inhibition with linagliptin would improve impaired cerebral blood flow in diabetic rats through improved insulin-induced cerebrovascular relaxation and reversal of pathological cerebrovascular remodeling that subsequently leads to improvement of cognitive function. Male type-2 diabetic Goto-Kakizaki (GK) and nondiabetic Wistar rats were treated with linagliptin, and ET-1 plasma levels and dose response curves to ET-1 (0.1-100nM) in basilar arteries were assessed. The impact of TLR2 antagonism on ET-1 mediated basilar contraction and endothelium-dependent relaxation to acetylcholine (ACh, 1nM-1M) in diabetic GK rats was examined with antibody directed against the TLR2 receptor (Santa Cruz, 5μg/mL). The expression of TLR2 in middle cerebral arteries (MCAs) from treated rats and in brain microvascular endothelial cells (BMVEC) treated with 100nM linagliptin was assessed. Linagliptin lowered plasma ET-1 levels in diabetes, and reduced ET-1-induced vascular contraction. TLR2 antagonism in diabetic basilar arteries reduced ET-1-mediated cerebrovascular dysfunction and improved endothelium-dependent vasorelaxation. Linagliptin treatment in the BMVEC was able to reduce TLR2 expression in cells from both diabetic and nondiabetic rats. These results suggest that inhibition of DPPIV using linagliptin improves the ET-1-mediated cerebrovascular dysfunction observed in diabetes through a reduction in ET-1 plasma levels and reduced cerebrovascular hyperreactivity. This effect is potentially a result of linagliptin causing a decrease in endothelial TLR2 expression and a subsequent increase in NO bioavailability. Copyright © 2016 Elsevier Inc. All rights reserved.

Potentiation of angiogenic switch in capillary endothelial cells by cAMP: A cross-talk between up-regulated LLO biosynthesis and the HSP-70 expression.

PubMed

Martínez, Juan A; Tavárez, José J; Oliveira, Caroline M; Banerjee, Dipak K

2006-05-01

During tumor growth and invasion, the endothelial cells from a relatively quiescent endothelium start proliferating. The exact mechanism of switching to a new angiogenic phenotype is currently unknown. We have examined the role of intracellular cAMP in this process. When a non-transformed capillary endothelial cell line was treated with 2 mM 8Br-cAMP, cell proliferation was enhanced by approximately 70%. Cellular morphology indicated enhanced mitosis after 32-40 h with almost one-half of the cell population in the S phase. Bcl-2 expression and caspase-3, -8, and -9 activity remained unaffected. A significant increase in the Glc(3)Man(9)GlcNAc(2)-PP-Dol biosynthesis and turnover, Factor VIIIC N-glycosylation, and cell surface expression of N-glycans was observed in cells treated with 8Br-cAMP. Dol-P-Man synthase activity in the endoplasmic reticulum membranes also increased. A 1.4-1.6-fold increase in HSP-70 and HSP-90 expression was also observed in 8Br-cAMP treated cells. On the other hand, the expression of GRP-78/Bip was 2.3-fold higher compared to that of GRP-94 in control cells, but after 8Br-cAMP treatment for 32 h, it was reduced by 3-fold. GRP-78/Bip expression in untreated cells was 1.2-1.5-fold higher when compared with HSP-70 and HSP-90, whereas that of the GRP-94 was 1.5-1.8-fold lower. After 8Br-cAMP treatment, GRP-78/Bip expression was reduced 4.5-4.8-fold, but the GRP-94 was reduced by 1.5-1.6-fold only. Upon comparison, a 2.9-fold down-regulation of GRP-78/Bip was observed compared to GRP-94. We, therefore, conclude that a high level of Glc(3)Man(9)GlcNAc(2)-PP-Dol, resulting from 8Br-cAMP stimulation up-regulated HSP-70 expression and down-regulated that of the GRP-78/Bip, maintained adequate protein folding, and reduced endoplasmic reticulum stress. As a result capillary endothelial cell proliferation was induced.

Sativex® effects on promoter methylation and on CNR1/CNR2 expression in peripheral blood mononuclear cells of progressive multiple sclerosis patients.

PubMed

Santoro, Massimo; Mirabella, Massimiliano; De Fino, Chiara; Bianco, Assunta; Lucchini, Matteo; Losavio, Francesco; Sabino, Andrea; Nociti, Viviana

2017-08-15

Multiple sclerosis (MS) is a chronic demyelinating central nervous system (CNS) disease that involve oligodendrocyte loss and failure to remyelinate damaged brain areas causing a progressive neurological disability. Studies in MS mouse model suggest that cannabinoids ameliorate symptoms as spasticity, tremor and pain reducing inflammation via cannabinoid-mediated system. The aim of our study is to investigate the changes in cannabinoid type 1 (CNR1) and 2 (CNR2) receptors mRNA expression levels and promoter methylation in peripheral blood mononuclear cells (PBMCs) of MS secondary progressive (MSS-SP) patients treated with Sativex®. Our cohort included MSS-SP patients, that at the time of Sativex® treatment, are treated (n=7), not treated (n=11) or that had terminated interferon-β-1b (IFN-β-1b) therapy (n=12). By Methylation Sensitive High Resolution Melting (MS-HRM), we characterized the methylation profile of CNR1 and CNR2 promoter region, while the relative mRNA transcript levels of these two genes were evaluated in the same samples by Quantitative Real-Time PCR (qRT-PCR) analysis. We did not find different pattern of cytosine-phosphate-guanine (CpG) methylation in the CNR1/CNR2 promoter region of all MSS-SP patients treated with Sativex®. In addition, CNR1 and CNR2 expression did not significantly differ in MSS-SP patients not treated with IFN-β-1b vs. them that have suspended, while in MSS-SP patients treated with IFN-β-1b during Sativex® therapy we found a specific decrease of the CNR2 expression levels. These results suggest that the different expression of cannabinoid receptors by Sativex® treatment in leukocytes might be regulated through a molecular mechanism that involve interferon modulation. Copyright © 2017 Elsevier B.V. All rights reserved.

Concomitant treatment with pertussis toxin plus temozolomide increases the survival of rats bearing intracerebral RG2 glioma.

PubMed

Magaña-Maldonado, Roxana; Manoutcharian, Karen; Hernández-Pedro, Norma Y; Rangel-López, Edgar; Pérez-De la Cruz, Verónica; Rodríguez-Balderas, César; Sotelo, Julio; Pineda, Benjamín

2014-02-01

Glioblastoma multiforme is the most frequent primary brain tumor, it has poor prognosis, and it remains refractory to current treatment. The success of temozolomide (TMZ) appears to be limited by the occurrence of chemoresistance. Recently, we report the use of pertussis toxin as adjuvant immunotherapy in a C6 glioma model; showing a decrease in tumoral size, it induced selective cell death in Treg cells, and it elicited less infiltration of tumoral macrophages. Here, we evaluated the cytotoxic effect of pertussis toxin in combination with TMZ for glioma treatment, both in vitro and in vivo RG2 glioma model. We determined cell viability, cell cycle, apoptosis, and autophagy on treated RG2 cells through flow cytometry, immunofluorescence, and Western blot assays. Twenty-eight rats were divided in four groups (n = 7) for each treatment. After intracranial implantation of RG2 cells, animals were treated with TMZ (10 mg/Kg/200 μl of apple juice), PTx (2 μg/200 μl of saline solution), and TMZ + PTx. Animals without treatment were considered as control. We found an induction of apoptosis in around 20 % of RG2 cells, in both single treatments and in their combination. Also, we determined the presence of autophagy vesicles, without any modifications in the cell cycle in the TMZ - PTx-treated groups. The survival analyses showed an increase due to individual treatments; while in the group treated with the combination TMZ - PTx, this effect was enhanced. We show that the concomitant use of pertussis toxin plus TMZ could represent an advantage to improve the glioma treatment.

The all-trans retinoic acid (atRA)-regulated gene Calmin (Clmn) regulates cell cycle exit and neurite outgrowth in murine neuroblastoma (Neuro2a) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marzinke, Mark A.; Clagett-Dame, Margaret, E-mail: dame@biochem.wisc.edu; Pharmaceutical Science Division, University of Wisconsin-Madison, Madison, WI 53705-2222

2012-01-01

The vitamin A metabolite all-trans retinoic acid (atRA) functions in nervous system development and regulates cell proliferation and differentiation. Neuroblastoma cells (SH-SY5Y and Neuro2a or N2A) exposed to atRA undergo growth inhibition and neuronal differentiation, both of which are preceded by an increase in Clmn mRNA. Treatment of N2A cells with atRA produces a reduction in phosphohistone 3 immunostaining and BrdU incorporation, both indicators of a reduction in cell proliferation. These effects are nearly eliminated in atRA-treated shClmn knockdown cells. Loss of Clmn in the mouse N2A cell line also results in a significant reduction of atRA-mediated neurite outgrowth, amore » response that can be rescued by reintroduction of the Clmn sequence. In contrast, ectopic overexpression of Clmn produces an increase in the cyclin dependent kinase inhibitor, p21{sup Cip1}, a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G{sub 1}/S arrest. Clmn overexpression alone is sufficient to inhibit N2A cell proliferation, whereas both Clmn and atRA must be present to induce neurite outgrowth. This study shows that the atRA-responsive gene Clmn promotes exit from the cell cycle, a requisite event for neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer Calmin is a retinoic acid-responsive gene. Black-Right-Pointing-Pointer Calmin promotes cell cycle exit in N2A cells. Black-Right-Pointing-Pointer Calmin overexpression increases p21Cip1 and decreases cyclin D1. Black-Right-Pointing-Pointer Calmin is required for RA-induced growth inhibition and neurite outgrowth.« less

Modifying effects of lemongrass essential oil on specific tissue response to the carcinogen N-methyl-N-nitrosurea in female BALB/c mice.

PubMed

Bidinotto, Lucas T; Costa, Celso A R A; Costa, Mirtes; Rodrigues, Maria A M; Barbisan, Luís F

2012-02-01

Lemongrass (Cymbopogon citratus Stapf) essential oil has been used worldwide because of its ethnobotanical and medicinal usefulness. Regarding its medicinal usefulness, the present study evaluated the beneficial effects of lemongrass essential oil (LGEO) oral treatment on cell proliferation and apoptosis events and on early development of hyperplastic lesions in the mammary gland, colon, and urinary bladder induced by N-methyl-N-nitrosourea (MNU) in female BALB/c mice. The animals were allocated into three groups: G1, treated with LGEO vehicle for 5 weeks (five times per week); G2, treated with LGEO vehicle as for G1 and MNU (two injections each of 30 mg/kg of body weight at weeks 3 and 5); and G3, treated with LGEO (five times each with 500 mg/kg of body weight per week) and MNU as for G2. Twenty-four hours after the last MNU application, all animals were euthanized, and mammary glands, colon, and urinary bladder were collected for histological and immunohistochemical analysis. LGEO oral treatment significantly changed the indexes of apoptosis and/or cellular proliferation for the tissues analyzed. In particular, the treatment reduced the incidence of hyperplastic lesions and increased apoptosis in mammary epithelial cells. This increment in the apoptosis response may be related to a favorable balance in Bcl-2/Bax immunoreactivity in mammary epithelial cells. These findings indicate that LGEO presented a protective role against early MNU-induced mammary gland alterations in BALB/c mice.

Growth Suppression and Therapy Sensitization of Breast Cancer

DTIC Science & Technology

1999-07-01

northern analysis, the same membrane was hybridized successively with JNKI probe, JNK2 probe and the G3PDH probe. Figure 10. A549 cells were treated with the...90 0 00 z SO E 40 04. z 20 to JNK2 Oligonucleotide n* 47 JNK1AS TREATED 2 JNlK2AS TREATED. 0) c a 0 mRNA mRNA JNK1 J&,I ~R JNK1 JNK2 JNK2 G3PDH SI... G3PDH 48 iil vý 0 z z Z O! zz C13~ A.LOR 496 Reference #28 PREFERENTIAL PLATINATION OF AN ACTIVATED CELLULAR PROMOTER BY CIS-DIAMMINEDICHLOROPLATINUM

Ipsilateral Irradiation for Oral and Oropharyngeal Carcinoma Treated With Primary Surgery and Postoperative Radiotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vergeer, Marije R., E-mail: mr.vergeer@vumc.n; Doornaert, Patricia; Jonkman, Anja

Purpose: The purpose was to evaluate the contralateral nodal control (CLNC) in postoperative patients with oral and oropharyngeal cancer treated with ipsilateral irradiation of the neck and primary site. Late radiation-induced morbidity was also evaluated. Methods and Materials: The study included 123 patients with well-lateralized squamous cell carcinomas treated with surgery and unilateral postoperative irradiation. Most patients had tumors of the gingiva (41%) or buccal mucosa (21%). The majority of patients underwent surgery of the ipsilateral neck (n = 102 [83%]). The N classification was N0 in 73 cases (59%), N1 or N2a in 23 (19%), and N2b in 27more » cases (22%). Results: Contralateral metastases developed in 7 patients (6%). The 5-year actuarial CLNC was 92%. The number of lymph node metastases was the only significant prognostic factor with regard to CLNC. The 5-year CLNC was 99% in N0 cases, 88% in N1 or N2a cases, and 73% in N2b cases (p = 0.008). Borderline significance (p = 0.06) was found for extranodal spread. Successful salvage could be performed in 71% of patients with contralateral metastases. The prevalence of Grade 2 or higher xerostomia was 2.6% at 5 years. Conclusions: Selected patients with oral or oropharyngeal carcinoma treated with primary surgery and postoperative ipsilateral radiotherapy have a very high CLNC with a high probability of successful salvage in case of contralateral metastases. However, bilateral irradiation should be applied in case of multiple lymph node metastases in the ipsilateral neck, particularly in the presence of extranodal spread. The incidence of radiation-induced morbidity is considerably lower as observed after bilateral irradiation.« less

Myricetin Protects Cells against Oxidative Stress-Induced Apoptosis via Regulation of PI3K/Akt and MAPK Signaling Pathways

PubMed Central

Kang, Kyoung Ah; Wang, Zhi Hong; Zhang, Rui; Piao, Mei Jing; Kim, Ki Cheon; Kang, Sam Sik; Kim, Young Woo; Lee, Jongsung; Park, Deokhoon; Hyun, Jin Won

2010-01-01

Recently, we demonstrated that myricetin exhibits cytoprotective effects against H2O2-induced cell damage via its antioxidant properties. In the present study, myricetin was found to inhibit H2O2-induced apoptosis in Chinese hamster lung fibroblast (V79-4) cells, as shown by decreased apoptotic bodies, nuclear fragmentation, sub-G1 cell population, and disruption of mitochondrial membrane potential (Δψm), which are increased in H2O2-treated cells. Western blot data showed that in H2O2-treated cells, myricetin increased the level of Bcl-2, which is an anti-apoptotic factor, and decreased the levels of Bax, active caspase-9 and -3, which are pro-apoptotic factors. And myricetin inhibited release of cytochrome c from mitochondria to cytosol in H2O2-treated cells. Myricetin-induced survival correlated with Akt activity, and the rescue of cells by myricetin treatment against H2O2-induced apoptosis was inhibited by the specific PI3K (phosphoinositol-3-kinase) inhibitor. Myricetin-mediated survival also inhibited the activation of p38 mitogen activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), which are members of MAPK. Our studies suggest that myricetin prevents oxidative stress-induced apoptosis via regulation of PI3K/Akt and MAPK signaling pathways. PMID:21151442

TRANSCUTANEOUS CERVICAL VAGUS NERVE STIMULATION AMELIORATES ACUTE ISCHEMIC INJURY IN RATS

PubMed Central

Ay, Ilknur; Nasser, Rena; Simon, Bruce; Ay, Hakan

2016-01-01

Background Direct stimulation of the vagus nerve in the neck via surgically implanted electrodes is protective in animal models of stroke. We sought to determine the safety and efficacy of a non-invasive cervical VNS (nVNS) method using surface electrodes applied to the skin overlying the vagus nerve in the neck in a model of middle cerebral artery occlusion (MCAO). Methods nVNS was initiated variable times after MCAO hour in rats (n=33). Control animals received sham stimulation (n=33). Infarct volume and functional outcome were assessed on day 7. Brains were processed by immunohistochemistry for microglial activation and cytokine levels. The ability of nVNS to activate the nucleus tractus solitarius (NTS) was assessed using c-Fos immunohistochemistry. Results Infarct volume was 43.15±3.36 percent of the contralateral hemisphere (PCH) in control and 28.75±4.22 PCH in nVNS-treated animals (p<0.05). The effect of nVNS on infarct size was consistent when stimulation was initiated up to 4 hours after MCAO. There was no difference in heart rate and blood pressure between control and nVNS-treated animals. The number of c-Fos positive cells was 32.4±10.6 and 6.2±6.3 in the ipsilateral NTS (p<0.05) and 30.4±11.2 and 5.8±4.3 in the contralateral NTS (p<0.05) in nVNS-treated and control animals, respectively. nVNS reduced the number of Iba-1, CD68, and TNF-α positive cells and increased the number of HMGB1 positive cells. Conclusions nVNS inhibits ischemia-induced immune activation and reduces the extent of tissue injury and functional deficit in rats without causing cardiac or hemodynamic adverse effects when initiated up to 4 hours after MCAO. PMID:26723020

Studies of heat treated CSS CdS films

NASA Astrophysics Data System (ADS)

Marinskiy, D.; Marinskaya, S.; Viswanathan, V.; Morel, D. L.; Ferekides, C. S.

1999-03-01

Cadmium sulfide continues to be the most successful and widely used n-type heterojunction partner in thin film CdTe solar cells. In most cases solar cell performance is enhanced if the CdS films are heat treated prior to the deposition of the CdTe. This paper discusses the effect of H2 annealing on the resistivity of CSS-CdS films and the use of a mobility activation model to explain the observed changes in resistivity. Photoluminescence measurements of CSS CdS films heat-treated in He and in CdCl2 vapor have also been carried out. In all cases the heat treatments lead to an increase in the intensity of a photoluminescence band believed to be associated with sulfur vacancies.

2-Deoxy-d-glucose increases GFAT1 phosphorylation resulting in endoplasmic reticulum-related apoptosis via disruption of protein N-glycosylation in pancreatic cancer cells.

PubMed

Ishino, Kousuke; Kudo, Mitsuhiro; Peng, Wei-Xia; Kure, Shoko; Kawahara, Kiyoko; Teduka, Kiyoshi; Kawamoto, Yoko; Kitamura, Taeko; Fujii, Takenori; Yamamoto, Tetsushi; Wada, Ryuichi; Naito, Zenya

2018-06-27

The glycolytic inhibitor 2-deoxy-d-glucose (2DG) causes energy starvation, affecting cell viability in a wide range of cancer cell lines. To determine the action of 2DG in pancreatic cancer, we performed proteomic analysis of pancreatic cancer cell line after 2DG treatment. Eighty proteins showed differential expression and among these, proteins involved in phosphohexose metabolism were upregulated. Up-regulation of glutamine: fructose 6-phosphate aminotransferase 1 (GFAT1), which belongs to the hexosamine biosynthesis pathway (HBP) that produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to maintain glycoprotein, was validated by evaluation of mRNA and protein levels. Therefore, we assessed the amounts of total N-glycoproteins. Unexpectedly, we found a reduction of total N-glycoproteins and phosphorylation of GFAT1 by AMP-activated protein kinase (AMPK). These data may shed light on HBP dysfunction. Furthermore, we found endoplasmic reticulum (ER) stress accompanied by increased expression of ER stress markers, such as glucose response protein 78 (GRP78) and C/EBP-homologous protein (CHOP), in 2DG-treated cells. Moreover, the additive activation of AMPK by metformin (Met) synergistically enhanced the reduction of protein N-glycosylation and cell growth inhibition in the presence of 2DG. These results suggest that 2DG reduces N-glycosylation of proteins following the increase of phosphorylation of GFAT1 and results in the inhibition of cell growth mediated by ER stress in pancreatic cancer cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Effects of the novel angiotensin II receptor type I antagonist, fimasartan on myocardial ischemia/reperfusion injury.

PubMed

Han, Jin; Park, Sung-Ji; Thu, Vu Thi; Lee, Sung-Ryul; Long, Le Thanh; Kim, Hyoung Kyu; Kim, Nari; Park, Seung Woo; Jeon, Eun-Seok; Kim, Eun-Ji; Yoon, Chang-Hwan; Cho, Goo-Young; Choi, Dong-Ju

2013-10-03

The aim of this study was to investigate the cardioprotective effect of fimasartan, a newly developed angiotensin II receptor type I blocker (ARB), against myocardial ischemia/reperfusion (I/R) injury and to identify the mechanism by which it reduces mitochondrial damage. Fimasartan was administered intravenously to Sprague-Dawley rats (3mg/kg), cardiomyocytes (50 μM), and H9c2 cells (50 μM) before ischemia or hypoxia. Myocardial infarction (MI), echocardiograms, DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling, immunoblotting, oxygen consumption, confocal microscopic appearance, and L-type Ca(2+) current (ICa,L) were then assessed. Fimasartan pretreatment remarkably reduced the rate of MI and improved cardiac performance well after I/R (n = 9/group). Fimasartan also reduced apoptotic cell death both in vivo and in hypoxia/reoxygenation (H/R)-treated H9c2 cells (n = 5~8/group). H/R-induced mitochondrial O2(-) production and collapse of membrane potential were markedly attenuated in fimasartan-treated cardiomyocytes (n = 4 ~ 6/group). Additionally, mitochondrial Ca(2+) overload during reoxygenation was suppressed by fimasartan (n = 4~6/group), and this was found to be possibly related to the inhibition of ICa,L and mitochondrial Ca(2+) uniporter. Furthermore, fimasartan pretreatment increased phosphorylations of Akt and glycogen synthase kinase-3β (n = 5 ~ 7/group), decreased pro-apoptotic p53 levels, and increased anti-apoptotic Bcl-2 levels (n = 4) during reperfusion. Fimasartan preconditioning has the potential to modulate Bcl-2 and suppress I/R-induced Ca(2+) overload by inhibiting ICa,L and MCU. These beneficial effects could prevent the mitochondrial dysfunction and apoptosis accompanied by I/R. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Steatosis induced CCL5 contributes to early-stage liver fibrosis in nonalcoholic fatty liver disease progress.

PubMed

Li, Bing-Hang; He, Fang-Ping; Yang, Xin; Chen, Yuan-Wen; Fan, Jian-Gao

2017-02-01

The rapidly increasing prevalence of nonalcoholic fatty liver disease (NAFLD) has become one of the major public health threats in China and worldwide. However, during the development of NAFLD, the key mechanism underlying the progression of related fibrosis remains unclear, which greatly impedes the development of optimal NAFLD therapy. In the current study, we were endeavored to characterize a proinflammatory cytokine, CCL5, as a major contributor for fibrosis in NAFLD. The results showed that CCL5 was highly expressed in fatty liver and NASH patients. In NAFLD rats induced by 8-week-HFD, CCL5 and its receptor, CCR5, were significantly up-regulated and liver fibrosis exclusively occurred in this group. In addition, we showed that hepatocytes are the major source contributing to this CCL5 elevation. Interestingly, a CCL5 inhibitor Met-CCL5, significantly decreased liver fibrosis but not hepatic steatosis. Using a cell model of hepatic steatosis, we found that the conditioned medium of lipid-overloaded hepatocytes (Fa2N-4 cells) which produced excessive CCL5 stimulated the profibrotic activities of hepatic stellate cells (LX-2) as manifested by increased migration rate, proliferation and collagen production of LX-2 cells. CCL5 knockdown in Fa2N-4 cells, Met-CCL5 or CCR5 antibody treatment on LX-2 cells all significantly inhibited the conditioned medium of FFA-treated Fa2N-4 cells to exert stimulatory effects on LX-2 cells. Consistently, the conditioned medium of Fa2N-4 cells with CCL5 over-expression significantly enhanced migration rate, cell proliferation and collagen production of LX-2 cells. All these results support that CCL5 produced by steatotic hepatocytes plays an essential role in fibrotic signaling machinery of NAFLD. In addition, we were able to identify C/EBP-β as the up-stream regulator of CCL5 gene transcription in hepatocytes treated with free fatty acid (FFA). Our data strongly supported that CCL5 plays a pivotal regulatory role in hepatic fibrosis during NAFLD, which constitutes a novel and exciting observation that may call for potential future development of specific CCL5-targeted NAFLD therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

Topical N-Acetylcysteine Accelerates Wound Healing in Vitro and in Vivo via the PKC/Stat3 Pathway

PubMed Central

Tsai, Min-Ling; Huang, Hui-Pei; Hsu, Jeng-Dong; Lai, Yung-Rung; Hsiao, Yu-Ping; Lu, Fung-Jou; Chang, Horng-Rong

2014-01-01

N-Acetylcysteine (Nac) is an antioxidant administered in both oral and injectable forms. In this study, we used Nac topically to treat burn wounds in vitro and in vivo to investigate mechanisms of action. In vitro, we monitored glutathione levels, cell proliferation, migration, scratch-wound healing activities and the epithelialization-related proteins, matrixmetalloproteinase-1 (MMP-1) and proteins involved in regulating the expression of MMP-1 in CCD-966SK cells treated with Nac. Various Nac concentrations (0.1, 0.5, and 1.0 mM) increased glutathione levels, cell viability, scratch-wound healing activities and migration abilities of CCD-966SK cells in a dose-dependent manner. The MMP-1 expression of CCD-966SK cells treated with 1.0 mM Nac for 24 h was significantly increased. Levels of phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), janus kinase 1 (Jak1), signal transducer and activator of transcription 3 (Stat3), c-Fos and Jun, but not extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), were also significantly increased in a dose-dependent manner compared to the controls. In addition, Nac induced collagenous expression of MMP-1 via the PKC/Stat3 signaling pathway. In vivo, a burn wound healing rat model was applied to assess the stimulation activity and histopathological effects of Nac, with 3.0% Nac-treated wounds being found to show better characteristics on re-epithelialization. Our results demonstrated that Nac can potentially promote wound healing activity, and may be a promising drug to accelerate burn wound healing. PMID:24798751

Notch3 overexpression causes arrest of cell cycle progression by inducing Cdh1 expression in human breast cancer cells.

PubMed

Chen, Chun-Fa; Dou, Xiao-Wei; Liang, Yuan-Ke; Lin, Hao-Yu; Bai, Jing-Wen; Zhang, Xi-Xun; Wei, Xiao-Long; Li, Yao-Chen; Zhang, Guo-Jun

2016-01-01

Uncontrolled cell proliferation, genomic instability and cancer are closely related to the abnormal activation of the cell cycle. Therefore, blocking the cell cycle of cancer cells has become one of the key goals for treating malignancies. Unfortunately, the factors affecting cell cycle progression remain largely unknown. In this study, we have explored the effects of Notch3 on the cell cycle in breast cancer cell lines by 3 methods: overexpressing the intra-cellular domain of Notch3 (N3ICD), knocking-down Notch3 by RNA interference, and using X-ray radiation exposure. The results revealed that overexpression of Notch3 arrested the cell cycle at the G0/G1 phase, and inhibited the proliferation and colony-formation rate in the breast cancer cell line, MDA-MB-231. Furthermore, overexpressing N3ICD upregulated Cdh1 expression and resulted in p27(Kip) accumulation by accelerating Skp2 degradation. Conversely, silencing of Notch3 in the breast cancer cell line, MCF-7, caused a decrease in expression levels of Cdh1 and p27(Kip) at both the protein and mRNA levels, while the expression of Skp2 only increased at the protein level. Correspondingly, there was an increase in the percentage of cells in the G0/G1 phase and an elevated proliferative ability and colony-formation rate, which may be caused by alterations of the Cdh1/Skp2/p27 axis. These results were also supported by exposing MDA-MB-231 cells or MCF-7 treated with siN3 to X-irradiation at various doses. Overall, our data showed that overexpression of N3ICD upregulated the expression of Cdh1 and caused p27(Kip) accumulation by accelerating Skp2 degradation, which in turn led to cell cycle arrest at the G0/G1 phase, in the context of proliferating breast cancer cell lines. These findings help to illuminate the precision therapy targeted to cell cycle progression, required for cancer treatment.

Multicenter study of banked third-party virus-specific T cells to treat severe viral infections after hematopoietic stem cell transplantation.

PubMed

Leen, Ann M; Bollard, Catherine M; Mendizabal, Adam M; Shpall, Elizabeth J; Szabolcs, Paul; Antin, Joseph H; Kapoor, Neena; Pai, Sung-Yun; Rowley, Scott D; Kebriaei, Partow; Dey, Bimalangshu R; Grilley, Bambi J; Gee, Adrian P; Brenner, Malcolm K; Rooney, Cliona M; Heslop, Helen E

2013-06-27

Virus-specific T cell (VST) lines could provide useful antiviral prophylaxis and treatment of immune-deficient patients if it were possible to avoid the necessity of generating a separate line for each patient, often on an emergency basis. We prepared a bank of 32 virus-specific lines from individuals with common HLA polymorphisms who were immune to Epstein-Barr virus (EBV), cytomegalovirus, or adenovirus. A total of 18 lines were administered to 50 patients with severe, refractory illness because of infection with one of these viruses after hematopoietic stem cell transplant. The cumulative rates of complete or partial responses at 6 weeks postinfusion were 74.0% (95% CI, 58.5%-89.5%) for the entire group (n = 50), 73.9% (95% CI, 51.2% -96.6%) for cytomegalovirus (n = 23), 77.8% for adenovirus (n = 18), and 66.7% (95% CI, 36.9%-96.5%) for EBV (n = 9). Only 4 responders had a recurrence or progression. There were no immediate infusion-related adverse events, and de novo graft-versus-host disease developed in only 2 patients. Despite the disparity between the lines and their recipients, the mean frequency of VSTs increased significantly postinfusion, coincident with striking decreases in viral DNA and resolution of clinical symptoms. The use of banked third-party VSTs is a feasible and safe approach to rapidly treat severe or intractable viral infections after stem cell transplantation. This study is registered at www.clinicaltrials.gov as NCT00711035.

Hot water-extracted Lycium barbarum and Rehmannia glutinosa inhibit proliferation and induce apoptosis of hepatocellular carcinoma cells

PubMed Central

Chao, Jane C-J; Chiang, Shih-Wen; Wang, Ching-Chiung; Tsai, Ya-Hui; Wu, Ming-Shun

2006-01-01

AIM: To investigate the effect of hot water-extracted Lycium barbarum (LBE) and Rehmannia glutinosa (RGE) on cell proliferation and apoptosis in rat and/or human hepatocellular carcinoma (HCC) cells. METHODS: Rat (H-4-II-E) and human HCC (HA22T/VGH) cell lines were incubated with various concentrations (0-10 g/L) of hot water-extracted LBE and RGE. After 6-24 h incubation, cell proliferation (n = 6) was measured by a colorimetric method. The apoptotic cells (n = 6) were detected by flow cytometry. The expression of p53 protein (n = 3) was determined by SDS-PAGE and Western blotting. RESULTS: Crude LBE (2-5 g/L) and RGE (2-10 g/L) dose-dependently inhibited proliferation of H-4-II-E cells by 11% (P < 0.05) to 85% (P < 0.01) after 6-24 h treatment. Crude LBE at a dose of 5 g/L suppressed cell proliferation of H-4-II-E cells more effectively than crude RGE after 6-24 h incubation (P < 0.01). Crude LBE (2-10 g/L) and RGE (2-5 g/L) also dose-dependently inhibited proliferation of HA22T/VGH cells by 14%-43% (P < 0.01) after 24 h. Crude LBE at a dose of 10 g/L inhibited the proliferation of HA22T/VGH cells more effectively than crude RGE (56.8% ± 1.6% vs 70.3% ± 3.1% of control, P = 0.0003 < 0.01). The apoptotic cells significantly increased in H-4-II-E cells after 24 h treatment with higher doses of crude LBE (2-5 g/L) and RGE (5-10 g/L) (P < 0.01). The expression of p53 protein in H-4-II-E cells was 119% and 143% of the control group compared with the LBE-treated (2, 5 g/L) groups, and 110% and 132% of the control group compared with the RGE -treated (5, 10 g/L) groups after 24 h. CONCLUSION: Hot water-extracted crude LBE (2-5 g/L) and RGE (5-10 g/L) inhibit proliferation and stimulate p53-mediated apoptosis in HCC cells. PMID:16874858

Discovery of 4-aryl-N-arylcarbonyl-2-aminothiazoles as Hec1/Nek2 inhibitors. Part I: optimization of in vitro potencies and pharmacokinetic properties.

PubMed

Lee, Ying-Shuan E; Chuang, Shih-Hsien; Huang, Lynn Y L; Lai, Chun-Liang; Lin, Yu-Hsiang; Yang, Ju-Ying; Liu, Chia-Wei; Yang, Sheng-chuan; Lin, Her-Sheng; Chang, Chia-chi; Lai, Jun-Yu; Jian, Pei-Shiou; Lam, King; Chang, Jia-Ming; Lau, Johnson Y N; Huang, Jiann-Jyh

2014-05-22

A series of 4-aryl-N-arylcarbonyl-2-aminothiazoles of scaffold 4 was designed and synthesized as Hec1/Nek2 inhibitors. Structural optimization of 4 led to compound 32 bearing C-4' 4-methoxyphenoxy and 4-(o-fluoropyridyl)carbonyl groups that showed low nanomolar in vitro antiproliferative activity (IC50: 16.3-42.7 nM), high intravenous AUC (64.9 μM·h, 2.0 mg/kg) in SD rats, and significant in vivo antitumor activity (T/C = 32%, 20 mg/kg, IV) in mice bearing human MDA-MB-231 xenografts. Cell responses resulting from Hec1/Nek2 inhibition were observed in cells treated with 32, including a reduced level of Hec1 coimmunoprecipitated with Nek2, degradation of Nek2, mitotic abnormalities, and apoptosis. Compound 32 showed selectivity toward cancer cells over normal phenotype cells and was inactive in a [(3)H]astemizole competitive binding assay for hERG liability screening. Therefore, 32 is as a good lead toward the discovery of a preclinical candidate targeting Hec1/Nek2 interaction.

Inhibition of mitochondrial calcium efflux by clonazepam in intact single rat cardiomyocytes and effects on NADH production.

PubMed

Griffiths, E J; Wei, S K; Haigney, M C; Ocampo, C J; Stern, M D; Silverman, H S

1997-04-01

The aims of this study were to determine: (i) whether clonazepam and CGP37157, which inhibit the Na+/Ca2+ exchanger of isolated mitochondria, could inhibit mitochondrial Ca2+ efflux in intact cells; and (ii) whether any sustained increase in mitochondrial [Ca2+] ([Ca2+]m) could alter mitochondrial NADH levels. [Ca2+]m was measured in Indo-1/AM loaded rat ventricular myocytes where the cytosolic fluorescence signal was quenched by superfusion with Mn2+. NADH levels were determined from cell autofluorescence. Upon exposure of myocytes to 50 nM norepinephrine (NE) and a stimulation rate of 3 Hz, [Ca2+]m increased from 59 +/- 3 nM to a peak of 517 +/- 115 nM (n = 8) which recovered rapidly upon return to low stimulation rate (0.2 Hz) and washout of NE. In the presence of clonazepam, the peak increase in [Ca2+]m was 937 +/- 192 nM (n = 5) which remained elevated at 652 +/- 131 nM upon removal of the stimulus. CGP37157 in some cells did give the same inhibition of mitochondrial Ca2+ efflux as clonazepam, but the effect was inconsistent since not all cells were capable of following the stimulation rate in presence of this compound. NADH levels increased upon exposure to rapid stimulation in the presence of NE alone and recovered upon return to low stimulation rates, whereas in clonazepam treated cells the recovery of NADH was prevented. We conclude that clonazepam is an effective inhibitor of mitochondrial [Ca2+] efflux in intact cells and also maintains the increase in NADH levels which occurs upon rapid stimulation of cells.

Apple polyphenol phloretin potentiates the anticancer actions of paclitaxel through induction of apoptosis in human hep G2 cells.

PubMed

Yang, Kuo-Ching; Tsai, Chia-Yi; Wang, Ying-Jan; Wei, Po-Li; Lee, Chia-Hwa; Chen, Jui-Hao; Wu, Chih-Hsiung; Ho, Yuan-Soon

2009-05-01

Phloretin (Ph), which can be obtained from apples, apple juice, and cider, is a known inhibitor of the type II glucose transporter (GLUT2). In this study, real-time PCR analysis of laser-capture microdissected (LCM) human hepatoma cells showed elevated expression (>5-fold) of GLUT2 mRNA in comparison with nonmalignant hepatocytes. In vitro and in vivo studies were performed to assess Ph antitumor activity when combined with paclitaxel (PTX) for treatment of human liver cancer cells. Inhibition of GLUT2 by Ph potentiated the anticancer effects of PTX, resensitizing human liver cancer cells to drugs. These results demonstrate that 50-150 microM Ph significantly potentiates DNA laddering induced in Hep G2 cells by 10 nM PTX. Activity assays showed that caspases 3, 8, and 9 are involved in this apoptosis. The antitumor therapeutic efficacy of Ph (10 mg/kg body weight) was determined in cells of the SCID mouse model that were treated in parallel with PTX (1 mg/kg body weight). The Hep G2-xenografted tumor volume was reduced more than fivefold in the Ph + PTX-treated mice compared to the PTX-treated group. These results suggest that Ph may be useful for cancer chemotherapy and chemoprevention.

Increased expression of cyclin B1 mRNA coincides with diminished G{sub 2}-phase arrest in irradiated HeLa cells treated with staurosporine or caffeine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernhard, E.J.; Maity, A.; McKenna, W.G.

1994-12-01

The irradiation of cells results in delayed progression through the G{sub 2} phase of the cell cycle. Treatment of irradiated HeLa cells with caffeine greatly reduces the G{sub 2}-phase delay, while caffeine does not alter progression of cells through the cell cycle in unirradiated cells. In this report we demonstrate that treatment of HeLa cells with the kinase inhibitor staurosporine, but not with the inhibitor H7, also results in a reduction of the G{sub 2}-phase arrest after irradiation. Cell cycle progression in unirradiated cells is unaffected by 4.4 nM (2ng/ml) staurosporine, which releases the radiation-induced G{sub 2}-phase arrest. In HeLamore » cells, the G{sub 2}-phase delay after irradiation in S phase is accompanied by decreased expression of cyclin B1 mRNA. Coincident with the reduction in G{sub 2}-phase delay, we observed an increase in cyclin B1 mRNA accumulation in irradiated, staurosporine-treated cells compared to cells treated with irradiation alone. Caffeine treatment of irradiated HeLa cells also resulted in an elevation in the levels of cyclin B1 message. These results support the hypothesis that diminished cyclin B1 mRNA levels influence G{sub 2}-phase arrest to some degree. The findings that both staurosporine and caffeine treatments reverse the depression in cyclin B1 expression suggest that these two compounds may act on a common pathway of cell cycle control in response to radiation injury. 33 refs., 6 figs.« less

Using Interferon Alfa Before Tyrosine Kinase Inhibitors May Increase Survival in Patients With Metastatic Renal Cell Carcinoma: A Turkish Oncology Group (TOG) Study.

PubMed

Artaç, Mehmet; Çoşkun, Hasan Şenol; Korkmaz, Levent; Koçer, Murat; Turhal, Nazım Serdar; Engin, Hüseyin; Dede, İsa; Paydaş, Semra; Öksüzoğlu, Berna; Bozcuk, Hakan; Demirkazık, Ahmet

2016-08-01

We aimed to investigate the outcomes of interferon alfa and sequencing tyrosine kinase inhibitors (TKIs) in patients with metastatic renal cell carcinoma. This multicenter study assessing the efficacy of TKIs after interferon alfa therapy in the first-line setting in patients with metastatic renal cell carcinoma. Patients (n = 104) from 8 centers in Turkey, who had been treated with interferon alfa in the first-line setting, were included in the study. Prognostic factors were evaluated for progression-free survival (PFS). The median age of the patients was 57 years. The median PFS of the patients treated with interferon alfa in the first-line was 3.6 months. A total of 61 patients received TKIs (sunitinib, n = 58; sorafenib, n = 3) after progression while on interferon alfa. The median PFS among the TKI-treated patients was 13.2 months. In the univariate analysis for interferon alfa treatment, neutrophil and hemoglobin level, platelet count, and Karnofsky performance status were the significant factors associated with PFS. In the univariate analysis for TKI treatment, neutrophil and hemoglobin levels were the significant factors for PFS. The median total PFS of the patients who had been treated with first-line interferon alfa and second-line TKIs was 24.9 months. This study showed that first-line interferon alfa treatment before TKIs may improve the total PFS in patients with metastatic renal cell carcinoma. Copyright © 2016 Elsevier Inc. All rights reserved.

CRACC-CRACC Interaction between Kupffer and NK Cells Contributes to Poly I:C/D-GalN Induced Hepatitis

PubMed Central

Li, Yangxi; Cao, Guoshuai; Zheng, Xiaodong; Wang, Jun; Wei, Haiming; Tian, Zhigang; Sun, Rui

2013-01-01

CD2-like receptor activating cytotoxic cells (CRACC) is known as a critical activating receptor of natural killer (NK) cells. We have previously reported that NK cells contribute to Poly I:C/D-galactosamine (D-GalN)-induced fulminant hepatitis. Since natural killer group 2, member D (NKG2D) is considered critical but not the only activating receptor for NK cells, we investigated the role of CRACC in this model. We found that CRACC was abundant on hepatic NK cells but with low expression levels on Kupffer cells under normal conditions. Expression of CRACC on NK cells and Kupffer cells was remarkably upregulated after poly I:C injection. Hepatic CRACC mRNA levels were also upregulated in Poly I:C/D-GalN-treated mice, and correlated positively with the serum alanine aminotransferase (ALT) levels. CRACC expression on Kupffer cells was specifically silenced by nano-particle encapsulated siRNA in vivo, which significantly reduced Poly I:C/D-GalN-induced liver injury. In co-culture experiments, it was further verified that silencing CRACC expression or blockade of CRACC activation by mAb reduced the production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Collectively, our findings suggest that CRACC-CRACC interaction between NK cells and resident Kupffer cells contributes to Poly I:C/D-GalN-induced fulminant hepatitis. PMID:24098802

Metabolic Enhancer Piracetam Attenuates the Translocation of Mitochondrion-Specific Proteins of Caspase-Independent Pathway, Poly [ADP-Ribose] Polymerase 1 Up-regulation and Oxidative DNA Fragmentation.

PubMed

Verma, Dinesh Kumar; Gupta, Sonam; Biswas, Joyshree; Joshi, Neeraj; Sivarama Raju, K; Wahajuddin, Mu; Singh, Sarika

2018-03-12

Piracetam, a nootropic drug, has been clinically used for decades; however, its mechanism of action still remains enigmatic. The present study was undertaken to evaluate the role of mitochondrion-specific factors of caspase-independent pathway like apoptotic-inducing factor (AIF) and endonuclease-G (endo-G) in piracetam-induced neuroprotection. N2A cells treated with lipopolysaccharide (LPS) exhibited significant cytotoxicity, impaired mitochondrial activity, and reactive oxygen species generation which was significantly attenuated with piracetam co-treatment. Cells co-treated with LPS and piracetam exhibited significant uptake of piracetam in comparison to only piracetam-treated cells as estimated by liquid chromatography-mass spectrometry (LC-MSMS). LPS treatment caused significant translocation of AIF and endonuclease-G in neuronal N2A cells which were significantly attenuated with piracetam co-treatment. Significant over-expression of proinflammatory cytokines was also observed after treatment of LPS to cells which was inhibited with piracetam co-treatment demonstrating its anti-inflammatory property. LPS-treated cells exhibited significant oxidative DNA fragmentation and poly [ADP-ribose] polymerase-1 (PARP-1) up-regulation in nucleus, both of which were attenuated with piracetam treatment. Antioxidant melatonin but not z-VAD offered the inhibited LPS-induced DNA fragmentation indicating the involvement of oxidative DNA fragmentation. Further, we did not observe the altered caspase-3 level after LPS treatment initially while at a later time point, significantly augmented level of caspase-3 was observed which was not inhibited with piracetam treatment. In total, our findings indicate the interference of piracetam in mitochondrion-mediated caspase-independent pathway, as well as its anti-inflammatory and antioxidative properties. Graphical Abstract Graphical abstract indicating the novel interference of metabolic enhancer piracetam (P) in neuronal death mechanisms.

Development of Biochemical Cyanide Antidotes.

DTIC Science & Technology

1992-03-01

Additionally, known cyanide antidotes (e.g., pyruvate, mercaptopyruvate, alpha ketoglutarate , naloxone and 20. DISTRIBUTION /AVAILABILITY OF ABSTRACT...effective, whereas imipramine, bretylium and carbamazepine blocked between 61 and 71% of the effect (Figure 3, Table 1). Alpha - ketoglutarate also decreased...Generation of hydroperoxides in cyanide treated cells was 1.98 + 0.43 gmol H2 0 2 /10 cells per 5 min. N = 2 to 8. a-KG = alpha - ketoglutarate . 15 Cytochrome

Differential effect of EGFR inhibitors on tamoxifen-resistant breast cancer cells.

PubMed

Kim, Sangmin; Lee, Jeongmin; Oh, Soo Jin; Nam, Seok Jin; Lee, Jeong Eon

2015-09-01

Although tamoxifen is the most common and effective therapy for treatment of estrogen receptor-α (ER-α) breast cancer patients, resistance of endocrine therapy occurs, either de novo or acquired during therapy. Here, we investigated the clinical value of epidermal growth factor receptor (EGFR) in tamoxifen-resistant (TamR) patients and the differential effect of EGFR inhibitors, neratinib and gefitinib, on TamR breast cancer cell model. The morphology of TamR MCF7 cells showed mesenchymal phenotypes and did not induce cell death by tamoxifen treatment compared with tamoxifen‑sensitive (TamS) MCF7 cells. In addition, mesenchymal marker proteins, including N-cadherin (N-cad), fibronectin (FN), and Slug, significantly increased in TamR cells. In contrast, ER-α and E-cadherin (E-cad) were greatly decreased. We also found that the levels of EGFR and HER2 expression were increased in TamR cells. Furthermore, we observed that EGFR expression was directly involved with poor prognosis of tamoxifen-treated breast cancer patients using the GSE1378 date set. Thus, we treated TamR and TamS cells with EGFR inhibitors, neratinib and gefitinib, respectively. Interestingly, neratinib induced apoptotic cell death of TamR but not gefitinib. Cleaved PARP-1 expression was also increased by neratinib treatment in TamR cells. Therefore, we suggest that neratinib may be a potential therapeutic drug for treating TamR breast cancer.

Association Studies of Fcγ Receptor Polymorphisms with Outcome in HER2+ Breast Cancer Patients Treated with Trastuzumab in NCCTG (Alliance) Trial N9831

PubMed Central

Norton, Nadine; Olson, Rebecca M.; Pegram, Mark; Tenner, Kathleen; Ballman, Karla V.; Clynes, Raphael; Knutson, Keith L.; Perez, Edith A.

2014-01-01

Patients with HER2+ breast cancer treated with trastuzumab and chemotherapy have superior survival compared to patients treated with chemotherapy alone. Polymorphisms within FCGR2A and FCGR3A are associated with binding affinity of natural killer cells to the IgG1 portion of trastuzumab and a polymorphism in FCGR2B (I232T) is associated with impaired regulatory activity. The association of these polymorphisms with clinical response among trastuzumab-treated patients is equivocal with both positive and negative associations. We performed genotyping analysis on the FCGR3A V158F, FCGR2A R131H, and FCGR2B I232T genes in 1,325 patients from the N9831 clinical trial. Patients in Arm A (N=419) received chemotherapy only. Patients in Arms B (N=469) and C (N=437) were treated with chemotherapy and trastuzumab, (sequentially in Arm B and concurrently in Arm C). Using log-rank test and Cox proportional hazard models, we compared disease-free survival (DFS) among genotypic groups within pooled Arms B/C. We found no differences in DFS between trastuzumab-treated patients who had the FCGR3A 158 V/V and/or FCGR2A 131 H/H high affinity genotypes and patients without those genotypes. Furthermore, there was no significant interaction between FCGR3A and FCGR2A and treatment. However, there was a difference in DFS for FCGR2B I232T with I/I patients deriving benefit from trastuzumab (p<0.001) compared to the T carriers who did not (p=0.81). The interaction between FCGR2B genotype and treatment was statistically significant (p=0.03). Our analysis did not reveal an association between FcγR high affinity genotypes and outcomes. However, it appears that the FCGR2B inhibitory gene may be predictive of adjuvant trastuzumab benefit. PMID:24989892

Pomegranate inhibits neuroinflammation and amyloidogenesis in IL-1β-stimulated SK-N-SH cells.

PubMed

Velagapudi, Ravikanth; Baco, Gina; Khela, Sunjeet; Okorji, Uchechukwu; Olajide, Olumayokun

2016-06-01

Pomegranate fruit, Punica granatum L. (Punicaceae), and its constituents have been shown to inhibit inflammation. In this study, we aimed to assess the effects of freeze-dried pomegranate (PWE) on PGE2 production in IL-1β-stimulated SK-N-SH cells. An enzyme immunoassay (EIA) was used to measure prostaglandin E2 (PGE2) production from supernatants of IL-1β-stimulated SK-N-SH cells. Expression of COX-2, phospho-IκB, and phospho-IKK proteins was evaluated, while NF-κB reporter gene assay was carried out in TNFα-stimulated HEK293 cells to determine the effect of PWE on NF-κB transactivation. Levels of BACE-1 and Aβ in SK-N-SH cells stimulated with IL-1β were measured with an in cell ELISA. PWE (25-200 μg/ml) dose dependently reduced COX-2-dependent PGE2 production in SK-N-SH cells stimulated with IL-1β. Phosphorylation of IκB and IKK was significantly (p < 0.001) inhibited by PWE (50-200 μg/ml). Our studies also show that PWE (50-200 μg/ml) significantly (p < 0.01) inhibited NF-κB transactivation in TNFα-stimulated HEK293 cells. Furthermore, PWE inhibited BACE-1 and Aβ expression in SK-N-SH cells treated with IL-1β. Taken together, our study demonstrates that pomegranate inhibits inflammation, as well as amyloidogenesis in IL-1β-stimulated SK-N-SH cells. We propose that pomegranate is a potential nutritional strategy in slowing the progression of neurodegenerative disorders such as Alzheimer's disease.

Role of endothelial nitric oxide synthetase in arteriogenesis after stroke in mice.

PubMed

Cui, X; Chopp, M; Zacharek, A; Zhang, C; Roberts, C; Chen, J

2009-03-17

Arteriogenesis supports restored perfusion in the ischemic brain and improves long-term functional outcome after stroke. We investigate the role of endothelial nitric oxide synthetase (eNOS) and a nitric oxide (NO) donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETA-NONOate), in promoting arteriogenesis after stroke. Adult wild-type (WT, n=18) and eNOS-knockout (eNOS(-/-), n=36) mice were subjected to transient (2.5 h) right middle cerebral artery occlusion (MCAo) and were treated with or without DETA-NONOate (0.4 mg/kg) 24 h after MCAo. Functional evaluation was performed. Animals were sacrificed 3 days after MCAo for arterial cell culture studies, or 14 days for immunohistochemical analysis. Consistent with previous studies, eNOS(-/-) mice exhibited a higher mortality rate (P<0.05, n=18/group) and more severe neurological functional deficit after MCAo than WT mice (P<0.05, n=12/group). Decreased arteriogenesis, was evident in eNOS(-/-) mice compared with WT mice, as demonstrated by reduced vascular smooth muscle cell (VSMC) proliferation, arterial density and diameter in the ischemic brain. eNOS(-/-) mice treated with DETA-NONOate had a significantly decreased mortality rate and improved functional recovery, and exhibited enhanced arteriogenesis identified by increased VSMC proliferation, and upregulated arterial density and diameter compared to eNOS(-/-) mice after stroke (P<0.05, n=12/group). To elucidate the mechanisms underlying eNOS/NO mediated arteriogenesis, VSMC migration was measured in vitro. Arterial cell migration significantly decreased in the cultured common carotid artery (CCA) derived from eNOS(-/-) mice 3 days after MCAo compared to WT arterial cells. DETA-NONOate-treatment significantly attenuated eNOS(-/-)-induced decrease of arterial cell migration compared to eNOS(-/-) control artery (P<0.05; n=6/group). Using VSMC culture, DETA-NONOate significantly increased VSMC migration, while inhibition of NOS significantly decreased VSMC migration (P<0.05; n=6/group). Our data indicated that eNOS not only promotes vascular dilation but also increases VSMC proliferation and migration, and thereby enhances arteriogenesis after stroke. Therefore, increase eNOS may play an important role in regulating of arteriogenesis after stroke.

A Model for Spheroid versus Monolayer Response of SK-N-SH Neuroblastoma Cells to Treatment with 15-Deoxy-PGJ 2

PubMed Central

Dunham, Ann; Chen, Paula X.; Chen, Michelle; Huynh, Milan; Rheingold, Evan; Prosper, Olivia

2016-01-01

Researchers have observed that response of tumor cells to treatment varies depending on whether the cells are grown in monolayer, as in vitro spheroids or in vivo. This study uses data from the literature on monolayer treatment of SK-N-SH neuroblastoma cells with 15-deoxy-PGJ 2 and couples it with data on growth rates for untreated SK-N-SH neuroblastoma cells grown as multicellular spheroids. A linear model is constructed for untreated and treated monolayer data sets, which is tuned to growth, death, and cell cycle data for the monolayer case for both control and treatment with 15-deoxy-PGJ 2. The monolayer model is extended to a five-dimensional nonlinear model of in vitro tumor spheroid growth and treatment that includes compartments of the cell cycle (G 1, S, G 2/M) as well as quiescent (Q) and necrotic (N) cells. Monolayer treatment data for 15-deoxy-PGJ 2 is used to derive a prediction of spheroid response under similar treatments. For short periods of treatment, spheroid response is less pronounced than monolayer response. The simulations suggest that the difference in response to treatment of monolayer versus spheroid cultures observed in laboratory studies is a natural consequence of tumor spheroid physiology rather than any special resistance to treatment. PMID:28044089

Improvement in Direct Methanol Fuel Cell Performance by Treating the Anode at High Anodic Potential

DTIC Science & Technology

2014-01-01

stripping voltammetryReorganization of Na? on ionomer Prabhuram Joghee, Svitlana Pylypenko, Kevin Wood , April Corpuz, Guido Bender, Huyen N. Dinh...methanol fuel cell performance by treating the anode at high anodic potential Prabhuram Joghee a,*, Svitlana Pylypenko a,b, Kevin Wood a, April Corpuz c...References [1] B. McNicol, D.A.J. Rand, K.R. Williams, J. Power Sources 83 (1999) 15e31. [2] K. Scot , W.M. Taama, J. Power Sources 79 (1999) 43e59. [3] P

Citral, A Monoterpene Protect Against High Glucose Induced Oxidative Injury in HepG2 Cell In Vitro-An Experimental Study.

PubMed

Subramaniyan, Sri Devi; Natarajan, Ashok Kumar

2017-08-01

Diabetes mellitus, a major metabolic disorder associated with hyperglycaemia is one of the leading cause of death in many developed countries. However, use of natural phytochemicals have been proved to have a protective effect against oxidative damage. To investigate the effect of citral, a monoterpene on high glucose induced cytotoxicity and oxidative stress in human hepatocellular liver carcinoma (Hep G2) cell line. Cells were treated with 50 mM concentration of glucose for 24 hours incubation following citral (30 μM) was added to confluent HepG2 cells. Cell viability, Reactive Oxygen Species (ROS) generation, DNA damage, lipid peroxidation, antioxidants and Mitogen Activated Protein Kinases (MAPKs) signaling were assessed in citral and/or high glucose induced HepG2 cells. Cells treated with glucose (50 mM), resulted in increased cytotoxicity, ROS generation, DNA damage, lipid peroxidation and depletion of enzymatic and non enzymatic antioxidants. In contrast, treatment with citral (30 μM) significantly decreased cell cytotoxicity, ROS generation, DNA damage, lipid peroxidation and increased antioxidants enzymes in high glucose induced HepG2 cells. In addition, the present study highlighted that high glucose treated cells showed increased expression of Extracellular Signal Regulated Protein Kinase-1 (ERK-1), c-Jun N-terminal Kinase (JNK) and p38 in HepG2 cells. On the other hand treatment with citral significantly suppressed the expression of ERK-1, JNK and p38 in high glucose induced HepG2 cells. Citral protects against high glucose induced oxidative stress through inhibiting ROS activated MAPK signaling pathway in HepG2 cells.

The Effects of Periostin in a Rat Model of Isoproterenol: Mediated Cardiotoxicity.

PubMed

Sözmen, Mahmut; Devrim, Alparslan K; Kabak, Yonca B; Devrim, Tuba; Sudagidan, Mert

2018-04-01

Periostin is an extracellular matrix protein from fasciclin family, and it plays an important role in the cell adhesion, migration, and growth of the organism. Periostin prevents apoptosis while stimulating cardiomyocytes. The present study was designed to investigate cardioprotective effects of the recombinant murine periostin peptide administration in a rat model of isoproterenol (ISO)-induced myocardial injury. The experiment was performed on 84 adult male Sprague Dawley rats in 4 groups (n = 21): control group (1), periostin-treated group (2), ISO-treated group (3), and ISO + periostin-treated group (4). The groups were further divided into three subgroups based on the duration of the experiment in which rats were killed on days 1, 7, and 28 (n = 7). Growth factors (VEGF, ANGPT, FGF-2, TGFβ), mitosis and apoptosis (Bcl-2, Bax, PCNA, Ki-67, phospho-histone H3), cell cycle activators and inhibitors (cyclin D1, D2, A2, Cdc2), the origin of regenerating cells (cKit and CD45) mRNA were detected using quantitative real-time polymerase chain reaction (PCR) and PCR array. Immunohistochemistry staining was used to directly detect protein level and distribution in the heart tissues. Administration of periostin following ISO-induced cardiotoxicity revealed that periostin alleviated deleterious effects of ISO through several pathways: (1) periostin induced mitotic activity of endothelial/fibroblastic cells, (2) periostin drives cardiomyocytes into S and M phases, thus promoting proliferation of cardiomyocytes, (3) periostin contributed to collagen degradation, tissue remodeling, and reduced cardiac fibrosis during the healing process following myocardial damage while preserving tissue matrix, (4) periostin stimulated angiogenesis by upregulating THBS1, TGFB2, and HGF genes, (5) periostin regulated cell growth and proliferation while maintaining cell shape and cellular muscle contractions (ACTB) and functioned as chemoattractant factor (CCL2) at the beginning of myocardial damage.

pH-Sensitive PEGylated liposomes for delivery of an acidic dinitrobenzamide mustard prodrug: Pathways of internalization, cellular trafficking and cytotoxicity to cancer cells.

PubMed

Yang, Mimi M; Wilson, William R; Wu, Zimei

2017-01-10

This paper aims to develop and evaluate a pH-sensitive PEGylated liposomal (pPSL) system for tumor-targeted intracellular delivery of SN25860, a weakly acidic, poorly water-soluble dinitrobenzamide mustard prodrug which is activated by the E. coli nitroreductase nfB. pPSL and non pH-sensitive liposomes (nPSL), as reference, were formulated by thin-film hydration; an active drug loading method was developed with the aid of solubilizers. Cytotoxicity was evaluated in an nfsB-transfected EMT6 mouse mammary carcinoma cell line. Cellular uptake of liposomes was evaluated by both high performance liquid chromatography and flow cytometry. Intracellular trafficking was visualised by confocal microscopy. High drug loading (7.0±0.2% w/w) was achieved after systematic optimization of drug loading conditions. pPSL-SN25860 demonstrated a 21 and 24- fold increase in antiproliferative potency compared to nPSL-SN25860 and free drug, respectively. Cells treated with pPSL had a 1.6-2.5- fold increase in intracellular drug concentration compared to nPSL. This trend was consistent with flow cytometry results. Cells treated with chlorpromazine demonstrated reduced uptake of both nPSL (40%) and pPSL (46%), indicating clathrin-mediated endocytosis was the major pathway. Confocal microscopy showed that pPSL had not only undergone faster and greater endocytosis than nPSL but was also homogeneously distributed in the cytosol and nuclei suggesting endosome escape, in contrast to nPSL. Copyright © 2016 Elsevier B.V. All rights reserved.

Coordinated modulation of albumin synthesis and mRNA levels in cultured hepatoma cells by hydrocortisone and cyclic AMP analogs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, P.C.; Papaconstantinou, J.

The treatment of Hepa-2 cells, a permanent mouse hepatoma cell line, for 72 h with hydrocortisone (10/sup -6/ M), N/sup 6/,O/sup 2/-dibutyryl cyclic AMP (10/sup -3/ M), or 8-bromo cyclic AMP(10/sup -3/ M) results in a 2-, 3-, or 4-fold increase, respectively, in rates of synthesis and secretion of mouse serum albumin. Simultaneous treatment with hydrocortisone and N/sup 6/,O/sup 2/-dibutyryl cyclic AMP results in a 10-fold stimulation in these parameters, an effect that is significantly more than additive for the two compounds tested. The number of albumin mRNA sequences, determined by hybridization of total cell RNA to albumin complementary DNA,more » was increased in direct proportion to the increases in albumin synthesis in all experiments. The relative rate of albumin synthesis approaches in vivo levels in cells treated simultaneously with hydrocortisone and N/sup 6/,O/sup 2/-dibutyryl cyclic AMP. We propose that these factors may be necessary to maintain the maximal level of differentiated function in the continuous culture of Hepa-2 cells.« less

ROLE OF ENDOTHELIAL NITRIC OXIDE SYNTHETASE IN ARTERIOGENESIS AFTER STROKE IN MICE

PubMed Central

CUI, X.; CHOPP, M.; ZACHAREK, A.; ZHANG, C.; ROBERTS, C.; CHEN, J.

2009-01-01

Arteriogenesis supports restored perfusion in the ischemic brain and improves long-term functional outcome after stroke. We investigate the role of endothelial nitric oxide synthetase (eNOS) and an NO donor, [(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) aminio] diazen-1-ium-1, 2-diolate (DETA-NONOate), in promoting arteriogenesis after stroke. Adult wild-type (WT, n=18) and eNOS-knockout (eNOS-/-, n=36) mice were subjected to transient (2.5 hours) right middle cerebral artery occlusion (MCAo) and were treated with or without DETA-NONOate (0.4 mg/kg) 24 hours after MCAo. Functional evaluation was performed. Animals were sacrificed 3 days after MCAo for arterial cell culture studies, or 14 days for immunohistochemical analysis. Consistent with previous studies, eNOS-/- mice exhibited a higher mortality rate (p<0.05, n=18/group) and more severe neurological functional deficit after MCAo than WT mice (p<0.05, n=12/group). Decreased arteriogenesis, was evident in eNOS-/- mice compared with WT mice, as demonstrated by reduced vascular smooth muscle cell (VSMC) proliferation, arterial density and diameter in the ischemic brain. eNOS-/- mice treated with DETA-NONOate had a significantly decreased mortality rate and improved functional recovery, and exhibited enhanced arteriogenesis identified by increased VSMC proliferation, and upregulated arterial density and diameter compared to eNOS-/- mice after stroke (p<0.05, n=12/group). To elucidate the mechanisms underlying eNOS/NO mediated arteriogenesis, VSMC migration was measured in vitro. Arterial cell migration significantly decreased in the cultured common carotid artery (CCA) derived from eNOS-/- mice 3 days after MCAo compared to WT arterial cells. DETA-NONOate-treatment significantly attenuated eNOS-/--induced decrease of arterial cell migration compared to eNOS-/- control artery (p<0.05. n=6/group). Using VSMC culture, DETA-NONOate significantly increased VSMC migration, while inhibition of NOS significantly decreased VSMC migration (p<0.05. n=6/group). Our data indicated that eNOS not only promotes vascular dilation but also increases VSMC proliferation and migration, and thereby enhances arteriogenesis after stroke. Therefore, increase eNOS may play an important role in regulating of arteriogenesis after stroke. PMID:19154781

Polyethylene glycol-conjugated HER2-targeted peptides as a nuclear imaging probe for HER2-overexpressed gastric cancer detection in vivo.

PubMed

Guan, Siao-Syun; Wu, Cheng-Tien; Chiu, Chen-Yuan; Luo, Tsai-Yueh; Wu, Jeng-Yih; Liao, Tse-Zung; Liu, Shing-Hwa

2018-06-19

The human epidermal growth factor receptor 2 (HER2) involved proliferation, angiogenesis, and reduced apoptosis in gastric cancer (GC), which is a common target for tumor therapy. HER2 is usually overexpressed in more than 15% GC patients, developing a reliable diagnostic tool for tumor HER2 detection is important. In this study, we attend to use polyethylene glycol (PEG) linked anti-HER2/neu peptide (AHNP-PEG) as a nuclear imaging agent probe for HER2 detection in GC xenograft animal model. The HER2 expression of human sera and tissues were detected in GC patients and normal subjects. GC cell lines NCI-N87 (high HER2 levels) and MKN45 (low HER2 levels) were treated with AHNP-PEG to assess the cell viability and HER2 binding ability. The NCI-N87 was treated with AHNP-PEG to observe the level and phosphorylation of HER2. The MKN45 and NCI-N87-induced xenograft mice were intravenous injection with fluorescence labeled AHNP-PEG for detecting in vivo fluorescence imaging properties and biodistribution. The AHNP-PEG was conjugated with diethylenetriaminopentaacetic acid (DTPA) for indium-111 labeling ( 111 In-DTPA-AHNP-PEG). The stability of was assessed in vitro. The imaging properties and biodistribution of 111 In-DTPA-AHNP-PEG were observed in NCI-N87-induced xenograft mice. The serum HER2 (sHER2) levels in GC patients were significantly higher than the normal subjects. The sHER2 levels were correlated with the tumor HER2 levels in different stages of GC patients. The AHNP-PEG inhibited the cell growth and down-regulated HER2 phosphorylation in HER2-overexpressed human GC cells (NCI-N87) via specific HER2 interaction of cell surface. In addition, the GC tumor tissues from HER2-postive xenograft mice presented higher HER2 fluorescence imaging as compared to HER2-negative group. The HER2 levels in the tumor tissues were also higher than other organs in NCI-N87-induced xenograft mice. Finally, we further observed that the 111 In-DTPA-AHNP-PEG was significantly enhanced in tumor tissues of NCI-N87-induced xenograft mice compared to control. These findings suggest that the sHER2 measurement may be as a potential tool for detecting HER2 expressions in GC patients. The radioisotope-labeled AHNP-PEG may be useful to apply in GC patients for HER2 nuclear medicine imaging.

The effects of cetrorelix and triptorelin on the viability and steroidogenesis of cultured human granulosa luteinized cells.

PubMed

Metallinou, Chryssa; Köster, Frank; Diedrich, Klaus; Nikolettos, Nikos; Asimakopoulos, Byron

2012-01-01

We investigated the effects of the gonadotropin-releasing hormone (GnRH) agonist triptorelin as well the GnRH antagonist cetrorelix those of on the viability and steroidogenesis in human granulosa luteinized (hGL) cell cultures. The hGL cells were obtained from 34 women undergoing ovarian stimulation for IVF treatment. The cells were cultured for 48 h with or without 1 nM or 3 nM of cetrorelix or triptorelin in serum-free media. The cell viability was evaluated by the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. The concentrations of estradiol and progesterone in culture supernatants were measured by ELISA. Treatment with triptorelin slightly increased cell viability, whereas treatment with 3 nM cetrorelix led to a significant decrease. Estradiol concentrations were reduced with 3 nM triptorelin. Cultures treated with high-dose of either cetrorelix or triptorelin tended to secrete less progesterone than controls. Cetrorelix significantly reduces the viability of hGL cells. Triptorelin and cetrorelix may have minor effects on steroidogenesis. These results suggest that GnRH analogues may influence ovarian functions.

Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model

PubMed Central

Chatterjee, Aparajita; Ratner, Daniel M.; Ryan, Christopher M.; Johnson, Patricia J.; O’Keefe, Barry R.; Secor, W. Evan; Anderson, Deborah J.; Robbins, Phillips W.; Samuelson, John

2015-01-01

Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas. PMID:26252012

Ecdysteroid-stimulated synthesis and secretion of an N-acetyl-D-glucosamine-rich glycopeptide in a lepidopteran cell line derived from imaginal discs.

PubMed

Porcheron, P; Morinière, M; Coudouel, N; Oberlander, H

1991-01-01

Hormone-regulated processing of N-acetyl-D-glucosamine was studied in an insect cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella (Hübner). The cell line, IAL-PID2, responded to treatment with 20-hydroxyecdysone with increased incorporation of GlcNAc into glycoproteins. Cycloheximide and tunicamycin counteracted the action of the hormone. In particular, treatment with 20-hydroxyecdysone resulted in the secretion of a 5,000 dalton N-acetyl-D-glucosamine-rich glycopeptide by the IAL-PID2 cells. Accumulation of this peptide was prevented by the use of teflubenzuron, a potent chitin synthesis inhibitor. A glycopeptide of similar molecular weight was observed in imaginal discs of P. interpunctella treated with 20-hydroxyecdysone in vitro, under conditions that induce chitin synthesis. Although the function of the 5,000 dalton glycopeptide is not known, we believe that the PID2 cell line is a promising model for molecular analysis of ecdysteroid-regulated processing of aminosugars by epidermal cells during insect development.

Effect of N-Acetylcysteine on Adipose-Derived Stem Cell and Autologous Fat Graft Survival in a Mouse Model.

PubMed

Gillis, Joshua; Gebremeskel, Simon; Phipps, Kyle D; MacNeil, Lori A; Sinal, Christopher J; Johnston, Brent; Hong, Paul; Bezuhly, Michael

2015-08-01

Autologous fat grafting is a popular reconstructive technique, but is limited by inconsistent graft retention. The authors examined whether a widely available, clinically safe antioxidant, N-acetylcysteine, could improve adipose-derived stem cell survival and graft take when added to tumescent solution during fat harvest. Inguinal fat pads were harvested from C57BL/6 mice using tumescent solution with or without N-acetylcysteine. Flow cytometric, proliferation, and differentiation assays were performed on isolated primary adipose-derived stem cells and 3T3-L1 preadipocytes treated with or without hydrogen peroxide and/or N-acetylcysteine. N-Acetylcysteine-treated or control grafts were injected under recipient mouse scalps and assessed by serial micro-computed tomographic volumetric analysis. Explanted grafts underwent immunohistochemical analysis. In culture, N-acetylcysteine protected adipose-derived stem cells from oxidative stress and improved cell survival following hydrogen peroxide treatment. Combined exposure to both N-acetylcysteine and hydrogen peroxide led to a 200-fold increase in adipose-derived stem cell proliferation, significantly higher than with either agent alone. N-Acetylcysteine decreased differentiation of adipose-derived stem cells into mature adipocytes, as evidenced by decreased transcription of adipocyte differentiation markers and reduced Oil Red-O staining. In vivo, N-acetylcysteine treatment resulted in improved graft retention at 3 months compared with control (46 versus 17 percent; p = 0.027). N-Acetylcysteine-treated grafts demonstrated less fibrosis and inflammation, and a 33 percent increase in adipocyte density compared with controls (p < 0.001) that was not associated with increased vascularity. These findings provide proof of principle for the addition of N-acetylcysteine to tumescent harvest solution in the clinical setting to optimize fat graft yields.

Insulin-like growth factor-1 enhances rat skeletal muscle charge movement and L-type Ca2+ channel gene expression

PubMed Central

Wang, Zhong-Min; Laura Messi, María; Renganathan, Muthukrishnan; Delbono, Osvaldo

1999-01-01

We investigated whether insulin-like growth factor-1 (IGF-1), an endogenous potent activator of skeletal muscle proliferation and differentiation, enhances L-type Ca2+ channel gene expression resulting in increased functional voltage sensors in single skeletal muscle cells. Charge movement and inward Ca2+ current were recorded in primary cultured rat myoballs using the whole-cell configuration of the patch-clamp technique. Ca2+ current and maximum charge movement (Qmax) were potentiated in cells treated with IGF-1 without significant changes in their voltage dependence. Peak Ca2+ current in control and IGF-1-treated cells was -7·8 ± 0·44 and -10·5 ± 0·37 pA pF−1, respectively (P < 0·01), whilst Qmax was 12·9 ± 0·4 and 22·0 ± 0·3 nC μF−1, respectively (P < 0·01). The number of L-type Ca2+ channels was found to increase in the same preparation. The maximum binding capacity (Bmax) of the high-affinity radioligand [3H]PN200-110 in control and IGF-1-treated cells was 1·21 ± 0·25 and 3·15 ± 0·5 pmol (mg protein)−1, respectively (P < 0·01). No significant change in the dissociation constant for [3H]PN200-110 was found. Antisense RNA amplification showed a significant increase in the level of mRNA encoding the L-type Ca2+ channel α1-subunit in IGF-1-treated cells. This study demonstrates that IGF-1 regulates charge movement and the level of L-type Ca2+ channel α1-subunits through activation of gene expression in skeletal muscle cells. PMID:10087334

Proteomic Investigation to Identify Anticancer Targets of Nemopilema nomurai Jellyfish Venom in Human Hepatocarcinoma HepG2 Cells

PubMed Central

Choudhary, Indu; Lee, Hyunkyoung; Pyo, Min Jung; Heo, Yunwi; Chae, Jinho; Yum, Seung Shic; Kang, Changkeun; Kim, Euikyung

2018-01-01

Nemopilema nomurai is a giant jellyfish that blooms in East Asian seas. Recently, N. nomurai venom (NnV) was characterized from a toxicological and pharmacological point of view. A mild dose of NnV inhibits the growth of various kinds of cancer cells, mainly hepatic cancer cells. The present study aims to identify the potential therapeutic targets and mechanism of NnV in the growth inhibition of cancer cells. Human hepatocellular carcinoma (HepG2) cells were treated with NnV, and its proteome was analyzed using two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS). The quantity of twenty four proteins in NnV-treated HepG2 cells varied compared to non-treated control cells. Among them, the amounts of fourteen proteins decreased and ten proteins showed elevated levels. We also found that the amounts of several cancer biomarkers and oncoproteins, which usually increase in various types of cancer cells, decreased after NnV treatment. The representative proteins included proliferating cell nuclear antigen (PCNA), glucose-regulated protein 78 (GRP78), glucose-6-phosphate dehydrogenase (G6PD), elongation factor 1γ (EF1γ), nucleolar and spindle-associated protein (NuSAP), and activator of 90 kDa heat shock protein ATPase homolog 1 (AHSA1). Western blotting also confirmed altered levels of PCNA, GRP78, and G6PD in NnV-treated HepG2 cells. In summary, the proteomic approach explains the mode of action of NnV as an anticancer agent. Further characterization of NnV may help to unveil novel therapeutic agents in cancer treatment. PMID:29748501

Deciphering allogeneic antibody response against native and denatured HLA epitopes in organ transplantation.

PubMed

Visentin, Jonathan; Guidicelli, Gwendaline; Moreau, Jean-François; Lee, Jar-How; Taupin, Jean-Luc

2015-07-01

Anti-HLA donor-specific antibodies are deleterious for organ transplant survival. Class I HLA donor-specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti-denatured HLA antibodies (anti-dHLAs). Anti-dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti-dHLAs to be discriminated from anti-nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid-treated (D for denaturation) and nontreated (N) LSAB, D ≥ 1.2 N identifying the anti-dHLAs. However, some anti-dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti-nHLAs conserved significant reactivity toward acid-treated LSAB. After depleting serum anti-nHLA reactivity with HLA-typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid-treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti-nHLAs and anti-dHLAs, or anti-nHLAs recognizing acid-resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti-HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti-HLA antibodies in human serum. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer.

PubMed

Jin, Jun-Xue; Li, Suo; Gao, Qing-Shan; Hong, Yu; Jin, Long; Zhu, Hai-Ying; Yan, Chang-Guo; Kang, Jin-Dan; Yin, Xi-Jun

2013-10-01

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Differences in temporal aspects of mutagenesis and cytotoxicity in Chinese hamster cells treated with methylating agents and thymidine.

PubMed Central

Peterson, A R; Peterson, H

1982-01-01

Equitoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MeMes) produced different frequencies of 8-azaguanine-resistant mutants and different amounts of N7-methylguanine, O6-methylguanine (m6G), and N3-methyladenine in the DNA of V79 Chinese hamster cells. Thus, neither the cytotoxicities nor the mutagenicities of these methylating agents could be attributed solely to nitrogen or to oxygen methylations in the DNA. However, MNNG produced 12-fold more m6G and 5-fold more mutants than did MeMes, indicating that a substantial part of the MNNG-induced mutations resulted from m6G--thymine mispairing during DNA replication. The expression as mutants of mutagenic oxygen methylations in the DNA of cells treated with MNNG was enhanced by thymidine (dThd) and deoxycytidine (dCyd), but these nucleosides did not significantly enhance MeMes-induced mutagenesis. The cytotoxicities of MNNG and MeMes were also increased by 10 microM dThd in proportion to the amount of m6G in the DNA. These increases in cytotoxicity were abolished by dCyd, which did not greatly reduce the dThd-induced enhancements of mutagenesis. Moreover, when dThd was present only during the 2-hr treatment with MNNG, maximal cytotoxicity occurred, but MNNG-induced mutagenesis was not increased. Maximal mutagenesis occurred when the dThd was present throughout the first doubling time of the MNNG-treated cells. Thus, the expression of the cytotoxicity and the mutagenicity associated with m6G in the DNA of V79 cells occurred by quite different mechanisms. PMID:6951203

Differences in temporal aspects of mutagenesis and cytotoxicity in Chinese hamster cells treated with methylating agents and thymidine.

PubMed

Peterson, A R; Peterson, H

1982-03-01

Equitoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MeMes) produced different frequencies of 8-azaguanine-resistant mutants and different amounts of N7-methylguanine, O6-methylguanine (m6G), and N3-methyladenine in the DNA of V79 Chinese hamster cells. Thus, neither the cytotoxicities nor the mutagenicities of these methylating agents could be attributed solely to nitrogen or to oxygen methylations in the DNA. However, MNNG produced 12-fold more m6G and 5-fold more mutants than did MeMes, indicating that a substantial part of the MNNG-induced mutations resulted from m6G--thymine mispairing during DNA replication. The expression as mutants of mutagenic oxygen methylations in the DNA of cells treated with MNNG was enhanced by thymidine (dThd) and deoxycytidine (dCyd), but these nucleosides did not significantly enhance MeMes-induced mutagenesis. The cytotoxicities of MNNG and MeMes were also increased by 10 microM dThd in proportion to the amount of m6G in the DNA. These increases in cytotoxicity were abolished by dCyd, which did not greatly reduce the dThd-induced enhancements of mutagenesis. Moreover, when dThd was present only during the 2-hr treatment with MNNG, maximal cytotoxicity occurred, but MNNG-induced mutagenesis was not increased. Maximal mutagenesis occurred when the dThd was present throughout the first doubling time of the MNNG-treated cells. Thus, the expression of the cytotoxicity and the mutagenicity associated with m6G in the DNA of V79 cells occurred by quite different mechanisms.

Nitric oxide donors attenuate clongenic potential in rat C6 glioma cells treated with alkylating chemotherapeutic agents.

PubMed

Yang, Jir-Jei; Yin, Jiu-Haw; Yang, Ding-I

2007-05-11

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) kills tumor cells via multiple actions including alkylation and carbamoylation. Previously, we have reported that formation of S-nitrosoglutathione (GSNO) in glioma cells overexpressing inducible nitric oxide synthase (iNOS) contributed to nitric oxide (NO)-dependent carbamoylating chemoresistance against BCNU. To further characterize the effects of NO on alkylating cytotoxicity, colony formation assay was applied to evaluate the effects of various NO donors on rat C6 glioma cells challenged with alkylating agents. We demonstrate that NO donors including GSNO, diethylamine NONOate (DEA/NO), and sodium nitroprusside (SNP) substantially reduced the extent of colony formation in glioma cells treated with alkylating agents, namely methyl methanesulfonate (MMS), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU). Without alkylating agents these NO-releasing agents alone had no effects on clongenic potential of rat C6 glioma cells. Among these three NO donors used, the effectiveness in potentiating alkylating cytotoxicity is in the order of "GSNO>DEA/NO>SNP" when applied at the same dosages. GSNO also exerted similar synergistic actions reducing the extents of colony formation when co-administrated with 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-hydrazine (compound #1), another alkylating agent that mimics the chloroethylating action of BCNU. Together with our previous findings, we propose that NO donors may be used as adjunct chemotherapy with alkylating agents for such malignant brain tumors as glioblastoma multiforme (GBM). In contrast, production of NO as a result of iNOS induction, such as that occurring after surgical resection of brain tumors, may compromise the efficacy of carbamoylating chemotherapy.

N-Acetylcysteine Increases Corneal Endothelial Cell Survival in a Mouse Model of Fuchs Endothelial Corneal Dystrophy

PubMed Central

Kim, Eun Chul; Meng, Huan; Jun, Albert S.

2014-01-01

The present study evaluated survival effects of N-acetylcysteine (NAC) on cultured corneal endothelial cells exposed to oxidative and endoplasmic reticulum (ER) stress and in a mouse model of early-onset Fuchs endothelial corneal dystrophy (FECD). Cultured bovine corneal endothelial cell viability against oxidative and ER stress was determined by CellTiter-Glo® luminescent reagent. Two-month-old homozygous knock-in Col8a2L450W/L450W mutant (L450W) and C57/Bl6 wild-type (WT) animals were divided into two groups of 15 mice. Group I received 7 mg/mL NAC in drinking water and Group II received control water for 7 months. Endothelial cell density and morphology were evaluated with confocal microscopy. Antioxidant gene (iNos) and ER stress/unfolded protein response gene (Grp78 and Chop) mRNA levels and protein expression were measured in corneal endothelium by real time PCR and Western blotting. Cell viability of H2O2 and thapsigargin exposed cells pre-treated with NAC was significantly increased compared to untreated controls (pitalic>0.01). Corneal endothelial cell density (CD) was higher (p=0.001) and percent polymegathism was lower (p=0.04) in NAC treated L450W mice than in untreated L450W mice. NAC treated L450W endothelium showed significant upregulation of iNos, whereas Grp78 and Chop were downregulated compared to untreated L450W endothelium by real time PCR and Western blotting. NAC increases survival in cultured corneal endothelial cells exposed against ER and oxidative stress. Systemic NAC ingestion increases corneal endothelial cell survival which is associated with increased antioxidant and decreased ER stress markers in a mouse model of early-onset FECD. Our study presents in vivo evidence of a novel potential medical treatment for FECD. PMID:24952277

N-Acetylcysteine increases corneal endothelial cell survival in a mouse model of Fuchs endothelial corneal dystrophy.

PubMed

Kim, Eun Chul; Meng, Huan; Jun, Albert S

2014-10-01

The present study evaluated survival effects of N-acetylcysteine (NAC) on cultured corneal endothelial cells exposed to oxidative and endoplasmic reticulum (ER) stress and in a mouse model of early-onset Fuchs endothelial corneal dystrophy (FECD). Cultured bovine corneal endothelial cell viability against oxidative and ER stress was determined by CellTiter-Glo(®) luminescent reagent. Two-month-old homozygous knock-in Col8a2(L450W/L450W) mutant (L450W) and C57/Bl6 wild-type (WT) animals were divided into two groups of 15 mice. Group I received 7 mg/mL NAC in drinking water and Group II received control water for 7 months. Endothelial cell density and morphology were evaluated with confocal microscopy. Antioxidant gene (iNos) and ER stress/unfolded protein response gene (Grp78 and Chop) mRNA levels and protein expression were measured in corneal endothelium by real time PCR and Western blotting. Cell viability of H2O2 and thapsigargin exposed cells pre-treated with NAC was significantly increased compared to untreated controls (p < 0.01). Corneal endothelial cell density (CD) was higher (p = 0.001) and percent polymegathism was lower (p = 0.04) in NAC treated L450W mice than in untreated L450W mice. NAC treated L450W endothelium showed significant upregulation of iNos, whereas Grp78 and Chop were downregulated compared to untreated L450W endothelium by real time PCR and Western blotting. NAC increases survival in cultured corneal endothelial cells exposed against ER and oxidative stress. Systemic NAC ingestion increases corneal endothelial cell survival which is associated with increased antioxidant and decreased ER stress markers in a mouse model of early-onset FECD. Our study presents in vivo evidence of a novel potential medical treatment for FECD. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of lapatinib on the outgrowth of metastatic breast cancer cells to the brain.

PubMed

Gril, Brunilde; Palmieri, Diane; Bronder, Julie L; Herring, Jeanne M; Vega-Valle, Eleazar; Feigenbaum, Lionel; Liewehr, David J; Steinberg, Seth M; Merino, Maria J; Rubin, Stephen D; Steeg, Patricia S

2008-08-06

The brain is increasingly being recognized as a sanctuary site for metastatic tumor cells in women with HER2-overexpressing breast cancer who receive trastuzumab therapy. There are no approved or widely accepted treatments for brain metastases other than steroids, cranial radiotherapy, and surgical resection. We examined the efficacy of lapatinib, an inhibitor of the epidermal growth factor receptor (EGFR) and HER2 kinases, for preventing the outgrowth of breast cancer cells in the brain in a mouse xenograft model of brain metastasis. EGFR-overexpressing MDA-MB-231-BR (231-BR) brain-seeking breast cancer cells were transfected with an expression vector that contained or lacked the HER2 cDNA and used to examine the effect of lapatinib on the activation (ie, phosphorylation) of cell signaling proteins by immunoblotting, on cell growth by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and on cell migration using a Boyden chamber assay. The outgrowth of large (ie, >50 microm(2)) and micrometastases was counted in brain sections from nude mice that had been injected into the left cardiac ventricle with 231-BR cells and, beginning 5 days later, treated by oral gavage with lapatinib or vehicle (n = 22-26 mice per treatment group). All statistical tests were two-sided. In vitro, lapatinib inhibited the phosphorylation of EGFR, HER2, and downstream signaling proteins; cell proliferation; and migration in 231-BR cells (both with and without HER2). Among mice injected with 231-BR-vector cells, those treated with 100 mg lapatinib/kg body weight had 54% fewer large metastases 24 days after starting treatment than those treated with vehicle (mean number of large metastases per brain section: 1.56 vs 3.36, difference = 1.80, 95% confidence interval [CI] = 0.92 to 2.68, P < .001), whereas treatment with 30 mg lapatinib/kg body weight had no effect. Among mice injected with 231-BR-HER2 cells, those treated with either dose of lapatinib had 50%-53% fewer large metastases than those treated with vehicle (mean number of large metastases per brain section, 30 mg/kg vs vehicle: 3.21 vs 6.83, difference = 3.62, 95% CI = 2.30 to 4.94, P < .001; 100 mg/kg vs vehicle: 3.44 vs 6.83, difference = 3.39, 95% CI = 2.08 to 4.70, P < .001). Immunohistochemical analysis revealed reduced phosphorylation of HER2 in 231-BR-HER2 cell-derived brain metastases from mice treated with the higher dose of lapatinib compared with 231-BR-HER2 cell-derived brain metastases from vehicle-treated mice (P < .001). Lapatinib is the first HER2-directed drug to be validated in a preclinical model for activity against brain metastases of breast cancer.

Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Meng; He, Hong-wei; Sun, Huan-xing

2009-09-18

Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, wemore » evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.« less

Use of Adipose Derived Stem Cells to Treat Large Bone Defects

DTIC Science & Technology

2009-03-01

and activates Sirt1 , which inhibit adipogenesis and induce apoptosis in adipocytes. This suggested that resveratrol could reduce the number of...5000 cells/cm2 with either growth media (GM) or osteogenic media (OM) consisting of GM supplemented with 1nM dexamethasone, 3mM beta-glycerophosphate

4-Methoxyestradiol-induced oxidative injuries in human lung epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng Yahsin; Chang, Louis W.; Cheng Lichuan

2007-05-01

Epidemiological studies indicated that people exposed to dioxins were prone to the development of lung diseases including lung cancer. Animal studies demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased liver tumors and promoted lung metaplasia in females. Metabolic changes in 17{beta}-estradiol (E{sub 2}) resulted from an interaction between TCDD and E{sub 2} could be associated with gender difference. Previously, we reported that methoxylestradiols (MeOE{sub 2}), especially 4-MeOE{sub 2}, accumulated in human lung cells (BEAS-2B) co-treated with TCDD and E{sub 2}. In the present study, we demonstrate unique accumulation of 4-MeOE{sub 2}, as a result of TCDD/E{sub 2} interaction and revealed its bioactivity inmore » human lung epithelial cell line (H1355). 4-Methoxyestradiol treatment significantly decreased cell growth and increased mitotic index. Elevation of ROS and SOD activity, with a concomitant decrease in the intracellular GSH/GSSG ratio, was also detected in 4-MeOE{sub 2}-treated cells. Quantitative comet assay showed increased oxidative DNA damage in the 4-MeOE{sub 2}-treated H1355 cells, which could be significantly reduced by the anti-oxidant N-acetylcysteine (NAC). However, inhibition of cell growth and increase in mitotic arrest induced by 4-MeOE{sub 2} were unaffected by NAC. We concluded that 4-MeOE{sub 2} accumulation resulting from TCDD and E{sub 2} interaction would contribute to the higher vulnerability on lung pathogenesis in females when exposed to TCDD.« less

Laminar distribution of GABAA- and glycine-receptor mediated tonic inhibition in the dorsal horn of the rat lumbar spinal cord: effects of picrotoxin and strychnine on expression of Fos-like immunoreactivity.

PubMed

Cronin, John N; Bradbury, Elizabeth J; Lidierth, Malcolm

2004-11-01

Inhibitory mechanisms are essential in suppressing the development of allodynia and hyperalgesia in the normal animal and there is evidence that loss of inhibition can lead to the development of neuropathic pain. We used Fos expression to map the distribution of tonically inhibited cells in the healthy rat lumbar spinal cord. In a control group, Fos-like immunoreactive (Fos-LI) cells were rare, averaging 7.5+/-2.2 cells (mean+/-SEM; N=13 sections) per 20 microm thick section of dorsal horn. This rose to 103+/-11 (mean+/-SEM; N=20) in picrotoxin-treated rats and to 88+/-11 (mean+/-SEM; N=18) in strychnine-treated rats. These changes were significant (ANOVA; P<0.001). There were marked regional variations in the distribution of Fos-LI cells between picrotoxin- and strychnine-treated animals. Picrotoxin induced a significant increase in the number of Fos-LI cells throughout the dorsal horn (lamina I-VI) while strychnine significantly elevated Fos-like immunoreactivity only in deep laminae (III-VI). For both picrotoxin and strychnine, the increase in Fos-like immunoreactivity peaked in lamina V (at 3579+/-319 and 3649+/-375% of control, respectively; mean+/-SEM) but for picrotoxin an additional peak was observed in the outer part of lamina II (1959+/-196%). Intrathecal administration of both GABAA and glycine receptor antagonists has been shown elsewhere to induce tactile allodynia. The present data suggest that this allodynia could arise due to blockade of tonic GABAA and glycine-receptor mediated inhibition in the deep dorsal horn. GABAA antagonists also induce hypersensitivity to noxious inputs. The blockade of tonic inhibition in the superficial dorsal horn shown here may underlie this hyperalgesia.

Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes in Advanced Melanoma Patients

PubMed Central

Saint-Jean, Mélanie; Volteau, Christelle; Quéreux, Gaëlle; Peuvrel, Lucie; Brocard, Anabelle; Saiagh, Soraya; Nguyen, Jean-Michel; Bedane, Christophe; Basset-Seguin, Nicole

2018-01-01

Immunotherapy for melanoma includes adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TILs). This monocenter retrospective study was undertaken to evaluate the efficacy and safety of this treatment of patients with advanced melanoma. All advanced melanoma patients treated with TILs using the same TIL expansion methodology and same treatment interleukin-2 (IL-2) regimen between 2009 and 2012 were included. After sterile intralesional excision of a cutaneous or subcutaneous metastasis, TILs were produced according to a previously described method and then infused into the patient who also received a complementary subcutaneous IL-2 regimen. Nine women and 1 man were treated for unresectable stage IIIC (n = 4) or IV (n = 6) melanoma. All but 1 patient with unresectable stage III melanoma (1st line) had received at least 2 previous treatments, including anti-CTLA-4 antibody for 4. The number of TILs infused ranged from 0.23 × 109 to 22.9 × 109. Regarding safety, no serious adverse effect was reported. Therapeutic responses included a complete remission, a partial remission, 2 stabilizations, and 6 progressions. Among these 4 patients with clinical benefit, 1 is still alive with 9 years of follow-up and 1 died from another cause after 8 years of follow-up. Notably, patients treated with high percentages of CD4 + CD25 + CD127lowFoxp3+ T cells among their TILs had significantly shorter OS. The therapeutic effect of combining TILs with new immunotherapies needs further investigation. PMID:29750176

Proton induces apoptosis of hypoxic tumor cells by the p53-dependent and p38/JNK MAPK signaling pathways.

PubMed

Lee, Kheun Byeol; Kim, Kye-Ryung; Huh, Tae-Lin; Lee, You Mie

2008-12-01

Tumor hypoxia is a main obstacle for radiation therapy. To investigate whether exposure to a proton beam can overcome radioresistance in hypoxic tumor cells, three kinds of cancer cells, Lewis lung carcinoma (LLC) cells, hepatoma HepG2 and Molt-4 leukemia cells, were treated with a proton beam (35 MeV, 1, 2, 5, 10 Gy) in the presence or absence of hypoxia. Cell death rates were determined 72 h after irradiation. Hypoxic cells exposed to the proton beam underwent a typical apoptotic program, showing condensed nuclei, fragmented DNA ladders, and poly-ADP-ribose polymerase (PARP) cleavage. Fluorescence-activated cell sorter analysis revealed a significant increase in Annexin-V-positive cells. Cells treated with the proton beam and hypoxia displayed increased expression of p53, p21 and Bax, but decreased levels of phospho-Rb, Bcl-2 and XIAP, as well as activated caspase-9 and -3. The proton beam with hypoxia induced cell death in wild-type HCT116 cells, but not in a p53 knockout cell line, demonstrating a requirement for p53. As reactive oxygen species (ROS) were also significantly increased, apoptosis could also be abolished by treatment with the anti-oxidant N-acetyl cysteine (NAC). P38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) were activated by the treatment, and their respective DN mutants restored the cell death induced by either proton therapy alone or with hypoxia. In conclusion, proton beam treatment did not differently regulate cancer cell apoptosis either in normoxic or hypoxic conditions via a p53-dependent mechanism and by the activation of p38/JNK MAPK pathways through ROS.

Treatment with specific soluble factors promotes the functional maturation of transcription factor-mediated, pancreatic transdifferentiated cells.

PubMed

Motoyama, Hiroaki; Kobayashi, Akira; Yokoyama, Takahide; Shimizu, Akira; Sakai, Hiroshi; Notake, Tsuyoshi; Fukushima, Kentaro; Miyagawa, Shin-Ichi

2018-01-01

Pancreatic lineage-specific transcription factors (TFs) display instructive roles in converting adult cells to endocrine pancreatic cells through a process known as transdifferentiation. However, little is known about potential factors capable of accelerating transdifferentiation following transduction to achieve the functional maturation of transdifferentiated cells. In this study, we demonstrated, using adult liver-derived progenitor cells, that soluble factors utilized in pancreatic differentiation protocols of pluripotent stem cells promote functional maturation of TFs-mediated transdifferentiated cells. Treatment with an N2 supplement in combination with three soluble factors (glucagon-like peptide-1 [GLP-1] receptor agonist, notch inhibitor, and transforming growth factor-β [TGF-β] inhibitor) enhanced liver-to-pancreas transdifferentiation based on the following findings: i) the incidence of c-peptide-positive cells increased by approximately 1.2-fold after the aforementioned treatment; ii) the c-peptide expression level in the treated cells increased by approximately 12-fold as compared with the level in the untreated cells; iii) the treated cells secreted insulin in a glucose-dependent manner, whereas the untreated cells did not; and iv) transplantation of treated-transdifferentiated cells into streptozotocin-induced immunodeficient diabetic mice led to the amelioration of hyperglycemia. These results suggest that treatment with specific soluble factors promotes the functional maturation of transdifferentiated cells. Our findings could facilitate the development of new modalities for cell-replacement therapy for patients with diabetes.

The α7-nicotinic acetylcholine receptor mediates the sensitivity of gastric cancer cells to taxanes.

PubMed

Tu, Chao-Chiang; Huang, Chien-Yu; Cheng, Wan-Li; Hung, Chin-Sheng; Uyanga, Batzorig; Wei, Po-Li; Chang, Yu-Jia

2016-04-01

Gastric cancer is difficult to cure because most patients are diagnosed at an advanced disease stage. Systemic chemotherapy remains an important therapy for gastric cancer, but both progression-free survival and disease-free survival associated with various combination regimens are limited because of refractoriness and chemoresistance. Accumulating evidence has revealed that the homomeric α7-nicotinic acetylcholine receptor (A7-nAChR) promotes human gastric cancer by driving cancer cell proliferation, migration, and metastasis. Therefore, A7-nAChR may serve as a potential therapeutic target for gastric cancer. However, the role of A7-nAChR in taxane therapy for gastric cancer was unclear. Cells were subjected to A7-nAChR knockdown (A7-nAChR KD) using short interfering RNA (siRNA). The anti-proliferative effects of taxane were assessed via 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL), and cell cycle distribution assays. A7-nAChR-KD cells exhibited low resistance to docetaxel and paclitaxel treatment, as measured by the MTT assay. Following paclitaxel treatment, the proportion of apoptotic cells was higher among A7-nAChR-KD cells than among scrambled control cells, as measured by cell cycle distribution and TUNEL assays. Further molecular analyses showed a reduction in the pAKT levels and a dramatic increase in the Bad levels in paclitaxel-treated A7-nAChR-KD cells but not in scrambled control cells. Following paclitaxel treatment, the level of Bax was slightly increased in both cell populations, whereas Poly (ADP-ribose) polymerase (PARP) cleavage was increased only in A7-nAChR-KD cells. These findings indicate that A7-nAChR-KD cells are more sensitive to paclitaxel treatment. We conclude that A7-nAChR may be a key biomarker for assessing the chemosensitivity of gastric cancer cells to taxane.

Multicenter study of banked third-party virus-specific T cells to treat severe viral infections after hematopoietic stem cell transplantation

PubMed Central

Leen, Ann M.; Bollard, Catherine M.; Mendizabal, Adam M.; Shpall, Elizabeth J.; Szabolcs, Paul; Antin, Joseph H.; Kapoor, Neena; Pai, Sung-Yun; Rowley, Scott D.; Kebriaei, Partow; Dey, Bimalangshu R.; Grilley, Bambi J.; Gee, Adrian P.; Brenner, Malcolm K.; Rooney, Cliona M.; Heslop, Helen E.

2013-01-01

Virus-specific T cell (VST) lines could provide useful antiviral prophylaxis and treatment of immune-deficient patients if it were possible to avoid the necessity of generating a separate line for each patient, often on an emergency basis. We prepared a bank of 32 virus-specific lines from individuals with common HLA polymorphisms who were immune to Epstein-Barr virus (EBV), cytomegalovirus, or adenovirus. A total of 18 lines were administered to 50 patients with severe, refractory illness because of infection with one of these viruses after hematopoietic stem cell transplant. The cumulative rates of complete or partial responses at 6 weeks postinfusion were 74.0% (95% CI, 58.5%-89.5%) for the entire group (n = 50), 73.9% (95% CI, 51.2% -96.6%) for cytomegalovirus (n = 23), 77.8% for adenovirus (n = 18), and 66.7% (95% CI, 36.9%-96.5%) for EBV (n = 9). Only 4 responders had a recurrence or progression. There were no immediate infusion-related adverse events, and de novo graft-versus-host disease developed in only 2 patients. Despite the disparity between the lines and their recipients, the mean frequency of VSTs increased significantly postinfusion, coincident with striking decreases in viral DNA and resolution of clinical symptoms. The use of banked third-party VSTs is a feasible and safe approach to rapidly treat severe or intractable viral infections after stem cell transplantation. This study is registered at www.clinicaltrials.gov as NCT00711035. PMID:23610374

Suppression of ErbB-2 in androgen-independent human prostate cancer cells enhances cytotoxic effect by gemcitabine in an androgen-reduced environment.

PubMed

Zhang, Li; Davis, Jeffrey S; Zelivianski, Stanislav; Lin, Fen-Fen; Schutte, Rachel; Davis, Thomas L; Hauke, Ralph; Batra, Surinder K; Lin, Ming-Fong

2009-11-18

We examined the efficacy of combination treatments utilizing cytotoxic drugs plus inhibitors to members of the ErbB-ERK signal pathway in human prostate cancer (PCa) LNCaP C-81 cells. Under an androgen-reduced condition, 50nM gemcitabine caused about 40% growth suppression on C-81 cells. Simultaneous treatment of gemcitabine plus 10microM AG825 produced 60% suppression (p<0.03); while, 85% growth inhibition (p<0.02) was seen if AG825 was added to gemcitabine-treated cells after a 24h-interval. Our data thus showed that in androgen-reduced conditions, inhibition of ErbB-2 increases the cytotoxic efficacy of gemcitabine in PCa cells. This finding has significant implications in the choice of drugs for combination therapy as well as the order of administration for treating cancer patients.

Androgen deprivation results in time-dependent hypoxia in LNCaP prostate tumours: informed scheduling of the bioreductive drug AQ4N improves treatment response.

PubMed

Ming, Louise; Byrne, Niall M; Camac, Sarah Nicole; Mitchell, Christopher A; Ward, Claire; Waugh, David J; McKeown, Stephanie R; Worthington, Jenny

2013-03-15

Androgen withdrawal induces hypoxia in androgen-sensitive tissue; this is important as in the tumour microenvironment, hypoxia is known to drive malignant progression. Our study examined the time-dependent effect of androgen deprivation therapy (ADT) on tumour oxygenation and investigated the role of ADT-induced hypoxia on malignant progression in prostate tumours. LNCaP xenografted tumours were treated with anti-androgens and tumour oxygenation measured. Dorsal skin fold (DSF) chambers were used to image tumour vasculature in vivo. Quantitative PCR (QPCR) identified differential gene expression following treatment with bicalutamide. Bicalutamide-treated and vehicle-only-treated tumours were re-established in vitro, and invasion and sensitivity to docetaxel were measured. Tumour growth delay was calculated following treatment with bicalutamide combined with the bioreductive drug AQ4N. Tumour oxygenation measurements showed a precipitate decrease following initiation of ADT. A clinically relevant dose of bicalutamide (2 mg/kg/day) decreased tumour oxygenation by 45% within 24 hr, reaching a nadir of 0.09% oxygen (0.67 ± 0.06 mmHg) by Day 7; this persisted until Day 14 when it increased up to Day 28. Using DSF chambers, LNCaP tumours treated with bicalutamide showed loss of small vessels at Days 7 and 14 with revascularisation occurring by Day 21. QPCR showed changes in gene expression consistent with the vascular changes and malignant progression. Cells from bicalutamide-treated tumours were more malignant than vehicle-treated controls. Combining bicalutamide with AQ4N (50 mg/kg, single dose) caused greater tumour growth delay than bicalutamide alone. Our study shows that bicalutamide-induced hypoxia selects for cells that show malignant progression; targeting hypoxic cells may provide greater clinical benefit. Copyright © 2012 UICC.

Inferior progression-free survival for Thai patients with diffuse large B-cell lymphoma treated under Universal Coverage Scheme: the impact of rituximab inaccessability.

PubMed

Intragumtornchai, Tanin; Bunworasate, Udomsak; Siritanaratkul, Noppadol; Khuhapinant, Archrob; Nawarawong, Weerasak; Norasetthada, Lalita; Lekhakula, Arnuparp; Rujirojindakul, Pairaya; Sirijerachai, Chittima; Chansung, Kanjana; Suwanban, Tawatchai; Chuncharunee, Suporn; Niparuck, Pimjai; Wongkhantee, Somchai; Mongkonsritragoon, Wichean; Numbenjapon, Tontanai

2013-01-01

The impact of health insurance with inequitable rituximab coverage on the survival of patients with diffuse large B-cell lymphoma (DLBCL) has never been reported. We conducted a nationwide multicenter analysis on the outcome of 553 adult patients consecutively diagnosed with DLBCL between July 2003 and June 2006, in whom treatment cost was reimbursed under the Civil Servant Medical Benefit Scheme (CSMBS) (n =201) or the Universal Coverage Scheme (UCS) (n =352). The international prognostic index was comparable between the two payment groups. Rituximab-based therapy was administered in 45.3% and 3.1% of CSMBS and UCS patients, respectively (p <0.001). With a median follow-up of 24.6 months, the 6-year progression-free survival (PFS) was superior for CSMBS patients (34.2 vs. 23.2%, p =0.005). "Not treated with rituximab-based therapy" was the strongest adverse prognostic feature indicating a short PFS (hazard ratio 2.1, p <0.001). It is concluded that lack of access to rituximab is the principal factor accounting for the inferior PFS observed in Thai patients with DLBCL who are treated under the UCS.

Effects of phenobarbital on aniline metabolism in primary liver cell culture of rats with ethionine-induced liver disorder.

PubMed

Noguchi, M; Nitoh, S; Mabuchi, M; Kawai, Y

1996-04-01

In experiment 1, the amount of aniline (AN) metabolites in the primary cell culture medium of the liver cells obtained from ethionine (ET)-treated rats was compared with that of the control (normal) rats. Although the metabolites detected in both groups were p-aminophenol (p-AP), N-acetyl-p-AP (AAP), acetoanilide (AAN), AAP-glucuronide (AAPG), phenylhydroxylamine sulfate (PHAS) and p-AP-glucuronide (p-APG), the amount of AAP was lower and that of p-APG was markedly higher in the ET-treated rats than in the control rats. In experiment 2, phenobarbital (PB) was orally administered to the ET-treated and control rats at a dose of 100 mg/kg. The time course changes in AN metabolites in the primary cell culture medium of liver cells obtained at 2 or 48 hr after PB treatment were compared with those without PB treatment. In the ET-treated rats, the amount of PHAS was slightly higher at 2 hr after PB treatment, and that of AAP was lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment. In the control rats, the amounts of AAP, AAN, p-AP and p-APG at 2 hr after PB treatment remained lower than those without PB treatment, and that of AAP was markedly lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment. These findings indicated greater detoxication in the primary liver cell culture in the ET-treated rats than in the control rats. Furthermore, detoxication was greater in the primary cell culture of liver cell obtained from the ET-treated rats after PB treatment than from those without PB treatment, because the production of acetylates (AAP) decreased and p-APG increased (induction of conjugated enzyme) in the PB treatment group.

Pre-emptive and therapeutic adoptive immunotherapy for nasopharyngeal carcinoma: Phenotype and effector function of T cells impact on clinical response.

PubMed

Smith, Corey; Lee, Victor; Schuessler, Andrea; Beagley, Leone; Rehan, Sweera; Tsang, Janice; Li, Vivian; Tiu, Randal; Smith, David; A Neller, Michelle; Matthews, Katherine K; Gostick, Emma; Price, David A; Burrows, Jacqueline; Boyle, Glen M; Chua, Daniel; Panizza, Benedict; Porceddu, Sandro V; Nicholls, John; Kwong, Dora; Khanna, Rajiv

2017-01-01

Adoptive T cell therapy has emerged as a powerful strategy to treat human cancers especially haematological malignancies. Extension of these therapies to solid cancers remains a significant challenge especially in the context of defining immunological correlates of clinical responses. Here we describe results from a clinical study investigating autologous Epstein-Barr virus (EBV)-specific T cells generated using a novel AdE1-LMPpoly vector to treat patients with nasopharyngeal carcinoma (NPC) either pre-emptively in at-risk patients with no or minimal residual disease (N/MRD) or therapeutically in patients with active recurrent/metastatic disease (ARMD). Tolerability, safety and efficacy, including progression-free survival (PFS) and overall survival (OS), were evaluated following adoptive T-cell immunotherapy. Twenty-nine patients, including 20 with ARMD and nine with N/MRD, successfully completed T-cell therapy. After a median follow-up of 18.5 months, the median PFS was 5.5 months (95% CI 2.1 to 9.0 months) and the median OS was 38.1 months (95% CI 17.2 months to not reached). Post-immunotherapy analyses revealed that disease stabilization in ARMD patients was significantly associated with the functional and phenotypic composition of in vitro -expanded T cell immunotherapy. These included a higher proportion of effector CD8 + T-cells and an increased number of EBV-specific T-cells with broader antigen specificity. These observations indicate that adoptive immunotherapy with AdE1-LMPpoly-expanded T cells stabilizes relapsed, refractory NPC without significant toxicity. Promising clinical outcomes in N/MRD patients further suggest a potential role for this approach as a consolidation treatment following first-line chemotherapy.

Association of Vedolizumab Level, Anti-Drug Antibodies, and α4β7 Occupancy With Response in Patients With Inflammatory Bowel Diseases.

PubMed

Ungar, Bella; Kopylov, Uri; Yavzori, Miri; Fudim, Ella; Picard, Orit; Lahat, Adi; Coscas, Daniel; Waterman, Matti; Haj-Natour, Ola; Orbach-Zingboim, Noam; Mao, Ren; Chen, Minhu; Chowers, Yehuda; Eliakim, Rami; Ben-Horin, Shomron

2018-05-01

There are few data available on the real-life pharmacokinetic and pharmacodynamics features of vedolizumab, a monoclonal antibody against integrin α4β7. We performed a prospective study of patients with inflammatory bowel diseases (IBDs) treated with vedolizumab to determine serum drug concentrations, formation of antivedolizumab antibodies (AVAs), and integrin α4β7 saturation. We performed a prospective study of 106 patients with IBD (67 with Crohn's disease and 39 with ulcerative colitis) treated with vedolizumab from September 2014 through March 2017 at 2 tertiary medical centers in Israel. Clinical data and serum samples were collected before and during induction and maintenance therapy. Clinical remission was defined as Harvey-Bradshaw index scores below 5 or as Simple Clinical Colitis Activity Index scores of 3 or less. We measured serum levels of vedolizumab, AVAs, and markers of inflammation. Peripheral blood mononuclear cells were obtained from some patients at designated trough time points and CD3+ CD45RO+ T cells were isolated from 36 samples. Cells were incubated with fluorescent-conjugated vedolizumab and flow cytometry was used to quantify α4β7 integrin saturation. We also performed flow cytometry analyses of CD3+ CD45RO+ lamina propria T cells isolated from intestinal mucosa of patients without IBD (non-IBD controls, n = 6), patients with IBD not treated with vedolizumab (untreated IBD controls, n = 8), and patients with IBD treated with vedolizumab (n = 15). Clinical remission was achieved by 48 of 106 patients (45%) by week 6 and 50 of 106 patients (48%) by week 14 of treatment. The median level of vedolizumab at week 6 was higher in patients in clinical remission (40.2 μg/mL) than in patients with active disease (29.7 μg/mL; P = .05). The median serum level of vedolizumab was significantly higher in patients with a normal level of C-reactive protein (21.8 μg/mL vedolizumab) vs the level in those with a high level of C-reactive protein (11.9 μg/mL vedolizumab) during maintenance treatment (P = .0006). The other clinical outcomes measured were not associated with median serum level of vedolizumab at any time point examined. AVAs were detected in 17% of patients during induction therapy and 3% of patients during maintenance therapy, but did not correlate with clinical outcomes. Flow-cytometry analysis of peripheral blood memory T cells (n = 36) showed near-complete occupancy of α4β7 integrin at weeks 2 and 14 and during the maintenance phase, regardless of response status or drug levels. Most intestinal CD3+CD45RO+ memory T cells of healthy and IBD controls expressed α4β7 (72%; interquartile range, 56%-81%). In contrast, free α4β7 was detectable on only 5.6% of intestinal memory cells (interquartile range, 4.4%-11.2%) (P < .0001) from vedolizumab-treated patients, regardless of response. In a prospective study of real-life patients with IBD, we associated vedolizumab drug levels with remission and inflammatory marker level. Integrin α4β7 was blocked in almost all T cells from patients treated with vedolizumab, regardless of serum level of the drug or response to treatment. These findings indicate a need to explore alternative mechanisms that prevent response to vedolizumab. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Minocycline Blocks Asthma-associated Inflammation in Part by Interfering with the T Cell Receptor-Nuclear Factor κB-GATA-3-IL-4 Axis without a Prominent Effect on Poly(ADP-ribose) Polymerase*

PubMed Central

Naura, Amarjit S.; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C.; Jordan, Joaquin; Catling, Andrew D.; Rezk, Bashir M.; Elmageed, Zakaria Y. Abd; Pyakurel, Kusma; Tarhuni, Abdelmetalab F.; Abughazleh, Mohammad Q.; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C.; Boulares, A. Hamid

2013-01-01

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N′-nitro-N-nitroso-guanidine-treated mice or H2O2-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production. PMID:23184953

Effect of aqueous and lipophilic mullet (Mugil cephalus) Bottarga extracts on the growth and lipid profile of intestinal Caco-2 cells.

PubMed

Rosa, Antonella; Atzeri, Angela; Deiana, Monica; Melis, M Paola; Loru, Debora; Incani, Alessandra; Cabboi, Barbara; Dessì, M Assunta

2011-03-09

The importance of n-3 polyunsaturated fatty acid (n-3 PUFA) intake has long been recognized in human nutrition. Although health benefits, n-3 PUFA are subject to rapid and/or extensive oxidation during processing and storage, resulting in potential alteration in nutritional composition and quality of food. Bottarga, a salted and semi-dried mullet ( Mugil cephalus ) ovary product, is proposed as an important source of n-3 PUFA, having high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we investigated the extent of lipid oxidation of grated bottarga samples during 7 months of storage at -20 °C and room temperature under light exposure. Cell viability, lipid composition, and lipid peroxidation were measured in intestinal differentiated Caco-2 cell monolayers after 6-48 h of incubation with lipid and hydrophilic extracts obtained from bottarga samples at different storage conditions. The storage of bottarga did not affect the n-3 PUFA level, but differences were observed in hydroperoxide levels in samples from different storage conditions. All tested bottarga extracts did not show a toxic effect on cell viability of differentiated Caco-2 cells. Epithelial cells incubated with bottarga oil had significant changes in fatty acid composition but not in cholesterol levels with an accumulation of EPA, DHA, and 22:5. Cell hydroperoxides were higher in treated cells, in relation to the oxidative status of bottarga oil. Moreover, the bottarga lipid extract showed an in vitro inhibitory effect on the growth of a colon cancer cell line (undifferentiated Caco-2 cells).

Recurrence Patterns and Second Primary Lung Cancers After Stereotactic Body Radiation Therapy for Early-Stage Non-Small-Cell Lung Cancer: Implications for Surveillance.

PubMed

Spratt, Daniel E; Wu, Abraham J; Adeseye, Victoria; Din, Shaun U; Shaikh, Fauzia; Woo, Kaitlin M; Zhang, Zhigang; Foster, Amanda; Rosenzweig, Kenneth E; Gewanter, Richard; Huang, James; Rimner, Andreas

2016-05-01

Patients treated with stereotactic body radiation therapy (SBRT) for early-stage non-small-cell lung cancer (NSCLC) are subject to locoregional and distant recurrence, as well as the formation of second primary lung cancers (SPLCs). The optimal surveillance regimen for patients treated with SBRT for early-stage NSCLC remains unclear; we therefore investigated the posttreatment recurrence patterns and development of SPLCs. Three hundred sixty-six patients with pathologically proven inoperable early-stage NSCLC treated with SBRT between 2006 and 2013 were assessed. Patients underwent a computed tomographic (CT) scan of the chest every 3 months during years 1 and 2, every 6 months during years 3 and 4, and annually thereafter. Competing risk analysis was used for all time-to-event analyses. With a median follow-up of 23 months, the 2-year cumulative incidence of local, nodal, and distant treatment failures were 12.2%, 16.1%, and 15.5%, respectively. In patients with disease progression after SBRT (n = 108), 84% (n = 91) of cases occurred within the first 2 years. Five percent (n = 19) of patients experienced SPLCs. The median time to development of an SPLC was 16.5 months (range, 6.5-71.1 months), with 33% (n = 6) of these patients experiencing SPLCs after 2 years. None of the never smokers, but 4% of former tobacco smokers and 15% of current tobacco smokers, experienced an SPLC (P = .005). Close monitoring with routine CT scans within the first 2 years after SBRT is effective in detecting early disease progression. In contrast, the risk for the development of an SPLC remains elevated beyond 2 years, particularly in former and current smokers. Copyright © 2015 Elsevier Inc. All rights reserved.

Pharmacological chaperones facilitate the post-ER transport of recombinant N370S mutant β-glucocerebrosidase in plant cells: Evidence that N370S is a folding mutant

PubMed Central

Babajani, Gholamreza; Tropak, Michael B.; Mahuran, Don J.; Kermode, Allison R.

2012-01-01

Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a N370S missense mutation in the GCase protein. As part of a larger endeavor to study the fate of mutant human proteins expressed in plant cells, the N370S mutant protein along with the wild-type- (WT)-GCase, both equipped with a signal peptide, were synthesized in transgenic tobacco BY2 cells, which do not possess lysosomes. The enzymatic activity of plant-recombinant N370S GCase lines was significantly lower (by 81–95%) than that of the WT-GCase lines. In contrast to the WT-GCase protein, which was efficiently secreted from tobacco BY2 cells, and detected in large amounts in the culture medium, only a small proportion of the N370S GCase was secreted. Pharmacological chaperones such as N-(n-nonyl) deoxynojirimycin and ambroxol increased the steady-state mutant protein levels both inside the plant cells and in the culture medium. These findings contradict the assertion that small molecule chaperones increase N370S GCase activity (as assayed in treated patient cell lysates) by stabilizing the enzyme in the lysosome, and suggest that the mutant protein is impaired in its ability to obtain its functional folded conformation, which is a requirement for exiting the lumen of the ER. PMID:22592100

Sufficient progesterone-priming prior to estradiol stimulation is required for optimal induction of the cervical prostaglandin system in pregnant sheep at 0.7 gestations.

PubMed

Wu, Wen Xuan; Coksaygan, Turhan; Chakrabarty, Kaushik; Collins, Valta; Rose, James C; Nathanielsz, Peter W

2005-08-01

The purposes of this study were to determine the separate and interactive functions of progesterone and estradiol in regulating the cervical prostaglandin (PG) system in pregnant sheep at 0.7 gestations. At 106-108 days of gestational age (dGA), ewes were treated with vehicle for 14 days (n = 5) or vehicle for 12 days followed by estradiol 5 mg twice a day, intramuscularly for 2 days (n = 5) or progesterone 100 mg, twice a day, intramuscularly for 14 days (n = 5) or progesterone 100 mg twice a day, intramuscularly for 10 days and then 2 days vehicle followed by estradiol 5 mg twice a day intramuscularly for 2 days (n = 5). At 121-123 dGA, cervical tissues were obtained under halothane anesthesia. Cervical RNA and protein were extracted and analyzed for prostaglandin-endoperoxide synthase 2 (COX2), two PGE(2) receptors, PTGER2 and PTGER4, and estrogen receptor alpha (ESR1) by Northern and Western blot analysis. Immunocytochemistry and in situ hybridization were applied to localize cellular distribution of COX2, PTGER2, and PTGER4 in the cervix. Data were analyzed by ANOVA. COX2 and PTGER4 mRNAs and proteins were increased (P < 0.05) in ewes treated with combined estradiol and progesterone but not in ewes treated with estradiol or progesterone alone compared with controls. ESR1 mRNA was increased in ewes treated with progesterone and estradiol plus progesterone. In contrast, PTGER2 mRNA and protein remained the same after all treatments. COX2 mRNA and protein were localized only in cervical glandular epithelial cells, whereas PTGER2 and PTGER4 were localized in both cervical glandular epithelial and smooth muscle cells. In conclusion, these data suggest that additional progesterone priming at 0.7 gestations synergizes with estradiol to induce cervical COX2, PTGER4, and ESR1 and support our hypothesis that stimulation of the cervical PG system by estradiol is optimized by sufficient progesterone priming in the pregnant sheep cervix.

Bone-Derived Stem Cells Repair the Heart after Myocardial Infarction Through Transdifferentiation and Paracrine Signaling Mechanisms

PubMed Central

Duran, Jason M.; Makarewich, Catherine A.; Sharp, Thomas E.; Starosta, Timothy; Fang, Zhu; Hoffman, Nicholas E.; Chiba, Yumi; Madesh, Muniswamy; Berretta, Remus M.; Kubo, Hajime; Houser, Steven R.

2013-01-01

Rationale Autologous bone marrow- or cardiac-derived stem cell therapy for heart disease has demonstrated safety and efficacy in clinical trials but functional improvements have been limited. Finding the optimal stem cell type best suited for cardiac regeneration is key toward improving clinical outcomes. Objective To determine the mechanism by which novel bone-derived stem cells support the injured heart. Methods and Results Cortical bone stem cells (CBSCs) and cardiac-derived stem cells (CDCs) were isolated from EGFP+ transgenic mice and were shown to express c-kit and Sca-1 as well as 8 paracrine factors involved in cardioprotection, angiogenesis and stem cell function. Wild-type C57BL/6 mice underwent sham operation (n=21) or myocardial infarction (MI) with injection of CBSCs (n=67), CDCs (n=36) or saline (n=60). Cardiac function was monitored using echocardiography. Only 2/8 paracrine factors were detected in EGFP+ CBSCs in vivo (basic fibroblast growth factor and vascular endothelial growth factor) and this expression was associated with increased neovascularization of the infarct border zone. CBSC therapy improved survival, cardiac function, regional strain, attenuated remodeling, and decreased infarct size relative to CDC- or saline-treated MI controls. By 6 weeks, EGFP+ cardiomyocytes, vascular smooth muscle and endothelial cells could be identified in CBSC- but not in CDC-treated animals. EGFP+ CBSC-derived isolated myocytes were smaller and more frequently mononucleated, but were functionally indistinguishable from EGFP- myocytes. Conclusions CBSCs improve survival, cardiac function, and attenuate remodeling through two mechanisms:1) secretion of pro-angiogenic factors that stimulate endogenous neovascularization, and 2) differentiation into functional adult myocytes and vascular cells. PMID:23801066

Effect of aminoguanidine and albendazole on inducible nitric oxide synthase (iNOS) activity in T. spiralis-infected mice muscles.

PubMed

Zeromski, Jan; Boczoń, Krystyna; Wandurska-Nowak, Elzbieta; Mozer-Lisewska, Iwona

2005-01-01

The aim of this study was to provide evidence for the expression of iNOS in the cells of inflammatory infiltrates around larvae in skeletal muscles of T. spiralis infected mice. The BALB/c mice (n = 8) divided into subgroups, received either aminoguanidine (AMG)--a specific iNOS inhibitor or albendazole (ALB)--an antiparasitic drug of choice in trichinellosis treatment. Control animals (n = 2 in each subgroup) were either uninfected and treated or uninfected and untreated. Frozen sections of hind leg muscles from mice sacrificed at various time intervals after infection were cut and subjected to immunohistochemistry, using monoclonal anti-iNOS antibody. The ALB-treated mice revealed stronger iNOS staining in the infiltrating cells around larvae than the infected and untreated animals. On the contrary, in the AMG-treated animals, the infiltrating cells did not show any specific iNOS reaction. These data confirm the specificity of iNOS staining in the cellular infiltrates around T. spiralis larvae and shed some light on the role of nitric oxide during ALB treatment in experimental trichinellosis.

Observation of Significant enhancement in the efficiency of a DSSC by InN nanoparticles over TiO 2-nanoparticle films

NASA Astrophysics Data System (ADS)

Wang, Tsai-Te; Raghunath, P.; Lu, Yun-Fang; Liu, Yu-Chang; Chiou, Chwei-Huawn; Lin, M. C.

2011-06-01

We have studied the effect of InN deposited over TiO2 nanoparticle (NP) films on the performance of dye-sensitized solar cells (DSSCs) using N3 dye with I/I3- electrolyte. A 10-20% increase in efficiency was observed for InN deposited, N3 sensitized 5-8.5 μm thick TiO2 films as compared to similar non-treated films. The deposition of InN was carried out in the temperature range of 573-723 K organometallic chemical vapor deposition (OMCVD). Spectral shifts and DFT calculations with a model anchoring group (R‧COOH) both suggest binding of the N3 dye directly to both InN and the InN/TiO2 sites.

Effect of Lapatinib on the Outgrowth of Metastatic Breast Cancer Cells to the Brain

PubMed Central

Gril, Brunilde; Palmieri, Diane; Bronder, Julie L.; Herring, Jeanne M.; Vega-Valle, Eleazar; Feigenbaum, Lionel; Liewehr, David J.; Steinberg, Seth M.; Merino, Maria J.; Rubin, Stephen D.

2008-01-01

Background The brain is increasingly being recognized as a sanctuary site for metastatic tumor cells in women with HER2-overexpressing breast cancer who receive trastuzumab therapy. There are no approved or widely accepted treatments for brain metastases other than steroids, cranial radiotherapy, and surgical resection. We examined the efficacy of lapatinib, an inhibitor of the epidermal growth factor receptor (EGFR) and HER2 kinases, for preventing the outgrowth of breast cancer cells in the brain in a mouse xenograft model of brain metastasis. Methods EGFR-overexpressing MDA-MB-231-BR (231-BR) brain-seeking breast cancer cells were transfected with an expression vector that contained or lacked the HER2 cDNA and used to examine the effect of lapatinib on the activation (ie, phosphorylation) of cell signaling proteins by immunoblotting, on cell growth by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and on cell migration using a Boyden chamber assay. The outgrowth of large (ie, >50 μm2) and micrometastases was counted in brain sections from nude mice that had been injected into the left cardiac ventricle with 231-BR cells and, beginning 5 days later, treated by oral gavage with lapatinib or vehicle (n = 22–26 mice per treatment group). All statistical tests were two-sided. Results In vitro, lapatinib inhibited the phosphorylation of EGFR, HER2, and downstream signaling proteins; cell proliferation; and migration in 231-BR cells (both with and without HER2). Among mice injected with 231-BR-vector cells, those treated with 100 mg lapatinib/kg body weight had 54% fewer large metastases 24 days after starting treatment than those treated with vehicle (mean number of large metastases per brain section: 1.56 vs 3.36, difference = 1.80, 95% confidence interval [CI] = 0.92 to 2.68, P < .001), whereas treatment with 30 mg lapatinib/kg body weight had no effect. Among mice injected with 231-BR-HER2 cells, those treated with either dose of lapatinib had 50%–53% fewer large metastases than those treated with vehicle (mean number of large metastases per brain section, 30 mg/kg vs vehicle: 3.21 vs 6.83, difference = 3.62, 95% CI = 2.30 to 4.94, P < .001; 100 mg/kg vs vehicle: 3.44 vs 6.83, difference = 3.39, 95% CI = 2.08 to 4.70, P < .001). Immunohistochemical analysis revealed reduced phosphorylation of HER2 in 231-BR-HER2 cell–derived brain metastases from mice treated with the higher dose of lapatinib compared with 231-BR-HER2 cell–derived brain metastases from vehicle-treated mice (P < .001). Conclusions Lapatinib is the first HER2-directed drug to be validated in a preclinical model for activity against brain metastases of breast cancer. PMID:18664652

ROS-dependent Atg4 upregulation mediated autophagy plays an important role in Cd-induced proliferation and invasion in A549 cells.

PubMed

Lv, Wei; Sui, Linlin; Yan, Xiaona; Xie, Huaying; Jiang, Liping; Geng, Chengyan; Li, Qiujuan; Yao, Xiaofeng; Kong, Ying; Cao, Jun

2018-01-05

Cadmium (Cd) is a toxic heavy metal that is widely used in industry and agriculture. In this study the role of autophagy in Cd-induced proliferation, migration and invasion was investigated in A549 cells. Exposure to Cd (2 μM) significantly increased reactive oxygen species (ROS) production, induced autophagy and enhanced cell growth, migration and invasion in A549 cells. Western blot analysis showed that the expression of autophagy-related proteins, LC3-II, Beclin-1 and Atg4 and invasion-related protein MMP-9 were upregulated in Cd-treated cells. N-acetyl cysteine (NAC) markedly prevented Cd-induced proliferation of A549 cells and the increasing protein level of LC3-II and Atg4. Blocking Atg4 expression by siRNA strongly reduced Beclin-1 and LC3-II protein expression and the number of autophagosome positive cells induced by Cd. Furthermore, Atg4 siRNA increased the number of cells at G0/G1 phase, reduced the number of S and G2/M phase cells, and inhibited Cd-induced cell growth significantly compared with that of Cd-treated Control siRNA cells. 3-MA pretreatment increased the percentage of G0/G1 phase cells, decreased S phase and G2/M phase percentage, and inhibited Cd-induced cell growth remarkably compared with that of only Cd-treated cells. Knocking down Atg4 reduced the number of cells that migrated and invaded through the Matrigel matrix significantly and led to a significant decrease of MMP-9 expression. In addition, in lung tissues of Cd-treated BALB/c mice, the increased expression of LC3-II, Beclin-1 and Atg4 were observed. Taken together, our results demonstrated that ROS-dependent Atg4-mediated autophagy plays an important role in Cd-induced cell growth, migration and invasion in A549 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Chloroform-Treated Filamentous Phage as a Bioreceptor for Piezoelectric Sensors

DTIC Science & Technology

2005-01-01

Gels were rinsed in double-deionized water (DDH2O) then treated by immersion in 0.2 N NaOH for 1 h, 1 M Tris-HCl (pH 7.5) for 15 min, and 0.05 M...Filamentous bacteriophage contract into hollow spherical particles upon exposure to a chloroform- water interface. Cell 23, 747- 753. Manning, M...Chrysogelos, S., Griffith, J., 1981. Mechanism of coliphage M13 contraction: intermediate structures trapped at low temperatures. J. Virol. 40, 912-919. Naylor

Taxol induces concentration-dependent phosphatidylserine (PS) externalization and cell cycle arrest in ASTC-a-1 cells

NASA Astrophysics Data System (ADS)

Guo, Wen-jing; Chen, Tong-sheng

2010-02-01

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Different concentrations of taxol can trigger distinct effects on both the cellular microtubule network and biochemical pathways. Apoptosis induced by low concentrations (5-30 nM) of taxol was associated with mitotic arrest, alteration of microtubule dynamics and/or G2/M cell cycle arrest, whereas high concentrations of this drug (0.2-30 μM) caused significant microtubule damage, and was found recently to induce cytoplasm vacuolization in human lung adenocarcinoma (ASTC-a-1) cells. In present study, cell counting kit (CCK-8) assay, confocal microscope, and flow cytometry analysis were used to analyze the cell death form induced by 35 nM and 70 μM of taxol respectively in human lung adenocarcinoma (ASTC-a-1) cells. After treatment of 35 nM taxol for 48 h, the OD450 value was 0.80, and 35 nM taxol was found to induce dominantly cell death in apoptotic pathway such as phosphatidylserine (PS) externalization, G2/M phase arrest after treatment for 24 h, and nuclear fragmentation after treatment for 48 h. After 70 μM taxol treated the cell for 24 h, the OD450 value was 1.01, and 70 μM taxol induced cytoplasm vacuolization programmed cell death (PCD) and G2/M phase as well as the polyploidy phase arrest in paraptotic-like cell death. These findings imply that the regulated signaling pathway of cell death induced by taxol is dependent on taxol concentration in ASTC-a-1 cells.

Methamphetamine induces autophagy and apoptosis in a mesencephalic dopaminergic neuronal culture model: role of cathepsin-D in methamphetamine-induced apoptotic cell death.

PubMed

Kanthasamy, Arthi; Anantharam, V; Ali, Syed F; Kanthasamy, A G

2006-08-01

Autophagy is a phylogenetically conserved process that plays a critical role in the degradation of oxidatively damaged proteins and organelle turnover. The role of oxidative stress and apoptosis in methamphetamine (METH)-induced neurotoxicity is well known; however, the potential contribution of autophagy to METH-induced oxidative damage in dopaminergic neuronal systems remains unclear. The goals of the present article were twofold: (a) to develop an in vitro dopaminergic cell culture model to study cellular and molecular mechanisms underlying METH-induced autophagy and apoptosis, and (b) to determine whether lysosomal protease cathepsin-D activation, resulting from the loss of lysosomal membrane integrity, contributes to METH-induced apoptosis. To accomplish these goals, we characterized morphological and biochemical changes in an immortalized mesencephalic dopaminergic neuronal cell line (N27 cells) following treatment with METH. Exposure of METH (2 mM) to N27 cells resulted in the appearance of cytoplasmic vacuolar structures reminiscent of autophagic vacuoles within 3 h. In order to ascertain the identity of the vacuolar structures that are formed following METH exposure, immunohistochemical staining for markers of autophagy were performed. LAMP 2, a classical marker of autophagolysosomes, revealed an extensive punctuate pattern of distribution on the vacuolar membrane surface, with exclusive localization in the cytoplasm. Additionally, using DNA fragmentation analysis we showed a dose-dependent increase in fragmented DNA in METH treated N27 cells. Since METH-induced autophagy preceded DNA fragmentation, we tested whether dysfunction of the autophagolysosomal system contributes to nuclear damage. Immunofluorescence studies with cathepsin-d demonstrated a granular pattern of staining in untreated cells, whereas an increased cathepsin- D immunoreactivity with a globular pattern of staining was observed in METH-treated cells. Nevertheless, blockade of cathepsin-D activation by pepstatin-A, cathepsin-D inhibitor, failed to alter METH-induced DNA fragmentation. Collectively, these results demonstrate that N27 dopaminergic neuronal cell model may serve as an excellent in vitro model to study the mechanisms of METH-induced autophagy and apoptosis. Furthermore, it is less likely that cathepsin-D may serve as a trigger for the induction of apoptosis subsequent to exposure of N27 dopaminergic neuronal cells to METH.

Enhancement of NAD+-dependent SIRT1 deacetylase activity by methylselenocysteine resets the circadian clock in carcinogen-treated mammary epithelial cells

PubMed Central

Fang, Mingzhu; Guo, Wei-Ren; Park, Youngil; Kang, Hwan-Goo; Zarbl, Helmut

2015-01-01

We previously reported that dietary methylselenocysteine (MSC) inhibits N-methyl-N-nitrosourea (NMU)-induced mammary tumorigenesis by resetting circadian gene expression disrupted by the carcinogen at the early stage of tumorigenesis. To investigate the underlying mechanism, we developed a circadian reporter system comprised of human mammary epithelial cells with a luciferase reporter driven by the promoter of human PERIOD 2 (PER2), a core circadian gene. In this in vitro model, NMU disrupted cellular circadian rhythm in a pattern similar to that observed with SIRT1-specific inhibitors; in contrast, MSC restored the circadian rhythms disrupted by NMU and protected against SIRT1 inhibitors. Moreover, NMU inhibited intracellular NAD+/NADH ratio and reduced NAD+-dependent SIRT1 activity in a dose-dependent manner, while MSC restored NAD+/NADH and SIRT1 activity in the NMU-treated cells, indicating that the NAD+-SIRT1 pathway was targeted by NMU and MSC. In rat mammary tissue, a carcinogenic dose of NMU also disrupted NAD+/NADH oscillations and decreased SIRT1 activity; dietary MSC restored NAD+/NADH oscillations and increased SIRT1 activity in the mammary glands of NMU-treated rats. MSC-induced SIRT1 activity was correlated with decreased acetylation of BMAL1 and increased acetylation of histone 3 lysine 9 at the Per2 promoter E-Box in mammary tissue. Changes in SIRT1 activity were temporally correlated with loss or restoration of rhythmic Per2 mRNA expression in NMU-treated or MSC-rescued rat mammary glands, respectively. Together with our previous findings, these results suggest that enhancement of NAD+-dependent SIRT1 activity contributes to the chemopreventive efficacy of MSC by restoring epigenetic regulation of circadian gene expression at early stages of mammary tumorigenesis. PMID:26544624

Low-dose lisinopril in normotensive men with idiopathic oligospermia and infertility: a 5-year randomized, controlled, crossover pilot study.

PubMed

Mbah, A U; Ndukwu, G O; Ghasi, S I; Shu, E N; Ozoemena, F N; Mbah, J O; Onodugo, O D; Ejim, E C; Eze, M I; Nkwo, P O; Okonkwo, P O

2012-04-01

The outcomes of drug treatment for male infertility remain conjectural, with controversial study results. Our pilot study employed a randomized, placebo-controlled, crossover methodology with intention-to-treat analysis. Thirty-three men with idiopathic oligospermia were randomized to start either daily oral lisinopril 2.5 mg (n = 17) or daily oral placebo (n = 16). Lisinopril was found to cause a normalization of seminal parameters in 53.6% of the participants. Although the mean ejaculate volume was unchanged (P ≥ 0.093), the total sperm cell count and the percentage of motile sperm cells increased (P ≤ 0.03 and P < 0.001, respectively), whereas the percentage of sperm cells with abnormal morphology decreased (P ≤ 0.04). The pregnancy rate was 48.5%, and there was no serious adverse drug event. It is concluded, albeit cautiously, that prolonged treatment with 2.5 mg/day of oral lisinopril may be well tolerated in normotensive men with idiopathic oligospermia, may improve sperm quantity and quality, and may enhance fertility in approximately half of those treated.

Role of cerium oxide nanoparticle-induced autophagy as a safeguard to exogenous H2O2-mediated DNA damage in tobacco BY-2 cells.

PubMed

Sadhu, Abhishek; Ghosh, Ilika; Moriyasu, Yuji; Mukherjee, Anita; Bandyopadhyay, Maumita

2018-04-13

The effect of cerium oxide nanoparticle (CeNP) in plants has elicited substantial controversy. While some investigators have reported that CeNP possesses antioxidant properties, others observed CeNP to induce reactive oxygen species (ROS). In spite of considerable research carried out on the effects of CeNP in metazoans, fundamental studies that can unveil its intracellular consequences linking ROS production, autophagy and DNA damage are lacking in plants. To elucidate the impact of CeNP within plant cells, tobacco BY-2 cells were treated with 10, 50 and 250 µg ml-1 CeNP (Ce10, Ce50 and Ce250), for 24 h. Results demonstrated concentration-dependent accumulation of Ca2+ and ROS at all CeNP treatment sets. However, significant DNA damage and alteration in antioxidant defence systems were noted prominently at Ce50 and Ce250. Moreover, Ce50 and Ce250 induced DNA damage, analysed by comet assay and DNA diffusion experiments, complied with the concomitant increase in ROS. Furthermore, to evaluate the antioxidant property of CeNP, treated cells were washed after 24 h (to minimise CeNP interference) and challenged with H2O2 for 3 h. Ce10 did not induce genotoxicity and H2O2 exposure to Ce10-treated cells showed lesser DNA breakage than cells treated with H2O2 only. Interestingly, Ce10 provided better protection over N-acetyl-L-cysteine against exogenous H2O2 in BY-2 cells. CeNP exposure to transgenic BY-2 cells expressing GFP-Atg8 fusion protein exhibited formation of autophagosomes at Ce10. Application of vacuolar protease inhibitor E-64c and fluorescent basic dye acridine orange, further demonstrated accumulation of particulate matters in the vacuole and occurrence of acidic compartments, the autophagolysosomes, respectively. BY-2 cells co-treated with CeNP and autophagy inhibitor 3-methyladenine exhibited increased DNA damage in Ce10 and cell death at all assessed treatment sets. Thus, current results substantiate an alternative autophagy-mediated, antioxidant and geno-protective role of CeNP, which will aid in deciphering novel phenomena of plant-nanoparticle interaction at cellular level.

γ-Tocotrienol prevents 5-FU-induced reactive oxygen species production in human oral keratinocytes through the stabilization of 5-FU-induced activation of Nrf2.

PubMed

Takano, Hideyuki; Momota, Yukihiro; Kani, Kouichi; Aota, Keiko; Yamamura, Yoshiko; Yamanoi, Tomoko; Azuma, Masayuki

2015-04-01

Chemotherapy-induced oral mucositis is a common adverse event in patients with oral squamous cell carcinoma, and is initiated through a variety of mechanisms, including the generation of reactive oxygen species (ROS). In this study, we examined the preventive effect of γ-tocotrienol on the 5-FU-induced ROS production in human oral keratinocytes (RT7). We treated RT7 cells with 5-FU and γ-tocotrienol at concentrations of 10 µg/ml and 10 nM, respectively. When cells were treated with 5-FU alone, significant growth inhibition was observed as compared to untreated cells. This inhibition was, in part, due to the ROS gene-rated by 5-FU treatment, because N-acetyl cysteine (NAC), a ROS scavenger, significantly ameliorated the growth of RT7 cells. γ-tocotrienol showed no cytotoxic effect on the growth of RT7 cells. Simultaneous treatment of cells with these agents resulted in the significant recovery of cell growth, owing to the suppression of ROS generation by γ-tocotrienol. Whereas 5-FU stimulated the expression of NF-E2-related factor 2 (Nrf2) protein in the nucleus up to 12 h after treatment of RT7 cells, γ-tocotrienol had no obvious effect on the expression of nuclear Nrf2 protein. Of note, the combined treatment with both agents stabilized the 5-FU-induced nuclear Nrf2 protein expression until 24 h after treatment. In addition, expression of Nrf2-dependent antioxidant genes, such as heme oxygenase-1 (HO-1) and quinone oxidoreductase-1 (NQO-1), was significantly augmented by treatment of cells with both agents. These findings suggest that γ-tocotrienol could prevent 5-FU-induced ROS generation by stabilizing Nrf2 activation, thereby leading to ROS detoxification and cell survival in human oral keratinocytes.

γ-tocotrienol prevents 5-FU-induced reactive oxygen species production in human oral keratinocytes through the stabilization of 5-FU-induced activation of Nrf2

PubMed Central

TAKANO, HIDEYUKI; MOMOTA, YUKIHIRO; KANI, KOUICHI; AOTA, KEIKO; YAMAMURA, YOSHIKO; YAMANOI, TOMOKO; AZUMA, MASAYUKI

2015-01-01

Chemotherapy-induced oral mucositis is a common adverse event in patients with oral squamous cell carcinoma, and is initiated through a variety of mechanisms, including the generation of reactive oxygen species (ROS). In this study, we examined the preventive effect of γ-tocotrienol on the 5-FU-induced ROS production in human oral keratinocytes (RT7). We treated RT7 cells with 5-FU and γ-tocotrienol at concentrations of 10 μg/ml and 10 nM, respectively. When cells were treated with 5-FU alone, significant growth inhibition was observed as compared to untreated cells. This inhibition was, in part, due to the ROS generated by 5-FU treatment, because N-acetyl cysteine (NAC), a ROS scavenger, significantly ameliorated the growth of RT7 cells. γ-tocotrienol showed no cytotoxic effect on the growth of RT7 cells. Simultaneous treatment of cells with these agents resulted in the significant recovery of cell growth, owing to the suppression of ROS generation by γ-tocotrienol. Whereas 5-FU stimulated the expression of NF-E2-related factor 2 (Nrf2) protein in the nucleus up to 12 h after treatment of RT7 cells, γ-tocotrienol had no obvious effect on the expression of nuclear Nrf2 protein. Of note, the combined treatment with both agents stabilized the 5-FU-induced nuclear Nrf2 protein expression until 24 h after treatment. In addition, expression of Nrf2-dependent antioxidant genes, such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO-1), was significantly augmented by treatment of cells with both agents. These findings suggest that γ-tocotrienol could prevent 5-FU-induced ROS generation by stabilizing Nrf2 activation, thereby leading to ROS detoxification and cell survival in human oral keratinocytes. PMID:25625649

A comparative study on the adverse effects of flunixin, ketoprofen and phenylbutazone in miniature donkeys: haematological, biochemical and pathological findings.

PubMed

Mozaffari, A A; Derakhshanfar, A; Alinejad, A; Morovati, M

2010-10-01

To evaluate the adverse effects of flunixin, ketoprofen and phenylbutazone when administered I/V to clinically normal miniature donkeys. Twenty clinically normal adult (2.0-2.5 years old) male miniature donkeys weighing 113-136 kg and 0.81- 0.86 m tall were randomly assigned to one of four groups, and administered either saline (n=5), 1.0 mg/kg flunixin (n=5), 2.2 mg/kg ketoprofen (n=5), or 4.4 mg/kg phenylbutazone (n=5) I/V at 0800 hours on Day 1, then every 12 h, for 12 days. The animals were observed every 8 h, and examined physically daily. Blood, faeces and urine samples were collected daily from all donkeys, for haematological indices and enzyme activities, occult blood, and urinalysis, respectively. Immediately after euthanasia, complete post-mortem examinations were performed on all donkeys, and gross lesions recorded. Histopathology was conducted on a wide range of tissues. Clinically, mild anorexia and diarrhoea were observed during the study only in donkeys treated with phenylbutazone. There was an effect of treatment with the non-steroidal anti-infl ammatory drugs (NSAID) on red blood cell (RBC) counts, packed cell volume (PCV) and enzyme activities, but not on urine. Lesions were observed in the glandular mucosa of the stomach of all donkeys treated with NSAID, including ulceration in most. Also, in donkeys treated with NSAID, hyperaemia, erosion and ulceration of the gastrointestinal tract, and congestion of the liver, kidney and spleen, were observed. Microscopically, hepatic and renal lesions comprised biliary hyperplasia and interstitial nephritis, respectively. The gastrointestinal, hepatic and renal lesions observed in the donkeys treated with NSAID demonstrated the toxic potential of NSAID, which was greatest for animals treated with phenylbutazone, less for flunixin, and least for ketoprofen. When use of these compounds is contemplated in clinical cases, the risk of adverse effects and the comparative toxic potential should be considered, together with the efficacy of the compound for the condition being treated.

Simultaneous inhibition of multiple steps in the processing of N-linked oligosaccharides does not impair immunoglobulin secretion from rat hybridoma cells.

PubMed Central

Hashim, O H; Cushley, W

1988-01-01

The effects of inhibiting selected pairs of oligosaccharide-processing activities upon the secretion of IgM and IgG molecules have been investigated. In the presence of castanospermine (CSP) plus swainsonine (SW) or deoxynojirimycin (dNM) plus deoxymannojirimycin (dMM), secretion of IgM and IgG from rat hybridoma cells was unimpaired relative to control cultures. The structures of the N-linked oligosaccharides found on the Ig heavy chains isolated from treated cells or culture supernatants were shown to be qualitatively different from those associated with control Ig by persistent sensitivity to digestion by endo H. Furthermore, the electrophoretic mobilities of mu and gamma chains on SDS-PAGE derived from treated cells were consistently slower than those of control heavy chains. IgM and IgG were also efficiently secreted when all glucosidase and mannosidase activities were blocked, and the secreted heavy chains bore endo H-sensitive oligosaccharides. The data suggest that Ig secretion from hybridomas can proceed in the absence of N-linked oligosaccharide processing. Images Figure 1 Figure 2 Figure 3 PMID:3350578

Copper ions stimulate the proliferation of hepatic stellate cells via oxygen stress in vitro.

PubMed

Xu, San-qing; Zhu, Hui-yun; Lin, Jian-guo; Su, Tang-feng; Liu, Yan; Luo, Xiao-ping

2013-02-01

This study examined the effect of copper ions on the proliferation of hepatic stellate cells (HSCs) and the role of oxidative stress in this process in order to gain insight into the mechanism of hepatic fibrosis in Wilson's disease. LX-2 cells, a cell line of human HSCs, were cultured in vitro and treated with different agents including copper sulfate, N-acetyl cysteine (NAC) and buthionine sulfoximine (BSO) for different time. The proliferation of LX-2 cells was measured by non-radioactive cell proliferation assay. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of platelet-derived growth factor receptor β subunit (PDGFβR), ELISA to determine the level of glutathione (GSH) and oxidized glutathione (GSSG), dichlorofluorescein assay to measure the level of reactive oxygen species (ROS), and lipid hydroperoxide assay to quantify the level of lipid peroxide (LPO). The results showed that copper sulfate over a certain concentration range could promote the proliferation of LX-2 cells in a time- and dose-dependent manner. The effect was most manifest when LX-2 cells were treated with copper sulfate at a concentration of 100 μmol/L for 24 h. Additionally, copper sulfate could dose-dependently increase the levels of ROS and LPO, and decrease the ratio of GSH/GSSG in LX-2 cells. The copper-induced increase in mRNA and protein expression of PDGFβR was significantly inhibited in LX-2 cells pre-treated with NAC, a precursor of GSH, and this phenomenon could be reversed by the intervention of BSO, an inhibitor of NAC. It was concluded that copper ions may directly stimulate the proliferation of HSCs via oxidative stress. Anti-oxidative stress therapies may help suppress the copper-induced activation and proliferation of HSCs.

Sodium Orthovanadate Effect on Outflow Facility and Intraocular Pressure in Live Monkeys

PubMed Central

Tan, James C.H.; Kiland, Julie A.; Gonzalez, Jose M.; Gabelt, B’Ann T.; Peters, Donna M.; Kaufman, Paul L.

2010-01-01

Sodium orthovanadate (Na3VO4) is reported to reduce IOP by affecting aqueous formation, but whether it also affects outflow facility (OF) is unclear. We tested the effect of Na3VO4 on OF and intraocular pressure (IOP) in live cynomolgus monkeys, and on actin and cell adhesion organization in cultured human trabecular meshwork (HTM) cells. Total OF (n = 12) was measured by 2-level constant pressure perfusion of the monkey anterior chamber (AC) before and after exchange with 1 mM Na3VO4 or vehicle in opposite eyes. Topical 1% Na3VO4 or vehicle only was given twice daily (each 2×20 μL drops) for 4 days to opposite eyes (n = 8), and Goldmann IOP was measured before and hourly after treatment for 6 hours on Days 1 and 4. Filamentous actin and vinculin-containing cell adhesions were examined by epifluorescence microscopy after the cells had been incubated with 1 mM Na3VO4 for 24 hours. A) In monkeys, Na3VO4 increased OF by 29.3 ± 8.8% (mean ± s.e.m.) over the perfusion interval when adjusted for baseline and contralateral eye washout (p = 0.01; n = 12). B) Day 1 baseline IOP was 16.2 ± 1.5 mmHg in treated eyes and 15.9 ± 1.3 mmHg in the contralateral control eyes. Following treatment on Day 1, IOP was no different (p>0.05) between treated eyes and control eyes at any time-point or compared to baseline. Day 4 mean IOP averaged over hours 2–6 was 13.5 ± 0.8 mmHg in treated eyes and 16.1 ± 0.2 mmHg in control eyes. Treated eye IOP was lower than its Day 4 baseline (p<0.005), lower than control eyes for the same Day 4 interval (p = 0.009), and lower than the Day 1 baseline (p = 0.0000). Control eye IOP on Day 4 was not significantly different from baseline on Day 1. C) Incubation of HTM cells with 1 mM Na3VO4 for 24 hours caused a loss of actin stress fibers and vinculin-containing adhesions. Cell retraction and separation was also observed in vanadate-treated cultures. Reformation of actin stress fibers, vinculin-containing adhesions and confluent monolayers occurred within 24 hours after Na3VO4-containing culture medium was replaced with Na3VO4-free medium. Ocular administration of Na3VO4 to live monkeys significantly increases OF and reduces IOP. Na3VO4 reversibly disrupts actin and cell adhesion organization and causes retraction and separation of cultured HTM cells. Na3VO4 increases pressure-dependent outflow in live monkeys. Altered actin architecture in the TM may play a part in this increased OF. PMID:20620138

Nano-technology based carriers for nitrogen-containing bisphosphonates delivery as sensitisers of γδ T cells for anticancer immunotherapy.

PubMed

Hodgins, Naomi O; Wang, Julie Tzu-Wen; Al-Jamal, Khuloud T

2017-05-15

Nitrogen containing bisphosphonates (N-BPs) including zoledronate (ZOL) and alendronate (ALD) inhibit farnesyl diphosphate synthase, and have been shown to have a cytotoxic affect against cancer cells as a monotherapy and to also sensitise tumour cells to destruction by γδ T cells. γδ T cells are a subset of human T lymphocytes and have a diverse range of roles in the immune system including the recognition and destruction of cancer cells. This property of γδ T cells can be harnessed for use in cancer immunotherapy through in vivo expansion or the adoptive transfer of ex vivo activated γδ T cells. The use of N-BPs with γδ T cells has been shown to have a synergistic effect in in vitro, animal and clinical studies. N-BPs have limited in vivo activity due to rapid clearance from the circulation. By encapsulating N-BPs in liposomes (L) it is possible to increase the levels of N-BPs at non-osseous tumour sites. L-ZOL and L-ALD have been shown to have different toxicological profiles than free ZOL or ALD. Both L-ALD and L-ZOL led to increased spleen weight, leucocytosis, neutrophilia and lymphocytopenia in mice after intravenous injection. L-ALD was shown to be better tolerated than L-ZOL in murine studies. Biodistribution studies have been performed in order to better understand the interaction of N-BPs and γδ T cells in vivo. Additionally, in vivo therapy studies have shown that mice treated with both L-ALD and γδ T cells had a significant reduction in tumour growth compared to mice treated with L-ALD or γδ T cells alone. The use of ligand-targeted liposomes may further increase the efficacy of this combinatory immunotherapy. Liposomes targeting the αvβ6 integrin receptor using the peptide A20FMDV2 had a greater ability than untargeted liposomes in sensitising cancer cells to destruction by γδ T cells in αvβ6 positive cancer cell lines. Copyright © 2017 Elsevier B.V. All rights reserved.

Neuroglobin overexpression plays a pivotal role in neuroprotection through mitochondrial raft-like microdomains in neuroblastoma SK-N-BE2 cells.

PubMed

Garofalo, Tina; Ferri, Alberto; Sorice, Maurizio; Azmoon, Pardis; Grasso, Maria; Mattei, Vincenzo; Capozzi, Antonella; Manganelli, Valeria; Misasi, Roberta

2018-04-01

Since stressing conditions induce a relocalization of endogenous human neuroglobin (NGB) to mitochondria, this research is aimed to evaluate the protective role of NGB overexpression against neurotoxic stimuli, through mitochondrial lipid raft-associated complexes. To this purpose, we built a neuronal model of oxidative stress by the use of human dopaminergic neuroblastoma cells, SK-N-BE2, stably overexpressing NGB by transfection and treated with 1-methyl-4-phenylpyridinium ion (MPP+). We preliminary observed the redistribution of NGB to mitochondria following MPP+ treatment. The analysis of mitochondrial raft-like microdomains revealed that, following MPP+ treatment, NGB translocated to raft fractions (Triton X-100-insoluble), where it interacts with ganglioside GD3. Interestingly, the administration of agents capable of perturbating microdomain before MPP+ treatment, significantly affected viability in SK-N-BE2-NGB cells. The overexpression of NGB was able to abrogate the mitochondrial injuries on complex IV activity or mitochondrial morphology induced by MPP+ administration. The protective action of NGB on mitochondria only takes place if the mitochondrial lipid(s) rafts-like microdomains are intact, indeed NGB fails to protect complex IV activity when purified mitochondria were treated with the lipid rafts disruptor methyl-β-cyclodextrin. Thus, our unique in vitro model of stably transfected cells overexpressing endogenous NGB allowed us to suggest that the role in neuroprotection played by NGB is reliable only through interaction with mitochondrial lipid raft-associated complexes. Copyright © 2018 Elsevier Inc. All rights reserved.

Regulation of sphingomyelin phosphodiesterase acid-like 3A gene (SMPDL3A) by liver X receptors.

PubMed

Noto, Paul B; Bukhtiyarov, Yuri; Shi, Meng; McKeever, Brian M; McGeehan, Gerard M; Lala, Deepak S

2012-10-01

Liver X receptor (LXR) α and LXRβ function as physiological sensors of cholesterol metabolites (oxysterols), regulating key genes involved in cholesterol and lipid metabolism. LXRs have been extensively studied in both human and rodent cell systems, revealing their potential therapeutic value in the contexts of atherosclerosis and inflammatory diseases. The LXR genome landscape has been investigated in murine macrophages but not in human THP-1 cells, which represent one of the frequently used monocyte/macrophage cell systems to study immune responses. We used a whole-genome screen to detect direct LXR target genes in THP-1 cells treated with two widely used LXR ligands [N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317) and 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy] phenylacetic acid hydrochloride (GW3965)]. This screen identified the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene as a novel LXR-regulated gene, with an LXR response element within its promoter. We investigated the regulation of SMPDL3A gene expression by LXRs across several human and mouse cell types. These studies indicate that the induction of SMPDL3A is LXR-dependent and is restricted to human blood cells with no induction observed in mouse cellular systems.

[Zn(phen)(O,N,O)(H2O)] and [Zn(phen)(O,N)(H2O)] with O,N,O is 2,6-dipicolinate and N,O is L-threoninate: synthesis, characterization, and biomedical properties.

PubMed

Chin, Lee-Fang; Kong, Siew-Ming; Seng, Hoi-Ling; Tiong, Yee-Lian; Neo, Kian-Eang; Maah, Mohd Jamil; Khoo, Alan Soo-Beng; Ahmad, Munirah; Hor, Tzi-Sum Andy; Lee, Hong-Boon; San, Swee-Lan; Chye, Soi-Moi; Ng, Chew-Hee

2012-10-01

Two ternary Zn(II) complexes, with 1,10-phenanthroline (phen) as the main ligand and a carboxylate-containing ligand [dipicolinate (dipico) or L-threoninate (L-Thr)] as the subsidiary ligand, were prepared and characterized by elemental analysis, Fourier transform IR, UV, and fluorescence spectroscopy, X-ray diffraction, molar conductivity, and electrospray ionization mass spectrometry. X-ray structure analysis shows that both [Zn(phen)(dipico)(H(2)O)]·H(2)O (1) and [Zn(phen)(L-Thr)(H(2)O)Cl]·2H(2)O (2) have octahedral geometry about the Zn(II) atom. Both complexes can inhibit topoisomerase I, and have better anticancer activity than cisplatin against nasopharyngeal cancer cell lines, HK1 and HONE-1, with concentrations causing 50 % inhibition of cell proliferation (IC(50)) in the low micromolar range. Complex 2 has the highest therapeutic index for HK1. Both Zn(II) complexes can induce cell death by apoptosis. Changing the subsidiary ligand in the Zn(II) complexes affects the UV-fluorescence spectral properties of the coordinated phen ligand, the binding affinity for some DNA sequences, nucleobase sequence-selective binding, the phase at which cell cycle progression was arrested for treated cancer cells, and their therapeutic index.

N-hydroxycinnamide derivatives of osthole ameliorate hyperglycemia through activation of AMPK and p38 MAPK.

PubMed

Lee, Wei-Hwa; Wu, Hsueh-Hsia; Huang, Wei-Jan; Li, Yi-Ning; Lin, Ren-Jye; Lin, Shyr-Yi; Liang, Yu-Chih

2015-03-11

Our previous studies found that osthole markedly reduced blood glucose levels in both db/db and ob/ob mice. To improve the antidiabetic activity of osthole, a series of N-hydroxycinnamide derivatives of osthole were synthesized, and their hypoglycemia activities were examined in vitro and in vivo. Both N-hydroxycinnamide derivatives of osthole, OHC-4p and OHC-2m, had the greatest potential for activating AMPK and increasing glucose uptake by L6 skeletal muscle cells. In addition, OHC-4p and OHC-2m time- and dose-dependently increased phosphorylation levels of AMPK and p38 MAPK. The AMPK inhibitor, compound C, and the p38 MAPK inhibitor, SB203580, significantly reversed activation of AMPK and p38 MAPK, respectively, in OHC-4p- and OHC-2m-treated cells. Compound C and SB203580 also inhibited glucose uptake induced by OHC-4p and OHC-2m. Next, we found that OHC-4p and OHC-2m significantly increased glucose transporter 4 (GLUT4) translocation to plasma membranes and counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that activation of AMPK and p38 MAPK by OHC-4p and OHC-2m is associated with increased glucose uptake and GLUT4 translocation and subsequently led to amelioration of hyperglycemia. Therefore, OHC-4p and OHC-2m might have potential as antidiabetic agents for treating type 2 diabetes. Our previous studies found that osthole markedly reduced blood glucose levels in both db/db and ob/ob mice. To improve the antidiabetic activity of osthole, a series of N-hydroxycinnamide derivatives of osthole were synthesized, and their hypoglycemia activities were examined in vitro and in vivo. Both N-hydroxycinnamide derivatives of osthole, OHC-4p and OHC-2m, had the greatest potential for activating AMPK and increasing glucose uptake by L6 skeletal muscle cells. In addition, OHC-4p and OHC-2m time- and dose-dependently increased phosphorylation levels of AMPK and p38 MAPK. The AMPK inhibitor, compound C, and the p38 MAPK inhibitor, SB203580, significantly reversed activation of AMPK and p38 MAPK, respectively, in OHC-4p- and OHC-2m-treated cells. Compound C and SB203580 also inhibited glucose uptake induced by OHC-4p and OHC-2m. Next, we found that OHC-4p and OHC-2m significantly increased glucose transporter 4 (GLUT4) translocation to plasma membranes and counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that activation of AMPK and p38 MAPK by OHC-4p and OHC-2m is associated with increased glucose uptake and GLUT4 translocation and subsequently led to amelioration of hyperglycemia. Therefore, OHC-4p and OHC-2m might have potential as antidiabetic agents for treating type 2 diabetes.

Genotoxicity of 2,6- and 3,5-Dimethylaniline in Cultured Mammalian Cells: The Role of Reactive Oxygen Species

PubMed Central

Chao, Ming-Wei; Kim, Min Young; Wogan, Gerald N.

2012-01-01

Several alkylanilines with structures more complex than toluidines have been associated epidemiologically with human cancer. Their mechanism of action remains largely undetermined, and there is no reported evidence that it replicates that of multicyclic aromatic amines even though the principal metabolic pathways of P450-mediated hydroxylation and phase II conjugation are very similar. As a means to elucidate their mechanisms of action, lethality and mutagenicity in the adenine phosphoribosyltransferase (aprt +/−) gene induced in several Chinese hamster ovary cell types by 2,6- and 3,5-dimethylaniline (2,6-DMA, 3,5-DMA) and their N- and ring-hydroxyl derivatives (N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP) were assessed. Dose-response relationships were determined in the parental AA8 cell line, its repair-deficient UV5 subclone and other repair-deficient 5P3NAT2 or -proficient 5P3NAT2R9 subclones engineered to express mouse cytochrome P4501A2 (CYP1A2) and human N-acetyltransferase (NAT2), and also in AS52 cells harboring the bacterial guanine-hypoxanthine phosphoribosyltransferase (gpt) gene. Mutations in the gpt gene of AS52 cells were characterized and found to be dominated by G:C to A:T and A:T to G:C transitions. Separately, treatment of AS52 cells with N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP, and 3,5-DMAP led to intracellular production of reactive oxygen species (ROS) for at least 24h after removal of the mutagens in every case. Using the comet assay, DNA strand breaks were observed in a dose-dependent manner in AS52 cells when treated with each of the four N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, and 3,5-DMAP derivatives. Comparative evaluation of the results indicates that the principal mechanism of mutagenic action is likely to be through redox cycling of intracellularly bound aminophenol/quinone imine structures to generate ROS rather than through formation of covalent DNA adducts. PMID:22831970

Atmospheric plasma surface modifications of electrospun PCL/chitosan/PCL hybrid scaffolds by nozzle type plasma jets for usage of cell cultivation

NASA Astrophysics Data System (ADS)

Surucu, Seda; Masur, Kai; Turkoglu Sasmazel, Hilal; Von Woedtke, Thomas; Weltmann, Klaus Dieter

2016-11-01

This paper reports Ar gas, Ar + O2, Ar + O2 + N2 gas mixtures and dry air plasma modifications by atmospheric pressure argon driven kINPen and air driven Diener (PlasmaBeam) plasma jets to alter surface properties of three dimensional (3D), electrospun PCL/Chitosan/PCL layer by layer hybrid scaffolds to improve human fibroblast (MRC5) cell attachment and growth. The characterizations of the samples were done by contact angle (CA) measurements, scanning electron microscopy (SEM), X-Ray Photoelectron spectroscopy (XPS) analysis. The results showed that the plasma modification carried out under dry air and Ar + O2 + N2 gas mixtures were altered effectively the nanotopography and the functionality of the material surfaces. It was found that the samples treated with Ar + O2 + N2 gas mixtures for 1 min and dry air for 9 min have better hydrophilicity 78.9° ± 1.0 and 75.6° ± 0.1, respectively compared to the untreated samples (126.5°). Biocompatibility performance of the scaffolds was determined with alamarBlue (aB) assay and MTT assay methods, Giemsa staining, fluorescence microscope, confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) analyses. The results showed that plasma treated samples increased the hydrophilicity and oxygen functionality and topography of the surfaces significantly, thus affecting the cell viability and proliferation on/within scaffolds.

Monte Carlo N-Particle (MCNP) Modeling of the Cellular Dosimetry of 64Cu: Comparison with MIRDcell S Values and Implications for Studies of Its Cytotoxic Effects.

PubMed

Cai, Zhongli; Kwon, Yongkyu Luke; Reilly, Raymond M

2017-02-01

64 Cu emits positrons as well as β - particles and Auger and internal conversion electrons useful for radiotherapy. Our objective was to model the cellular dosimetry of 64 Cu under different geometries commonly used to study the cytotoxic effects of 64 Cu. Monte Carlo N-Particle (MCNP) was used to simulate the transport of all particles emitted by 64 Cu from the cell surface (CS), cytoplasm (Cy), or nucleus (N) of a single cell; monolayer in a well (radius = 0.32-1.74 cm); or a sphere (radius = 50-6,000 μm) of cells to calculate S values. The radius of the cell and N ranged from 5 to 12 μm and 2 to 11 μm, respectively. S values were obtained by MIRDcell for comparison. MCF7/HER2-18 cells were exposed in vitro to 64 Cu-labeled trastuzumab. The subcellular distribution of 64 Cu was measured by cell fractionation. The surviving fraction was determined in a clonogenic assay. The relative differences of MCNP versus MIRDcell self-dose S values (S self ) for 64 Cu ranged from -0.2% to 3.6% for N to N (S N←N ), 2.3% to 8.6% for Cy to N (S N←Cy ), and -12.0% to 7.3% for CS to N (S N←CS ). The relative differences of MCNP versus MIRDcell cross-dose S values were 25.8%-30.6% for a monolayer and 30%-34% for a sphere, respectively. The ratios of S N←N versus S N←Cy and S N←Cy versus S N←CS decreased with increasing ratio of the N of the cell versus radius of the cell and the size of the monolayer or sphere. The surviving fraction of MCF7 /: HER2-18 cells treated with 64 Cu-labeled trastuzumab (0.016-0.368 MBq/μg, 67 nM) for 18 h versus the absorbed dose followed a linear survival curve with α = 0.51 ± 0.05 Gy -1 and R 2 = 0.8838. This is significantly different from the linear quadratic survival curve of MCF7 /: HER2-18 cells exposed to γ-rays. MCNP- and MIRDcell-calculated S values agreed well. 64 Cu in the N increases the dose to the N in isolated single cells but has less effect in a cell monolayer or small cluster of cells simulating a micrometastasis, and little effect in a sphere analogous to a tumor xenograft compared with 64 Cu in the Cy or on the CS. The dose deposited by 64 Cu is less effective for cell killing than γ-rays. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Systemic administration of low dosage of tetanus toxin decreases cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus

PubMed Central

Yan, Bing Chun; Kim, In Hye; Park, Joon Ha; Ahn, Ji Hyeon; Cho, Jeong-Hwi; Chen, Bai Hui; Lee, Jae-Chul; Choi, Jung Hoon; Yoo, Ki-Yeon; Lee, Choong Hyun; Cho, Jun Hwi

2013-01-01

In the present study, we investigated the effect of Tetaus toxin (TeT) on cell proliferation and neuroblast differentiation using specific markers: 5-bromo-2-deoxyuridine (BrdU) as an exogenous marker for cell proliferation, Ki-67 as an endogenous marker for cell proliferation and doublecortin (DCX) as a marker for neuroblasts in the mouse hippocampal dentate gyrus (DG) after TeT treatment. Mice were intraperitoneally administered 2.5 and 10 ng/kg TeT and sacrificed 15 days after the treatment. In both the TeT-treated groups, no neuronal death occurred in any layers of the DG using neuronal nuclei (NeuN, a neuron nuclei maker) and Fluoro-Jade B (F-J B, a high-affinity fluorescent marker for the localization of neuronal degeneration). In addition, no significant change in glial activation in both the 2.5 and 10 ng/kg TeT-treated-groups was found by GFAP (a marker for astrocytes) and Iba-1 (a marker for microglia) immunohistochemistry. However, in the 2.5 ng/kg TeT-treated-group, the mean number of BrdU, Ki-67 and DCX immunoreactive cells, respectively, were apparently decreased compared to the control group, and the mean number of each in the 10 ng/kg TeT-treated-group was much more decreased. In addition, processes of DCX-immunoreactive cells, which projected into the molecular layer, were short compared to those in the control group. In brief, our present results show that low dosage (10 ng/kg) TeT treatment apparently decreased cell proliferation and neuroblast differentiation in the mouse hippocampal DG without distinct gliosis as well as any loss of adult neurons. PMID:24106509

Bmi-1 plays a critical role in protection from renal tubulointerstitial injury by maintaining redox balance

PubMed Central

Jin, Jianliang; Lv, Xianhui; Chen, Lulu; Zhang, Wei; Li, Jinbo; Wang, Qian; Wang, Rong; Lu, Xiang; Miao, Dengshun

2014-01-01

To determine whether Bmi-1 deficiency could lead to renal tubulointerstitial injury by mitochondrial dysfunction and increased oxidative stress in the kidney, 3-week-old Bmi-1-/- mice were treated with the antioxidant N-acetylcysteine (NAC, 1 mg mL−1) in their drinking water, or pyrro-quinoline quinone (PQQ, 4 mg kg−1 diet) in their diet for 2 weeks, and their renal phenotypes were compared with vehicle-treated Bmi1-/- and wild-type mice. Bmi-1 was knocked down in human renal proximal tubular epithelial (HK2) cells which were treated with 1 mm NAC for 72 or 96 h, and their phenotypes were compared with control cells. Five-week-old vehicle-treated Bmi-1-/- mice displayed renal interstitial fibrosis, tubular atrophy, and severe renal function impairment with decreased renal cell proliferation, increased renal cell apoptosis and senescence, and inflammatory cell infiltration. Impaired mitochondrial structure, decreased mitochondrial numbers, and increased oxidative stress occurred in Bmi-1-/- mice; subsequently, this caused DNA damage, the activation of TGF-β1/Smad signaling, and the imbalance between extracellular matrix synthesis and degradation. Oxidative stress-induced epithelial-to-mesenchymal transition of renal tubular epithelial cells was enhanced in Bmi-1 knocked down HK2 cells. All phenotypic alterations caused by Bmi-1 deficiency were ameliorated by antioxidant treatment. These findings indicate that Bmi-1 plays a critical role in protection from renal tubulointerstitial injury by maintaining redox balance and will be a novel therapeutic target for preventing renal tubulointerstitial injury. PMID:24915841

Rotavirus and Serotonin Cross-Talk in Diarrhoea

PubMed Central

Nordgren, Johan; Karlsson, Thommie; Sharma, Sumit; Magnusson, Karl-Eric; Svensson, Lennart

2016-01-01

Rotavirus (RV) has been shown to infect and stimulate secretion of serotonin from human enterochromaffin (EC) cells and to infect EC cells in the small intestine of mice. It remains to identify which intracellularly expressed viral protein(s) is responsible for this novel property and to further establish the clinical role of serotonin in RV infection. First, we found that siRNA specifically silencing NSP4 (siRNANSP4) significantly attenuated secretion of serotonin from Rhesus rotavirus (RRV) infected EC tumor cells compared to siRNAVP4, siRNAVP6 and siRNAVP7. Second, intracellular calcium mobilization and diarrhoeal capacity from virulent and avirulent porcine viruses correlated with the capacity to release serotonin from EC tumor cells. Third, following administration of serotonin, all (10/10) infants, but no (0/8) adult mice, responded with diarrhoea. Finally, blocking of serotonin receptors using Ondansetron significantly attenuated murine RV (strain EDIM) diarrhoea in infant mice (2.9 vs 4.5 days). Ondansetron-treated mice (n = 11) had significantly (p < 0.05) less diarrhoea, lower diarrhoea severity score and lower total diarrhoea output as compared to mock-treated mice (n = 9). Similarly, Ondansetron-treated mice had better weight gain than mock-treated animals (p < 0.05). A most surprising finding was that the serotonin receptor antagonist significantly (p < 0.05) also attenuated total viral shedding. In summary, we show that intracellularly expressed NSP4 stimulates release of serotonin from human EC tumor cells and that serotonin participates in RV diarrhoea, which can be attenuated by Ondansetron. PMID:27459372

Omega-3 fatty acids as adjunctive treatment for bexarotene-induced hypertriglyceridaemia in patients with cutaneous T-cell lymphoma.

PubMed

Cabello, I; Servitje, O; Corbella, X; Bardés, I; Pintó, X

2017-04-01

Bexarotene is an oral retinoid approved for treating cutaneous T-cell lymphoma (CTCL) in patients resistant to first-line systemic treatment. Hypertriglyceridaemia is an unavoidable adverse effect of bexarotene therapy, and requires monitoring because of the risk of developing pancreatitis. Therefore, prophylactic hypolipidaemic therapy, usually with a fibrate alone, is required for preventing bexarotene-induced hypertriglyceridaemia. Despite these measures, a large number of patients develop very severe hypertriglyceridaemia. To assess the lipid metabolism changes before and after the use of a combination of omega-3 fatty acids (n-3 FA) plus fenofibrate compared with fenofibrate alone as a more effective lipid-lowering therapy in patients with CTCL treated with bexarotene. From January 2005 to January 2013, we analysed all 25 patients with CTCL treated with bexarotene. The first 18 consecutively enrolled patients received fenofibrate alone as a lipid-lowering therapy, and the next 7 consecutively enrolled patients received a combination of fenofibrate and n-3 FA. Data for all 25 consecutive patients with CTCL treated with bexarotene were evaluated. Of these, 24 patients (96%) developed hypertriglyceridaemia despite the hypolipidaemic therapy, with this being very severe (> 11.2 mmol/L) in 20% of the cases. Of the 18 patients receiving fenofibrate alone, 5 (28%) developed very severe hypertriglyceridaemia, compared with none of the 7 patients treated with the n-3 FA combination. Our results suggest that the n-3 FA combination may be more effective than fibrate alone for preventing bexarotene-induced hypertriglyceridaemia. © 2017 British Association of Dermatologists.

Glucosamine Treatment-mediated O-GlcNAc Modification of Paxillin Depends on Adhesion State of Rat Insulinoma INS-1 Cells*

PubMed Central

Kwak, Tae Kyoung; Kim, Hyeonjung; Jung, Oisun; Lee, Sin-Ae; Kang, Minkyung; Kim, Hyun Jeong; Park, Ji-Min; Kim, Sung-Hoon; Lee, Jung Weon

2010-01-01

Protein-protein interactions and/or signaling activities at focal adhesions, where integrin-mediated adhesion to extracellular matrix occurs, are critical for the regulation of adhesion-dependent cellular functions. Although the phosphorylation and activities of focal adhesion molecules have been intensively studied, the effects of the O-GlcNAc modification of their Ser/Thr residues on cellular functions have been largely unexplored. We investigated the effects of O-GlcNAc modification on actin reorganization and morphology of rat insulinoma INS-1 cells after glucosamine (GlcN) treatment. We found that paxillin, a key adaptor molecule in focal adhesions, could be modified by O-GlcNAc in INS-1 cells treated with GlcN and in pancreatic islets from mice treated with streptozotocin. Ser-84/85 in human paxillin appeared to be modified by O-GlcNAc, which was inversely correlated to Ser-85 phosphorylation (Ser-83 in rat paxillin). Integrin-mediated adhesion signaling inhibited the GlcN treatment-enhanced O-GlcNAc modification of paxillin. Adherent INS-1 cells treated with GlcN showed restricted protrusions, whereas untreated cells showed active protrusions for multiple-elongated morphologies. Upon GlcN treatment, expression of a triple mutation (S83A/S84A/S85A) resulted in no further restriction of protrusions. Together these observations suggest that murine pancreatic β cells may have restricted actin organization upon GlcN treatment by virtue of the O-GlcNAc modification of paxillin, which can be antagonized by a persistent cell adhesion process. PMID:20829364

Effect of synthetic antimicrobial peptides on Naegleria fowleri trophozoites.

PubMed

Tiewcharoen, Supathra; Phurttikul, Watchara; Rabablert, Jundee; Auewarakul, Prasert; Roytrakul, Sittiruk; Chetanachan, Pruksawan; Atithep, Thassanant; Junnu, Virach

2014-05-01

We evaluated the effect of tritrpticin, lactoferrin, killer decapeptide and scrambled peptide in vitro against Naegleria fowleri trophozoites compared with amphotericin B. Tritrpticin (100 microg/ml) caused apoptosis of N. fowleri trophozoites (2x10(5) cells/ml), while lactoferrin, killer decapeptide and scrambled peptide did not. On Gormori trichrome staining, tritrpticin affected the elasticity of the surface membrane and reduced the size of the nuclei of N. fowleri trophozoites. The ultrastructure surface membrane and food cup formation of the trophozoites were 100% inhibited. These results are consistent with inhibition of the nfa1, Mp2CL5 of the treated trophozoite, which plays a role in food cup formation. Tritrpticin 100 microg/ml was not toxic against SK-N-MC cells. Our findings suggest tritrpticin has activity against the surface membrane and nfa1 and Mp2CL5 of N. fowleri trophozoites and could be developed as a potential therapeutic agent.

Effect of N-benzoyl-D-phenylalanine and metformin on insulin receptors in neonatal streptozotocin-induced diabetic rats: studies on insulin binding to erythrocytes.

PubMed

Ashokkumar, N; Pari, L; Rao, Ch Appa

2006-07-01

In the present study, we focused on the insulin-receptor binding in circulating erythrocytes of N-benzoyl-D-phenylalanine (NBDP) and metformin in neonatal streptozotocin (nSTZ)-induced male Wistar rats. We measured blood levels of glucose and plasma insulin and the binding of insulin to cell-membrane ER receptors in NBDP and metformin-treated diabetic rats. The mean specific binding of insulin to ER was significantly lower in diabetic control rats (DC) (53.0 +/- 3.1%) than in NBDP (62.0 +/- 3.1%), metformin (66.0 +/- 3.3%) and NBDP and metformin combination-treated (72.0 +/- 4.2%) diabetic rats, resulting in a significant decrease in plasma insulin. Scatchard plot analysis demonstrated that the decrease in insulin binding was accounted for by a lower number of insulin receptor sites per cell in DC rats when compared with NBDP and metformin-treated rats. High-affinity (Kd1), low-affinity (Kd2), and kinetic analysis revealed an increase in the average receptor affinity in ER from NBDP and metformin-treated diabetic rats having NBDP 2.0 +/- 0.10 x 10(-10) M(-1) (Kd1); 12.0 +/- 0.85 x 10(-8) M(-1) (Kd2), Metformin 2.1 +/- 0.15 x 10(-10) M(-1) (Kd1); 15.0 +/- 0.80 x 10(-8) M(-1) (Kd2), NBDP and metformin 2.7 +/- 0.10 x 10(-10) M(-1) (Kd1); 20.0 +/- 1.2 x 10(-8) M(-1) (Kd2) compared with 0.9 +/- 0.06 x 10(-10) M(-1) (Kd1); 6.0 +/- 0.30 x 10(-8) M(-1) (Kd2) in DC rats. The results suggest an acute alteration in the number of insulin receptors on ER membranes in nSTZ induced diabetic control rats. Treatment with NBDP along with metformin significantly improved specific insulin binding, with receptor number and affinity binding reaching almost normal non-diabetic levels. The data presented here show that NBDP along with metformin increase total ER membrane insulin binding sites with a concomitant significant increase in plasma insulin.

Peroxisome proliferator-activated receptor-β/δ inhibits human neuroblastoma cell tumorigenesis by inducing p53- and SOX2-mediated cell differentiation.

PubMed

Yao, Pei-Li; Chen, Liping; Dobrzański, Tomasz P; Zhu, Bokai; Kang, Boo-Hyon; Müller, Rolf; Gonzalez, Frank J; Peters, Jeffrey M

2017-05-01

Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-β/δ, (PPARβ/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARβ/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARβ/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARβ/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARβ/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARβ/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARβ/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients. Published [2016]. This article is a U.S. Government work and is in the public domain in the USA.

Dynorphin up-regulation in the dentate granule cell mossy fiber pathway following chronic inhibition of GluN2B-containing NMDAR is associated with increased CREB (Ser 133) phosphorylation, but is independent of BDNF/TrkB signaling pathways.

PubMed

Rittase, W Bradley; Dong, Yu; Barksdale, DaRel; Galdzicki, Zygmunt; Bausch, Suzanne B

2014-05-01

Emerging evidence suggests that neuronal responses to N-methyl-d-aspartate (NMDAR) activation/inactivation are influenced by subunit composition. For example, activation of synaptic NMDAR (comprised of GluN2A>GluN2B) phosphorylates cAMP-response-element-binding protein (CREB) at Ser 133, induces BDNF expression and promotes neuronal survival. Activation of extrasynaptic NMDAR (comprised of GluN2B>GluN2) dephosphorylates CREB (Ser 133), reduces BDNF expression and triggers neuronal death. These results led us to hypothesize that chronic inhibition of GluN2B-containing NMDAR would increase CREB (Ser 133) phosphorylation, increase BDNF levels and subsequently alter downstream dynorphin (DYN) and neuropeptide Y (NPY) expression. We focused on DYN and NPY because these neuropeptides can decrease excitatory neurotransmission and seizure occurrence and we reported previously that seizure-like events are reduced following chronic treatment with GluN2B antagonists. Consistent with our hypothesis, chronic treatment (17-21days) of hippocampal slice cultures with the GluN2B-selective antagonists ifenprodil or Ro25,6981 increased both CREB (Ser 133) phosphorylation and granule cell mossy fiber pathway DYN expression. Similar treatment with the non-subtype-selective NMDAR antagonists d-APV or memantine had no significant effect on either CREB (Ser 133) phosphorylation or DYN expression. In contrast to our hypothesis, BDNF levels were decreased following chronic treatment with Ro25,6981, but not ifenprodil, d-APV or memantine. Blockade of BDNF actions and TrkB activation did not significantly augment hilar DYN expression in vehicle-treated cultures and had no effect in Ro25,6981 treated cultures. These findings suggest that chronic exposure to GluN2B-selective NMDAR antagonists increased DYN expression through a putatively pCREB-dependent, but BDNF/TrkB-independent mechanism. Published by Elsevier Inc.

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

Nitrogen mass balance in a constructed wetland treating piggery wastewater effluent.

PubMed

Lee, Soyoung; Maniquiz-Redillas, Marla C; Choi, Jiyeon; Kim, Lee-Hyung

2014-06-01

The nitrogen changes and the nitrogen mass balance in a free water surface flow constructed wetland (CW) using the four-year monitoring data from 2008 to 2012 were estimated. The CW was composed of six cells in series that include the first settling basin (Cell 1), aeration pond (Cell 2), deep marsh (Cell 3), shallow marsh (Cell 4), deep marsh (Cell 5) and final settling basin (Cell 6). Analysis revealed that the NH(+)4-N concentration decreased because of ammonification which was then followed by nitrification. The NO(-)2-N and NO(-)2-N were also further reduced by means of microbial activities and plant uptake during photosynthesis. The average nitrogen concentration at the influent was 37,819 kg/year and approximately 45% of that amount exited the CW in the effluent. The denitrification amounted to 34% of the net nitrogen input, whereas the accretion of sediment was only 7%. The biomass uptake of plants was able to retain only 1% of total nitrogen load. In order to improve the nutrient removal by plant uptake, plant coverage in four cells (i.e., Cells 1, 3, 4 and 5) could be increased. Copyright © 2014 The Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved.

Room-Temperature and Aqueous Solution-Processed Two-Dimensional TiS2 as an Electron Transport Layer for Highly Efficient and Stable Planar n-i-p Perovskite Solar Cells.

PubMed

Huang, Peng; Yuan, Ligang; Zhang, Kaicheng; Chen, Qiaoyun; Zhou, Yi; Song, Bo; Li, Yongfang

2018-05-02

In this study, a room-temperature and aqueous solution-processed two-dimensional (2D) transition-metal dichalcogenide TiS 2 was applied as an electron transport layer (ETL) in planar n-i-p perovskite solar cells (Pero-SCs). Upon insertion of the 2D TiS 2 ETL with UV-ozone (UVO) treatment, the power conversion efficiency (PCE) of the planar Pero-SCs was optimized to 18.79%. To the best of our knowledge, this value should be the highest efficiency to date among those PCEs of the n-i-p Pero-SCs with room-temperature-processed metal compound ETLs. More importantly, the n-i-p Pero-SCs with the UVO-treated 2D TiS 2 as an ETL also show extremely high stability, where the average PCE remained over 95% of its initial value after 816 h storage without encapsulation.

4β-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

PubMed

Tang, Jen-Yang; Huang, Hurng-Wern; Wang, Hui-Ru; Chan, Ya-Ching; Haung, Jo-Wen; Shu, Chih-Wen; Wu, Yang-Chang; Chang, Hsueh-Wei

2018-03-01

Reactive oxygen species (ROS) induction had been previously reported in 4β-hydroxywithanolide (4βHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4βHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4βHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4βHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4βHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4βHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4βHWE-treated oral cancer cells than in oral normal cells. All the 4βHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4βHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells. © 2017 Wiley Periodicals, Inc.

β2-Adrenergic Receptor Agonists Inhibit the Proliferation of 1321N1 Astrocytoma CellsS⃞

PubMed Central

Toll, L.; Jimenez, L.; Waleh, N.; Jozwiak, K.; Woo, A.Y.-H.; Xiao, R.-P.; Bernier, M.

2011-01-01

Astrocytomas and glioblastomas have been particularly difficult to treat and refractory to chemotherapy. However, significant evidence has been presented that demonstrates a decrease in astrocytoma cell proliferation subsequent to an increase in cAMP levels. The 1321N1 astrocytoma cell line, as well as other astrocytomas and glioblastomas, expresses β2-adrenergic receptors (β2-ARs) that are coupled to Gs activation and consequent cAMP production. Experiments were conducted to determine whether the β2-AR agonist (R,R′)-fenoterol and other β2-AR agonists could attenuate mitogenesis and, if so, by what mechanism. Receptor binding studies were conducted to characterize β2-AR found in 1321N1 and U118 cell membranes. In addition, cells were incubated with (R,R′)-fenoterol and analogs to determine their ability to stimulate intracellular cAMP accumulation and inhibit [3H]thymidine incorporation into the cells. 1321N1 cells contain significant levels of β2-AR as determined by receptor binding. (R,R′)-fenoterol and other β2-AR agonists, as well as forskolin, stimulated cAMP accumulation in a dose-dependent manner. Accumulation of cAMP induced a decrease in [3H]thymidine incorporation. There was a correlation between concentration required to stimulate cAMP accumulation and inhibit [3H]thymidine incorporation. U118 cells have a reduced number of β2-ARs and a concomitant reduction in the ability of β2-AR agonists to inhibit cell proliferation. These studies demonstrate the efficacy of β2-AR agonists for inhibition of growth of the astrocytoma cell lines. Because a significant portion of brain tumors contain β2-ARs to a greater extent than whole brain, (R,R′)-fenoterol, or some analog, may be useful in the treatment of brain tumors after biopsy to determine β2-AR expression. PMID:21071556

KML001 Induces Apoptosis and Autophagic Cell Death in Prostate Cancer Cells via Oxidative Stress Pathway

PubMed Central

You, Dalsan; Kim, Yunlim; Jang, Myoung Jin; Lee, Chunwoo; Jeong, In Gab; Cho, Yong Mee; Hwang, Jung Jin; Hong, Jun Hyuk; Ahn, Hanjong; Kim, Choung-Soo

2015-01-01

We investigated the effects of KML001 (NaAsO2, sodium metaarsenite, Kominox), an orally bioavailable arsenic compound, on the growth and death of human prostate cancer cells and its mechanism of action. Growth inhibition was assessed by cytotoxicity assays in the presence or absence of inhibitor of apoptosis, inhibitor of autophagy or antioxidant N-Acetyl-L-cysteine to study mechanism of cell death induced by KML001 in PC3, DU145 and LNCaP prostate cancer cell lines. Electron microscopy, flow cytometry and Western blotting were used to study apoptotic and autophagic mechanisms. The DU145 xenograft model was used to determine the efficacy of KML001 in vivo. KML001 decreased the viability of cells and increased the percentage of annexin V-positive cells dose-dependently in prostate cancer cells, and LNCaP cells were more sensitive to KML001 than PC3 or DU145 cells. Electron microscopy revealed typical apoptotic characters and autophagic vacuoles in cells treated with KML001. Exposure to KML001 in prostate cancer cells induced apoptosis and autophagy in a time- and dose-dependent manner. KML001 induced dose-dependent accumulation of reactive oxygen species, and scavenging the reactive oxygen species with N-Acetyl-L-cysteine reduced LC3 and cleaved poly (ADP-ribose) polymerase. KML001 significantly inhibited tumor growth in the DU145 xenograft model. In addition, significant decrease of proliferation and significant increases of apoptosis and autophagy were observed in KML001-treated tumors than in vehicle-treated tumors. Exposure of human prostate cancer cells to KML001 induced both apoptosis and autophagic cell death via oxidative stress pathway. And KML001 had an antiproliferative effect on DU145 cells in xenograft mice. PMID:26352139

KML001 Induces Apoptosis and Autophagic Cell Death in Prostate Cancer Cells via Oxidative Stress Pathway.

PubMed

You, Dalsan; Kim, Yunlim; Jang, Myoung Jin; Lee, Chunwoo; Jeong, In Gab; Cho, Yong Mee; Hwang, Jung Jin; Hong, Jun Hyuk; Ahn, Hanjong; Kim, Choung-Soo

2015-01-01

We investigated the effects of KML001 (NaAsO2, sodium metaarsenite, Kominox), an orally bioavailable arsenic compound, on the growth and death of human prostate cancer cells and its mechanism of action. Growth inhibition was assessed by cytotoxicity assays in the presence or absence of inhibitor of apoptosis, inhibitor of autophagy or antioxidant N-Acetyl-L-cysteine to study mechanism of cell death induced by KML001 in PC3, DU145 and LNCaP prostate cancer cell lines. Electron microscopy, flow cytometry and Western blotting were used to study apoptotic and autophagic mechanisms. The DU145 xenograft model was used to determine the efficacy of KML001 in vivo. KML001 decreased the viability of cells and increased the percentage of annexin V-positive cells dose-dependently in prostate cancer cells, and LNCaP cells were more sensitive to KML001 than PC3 or DU145 cells. Electron microscopy revealed typical apoptotic characters and autophagic vacuoles in cells treated with KML001. Exposure to KML001 in prostate cancer cells induced apoptosis and autophagy in a time- and dose-dependent manner. KML001 induced dose-dependent accumulation of reactive oxygen species, and scavenging the reactive oxygen species with N-Acetyl-L-cysteine reduced LC3 and cleaved poly (ADP-ribose) polymerase. KML001 significantly inhibited tumor growth in the DU145 xenograft model. In addition, significant decrease of proliferation and significant increases of apoptosis and autophagy were observed in KML001-treated tumors than in vehicle-treated tumors. Exposure of human prostate cancer cells to KML001 induced both apoptosis and autophagic cell death via oxidative stress pathway. And KML001 had an antiproliferative effect on DU145 cells in xenograft mice.

Interleukin-6 and vascular endothelial growth factor release by renal cell carcinoma cells impedes lymphocyte-dendritic cell cross-talk.

PubMed

Cabillic, F; Bouet-Toussaint, F; Toutirais, O; Rioux-Leclercq, N; Fergelot, P; de la Pintière, C Thomas; Genetet, N; Patard, J-J; Catros-Quemener, V

2006-12-01

Anti-tumour T cell response requires antigen presentation via efficient immunological synapse between antigen presenting cells, e.g. dendritic cells (DC), and specific T cells in an adapted Th1 cytokine context. Nine renal cell carcinoma (RCC) primary culture cells were used as sources of tumour antigens which were loaded on DC (DC-Tu) for autologous T cell activation assays. Cytotoxic activity of lymphocytes stimulated with DC-Tu was evaluated against autologous tumour cells. Assays were performed with 75 grays irradiated tumour cells (Tu irr) and with hydrogen peroxide +/- heat shock (Tu H(2)O(2) +/- HS) treated cells. DC-Tu irr failed to enhance cytotoxic activity of autologous lymphocytes in seven of 13 assays. In all these defective assays, irradiated tumour cells displayed high interleukin (IL)-6 and vascular endothelial growth factor (VEGF) release. Conversely, when tumour cells released low IL-6 levels (n = 4), DC-Tu irr efficiently enhanced CTL activity. When assays were performed with the same RCC cells treated with H(2)O(2) + HS, DC-Tu stimulation resulted in improved CTL activity. H(2)O(2) + HS treatment induced post-apoptotic cell necrosis of tumour cells, totally abrogated their cytokine release [IL-6, VEGF, transforming growth factor (TGF)-beta1] and induced HSP70 expression. Taken together, data show that reduction in IL-6 and VEGF release in the environment of the tumour concomitantly to tumour cell HSP expression favours induction of a stronger anti-tumour CTL response.

Induced Polyploidy in Diploid Ornamental Ginger (Hedychium muluense) Using Colchicine and Oryzalin

USDA-ARS?s Scientific Manuscript database

The ploidy level of H. muluense, a diploid (2n = 2x = 34) and dwarf ornamental ginger species, has been determined and is reported for the first time. Oryzalin and colchicine were successfully used to induce polyploidy in Hedychium muluense in vitro. Embryogenic cell lines were treated with oryzalin...

Efficacy of mitoxantrone against various neoplasms in dogs.

PubMed

Ogilvie, G K; Obradovich, J E; Elmslie, R E; Vail, D M; Moore, A S; Straw, R C; Dickinson, K; Cooper, M F; Withrow, S J

1991-05-01

One hundred twenty-six dogs with histologically confirmed, measurable malignant tumors were evaluated in a prospective study to determine the response to the antineoplastic drug mitoxantrone. Ninety-five dogs had been refractory to one or more treatment modalities (surgery, n = 57; chemotherapy other than mitoxantrone, n = 37; radiation, n = 4; whole body hyperthermia, n = 1). The extent of neoplastic disease was determined immediately before each dose of mitoxantrone was administered (1 to 10 doses, 2.5 to 5 mg/m2 of body surface area, IV) 21 days apart. Each dog was treated with mitoxantrone until the dog developed progressive disease or until the dog's quality of life diminished to an unacceptable level as determined by the owner or attending veterinarian. A partial or complete remission (greater than 50% volume reduction) was obtained in 23% (29/126) of all dogs treated. Tumors in which there was a partial or complete remission included lymphoma (11/32), squamous cell carcinoma (4/9), fibrosarcoma (2/9), thyroid carcinoma (1/10), transitional cell carcinoma (1/6), mammary adenocarcinoma (1/6), hepatocellular carcinoma (1/4), renal adenocarcinoma (1/1), rectal carcinoma (1/1), chondrosarcoma (1/2), oral malignant melanoma (1/12), cutaneous malignant melanoma (1/1), myxosarcoma (1/1), mesothelioma (1/1), and hemangiopericytoma (1/1). Our results indicated that mitoxantrone induces measurable regression in various malignant tumors in dogs.

Depletion of intracellular GSH and NPSH by buthionine sulfoximine and diethyl maleate: factors that influence enhancement of aerobic radiation response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varnes, M.E.; Biaglow, J.E.; Roizin-Towle, L.

1984-08-01

Many investigators have observed aerobic sensitization of V79, CHO and A549 (human lung carcinoma) cells upon depletion of GSH using buthionine sulfoximine (BSO). Recently the authors discovered that this aerobic sensitization can be reversed if WR-2721 or N-acetylcysteine is added to the cells just prior to irradiation. Reversal requires that the exogenous thiols be present during the time of irradiation. One possible explanation was that these thiols entered the cells and either increased the pool of cellular nonprotein thiols or reversed the thiol-depleted state by stimulation of GSH synthesis. Cells treated with BSO do not readily regenerate intracellular GSH becausemore » this agent irreversibly inhibits ..gamma..-glutamyl synthetase. They found that addition of WR-2721 or N-acetylcysteine to BSO-treated cells did not affect the rate of regeneration of intracellular GSH. Thus, reversal of the aerobic sensitization of A549 cells by BSO cannot be explained on the basis of intracellular thiol levels alone, or by rapid reversal of BSO inhibition. In addition, diethylmaleate (DEM)-treated cells are considerably different from BSO-treated cells with respect to the ability to regenerate GSH.« less

A comparison of human papillomavirus testing of clinician-collected and self-collected samples during follow-up after screen-and-treat.

PubMed

Taylor, Sylvia; Wang, Chunhui; Wright, Thomas C; Denny, Lynette; Kuhn, Louise

2011-08-15

Screen-and-treat cervical cancer prevention programs based on high-risk human papillomavirus (HPV) testing and cryotherapy have been shown to be effective in resource-limited settings. However, because cryotherapy is not 100% effective, follow-up is needed after treatment to detect post-treatment failures. We compared the test performances of high-risk HPV testing (Hybrid Capture 2) using self-collected and clinician-collected samples as well as cervical cytology for identifying cervical intraepithelial neoplasia grades 2 or 3 or invasive cervical cancer (CIN2+) among women who did (n=812) and did not (n=1858) undergo cryotherapy in a South African screen-and-treat trial. At 6 months after enrolment (and after cryotherapy, if performed), women were tested using all three screening methods and then underwent colposcopy/biopsy. A predefined subset of women (n=1,455) had extended follow-up with colposcopy/biopsy at 12 months. A total of 33 and 91 cases of CIN2+ were detected among treated and untreated women, respectively. The sensitivity of HPV testing using clinician-collected samples and cervical cytology did not differ by treatment status. HPV testing of clinician-collected samples detected the most cases of CIN2+ among both treated (85%) and untreated (91%) women (p=0.31). Cytology (at a cutoff of atypical squamous cells of undetermined significance or greater) detected 76% of cases among both treated and untreated women. However, the sensitivity of HPV testing using self-collected samples was significantly lower among treated versus untreated women (55% vs. 78%, p=0.01). HPV testing using self-collected vaginal specimens may be useful in primary screening but performs poorly for detecting post-treatment failures. Copyright © 2010 UICC.

Different induction of LPA receptors by chemical liver carcinogens regulates cellular functions of liver epithelial WB-F344 cells.

PubMed

Hirane, Miku; Ishii, Shuhei; Tomimatsu, Ayaka; Fukushima, Kaori; Takahashi, Kaede; Fukushima, Nobuyuki; Honoki, Kanya; Tsujiuchi, Toshifumi

2016-11-01

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA 1 to LPA 6 ) mediates a variety of cellular functions, including cell motility. In the present study, we investigated the effects of LPA receptors on cell motile activity during multi-stage hepatocarcinogenesis in rat liver epithelial WB-F344 cells treated with chemical liver carcinogens. Cells were treated with a initiator (N-nitrosodiethylamine (DEN)) and three promoters (phenobarbital (PB), okadaic acid (OA) and clofibrate) every 24 h for 2 days. Cell motile activity was elevated by DEN, correlating with Lpar3 expression. PB, OA, and clofibrate elevated Lpar1 expression and inhibited cell motile activity. To evaluate the effects of long-term treatment on cell motility, cells were treated with DEN and/or PB for at least 6 months. Lpar3 expression and cell motile activity were significantly elevated by the long-term DEN treatment with or without further PB treatment. In contrast, long-term PB treatment with or without further DEN elevated Lpar1 expression and inhibited cell motility. When the synthesis of extracellular LPA was blocked by a potent ATX inhibitor S32826 before cell motility assay, the cell motility induced by DEN and PB was markedly suppressed. These results suggest that activation of the different LPA receptors may regulate the biological functions of cells treated with chemical carcinogens. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

The prognostic impact of supraclavicular lymph node in N3-IIIB stage non-small cell lung cancer patients treated with definitive concurrent chemo-radiotherapy.

PubMed

Oh, Dongryul; Ahn, Yong Chan; Park, Hee Chul; Lim, Do Hoon; Noh, Jae Myoung; Cho, Won Kyung; Pyo, Hongryull

2017-05-30

This study aimed to investigate the prognostic impact of supraclavicular lymph node (SCN) metastasis in patients who were treated with definitive chemoradiotherapy for N3-IIIB stage non-small cell lung cancer (NSCLC). The 2- and 5-year overall survival (OS) rates were 57.3% and 35.7% in patients without SCN metastasis and 56.4% and 26.7% in patients with SCN metastasis, respectively. The median OS was 34 months in both groups. There was no significant difference in OS between the two groups (p = 0.679). The 2- and 5-year progression-free survival (PFS) rates were 24.1% and 12.6% in patients without SCN metastasis and 18.0% and 16.0% in patients with SCN metastasis, respectively. Patients without SCN metastasis had slightly longer median PFS (10 months vs. 8 months), but the difference was not statistically significant (p = 0.223). In multivariate analysis, SCN metastasis was not a significant factor for OS (p = 0.391) and PFS (p = 0.149). This retrospective analysis included 204 consecutive patients who were treated with chemoradiotherapy for N3-IIIB stage NSCLC between May 2003 and December 2012. A median RT dose of 66 Gy was administered over 6.5 weeks. Of these, 119 patients (58.3%) had SCN metastasis and 85 (41.7%) had another type of N3 disease: mediastinal N3 nodes in 84 patients (98.8%) and contralateral hilar node in one (1.2%). The patients were divided into two groups according to SCN metastasis. SCN metastasis does not compromise treatment outcomes compared to other mediastinal metastasis in the setting of definitive chemoradiotherapy.

Acrolein oxidizes the cytosolic and mitochondrial thioredoxins in human endothelial cells.

PubMed

Szadkowski, Adam; Myers, Charles R

2008-01-14

Acrolein is a reactive aldehyde that is a widespread environmental pollutant and can be generated endogenously from lipid peroxidation. The thioredoxin (Trx) system in endothelial cells plays a major role in the maintenance of cellular thiol redox balance, and is critical for cell survival. Normally, cells maintain the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins largely in the reduced state. In human microvascular endothelial cells, Trx1 was more sensitive than Trx2 to oxidation by acrolein. A 30-min exposure to 2.5 microM acrolein caused partial oxidation of Trx1 but not Trx2. The active site dithiol of Trx1 was essentially completely oxidized by 5 microM acrolein whereas 12.5 microM was required for complete oxidation of Trx2. Partial recovery of the Trx1 redox status was observed over a 4h acrolein-free recovery period, with increases in the reduced form and decreases in the fully oxidized form. For cells treated with 2.5 or 5 microM acrolein the recovery did not require protein synthesis, whereas protein synthesis was required for the return of reduced Trx1 in cells treated with 12.5 microM acrolein. Pretreatment of cells with N-acetylcysteine (NAC) resulted in partial protection of Trx1 from oxidation by acrolein. In cells treated with acrolein for 30 min, followed by a 14- to 16-h acrolein-free period, small but significant cytotoxic effects were observed with 2.5 microM acrolein whereas all cells were adversely affected by >or= 12.5 microM. NAC pretreatment significantly decreased the percentage of stressed cells subsequently exposed to 5 or 12.5 microM acrolein. Given the critical role of the thioredoxins in cell survival, the ability of acrolein to oxidize both thioredoxins should be taken into account for a thorough understanding of its cytotoxic effects.

Modulation of gene expression and cell-cycle signaling pathways by the EGFR inhibitor gefitinib (Iressa) in rat urinary bladder cancer.

PubMed

Lu, Yan; Liu, Pengyuan; Van den Bergh, Francoise; Zellmer, Victoria; James, Michael; Wen, Weidong; Grubbs, Clinton J; Lubet, Ronald A; You, Ming

2012-02-01

The epidermal growth factor receptor inhibitor Iressa has shown strong preventive efficacy in the N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) model of bladder cancer in the rat. To explore its antitumor mechanism, we implemented a systems biology approach to characterize gene expression and signaling pathways in rat urinary bladder cancers treated with Iressa. Eleven bladder tumors from control rats, seven tumors from rats treated with Iressa, and seven normal bladder epithelia were profiled by the Affymetrix Rat Exon 1.0 ST Arrays. We identified 713 downregulated and 641 upregulated genes in comparing bladder tumors versus normal bladder epithelia. In addition, 178 genes were downregulated and 96 genes were upregulated when comparing control tumors versus Iressa-treated tumors. Two coexpression modules that were significantly correlated with tumor status and treatment status were identified [r = 0.70, P = 2.80 × 10(-15) (bladder tumor vs. normal bladder epithelium) and r = 0.63, P = 2.00 × 10(-42) (Iressa-treated tumor vs. control tumor), respectively]. Both tumor module and treatment module were enriched for genes involved in cell-cycle processes. Twenty-four and twenty-one highly connected hub genes likely to be key drivers in cell cycle were identified in the tumor module and treatment module, respectively. Analysis of microRNA genes on the array chips showed that tumor module and treatment module were significantly associated with expression levels of let-7c (r = 0.54, P = 3.70 × 10(-8) and r = 0.73, P = 1.50 × 10(-65), respectively). These results suggest that let-7c downregulation and its regulated cell-cycle pathway may play an integral role in governing bladder tumor suppression or collaborative oncogenesis and that Iressa exhibits its preventive efficacy on bladder tumorigenesis by upregulating let-7 and inhibiting the cell cycle. Cell culture study confirmed that the increased expression of let-7c decreases Iressa-treated bladder tumor cell growth. The identified hub genes may also serve as pharmacodynamic or efficacy biomarkers in clinical trials of chemoprevention in human bladder cancer. ©2011 AACR.

In vitro fatty acid enrichment of macrophages alters inflammatory response and net cholesterol accumulation

PubMed Central

Wang, Shu; Wu, Dayong; Lamon-Fava, Stefania; Matthan, Nirupa R.; Honda, Kaori L.; Lichtenstein, Alice H.

2010-01-01

Dietary long-chain PUFA, both n-3 and n-6, have unique benefits with respect to CVD risk. The aim of the present study was to determine the mechanisms by which n-3 PUFA (EPA, DHA) and n-6 PUFA (linoleic acid (LA), arachidonic acid (AA)) relative to SFA (myristic acid (MA), palmitic acid (PA)) alter markers of inflammation and cholesterol accumulation in macrophages (MΦ). Cells treated with AA and EPA elicited significantly less inflammatory response than control cells or those treated with MA, PA and LA, with intermediate effects for DHA, as indicated by lower levels of mRNA and secretion of TNFα, IL-6 and monocyte chemoattractant protein-1. Differences in cholesterol accumulation after exposure to minimally modified LDL were modest. AA and EPA resulted in significantly lower MΦ scavenger receptor 1 mRNA levels relative to control or MA-, PA-, LA- and DHA-treated cells, and ATP-binding cassette A1 mRNA levels relative to control or MA-, PA- and LA-treated cells. These data suggest changes in the rate of bidirectional cellular cholesterol flux. In summary, individual long-chain PUFA have differential effects on inflammatory response and markers of cholesterol flux in MΦ which are not related to the n position of the first double bond, chain length or degree of saturation. PMID:19660150

Cattle with increased severity of bovine respiratory disease complex exhibit decreased capacity to protect against histone cytotoxicity.

PubMed

Matera, J A; Wilson, B K; Hernandez Gifford, J A; Step, D L; Krehbiel, C R; Gifford, C A

2015-04-01

Bovine respiratory disease complex (BRDC) is the leading cause of morbidity and mortality in feedlot cattle. Significant inflammation and lesions are often observed in lungs of infected cattle. During acute inflammatory responses, histones contribute to mortality in rodents and humans and serum proteins can protect against histone-induced cytotoxicity. We hypothesized that cattle experiencing chronic or fatal cases of BRDC have reduced ability to protect against cytotoxic effects of histones. Serum samples were collected from 66 bull calves at the time of normal feedlot processing procedures. Animals were retrospectively assigned to groups consisting of calves never treated for BRDC (control [CONT]; n = 10), calves treated with antimicrobials once for BRDC (1T; n = 16), calves treated twice for BRDC (2T; n = 13), calves treated 3 times for BRDC (3T; n = 14), or calves treated 4 times for BRDC (4T; n = 13). Samples were also collected each time animals received antimicrobial treatment; animals within a group were further sorted by calves that recovered and calves that died to test histone cytotoxicity. Bovine kidney cells were cultured in duplicate in 96-well plates and exposed to 0 or 50 μg/mL of total histones for 18 h with 1% serum from each animal. Cell viability was assessed by the addition of resazurin for 6 h followed by fluorescent quantification. Fluorescent values from serum alone were subtracted from values obtained for histone treatment for each animal. Serum from CONT, 1T, and 2T at initial processing all exhibited a similar (P > 0.10) response to histone treatment with fluorescent values of -312 ± 557, -1,059 ± 441, and -975 ± 489, respectively. However, 3T and 4T demonstrated an impaired capacity (P < 0.05) to protect against histones (-2,778 ± 471 and -3,026 ± 489) at initial processing when compared to the other groups. When sorted by mortality within group, calves that were treated twice and recovered (-847 ± 331) demonstrated a greater (P < 0.05) protective capacity than calves that were treated twice and died (-2,264 ± 412), indicating that calves that contract BRDC and ultimately die might have reduced protective capacity against histone cytotoxicity. Results suggest that calves that require multiple treatments for BRDC have reduced ability to protect against cytotoxicity of histones. Understanding the primary mechanism responsible for protecting against histone cytotoxicity could lead to improved identification of animals susceptible to severe cases of BRDC, improved focus and use of available resources, or better treatments for severe cases of BRDC

Coniferyl alcohol hinders the growth of tobacco BY-2 cells and Nicotiana benthamiana seedlings.

PubMed

Väisänen, Enni E; Smeds, Annika I; Fagerstedt, Kurt V; Teeri, Teemu H; Willför, Stefan M; Kärkönen, Anna

2015-09-01

Externally added coniferyl alcohol at high concentrations reduces the growth of Nicotiana cells and seedlings. Coniferyl alcohol is metabolized by BY-2 cells to several compounds. Coniferyl alcohol (CA) is a common monolignol and a building block of lignin. The toxicity of monolignol alcohols has been stated in the literature, but there are only few studies suggesting that this is true. We investigated the physiological effects of CA on living plant cells in more detail. Tobacco (Nicotiana tabacum) Bright yellow-2 cells (BY-2) and Nicotiana benthamiana seedlings both showed concentration-dependent growth retardation in response to 0.5-5 mM CA treatment. In some cases, CA addition caused cell death in BY-2 cultures, but this response was dependent on the growth stage of the cells. Based on LC-MS/MS analysis, BY-2 cells did not accumulate the externally supplemented CA, but metabolized it to ferulic acid, ferulic acid glycoside, coniferin, and to some other phenolic compounds. In addition to growth inhibition, CA caused the formation of a lignin-like compound detected by phloroglucinol staining in N. benthamiana roots and occasionally in BY-2 cells. To prevent this, we added potassium iodide (KI, at 5 mM) to overcome the peroxidase-mediated CA polymerization to lignin. KI had, however, toxic effects on its own: in N. benthamiana seedlings, it caused reduction in growth; in BY-2 cells, reduction in growth and cell viability. Surprisingly, CA restored the growth of KI-treated BY-2 cells and N. benthamiana seedlings. Our results suggest that CA at high concentrations is toxic to plant cells.

Differential apoptotic effect of wogonin and nor-wogonin via stimulation of ROS production in human leukemia cells.

PubMed

Chow, Jyh-Ming; Huang, Guan-Cheng; Shen, Shing-Chuan; Wu, Chin-Yen; Lin, Cheng-Wei; Chen, Yen-Chou

2008-04-01

We investigate the roles of methoxyl (OCH(3)) and hydroxyl (OH) substitutions at C8 of flavonoids on their apoptosis-inducing activities. Wogonin (Wog) and nor-wogonin (N-Wog) are structurally related flavonoids, and respectively contain an OH and OCH(3) at C8. In leukemia HL-60 cells, N-Wog exhibited more-potent cytotoxicity than Wog according to the MTT and LDH release assays, and the IC(50) values of Wog and N-Wog in HL-60 cells were 67.5 +/- 2.1 and 21.7 +/- 1.5 microM, respectively. Apoptotic characteristics including DNA ladders, apoptotic bodies, and hypodiploid cells accompanied by the induction of caspase 3 protein processing appeared in Wog- and N-Wog-treated HL-60 cells. Interestingly, an increase in intracellular peroxide production was detected in N-Wog- but not Wog-treated HL-60 cells by the DCHF-DA assay, and the reduction of intracellular peroxide by catalase (CAT) induced by N-Wog significantly reduced the N-Wog- but not the Wog-induced cytotoxic effect according to the MTT assay in accordance with the blocking of DNA ladder formation and caspase 3 and PARP protein processing elicited by N-Wog. We further analyzed the effect of six structurally related compounds, including 5-OH, 7-OH, 5,7-diOH, 5,7-diOCH(3), 7,8-diOCH(3), and 7-OCH(3)-8-OH flavones, on apoptosis induction in HL-60 cells. Results suggested that OH at C5 and C7 is essential for both the apoptosis-inducing activity of flavonoids, and OH at C8 may contribute to apoptosis induction ability. Evidence to support a distinct structure-activity relationship in apoptosis induction of flavonoids is provided for the first time in this study. 2007 Wiley-Liss, Inc.

Per a 10 activates human derived epithelial cell line in a protease dependent manner via PAR-2.

PubMed

Kale, Sagar L; Arora, Naveen

2015-04-01

Protease activity of Per a 10 has been shown to modulate dendritic cells toward Th-2 polarization and to induce airway inflammation. To elucidate the role of serine protease activity of Per a 10 in inducing biochemical responses in epithelial cells. Per a 10 was inactivated by heat treatment (ΔPer a 10) or AEBSF (iPer a 10). A549 cells were exposed to either enzymatically active/inactive Per a 10. The supernatant was analyzed for the secretion of proinflammatory cytokines by ELISA. Ca(2+) mobilization was analyzed by flow cytometry. A PAR-2 derived synthetic peptide 28GTNRSSKGRSLIGKVDGTSHVTGKGVTC54 was incubated with Per a 10 and the resultant cleaved products were analyzed by LC-MS. PAR-2 activation was inhibited by PAR-2 cleavage inhibiting antibody. ΔPer a 10 was completely inactivated whereas iPer a 10 showed some residual activity. nPer a 10 having protease activity increased the secretion of IL-6, IL-8 and GMCSF from A549 in a dose and time dependent manner whereas iPer a 10 has reduced cytokine secretion. ΔPer a 10 and rPer a 10 were unable to activate the cells. nPer a 10 mobilized intracellular Ca(2+). nPer a 10 cleaved the PAR-2 derived peptide between arginine and serine residues (36R-S37) to expose PAR-2 ligand SLIGKV, as determined by LC-MS. Incubating with anti-PAR-2 cleavage antibody showed diminished cytokine secretion when treated with nPer a 10. Serine protease activity of Per a 10 activates A549 cells to secrete proinflammatory cytokines by PAR-2 activation and Ca(2+)mobilization and can be exploited therapeutically. Copyright © 2014 Elsevier GmbH. All rights reserved.

Examination by EPR spectroscopy of free radicals in melanins isolated from A-375 cells exposed on valproic acid and cisplatin.

PubMed

Chodurek, Ewa; Zdybel, Magdalena; Pilawa, Barbara; Dzierzewicz, Zofia

2012-01-01

Drug binding by melanin biopolymers influence the effectiveness of the chemotherapy, radiotherapy and photodynamic therapy. Free radicals of melanins take part in formation of their complex with drugs. The aim of this work was to determine the effect of the two compounds: valproic acid (VPA) and cisplatin (CPT) on free radicals properties of melanin isolated from A-375 melanoma cells. Free radicals were examined by an X-band (9.3 GHz) electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were measured for the model synthetic eumelanin - DOPA-melanin, the melanin isolated from the control A-375 cells and these cells treated by VPA, CPT and both VPA and CPT. For all the examined samples broad EPR lines (deltaBpp: 0.48-0.68 mT) with g-factors of 2.0045-2.0060 characteristic for o-semiquinone free radicals were observed. Free radicals concentrations (N) in the tested samples, g-factors, amplitudes (A), integral intensities (I) and linewidths (deltaBpp) of the EPR spectra, were analyzed. The EPR lines were homogeneously broadened. Continuous microwave saturation of the EPR spectra indicated that slow spin-lattice relaxation processes existed in all the tested melanin samples. The relatively slowest spin-lattice relaxation processes characterized melanin isolated from A-375 cells treated with both VPA and CPT. The changes of the EPR spectra with increasing microwave power in the range of 2.2-70 mW were evaluated. Free radicals concentrations in the melanin from A-375 cells were higher than in the synthetic DOPA-melanin. The strong increase of free radicals concentration in the melanin from A-375 cells was observed after their treating by VPA. CPT also caused the increase of free radicals concentrations in the examined natural melanin. The free radicals concentration in melanin isolated from A-375 cells treated with both VPA and CPT was slightly higher than those in melanin from the control cells.

A NEW COPPER (II)-IMIDAZOLE DERIVATIVE EFFECTIVELY INHIBITS REPLICATION OF DENV-2 IN VERO CELL

PubMed Central

Sucipto, Teguh Hari; Churrotin, Siti; Setyawati, Harsasi; Martak, Fahimah; Mulyatno, Kris Cahyo; Amarullah, Ilham Harlan; Kotaki, Tomohiro; Kameoka, Masanori; Yotopranoto, Subagyo; Soegijanto, and Soegeng

2018-01-01

Background: Dengue is a kind of infectious disease that was distributed in the tropical and sub-tropical areas. To date, there is no clinically approved dengue vaccine or antiviral for humans, even though there have been great efforts towards this end. Therefore, finding the effective compound against dengue virus (DENV) replication is very important. Among the complex compounds, copper(II)-imidazole derivatives are of interest because of their biological and medicinal benefits. Materials and Methods: In the present study, antiviral activity of [Cu(2,4,5-triphenylimidazole)2]n, was evaluated against different stages of dengue virus type 2 (DENV-2) replication in Vero cell using focus forming unit reduction assay and quantitative ELISA. Results: [Cu(2,4,5-triphenylimidazole)2]n inhibited DENV-2 replication in Vero cells with IC50 = 2.3 μg/ml and SI= 19.42 when cells were treated 2 days after virus infection, whereas its CC50 for cytotoxicity to Vero cells was 44.174 μg/ml. Conclusion: The compound has high anti-DENV2 activity, less toxicity, and a high possibility to be considered a drug candidate. PMID:29619441

A field trial of production and financial consequences of helminthosis control in sheep production in Ethiopia.

PubMed

Tibbo, M; Aragaw, K; Philipsson, J; Malmfors, B; Näsholm, A; Ayalew, W; Rege, J E O

2008-04-17

We used a partial-budget analysis to evaluate profitability of different management strategies of three genotypes of sheep in a 2 x 2 x 3 factorial experiment conducted at Debre Berhan research station in the central highlands of Ethiopia. This involved two anthelmintic-treatment levels (treated vs. non-treated), two supplementary nutrition levels (protein-energy supplementation yes/no) and three genotypes: indigenous Menz (n=40), 50% Awassi x 50% Menz crosses (n=38) and 75% Awassi x 25% Menz crosses (n=31). All sheep were exposed to natural sub-clinical helminthosis challenge. Supplemented sheep were offered a concentrate mix daily on an individual basis. Anthelmintic-treated sheep were drenched with fenbendazole against nematodes and with triclabendazole against trematodes. Data were collected during the experimental period (for 10 months from approximately 1 year of age) on feed intake, live weight, eggs per gram (EPG) of faeces, packed-cell volume (PCV), wool weight, and adult-worm burden. Actual market input and output prices were recorded. Supplemented sheep had significantly higher marginal profit (MP) per sheep than non-supplemented sheep (ETB 33 vs. 4). Likewise, anthelmintic treated sheep performed significantly better than their non-treated contemporaries (MP=ETB 28 vs. 8). The 75% Awassi crosses were least profitable.

Sodium-Glucose Transporter-2 (SGLT2; SLC5A2) Enhances Cellular Uptake of Aminoglycosides

PubMed Central

Jiang, Meiyan; Wang, Qi; Karasawa, Takatoshi; Koo, Ja-Won; Li, Hongzhe; Steyger, Peter S.

2014-01-01

Aminoglycoside antibiotics, like gentamicin, continue to be clinically essential worldwide to treat life-threatening bacterial infections. Yet, the ototoxic and nephrotoxic side-effects of these drugs remain serious complications. A major site of gentamicin uptake and toxicity resides within kidney proximal tubules that also heavily express electrogenic sodium-glucose transporter-2 (SGLT2; SLC5A2) in vivo. We hypothesized that SGLT2 traffics gentamicin, and promotes cellular toxicity. We confirmed in vitro expression of SGLT2 in proximal tubule-derived KPT2 cells, and absence in distal tubule-derived KDT3 cells. D-glucose competitively decreased the uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent analog of glucose, and fluorescently-tagged gentamicin (GTTR) by KPT2 cells. Phlorizin, an SGLT2 antagonist, strongly inhibited uptake of 2-NBDG and GTTR by KPT2 cells in a dose- and time-dependent manner. GTTR uptake was elevated in KDT3 cells transfected with SGLT2 (compared to controls); and this enhanced uptake was attenuated by phlorizin. Knock-down of SGLT2 expression by siRNA reduced gentamicin-induced cytotoxicity. In vivo, SGLT2 was robustly expressed in kidney proximal tubule cells of heterozygous, but not null, mice. Phlorizin decreased GTTR uptake by kidney proximal tubule cells in Sglt2+/− mice, but not in Sglt2−/− mice. However, serum GTTR levels were elevated in Sglt2−/− mice compared to Sglt2+/− mice, and in phlorizin-treated Sglt2+/− mice compared to vehicle-treated Sglt2+/− mice. Loss of SGLT2 function by antagonism or by gene deletion did not affect gentamicin cochlear loading or auditory function. Phlorizin did not protect wild-type mice from kanamycin-induced ototoxicity. We conclude that SGLT2 can traffic gentamicin and contribute to gentamicin-induced cytotoxicity. PMID:25268124

Analysis of lymphocyte cell death and apoptosis in HIV-2-infected patients.

PubMed

Jaleco, A C; Covas, M J; Victorino, R M

1994-11-01

Recent evidence suggests that T cell apoptosis could be involved in the pathogenesis of HIV-1 infection. As the progression of HIV-2 associated disease appears to be slower than that of HIV-1, we investigated whether there were differences in the degree of T cell death and apoptosis in peripheral blood mononuclear cell (PBMC) cultures from patients with HIV-1 or HIV-2 infection. PBMC from healthy controls (n = 28) and patients infected with HIV-1 (n = 26: asymptomatic (ASY)/persistent generalized lymphadenopathy (PGL), n = 16; and AIDS-related complex (ARC)/AIDS n = 10) or HIV-2 (n = 30: ASY/PGL, n = 16; ARC/AIDS, n = 14) were cultured in the absence or presence of mitogens (PHA, PWM) or superantigen (SEB). After 48 h, cell death (CD) was assessed by trypan blue exclusion and in some patients programmed cell death (PCD) was quantified in flow cytometry by measuring the percentage of hypodiploid nuclei corresponding to fragmented DNA, after treating the cells with a propidium iodide hypotonic solution. HIV-1 and HIV-2 ARC/AIDS patients and ASY/PGL HIV-1+ patients had significant increases in cell death percentages compared with controls, both in unstimulated and stimulated lymphocyte cultures. However, HIV-2+ ASY/PGL patients did not exhibit significant increases of cell death in unstimulated cultures. In addition, the comparison between HIV-1 and HIV-2 infected subjects in similar stages of disease, showed no significant differences in CD in the ARC/AIDS patients, although ASY/PGL HIV-2 infected subjects had lower levels of CD than the HIV-1+ ASY/PGL (3.4% +/- 0.6 s.e.m. versus 6.8% +/- 1.1 s.e.m., P < 0.01). PCD was significantly increased both in ASY/PGL (14.3% +/- 2.2 s.e.m., n = 8, P < 0.005) and in ARC/AIDS (25.3% +/- 4.5 s.e.m., n = 9, P < 0.001) HIV-1+ patients compared with healthy controls (5.8% +/- 1.7 s.e.m., n = 11). This contrasts with HIV-2 infected subjects where the ASY/PGL patients (10.0% +/- 2.8 s.e.m., n = 6) did not differ significantly from healthy controls, although ARC/AIDS patients (27.2% +/- 4.2 s.e.m., n = 9, P < 0.001) had significantly increased levels of PCD. In conclusion, this is the first report describing the occurrence of spontaneous and activation-induced lymphocyte death by apoptosis in HIV-1 infected subjects.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of lymphocyte cell death and apoptosis in HIV-2-infected patients.

PubMed Central

Jaleco, A C; Covas, M J; Victorino, R M

1994-01-01

Recent evidence suggests that T cell apoptosis could be involved in the pathogenesis of HIV-1 infection. As the progression of HIV-2 associated disease appears to be slower than that of HIV-1, we investigated whether there were differences in the degree of T cell death and apoptosis in peripheral blood mononuclear cell (PBMC) cultures from patients with HIV-1 or HIV-2 infection. PBMC from healthy controls (n = 28) and patients infected with HIV-1 (n = 26: asymptomatic (ASY)/persistent generalized lymphadenopathy (PGL), n = 16; and AIDS-related complex (ARC)/AIDS n = 10) or HIV-2 (n = 30: ASY/PGL, n = 16; ARC/AIDS, n = 14) were cultured in the absence or presence of mitogens (PHA, PWM) or superantigen (SEB). After 48 h, cell death (CD) was assessed by trypan blue exclusion and in some patients programmed cell death (PCD) was quantified in flow cytometry by measuring the percentage of hypodiploid nuclei corresponding to fragmented DNA, after treating the cells with a propidium iodide hypotonic solution. HIV-1 and HIV-2 ARC/AIDS patients and ASY/PGL HIV-1+ patients had significant increases in cell death percentages compared with controls, both in unstimulated and stimulated lymphocyte cultures. However, HIV-2+ ASY/PGL patients did not exhibit significant increases of cell death in unstimulated cultures. In addition, the comparison between HIV-1 and HIV-2 infected subjects in similar stages of disease, showed no significant differences in CD in the ARC/AIDS patients, although ASY/PGL HIV-2 infected subjects had lower levels of CD than the HIV-1+ ASY/PGL (3.4% +/- 0.6 s.e.m. versus 6.8% +/- 1.1 s.e.m., P < 0.01). PCD was significantly increased both in ASY/PGL (14.3% +/- 2.2 s.e.m., n = 8, P < 0.005) and in ARC/AIDS (25.3% +/- 4.5 s.e.m., n = 9, P < 0.001) HIV-1+ patients compared with healthy controls (5.8% +/- 1.7 s.e.m., n = 11). This contrasts with HIV-2 infected subjects where the ASY/PGL patients (10.0% +/- 2.8 s.e.m., n = 6) did not differ significantly from healthy controls, although ARC/AIDS patients (27.2% +/- 4.2 s.e.m., n = 9, P < 0.001) had significantly increased levels of PCD. In conclusion, this is the first report describing the occurrence of spontaneous and activation-induced lymphocyte death by apoptosis in HIV-1 infected subjects.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7955520

Circulating Tumor Cells in Breast Cancer Patients Treated by Neoadjuvant Chemotherapy: A Meta-analysis.

PubMed

Bidard, François-Clément; Michiels, Stefan; Riethdorf, Sabine; Mueller, Volkmar; Esserman, Laura J; Lucci, Anthony; Naume, Bjørn; Horiguchi, Jun; Gisbert-Criado, Rafael; Sleijfer, Stefan; Toi, Masakazu; Garcia-Saenz, Jose A; Hartkopf, Andreas; Generali, Daniele; Rothé, Françoise; Smerage, Jeffrey; Muinelo-Romay, Laura; Stebbing, Justin; Viens, Patrice; Magbanua, Mark Jesus M; Hall, Carolyn S; Engebraaten, Olav; Takata, Daisuke; Vidal-Martínez, José; Onstenk, Wendy; Fujisawa, Noriyoshi; Diaz-Rubio, Eduardo; Taran, Florin-Andrei; Cappelletti, Maria Rosa; Ignatiadis, Michail; Proudhon, Charlotte; Wolf, Denise M; Bauldry, Jessica B; Borgen, Elin; Nagaoka, Rin; Carañana, Vicente; Kraan, Jaco; Maestro, Marisa; Brucker, Sara Yvonne; Weber, Karsten; Reyal, Fabien; Amara, Dominic; Karhade, Mandar G; Mathiesen, Randi R; Tokiniwa, Hideaki; Llombart-Cussac, Antonio; Meddis, Alessandra; Blanche, Paul; d'Hollander, Koenraad; Cottu, Paul; Park, John W; Loibl, Sibylle; Latouche, Aurélien; Pierga, Jean-Yves; Pantel, Klaus

2018-04-12

We conducted a meta-analysis in nonmetastatic breast cancer patients treated by neoadjuvant chemotherapy (NCT) to assess the clinical validity of circulating tumor cell (CTC) detection as a prognostic marker. We collected individual patient data from 21 studies in which CTC detection by CellSearch was performed in early breast cancer patients treated with NCT. The primary end point was overall survival, analyzed according to CTC detection, using Cox regression models stratified by study. Secondary end points included distant disease-free survival, locoregional relapse-free interval, and pathological complete response. All statistical tests were two-sided. Data from patients were collected before NCT (n = 1574) and before surgery (n = 1200). CTC detection revealed one or more CTCs in 25.2% of patients before NCT; this was associated with tumor size (P < .001). The number of CTCs detected had a detrimental and decremental impact on overall survival (P < .001), distant disease-free survival (P < .001), and locoregional relapse-free interval (P < .001), but not on pathological complete response. Patients with one, two, three to four, and five or more CTCs before NCT displayed hazard ratios of death of 1.09 (95% confidence interval [CI] = 0.65 to 1.69), 2.63 (95% CI = 1.42 to 4.54), 3.83 (95% CI = 2.08 to 6.66), and 6.25 (95% CI = 4.34 to 9.09), respectively. In 861 patients with full data available, adding CTC detection before NCT increased the prognostic ability of multivariable prognostic models for overall survival (P < .001), distant disease-free survival (P < .001), and locoregional relapse-free interval (P = .008). CTC count is an independent and quantitative prognostic factor in early breast cancer patients treated by NCT. It complements current prognostic models based on tumor characteristics and response to therapy.

Nonlytic Fc-fused IL-7 synergizes with Mtb32 DNA vaccine to enhance antigen-specific T cell responses in a therapeutic model of tuberculosis.

PubMed

Ahn, So-Shin; Jeon, Bo-Young; Park, Seong-Jeong; Choi, Dong-Hoon; Ku, Sun-Hwa; Cho, Sang-Nae; Sung, Young-Chul

2013-06-12

Improvement to the immunogenicity of DNA vaccines was evaluated in a Mycobacterium tuberculosis (MTB) infection mouse model examining the combined effects of nonlytic Fc-fused IL-7 DNA (IL-7-nFc) and Flt3-ligand fused Mtb32 (F-Mtb32) DNA. Mice were treated with conventional chemotherapy for 6 weeks from 4 weeks after aerosol infection of MTB. Following the start of chemotherapy, DNA immunizations were administered five times with 2-week intervals. Coadministration of IL-7-nFc and F-Mtb32 DNA given during chemotherapy synergistically enhanced the magnitude of Mtb32-specific T cell responses and sustained for one-year after the last immunization assessed by IFN-γ ELISPOT assay. After dexamethasone treatment, a significantly reduced MTB reactivation was observed in mice received both IL-7-nFc and F-Mtb32 DNA, compared with F-MTb32 DNA alone or with control mice. In addition, mice treated with IL-7-nFc and F-Mtb32 DNA together showed improved lung pathology and reduced pulmonary inflammation values relative to F-Mtb32 DNA or saline injected mice. Intracellular cytokine staining revealed that the protection levels induced by combination therapy with IL-7-nFc and F-Mtb32 DNA was associated with enhanced Mtb32-specific IFN-γ secreting CD4(+) T cell responses and CD8(+) T cell responses stimulated with CTL epitope peptide in the lungs and spleens. These data suggest that IL-7-nFc as a novel TB adjuvant may facilitate therapeutic TB DNA vaccine to the clinics through significant enhancement of codelivered DNA vaccine-induced T cell immunity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Monocyte NOTCH2 expression predicts IFN-β immunogenicity in multiple sclerosis patients.

PubMed

Adriani, Marsilio; Nytrova, Petra; Mbogning, Cyprien; Hässler, Signe; Medek, Karel; Jensen, Poul Erik H; Creeke, Paul; Warnke, Clemens; Ingenhoven, Kathleen; Hemmer, Bernhard; Sievers, Claudia; Lindberg Gasser, Raija Lp; Fissolo, Nicolas; Deisenhammer, Florian; Bocskei, Zsolt; Mikol, Vincent; Fogdell-Hahn, Anna; Kubala Havrdova, Eva; Broët, Philippe; Dönnes, Pierre; Mauri, Claudia; Jury, Elizabeth C

2018-06-07

Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-β is an established treatment for MS; however, up to 30% of IFN-β-treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14+ monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-β. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-β administration. Reduced monocyte NOTCH2 expression in nADA+ MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA+ patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-β administration.

Postoperative Radiotherapy for Pathologic N2 Non–Small-Cell Lung Cancer Treated With Adjuvant Chemotherapy: A Review of the National Cancer Data Base

PubMed Central

Robinson, Cliff G.; Patel, Aalok P.; Bradley, Jeffrey D.; DeWees, Todd; Waqar, Saiama N.; Morgensztern, Daniel; Baggstrom, Maria Q.; Govindan, Ramaswamy; Bell, Jennifer M.; Guthrie, Tracey J.; Colditz, Graham A.; Crabtree, Traves D.; Kreisel, Daniel; Krupnick, Alexander S.; Patterson, G. Alexander; Meyers, Bryan F.; Puri, Varun

2015-01-01

Purpose To investigate the impact of modern postoperative radiotherapy (PORT) on overall survival (OS) for patients with N2 non–small-cell lung cancer (NSCLC) treated nationally with surgery and adjuvant chemotherapy. Patients and Methods Patients with pathologic N2 NSCLC who underwent complete resection and adjuvant chemotherapy from 2006 to 2010 were identified from the National Cancer Data Base and stratified by use of PORT (≥ 45 Gy). A total of 4,483 patients were identified (PORT, n = 1,850; no PORT, n = 2,633). The impact of patient and treatment variables on OS was explored using Cox regression. Results Median follow-up time was 22 months. On univariable analysis, improved OS correlated with younger age, treatment at an academic facility, female sex, urban population, higher income, lower Charlson comorbidity score, smaller tumor size, multiagent chemotherapy, resection with at least a lobectomy, and PORT. On multivariable analysis, improved OS remained independently predicted by younger age, female sex, urban population, lower Charlson score, smaller tumor size, multiagent chemotherapy, resection with at least a lobectomy, and PORT (hazard ratio, 0.886; 95% CI, 0.798 to 0.988). Use of PORT was associated with an increase in median and 5-year OS compared with no PORT (median OS, 45.2 v 40.7 months, respectively; 5-year OS, 39.3% [95% CI, 35.4% to 43.5%] v 34.8% [95% CI, 31.6% to 38.3%], respectively; P = .014). Conclusion For patients with N2 NSCLC after complete resection and adjuvant chemotherapy, modern PORT seems to confer an additional OS advantage beyond that achieved with adjuvant chemotherapy alone. PMID:25667283

HYPOTONIC SWELLING OF SALICYLATE-TREATED COCHLEAR OUTER HAIR CELLS

PubMed Central

Zhi, Man; Ratnanather, J. Tilak; Ceyhan, Elvan; Popel, Aleksander S.; Brownell, William E.

2007-01-01

The outer hair cell (OHC) is a hydrostat with a low hydraulic conductivity of Pf = 3×10−4 cm/s across the plasma membrane (PM) and subsurface cisterna (SSC) that make up the OHC's lateral wall. The SSC is structurally and functionally a transport barrier in normal cells that is known to be disrupted by salicylate. The effect of sodium salicylate on Pf is determined from osmotic experiments in which isolated, control and salicylate-treated OHCs were exposed to hypotonic solutions in a constant flow chamber. The value of Pf = 3.5±0.5 ×10−4 cm/s (mean ± s.e.m, n = 34) for salicylate-treated OHCs was not significantly different from Pf = 2.4±0.3 ×10−4 cm/s (mean ± s.e.m, n = 31) for untreated OHCs (p=.3302). Thus Pf is determined by the PM and is unaffected by salicylate treatment. The ratio of longitudinal strain to radial strain εz/εc = −0.76 for salicylate-treated OHCs was significantly smaller (p = .0143) from −0.72 for untreated OHCs, and is also independent of the magnitude of the applied osmotic challenge. Salicylate-treated OHCs took longer to attain a steady-state volume which is larger than that for untreated OHCs and increased in volume by 8-15% prior to hypotonic perfusion unlike sodium α-ketoglutarate treated OHCs. It is suggested that depolymerization of cytoskeletal proteins and/or glycogen maybe responsible for the large volume increase in salicylate-treated OHCs as well as the different responses to different modes of application of the hypotonic solution. PMID:17400411

Kidins220/ARMS depletion is associated with the neural-to Schwann-like transition in a human neuroblastoma cell line model.

PubMed

Rogers, Danny A; Schor, Nina F

2013-03-10

Peripheral neuroblastic tumors exist as a heterogeneous mixture of neuroblastic (N-type) cells and Schwannian stromal (S-type) cells. These stromal cells not only represent a differentiated and less aggressive fraction of the tumor, but also have properties that can influence the further differentiation of nearby malignant cells. In vitro neuroblastoma cultures exhibit similar heterogeneity with N-type and S-type cells representing the neuroblastic and stromal portions of the tumor, respectively, in behavior, morphology, and molecular expression patterns. In this study, we deplete kinase D-interacting substrate of 220kD (Kidins220) with an shRNA construct and thereby cause morphologic transition of the human SH-SY5Y neuroblastoma cell line from N-type to S-type. The resulting cells have similar morphology and expression profile to SH-EP1 cells, a native S-type cell line from the same parent cell line, and to SH-SY5Y cells treated with BrdU, a treatment that induces S-type morphology. Specifically, both Kidins220-deficient SH-SY5Y cells and native SH-EP1 cells demonstrate down-regulation of the genes DCX and STMN2, markers for the neuronal lineage. We further show that Kidins220, DCX and STMN2 are co-down-regulated in cells of S-type morphology generated by methods other than Kidins220 depletion. Finally, we report that the association of low Kidins220 expression with S-type morphology and low DCX and STMN2 expression is demonstrated in spontaneously occurring human peripheral neuroblastic tumors. We propose that Kidins220 is critical in N- to S-type transition of neural crest tumor cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Protein synthesis in TE 671/RD (human rabdomiosarcoma) cells treated with thapsigargin and hyperthermia: impairment of HSP 70 induction.

PubMed

Delpino, A; Piselli, P; Mangano, G

1995-01-01

In this study we considered the quantitative and qualitative changes of protein synthetic activity occurring in TE 671/RD cells treated with thapsigargin (TG), with hyperthermia (HT) or with a combination of both these agents. In cells treated with TG (100 nM, continuous exposure), the overall protein synthetic activity was initially inhibited but subsequently recovered to about 60% of the initial level. Chronic TG exposure was also able to induce the expression of GRP 78. The rate of synthesis of GRP 78, after a lag period of about 2 h, increased gradually to reach a maximum (9-fold induction) after 6 h of TG-treatment and was then maintained at that level up to 18 h. A weak induction of GRP 94 was observed following 6-8 h of continuous exposure to TG. In cells treated with HT (43 degrees C for 30 min), a typical heat shock response was observed: in particular, the relative rate of synthesis of HSP 70 (the major heat-inducible mammalian heat shock protein) was increased 10-fold over the constitutive level. The heat-promoted induction of HSP 70 was significantly reduced by concomitant or previous exposure to TG. When TG and HT were administred simultaneously, the increase in HSP 70 synthesis was only 4.7-fold over the control level, while in cells pre-treated for 1 h with TG before the hyperthermic challenge the rate of HSP 70 synthesis was only stimulated 2-fold. In both these conditions, by contrast, it was apparent that HT did not affect the TG-promoted induction of GRP 78. The correlations between the TG-induced mobilization of cytosolic Ca2+ and the effects on protein synthesis are discussed.

Impairment of the Ubiquitin-Proteasome Pathway by Methyl N-(6-Phenylsulfanyl-1H-benzimidazol-2-yl)carbamate Leads to a Potent Cytotoxic Effect in Tumor Cells

PubMed Central

Dogra, Nilambra; Mukhopadhyay, Tapas

2012-01-01

In recent years, there has been a great deal of interest in proteasome inhibitors as a novel class of anticancer drugs. We report that fenbendazole (FZ) (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate) exhibits a potent growth-inhibitory activity against cancer cell lines but not normal cells. We show here, using fluorogenic substrates, that FZ treatment leads to the inhibition of proteasomal activity in the cells. Succinyl-Leu-Leu-Val-Tyr-methylcoumarinamide (MCA), benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-MCA, and t-butoxycarbonyl-Gln-Ala-Arg-7-amido-4-MCA fluorescent derivatives were used to assess chymotrypsin-like, post-glutamyl peptidyl-hydrolyzing, and trypsin-like protease activities, respectively. Non-small cell lung cancer cells transiently transfected with an expression plasmid encoding pd1EGFP and treated with FZ showed an accumulation of the green fluorescent protein in the cells due to an increase in its half-life. A number of apoptosis regulatory proteins that are normally degraded by the ubiquitin-proteasome pathway like cyclins, p53, and IκBα were found to be accumulated in FZ-treated cells. In addition, FZ induced distinct ER stress-associated genes like GRP78, GADD153, ATF3, IRE1α, and NOXA in these cells. Thus, treatment of human NSCLC cells with fenbendazole induced endoplasmic reticulum stress, reactive oxygen species production, decreased mitochondrial membrane potential, and cytochrome c release that eventually led to cancer cell death. This is the first report to demonstrate the inhibition of proteasome function and induction of endoplasmic reticulum stress/reactive oxygen species-dependent apoptosis in human lung cancer cell lines by fenbendazole, which may represent a new class of anticancer agents showing selective toxicity against cancer cells. PMID:22745125

Preferential cytotoxicity of bortezomib toward highly malignant human liposarcoma cells via suppression of MDR1 expression and function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Yamei; Wang, Lingxian; Wang, Lu

2015-02-15

Liposarcoma is the most common soft tissue sarcoma with a high risk of relapse. Few therapeutic options are available for the aggressive local or metastatic disease. Here, we report that the clinically used proteasome inhibitor bortezomib exhibits significantly stronger cytotoxicity toward highly malignant human liposarcoma SW872-S cells compared with its parental SW872 cells, which is accompanied by enhanced activation of apoptotic signaling both in vitro and in vivo. Treatment of cells with Jun-N-terminal kinase (JNK) inhibitor SP60015 or the translation inhibitor cycloheximide ameliorated this enhanced apoptosis. Bortezomib inhibited MDR1 expression and function more effectively in SW872-S cells than in SW872more » cells, indicating that the increased cytotoxicity relies on the degree of proteasome inhibition. Furthermore, the pharmacological or genetic inhibition of sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2, which is highly expressed in SW872-S cells, resulted in partial reversal of cell growth inhibition and increase of MDR1 expression in bortezomib-treated SW872-S cells. These results show that bortezomib exhibits preferential cytotoxicity toward SW872-S cells possibly via highly expressed SERCA2-associated MDR1 suppression and suggest that bortezomib may serve as a potent agent for treating advanced liposarcoma. - Highlights: • We compare the cytotoxicity of different drugs between SW872-S and SW872 cells. • Highly malignant liposarcoma cells SW872-S show hypersensitivity to bortezomib. • Apoptotic signaling is robustly enhanced in bortezomib-treated SW872-S cells. • Bortezomib has strong suppression on MDR1 expression and function in SW872-S cells. • Inhibition of SERCA2 protects SW872-S cells from bortezomib.« less

Antisnake Venom Activity of Hibiscus aethiopicus L. against Echis ocellatus and Naja n. nigricollis.

PubMed

Hasson, S S; Al-Jabri, A A; Sallam, T A; Al-Balushi, M S; Mothana, R A A

2010-01-01

The objective of the study is to investigate whether the Hibiscus aethiopicus L. plant has neutralization activity against venoms of two clinically important snakes. The H. aethiopicus was dried and extracted with water. Different assays were performed to evaluate the plant's acute toxicity and its anti-snake venom activities. The results showed that H. aethiopicus extract alone had no effect on the viability of C(2)C(12) muscle cells, but significantly (P < .05) protected muscle cells against the toxic effects of E. ocellatus venom at 55, 150, and 300 mug/mL. The maximum protective effect of the extract was exhibited at 75 mug/mL. The extract significantly (P < .001) inhibited the cytotoxic effects of E. ocellatus venom at 300 mug/mL. All rabbits (n = 10) and guinea pigs (n = 10) were alive after the two weeks of given the lethal dosage 16 g/Kg of the H. aethiopicus extract herbal solution. No abnormal behaviour was observed of both groups of animals. All guinea pigs (n = 3) treated with venoms alone (5 mg/kg) died. However, all guinea pigs (n = 21) treated with venom (5 mg/kg) and the extract (400 to 1000 mg/kg) survived. Guinea pigs (n = 3) treated with Naja n. nigricollis venom alone (2.5 mg/kg) and guinea pigs (n = 21) venom with the extract (400 to 1000 mg/kg) died. The H. aethiopicus completely (100%) blocked the haemorrhagic activity of E. ocellatus in the egg embryo at 3.3 mg/mL of extract. These findings suggest that H. aethiopicus may contain an endogenous inhibitor of venom-induced haemorrhage.

Involvement of H2O2 in fluazifop-P-butyl-induced cell death in bristly starbur seedlings.

PubMed

Luo, Xiaoyong; Liu, Zhihang; Sunohara, Yukari; Matsumoto, Hiroshi; Li, Pingliang

2017-11-01

In order to understand the action mechanism of fluazifop-P-butyl (FB) in bristly starbur (Acanthospermum hispidum D.C.), a susceptible plant, the role of active oxygen species (ROS) in herbicide-induced cell death in shoots was investigated. FB-induced phytotoxicity was not reduced by the antioxidants, 1,4-diazabicyclooctane (dabaco), sodium azide, l-tryptophan, d-tryptophan, hydroquinone and dimethyl pyridine N-oxide (DMPO). The activities of superoxide dismutase (SOD) and catalase (CAT), in bristly starbur seedlings were significantly increased by FB at 12 HAT and 24 HAT, while ascorbate peroxidase (APX) and glutathione reductase (GR) activities increased only at 12 HAT. The contents of H 2 O 2 in FB-treated bristly starbur seedlings were significantly higher to that of control between 8 and 24 HAT. According to the analysis of potassium iodide - starch or 3,3-diaminobenzidine, the accumulation of hydrogen peroxide was observed in the apical growing point, stem, petiole and veins of FB-treated bristly starbur seedlings at 24 HAT. The cell viability of bristly starbur seedlings treated by 10μM FB decreased at 18 HAT. These results suggested that FB-induced cell death in bristly starbur shoots may be caused by ROS (O 2 - and H 2 O 2 ) generation and lipid peroxidation. Copyright © 2016 Elsevier Inc. All rights reserved.

Graves' ophthalmopathy: low-dose dexamethasone reduces retinoic acid receptor-alpha gene expression in orbital fibroblasts.

PubMed

Cury, Sarah Santiloni; Oliveira, Miriane; Síbio, Maria Teresa; Clara, Sueli; Luvizotto, Renata De Azevedo Melo; Conde, Sandro; Jorge, Edson Nacib; Nunes, Vania Dos Santos; Nogueira, Célia Regina; Mazeto, Gláucia Maria Ferreira da Silva

2018-05-17

Graves' ophthalmopathy (GO) is an autoimmune disease that leads to ocular proptosis caused by fat accumulation and inflammation, and the main treatment is corticosteroid therapy. Retinoid acid receptor-alpha (RARα) seems to be associated with inflammation and adipocyte differentiation. This study aimed to assess the effect of glucocorticoid treatment on orbital fibroblasts of GO patient treated or not with different glucocorticoid doses. Orbital fibroblasts collected during orbital decompression of a female patient with moderately severe/severe GO were cultivated and treated with 10 nM and 100 nM dexamethasone (Dex). rRARα gene expression in the treated and untreated cells was then compared. Fibroblast RARα expression was not affected by 100 nM Dex. On the other hand, RARα expression was 24% lower in cells treated with 10 nM Dex (p < 0.05). Orbital fibroblasts from a GO patient expressed the RARα gene, which was unaffected by higher, but decreased with lower doses of glucocorticoid.

Gomisin N Inhibits Melanogenesis through Regulating the PI3K/Akt and MAPK/ERK Signaling Pathways in Melanocytes

PubMed Central

Chae, Jae Kyoung; Subedi, Lalita; Jeong, Minsun; Park, Yong Un; Kim, Chul Young; Kim, Hakwon; Kim, Sun Yeou

2017-01-01

Gomisin N, one of the lignan compounds found in Schisandra chinensis has been shown to possess anti-oxidative, anti-tumorigenic, and anti-inflammatory activities in various studies. Here we report, for the first time, the anti-melenogenic efficacy of Gomisin N in mammalian cells as well as in zebrafish embryos. Gomisin N significantly reduced the melanin content without cellular toxicity. Although it was not capable of modulating the catalytic activity of mushroom tyrosinase in vitro, Gomisin N downregulated the expression levels of key proteins that function in melanogenesis. Gomisin N downregulated melanocortin 1 receptor (MC1R), adenylyl cyclase 2, microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). In addition, Gomisin N-treated Melan-A cells exhibited increased p-Akt and p-ERK levels, which implies that the activation of the PI3K/Akt and MAPK/ERK pathways may function to inhibit melanogenesis. We also validated that Gomisin N reduced melanin production by repressing the expression of MITF, tyrosinase, TRP-1, and TRP-2 in mouse and human cells as well as in developing zebrafish embryos. Collectively, we conclude that Gomisin N inhibits melanin synthesis by repressing the expression of MITF and melanogenic enzymes, probably through modulating the PI3K/Akt and MAPK/ERK pathways. PMID:28241436

Ovarian folliculogenesis: detrimental effect of prenatal exposure to cyclophosphamide: a preliminary study.

PubMed

Ray, B; Potu, B K

2010-01-01

To investigate whether cyclophosphamide interferes with ovarian folliculogenesis. In this experiment, pregnant rats (n=12) were randomly assigned into two groups, control group (n=6) and cyclophosphamide treatment group (n=6). In the cyclophosphamide treatment group cyclophosphamide was injected intraperitoneally from day 10 of gestation till 20th day, at 2 mg/kg of body weight. The pregnant rats were sacrificed on gestation day 20 and the fetus was collected. The collected fetuses were processed for sectioning and stained with haematoxyline and eosin for microscopic observation of the ovaries. A meshwork-like appearance of mesenchyme with decreased number of somatic cells and absence of the majority of the germ cells in the ovarian follicles were found in treated fetus. Non-availability of primordial germ cells stopped the interaction between primordial germ cells and somatic supporting cells leading to nonproliferation and degeneration of somatic cells and fluid-filled vacant spaces in the meshwork -like arrangement of mesenchymal cells. We conclude that cyclophosphamide exposure prevents folliculogenesis by causing anovulation and results in infertility. The same detrimental effect might be seen in human fertility with environmental pollutants which are also metabolites of the drug (Fig. 2, Ref. 25).

Nitric oxide regulates Angiopoietin1/Tie2 expression after stroke

PubMed Central

Zacharek, Alex; Chen, Jieli; Zhang, Chunling; Cui, Xu; Roberts, Cynthia; Jiang, Hao; Teng, Hua; Chopp, Michael

2009-01-01

We tested whether the nitric oxide donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) aminio] diazen-1-ium-1,2-diolate (DETA-NONOate), increases expression of Angiopoietin (Ang1)/Tie2, which may play a role in regulating angiogenesis and vascular integrity after stroke in rats. Wistar rats were subjected to middle cerebral artery occlusion and treated with or without DETA-NONOate. Stroke rats treated with DETA-NONOate show significantly increased Ang1, Tie2 and Occludin expression in the ischemic border compared with control stroke animals (p < 0.05). Consistent with in vivo data, DETA-NONOate promotes capillary tube formation in cultured brain endothelial cells. Neutralizing Ang1 antibody attenuates DETA-NONOate-induced capillary tube formation. The data suggest that the Ang1/Tie2 axis promotes DETA-NONOate-induced angiogenesis and stabilizes of angiogenic vessels after stroke. PMID:16762501

Elovl5 regulates the mTORC2-Akt-FOXO1 pathway by controlling hepatic cis-vaccenic acid synthesis in diet-induced obese mice[S

PubMed Central

Tripathy, Sasmita; Jump, Donald B.

2013-01-01

Elevated hepatic expression of fatty acid elongase-5 (Elovl5) induces FoxO1 phosphorylation, lowers FoxO1 nuclear content, and suppresses expression of genes involved in gluconeogenesis (GNG). In this report, we define the molecular and metabolic basis of Elovl5 control of FoxO1 phosphorylation. Adenoviral-mediated (Ad-Elovl5) induction of hepatic Elovl5 in diet-induced obese, glucose-intolerant mice and HepG2 cells increased the phosphorylation of Akt2-S473 [mammalian target of rapamycin complex-2 (mTORC2) site], but not Akt2-T308 (PDK1 site). The Akt2 inhibitor Akti1/2 blocked Elovl5 induction of FoxO1-S256 phosphorylation in HepG2 cells. Elevated Elovl5 activity in liver and HepG2 cells induced rictor mRNA, rictor protein, and rictor-mTOR interaction, whereas rictor knockdown (siRNA) attenuated Elovl5 induction of Akt2-S473 and FoxO1-S256 phosphorylation in HepG2 cells. FA analysis revealed that the abundance of cis-vaccenic acid (18:1,n-7) was increased in livers of obese mice and HepG2 cells following Ad-Elovl5 infection. Treating HepG2 cells with Elovl5 substrates established that palmitoleic acid (16:1,n-7), but not γ-linolenic acid (18:3,n-6), induced rictor protein, Akt-S473, and FoxO1-S256 phosphorylation. Inhibition of FA elongation blocked 16:1,n-7 but not 18:1,n-7 induction of rictor protein and Akt-S473 and FoxO1-S256 phosphorylation. These results establish a novel link between Elovl5-mediated synthesis of 18:1,n-7 and GNG through the control of the mTORC2-Akt-FoxO1 pathway. PMID:23099444

Electronic waste leachate-mediated DNA fragmentation and cell death by apoptosis in mouse fibroblast (NIH/3T3) cell line.

PubMed

Alabi, Okunola A; Bakare, Adekunle A; Filippin-Monteiro, Fabíola B; Sierra, Jelver A; Creczynski-Pasa, Tânia B

2013-08-01

This study investigated the apoptotic effect of electronic waste on fibroblast cell line. Cells were treated with different concentrations of the leachate for 24h. Cell viability was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, nuclear morphology of cells was explored by acridine orange (AO)/ethidium bromide (EB) double staining, mitochondrial membrane potential was evaluated using JC-1 probe while cell cycle analysis was conducted using flow cytometry. The oxidative status was detected using DCFH-DA (dichlorofluorescin diacetate) probe and the relationship between cell death and ROS (reactive oxygen species) was investigated using N-acetylcysteine. Results showed an increased cell death as detected by MTT assay and AO/EB staining. Cell cycle analysis indicated an induction of sub/G1 events while JC-1 probe showed significant disruption of mitochondrial membrane potential. There was significant induction of ROS, while N-acetylcysteine protected the cells from DNA damage. These suggest apoptotic pathway as a possible mechanism of e-waste induced cyto-genotoxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

Intracoronary Administration of Allogeneic Adipose Tissue-Derived Mesenchymal Stem Cells Improves Myocardial Perfusion But Not Left Ventricle Function, in a Translational Model of Acute Myocardial Infarction.

PubMed

Bobi, Joaquim; Solanes, Núria; Fernández-Jiménez, Rodrigo; Galán-Arriola, Carlos; Dantas, Ana Paula; Fernández-Friera, Leticia; Gálvez-Montón, Carolina; Rigol-Monzó, Elisabet; Agüero, Jaume; Ramírez, José; Roqué, Mercè; Bayés-Genís, Antoni; Sánchez-González, Javier; García-Álvarez, Ana; Sabaté, Manel; Roura, Santiago; Ibáñez, Borja; Rigol, Montserrat

2017-05-03

Autologous adipose tissue-derived mesenchymal stem cells (ATMSCs) therapy is a promising strategy to improve post-myocardial infarction outcomes. In a porcine model of acute myocardial infarction, we studied the long-term effects and the mechanisms involved in allogeneic ATMSCs administration on myocardial performance. Thirty-eight pigs underwent 50 minutes of coronary occlusion; the study was completed in 33 pigs. After reperfusion, allogeneic ATMSCs or culture medium (vehicle) were intracoronarily administered. Follow-ups were performed at short (2 days after acute myocardial infarction vehicle-treated, n=10; ATMSCs-treated, n=9) or long term (60 days after acute myocardial infarction vehicle-treated, n=7; ATMSCs-treated, n=7). At short term, infarcted myocardium analysis showed reduced apoptosis in the ATMSCs-treated animals (48.6±6% versus 55.9±5.7% in vehicle; P =0.017); enhancement of the reparative process with up-regulated vascular endothelial growth factor, granulocyte macrophage colony-stimulating factor, and stromal-derived factor-1α gene expression; and increased M2 macrophages (67.2±10% versus 54.7±10.2% in vehicle; P =0.016). In long-term groups, increase in myocardial perfusion at the anterior infarct border was observed both on day-7 and day-60 cardiac magnetic resonance studies in ATMSCs-treated animals, compared to vehicle (87.9±28.7 versus 57.4±17.7 mL/min per gram at 7 days; P =0.034 and 99±22.6 versus 43.3±14.7 22.6 mL/min per gram at 60 days; P =0.0001, respectively). At day 60, higher vascular density was detected at the border zone in the ATMSCs-treated animals (118±18 versus 92.4±24.3 vessels/mm 2 in vehicle; P =0.045). Cardiac magnetic resonance-measured left ventricular ejection fraction of left ventricular volumes was not different between groups at any time point. In this porcine acute myocardial infarction model, allogeneic ATMSCs-based therapy was associated with increased cardioprotective and reparative mechanisms and with better cardiac magnetic resonance-measured perfusion. No effect on left ventricular volumes or ejection fraction was observed. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Detection of immune-related adverse events by medical imaging in patients treated with anti-programmed cell death 1.

PubMed

Mekki, Ahmed; Dercle, Laurent; Lichtenstein, Philip; Marabelle, Aurélien; Michot, Jean-Marie; Lambotte, Olivier; Le Pavec, Jérôme; De Martin, Eleonora; Balleyguier, Corinne; Champiat, Stéphane; Ammari, Samy

2018-06-01

Programmed death receptor-1 blocking antibodies (anti-PD1) are a new standard of care in many cancer types. Patients benefit from improved survival but have the risk of immune-related adverse events (irAE). We evaluated if medical imaging procedures, used for anti-tumour response assessment, can detect irAEs. All consecutive patients treated with anti-PD1 and with a medical imaging acquisition performed within 2 weeks with irAEs ≥2 were retrospectively included. Data were gathered from June 2014 to February 2017, and a central review was performed. The primary and secondary end-points were i) to evaluate the overall detection rate of irAEs by medical imaging and ii) to provide a comprehensive radiological description of irAEs. Fifty-three patients (31 women, 22 men; average age: 61 years) were included. The primary tumour was melanoma (n = 32), lung cancer (n = 18) and other (n = 3). Patients were treated with nivolumab (n = 27) or pembrolizumab (n = 26). Of 74 medical imaging procedures analysed (ratio = 1.4 medical imaging per patient), 55 irAE were detected. The detection rate was overall: 74% (95 confidence interval: 63-84%), positron emission tomography with 18F-fludeoxyglucose integrated with computed tomography (18F-FDG PET/CT): 83% (n = 10/12), magnetic resonance imaging: 83% (n = 5/6), computed tomography scan: 79% (n = 19/24), ultrasonography: 70% (n = 19/27), standard X-rays: 40% (n = 2/5), lung/mediastinum: 100% (n = 7/7), enterocolitis: 100% (n = 8/8), hypophysitis: 100% (n = 3/3), thyroiditis: 75% (n = 15/20), hepatitis: 67% (n = 2/3), arthralgia or arthritis: 40% (n = 2/5) and pancreas: 28% (n = 2/7). Medical imaging detected 74% of irAE in patients treated with anti-PD1. Beyond response assessment, medical imaging can detect irAE and guide towards specific management. We described the most frequent sites and patterns of imaging findings. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effect of low temperature baking in nitrogen on the performance of a niobium superconducting radio frequency cavity

DOE PAGES

Dhakal, Pashupati; Chetri, Santosh; Balachandran, Shreyas; ...

2018-03-08

Here, we report the rf performance of a single-cell superconducting radiofrequency cavity after low temperature baking in a nitrogen environment. A significant increase in quality factor has been observed when the cavity was heat treated in the temperature range of 120-160 °C with a nitrogen partial pressure of ~25 mTorr. This increase in quality factor as well as the Q-rise phenomenon (anti-Q-slope) is similar to those previously obtained with high temperature nitrogen doping as well as titanium doping. In this study, a cavity N 2-treated at 120 °C and at 140 °C, showed no degradation in accelerating gradient, however themore » accelerating gradient was reduced by ~25% with a 160 °C N 2 treatment, compared to the baseline tests after electropolishing. Sample coupons treated in the same conditions as the cavity were analyzed by scanning electron microscope, x-ray photoelectron spectroscopy and secondary ion mass spectroscopy revealed a complex surface composition of Nb 2O 5, NbO and NbN (1-x)O x within the rf penetration depth. Furthermore, magnetization measurements showed no significant change on bulk superconducting properties.« less

Effect of low temperature baking in nitrogen on the performance of a niobium superconducting radio frequency cavity

NASA Astrophysics Data System (ADS)

Dhakal, Pashupati; Chetri, Santosh; Balachandran, Shreyas; Lee, Peter J.; Ciovati, Gianluigi

2018-03-01

We report the rf performance of a single cell superconducting radiofrequency cavity after low temperature baking in a nitrogen environment. A significant increase in quality factor has been observed when the cavity was heat treated in the temperature range of 120 - 160 °C with a nitrogen partial pressure of ˜25 m Torr . This increase in quality factor as well as the Q -rise phenomenon (anti-Q -slope) is similar to those previously obtained with high temperature nitrogen doping as well as titanium doping. In this study, a cavity N2 -treated at 120 °C and at 140 °C showed no degradation in accelerating gradient, however the accelerating gradient was reduced by ˜25 % with a 160 °C N2 treatment, compared to the baseline tests after electropolishing. Sample coupons treated in the same conditions as the cavity were analyzed by scanning electron microscope, x-ray photoelectron spectroscopy and secondary ion mass spectroscopy revealed a complex surface composition of Nb2O5 , NbO and NbN(1 -x )Ox within the rf penetration depth. Furthermore, magnetization measurements showed no significant change on bulk superconducting properties.

Effect of low temperature baking in nitrogen on the performance of a niobium superconducting radio frequency cavity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhakal, Pashupati; Chetri, Santosh; Balachandran, Shreyas

Here, we report the rf performance of a single-cell superconducting radiofrequency cavity after low temperature baking in a nitrogen environment. A significant increase in quality factor has been observed when the cavity was heat treated in the temperature range of 120-160 °C with a nitrogen partial pressure of ~25 mTorr. This increase in quality factor as well as the Q-rise phenomenon (anti-Q-slope) is similar to those previously obtained with high temperature nitrogen doping as well as titanium doping. In this study, a cavity N 2-treated at 120 °C and at 140 °C, showed no degradation in accelerating gradient, however themore » accelerating gradient was reduced by ~25% with a 160 °C N 2 treatment, compared to the baseline tests after electropolishing. Sample coupons treated in the same conditions as the cavity were analyzed by scanning electron microscope, x-ray photoelectron spectroscopy and secondary ion mass spectroscopy revealed a complex surface composition of Nb 2O 5, NbO and NbN (1-x)O x within the rf penetration depth. Furthermore, magnetization measurements showed no significant change on bulk superconducting properties.« less

Efficiency Enhancement of Nanotextured Black Silicon Solar Cells Using Al2O3/TiO2 Dual-Layer Passivation Stack Prepared by Atomic Layer Deposition.

PubMed

Wang, Wei-Cheng; Tsai, Meng-Chen; Yang, Jason; Hsu, Chuck; Chen, Miin-Jang

2015-05-20

In this study, efficient nanotextured black silicon (NBSi) solar cells composed of silicon nanowire arrays and an Al2O3/TiO2 dual-layer passivation stack on the n(+) emitter were fabricated. The highly conformal Al2O3 and TiO2 surface passivation layers were deposited on the high-aspect-ratio surface of the NBSi wafers using atomic layer deposition. Instead of the single Al2O3 passivation layer with a negative oxide charge density, the Al2O3/TiO2 dual-layer passivation stack treated with forming gas annealing provides a high positive oxide charge density and a low interfacial state density, which are essential for the effective field-effect and chemical passivation of the n(+) emitter. In addition, the Al2O3/TiO2 dual-layer passivation stack suppresses the total reflectance over a broad range of wavelengths (400-1000 nm). Therefore, with the Al2O3/TiO2 dual-layer passivation stack, the short-circuit current density and efficiency of the NBSi solar cell were increased by 11% and 20%, respectively. In conclusion, a high efficiency of 18.5% was achieved with the NBSi solar cells by using the n(+)-emitter/p-base structure passivated with the Al2O3/TiO2 stack.

Stereotactic radiation therapy in the strategy of treatment of metastatic renal cell carcinoma: A study of the Getug group.

PubMed

Meyer, Emmanuel; Pasquier, David; Bernadou, Guillemette; Calais, Gilles; Maroun, Pierre; Bossi, Alberto; Theodore, Christine; Albiges, Laurence; Stefan, Dinu; Crevoisier, Renaud D E; Hennequin, Christophe; Lagrange, Jean-Léon; Grellard, Jean-Michel; Clarisse, Bénédicte; Licaj, Idlir; Habrand, Jean-Louis; Carrie, Christian; Joly, Florence

2018-06-01

Renal cell carcinoma (RCC) is usually considered radioresistant, but stereotactic radiation therapy (SRT) may increase local disease control. This study aimed to assess the benefit of SRT in the management of metastatic RCC patients. Data of all RCC patients who received SRT between 2008 and 2015 with curative intent were retrospectively collected in six French referral centres. Local control (LC), progression-free survival (PFS), local recurrence-free survival (LRFS), time to systemic therapy (TTS) and overall survival (OS) were assessed. One hundred and eighty-eight patients treated with SRT for 252 RCC metastases (brain [n = 120]; spine [n = 75]; and others [n = 57]) were recensed. SRT was performed for oligoprogressive disease (101 patients), oligometastatic disease (80 patients) or residual tumour after a partial response to systemic treatment (7 patients). The median biologically effective dose was 78 Gy. For the whole population, local control rates at 6, 12 and 24 months were 87.5%, 82.9% and 77.6%, respectively; median PFS, LRFS, TTS and OS were 8.5, 23.2, 13.2 and 29.2 months, respectively. Among patients treated for oligoprogressive/oligometastatic disease, the median PFS, TTS, and OS were 8.6/7.6, 10.5/14.2 and 23.2/33.9 months, respectively. Among the 7 patients treated with SRT after partial response to systemic treatment, no relapse occurred for 3 of them after a median follow-up of 22 months. Acute and late severe toxicities were noted in 5 (2.6%) patients. SRT is effective and safe for oligometastatic and oligoprogressive RCC patients and may delay introduction or change of systemic therapy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Glycophenotype of breast and prostate cancer stem cells treated with thieno[2,3-b]pyridine anticancer compound.

PubMed

Mastelić, Angela; Čikeš Čulić, Vedrana; Režić Mužinić, Nikolina; Vuica-Ross, Milena; Barker, David; Leung, Euphemia Y; Reynisson, Jóhannes; Markotić, Anita

2017-01-01

Tumor progression may be driven by a small subpopulation of cancer stem cells (CSCs characterized by CD44 + /CD24 - phenotype). We investigated the influence of a newly developed thienopyridine anticancer compound (3-amino-5-oxo- N -naphthyl-5,6,7, 8-tetrahydrothieno[2,3- b ]quinoline-2-carboxamide, 1 ) on the growth, survival and glycophenotype (CD15s and GM3 containing neuraminic acid substituted with acetyl residue, NeuAc) of breast and prostate cancer stem/progenitor-like cell population. MDA-MB-231 and Du-145 cells were incubated with compound 1 alone or in combination with paclitaxel. The cellular metabolic activity was determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. The type of cell death induced by 48-h treatment was assessed using a combination of Annexin-V-FITC and propidium iodide staining. Flow cytometric analysis was performed to detect the percentage of CD44 + /CD24 - cells, and GM3 and CD15s positive CSCs, as well as the expression of GM3 and CD15s per one CSC, in both cell lines. Compound 1 produces a dose- and time-dependent cytotoxicity, mediated mainly by apoptosis in breast cancer cells, and slightly (2.3%) but statistically significant lowering breast CSC subpopulation. GM3 expression per one breast CSC was increased, and the percentage of prostate GM3 + CSC subpopulation was decreased in cells treated with compound 1 compared with non-treated cells. The percentage of CD15s + CSCs was lower in both cell lines after treatment with compound 1 . Considering that triple-negative breast cancers are characterized by an increased percentage of breast CSCs and knowing their association with an increased risk of metastasis and mortality, compound 1 is a potentially effective drug for triple-negative breast cancer treatment.

Improved survival of mice bearing liver metastases of colon cancer cells treated with a combination of radioimmunotherapy and antiangiogenic therapy.

PubMed

Kinuya, Seigo; Yokoyama, Kunihiko; Koshida, Kiyoshi; Mori, Hirofumi; Shiba, Kazuhiro; Watanabe, Naoto; Shuke, Noriyuki; Bai, Jingming; Michigishi, Takatoshi; Tonami, Norihisa

2004-07-01

We attempted to determine whether the combined regimen of radioimmunotherapy (RIT) and antiangiogenic therapy would favorably affect the survival of animals bearing liver metastases of colon cancer cells. Daily antiangiogenic therapy with 2-methoxyestradiol (2-ME), 75 mg/kg, was initiated at 3 days following intrasplenic cell inoculation of LS180 colon cancer cells. RIT with 7 MBq of (131)I-A7, an IgG1 anti-colorectal monoclonal antibody, or (131)I-HPMS-1, an irrelevant IgG1, was conducted at 7 days. Production of vascular endothelial growth factor (VEGF) by LS180 cells was assessed in vitro. All nontreated mice died by 31 days following cell inoculation ( n=5). Monotherapy comprising 2-ME treatment resulted in slightly better survival of mice ( n=8) ( P<0.05). (131)I-A7 RIT displayed a marked therapeutic effect ( n=8) ( P<0.001); however, all animals eventually died due to metastases by 99 days. The combined regimen of (131)I-A7 RIT and antiangiogenic therapy demonstrated a superior therapeutic effect in comparison to monotherapy consisting of either RIT or antiangiogenic therapy ( n=10) ( P<0.05); three mice survived the entire 160-day observation period. The combination of antiangiogenic therapy and (131)I-HPMS-1 RIT failed to provide an appreciable benefit ( n=5). Treatment with 2-ME decreased VEGF production by LS180 cells in a dose-dependent fashion. In conclusion, a combination regimen comprising RIT and antiangiogenic therapy initiated at the early stage of metastasis would be of great benefit in terms of improvement of the therapeutic efficacy with respect to liver metastases.

CHECKPOINT INHIBITOR IMMUNE THERAPY: Systemic Indications and Ophthalmic Side Effects.

PubMed

Dalvin, Lauren A; Shields, Carol L; Orloff, Marlana; Sato, Takami; Shields, Jerry A

2018-06-01

To review immune checkpoint inhibitor indications and ophthalmic side effects. A literature review was performed using a PubMed search for publications between 1990 and 2017. Immune checkpoint inhibitors are designed to treat system malignancies by targeting one of three ligands, leading to T-cell activation for attack against malignant cells. These ligands (and targeted drug) include cytotoxic T-lymphocyte antigen-4 (CTLA-4, ipilimumab), programmed death protein 1 (PD-1, pembrolizumab, nivolumab), and programmed death ligand-1 (PD-L1, atezolizumab, avelumab, durvalumab). These medications upregulate the immune system and cause autoimmune-like side effects. Ophthalmic side effects most frequently manifest as uveitis (1%) and dry eye (1-24%). Other side effects include myasthenia gravis (n = 19 reports), inflammatory orbitopathy (n = 11), keratitis (n = 3), cranial nerve palsy (n = 3), optic neuropathy (n = 2), serous retinal detachment (n = 2), extraocular muscle myopathy (n = 1), atypical chorioretinal lesions (n = 1), immune retinopathy (n = 1), and neuroretinitis (n = 1). Most inflammatory side effects are managed with topical or periocular corticosteroids, but advanced cases require systemic corticosteroids and cessation of checkpoint inhibitor therapy. Checkpoint inhibitors enhance the immune system by releasing inhibition on T cells, with risk of autoimmune-like side effects. Ophthalmologists should include immune-related adverse events in their differential when examining cancer patients with new ocular symptoms.

Dielectric barrier discharge and jet type plasma surface modifications of hybrid polymeric poly (ε-caprolactone)/chitosan scaffolds.

PubMed

Ozkan, Ozan; Turkoglu Sasmazel, Hilal

2018-04-01

In this study, dry air plasma jet and dielectric barrier discharge Ar + O 2 or Ar + N 2 plasma modifications and their effects on wettability, topography, functionality and biological efficiency of the hybrid polymeric poly (ε-caprolactone)/chitosan scaffolds were reported. The samples treated with Ar + O 2 dielectric barrier discharge plasma (80 sccm O 2 flow rate, 3-min treatment) or with dry air plasma jet (15-cm nozzle-sample distance, 13-min treatment) had the closest wettability (49.11 ± 1.83 and 53.60 ± 0.95, respectively) to the commercial tissue culture polystyrene used for cell cultivation. Scanning electron microscopy images and X-ray photoelectron spectrometry analysis showed increase in topographical roughness and OH/NH 2 functionality, respectively. Increased fluid uptake capacity for the scaffolds treated with Ar + O 2 dielectric barrier discharge plasma (73.60% ± 1.78) and dry air plasma jet (72.48% ± 0.75) were also noted. Finally, initial cell attachment as well as seven-day cell viability, growth and proliferation performances were found to be significantly better for both plasma treated scaffolds than for untreated scaffolds.

Renal Allograft Survival in Nonhuman Primates Infused with Donor Antigen-Pulsed Autologous Regulatory Dendritic Cells

PubMed Central

Ezzelarab, M.B.; Raich-Regue, D.; Lu, L.; Zahorchak, A.F.; Perez-Gutierrez, A.; Humar, A.; Wijkstrom, M.; Minervini, M.; Wiseman, R.W.; Cooper, D.K.C.; Morelli, A.E.; Thomson, A.W.

2017-01-01

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte-derived DCreg pre-loaded with cell membrane vesicles from allogeneic PBMC, induce T cell hyporesponsiveness to donor alloAg in vitro. These donor alloAg-pulsed autologous DCreg (1.4–3.6 x 106/kg) were administered intravenously, one day before MHC-mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n=6 historical controls) and 29 days with control unpulsed DCreg (n=2), to 56 days with donor Ag-pulsed DCreg (n=5), was associated with evidence of modulated host CD4+ and CD8+ T cell responses to donor Ag and attenuation of systemic IL-17 production. Circulating anti-donor antibody (Ab) was not detected until CTLA4Ig withdrawal. One monkey treated with donor Ag-pulsed DCreg rejected its graft in association with progressively elevated anti-donor Ab, 525 days post-transplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag-pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen. PMID:28009481

Predictors of CD34+ cell mobilization and collection in adult men with germ cell tumors: implications for the salvage treatment strategy.

PubMed

Necchi, Andrea; Miceli, Rosalba; Pedrazzoli, Paolo; Giannatempo, Patrizia; Secondino, Simona; Di Nicola, Massimo; Farè, Elena; Raggi, Daniele; Magni, Michele; Matteucci, Paola; Longoni, Paolo; Milanesi, Marco; Paternò, Emanuela; Ravagnani, Fernando; Arienti, Flavio; Nicolai, Nicola; Salvioni, Roberto; Carlo-Stella, Carmelo; Gianni, Alessandro M

2014-06-01

High-dose chemotherapy with tandem or triple carboplatin and etoposide course is currently the first curative choice for relapsing GCT. The collection of an adequate amount of hematopoietic (CD34(+)) stem cells is a priority. We analyzed data of patients who underwent HDCT at 2 referral institutions. Chemotherapy followed by myeloid growth factors was applied in all cases. Uni- and multivariable models were used to evaluate the association between 2 prespecified variables and mobilization parameters. Analyses included only the first mobilizing course of chemotherapy and mobilization failures. A total of 116 consecutive patients underwent a mobilization attempt from December 1995 to November 2012. Mobilizing regimens included cyclophosphamide (CTX) 7 gr/m(2) (n = 39), cisplatin, etoposide, and ifosfamide (PEI) (n = 42), paclitaxel, cisplatin, and gemcitabine (TPG) (n = 11), and mixed regimens (n = 24). Thirty-seven percent were treated in first-line, 50% (n = 58) in second-line, 9.5% (n = 11) and 3.4% (n = 4) in third- and fourth-line settings, respectively. Six patients did not undergo HDCT because they were poor mobilizers, 2 in first- and second-line (1.9%), and 4 beyond the second-line (26.7%). In the multivariable model, third-line or later setting was associated with a lower CD34(+) cell peak/μL (P = .028) and a lower total CD34(+)/kg collected (P = .008). The latter was also influenced by the type of mobilizing regimen (P < .001). A decline in significant mobilization parameters was found, primarily depending on the pretreatment load. Results lend support to the role of CD34(+) cell mobilization in the therapeutic algorithm of relapsing GCT, for whom multiple HDCT courses are still an option, and potentially a cure. Copyright © 2014 Elsevier Inc. All rights reserved.

Fluorophore:dendrimer ratio impacts cellular uptake and intracellular fluorescence lifetime.

PubMed

Dougherty, Casey A; Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M

2015-02-18

G5-NH2-TAMRAn (n = 1-4, 5+, and 1.5(avg)) were prepared with n = 1-4 as a precise dye:dendrimer ratio, 5+ as a mixture of dendrimers with 5 or more dye per dendrimer, and 1.5(avg) as a Poisson distribution of dye:dendrimer ratios with a mean of 1.5 dye per dendrimer. The absorption intensity increased sublinearly with n whereas the fluorescence emission and lifetime decreased with an increasing number of dyes per dendrimer. Flow cytometry was employed to quantify uptake into HEK293A cells. Dendrimers with 2-4 dyes were found to have greater uptake than dendrimer with a single dye. Fluorescence lifetime imaging microscopy (FLIM) showed that the different dye:dendrimer ratio alone was sufficient to change the fluorescence lifetime of the material observed inside cells. We also observed that the lifetime of G5-NH2-TAMRA5+ increased when present in the cell as compared to solution. However, cells treated with G5-NH2-TAMRA1.5(avg) did not exhibit the high lifetime components present in G5-NH2-TAMRA1 and G5-NH2-TAMRA5+. In general, the effects of the dye:dendrimer ratio on fluorescence lifetime were of similar magnitude to environmentally induced lifetime shifts.

In Vitro Evaluation of the Impact of the Probiotic E. coli Nissle 1917 on Campylobacter jejuni's Invasion and Intracellular Survival in Human Colonic Cells.

PubMed

Helmy, Yosra A; Kassem, Issmat I; Kumar, Anand; Rajashekara, Gireesh

2017-01-01

Campylobacter jejuni is a leading cause of bacterial food poisoning in humans. Due to the rise in antibiotic-resistant Campylobacter , there exists a need to develop antibiotic-independent interventions to control infections in humans. Here, we evaluated the impact of Escherichia coli Nissle 1917 (EcN), a probiotic strain, on C. jejuni's invasion and intracellular survival in polarized human colonic cells (HT-29). To further understand how EcN mediates its impact, the expression of 84 genes associated with tight junctions and cell adhesion was profiled in HT-29 cells after treatment with EcN and challenge with C. jejuni . The pre-treatment of polarized HT-29 cells with EcN for 4 h showed a significant effect on C. jejuni 's invasion (∼2 log reduction) of the colonic cells. Furthermore, no intracellular C. jejuni were recovered from EcN pre-treated HT-29 cells at 24 h post-infection. Other probiotic strains tested had no significant impact on C. jejuni invasion and intracellular survival. C. jejuni decreased the expression of genes associated with epithelial cells permeability and barrier function in untreated HT-29 cells. However, EcN positively affected the expression of genes that are involved in enhanced intestinal barrier function, decreased cell permeability, and increased tight junction integrity. The results suggest that EcN impedes C. jejuni invasion and subsequent intracellular survival by affecting HT-29 cells barrier function and tight junction integrity. We conclude that EcN might be a viable alternative for controlling C. jejuni infections.

Exploring the Role of Ubiquitination in Progesterone Receptor Transcriptional Activation and Turnover in Breast Cancer Cells

DTIC Science & Technology

2006-06-01

factors. T47DY cells were cotransfected with a PR construct, a PRE- luciferase plasmid and a renilla plasmid, for transfection control. The cells...PR-B or S294A PR-B, PRE-luciferase reporter constructs and a Renilla control plasmid. Cells were treated for 24hrs with or without R5020 (10nM...plasmid and a plasmid constitutively expressing renilla luciferase for transfection control. Cell were starved for one day and treated with or without

Inhibition of placental 11beta-hydroxysteroid dehydrogenase type 2 by lead.

PubMed

St-Pierre, Joey; Fraser, Marc; Vaillancourt, Cathy

2016-10-01

Lead interferes with cortisol blood concentration, increases the risk of obstetrical complications, and could alter fetal development. The placenta controls maternal cortisol transfer to the fetus by the activity of the type 2 11β-hydroxysteroid dehydrogenase (11β-HSD2), which converts cortisol into inactive cortisone. This study determines the effect of lead on the expression and activity of the placental 11β-HSD2 in human trophoblast-like BeWo cells. Cells were treated with increasing concentration (0-1000nM) of PbCl2 for 24h. 11β-HSD2 protein expression was reduced by 45% at 1000nM of PbCl 2 compared to untreated cells, while the activity was significantly reduced by PbCl 2 at 10, 100 and 1000nM. This study shows the direct inhibitory action of lead on placental 11β-HSD2 activity and suggests that this heavy metal reduces the efficiency of the placental protection against the adverse effects of high cortisol level during fetal development. Copyright © 2016 Elsevier Inc. All rights reserved.

Intramammary infusion of a live culture of Lactococcus lactis in ewes to treat staphylococcal mastitis.

PubMed

Mignacca, Sebastian Alessandro; Dore, Simone; Spuria, Liliana; Zanghì, Pietro; Amato, Benedetta; Duprè, Ilaria; Armas, Federica; Biasibetti, Elena; Camperio, Cristina; Lollai, Stefano A; Capucchio, Maria Teresa; Cannas, Eugenia Agnese; Di Marco Lo Presti, Vincenzo; Marianelli, Cinzia

2017-12-01

Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an evaluation, in two trials, of the efficacy of an intramammary infusion of a live culture of Lactococcus lactis for the treatment of subclinical and clinical mastitis in ewes. In total, 67 animals were enrolled: 19 lactating ewes (study 1), including healthy (N=6) and coagulase-negative staphylococci (CNS)-infected ewes (N=13); and 48 lactating ewes (study 2) with either CNS mastitis (N=32), or Staphylococcus aureus mastitis (N=16), for a total of 123 mammary glands. Intramammary infusions were performed with either L. lactis or PBS for 3 (study 1) or 7 (study 2) consecutive days. Antibiotic-treated and untreated control glands were included. Milk samples for microbiology, somatic cell analysis and milk production were collected before and after treatment.Results/Key findings.L. lactis rapidly activated the mammary glands' innate immune response and initiated an inflammatory response as evidenced by the recruitment of polymorphonuclear neutrophils and increased somatic cell counts. But while leading to a transient clearance of CNS in the gland, this response caused mild to moderate clinical cases of mastitis characterized by abnormal milk secretions and udder inflammation. Moreover, S. aureus infections did not improve, and CNS infections tended to relapse. Under our experimental conditions, the L. lactis treatment led to a transient clearance of the pathogen in the gland, but also caused mild to moderate clinical cases of mastitis. We believe it is still early to implement bacterial formulations as alternatives in treating mastitis in ruminants and further experimentation is needed.

Mechanisms of Cellular Membrane Effects of TCDD (2,3,7,8-Tetrachlorodibenzo-p-Dioxin) Selected Perfluorinated Acids and Polyhalogenated Aromatic Hydrocarbons.

DTIC Science & Technology

1986-10-15

tetrachloro dibenzo-p-dioxin (TCDD); dioxin; perfluoro -n-decanoic acid ( PFDA ); perfluoro -n-octanoic acid ( PFOA ); 3,31,4,41,5,51 hexachlorobiphenyl (345PCB) and...analysis of livers from rats treated with perfluoro -n-decanoic acid ( PFDA ) showed a decrease in stearic acid and an increase in 18 carbon unsaturates...effects of TCDD, PHAH and perfluorinated acids ( PFA ) on mammalian cell membrane function by examing the phenomenon of metabolic cooperation. Metabolic

[Correlations between cellular immunity and invasiveness in differentiated thyroid cancer].

PubMed

Han, Ting; Liang, Jun; Meng, Chao; Yang, Ke; Li, Xiao-yi; Lin, Yan-song

2014-02-01

To investigate the relationship between the immunity and invasiveness in differentiated thyroid cancer (DTC). Totally 74 DTC who were treated in Peking Union Medical College Hospital from September 2012 to December 2012 were enrolled in this study. These 74 patients were divided into membrane invasion group (n=36) and without membrane invasion group (n=38); also, they were divided into distant metastasis group (n=18) and without distant metastasis group (n=56). Natural killer (NK) cells and T-cell subsets were chosen as indicators for cellular immunity to investigate the correlation between cellular immunity and invasiveness in DTC. Univariate analysis showed that the membrane invasion (Χ(2)=12.175, P=0.000) and distant metastasis (Χ(2)=8.139, P=0.006) correlated with cell immunity, whereas distant metastasis correlated with lymphocytic thyroiditis (Χ(2)=7.094, P=0.008). Further investigation shows that distant metastasis was associated with the percentage of CD8+T cell subgroup (Χ(2)=5.429, P=0.020), and membrane invasion was significantly associated with NK cells (Χ(2)=2.445, P=0.018) and CD4/CD8 disorder subgroup (Χ(2)=8.079, P=0.002). Multivariate analysis showed that cell immunity disorder was a risk factor for membrane invasion [OR=5.701,95%CI(2.075~15.666), P=0.001] and distant metastasis [OR=5.063,95%CI (1.571~16.320), P=0.008]. Further analysis showed that CD8+T cell was a risk factor for metastasis [OR=2.236,95%CI( 1.084~4.613), P=0.029], and CD4/CD8 disorders were the risk factors for membrane invasion [OR=2.802,95%CI(1.257~6.244), P=0.012]. Cell immunity in thyroid cancer has close relationship with membrane invasion and distant metastasis, especially when the percentage of CD8+T cells decreases and when the NK cells and CD4/CD8 are abnormal, which may lead to membrane invasion and distant metastasis.

Perovskite solar cells based on nanocolumnar plasma-deposited ZnO thin films.

PubMed

Ramos, F Javier; López-Santos, Maria C; Guillén, Elena; Nazeeruddin, Mohammad Khaja; Grätzel, Michael; Gonzalez-Elipe, Agustin R; Ahmad, Shahzada

2014-04-14

ZnO thin films having a nanocolumnar microstructure are grown by plasma-enhanced chemical vapor deposition at 423 K on pre-treated fluorine-doped tin oxide (FTO) substrates. The films consist of c-axis-oriented wurtzite ZnO nanocolumns with well-defined microstructure and crystallinity. By sensitizing CH3NH3PbI3 on these photoanodes a power conversion of 4.8% is obtained for solid-state solar cells. Poly(triarylamine) is found to be less effective when used as the hole-transport material, compared to 2,2',7,7'-tetrakis(N,N-di-p-methoxyphenylamine)-9,9'-spirobifluorene (spiro-OMeTAD), while the higher annealing temperature of the perovskite leads to a better infiltration in the nanocolumnar structure and an enhancement of the cell efficiency. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Anti-Diabetic Potential of Ocimum gratissimum Leaf Fractions in Fortified Diet-Fed Streptozotocin Treated Rat Model of Type-2 Diabetes

PubMed Central

Umar, Isamila A.; James, Dorcas B.; Inuwa, Hajiya M.

2017-01-01

Background: Ocimum gratissimum (OG) is used in the traditional management of diabetes in Nigeria. This study investigated the anti-diabetic potential of OG leaf fractions (OGLF) in a rat model of Type-2 diabetes (T2D). Methods: Methanol crude extract of OG leaf was fractionated with solvents of increasing order of polarity (n-hexane, chloroform, ethyl-acetate, n-butanol and water). The anti-diabetic potential of the fractions was evaluated in vivo. T2D was induced in Albino Wistar rats and treated with OGLF. Results: The T2D rats showed significant elevation in serum levels of fasting blood glucose (FBG), liver and kidney function biomarkers. At 4-week of intervention with OGLF, the untreated diabetic control group maintained severe hyperglycaemia in the presence of 61.7% serum insulin, 17.3% pancreatic β-cell function (HOMA-β) and 51.5% Insulin sensitivity. The glucose tolerance ability was enhanced in the n-butanol-fraction (OGb) treated group. With 74.8% available serum insulin and 38.6% improvement in insulin sensitivity, the OGb treated group had a 63.5% reduction in FBG and it was found to be most effective as it ameliorates a majority of the changes caused in the studied parameters in diabetic rats. Conclusions: The data from this study suggest that OGb fraction is a potential candidate for the development of an effective drug for the management of T2D. PMID:29019956

Anti-Diabetic Potential of Ocimum gratissimum Leaf Fractions in Fortified Diet-Fed Streptozotocin Treated Rat Model of Type-2 Diabetes.

PubMed

Okoduwa, Stanley I R; Umar, Isamila A; James, Dorcas B; Inuwa, Hajiya M

2017-10-11

Background : Ocimum gratissimum (OG) is used in the traditional management of diabetes in Nigeria. This study investigated the anti-diabetic potential of OG leaf fractions (OGLF) in a rat model of Type-2 diabetes (T2D). Method : Methanol crude extract of OG leaf was fractionated with solvents of increasing order of polarity ( n -hexane, chloroform, ethyl-acetate, n -butanol and water). The anti-diabetic potential of the fractions was evaluated in vivo. T2D was induced in Albino Wistar rats and treated with OGLF. Result : The T2D rats showed significant elevation in serum levels of fasting blood glucose (FBG), liver and kidney function biomarkers. At 4-weeks of intervention with OGLF, the untreated diabetic control group maintained severe hyperglycaemia in the presence of 61.7% serum insulin, 17.3% pancreatic β-cell function (HOMA-β) and 51.5% Insulin sensitivity. The glucose tolerance ability was enhanced in the n -butanol-fraction (OGb) treated group. With 74.8% available serum insulin and 38.6% improvement in insulin sensitivity, the OGb treated group had a 63.5% reduction in FBG and it was found to be most effective as it ameliorates a majority of the changes caused in the studied parameters in diabetic rats. Conclusions : The data from this study suggest that OGb fraction is a potential candidate for the development of an effective drug for the management of T2D.

Mineral trioxide aggregate upregulates odonto/osteogenic capacity of bone marrow stromal cells from craniofacial bones via JNK and ERK MAPK signalling pathways.

PubMed

Wang, Y; Li, J; Song, W; Yu, J

2014-06-01

The aim of this study was to investigate effects of mineral trioxide aggregate (MTA) on odonto/osteogenic differentiation of bone marrow stromal cells (BMSCs) from craniofacial bones. Craniofacial BMSCs were isolated from rat mandible and effects of MTA on their proliferation, differentiation and MAPK pathway involvement were subsequently investigated, in vitro. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,5-tetrazoliumbromide) assay was performed to evaluate proliferation of the MTA-treated cells. Alkaline phosphatase (ALP) activity, alizarin red staining, real-time reverse transcription polymerase chain reaction and western blot assays were used to assess differentiation capacity as well as MAPK pathway involvement. 0.02 mg/ml MTA-treated BMSCs had significantly higher ALP activity and formed more mineralized nodules than the untreated group. Odonto/osteoblastic marker genes/proteins (Alp, Runx2/RUNX2, Osx/OSX, Ocn/OCN and Dspp/DSP respectively) in MTA-treated cells were remarkably upregulated compared to untreated ones. Mechanistically, phosphorylated Jun N-terminal kinase (P-JNK) and phosphorylated extracellular regulated protein kinases (P-ERK) in MTA-treated BMSCs increased significantly in a time-dependent manner, while inhibition of JNK and ERK MAPK pathways dramatically blocked MTA-induced odonto/osteoblastic differentiation, as indicated by reduced ALP levels, weakened mineralization capacity and downregulated levels of odonto/osteoblastic marker genes (Alp, Runx2, Osx, Ocn and Dspp). Mineral trioxide aggregate promoted odonto/osteogenic capacity of craniofacial BMSCs via JNK and ERK MAPK signalling pathways. © 2014 John Wiley & Sons Ltd.

Photorefractive keratectomy with a small spot laser and tracker.

PubMed

Pallikaris, I G; Koufala, K I; Siganos, D S; Papadaki, T G; Katsanevaki, V J; Tourtsan, V; McDonald, M B

1999-01-01

The Autonomous Technologies LADARVision excimer laser system utilizes an eye tracking mechanism and a small spot for photorefractive keratectomy. One hundred and two eyes of 102 patients were treated for -1.50 to -6.25 D of spherical myopia at the spectacle plane using a 6-mm diameter ablation zone. One year follow-up was available for 93 eyes (91%). Uncorrected visual acuity for eyes treated for distance vision was 20/40 or better in 99% (n = 90), and 20/20 or better in 70% (n = 64) of eyes at 12 months. Spectacle-corrected visual acuity was 20/25 or better in all 92 eyes reported; no eye lost more than 2 lines of spectacle-corrected visual acuity, and only 1 eye (1.0%) experienced a loss of 2 lines (20/12.5 to 20/20) at 1 year. The refractive result was within +/- 0.50 D of the desired correction in 75% (n = 70), and within +/- 1.00 D in 93% (n = 86) of eyes at 12 months. Refractive stability was achieved between 3 and 6 months. Corneal haze was graded as trace or less in 100% of the 93 eyes. No significant reductions were noted in contrast sensitivity or endothelial cell density. Patients treated with the Autonomous Technologies LADARVision excimer laser system for -1.50 to -6.25 D of spherical myopia with 1 year follow-up had uncorrected visual acuity of 20/20 or better in 70%, no significant loss of spectacle-corrected visual acuity, no reduction of endothelial cell density or contrast sensitivity, and low levels of corneal haze.

The use of a sequential high dose recombinant interleukin 2 regimen after autologous bone marrow transplantation does not improve the disease free survival of patients with acute leukemia transplanted in first complete remission.

PubMed

Blaise, D; Attal, M; Pico, J L; Reiffers, J; Stoppa, A M; Bellanger, C; Molina, L; Nedellec, G; Vernant, J P; Legros, M; Gabus, R; Huguet, F; Brandely, M; Hercend, T; Olive, D; Maraninchi, D

1997-05-01

We report the outcome of 50 consecutive patients with CR1 acute leukemia (AML = 22; ALL = 28) treated with autologous BMT, after cyclophosphamide and TBI, followed with a sequential high dose rIL2 regimen. rIL-2 (RU 49637 from Roussel-Uclaf, Romainville, France) was started after hematological reconstitution an average of 72 +/- 22 days post transplant. The schedule consisted of a continuous infusion over 5 cycles (Cycle 1: 5 days starting on day 1; cycle 2-5: 2 days starting on day 15, 29, 43 and 57). Patients were treated at 4 different dosages (12 (N = 40), 16 (N = 3), 20 (N = 2), 24 (N = 5) x 10(6) IU/m2/day). Toxicities were mainly related to capillary leak syndrome and thrombocytopenia. Patients received an average of 122 +/- 49 10(6) IU/m2. Two patients with AML died from toxicity. rIL-2 infusion was associated with very a high level of immune stimu-lation of both T-cells (P < 0.05) and natural killer (NK) cells (P < 0.05) and associated cytolytic functions (P < 0.05). With a minimal and median follow-up of 21 and 46 months, 3 year leukemia free survival is 41 +/- 6% overall, 39 +/- 10% and 43 +/- 8% for AML and ALL respectively. Relapse probabilities at 3 years are 59 +/- 11% for AML and 57 +/- 8% for ALL. We conclude that this short infusion of rIL-2 over 2 months, resulting in an increased immune stimulation, is not associated with a better leukemic control for patients with acute leukemia transplanted early after reaching first complete remission.

Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS1

PubMed Central

Zhang, Hong-Hai; Lechuga, Thomas J.; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

2016-01-01

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with “light” (L-12C614N4-Arg and L-12C614N2-Lys) or “heavy” (L-13C615N4-Arg and L-13C615N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. PMID:27075618

GSK-3 mediates the okadaic acid-induced modification of collapsin response mediator protein-2 in human SK-N-SH neuroblastoma cells.

PubMed

Ni, Mei-Hui; Wu, Chih-Ching; Chan, Wen-Hsiung; Chien, Kun-Yi; Yu, Jau-Song

2008-04-15

Collapsin response mediator protein-2 (CRMP-2), a phosphoprotein involved in axonal outgrowth and microtubule dynamics, is aberrantly phosphorylated in Alzheimer's disease (AD) brain. Alteration of glycogen synthase kinase-3 (GSK-3) activity is associated with the pathogenesis of AD. Here, we show that CRMP-2 is one of the major substrates for GSK-3 in pig brain extracts. Both GSK-3alpha and 3beta phosphorylate purified pig brain CRMP-2 and significantly alter its mobility in SDS-gels, resembling the CRMP-2 modification observed in AD brain. Interestingly, this modification can be detected in SK-N-SH neuroblastoma cells treated with a phosphatase inhibitor, okadaic acid (OA), and GSK-3 inhibitors completely block this OA-induced event. Knockdown of both GSK-3alpha and 3beta, but not either kinase alone, impairs OA-induced modification of CRMP-2. Mutation of Ser-518 or Ser-522 of CRMP-2, which are highly phosphorylated in AD brain, to Ala blocks the OA-induced modification of CRMP-2 in SK-N-SH cells. Ser-522 prephosphorylated by Cdk5 is required for subsequent GSK-3alpha-mediated phosphorylation of CRMP-2 in vitro. Collectively, our results demonstrate for the first time that OA can induce phosphorylation of CRMP-2 in SK-N-SH cells at sites aberrantly phosphorylated in AD brain, and both GSK-3alpha and 3beta and Ser-522 kinase(s) are involved in this process.

Direct, rapid effects of 25-hydroxyvitamin D3 on isolated intestinal cells.

PubMed

Phadnis, Ruta; Nemere, Ilka

2003-10-01

Scattered reports in the literature have suggested that the metabolite 25-hydroxyvitamin D(3) [25(OH)D(3)] has biological activity. In the present work, perfusion of isolated duodenal loops of normal chickens with 100 nM 25(OH)D(3) resulted in enhanced transport of (45)Ca within 2 min relative to the vehicle controls. We then tested the effect of a range of 25(OH)D(3) concentrations on (45)Ca handling by isolated intestinal cells in time course studies. Following a basal uptake period, cell suspensions from 7-week old chicks were treated either with 25, 100, or 300 nM 25(OH)D(3), or the vehicle ethanol (0.01%, final concentration). Both 25 and 100 nM 25(OH)D(3) resulted in a significant (P < 0.05) reduction in (45)Ca levels, relative to controls, between 1-10 min after treatment, while 300 nM 25(OH)D(3) resulted in a significant increase in (45)Ca levels, relative to controls, after 10 min of incubation. The effect of 100 nM 25(OH)D(3) (a physiological level) on cell calcium was abolished by the presence of 6.5 nM 24,25-dihydroxyvitamin D(3). In cell preparations from 14- or 28-week old birds 100nM 25(OH)D(3) had no effect, relative to vehicle controls. Incubation of cells with 2 microM BAY K8644, a calcium channel activator, stimulated (45)Ca uptake within 3 min relative to vehicle controls (P < 0.05), while addition of either 20 microM forskolin or 100 nM phorbol ester (stimulators of the PKA and PKC pathways, respectively) resulted in enhanced radionuclide levels after 10 min of incubation (P < 0.05, relative to corresponding controls). Finally, cells were treated with 100 nM 25(OH)D(3) or vehicle and samples taken at various times for analyses of protein kinase C and A activities. No effect of 25(OH)D(3) on protein kinase C activity was observed, while protein kinase A activity was stimulated to nearly 200% of controls at 1 min after 25(OH)D(3) addition (P < 0.05, relative to corresponding controls) and began declining at 3 min, returning to control levels 5 min after additions. We conclude that 25(OH)D(3) has a direct effect on calcium handling in enterocytes of young animals that may in part be mediated by the protein kinase A signal transduction pathway. Copyright 2003 Wiley-Liss, Inc.

Activating autoantibodies to the angiotensin II type I receptor play an important role in mediating hypertension in response to adoptive transfer of CD4+ T lymphocytes from placental ischemic rats.

PubMed

Novotny, Sarah Richards; Wallace, Kedra; Heath, Judith; Moseley, Janae; Dhillon, Pushpinder; Weimer, Abram; Wallukat, Gerd; Herse, Florian; Wenzel, Katrin; Martin, James N; Dechend, Ralf; Lamarca, Babbette

2012-05-15

Hypertension in rats with chronic placental ischemia (reduced uterine perfusion pressure, RUPP) is associated with elevated inflammatory cytokines, agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) and CD4(+) T cells; all of which are elevated in preclamptic women. Additionally, we have shown that adoptive transfer of RUPP CD4(+) T cells increases blood pressure, inflammatory cytokines, and sFlt-1. The objective of this study was to determine the long-term effects of RUPP CD4(+) T cells on AT1-AA, renal and systemic hemodynamics in pregnant rats. To answer this question CD4(+) T splenocytes were magnetically isolated on day 19 of gestation from control RUPP and normal pregnant (NP) rats and injected into a new group of NP rats at day 13 of gestation. On day 19 of gestation mean arterial pressure (MAP) and renal function (glomerular filtration rates, GFR) were analyzed and serum collected for AT1-AA analysis. To determine a role for AT1-AA to mediate RUPP CD4(+) T cell-induced blood pressure increases, MAP was analyzed in a second group of rats treated with AT1 receptor blockade losartan (10 mg·kg(-1)·day(-1)) and in a third group of rats treated with rituximab, a B cell-depleting agent (250 mg/kg) we have shown previously to decrease AT1-AA production in RUPP rats. MAP increased from 101 ± 2 mmHg NP to 126 ± 2 mmHg in RUPP rats (P < 0.001) and to 123 ± 1 mmHg in NP rats injected with RUPP CD4(+) T cells (NP+RUPP CD4(+)T cells) (P < 0.001). Furthermore, GFR decreased from 2.2 ml/min (n = 7) in NP rats to 1.0 ml/min (n = 5) NP+RUPP CD4(+)T cell. Circulating AT1-AA increased from 0.22 ± 0.1 units in NP rats to 13 ± 0.7 (P < 0.001) units in NP+RUPP CD4(+)T cell-treated rats but decreased to 8.34 ± 1 beats/min in NP+RUPP CD4(+) T cells chronically treated with rituximab. Hypertension in NP+RUPP CD4(+)T cell group was attenuated by losartan (102 ± 4 mmHg) and with B cell depletion (101 ± 5 mmHg). Therefore, we conclude that one mechanism of hypertension in response to CD4(+) T lymphocytes activated during placental ischemia is via AT1 receptor activation, potentially via AT1-AA during pregnancy.

DNA precursor pool: a significant target for N-methyl-N-nitrosourea in C3H/10T1/2 clone 8 cells.

PubMed Central

Topal, M D; Baker, M S

1982-01-01

Synchronized C3H/10T1/2 clone 8 cells were treated in vitro with a nontoxic dose of N-methyl-N-nitrosourea during their S phase. Chromatographic isolation of the deoxyribonucleotide DNA precursor pool and measurement of the precursor content per cell showed that a nucleic acid residue in the precursor pool is 190-13,000 times more susceptible to methylation than a residue in the DNA duplex, depending on the site of methylation. This conclusion comes from measurements indicating that, for example, the N-1 position of adenine in dATP is 6.3 times more methylated than the same position in the DNA, even though the adenine content of the pool is only a fraction (0.0005) of the adenine content of the DNA helix. The comparative susceptibility between pool and DNA was found to vary with the site of methylation in the order the N-1 position of adenine greater than phosphate greater than the N-3 position of adenine greater than the O6 position of guanine greater than the N-7 position of guanine. The significance of these results for chemical mutagenesis and carcinogenesis is discussed. PMID:6954535

Differential expression of steroid 5alpha-reductase isozymes and association with disease severity and angiogenic genes predict their biological role in prostate cancer.

PubMed

Das, Kakoli; Lorena, Pia D N; Ng, Lai Kuan; Lim, Diana; Shen, Liang; Siow, Woei Yun; Teh, Ming; Reichardt, Juergen K V; Salto-Tellez, Manuel

2010-09-01

The biological role of steroid 5alpha-reductase isozymes (encoded by the SRD5A1 and SRD5A2 genes) and angiogenic factors that play important roles in the pathogenesis and vascularization of prostate cancer (PC) is poorly understood. The sub-cellular expression of these isozymes and vascular endothelial growth factor (VEGF) in PC tissue microarrays (n=62) was examined using immunohistochemistry. The effect of SRD5A inhibition on the angiogenesis pathway genes in PC was also examined in prostate cell lines, LNCaP, PC3, and RWPE-1, by treating them with the SRD5A inhibitors finasteride and dutasteride, followed by western blot, quantitative PCR, and ELISA chip array techniques. In PC tissues, nuclear SRD5A1 expression was strongly associated with higher cancer Gleason scores (P=0.02), higher cancer stage (P=0.01), and higher serum prostate specific antigen (PSA) levels (P=0.01), whereas nuclear SRD5A2 expression was correlated with VEGF expression (P=0.01). Prostate tumor cell viability was significantly reduced in dutasteride-treated PC3 and RWPE-1 cells compared with finasteride-treated groups. Expression of the angiogenesis pathway genes transforming growth factor beta 1 (TGFB1), endothelin (EDN1), TGFalpha (TGFA), and VEGFR1 was upregulated in LNCaP cells, and at least 7 out of 21 genes were upregulated in PC3 cells treated with finasteride (25 muM). Our findings suggest that SRD5A1 expression predominates in advanced PC, and that inhibition of SRD5A1 and SRD5A2 together was more effective in reducing cell numbers than inhibition of SRD5A2 alone. However, these inhibitors did not show any significant difference in prostate cell angiogenic response. Interestingly, some angiogenic genes remained activated after treatment, possibly due to the duration of treatment and tumor resistance to inhibitors.

Adjuvant chemotherapy versus chemoradiotherapy for small cell lung cancer with lymph node metastasis: a retrospective observational study with use of a national database in Japan.

PubMed

Urushiyama, Hirokazu; Jo, Taisuke; Yasunaga, Hideo; Yamauchi, Yasuhiro; Matsui, Hiroki; Hasegawa, Wakae; Takeshima, Hideyuki; Hiraishi, Yoshihisa; Mitani, Akihisa; Fushimi, Kiyohide; Nagase, Takahide

2017-09-02

The optimal postoperative treatment strategy for small cell lung cancer (SCLC) remains unclear, especially in patients with lymph node metastasis. We aimed to compare the outcomes of patients with SCLC and lymph node metastasis treated with postoperative adjuvant chemotherapy or chemoradiotherapy. We retrospectively collected data on patients with postoperative SCLC diagnosed with N1 and N2 lymph node metastasis from the Diagnosis Procedure Combination database in Japan, between July 2010 and March 2015. We extracted data on patient age, sex, comorbidities, and TNM classification at lung surgery; operative procedures, chemotherapy drugs, and radiotherapy during hospitalization; and discharge status. Recurrence-free survival was compared between the chemotherapy and chemoradiotherapy groups using multivariable Cox regression analysis. Median recurrence-free survival was 1146 days (95% confidence interval [CI], 885-1407) in the chemotherapy group (n = 489) and 873 days (95% CI, 464-1282) in the chemoradiotherapy group (n = 75). There was no significant difference between these after adjusting for patient backgrounds (hazard ratio, 1.29; 95% CI, 0.91-1.84). There was no significant difference in recurrence-free survival between patients with SCLC and N1-2 lymph node metastasis treated with postoperative adjuvant chemotherapy and chemoradiotherapy. Further randomized clinical trials are needed to address this issue.

Outcome of patients after treatment for a squamous cell carcinoma of the oropharynx.

PubMed

Röösli, Christof; Tschudi, Dominique C; Studer, Gabriela; Braun, Julia; Stoeckli, Sandro J

2009-03-01

This study evaluates the oncologic outcome with regard to survival and locoregional tumor control in a cohort of patients with oropharyngeal squamous cell carcinoma (OPSCC) treated according to a uniform algorithm. Retrospective chart review. A total of 427 consecutive patients with OPSCC were treated from 1990 to 2006. Treatment modalities were surgery alone (n = 102), surgery with adjuvant radio(chemo)therapy (n = 159), and primary radio(chemo)therapy (n = 166). Study endpoints were the five-year overall survival (OS) and disease-specific survival (DSS) stratified for primary tumor subsite, stage, T and N category, and age. The five-year OS and DSS for the entire cohort were 57.9% and 68.6%, respectively. OS and DSS for surgery alone were 70.3% and 76.5%, for surgery with radiation 66.6% and 78.9%, and for primary radiation 40.8% and 52.6%, respectively. Survival was significantly better for low stages (stage I/II vs. III/IV), small tumors (T1/2 vs. T3/4), limited nodal involvement (N0/1 vs. N2/3), and younger age at diagnosis. Together with our previous study on quality of life, we were able to show that our selection process gives excellent oncologic outcome in combination with high levels of function and quality of life. Surgery alone for early OPSCC and surgery followed by radiation for advanced OPSCC remain valuable treatment options. Primary radiochemotherapy is a strong alternative for patients who are not candidates for function-preserving surgery.

Effects of 2-methoxyestradiol on apoptosis and HIF-1α and HIF-2α expression in lung cancer cells under normoxia and hypoxia.

PubMed

Aquino-Gálvez, Arnoldo; González-Ávila, Georgina; Delgado-Tello, Javier; Castillejos-López, Manuel; Mendoza-Milla, Criselda; Zúñiga, Joaquín; Checa, Marco; Maldonado-Martínez, Héctor Aquiles; Trinidad-López, Axel; Cisneros, José; Torres-Espíndola, Luz María; Hernández-Jiménez, Claudia; Sommer, Bettina; Cabello-Gutiérrez, Carlos; Gutiérrez-González, Luis H

2016-01-01

Hypoxic tumor cells are known to be more resistant to conventional chemotherapy and radiation than normoxic cells. However, the effects of 2-methoxyestradiol (2-ME), an anti-angiogenic, antiproliferative and pro-apoptotic drug, on hypoxic lung cancer cells are unknown. The aim of the present study was to compare the effects of 2-ME on cell growth, apoptosis, hypoxia-inducible factor 1α (HIF-1α) and HIF-2α gene and protein expression in A549 cells under normoxic and hypoxic conditions. To establish the optimal 2-ME concentration with which to carry out the apoptosis assay and to examine mRNA and protein expression of HIFs, cell growth analysis was carried out through N-hexa-methylpararosaniline staining assays in A549 cell cultures treated with one of five different 2-ME concentrations at different times under normoxic or hypoxic growth conditions. The 2-ME concentration of 10 mM at 72 h was selected to perform all further experiments. Apoptotic cells were analyzed by flow cytometry. Western blotting was used to determine HIF-1α and HIF-2α protein expression in total cell extracts. Cellular localization of HIF-1α and HIF-2α was assessed by immunocytochemistry. HIF-1α and HIF-2α gene expression was determined by real-time PCR. A significant increase in the percentage of apoptosis was observed when cells were treated with 2-ME under a normoxic but not under hypoxic conditions (p=0.006). HIF-1α and HIF-2α protein expression levels were significantly decreased in cells cultured under hypoxic conditions and treated with 2-ME (p<0.001). Furthermore, 2-ME decreased the HIF-1α and HIF-2α nuclear staining in cells cultured under hypoxia. The HIF-1α and HIF-2α mRNA levels were significantly lower when cells were exposed to 2-ME under normoxia and hypoxia. Our results suggest that 2-ME could have beneficial results when used with conventional chemotherapy in an attempt to lower the invasive and metastatic processes during cancer development due to its effects on the gene expression and protein synthesis of HIFs.

Multiplex polymerase chain reaction-based prognostic models in diffuse large B-cell lymphoma patients treated with R-CHOP.

PubMed

Green, Tina M; Jensen, Andreas K; Holst, René; Falgreen, Steffen; Bøgsted, Martin; de Stricker, Karin; Plesner, Torben; Mourits-Andersen, Torben; Frederiksen, Mikael; Johnsen, Hans E; Pedersen, Lars M; Møller, Michael B

2016-09-01

We present a multiplex analysis for genes known to have prognostic value in an attempt to design a clinically useful classification model in patients with diffuse large B-cell lymphoma (DLBCL). Real-time polymerase chain reaction was used to measure transcript levels of 28 relevant genes in 194 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). Including International Prognostic Index (IPI) as a variable in a penalized Cox regression, we investigated the association with disease progression for single genes or gene combinations in four models. The best model was validated in data from an online available R-CHOP treated cohort. With progression-free survival (PFS) as primary endpoint, the best performing IPI independent model incorporated the LMO2 and HLADQA1 as well as gene interactions for GCSAMxMIB1, GCSAMxCTGF and FOXP1xPDE4B. This model assigned 33% of patients (n = 60) to poor outcome with an estimated 3-year PFS of 40% vs. 87% for low risk (n = 61) and intermediate (n = 60) risk groups (P < 0·001). However, a simpler, IPI independent model incorporated LMO2 and BCL2 and assigned 33% of the patients with a 3-year PFS of 35% vs. 82% for low risk group (P < 0·001). We have documented the impact of a few single genes added to IPI for assignment in new drug trials. © 2016 John Wiley & Sons Ltd.

Cervical lymph node metastases of squamous cell carcinoma from an unknown primary: outcomes and patterns of failure.

PubMed

Mistry, R C; Qureshi, S S; Talole, S D; Deshmukh, S

2008-01-01

Management of cervical lymph nodes metastases of squamous cell carcinoma (SCC) from primary of unknown origin (PUO) is contentious and there is insignificant data from India on this subject. To present experience of management of these patients treated with curative intent at a single institution. Retrospective study of patients treated between 1989-1994 in a tertiary referral cancer centre. Eighty-nine patients were evaluated in the study period and their survival compared with patients with common sites of primary in the head and neck with comparable node stage. Kaplan-Meier method. The clinical stage of the neck nodes at presentation was N1 in 11%, N2a in 28.5%, N2b in 22.5%, N3 in 35% and Nx in 3.4% patients. All patients underwent surgery and 70 patients received more than 40Gy postoperative radiotherapy. Twenty-nine (32.6%) patients had relapse of which 19 (21%) were in the neck. Postoperative radiotherapy did not influence the neck relapse (p=0.72). Primary was detected in 13 patients (14.6%) on subsequent follow up. The overall five and eight-years survival was 55% and 51% respectively. The overall five-year survival was better compared to patients with known primary with comparable node stage. Patients with cervical lymph nodes metastases of SCC from PUO have reasonable survival and low rate of development of subsequent primary when treated with surgery and radiotherapy. The overall survival is comparable to that of patients with known primary and hence an attempt at cure should always be made.

Neem leaf extract inhibits mammary carcinogenesis by altering cell proliferation, apoptosis, and angiogenesis

PubMed Central

Arumugam, Arunkumar; Agullo, Pamela; Boopalan, Thiyagarajan; Nandy, Sushmita; Lopez, Rebecca; Gutierrez, Christina; Narayan, Mahesh; Rajkumar, Lakshmanaswamy

2014-01-01

Plant-based medicines are useful in the treatment of cancer. Many breast cancer patients use complementary and alternative medicine in parallel with conventional treatments. Neem is historically well known in Asia and Africa as a versatile medicinal plant with a wide spectrum of biological activities. The experiments reported herein determined whether the administration of an ethanolic fraction of Neem leaf (EFNL) inhibits progression of chemical carcinogen-induced mammary tumorigenesis in rat models. Seven-week-old female Sprague Dawley rats were given a single intraperitoneal injection of N-methyl-N-nitrosourea (MNU). Upon the appearance of palpable mammary tumors, the rats were divided into vehicle-treated control groups and EFNL-treated groups. Treatment with EFNL inhibited MNU-induced mammary tumor progression. EFNL treatment was also highly effective in reducing mammary tumor burden and in suppressing mammary tumor progression even after the cessation of treatment. Further, we found that EFNL treatment effectively upregulated proapoptotic genes and proteins such as p53, B cell lymphoma-2 protein (Bcl-2)-associated X protein (Bax), Bcl-2-associated death promoter protein (Bad) caspases, phosphatase and tensin homolog gene (PTEN), and c-Jun N-terminal kinase (JNK). In contrast, EFNL treatment caused downregulation of anti-apoptotic (Bcl-2), angiogenic proteins (angiopoietin and vascular endothelial growth factor A [VEGF-A]), cell cycle regulatory proteins (cyclin D1, cyclin-dependent kinase 2 [Cdk2], and Cdk4), and pro-survival signals such as NFκB, mitogen-activated protein kinase 1 (MAPK1). The data obtained in this study demonstrate that EFNL exert a potent anticancer effect against mammary tumorigenesis by altering key signaling pathways. PMID:24146019

Antioxidative and apoptotic properties of polyphenolic extracts from edible part of artichoke (Cynara scolymus L.) on cultured rat hepatocytes and on human hepatoma cells.

PubMed

Miccadei, Stefania; Di Venere, Donato; Cardinali, Angela; Romano, Ferdinando; Durazzo, Alessandra; Foddai, Maria Stella; Fraioli, Rocco; Mobarhan, Sohrab; Maiani, Giuseppe

2008-01-01

Cultured rat hepatocytes and human hepatoma HepG2 cells were used to evaluate the hepatoprotective properties of polyphenolic extracts from the edible part of artichoke (AE). The hepatocytes were exposed to H2O2generated in situ by glucose oxidase and were treated with either AE, or pure chlorogenic acid (ChA) or with the well known antioxidant, N, N'-diphenyl-p-phenilenediamine (DPPD). Addition of glucose oxidase to the culture medium caused depletion of intracellular glutathione (GSH) content, accumulation of malondialdehyde (MDA) in the cultures, as a lipid peroxidation indicator, and cell death. These results demonstrated that AE protected cells from the oxidative stress caused by glucose oxidase, comparable to DPPD. Furthermore, AE, as well as ChA, prevented the loss of total GSH and the accumulation of MDA. Treatment of HepG2 cells for 24 h with AE reduced cell viability in a dose-dependent manner, however, ChA had no prominent effects on the cell death rate. Similarly, AE rather than ChA induced apoptosis, measured by flow cytometric analysis of annexin and by activation of caspase-3, in HepG2 cells. Our findings indicate that AE had a marked antioxidative potential that protects hepatocytes from an oxidative stress. Furthermore, AE reduced cell viability and had an apoptotic activity on a human liver cancer cell line.

Lamin A/C Is Required for ChAT-Dependent Neuroblastoma Differentiation.

PubMed

Guglielmi, Loredana; Nardella, Marta; Musa, Carla; Iannetti, Ilaria; Arisi, Ivan; D'Onofrio, Mara; Storti, Andrea; Valentini, Alessandra; Cacci, Emanuele; Biagioni, Stefano; Augusti-Tocco, Gabriella; D'Agnano, Igea; Felsani, Armando

2017-07-01

The mouse neuroblastoma N18TG2 clone is unable to differentiate and is defective for the enzymes of the biosynthesis of neurotransmitters. The forced expression of choline acetyltransferase (ChAT) in these cells results in the synthesis and release of acetylcholine (Ach) and hence in the expression of neurospecific features and markers. To understand how the expression of ChAT triggered neuronal differentiation, we studied the differences in genome-wide transcription profiles between the N18TG2 parental cells and its ChAT-expressing 2/4 derived clone. The engagement of the 2/4 cells in the neuronal developmental program was confirmed by the increase of the expression level of several differentiation-related genes and by the reduction of the amount of transcripts of cell cycle genes. At the same time, we observed a massive reorganization of cytoskeletal proteins in terms of gene expression, with the accumulation of the nucleoskeletal lamina component Lamin A/C in differentiating cells. The increase of the Lmna transcripts induced by ChAT expression in 2/4 cells was mimicked treating the parental N18TG2 cells with the acetylcholine receptor agonist carbachol, thus demonstrating the direct role played by this receptor in neuron nuclei maturation. Conversely, a treatment of 2/4 cells with the muscarinic receptor antagonist atropine resulted in the reduction of the amount of Lmna RNA. Finally, the hypothesis that Lmna gene product might play a crucial role in the ChAT-dependent molecular differentiation cascade was strongly supported by Lmna knockdown in 2/4 cells leading to the downregulation of genes involved in differentiation and cytoskeleton formation and to the upregulation of genes known to regulate self-renewal and stemness.

Effects of sub-lethal neurite outgrowth inhibitory concentrations of chlorpyrifos oxon on cytoskeletal proteins and acetylcholinesterase in differentiating N2a cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flaskos, J., E-mail: flaskos@vet.auth.gr; Nikolaidis, E.; Harris, W.

2011-11-15

Previous work in our laboratory has shown that sub-lethal concentrations (1-10 {mu}M) of chlorpyrifos (CPF), diazinon (DZ) and diazinon oxon (DZO) inhibit the outgrowth of axon-like neurites in differentiating mouse N2a neuroblastoma cells concomitant with altered levels and/or phosphorylation state of axonal cytoskeleton and growth-associated proteins. The aim of the present work was to determine whether chlorpyrifos oxon (CPO) was capable of inhibiting N2a cell differentiation in a similar manner. Using experimental conditions similar to our previous work, sub-lethal concentrations (1-10 {mu}M) of CPO were found to inhibit N2a cell differentiation. However, unlike previous studies with DZ and DZO, theremore » was a high level of sustained inhibition of acetylcholinesterase (AChE) in CPO treated cells. Impairment of neurite outgrowth was also associated with reduced levels of growth associated protein-43 and neurofilament heavy chain (NFH), and the distribution of NFH in cells stained by indirect immunofluorescence was disrupted. However, in contrast to previous findings for DZO, the absolute level of phosphorylated NFH was unaffected by CPO exposure. Taken together, the findings suggest that sub-lethal concentrations of CPO inhibit axon outgrowth in differentiating N2a cells and that this effect involves reduced levels of two proteins that play key roles in axon outgrowth and maintenance. Although the inhibition of neurite outgrowth is unlikely to involve AChE inhibition directly, further work will help to determine whether the persistent inhibition of AChE by CPO can account for the different effects induced by CPO and DZO on the levels of total and phosphorylated NFH. -- Highlights: Black-Right-Pointing-Pointer Sub-lethal levels of chlorpyrifos oxon inhibit neurite outgrowth in N2a cells Black-Right-Pointing-Pointer Acetylcholinesterase exhibits sustained inhibition throughout exposure Black-Right-Pointing-Pointer The levels of neurofilament heavy chain and GAP-43 protein are reduced Black-Right-Pointing-Pointer Neurofilament heavy chain forms aggregates in cell bodies Black-Right-Pointing-Pointer Thus at least two axon-associated cytoskeletal proteins are disrupted by this agent.« less

Renal Allograft Survival in Nonhuman Primates Infused With Donor Antigen-Pulsed Autologous Regulatory Dendritic Cells.

PubMed

Ezzelarab, M B; Raich-Regue, D; Lu, L; Zahorchak, A F; Perez-Gutierrez, A; Humar, A; Wijkstrom, M; Minervini, M; Wiseman, R W; Cooper, D K C; Morelli, A E; Thomson, A W

2017-06-01

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte-derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg-pulsed autologous DCreg (1.4-3.6 × 10 6 /kg) were administered intravenously, 1 day before MHC-mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag-pulsed DCreg (n = 5) was associated with evidence of modulated host CD4 + and CD8 + T cell responses to donor Ag and attenuation of systemic IL-17 production. Circulating anti-donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag-pulsed DCreg rejected its graft in association with progressively elevated anti-donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag-pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen. © 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

JAK2 mediates lung fibrosis, pulmonary vascular remodelling and hypertension in idiopathic pulmonary fibrosis: an experimental study.

PubMed

Milara, Javier; Ballester, Beatriz; Morell, Anselm; Ortiz, José L; Escrivá, Juan; Fernández, Estrella; Perez-Vizcaino, Francisco; Cogolludo, Angel; Pastor, Enrique; Artigues, Enrique; Morcillo, Esteban; Cortijo, Julio

2018-06-01

Pulmonary hypertension (PH) is a common disorder in patients with idiopathic pulmonary fibrosis (IPF) and portends a poor prognosis. Recent studies using vasodilators approved for PH have failed in improving IPF mainly due to ventilation ( V )/perfusion ( Q ) mismatching and oxygen desaturation. Janus kinase type 2 (JAK2) is a non-receptor tyrosine kinase activated by a broad spectrum of profibrotic and vasoactive mediators, but its role in PH associated to PH is unknown. The study of JAK2 as potential target to treat PH in IPF. JAK2 expression was increased in pulmonary arteries (PAs) from IPF (n=10; 1.93-fold; P=0.0011) and IPF+PH (n=9; 2.65-fold; P<0.0001) compared with PA from control subjects (n=10). PA remodelling was evaluated in human pulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells (HPASMCs) from patients with IPF in vitro treated with the JAK2 inhibitor JSI-124 or siRNA-JAK2 and stimulated with transforming growth factor beta. Both JSI-124 and siRNA-JAK2 inhibited the HPAEC to mesenchymal transition and the HPASMCs to myofibroblast transition and proliferation. JAK2 inhibition induced small PA relaxation in precision-cut lung slice experiments. PA relaxation was dependent of the large conductance calcium-activated potassium channel (BK Ca ). JAK2 inhibition activated BK Ca channels and reduced intracellular Ca 2+ . JSI-124 1 mg/kg/day, reduced bleomycin-induced lung fibrosis, PA remodelling, right ventricular hypertrophy, PA hypertension and V / Q mismatching in rats. The animal studies followed the ARRIVE guidelines. JAK2 participates in PA remodelling and tension and may be an attractive target to treat IPF associated to PH. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Neoadjuvant chemoradiation and surgery improves survival outcomes compared with definitive chemoradiation in the treatment of stage IIIA N2 non-small-cell lung cancer.

PubMed

Darling, Gail E; Li, Fei; Patsios, Demetris; Massey, Christine; Wallis, Adam G; Coate, Linda; Keshavjee, Shaf; Pierre, Andrew; De Perrot, Marc; Yasufuku, Kazuhiro; Cypel, Marcelo; Waddell, Tom

2015-11-01

The objective of this study was to compare survival in patients with stage IIIA (N2) non-small-cell lung cancer (NSCLC) treated with definitive chemoradiation (CRT) or surgery plus neoadjuvant chemoradiation or chemotherapy (CRTS). A retrospective analysis of 242 patients with stage IIIA (N2) NSCLC treated with curative intent between 1997 and 2007, identified 215 patients with surgically resectable disease. Overall survival outcomes were analysed using the Kaplan-Meier plots, log-rank tests and Cox proportional hazards models adjusting for age, gender, histology, smoking history and performance status. Recurrences were compared using competing risks methods, including the proportional subdistribution hazards regression model. CRTS was used to treat 104 patients and CRT in 111. Comparing CRTS with CRT patients, median age was 60 vs 62, 50 (48%) vs 69 (62%) were male and 65 (62.5%) vs 60 (54%) had adenocarcinoma. Of CRTS patients, 83 (80%) had a lobectomy. CRTS patients compared with CRT patients had decreased risk of recurrence at any site [hazard ratio (HR) = 0. 46, 95% confidence interval (CI): 0.32-0.64 P < 0.0001], local recurrence (HR = 0.50, 95% CI: 0.29-0.87, P = 0.013), loco--regional recurrence (HR = 0.51, 95% CI: 0.33-0.78, P = 0.002) and death (HR: 0.45, 95% CI: 0.33-0.62, P < 0.0001) with a median survival of 4.2 years vs 1.7 years). Risk of distant recurrence was also reduced in the surgical group (HR: 0.57; 95% CI: 0.38-0.87, P = 0.017). Treatment-related mortality was low in both cohorts. For patients with surgically resectable stage IIIA (N2) NSCLC, neoadjuvant therapy plus surgery reduces loco-regional and distant recurrence and improves survival. Treatment-related mortality was not significantly increased compared with the patients treated with CRT alone. © The Author 2015. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Sodium nitrite induces acute central nervous system toxicity in guinea pigs exposed to systemic cell-free hemoglobin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buehler, Paul W.; Butt, Omer I.; D'Agnillo, Felice, E-mail: felice.dagnillo@fda.hhs.gov

Highlights: {yields} Toxicological implications associated with the use of NaNO{sub 2} therapy to treat systemic cell-free Hb exposure are not well-defined. {yields} Systemic Hb exposure followed by NaNO{sub 2} infusion induces acute CNS toxicities in guinea pigs. {yields} These CNS effects were not reproduced by the infusion of cell-free Hb or NaNO{sub 2} alone. {yields} NaNO{sub 2}-mediated oxidation of cell-free Hb may play a causative role in the observed CNS changes. -- Abstract: Systemic cell-free hemoglobin (Hb) released via hemolysis disrupts vascular homeostasis, in part, through the scavenging of nitric oxide (NO). Sodium nitrite (NaNO{sub 2}) therapy can attenuate themore » hypertensive effects of Hb. However, the chemical reactivity of NaNO{sub 2} with Hb may enhance heme- or iron-mediated toxicities. Here, we investigate the effect of NaNO{sub 2} on the central nervous system (CNS) in guinea pigs exposed to systemic cell-free Hb. Intravascular infusion of NaNO{sub 2}, at doses sufficient to alleviate Hb-mediated blood pressure changes, reduced the expression of occludin, but not zona occludens-1 (ZO-1) or claudin-5, in cerebral tight junctions 4 h after Hb infusion. This was accompanied by increased perivascular heme oxygenase-1 expression, neuronal iron deposition, increased astrocyte and microglial activation, and reduced expression of neuron-specific nuclear protein (NeuN). These CNS changes were not observed in animals treated with Hb or NaNO{sub 2} alone. Taken together, these findings suggest that the use of nitrite salts to treat systemic Hb exposure may promote acute CNS toxicity.« less

Three-year Follow up of GMCSF/bi-shRNA(furin) DNA-transfected Autologous Tumor Immunotherapy (Vigil) in Metastatic Advanced Ewing's Sarcoma.

PubMed

Ghisoli, Maurizio; Barve, Minal; Mennel, Robert; Lenarsky, Carl; Horvath, Staci; Wallraven, Gladice; Pappen, Beena O; Whiting, Sam; Rao, Donald; Senzer, Neil; Nemunaitis, John

2016-08-01

Ewing's sarcoma is a devastating rare pediatric cancer of the bone. Intense chemotherapy temporarily controls disease in most patients at presentation but has limited effect in patients with progressive or recurrent disease. We previously described preliminary results of a novel immunotherapy, FANG (Vigil) vaccine, in which 12 advanced stage Ewing's patients were safely treated and went on to achieve a predicted immune response (IFNγ ELISPOT). We describe follow-up through year 3 of a prospective, nonrandomized study comparing an expanded group of Vigil-treated advanced disease Ewing's sarcoma patients (n = 16) with a contemporaneous group of Ewing's sarcoma patients (n = 14) not treated with Vigil. Long-term follow-up results show a survival benefit without evidence of significant toxicity (no ≥ grade 3) to Vigil when administered once monthly by intradermal injection (1 × 10e(6) cells/injection to 1 × 10e(7) cells/injection). Specifically, we report a 1-year actual survival of 73% for Vigil-treated patients compared to 23% in those not treated with Vigil. In addition, there was a 17.2-month difference in overall survival (OS; Kaplan-Meier) between the Vigil (median OS 731 days) and no Vigil patient groups (median OS 207 days). In conclusion, these results supply the rational for further testing of Vigil in advanced stage Ewing's sarcoma.

Prognostic Value of the Pretreatment Advanced Lung Cancer Inflammation Index (ALI) in Diffuse Large B Cell Lymphoma Patients Treated with R-CHOP Chemotherapy.

PubMed

Park, Young Hoon; Yi, Hyeon Gyu; Lee, Moon Hee; Kim, Chul Soo; Lim, Joo Han

2017-01-01

The Advanced Lung Cancer Inflammation Index (ALI, body mass index × albumin/neutrophil-to-lymphocyte ratio) has been demonstrated to be a prognostic factor of survival in some solid cancers. We retrospectively investigated the usefulness of the ALI to predict chemotherapy response and survival in 212 patients with diffuse large B cell lymphoma (DLBCL) treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy. Patients were allocated to a low ALI group (n = 82, 38.7%) or a high ALI group (n = 130, 61.3%) according to an optimal pretreatment ALI cut-off value of 15.5 as determined by receiver operating curve analysis. The low ALI group displayed more adverse clinical characteristics, lower rates of complete remission (54.9 vs. 75.4%, p = 0.008), and poorer 5-year progression-free (PFS, 58.1 vs. 77.3%, p = 0.006) and overall (OS, 64.2 vs. 80.2%, p = 0.008) survival. Multivariate analysis showed that low ALI was found to independently predict shorter PFS and OS. Interestingly, a low ALI reverted to a high ALI during treatment in 58 patients (27.4%), and the 5-year OS of these patients was better than that of patients whose ALI remained low (n = 24, 72.5 vs. 24%, p < 0.001). ALI might be an easily available marker for predicting clinical outcomes in DLBCL patients treated with R-CHOP chemotherapy. © 2017 S. Karger AG, Basel.

Sorafenib induced alteration of protein glycosylation in hepatocellular carcinoma cells

PubMed Central

Liu, Tianhua; Liu, Riqiang; Zhang, Shu; Guo, Kun; Zhang, Qinle; Li, Wei; Liu, Yinkun

2017-01-01

Sorafenib is a multikinase inhibitor and is effective in treating hepatocellular carcinoma (HCC). However, it remains unknown whether sorafenib induces the alteration of protein glycosylation. The present study treated HCC MHCC97L and MHCC97H cells with a 50% inhibitory concentration of sorafenib. Following this treatment, alteration of protein glycosylation was detected using a lectin microarray. Compared with the controls, the binding capacity of glycoproteins extracted from sorafenib-treated HCC cells to the lectins Bauhinia purpurea lectin, Dolichos biflorus agglutinin, Euonymus europaeus lectin, Helix aspersa lectin, Helix pomatia lectin, Jacalin, Maclura pomifera lectin and Vicia villosa lectin were enhanced; while, the binding capacities to the lectins Caragana arborescens lectin, Lycopersicon esculentum lectin, Limulus polyphemus lectin, Maackia amurensis lecin I, Phaseolus vulgaris leucoagglutinin, Ricinus communis agglutinin 60, Sambucus nigra lectin and Solanum tuberosum lectin were reduced (spot intensity median/background intensity median ≥2, P<0.05). This difference in glycoprotein binding capacity indicates that cells treated with sorafenib could increase α-1,3GalNAc/Gal, β-1,3 Gal, GalNAcα-Ser/Thr(Tn) and α-GalNAc structures and decrease GlcNAc, sialic acid, tetra-antennary complex-type N-glycan and β-1,4Gal structures. These results were additionally confirmed by lectin blotting. Expression levels of signaling molecules including erythroblastosis 26–1 (Ets-1), extracellular signal-related kinases (ERK) and phosphorylated-ERK were measured by western blotting. There was a reduction in the expression of Ets-1 and ERK phosphorylation in sorafenib or 1,4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene treated cells suggesting that sorafenib may reduce the expression levels of Ets-1 by blocking the Ras/Raf/mitogen activated protein kinase signaling pathway. In the present study, it was clear that sorafenib could inhibit the proliferation of HCC cells and alter protein glycosylation. The findings of this study may lead to providing a novel way of designing new anti-HCC drugs. PMID:28693200

Palm oil mill effluent treatment using a two-stage microbial fuel cells system integrated with immobilized biological aerated filters.

PubMed

Cheng, Jia; Zhu, Xiuping; Ni, Jinren; Borthwick, Alistair

2010-04-01

An integrated system of two-stage microbial fuel cells (MFCs) and immobilized biological aerated filters (I-BAFs) was used to treat palm oil mill effluent (POME) at laboratory scale. By replacing the conventional two-stage up-flow anaerobic sludge blanket (UASB) with a newly proposed upflow membrane-less microbial fuel cell (UML-MFC) in the integrated system, significant improvements on NH(3)-N removal were observed and direct electricity generation implemented in both MFC1 and MFC2. Moreover, the coupled iron-carbon micro-electrolysis in the cathode of MFC2 further enhanced treatment efficiency of organic compounds. The I-BAFs played a major role in further removal of NH(3)-N and COD. For influent COD and NH(3)-N of 10,000 and 125 mg/L, respectively, the final effluents COD and NH(3)-N were below 350 and 8 mg/L, with removal rates higher than 96.5% and 93.6%. The GC-MS analysis indicated that most of the contaminants were satisfactorily biodegraded by the integrated system. Copyright 2009 Elsevier Ltd. All rights reserved.

Large Cardiac Muscle Patches Engineered From Human Induced-Pluripotent Stem Cell-Derived Cardiac Cells Improve Recovery From Myocardial Infarction in Swine.

PubMed

Gao, Ling; Gregorich, Zachery R; Zhu, Wuqiang; Mattapally, Saidulu; Oduk, Yasin; Lou, Xi; Kannappan, Ramaswamy; Borovjagin, Anton V; Walcott, Gregory P; Pollard, Andrew E; Fast, Vladimir G; Hu, Xinyang; Lloyd, Steven G; Ge, Ying; Zhang, Jianyi

2018-04-17

Here, we generated human cardiac muscle patches (hCMPs) of clinically relevant dimensions (4 cm × 2 cm × 1.25 mm) by suspending cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human induced-pluripotent stem cells in a fibrin scaffold and then culturing the construct on a dynamic (rocking) platform. In vitro assessments of hCMPs suggest maturation in response to dynamic culture stimulation. In vivo assessments were conducted in a porcine model of myocardial infarction (MI). Animal groups included: MI hearts treated with 2 hCMPs (MI+hCMP, n=13), MI hearts treated with 2 cell-free open fibrin patches (n=14), or MI hearts with neither experimental patch (n=15); a fourth group of animals underwent sham surgery (Sham, n=8). Cardiac function and infarct size were evaluated by MRI, arrhythmia incidence by implanted loop recorders, and the engraftment rate by calculation of quantitative polymerase chain reaction measurements of expression of the human Y chromosome. Additional studies examined the myocardial protein expression profile changes and potential mechanisms of action that related to exosomes from the cell patch. The hCMPs began to beat synchronously within 1 day of fabrication, and after 7 days of dynamic culture stimulation, in vitro assessments indicated the mechanisms related to the improvements in electronic mechanical coupling, calcium-handling, and force generation, suggesting a maturation process during the dynamic culture. The engraftment rate was 10.9±1.8% at 4 weeks after the transplantation. The hCMP transplantation was associated with significant improvements in left ventricular function, infarct size, myocardial wall stress, myocardial hypertrophy, and reduced apoptosis in the periscar boarder zone myocardium. hCMP transplantation also reversed some MI-associated changes in sarcomeric regulatory protein phosphorylation. The exosomes released from the hCMP appeared to have cytoprotective properties that improved cardiomyocyte survival. We have fabricated a clinically relevant size of hCMP with trilineage cardiac cells derived from human induced-pluripotent stem cells. The hCMP matures in vitro during 7 days of dynamic culture. Transplantation of this type of hCMP results in significantly reduced infarct size and improvements in cardiac function that are associated with reduction in left ventricular wall stress. The hCMP treatment is not associated with significant changes in arrhythmogenicity. © 2017 American Heart Association, Inc.

MDM2 phenotypic and genotypic profiling, respective to TP53 genetic status, in diffuse large B-cell lymphoma patients treated with rituximab-CHOP immunochemotherapy: a report from the International DLBCL Rituximab-CHOP Consortium Program

PubMed Central

Xu-Monette, Zijun Y.; Møller, Michael B.; Tzankov, Alexander; Montes-Moreno, Santiago; Hu, Wenwei; Manyam, Ganiraju C.; Kristensen, Louise; Fan, Lei; Visco, Carlo; Dybkær, Karen; Chiu, April; Tam, Wayne; Zu, Youli; Bhagat, Govind; Richards, Kristy L.; Hsi, Eric D.; Choi, William W. L.; van Krieken, J. Han; Huang, Qin; Huh, Jooryung; Ai, Weiyun; Ponzoni, Maurilio; Ferreri, Andrés J. M.; Wu, Lin; Zhao, Xiaoying; Bueso-Ramos, Carlos E.; Wang, Sa A.; Go, Ronald S.; Li, Yong; Winter, Jane N.; Medeiros, L. Jeffrey

2013-01-01

MDM2 is a key negative regulator of the tumor suppressor p53, however, the prognostic significance of MDM2 overexpression in diffuse large B-cell lymphoma (DLBCL) has not been defined convincingly. In a p53 genetically–defined large cohort of de novo DLBCL patients treated with rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (R-CHOP) chemotherapy, we assessed MDM2 and p53 expression by immunohistochemistry (n = 478), MDM2 gene amplification by fluorescence in situ hybridization (n = 364), and a single nucleotide polymorphism in the MDM2 promoter, SNP309, by SNP genotyping assay (n = 108). Our results show that MDM2 overexpression, unlike p53 overexpression, is not a significant prognostic factor in overall DLBCL. Both MDM2 and p53 overexpression do not predict for an adverse clinical outcome in patients with wild-type p53 but predicts for significantly poorer survival in patients with mutated p53. Variable p53 activities may ultimately determine the survival differences, as suggested by the gene expression profiling analysis. MDM2 amplification was observed in 3 of 364 (0.8%) patients with high MDM2 expression. The presence of SNP309 did not correlate with MDM2 expression and survival. This study indicates that evaluation of MDM2 and p53 expression correlating with TP53 genetic status is essential to assess their prognostic significance and is important for designing therapeutic strategies that target the MDM2-p53 interaction. PMID:23982177

Ursodeoxycholic acid inhibits overexpression of P-glycoprotein induced by doxorubicin in HepG2 cells.

PubMed

Komori, Yuki; Arisawa, Sakiko; Takai, Miho; Yokoyama, Kunihiro; Honda, Minako; Hayashi, Kazuhiko; Ishigami, Masatoshi; Katano, Yoshiaki; Goto, Hidemi; Ueyama, Jun; Ishikawa, Tetsuya; Wakusawa, Shinya

2014-02-05

The hepatoprotective action of ursodeoxycholic acid (UDCA) was previously suggested to be partially dependent on its antioxidative effect. Doxorubicin (DOX) and reactive oxygen species have also been implicated in the overexpression of P-glycoprotein (P-gp), which is encoded by the MDR1 gene and causes antitumor multidrug resistance. In the present study, we assessed the effects of UDCA on the expression of MDR1 mRNA, P-gp, and intracellular reactive oxygen species levels in DOX-treated HepG2 cells and compared them to those of other bile acids. DOX-induced increases in reactive oxygen species levels and the expression of MDR1 mRNA were inhibited by N-acetylcysteine, an antioxidant, and the DOX-induced increase in reactive oxygen species levels and DOX-induced overexpression of MDR1 mRNA and P-gp were inhibited by UDCA. Cells treated with UDCA showed improved rhodamine 123 uptake, which was decreased in cells treated with DOX alone. Moreover, cells exposed to DOX for 24h combined with UDCA accumulated more DOX than that of cells treated with DOX alone. Thus, UDCA may have inhibited the overexpression of P-gp by suppressing DOX-induced reactive oxygen species production. Chenodeoxycholic acid (CDCA) also exhibited these effects, whereas deoxycholic acid and litocholic acid were ineffective. In conclusion, UDCA and CDCA had an inhibitory effect on the induction of P-gp expression and reactive oxygen species by DOX in HepG2 cells. The administration of UDCA may be beneficial due to its ability to prevent the overexpression of reactive oxygen species and acquisition of multidrug resistance in hepatocellular carcinoma cells. Copyright © 2013 Elsevier B.V. All rights reserved.

α-Hederin inhibits interleukin 6-induced epithelial-to-mesenchymal transition associated with disruption of JAK2/STAT3 signaling in colon cancer cells.

PubMed

Sun, Dongdong; Shen, Weixing; Zhang, Feng; Fan, Huisen; Xu, Changliang; Li, Liu; Tan, Jiani; Miao, Yunjie; Zhang, Haibin; Yang, Ye; Cheng, Haibo

2018-05-01

Colon cancer is the third most frequently diagnosed malignancy and has high morbidity worldwide. Epithelial-mesenchymal transition (EMT) has been increasingly implicated in colon cancer progression and metastasis. The present study was aimed to evaluate the potential antitumor activity of α-hederin, a monodesmosidic triterpenoid saponin isolated from Hedera helix, in human SW620 colon cancer cells stimulated with interleukin 6 (IL-6) for mimicking the tumor inflammatory microenvironment in vivo. Cell viability assay showed that IL-6 at 6.25 ng/ml significantly enhanced viability of SW620 cells, and thus this concentration was used to stimulate SW620 cells throughout this study. We observed that α-hederin concentration-dependently inhibited cell viability, migration and invasion in IL-6-treated SW620 cells. Moreover, α-hederin significantly restored IL-6-induced decrease in E-cadherin expression and abolished IL-6-induced increase in N-cadherin, vimentin, fibronectin, twist and snail at both mRNA and protein levels in SW620 cells. These data suggested that α-hederin suppressed IL-6-indcued EMT in colon cancer cells. Further molecular examinations showed that α-hederin inhibited phosphorylation of Janus Kinase 2 (JAK2) and Signal Transducer and Activator of Transcription 3(STAT3), and halted the nuclear translocation of phosphorylated STAT3 in IL-6-treated SW620 cells. In addition, JAK2/STAT3 signaling inhibitor AG490 not only produced similar inhibitory effects on EMT markers as α-hederin, but also synergistically enhanced α-hederin's inhibitory effects on EMT markers in IL-6-treated SW620 cells. Altogether, we demonstrated that α-hederin suppressed IL-6-induced EMT associated with disruption of JAK2/STAT3 signaling in colon cancer cells. Our data strongly suggested α-hederin as a promising candidate for intervention of colon cancer and metastasis. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

111In-BnDTPA-F3: an Auger electron-emitting radiotherapeutic agent that targets nucleolin.

PubMed

Cornelissen, Bart; Waller, Andrew; Target, Carol; Kersemans, Veerle; Smart, Sean; Vallis, Katherine A

2012-02-20

The F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), a fragment of the human high mobility group protein 2, binds nucleolin. Nucleolin is expressed in the nuclei of normal cells but is also expressed on the membrane of some cancer cells. The goal was to investigate the use of 111In-labeled F3 peptide for Auger electron-targeted radiotherapy. F3 was labeled with fluorescein isothiocyanate (FITC) for confocal microscopy and conjugated to p-SCN-benzyl-diethylenetriaminepentaacetic acid (BnDTPA) for labeling with 111In to form 111In-BnDTPA-F3. MDA-MB-231-H2N (231-H2N) human breast cancer cells were exposed to 111In-BnDTPA-F3 and used in cell fractionation, γH2AX immunostaining (a marker of DNA double-strand breaks), and clonogenic assays. In vivo, biodistribution studies of 111In-BnDTPA-F3 were performed in 231-H2N xenograft-bearing mice. In tumor growth delay studies, 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) was administered intravenously to 231-H2N xenograft-bearing mice once weekly for 3 weeks. Membrane-binding of FITC-F3 was observed in 231-H2N cells, and there was co-localization of FITC-F3 with nucleolin in the nuclei. After exposure of 231-H2N cells to 111In-BnDTPA-F3 for 2 h, 1.7% of 111In added to the medium was membrane-bound. Of the bound 111In, 15% was internalized, and of this, 37% was localized in the nucleus. Exposure of 231-H2N cells to 111In-BnDTPA-F3 (1 μM, 6 MBq/μg) resulted in a dose-dependent increase in γH2AX foci and in a significant reduction of clonogenic survival compared to untreated cells or cells exposed to unlabeled BnDTPA-F3 (46 ± 4.1%, 100 ± 1.8%, and 132 ± 7.7%, respectively). In vivo, tumor uptake of 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) at 3-h post-injection was 1% of the injected dose per gram (%ID/g), and muscle uptake was 0.5%ID/g. In tumor growth delay studies, tumor growth rate was reduced 19-fold compared to untreated or unlabeled BnDTPA-F3-treated mice (p = 0.023). 111In-BnDTPA-F3 is internalized into 231-H2N cells and translocates to the nucleus. 111In-BnDTPA-F3 has a potent cytotoxic effect in vitro and an anti-tumor effect in mice bearing 231-H2N xenografts despite modest total tumor accumulation.

111In-BnDTPA-F3: an Auger electron-emitting radiotherapeutic agent that targets nucleolin

PubMed Central

2012-01-01

Introduction The F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), a fragment of the human high mobility group protein 2, binds nucleolin. Nucleolin is expressed in the nuclei of normal cells but is also expressed on the membrane of some cancer cells. The goal was to investigate the use of 111In-labeled F3 peptide for Auger electron-targeted radiotherapy. Methods F3 was labeled with fluorescein isothiocyanate (FITC) for confocal microscopy and conjugated to p-SCN-benzyl-diethylenetriaminepentaacetic acid (BnDTPA) for labeling with 111In to form 111In-BnDTPA-F3. MDA-MB-231-H2N (231-H2N) human breast cancer cells were exposed to 111In-BnDTPA-F3 and used in cell fractionation, γH2AX immunostaining (a marker of DNA double-strand breaks), and clonogenic assays. In vivo, biodistribution studies of 111In-BnDTPA-F3 were performed in 231-H2N xenograft-bearing mice. In tumor growth delay studies, 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) was administered intravenously to 231-H2N xenograft-bearing mice once weekly for 3 weeks. Results Membrane-binding of FITC-F3 was observed in 231-H2N cells, and there was co-localization of FITC-F3 with nucleolin in the nuclei. After exposure of 231-H2N cells to 111In-BnDTPA-F3 for 2 h, 1.7% of 111In added to the medium was membrane-bound. Of the bound 111In, 15% was internalized, and of this, 37% was localized in the nucleus. Exposure of 231-H2N cells to 111In-BnDTPA-F3 (1 μM, 6 MBq/μg) resulted in a dose-dependent increase in γH2AX foci and in a significant reduction of clonogenic survival compared to untreated cells or cells exposed to unlabeled BnDTPA-F3 (46 ± 4.1%, 100 ± 1.8%, and 132 ± 7.7%, respectively). In vivo, tumor uptake of 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) at 3-h post-injection was 1% of the injected dose per gram (%ID/g), and muscle uptake was 0.5%ID/g. In tumor growth delay studies, tumor growth rate was reduced 19-fold compared to untreated or unlabeled BnDTPA-F3-treated mice (p = 0.023). Conclusion 111In-BnDTPA-F3 is internalized into 231-H2N cells and translocates to the nucleus. 111In-BnDTPA-F3 has a potent cytotoxic effect in vitro and an anti-tumor effect in mice bearing 231-H2N xenografts despite modest total tumor accumulation. PMID:22348532

Outcomes of surgically treated human papillomavirus-related oropharyngeal squamous cell carcinoma with N3 disease.

PubMed

Zenga, Joseph; Haughey, Bruce H; Jackson, Ryan S; Adkins, Douglas R; Aranake-Chrisinger, John; Bhatt, Neel; Gay, Hiram A; Kallogjeri, Dorina; Martin, Eliot J; Moore, Eric J; Paniello, Randal C; Rich, Jason T; Thorstad, Wade L; Nussenbaum, Brian

2017-09-01

To evaluate outcomes for patients with pathological N3 (pN3) neck disease from human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) and determine variables predictive of survival. Retrospective case series with chart review. This study was conducted between 1998 and 2013 and included patients with HPV-related OPSCC treated with surgery with or without adjuvant therapy and who had pN3 nodal disease. The primary outcome was disease-specific survival (DSS). Secondary outcomes included overall survival (OS), disease-free survival (DFS), adverse events, and gastrostomy tube rates. Thirty-nine patients were included, of whom 36 (90%) underwent adjuvant therapy. Median follow-up was 39 months (range, 2-147 months). Mean age was 56 years, and 87% were male. Seventeen patients (44%) underwent selective neck dissection, whereas six (15%) underwent radical (n = 2) or extended radical (n = 4) neck dissection. Ninety-two percent had extracapsular extension. Five-year Kaplan-Meier estimated DSS, OS, and DFS were 89% (95% confidence interval [CI]: 79%-99%), 87% (95% CI: 75%-99%), and 84% (95% CI: 72%-96%), respectively. The disease recurrence rate was 10% (5% regional, 5% distant metastasis). Patients with less than 5 pathologically positive lymph nodes (P = .041) had improved DFS. Patients with HPV-related OPSCC and pN3 nodal disease treated with surgery and adjuvant therapy have very favorable long-term survival and regional control. Patients with five or more pathologically positive lymph nodes may be at higher risk for recurrence. 4. Laryngoscope, 127:2033-2037, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Effects of O 2 and N 2/H 2 plasma treatments on the neuronal cell growth on single-walled carbon nanotube paper scaffolds

NASA Astrophysics Data System (ADS)

Yoon, Ok Ja; Lee, Hyun Jung; Jang, Yeong Mi; Kim, Hyun Woo; Lee, Won Bok; Kim, Sung Su; Lee, Nae-Eung

2011-08-01

The O 2 and N 2/H 2 plasma treatments of single-walled carbon nanotube (SWCNT) papers as scaffolds for enhanced neuronal cell growth were conducted to functionalize their surfaces with different functional groups and to roughen their surfaces. To evaluate the effects of the surface roughness and functionalization modifications of the SWCNT papers, we investigated the neuronal morphology, mitochondrial membrane potential, and acetylcholine/acetylcholinesterase levels of human neuroblastoma during SH-SY5Y cell growth on the treated SWCNT papers. Our results demonstrated that the plasma-chemical functionalization caused changes in the surface charge states with functional groups with negative and positive charges and then the increased surface roughness enhanced neuronal cell adhesion, mitochondrial membrane potential, and the level of neurotransmitter in vitro. The cell adhesion and mitochondrial membrane potential on the negatively charged SWCNT papers were improved more than on the positively charged SWCNT papers. Also, measurements of the neurotransmitter level showed an enhanced acetylcholine level on the negatively charged SWCNT papers compared to the positively charged SWCNT papers.

Hybrid- and complex-type N-glycans are not essential for Newcastle disease virus infection and fusion of host cells.

PubMed

Sun, Qing; Zhao, Lixiang; Song, Qingqing; Wang, Zheng; Qiu, Xusheng; Zhang, Wenjun; Zhao, Mingjun; Zhao, Guo; Liu, Wenbo; Liu, Haiyan; Li, Yunsen; Liu, Xiufan

2012-03-01

N-linked glycans are composed of three major types: high-mannose (Man), hybrid or complex. The functional role of hybrid- and complex-type N-glycans in Newcastle disease virus (NDV) infection and fusion was examined in N-acetylglucosaminyltransferase I (GnT I)-deficient Lec1 cells, a mutant Chinese hamster ovary (CHO) cell incapable of synthesizing hybrid- and complex-type N-glycans. We used recombinant NDV expressing green fluorescence protein or red fluorescence protein to monitor NDV infection, syncytium formation and viral yield. Flow cytometry showed that CHO-K1 and Lec1 cells had essentially the same degree of NDV infection. In contrast, Lec2 cells were found to be resistant to NDV infection. Compared with CHO-K1 cells, Lec1 cells were shown to more sensitive to fusion induced by NDV. Viral attachment was found to be comparable in both lines. We found that there were no significant differences in the yield of progeny virus produced by both CHO-K1 and Lec1 cells. Quantitative analysis revealed that NDV infection and fusion in Lec1 cells were also inhibited by treatment with sialidase. Pretreatment of Lec1 cells with Galanthus nivalis agglutinin specific for terminal α1-3-linked Man prior to inoculation with NDV rendered Lec1 cells less sensitive to cell-to-cell fusion compared with mock-treated Lec1 cells. Treatment of CHO-K1 and Lec1 cells with tunicamycin, an inhibitor of N-glycosylation, significantly blocked fusion and infection. In conclusion, our results suggest that hybrid- and complex-type N-glycans are not required for NDV infection and fusion. We propose that high-Man-type N-glycans could play an important role in the cell-to-cell fusion induced by NDV.

Comparison of transplantation of bone marrow stromal cells (BMSC) and stem cell mobilization by granulocyte colony stimulating factor after traumatic brain injury in rat.

PubMed

Bakhtiary, Mehrdad; Marzban, Mohsen; Mehdizadeh, Mehdi; Joghataei, Mohammad Taghi; Khoei, Samideh; Pirhajati Mahabadi, Vahid; Laribi, Bahareh; Tondar, Mahdi; Moshkforoush, Arash

2010-10-01

Recent clinical studies of treating traumatic brain injury (TBI) with autologous adult stem cells led us to compare effect of intravenous injection of bone marrow mesenchymal stem cells (BMSC) and bone marrow hematopoietic stem cell mobilization, induced by granulocyte colony stimulating factor (G-CSF), in rats with a cortical compact device. Forty adult male Wistar rats were injured with controlled cortical impact device and divided randomly into four groups. The treatment groups were injected with 2 × 106 intravenous bone marrow stromal stem cell (n = 10) and also with subcutaneous G-CSF (n = 10) and sham-operation group (n = 10) received PBS and "bromodeoxyuridine (Brdu)" alone, i.p. All injections were performed 1 day after injury into the tail veins of rats. All cells were labeled with Brdu before injection into the tail veins of rats. Functional neurological evaluation of animals was performed before and after injury using modified neurological severity scores (mNSS). Animals were sacrificed 42 days after TBI and brain sections were stained by Brdu immunohistochemistry. Statistically, significant improvement in functional outcome was observed in treatment groups compared with control group (P<0.01). mNSS showed no significant difference between the BMSC and G-CSF-treated groups during the study period (end of the trial). Histological analyses showed that Brdu-labeled (MSC) were present in the lesion boundary zone at 42nd day in all injected animals. In our study, we found that administration of a bone marrow-stimulating factor (G-CSF) and BMSC in a TBI model provides functional benefits.

Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile.

PubMed

Ortiz, María Carolina; Lefimil, Claudia; Rodas, Paula I; Vernal, Rolando; Lopez, Mercedes; Acuña-Castillo, Claudio; Imarai, Mónica; Escobar, Alejandro

2015-01-01

Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ) and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ) or alternative-M2 (M2-MФ) activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM) upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1), widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies.

G2 accumulation and melanin overproduction in malignant melanocytes treated with a new nitrosourea.

PubMed

Buchdahl, C; Papon, J; Communal, Y; Bourges, M; Madelmont, J C

1998-12-01

Cystemustine (N'-(2-chloroethyl)-N-(2-(methylsulphonyl)ethyl)-N'-nitrosourea), a new anticancer chloroethylnitrosourea (CENU) is being tested in a phase II clinical trial of disseminated melanoma. The antitumour effect of this drug is mainly due to DNA damage in malignant melanocytes. Recently, we have shown that this damage can induce apoptosis in some melanoma cell lines. In others, apoptosis is not clearly observed, although there is a strong cytostatic effect. In this paper, we have characterized the cytological effect of cystemustine on murine malignant melanocytes (B16 cell line) which are resistant to apoptosis induced by this CENU. The results show that 3 days after cystemustine treatment, these melanocytes had accumulated in phase G2 of the cell cycle. There was then a strong morphological modification during a long cytostatic phase up to 30 days after treatment. During this cytostatic phase, there was uncontrolled DNA synthesis and marked swelling. Also, tyrosinase activity, melanin content and the number of mature melanosomes were greatly increased. These results suggest that when malignant melanocytes are not able to undergo apoptosis after treatment with CENU, they accumulate in G2 and this is followed by enhancement of melanogenesis.

Vitamin K2 Induces Mitochondria-Related Apoptosis in Human Bladder Cancer Cells via ROS and JNK/p38 MAPK Signal Pathways

PubMed Central

Duan, Fengsen; Yu, Yuejin; Guan, Rijian; Xu, Zhiliang; Liang, Huageng; Hong, Ling

2016-01-01

The effects of vitamin K2 on apoptosis in a variety of cancer cells have been well established in previous studies. However, the apoptotic effect of vitamin K2 on bladder cancer cells has not been evaluated. The aim of this study is to examine the apoptotic activity of Vitamin K2 in bladder cancer cells and investigate the underlying mechanism. In this study, Vitamin K2 induced apoptosis in bladder cancer cells through mitochondria pathway including loss of mitochondria membrane potential, cytochrome C release and caspase-3 cascade. Furthermore, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK was detected in Vitamin K2-treated cells and both SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38 MAPK) completely abolished the Vitamin K2-induced apoptosis and loss of mitochondria membrane potential. Moreover, the generation of reactive oxygen species (ROS) was detected in bladder cancer cells, upon treatment of vitamin K2 and the anti-oxidant N-acetyl cysteine (NAC) almost blocked the Vitamin K2-triggered apoptosis, loss of mitochondria membrane potential and activation of JNK and p38 MAPK. Taken together, these findings revealed that Vitamin K2 induces apoptosis in bladder cancer cells via ROS-mediated JNK/p38 MAPK and Mitochondrial pathways. PMID:27570977

Vitamin K2 Induces Mitochondria-Related Apoptosis in Human Bladder Cancer Cells via ROS and JNK/p38 MAPK Signal Pathways.

PubMed

Duan, Fengsen; Yu, Yuejin; Guan, Rijian; Xu, Zhiliang; Liang, Huageng; Hong, Ling

2016-01-01

The effects of vitamin K2 on apoptosis in a variety of cancer cells have been well established in previous studies. However, the apoptotic effect of vitamin K2 on bladder cancer cells has not been evaluated. The aim of this study is to examine the apoptotic activity of Vitamin K2 in bladder cancer cells and investigate the underlying mechanism. In this study, Vitamin K2 induced apoptosis in bladder cancer cells through mitochondria pathway including loss of mitochondria membrane potential, cytochrome C release and caspase-3 cascade. Furthermore, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK was detected in Vitamin K2-treated cells and both SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38 MAPK) completely abolished the Vitamin K2-induced apoptosis and loss of mitochondria membrane potential. Moreover, the generation of reactive oxygen species (ROS) was detected in bladder cancer cells, upon treatment of vitamin K2 and the anti-oxidant N-acetyl cysteine (NAC) almost blocked the Vitamin K2-triggered apoptosis, loss of mitochondria membrane potential and activation of JNK and p38 MAPK. Taken together, these findings revealed that Vitamin K2 induces apoptosis in bladder cancer cells via ROS-mediated JNK/p38 MAPK and Mitochondrial pathways.

Myricetin-induced apoptosis of triple-negative breast cancer cells is mediated by the iron-dependent generation of reactive oxygen species from hydrogen peroxide.

PubMed

Knickle, Allison; Fernando, Wasundara; Greenshields, Anna L; Rupasinghe, H P Vasantha; Hoskin, David W

2018-05-06

Myricetin is a dietary phytochemical with anticancer activity; however, the effect of myricetin on breast cancer cells remains unclear. Here, we show that myricetin inhibited the growth of triple-negative breast cancer (TNBC) cells but was less inhibitory for normal cells. The effect of myricetin was comparable to epigallocatechin gallate and doxorubicin, and greater than resveratrol and cisplatin. Myricetin-treated TNBC cells showed evidence of early and late apoptosis/necrosis, which was associated with intracellular reactive oxygen species (ROS) accumulation, extracellular regulated kinase 1/2 and p38 mitogen-activated protein kinase activation, mitochondrial membrane destabilization and cytochrome c release, and double-strand DNA breaks. The antioxidant N-acetyl-cysteine protected myricetin-treated TNBC cells from cytotoxicity due to DNA damage. Myricetin also induced hydrogen peroxide (H 2 O 2 ) production in cell-free culture medium, as well as in the presence of TNBC cells and normal cells. In addition, deferriprone-mediated inhibition of intracellular ROS generation via the iron-dependent Fenton reaction and inhibition of extracellular ROS accumulation with superoxide dismutase plus catalase prevented myricetin-induced cytotoxicity in TNBC cell cultures. We conclude that the cytotoxic effect of myricetin on TNBC cells was due to oxidative stress initiated by extracellular H 2 O 2 formed by autoxidation of myricetin, leading to intracellular ROS production via the Fenton reaction. Copyright © 2018. Published by Elsevier Ltd.

Synthesis of FUDP-N-acetylglucosamine and FUDP-glucose in Saccharomyces cerevisiae cells treated with 5-fluorouracil.

PubMed

Günther Sillero, María A; Pérez-Zúñiga, Francisco; Gomes, Joana; de Carvalho, Ana Isabel; Martins, Susana; Silles, Eduardo; Sillero, Antonio

2008-03-01

Saccharomyces cerevisiae cells (strain W303-1A) treated with 5-fluorouracil and grown in 2% (fermentative conditions) or in 0.1% glucose (oxidative conditions) accumulated two types of 5-fluoro-UDP-sugars (FUDP-sugars): FUDP-N-acetylglucosamine and FUDP-glucose. No difference was observed in both conditions of culture. The viability of yeast cells on treatment with 5-fluorouracil was also followed. Both FUDP-sugars were partially purified by column chromatography (on Hypersil ODS and Mono Q columns) and characterized by: (i) treatment with alkaline phosphatase (EC 3.1.3.1), snake venom phosphodiesterase (EC 3.1.4.1) and UDP-glucose dehydrogenase (EC 1.1.1.22); (ii) UV spectra; and (iii) matrix-assisted laser desorption/ionization-time of flight mass analysis and 1H-nuclear magnetic resonance spectrometry. The syntheses of both FUDP-sugars were inversely related to the concentration of uracil and directly related to the concentration of 5-fluorouracil in the culture medium. The strain W303-1A, requiring uracil for growth, was useful as a tool to analyze the effect of 5-fluorouracil on nucleotide metabolism.

Permanently Hypoxic Cell Culture Yields Rat Bone Marrow Mesenchymal Cells with Higher Therapeutic Potential in the Treatment of Chronic Myocardial Infarction.

PubMed

Liu, Yihua; Yang, Xiaoxi; Maureira, Pablo; Falanga, Aude; Marie, Vanessa; Gauchotte, Guillaume; Poussier, Sylvain; Groubatch, Frederique; Marie, Pierre-Yves; Tran, Nguyen

2017-01-01

The mismatch between traditional in vitro cell culture conditions and targeted chronic hypoxic myocardial tissue could potentially hamper the therapeutic effects of implanted bone marrow mesenchymal stem cells (BMSCs). This study sought to address (i) the extent of change to BMSC biological characteristics in different in vitro culture conditions and (ii) the effectiveness of permanent hypoxic culture for cell therapy in treating chronic myocardial infarction (MI) in rats. rat BMSCs were harvested and cultured in normoxic (21% O2, n=27) or hypoxic conditions (5% O2, n=27) until Passage 4 (P4). Cell growth tests, flow cytometry, and Bio-Plex assays were conducted to explore variations in the cell proliferation, phenotype, and cytokine expression, respectively. In the in vivo set-up, P3-BMSCs cultured in normoxia (n=6) or hypoxia (n=6) were intramyocardially injected into rat hearts that had previously experienced 1-month-old MI. The impact of cell therapy on cardiac segmental viability and hemodynamic performance was assessed 1 month later by 2-Deoxy-2[18F]fluoro-D-glucose (18F-FDG) positron emission tomography (PET) imaging and pressure-volume catheter, respectively. Additional histomorphological examinations were conducted to evaluate inflammation, fibrosis, and neovascularization. Hypoxic preconditioning significantly enhanced rat BMSC clonogenic potential and proliferation without altering the multipotency. Different profiles of inflammatory, fibrotic, and angiogenic cytokine secretion were also documented, with a marked correlation observed between in vitro and in vivo proangiogenic cytokine expression and tissue neovessels. Hypoxic-preconditioned cells presented a beneficial effect on the myocardial viability of infarct segments and intrinsic contractility. Hypoxic-preconditioned BMSCs were able to benefit myocardial perfusion and contractility, probably by modulating the inflammation and promoting angiogenesis. © 2017 The Author(s). Published by S. Karger AG, Basel.

Topical administration of interleukin-1 receptor antagonist as a therapy for aqueous-deficient dry eye in autoimmune disease.

PubMed

Vijmasi, Trinka; Chen, Feeling Y T; Chen, Ying Ting; Gallup, Marianne; McNamara, Nancy

2013-01-01

Dry eye is commonly associated with autoimmune diseases such as Sjögren's syndrome (SS), in which exocrinopathy of the lacrimal gland leads to aqueous tear deficiency and keratoconjunctivitis sicca (KCS). KCS is among the most common and debilitating clinical manifestations of SS that is often recalcitrant to therapy. We established mice deficient in the autoimmune regulator (Aire) gene as a model for autoimmune-mediated aqueous-deficient dry eye. In Aire-deficient mice, CD4+ T cells represent the main effector cells and local signaling via the interleukin-1 (IL-1/IL-1R1) pathway provides an essential link between autoreactive CD4+ T cells and ocular surface disease. In the current study, we evaluated the efficacy of topical administration of IL-1R1 antagonist (IL-1RA) anakinra in alleviating ocular surface damage resulting from aqueous-deficient dry eye in the setting of autoimmune disease. We compared the effect of commercially available IL-1R1 antagonist, anakinra (50 μg/mL concentration) to that of carboxymethylcellulose (CMC) vehicle control as a treatment for dry eye. Age-matched, Aire-deficient mice were treated three times daily with anakinra or CMC vehicle for 14 days using side-by-side (n = 4 mice/group) and paired-eye (n = 5) comparisons. We assessed (1) ocular surface damage with lissamine green staining; (2) tear secretion with wetting of phenol-red threads; (3) goblet cell (GC) mucin glycosylation with lectin histochemistry; (4) immune cell infiltration using anti-F4/80, CD11c, and CD4 T cell antibodies; and (5) gene expression of cornified envelope protein, Small Proline-Rich Protein-1B (SPRR1B) with real-time quantitative polymerase chain reaction. Aire-deficient mice treated with anakinra experienced significant improvements in ocular surface integrity and tear secretion. After 7 days of treatment, lissamine green staining decreased in eyes treated with anakinra compared to an equivalent increase in staining following treatment with CMC vehicle alone. By day 14, lissamine green staining in anakinra-treated eyes remained stable while eyes treated with CMC vehicle continued to worsen. Accordingly, there was a progressive decline in tear secretion in eyes treated with the CMC vehicle compared to a progressive increase in the anakinra-treated eyes over the 2-week treatment period. Aberrant acidification of GC mucins and pathological keratinization of the ocular surface were significantly reduced in anakinra-treated eyes. Significantly fewer Maackia amurensis leukoagglutinin positive goblet cells were noted in the conjunctiva of anakinra-treated eyes with a corresponding decrease in the expression of the pathological keratinization marker, SPRR1B. Finally, there was a downward trend in the infiltration of each immune cell type following anakinra treatment, but the cell counts compared to eyes treated with the vehicle alone were not significantly different. IL-1R antagonist, anakinra, demonstrates therapeutic benefits as a topical treatment for aqueous-deficient dry eye in a spontaneous mouse model of autoimmune KCS that mimics the clinical characteristics of SS. Targeting the IL-1/IL-1R1 signaling pathway through topical administration of IL-1RA may provide a novel option to improve ocular surface integrity, increase tear secretion, and restore the normal glycosylation pattern of GC mucins in patients with SS.

Topical administration of interleukin-1 receptor antagonist as a therapy for aqueous-deficient dry eye in autoimmune disease

PubMed Central

Vijmasi, Trinka; Chen, Feeling YT; Chen, Ying Ting; Gallup, Marianne

2013-01-01

Purpose Dry eye is commonly associated with autoimmune diseases such as Sjögren’s syndrome (SS), in which exocrinopathy of the lacrimal gland leads to aqueous tear deficiency and keratoconjunctivitis sicca (KCS). KCS is among the most common and debilitating clinical manifestations of SS that is often recalcitrant to therapy. We established mice deficient in the autoimmune regulator (Aire) gene as a model for autoimmune-mediated aqueous-deficient dry eye. In Aire-deficient mice, CD4+ T cells represent the main effector cells and local signaling via the interleukin-1 (IL-1/IL-1R1) pathway provides an essential link between autoreactive CD4+ T cells and ocular surface disease. In the current study, we evaluated the efficacy of topical administration of IL-1R1 antagonist (IL-1RA) anakinra in alleviating ocular surface damage resulting from aqueous-deficient dry eye in the setting of autoimmune disease. Methods We compared the effect of commercially available IL-1R1 antagonist, anakinra (50 μg/mL concentration) to that of carboxymethylcellulose (CMC) vehicle control as a treatment for dry eye. Age-matched, Aire-deficient mice were treated three times daily with anakinra or CMC vehicle for 14 days using side-by-side (n=4 mice/group) and paired-eye (n=5) comparisons. We assessed (1) ocular surface damage with lissamine green staining; (2) tear secretion with wetting of phenol-red threads; (3) goblet cell (GC) mucin glycosylation with lectin histochemistry; (4) immune cell infiltration using anti-F4/80, CD11c, and CD4 T cell antibodies; and (5) gene expression of cornified envelope protein, Small Proline-Rich Protein-1B (SPRR1B) with real-time quantitative polymerase chain reaction. Results Aire-deficient mice treated with anakinra experienced significant improvements in ocular surface integrity and tear secretion. After 7 days of treatment, lissamine green staining decreased in eyes treated with anakinra compared to an equivalent increase in staining following treatment with CMC vehicle alone. By day 14, lissamine green staining in anakinra-treated eyes remained stable while eyes treated with CMC vehicle continued to worsen. Accordingly, there was a progressive decline in tear secretion in eyes treated with the CMC vehicle compared to a progressive increase in the anakinra-treated eyes over the 2-week treatment period. Aberrant acidification of GC mucins and pathological keratinization of the ocular surface were significantly reduced in anakinra-treated eyes. Significantly fewer Maackia amurensis leukoagglutinin positive goblet cells were noted in the conjunctiva of anakinra-treated eyes with a corresponding decrease in the expression of the pathological keratinization marker, SPRR1B. Finally, there was a downward trend in the infiltration of each immune cell type following anakinra treatment, but the cell counts compared to eyes treated with the vehicle alone were not significantly different. Conclusions IL-1R antagonist, anakinra, demonstrates therapeutic benefits as a topical treatment for aqueous-deficient dry eye in a spontaneous mouse model of autoimmune KCS that mimics the clinical characteristics of SS. Targeting the IL-1/IL-1R1 signaling pathway through topical administration of IL-1RA may provide a novel option to improve ocular surface integrity, increase tear secretion, and restore the normal glycosylation pattern of GC mucins in patients with SS. PMID:24068863

Effect of Saw Palmetto Supplements on Androgen-Sensitive LNCaP Human Prostate Cancer Cell Number and Syrian Hamster Flank Organ Growth

PubMed Central

Opoku-Acheampong, Alexander B.; Penugonda, Kavitha; Lindshield, Brian L.

2016-01-01

Saw palmetto supplements (SPS) are commonly consumed by men with prostate cancer. We investigated whether SPS fatty acids and phytosterols concentrations determine their growth-inhibitory action in androgen-sensitive LNCaP cells and hamster flank organs. High long-chain fatty acids-low phytosterols (HLLP) SPS ≥ 750 nM with testosterone significantly increased and ≥500 nM with dihydrotestosterone significantly decreased LNCaP cell number. High long-chain fatty acids-high phytosterols (HLHP) SPS ≥ 500 nM with dihydrotestosterone and high medium-chain fatty acids-low phytosterols (HMLP) SPS ≥ 750 nM or with androgens significantly decreased LNCaP cell number (n = 3; p < 0.05). Five- to six-week-old, castrated male Syrian hamsters were randomized to control (n = 4), HLLP, HLHP, and HMLP SPS (n = 6) groups. Testosterone or dihydrotestosterone was applied topically daily for 21 days to the right flank organ; the left flank organ was treated with ethanol and served as the control. Thirty minutes later, SPS or ethanol was applied to each flank organ in treatment and control groups, respectively. SPS treatments caused a notable but nonsignificant reduction in the difference between left and right flank organ growth in testosterone-treated SPS groups compared to the control. The same level of inhibition was not seen in dihydrotestosterone-treated SPS groups (p < 0.05). Results may suggest that SPS inhibit 5α-reductase thereby preventing hamster flank organ growth. PMID:27272436

Effect of Saw Palmetto Supplements on Androgen-Sensitive LNCaP Human Prostate Cancer Cell Number and Syrian Hamster Flank Organ Growth.

PubMed

Opoku-Acheampong, Alexander B; Penugonda, Kavitha; Lindshield, Brian L

2016-01-01

Saw palmetto supplements (SPS) are commonly consumed by men with prostate cancer. We investigated whether SPS fatty acids and phytosterols concentrations determine their growth-inhibitory action in androgen-sensitive LNCaP cells and hamster flank organs. High long-chain fatty acids-low phytosterols (HLLP) SPS ≥ 750 nM with testosterone significantly increased and ≥500 nM with dihydrotestosterone significantly decreased LNCaP cell number. High long-chain fatty acids-high phytosterols (HLHP) SPS ≥ 500 nM with dihydrotestosterone and high medium-chain fatty acids-low phytosterols (HMLP) SPS ≥ 750 nM or with androgens significantly decreased LNCaP cell number (n = 3; p < 0.05). Five- to six-week-old, castrated male Syrian hamsters were randomized to control (n = 4), HLLP, HLHP, and HMLP SPS (n = 6) groups. Testosterone or dihydrotestosterone was applied topically daily for 21 days to the right flank organ; the left flank organ was treated with ethanol and served as the control. Thirty minutes later, SPS or ethanol was applied to each flank organ in treatment and control groups, respectively. SPS treatments caused a notable but nonsignificant reduction in the difference between left and right flank organ growth in testosterone-treated SPS groups compared to the control. The same level of inhibition was not seen in dihydrotestosterone-treated SPS groups (p < 0.05). Results may suggest that SPS inhibit 5α-reductase thereby preventing hamster flank organ growth.

Macrophages promote coal tar pitch extract-induced tumorigenesis of BEAS-2B cells and tumor metastasis in nude mice mediated by AP-1.

PubMed

Zhang, Peng; Jin, Yue-Fei; Zhang, Qiao; Wu, Yi-Ming; Wu, Wei-Dong; Yao, Wu; Wu, Yong-Jun; Li, Zhi-Tao; Zhao, Yong; Liu, Yu; Feng, Fei-Fei

2014-01-01

We sought to evaluate the role of tumor associated macrophages (TAMs) on the promotion of coal tar pitch extract (CTPE)-induced tumorigenesis of human bronchial epithelial cells (BEAS-2B) and tumor metastasis in nude mice, and related mechanisms. BEAS-2B cells were first treated with 2.4 mg/mL CTPE for 72 hours. After removal of CTPE, the cells were continuously cultured and passaged using trypsin-EDTA. THP-1 cells were used as macrophage-like cells. BEAS-2B cells under different conditions (n=6/ group) were injected into the back necks of nude mice, and alterations of tumor xenograft growth, indicative of tumorigenicity, and tumor metastasis were determined. Pathological changes (tumor nests and microvascular lesions) of HE-stained tumor tissues were also evaluated. The expression of AP-1(c-Jun) in xenografts and metastatic tumors was determined using immunohistochemistry. Tumor size and weight in nude mice transplanted with the mixture of CTPE-induced passage 30 BEAS-2B and THP-1 cells (2:1) were increased compared to those from the CTPE-treated BEAS-2B cells at passage 30 alone at different observation time points. Tumor metastasis to lymph nodes and liver was only detected after transplantation of a mixture the two kinds of cells. The numbers of tumor nests and microvascular lesions, and the expression levels of AP-1 (c-Jun) in tumors from the mixture of two kinds of cells were increased apparently in contrast to those in tumor from the CTPE-treated BEAS-2B cells of passage 30 alone. In addition, there was positive correlation between AP-1 (c-Jun) expression level and the number of microvascular lesions, or between AP-1 (c-Jun) expression level and tumor metastasis in these two groups. TAMs not only facilitate tumorigenesis transformation of CTPE-induced BEAS-2B cells, but also promote tumor growth, angiogenesis and metastasis in nude mice in vivo, which may be mediated by AP-1.

Hydrogen peroxide-induced DNA damage is independent of nuclear calcium but dependent on redox-active ions.

PubMed Central

Jornot, L; Petersen, H; Junod, A F

1998-01-01

In cells undergoing oxidative stress, DNA damage may result from attack by .OH radicals produced by the Fenton reaction, and/or by nucleases activated by nuclear calcium. In the present study, the participation of these two mechanisms was investigated in HeLa cells. Nuclear-targeted aequorin was used for selectively monitoring Ca2+ concentrations within the nuclei ([Ca2+]n), in conjunction with the cell-permeant calcium chelator bis-(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), the lipid-soluble broad-spectrum metal chelator with low affinity for Ca2+ and Mg2+ N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and the high-affinity iron/copper chelator 1, 10-phenanthroline (PHE). In Ca2+-containing medium, H2O2 induced extensive DNA strand breaks and an increase in [Ca2+]n that was almost identical to that observed in the cytosol ([Ca2+]c). In cells bathed in Ca2+-free/EGTA medium, in which the increases in [Ca2+]n and [Ca2+]c due to H2O2 were significantly reduced, similar levels of DNA fragmentation also occurred. In cells preloaded with BAPTA/AM or TPEN, the small increase of [Ca2+]n normally elicited by H2O2 in Ca2+-free medium was completely buffered, and DNA damage was largely prevented. On the other hand, pretreatment with PHE did not affect the calcium response in the nuclei, but completely prevented DNA strand breakage induced by H2O2. Re-addition of 100 microM CuSO4 and 100 microM FeSO4 to TPEN- and PHE-treated cells prior to H2O2 challenge reversed the effect of TPEN and PHE, whereas 1 mM was necessary to negate the effect of BAPTA/AM. The levels of DNA strand breakage observed, however, did not correlate with the amounts of 8-hydroxy 2'-deoxyguanosine (8-OHdG): H2O2 did not produce 8-OHdG, whereas PHE alone slightly increased 8-OHdG levels. CuSO4 and FeSO4 enhanced the effects of PHE, particularly in the presence of H2O2. Exposure of cells to a mixture of CuSO4/FeSO4 also resulted in a significant increase in 8-OHdG levels, which was prevented in cells preloaded with BAPTA/AM. Similar results were obtained in a cell-free system using isolated calf thymus DNA exposed to CuSO4/FeSO4, regardless of whether H2O2 was present or not. These results suggest that BAPTA/AM prevents H2O2-induced DNA damage by acting as an iron/copper chelator. These data also indicate that caution must be exercised in using Ca2+ chelating agents as evidence for a role in cellular Ca2+ levels in experimental conditions in which transition-metal-ion-mediated oxidant production is also occurring. PMID:9742216

Comparison of Newly Diagnosed and Relapsed Patients with Acute Promyelocytic Leukemia Treated with Arsenic Trioxide: Insight into Mechanisms of Resistance

PubMed Central

Chendamarai, Ezhilarasi; Ganesan, Saravanan; Alex, Ansu Abu; Kamath, Vandana; Nair, Sukesh C.; Nellickal, Arun Jose; Janet, Nancy Beryl; Srivastava, Vivi; Lakshmi, Kavitha M.; Viswabandya, Auro; Abraham, Aby; Aiyaz, Mohammed; Mullapudi, Nandita; Mugasimangalam, Raja; Padua, Rose Ann; Chomienne, Christine; Chandy, Mammen; Srivastava, Alok; George, Biju; Balasubramanian, Poonkuzhali; Mathews, Vikram

2015-01-01

There is limited data on the clinical, cellular and molecular changes in relapsed acute promyeloytic leukemia (RAPL) in comparison with newly diagnosed cases (NAPL). We undertook a prospective study to compare NAPL and RAPL patients treated with arsenic trioxide (ATO) based regimens. 98 NAPL and 28 RAPL were enrolled in this study. RAPL patients had a significantly lower WBC count and higher platelet count at diagnosis. IC bleeds was significantly lower in RAPL cases (P=0.022). The ability of malignant promyelocytes to concentrate ATO intracellularly and their in-vitro IC50 to ATO was not significantly different between the two groups. Targeted NGS revealed PML B2 domain mutations in 4 (15.38%) of the RAPL subset and none were associated with secondary resistance to ATO. A microarray GEP revealed 1744 genes were 2 fold and above differentially expressed between the two groups. The most prominent differentially regulated pathways were cell adhesion (n=92), cell survival (n=50), immune regulation (n=74) and stem cell regulation (n=51). Consistent with the GEP data, immunophenotyping revealed significantly increased CD34 expression (P=0.001) in RAPL cases and there was in-vitro evidence of significant microenvironment mediated innate resistance (EM-DR) to ATO. Resistance and relapse following treatment with ATO is probably multi-factorial, mutations in PML B2 domain while seen only in RAPL may not be the major clinically relevant cause of subsequent relapses. In RAPL additional factors such as expansion of the leukemia initiating compartment along with EM-DR may contribute significantly to relapse following treatment with ATO based regimens. PMID:25822503

In vitro and in vivo anti-glioma activity of a chalcone-quinoxaline hybrid.

PubMed

Loch-Neckel, Gecioni; Bicca, Maíra Assunção; Leal, Paulo César; Mascarello, Alessandra; Siqueira, Jarbas Mota; Calixto, João B

2015-01-27

Chalcones are important compounds that exhibit multiple biological activities, including anti-inflammatory, antimitotic and antibacterial properties. In the present study, we have analyzed the potential anti-cancer activity of a chalcone named N9 (a hybrid chalcone-quinoxaline compound) using in vitro and in vivo experimental glioma models. Here, we report N9-induced inhibition of cell proliferation and also N9-induced cell death in a concentration-dependent manner in U87-MG glioma cells. These effects of N9 appear to be associated with its ability to inhibit the expression of cell cycle-associated proteins, and also the augmentation in the expression of the p21 (p21/Cip1) protein, a cyclin-dependent kinase inhibitor. Additionally, N9 also potentiates the production of the pro-apoptotic markers Bax and p53 via inhibition of MDM2. Moreover, our results show that N9 also significantly enhanced apoptosis of U87-MG cells with disruption of mitochondrial membrane potential, generation of ROS and caspase-9 activation. In vivo experiments carried out in a murine xenograft model of U87-MG revealed that N9 produced a significant reduction of tumors volume when compared to vehicle treated mice. Collectively, data demonstrate that N9 possess in vitro and in vivo anti-cancer activity, an effect that seems to involve the induction of p53 and p21 proteins, as well as, the activation of mitochondrial apoptosis pathway associated with the inhibition of protein MDM2. Overall, this study suggests N9 is affecting a variety of intracellular pathways related to tumor apoptosis. Perhaps N9 or derivate molecules could represent new potential drugs for cancer therapeutics. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

A 4-deoxy analogue of N-acetyl-D-glucosamine inhibits heparan sulphate expression and growth factor binding in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wijk, Xander M.R. van; Oosterhof, Arie; Broek, Sebastiaan A.M.W. van den

2010-09-10

Heparan sulphate (HS) is a long, linear polysaccharide, which has a basic backbone of -{beta}1-4GlcA-{alpha}1-4GlcNAc- units. The involvement of HS in many steps of tumourigenesis, including growth and angiogenesis, makes it an appealing target for cancer therapy. To target the biosynthesis of HS by interfering with its chain elongation, a 4-deoxy analogue of N-acetyl-D-glucosamine (4-deoxy-GlcNAc) was synthesized. Using immunocytochemistry and agarose gel electrophoresis it was shown that incubation with the 4-deoxysugar resulted in a dose dependent reduction of HS expression of MV3 melanoma cells, 1 mM resulting in an almost nullified HS expression. The parent sugar GlcNAc had no effect.more » 4-deoxysugar treated cells were viable and proliferated at the same rate as control cells. Other glycan structures appeared to be only mildly affected, as staining by various lectins was generally not or only modestly inhibited. At 1 mM of the 4-deoxysugar, the capacity of cells to bind the HS-dependent pro-angiogenic growth factors FGF-2 and VEGF was greatly compromised. Using an in vitro angiogenesis assay, 4-deoxysugar treated endothelial cells showed a sharp reduction of FGF-2-induced sprout formation. Combined, these data indicate that an inexpensive, easily synthesized, water-soluble monosaccharide analogue can interfere with HS expression and pro-angiogenic growth factor binding.« less

Protective effect of Korean Red Ginseng against glucocorticoid-induced osteoporosis in vitro and in vivo

PubMed Central

Kim, Jinhee; Lee, Hyejin; Kang, Ki Sung; Chun, Kwang-Hoon; Hwang, Gwi Seo

2014-01-01

Background Glucocorticoids (GCs) are commonly used in many chemotherapeutic protocols and play an important role in the normal regulation of bone remodeling. However, the prolonged use of GCs results in osteoporosis, which is partially due to apoptosis of osteoblasts and osteocytes. In this study, effects of Korean Red Ginseng (KRG) on GC-treated murine osteoblastic MC3T3-E1 cells and a GC-induced osteoporosis mouse model were investigated. Methods MC3T3-E1 cells were exposed to dexamethasone (Dex) with or without KRG and cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Real-time polymerase chain reaction was performed to evaluate the apoptotic gene expression; osteogenic gene expression and alkaline phosphatase (ALP) activity were also measured. Western blotting was performed to evaluate the mitogen-activated protein kinase (MAPK) proteins. A GC-induced osteoporosis animal model was used for in vivo study. Results and conclusion The MTT assay revealed that Korean Red Ginseng (KRG) prevents loss of cell viability caused by Dex-induced apoptosis in MC3T3E1 cells. Real-time polymerase chain reaction data showed that groups treated with both Dex and KRG exhibited lower mRNA levels of caspase-3 and -9, whereas the mRNA levels of Bcl2, IAPs, and XIAP increased. Moreover, groups treated with both Dex and KRG demonstrated increased mRNA levels of ALP, RUNX2, and bone morphogenic proteins as well as increased ALP activity in MC3T3-E1 cells, compared to cells treated with Dex only. In addition, KRG increased protein kinase B (AKT) phosphorylation and decreased c-Jun N-terminal kinase (JNK) phosphorylation. Moreover, microcomputed tomography analysis of the femurs showed that GC implantation caused trabecular bone loss. However, a significant reduction of bone loss was observed in the KRG-treated group. These results suggest that the molecular mechanism of KRG in the GC-induced apoptosis may lead to the development of therapeutic strategies to prevent and/or delay osteoporosis. PMID:25535476

The insect repellent DEET (N,N-diethyl-3-methylbenzamide) increases the synthesis of glutathione S-transferase in cultured mosquito cells.

PubMed

Hellestad, Vanessa J; Witthuhn, Bruce A; Fallon, Ann M

2011-04-01

DEET (N,N-diethyl-3-methylbenzamide) is the active ingredient used in many commonly used insect repellents, but its mode of action remains poorly understood. Efforts to identify properties that could lead to the development of more effective active ingredients have distinguished among DEET's repellent, deterrent, and insecticidal activities. We used an Aedes albopictus mosquito cell line to evaluate DEET's toxicological properties in the absence of sensory input mediated by the olfactory system. When cells were treated with DEET and labeled with [(35)S]methionine/cysteine, a single 25-kDa protein was induced, relative to other proteins, on SDS-polyacrylamide gels. The 25-kDa band from DEET-treated cells was enriched in peptides corresponding to glutathione S-transferase D10 and/or theta in the Aedes aegypti genome. Consistent with the increased expression of the labeled protein, DEET-treated cells had increased glutathione S-transferase activity, and the radiolabeled band bound to Sepharose 4B containing reduced glutathione. By analyzing partial tryptic digests, we established that DEET induces the homolog of A. aegypti glutathione S-transferase, class theta, corresponding to protein XP_001658009.1 in the NCBI database. This specific effect of DEET at the subcellular level suggests that DEET induces physiological responses that extend beyond recognition by the peripheral olfactory system.

CD34 EXPRESSION BY HAIR FOLLICLE STEM CELLS IS REQUIRED FOR SKIN TUMOR DEVELOPMENT IN MICE

EPA Science Inventory

We used knockout mice to show that a cell surface protein called CD34 is required for skin tumor formation in mice. Wild type mice treated with 7-12-Dimethylbenz(a)anthracene (DMBA) and a tumor promoter developed papillomas. When we treated CD34 knockout (KO) mice the same way, n...

N-(2-hydroxyphenyl)-2-propylpentanamide, a valproic acid aryl derivative designed in silico with improved anti-proliferative activity in HeLa, rhabdomyosarcoma and breast cancer cells.

PubMed

Prestegui-Martel, Berenice; Bermúdez-Lugo, Jorge Antonio; Chávez-Blanco, Alma; Dueñas-González, Alfonso; García-Sánchez, José Rubén; Pérez-González, Oscar Alberto; Padilla-Martínez, Itzia Irene; Fragoso-Vázquez, Manuel Jonathan; Mendieta-Wejebe, Jessica Elena; Correa-Basurto, Ana María; Méndez-Luna, David; Trujillo-Ferrara, José; Correa-Basurto, José

2016-01-01

Epigenetic alterations are associated with cancer and their targeting is a promising approach for treatment of this disease. Among current epigenetic drugs, histone deacetylase (HDAC) inhibitors induce changes in gene expression that can lead to cell death in tumors. Valproic acid (VPA) is a HDAC inhibitor that has antitumor activity at mM range. However, it is known that VPA is a hepatotoxic drug. Therefore, the aim of this study was to design a set of VPA derivatives adding the arylamine core of the suberoylanilide hydroxamic acid (SAHA) with different substituents at its carboxyl group. These derivatives were submitted to docking simulations to select the most promising compound. The compound 2 (N-(2-hydroxyphenyl)-2-propylpentanamide) was the best candidate to be synthesized and evaluated in vitro as an anti-cancer agent against HeLa, rhabdomyosarcoma and breast cancer cell lines. Compound 2 showed a better IC 50 (μM range) than VPA (mM range) on these cancer cells. And also, 2 was particularly effective on triple negative breast cancer cells. In conclusion, 2 is an example of drugs designed in silico that show biological properties against human cancer difficult to treat as triple negative breast cancer.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pomorska-Mól, Małgorzata, E-mail: mpomorska@piwet.pulawy.pl; Kwit, Krzysztof; Markowska-Daniel, Iwona

The effect of a seven-day antibiotic therapy with doxycycline was investigated on the postvaccinal humoral and cellular immune response in pigs. The selected parameters of non-specific immunity were also studied. Fifty pigs were used (control not vaccinated (C, n = 10), control vaccinated (CV, n = 20), and experimental — received doxycycline (DOXY, n = 20)). For vaccination live-attenuated vaccine against pseudorabies (PR) was used. From day − 1 to day 5 pigs from DOXY group received doxycycline orally with drinking water, at the recommended dose. Pigs from DOXY and CV groups were vaccinated at 8 and 10 weeks ofmore » age. The results of the present study showed that cell-mediated postvaccinal immune response can be modulated by oral treatment with doxycycline. Significantly lower values of stimulation index were observed after PRV restimulation in doxycycline-treated pigs. Moreover, in the DOXY group a significant decrease in IFN-γ production after PRV restimulation was noted. The significantly lower number of CD4+CD8 + cells was also observed in doxy-treated, vaccinated pigs, 2 weeks after final vaccination. Simultaneously, specific humoral response was not disturbed. This study demonstrated the importance of defining the immune modulatory activity of doxycycline because it may alter the immune responses to vaccines. The exact mechanism of T-cell response suppression by doxycycline remains to be elucidated, however the influence of doxycycline on the secretion of various cytokines, including IFN-γ, may be considered as a possible cause. The present observations should prompt further studies on the practical significance of such phenomena in terms of clinical implications. - Highlights: • We examine the postvaccinal immune response in pigs treated with doxycycline. • Doxycycline negatively influenced the specific proliferation after recall stimulation. • Doxycycline negatively influenced secretion of IFN-γ after recall stimulation. • The lower number of CD4+CD8 + cells was observed in doxy-treated pigs. • The development of specific humoral response was not disturbed by doxycycline.« less

Intraportal Infusion of Bone Marrow Mononuclear or CD133+ Cells in Patients With Decompensated Cirrhosis: A Double-Blind Randomized Controlled Trial

PubMed Central

Mohamadnejad, Mehdi; Vosough, Massoud; Moossavi, Shirin; Nikfam, Sepideh; Mardpour, Soura; Akhlaghpoor, Shahram; Ashrafi, Mandana; Azimian, Vajiheh; Jarughi, Neda; Hosseini, Seyedeh-Esmat; Moeininia, Fatemeh; Bagheri, Mohamad; Sharafkhah, Maryam; Aghdami, Nasser

2016-01-01

The present study assessed the effects of intraportal infusions of autologous bone marrow-derived mononuclear cells (MNCs) and/or CD133+ cells on liver function in patients with decompensated cirrhosis. We randomly assigned 27 eligible patients to a placebo, MNCs, and/or CD133+ cells. Cell infusions were performed at baseline and month 3. We considered the absolute changes in the Model for End-Stage Liver Disease (MELD) scores at months 3 and 6 after infusion as the primary outcome. The participants and those who assessed the outcomes were unaware of the treatment intervention assignments. After 6 months, 9 patients were excluded because of liver transplantation (n = 3), hepatocellular carcinoma (n = 1), loss to follow-up (n = 3), and death (n = 2). The final analysis included 4 patients from the CD133+ group, 8 from the MNC group, and 6 from the placebo group. No improvement was seen in the MELD score at month 6 using either CD133+ cells or MNC infusions compared with placebo. However, at month 3 after infusion, a trend was seen toward a higher mean absolute change in the MELD score in patients who had received CD133+ cells compared with placebo (−2.00 ± 1.87 vs. −0.13 ± 1.46; p = .08). No significant adverse events occurred in the present study. A transient improvement in the MELD score was observed in subjects treated with CD133+ cells but not in the MNC or placebo group. Although the study was not powered to make definitive conclusions, the data justify further study of CD133+ therapy in cirrhotic patients. Significance Cell therapy is a new approach in liver disease. Several clinical experiments have been reported on the safety of bone marrow-derived stem cells to treat liver disorders. However, the effectiveness of these approaches in the long-term follow-ups of patients initiated controversial discussions among the scientific community. A double-blind randomized controlled trial was designed to address this concern scientifically. A transient improvement in the patients’ signs occurred; however, for a sustainable result, more work is needed. The results of multiple administrations of cells reported in the present study can be compared with the results from other single-injection studies. PMID:26659833

Are we optimizing gestational diabetes treatment with glyburide? The pharmacologic basis for better clinical practice

PubMed Central

Hebert, MF; Ma, X; Naraharisetti, SB; Krudys, KM; Umans, JG; Hankins, GDV; Caritis, SN; Miodovnik, M; Mattison, DR; Unadkat, JD; Kelly, EJ; Blough, D; Cobelli, C; Ahmed, MS; Snodgrass, WR; Carr, DB; Easterling, TR; Vicini, P

2009-01-01

Glyburide’s PK and PD have not been studied in women with gestational diabetes mellitus (GDM). The objective was to assess steady-state PK of glyburide as well as insulin sensitivity, beta-cell responsivity and overall disposition indices following a mixed meal tolerance test (MMTT) in GDM (n=40), non-pregnant type 2 diabetic (T2DM) (n=26) and healthy pregnant (n=40, MMTT only) women. At equivalent doses, glyburide plasma concentrations were ~50% lower in pregnancy compared to non-pregnant women. Average glyburide umbilical cord to maternal plasma concentration ratio at the time of delivery was 0.7 ± 0.4. Insulin sensitivity was ~5-fold lower in women with GDM compared to healthy pregnancy. Despite comparable beta-cell responsivity index, average beta-cell function corrected for insulin resistance was >3.5- fold lower in women with glyburide-treated GDM than healthy pregnancy. Women with GDM that fail glyburide may benefit from alternate medication selection or dosage escalation, though fetal safety should be considered. PMID:19295505

Grape juice concentrate prevents oxidative DNA damage in peripheral blood cells of rats subjected to a high-cholesterol diet.

PubMed

Aguiar, Odair; Gollücke, Andréa Pittelli Boiago; de Moraes, Bárbara Bueno; Pasquini, Gabriela; Catharino, Rodrigo Ramos; Riccio, Maria Francesca; Ihara, Silvia Saiuli Miki; Ribeiro, Daniel Araki

2011-03-01

The goal of the present study was to investigate whether subchronic treatment with grape juice concentrate is able to protect liver and peripheral blood cells against cholesterol-induced injury in rats. The effects of the grape juice concentrate treatment on histopathological changes, immunohistochemistry for cyclo-oxygenase-2 (COX-2), and basal and oxidative DNA damage induced by H2O2 using a single-cell gel (comet) assay were evaluated. Male Wistar rats (n 18) were divided into three groups: group 1--negative control; group 2--cholesterol at 1 % (w/w) in their diet, treated for 5 weeks; group 3--cholesterol at 1 % in their chow, treated for 5 weeks, and grape juice concentrate at 222 mg/d in their drinking-water in the final week only. The results indicated that the treatment with grape juice concentrate did not show remarkable differences regarding liver tissue in group 3 compared with group 2. However, grape juice concentrate was able to decrease oxidative DNA damage induced by H2O2 in peripheral blood cells, as depicted by the tail moment results. COX-2 expression in the liver did not show statistically significant differences (P>0·05) between groups. Taken together, the present results suggest that the administration of subchronic grape juice concentrate prevents oxidative DNA damage in peripheral blood cells.

Modulation of notch signaling pathway to prevent H2O2/menadione-induced SK-N-MC cells death by EUK134.

PubMed

Kamarehei, Maryam; Yazdanparast, Razieh

2014-10-01

The brain in Alzheimer's disease is under increased oxidative stress, and this may have a role in the pathogenesis and neural death in this disorder. It has been verified that numerous signaling pathways involved in neurodegenerative disorders are activated in response to reactive oxygen species (ROS). EUK134, a synthetic salen-manganese antioxidant complex, has been found to possess many interesting pharmacological activities awaiting exploration. The present study is to characterize the role of Notch signaling in apoptotic cell death of SK-N-MC cells. The cells were treated with hydrogen peroxide (H2O2) or menadione to induce oxidative stress. The free-radical scavenging capabilities of EUK134 were studied through the MTT assay, glutathione peroxidase (GPx) enzyme activity assay, and glutathione (GSH) Levels. The extents of lipid peroxidation, protein carbonyl formation, and intracellular ROS levels, as markers of oxidative stress, were also studied. Our results showed that H2O2/menadione reduced GSH levels and GPx activity. However, EUK134 protected cells against ROS-induced cell death by down-regulation of lipid peroxidation and protein carbonyl formation as well as restoration of antioxidant enzymes activity. ROS induced apoptosis and increased NICD and HES1 expression. Inhibition of NICD production proved that Notch signaling is involved in apoptosis through p53 activation. Moreover, H2O2/menadione led to Numb protein down-regulation which upon EUK134 pretreatment, its level increased and subsequently prevented Notch pathway activation. We indicated that EUK134 can be a promising candidate in designing natural-based drugs for ROS-induced neurodegenerative diseases. Collectively, ROS activated Notch signaling in SK-N-MC cells leading to cell apoptosis.

The chance of small interfering RNAs as eligible candidates for a personalized treatment of prostate cancer.

PubMed

Pietschke, Katharina; Walker, Tobias; Krajewski, Stefanie; Kurz, Julia; Aufderklamm, Stefan; Schwentner, Christian; Schlensak, Christian; Stenzl, Arnulf; Wendel, Hans P; Nolte, Andrea

2014-01-01

Prostate cancer is one of the leading malignant tumors in men. Current therapies are associated with severe side effects making it problematic for many multi-morbid patients to receive treatment. Prostate specific antigen, serum response factor (SRF), signal transducer and activator of transcription-3 (STAT3), hypoxia-inducible factor-1α (HIF-1α), HIF-2α, E2F1 and Survivin are well known proteins being overexpressed in cancer cells, expediting cell growth and also demonstrated in prostate cancer cells. Targeting these genes using the RNA-Interference pathway could be a new approach for prostate cancer therapy with fewer side effects. Three prostate cancer cell lines were cultured under standard conditions and transfected with three different concentrations (25 nM, 50 nM, 100 nM) of specific small interfering RNAs (siRNAs) targeting SRF, STAT3, HIF1α, HIF2α, E2F1 and Survivin in a non-viral manner. Cells treated with non-specific siRNA (SCR-siRNA) served as control. Changes of messenger RNA (mRNA) levels were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The analysis of the effect of siRNA on the number of cells was detected using CASY cell counter system. Transfections of the PC-3 cell line with specific siRNA especially against Survivin, E2F1, HIF1α- and HIF2α-siRNA resulted in a significant reduction of intracellular mRNA concentration together with a significant decreased number of cells. In the LnCAP and DU-145 cell lines Survivin and E2F1 showed similar effects. The impact of silencing STAT3 or SRF showed little influence on the amount of cells in all three cell lines. This study shows that RNAi succeeds in silencing gene expression and reducing the number of cells in differing dimensions depending on the transfected cell line and used siRNA.

ANTI-AMEBIC ACTIVITY OF DIOSGENIN ON NAEGLERIA FOWLERI TROPHOZOITES.

PubMed

Rabablert, Jundee; Tiewcharoen, Supathra; Auewarakul, Prasert; Atithep, Thassanant; Lumlerdkij, Natchagorn; Vejaratpimol, Renu; Junnu, Virach

2015-09-01

The aim of this study was to investigate the activity of diosgenin against Naegleria fowleri trophozoites at the cellular and molecular levels. Diosgenin (100 μg/ml; 241.2 μM) had a 100% inhibitory effect on N. fowleri trophozoites (5 x 10(5) cell/ml). Scanning electron micrograph revealed diosgenin decreased the number of sucker-like apparatuses and food cup formation among N. fowleri trophozoites at 3 and 6 hours post-exposure, respectively. Diosgenin down-regulated the nf cysteine protease gene expression of N. fowleri trophozoites at 6 and 12 hours post-exposure. The toxicity to mammalian cells caused by diosgenin at therapeutic dose was less than amphotericin B, the current drug used to treat N. fowleri infections. Our findings suggest diosgenin has activity against the surface membrane and the nf cysteine pro tease of N. fowleri trophozoites. However, the other mechanisms of action of diosgenin against N. fowleri trophozoites require further exploration.

Induction of glandular stomach cancers in Helicobacter pylori-sensitive Mongolian gerbils treated with N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine in drinking water.

PubMed

Tatematsu, M; Yamamoto, M; Shimizu, N; Yoshikawa, A; Fukami, H; Kaminishi, M; Oohara, T; Sugiyama, A; Ikeno, T

1998-02-01

An animal model of stomach carcinogenesis was established using Mongolian gerbils with N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as the carcinogens. In addition, the sensitivity of these gerbils to Helicobacter pylori (H. pylori) was confirmed. One hundred and sixty specific pathogen-free male MGS/Sea animals, 7 weeks old, were treated with MNU in the drinking water (30 ppm for alternate weeks to give 10 weeks exposure, or 10 ppm or 3 ppm for 20 weeks continuous exposure), or given MNNG in the drinking water at 400 ppm or 200 ppm for 20 weeks, or orally inoculated with ATCC43504 H. pylori (1.7 x 10(8) CFUs/animal). Adenocarcinomas in the glandular stomach were found in 2 out of 12 effective animals (2/ 12) treated with 30 ppm MNU at week 20, although all were dead or moribund by week 30 due to MNU toxicity. At week 50, the incidences of gastric adenocarcinomas in groups treated with 10 ppm MNU, 3 ppm MNU, 400 ppm MNNG, and 200 ppm MNNG were 2/21 (9.5%), 1/23 (4.3%), 7/ 11 (63.6%), and 1/10 (10.0%). The lesions were generally well differentiated, although poorly differentiated adenocarcinoma was also found in a single gerbil in each of the 10 ppm MNU and 400 ppm MNNG groups. In control animals no tumors were found. In the infection study, the animals were killed at week 20, and H. pylori was detected in all cases, causing multiple erosions with marked inflammatory cell infiltration in the lamina propria and submucosa, and frequent formation of lymphoid follicles. Thus, MNU and MNNG in the drinking water induced neoplastic lesions in the glandular stomach epithelium of H. pylori-sensitive gerbils.

KPU-300, a Novel Benzophenone–Diketopiperazine–Type Anti-Microtubule Agent with a 2-Pyridyl Structure, Is a Potent Radiosensitizer That Synchronizes the Cell Cycle in Early M Phase

PubMed Central

Okuyama, Kohei; Kaida, Atsushi; Hayashi, Yoshiki; Hayashi, Yoshio; Harada, Kiyoshi; Miura, Masahiko

2015-01-01

KPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300. PMID:26716455

KPU-300, a Novel Benzophenone-Diketopiperazine-Type Anti-Microtubule Agent with a 2-Pyridyl Structure, Is a Potent Radiosensitizer That Synchronizes the Cell Cycle in Early M Phase.

PubMed

Okuyama, Kohei; Kaida, Atsushi; Hayashi, Yoshiki; Hayashi, Yoshio; Harada, Kiyoshi; Miura, Masahiko

2015-01-01

KPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

NHERF Links the N-Cadherin/Catenin Complex to the Platelet-derived Growth Factor Receptor to Modulate the Actin Cytoskeleton and Regulate Cell Motility

PubMed Central

Theisen, Christopher S.; Wahl, James K.; Johnson, Keith R.

2007-01-01

Using phage display, we identified Na+/H+ exchanger regulatory factor (NHERF)-2 as a novel binding partner for the cadherin-associated protein, β-catenin. We showed that the second of two PSD-95/Dlg/ZO-1 (PDZ) domains of NHERF interacts with a PDZ-binding motif at the very carboxy terminus of β-catenin. N-cadherin expression has been shown to induce motility in a number of cell types. The first PDZ domain of NHERF is known to bind platelet-derived growth factor-receptor β (PDGF-Rβ), and the interaction of PDGF-Rβ with NHERF leads to enhanced cell spreading and motility. Here we show that β-catenin and N-cadherin are in a complex with NHERF and PDGF-Rβ at membrane ruffles in the highly invasive fibrosarcoma cell line HT1080. Using a stable short hairpin RNA system, we showed that HT1080 cells knocked down for either N-cadherin or NHERF had impaired ability to migrate into the wounded area in a scratch assay, similar to cells treated with a PDGF-R kinase inhibitor. Cells expressing a mutant NHERF that is unable to associate with β-catenin had increased stress fibers, reduced lamellipodia, and impaired cell migration. Using HeLa cells, which express little to no PDGF-R, we introduced PDGF-Rβ and showed that it coimmunoprecipitates with N-cadherin and that PDGF-dependent cell migration was reduced in these cells when we knocked-down expression of N-cadherin or NHERF. These studies implicate N-cadherin and β-catenin in cell migration via PDGF-R–mediated signaling through the scaffolding molecule NHERF. PMID:17229887

Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus.

PubMed Central

Innaimo, S F; Seifer, M; Bisacchi, G S; Standring, D N; Zahler, R; Colonno, R J

1997-01-01

BMS-200475 is a novel carbocyclic 2'-deoxyguanosine analog found to possess potent and selective anti-hepatitis B virus (anti-HBV) activity. BMS-200475 is distinguished from guanosine by replacement of the natural furanose oxygen on the sugar moiety with an exo carbon-carbon double bond. In the HepG2 stably transfected cell line 2.2.15, BMS-200475 had a 50% effective concentration (EC50) of 3.75 nM against HBV, as determined by analysis of secreted HBV DNA. Structurally related compounds with adenine, iodouracil, or thymine base substitutions were significantly less potent or were inactive. Direct comparison of the antiviral activities of BMS-200475 with those of a variety of other nucleoside analogs, including lamivudine (EC50 = 116.26 nM), demonstrated the clearly superior in vitro potency of BMS-200475 in 2.2.15 cells. Intracellular HBV replicative intermediates were uniformly reduced when cells were treated with BMS-200475, but rebounded after treatment was terminated. The concentration of BMS-200475 causing 50% cytotoxicity in 2.2.15 cell cultures was 30 microM, approximately 8,000-fold greater than the concentration required to inhibit HBV replication in the same cell line. Treatment with BMS-200475 resulted in no apparent inhibitory effects on mitochondrial DNA content. PMID:9210663

Naive T cells are dispensable for memory CD4+ T cell homeostasis in progressive simian immunodeficiency virus infection.

PubMed

Okoye, Afam A; Rohankhedkar, Mukta; Abana, Chike; Pattenn, Audrie; Reyes, Matthew; Pexton, Christopher; Lum, Richard; Sylwester, Andrew; Planer, Shannon L; Legasse, Alfred; Park, Byung S; Piatak, Michael; Lifson, Jeffrey D; Axthelm, Michael K; Picker, Louis J

2012-04-09

The development of AIDS in chronic HIV/simian immunodeficiency virus (SIV) infection has been closely linked to progressive failure of CD4(+) memory T cell (T(M)) homeostasis. CD4(+) naive T cells (T(N)) also decline in these infections, but their contribution to disease progression is less clear. We assessed the role of CD4(+) T(N) in SIV pathogenesis using rhesus macaques (RMs) selectively and permanently depleted of CD4(+) T(N) before SIV infection. CD4(+) T(N)-depleted and CD4(+) T(N)-repleted RMs were created by subjecting juvenile RMs to thymectomy versus sham surgery, respectively, followed by total CD4(+) T cell depletion and recovery from this depletion. Although thymectomized and sham-treated RMs manifested comparable CD4(+) T(M) recovery, only sham-treated RMs reconstituted CD4(+) T(N). CD4(+) T(N)-depleted RMs responded to SIVmac239 infection with markedly attenuated SIV-specific CD4(+) T cell responses, delayed SIVenv-specific Ab responses, and reduced SIV-specific CD8(+) T cell responses. However, CD4(+) T(N)-depleted and -repleted groups showed similar levels of SIV replication. Moreover, CD4(+) T(N) deficiency had no significant effect on CD4(+) T(M) homeostasis (either on or off anti-retroviral therapy) or disease progression. These data demonstrate that the CD4(+) T(N) compartment is dispensable for CD4(+) T(M) homeostasis in progressive SIV infection, and they confirm that CD4(+) T(M) comprise a homeostatically independent compartment that is intrinsically capable of self-renewal.

Interferon induction by two 2'-modified double-helical RNAs, poly(2'-fluoro-2'-deoxyinosinic acid) x poly(cytidylic acid) and poly(2'-chloro-2'-deoxyinosinic acid) x poly(cytidylic acid).

PubMed

De Clercq, E; Stollar, B D; Hobbs, J; Fukui, T; Kakiuchi, N; Ikehara, M

1980-01-01

In addition to the 2'-azido analogue of (I)n x (C)n, (dIn3)n x (C)n, we have found two other (I)n x (C)n analogues, (dIfl)n x (C)n and (dIcl)n x (C)n, in which the 2'-hydroxyls of the (I)n strand are replaced by either fluorine or chlorine, to be highly effective in inducing interferon. This contrasted with the lack of interferon-inducing activity noted for various other 2'-halogeno analogues of (I)n x (C)n and (A)n x (U)n, i.e. (I)n x (dCcl)n, (dAfl)n x (U)n, (dAcl)n x (U)n, (A)n x (dUfl)n and (A)n x (dUcl)n. In most assay systems, viz. primary rabbit kidney cells, human diploid fibroblasts, HeLa cells, interferon-primed mouse L-929 cells, and intact rabbits, (dIfl)n x (C)n and (dIcl)n x (C)n induced interferon levels that were comparable to those induced by (I)n x (C)n. There was one particular system (L-929 cells treated with DEAE-dextran), however, in which (dIfl)n x (C)n and (dIcl)n x (C)n, unlike (I)n x (C)n, failed to stimulate interferon production. As monitored by both radiochemical and biological means, (dIfl)n x (C)n and, to a lesser extent, (dIcl)n x (C)n were more resistant to degradation by ribonuclease A, T1 and human serum nucleases than was (I)n x (C)n. In their reactivity towards antibodies to double-stranded RNA (dIfl)n x (C)n and (dIcl)n x (C)n conformed more closely to (I)n x (C)n than did other 2'-substituted (e.g. 2'-O-methyl or 2'-O-ethyl) analogues of (I)n x (C)n. The high interferon-inducing potency of (dIfl)n x (C)n and (dIcl)n x (C)n has both theoretical and practical implications. While our findings suggest that (dIfl)n x (C)n and (dIcl)n x (C)n should be further explored for their therapeutic potentials, they also strengthen the notion that the interferon-inducing capacity, and possibly other biological functions of double-stranded RNAs is dependent on the recognition of the overall conformation of the polynucleotide rather than on the binding of specific functional groups such as the 2'-hydroxyl group.

Differential effects of estradiol on carotid artery inflammation when administered early versus late after surgical menopause.

PubMed

Sophonsritsuk, Areepan; Appt, Susan E; Clarkson, Thomas B; Shively, Carol A; Espeland, Mark A; Register, Thomas C

2013-05-01

The aim of this study was to determine the effects of estrogen therapy (ET) on carotid artery inflammation when initiated early and late relative to surgical menopause. Female cynomolgus macaques consuming atherogenic diets were ovariectomized and randomized to control or oral estradiol (E2; human equivalent dose of 1 mg/d micronized E2) initiated at 1 month (early menopause, n = 24) or 54 months (late menopause, n = 40) after ovariectomy. The treatment period was 8 months. Carotid artery expression of the markers of monocyte/macrophages (CD68 and CD163), dendritic cells (CD83), natural killer cells (neural cell adhesion molecule-1), and interferon-γ was significantly lower in E2-treated animals in the early menopause group but not in the late menopause group (P < 0.05). In contrast, carotid artery transcripts for T-cell markers (CD3, CD4, CD8, and CD25), interleukin-10, type I collagen, monocyte chemoattractant protein-1, matrix metalloproteinase-9, and tumor necrosis factor-α were lower in E2-treated monkeys regardless of menopausal stage (P < 0.05). ET initiated soon after menopause inhibits macrophage accumulation in the carotid artery, an effect that is not observed when E2 is administered after several years of estrogen deficiency. No evidence for pro-inflammatory effects of late ET is observed. The results provide support for the timing hypothesis of postmenopausal ET with implications for the interpretation of outcomes in the Women's Health Initiative.

Differential effects of erufosine on proliferation, wound healing and apoptosis in colorectal cancer cell lines.

PubMed

Kaleağasıoğlu, Ferda; Berger, Martin R

2014-03-01

The alkylphosphocholine, erucylphospho-N,N, N-trimethylpropanolamine (erufosine), has demonstrated anticancer effects in various cell lines, including leukemia, multiple myeloma, bladder, breast and oral squamous cell carcinoma cells. The purpose of the present study was to investigate its antiproliferative, antimigratory and pro-apoptotic effects in colorectal cancer cell lines, SW480 and CC531. The antiproliferative effect was determined by (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) (MTT) dye reduction assay following exposure to erufosine (3.1-100 µM) for 24-72 h. The antimigratory effect of erufosine (1.6-6 µM) was investigated by a wound healing assay for 12-48 h. Caspase-3/-7 activity was measured to detect apoptotic cell death. Erufosine inhibited cell proliferation in a dose- and time-dependent manner. The IC50 values following 72 h of incubation were 3.4 and 25.4 µM for SW480 and CC531 cells, respectively. erufosine at concentrations of 50 and 100 µM induced caspase-3/-7 activity concentration-dependently in SW480 cells, but only at 100 µM in CC531 cells. Incubation of SW480 cells with erufosine (1.56 µM) for 48 h inhibited migration into the scratched area by 54% as compared to the untreated cells; whereas in CC531 cells, the wound width in the erufosine-treated (1.56-6.25 µM) cells following 48 h was closed 2-fold slower than the rate in the untreated group. Erufosine (25 µM) attenuated osteonectin expression and abolished COL1A1 expression in CC531 cells. Erufosine appears to be a promising treatment agent for colorectal cancer. Rat CC531 cells are less sensitive to erufosine than human SW480 cells.

Deregulation of miRNAs Contributes to Development and Progression of Prostate Cancer

DTIC Science & Technology

2011-09-01

blocking with 5% non- fat dry milk in Tris-buffered saline/0.05% Tween 20 (TBST), the membrane was incubated with a specific primary antibody...androgen R1881, and p53 protein was detected by Western blot analysis. Consistent with our previous observation (2), untreated LNCaP-R273H cells expressed...Cell Biochem 2009; 106(3): 363–371. 7 Appendices Figure 1. Western blot analysis of p53 protein in 5.0 nM R1881-treated LNCaP cells and LNCaP-R273H

Bone-Induced Chondroinduction in Sheep Jamshidi Biopsy Defects with and without Treatment by Subchondral Chitosan-Blood Implant: 1-Day, 3-Week, and 3-Month Repair.

PubMed

Bell, Angela D; Lascau-Coman, Viorica; Sun, Jun; Chen, Gaoping; Lowerison, Mark W; Hurtig, Mark B; Hoemann, Caroline D

2013-04-01

Delivery of chitosan to subchondral bone is a novel approach for augmented marrow stimulation. We evaluated the effect of 3 presolidified chitosan-blood implant formulations on osteochondral repair progression compared with untreated defects. In N = 5 adult sheep, six 2-mm diameter Jamshidi biopsy holes were created bilaterally in the medial femoral condyle and treated with presolidified chitosan-blood implant with fluorescent chitosan tracer (10 kDa, 40 kDa, or 150k Da chitosan, left knee) or left to bleed (untreated, right knee). Implant residency and osteochondral repair were assessed at 1 day (N = 1), 3 weeks (N = 2), or 3 months (N = 2) postoperative using fluorescence microscopy, histomorphometry, stereology, and micro-computed tomography. Chitosan implants were retained in 89% of treated Jamshidi holes up to 3 weeks postoperative. At 3 weeks, biopsy sites were variably covered by cartilage flow, and most bone holes contained cartilage flow fragments and heterogeneous granulation tissues with sparse leukocytes, stromal cells, and occasional adipocytes (volume density 1% to 3%). After 3 months of repair, most Jamshidi bone holes were deeper, remodeling at the edges, filled with angiogenic granulation tissue, and lined with variably sized chondrogenic foci fused to bone trabeculae or actively repairing bone plate. The 150-kDa chitosan implant elicited more subchondral cartilage formation compared with 40-kDa chitosan-treated and control defects (P < 0.05, N = 4). Treated defects contained more mineralized repair tissue than control defects at 3 months (P < 0.05, N = 12). Bone plate-induced chondroinduction is an articular cartilage repair mechanism. Jamshidi biopsy repair takes longer than 3 months and can be influenced by subchondral chitosan-blood implant.

Bone-Induced Chondroinduction in Sheep Jamshidi Biopsy Defects with and without Treatment by Subchondral Chitosan-Blood Implant

PubMed Central

Bell, Angela D.; Lascau-Coman, Viorica; Sun, Jun; Chen, Gaoping; Lowerison, Mark W.; Hurtig, Mark B.

2013-01-01

Objective: Delivery of chitosan to subchondral bone is a novel approach for augmented marrow stimulation. We evaluated the effect of 3 presolidified chitosan-blood implant formulations on osteochondral repair progression compared with untreated defects. Design: In N = 5 adult sheep, six 2-mm diameter Jamshidi biopsy holes were created bilaterally in the medial femoral condyle and treated with presolidified chitosan-blood implant with fluorescent chitosan tracer (10 kDa, 40 kDa, or 150k Da chitosan, left knee) or left to bleed (untreated, right knee). Implant residency and osteochondral repair were assessed at 1 day (N = 1), 3 weeks (N = 2), or 3 months (N = 2) postoperative using fluorescence microscopy, histomorphometry, stereology, and micro–computed tomography. Results: Chitosan implants were retained in 89% of treated Jamshidi holes up to 3 weeks postoperative. At 3 weeks, biopsy sites were variably covered by cartilage flow, and most bone holes contained cartilage flow fragments and heterogeneous granulation tissues with sparse leukocytes, stromal cells, and occasional adipocytes (volume density 1% to 3%). After 3 months of repair, most Jamshidi bone holes were deeper, remodeling at the edges, filled with angiogenic granulation tissue, and lined with variably sized chondrogenic foci fused to bone trabeculae or actively repairing bone plate. The 150-kDa chitosan implant elicited more subchondral cartilage formation compared with 40-kDa chitosan-treated and control defects (P < 0.05, N = 4). Treated defects contained more mineralized repair tissue than control defects at 3 months (P < 0.05, N = 12). Conclusion: Bone plate–induced chondroinduction is an articular cartilage repair mechanism. Jamshidi biopsy repair takes longer than 3 months and can be influenced by subchondral chitosan-blood implant. PMID:26069656

Morphine Antidependence of Erythroxylum cuneatum (Miq.) Kurz in Neurotransmission Processes In Vitro

PubMed Central

Adenan, Mohd Ilham; Amom, Zulkhairi

2016-01-01

Opiate abuse has been studied to cause adaptive changes observed in the presynaptic release and the mediated-synaptic plasticity proteins. The involvement of neuronal SNARE proteins reveals the role of the neurotransmitter release in expressing the opioid actions. The present study was designed to determine the effect of the alkaloid extract of Erythroxylum cuneatum (E. cuneatum) against chronic morphine and the influences of E. cuneatum on neurotransmission processes observed in vitro. The human neuroblastoma cell line, SK-N-SH, was treated with the morphine, methadone, or E. cuneatum. The cell lysates were collected and tested for α-synuclein, calmodulin, vesicle-associated membrane protein 2 (VAMP 2), and synaptotagmin 1. The extract of E. cuneatum was observed to upregulate the decreased expression of dependence proteins, namely, α-synuclein and calmodulin. The effects were comparable to methadone and control. The expressions of VAMP 2 and synaptotagmin 1 were normalised by the plant and methadone. The extract of E. cuneatum was postulated to treat dependence symptoms after chronic morphine and improve the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) protein involved in synaptic vesicle after. PMID:27974903

Tetrahydrocannabinolic acid is a potent PPARγ agonist with neuroprotective activity.

PubMed

Nadal, Xavier; Del Río, Carmen; Casano, Salvatore; Palomares, Belén; Ferreiro-Vera, Carlos; Navarrete, Carmen; Sánchez-Carnerero, Carolina; Cantarero, Irene; Bellido, Maria Luz; Meyer, Stefan; Morello, Gaetano; Appendino, Giovanni; Muñoz, Eduardo

2017-12-01

Phytocannabinoids are produced in Cannabis sativa L. in acidic form and are decarboxylated upon heating, processing and storage. While the biological effects of decarboxylated cannabinoids such as Δ 9 -tetrahydrocannabinol have been extensively investigated, the bioactivity of Δ 9 -tetahydrocannabinol acid (Δ 9 -THCA) is largely unknown, despite its occurrence in different Cannabis preparations. Here we have assessed possible neuroprotective actions of Δ 9 -THCA through modulation of PPARγ pathways. The effects of six phytocannabinoids on PPARγ binding and transcriptional activity were investigated. The effect of Δ 9 -THCA on mitochondrial biogenesis and PPARγ coactivator 1-α expression was investigated in Neuro-2a (N2a) cells. The neuroprotective effect was analysed in STHdh Q111/Q111 cells expressing a mutated form of the huntingtin protein and in N2a cells infected with an adenovirus carrying human huntingtin containing 94 polyQ repeats (mHtt-q94). The in vivo neuroprotective activity of Δ 9 -THCA was investigated in mice intoxicated with the mitochondrial toxin 3-nitropropionic acid (3-NPA). Cannabinoid acids bind and activate PPARγ with higher potency than their decarboxylated products. Δ 9 -THCA increased mitochondrial mass in neuroblastoma N2a cells and prevented cytotoxicity induced by serum deprivation in STHdh Q111/Q111 cells and by mutHtt-q94 in N2a cells. Δ 9 -THCA, through a PPARγ-dependent pathway, was neuroprotective in mice treated with 3-NPA, improving motor deficits and preventing striatal degeneration. In addition, Δ 9 -THCA attenuated microgliosis, astrogliosis and up-regulation of proinflammatory markers induced by 3-NPA. Δ 9 -THCA shows potent neuroprotective activity, which is worth considering for the treatment of Huntington's disease and possibly other neurodegenerative and neuroinflammatory diseases. © 2017 The British Pharmacological Society.

Anti-inflammatory activities of Ganoderma lucidum (Lingzhi) and San-Miao-San supplements in MRL/lpr mice for the treatment of systemic lupus erythematosus.

PubMed

Cai, Zhe; Wong, Chun Kwok; Dong, Jie; Jiao, Delong; Chu, Man; Leung, Ping Chung; Lau, Clara Bik San; Lau, Ching Po; Tam, Lai Shan; Lam, Christopher Wai Kei

2016-01-01

Ganoderma lucidum (Lingzhi; LZ) and San-Miao-San (SMS) are Chinese medicines (CMs) used to treat inflammatory ailments and numbing syndrome/arthralgia syndrome (Bi Zheng), respectively. Given that the main symptoms of systemic lupus erythematosus (SLE) include inflammation of the joints, joint pain, edema and palpitations of the heart because of problems associated with Bi Zheng, it was envisaged that LZ and SMS could be used as potential treatments for this autoimmune disease. This study aims to investigate the anti-inflammatory activity of a combination formulation containing LZ and SMS (LZ-SMS) in SLE mice. Female adult Balb/c mice of 20-24 weeks of age were used as normal mice (n = 10), whereas female MRL/lpr mice of 12-24 weeks of age were divided into three groups (n = 10 in each group), including mild, moderate and severe SLE mice groups. The clinical characteristics of the SLE and Babl/c mice (i.e., body weight, joint thickness, lupus flare, proteinuria, leukocyturia and lymphadenopathy) were assessed. The plasma concentrations of anti-nuclear antibody (ANA) and anti-double stranded DNA antibody (anti-ds-DNA) were analyzed by an enzyme-linked immunosorbent assay, whereas the concentration of several key cytokines (IFN-γ, TNF-α, IL-6, IL-10, IL-2, IL-27, IL-12P70, IL-17A and IL-21) were analyzed by a Luminex multiplex assay. The gene expression profiles for differentiation of the T helper (Th) lymphocytes in splenic CD4(+) Th cells were assessed by RT-qPCR. Flow cytometry was used to measure the percentages of CD4(+)CD25(+)Foxp3(+) Treg cells and CD19(+)CD5(+)CD1d(+)IL-10(+) regulatory B (Breg) cells (IL-10(+) Bregs). Concentrations of anti-ds-DNA in the plasma samples collected from the LZ-SMS-treated (500 mg/kg/day oral administration for 7 days followed with 50 mg/kg/day intraperitoneal administration for 7 days), moderate and severe SLE mice decreased significantly compared with the PBS treated mice (P < 0.05). The gene expression levels of the induced regulatory T (iTreg) and natural Treg (nTreg) cells were significantly higher than those of the Th17, Th1 and "conventional Th cells vs. Treg cells" regulated genes following the LZ-SMS treatment (P < 0.05). The percentages of CD4(+)CD25(+)Foxp3(+) Treg cells collected from the splenic, thymic and peripheral blood cells, as well as the percentages of IL-10(+) Bregs collected from the splenic and thymic cells increased significantly in the LZ-SMS-treated SLE mice (P < 0.05) compared with the untreated PBS group. The ratio of the percentage of CD4(+)CD25(+)Foxp3(+) Treg cells to the percentage of CD4(+)CD25(-) effector T cells collected from the splenic, thymic and peripheral blood cells in LZ-SMS-treated moderate and severe SLE mice increased significantly compared with the untreated PBS group (P < 0.05). Furthermore, a comparison with the PBS treatment group revealed significant decreases in the concentrations of several inflammatory cytokines, including IL-21, IL-10 and IL-17A (P < 0.05), as well as significant increases in the concentrations of IL-2 and IL-12P70 in the LZ-SMS treated SLE mice (P < 0.05). LZ-SMS treatment led to significant increases in the percentages of CD4(+)CD25(+)Foxp3(+) Treg and IL-10(+) Breg cells, together with a reduction in the plasma concentrations of several inflammatory cytokines and the down-regulated expression of the corresponding cytokine related genes in SLE mice. The clinical characteristics of the LZ-SMS-treated SLE mice also improved significantly.

l-N-acetylcysteine protects outer hair cells against TNFα initiated ototoxicity in vitro.

PubMed

Tillinger, Joshua A; Gupta, Chhavi; Ila, Kadri; Ahmed, Jamal; Mittal, Jeenu; Van De Water, Thomas R; Eshraghi, Adrien A

2018-08-01

The present study is aimed at determining the efficacy and exploring the mechanisms by which l-N-acetylcysteine (l-NAC) provides protection against tumor necrosis factor-alpha (TNFα)-induced oxidative stress damage and hair cell loss in 3-day-old rat organ of Corti (OC) explants. Previous work has demonstrated a high level of oxidative stress in TNFα-challenged OC explants. TNFα can potentially play a significant role in hair cell loss following an insult to the inner ear. l-NAC has shown to provide effective protection against noise-induced hearing loss in laboratory animals but mechanisms of this otoprotective effect are not well-defined. Rat OC explants were exposed to either: (1) saline control (N = 12); (2) TNFα (2 μg/ml, N = 12); (3) TNFα+l-NAC (5 mM, N = 12); (4) TNFα+l-NAC (10 mM, N = 12); or (5) l-NAC (10 mM, N = 12). Outer hair cell (OHC) density, levels of reactive oxygen species (ROS), lipid peroxidation of cell membranes, gluthathione activity, and mitochondrial viability were assayed. l-NAC (5 and 10 mM) provided protection for OHCs from ototoxic level of TNFα in OC explants. Groups treated with TNFα+l-NAC (5 mM) showed a highly significant reduction of both ROS (p < 0.01) and 4-hydroxy-2-nonenal immunostaining (p < 0.001) compared to TNFα-challenged explants. Total glutathione levels were low in TNFα-challenged explants compared to control and TNFα+l-NAC (5 mM) treated explants (p < 0.001). l-NAC is a promising treatment for protecting auditory HCs from TNFα-induced oxidative stress and subsequent loss via programmed cell death.

Rapamycin efficiently promotes cardiac differentiation of mouse embryonic stem cells.

PubMed

Lu, Qin; Liu, Yinan; Wang, Yang; Wang, Weiping; Yang, Zhe; Li, Tao; Tian, Yuyao; Chen, Ping; Ma, Kangtao; Jia, Zhuqing; Zhou, Chunyan

2017-06-30

To investigate the effects of rapamycin on cardiac differentiation, murine embryonic stem cells (ESCs) were induced into cardiomyocytes by 10 -4 M ascorbic acid (AA), 20 nM rapamycin alone or 0.01% solvent DMSO. We found that rapamycin alone was insufficient to initiate cardiomyogenesis. Then, the ESCs were treated with AA and rapamycin (20 nM) or AA and DMSO (0.01%) as a control. Compared with control, mouse ESCs (mESCs) treated with rapamycin (20 nM) and AA yielded a significantly higher percentage of cardiomyocytes, as confirmed by the percentage of beating embryonic bodies (EBs), the immunofluorescence and FACS analysis. Rapamycin significantly increased the expression of a panel of cardiac markers including Gata 4, α- Mhc , β- Mhc , and Tnnt 2. Additionally, rapamycin enhanced the expression of mesodermal and cardiac transcription factors such as Mesp 1, Brachyury T, Eomes, Isl 1 , Gata 4 , Nkx 2.5 , Tbx 5, and Mef2c. Mechanistic studies showed that rapamycin inhibits Wnt/β-catenin and Notch signaling but promotes the expression of fibroblast growth factor ( Fgf 8), Fgf 10, and Nodal at early stage, and bone morphogenetic protein 2 ( Bmp 2) at later stages. Sequential treatment of rapamycin showed that rapamycin promotes cardiac differentiation at the early and later stages. Interestingly, another mammalian target of rapamycin (mTOR) inhibitor Ku0063794 (1 µM) had similar effects on cardiomyogenesis. In conclusion, our results highlight a practical approach to generate cardiomyocytes from mESCs by rapamycin. © 2017 The Author(s).

Proapoptotic and Antimetastatic Properties of Supercritical CO2 Extract of Nigella sativa Linn. Against Breast Cancer Cells

PubMed Central

Baharetha, Hussein M.; Nassar, Zeyad D.; Aisha, Abdalrahim F.; Ahamed, Mohamed B. Khadeer; Al-Suede, Foaud Saleih R.; Kadir, Mohd Omar Abd; Ismail, Zhari

2013-01-01

Abstract Nigella sativa, commonly referred as black cumin, is a popular spice that has been used since the ancient Egyptians. It has traditionally been used for treatment of various human ailments ranging from fever to intestinal disturbances to cancer. This study investigated the apoptotic, antimetastatic, and anticancer activities of supercritical carbon dioxide (SC-CO2) extracts of the seeds of N. sativa Linn. against estrogen-dependent human breast cancer cells (MCF-7). Twelve extracts were prepared from N. sativa seeds using the SC-CO2 extraction method by varying pressure and temperature. Extracts were analyzed using FTIR and UV-Vis spectrometry. Cytotoxicity of the extracts was evaluated on various human cancer and normal cell lines. Of the 12 extracts, 1 extract (A3) that was prepared at 60°C and 2500 psi (∼17.24 MPa) showed selective antiproliferative activity against MCF-7 cells with an IC50 of 53.34±2.15 μg/mL. Induction of apoptosis was confirmed by evaluating caspases activities and observing the cells under a scanning electron microscope. In vitro antimetastatic properties of A3 were investigated by colony formation, cell migration, and cell invasion assays. The elevated levels of caspases in A3 treated MCF-7 cells suggest that A3 is proapoptotic. Further nuclear condensation and fragmentation studies confirmed that A3 induces cytotoxicity through the apoptosis pathway. A3 also demonstrated remarkable inhibition in migration and invasion assays of MCF-7 cells at subcytotoxic concentrations. Thus, this study highlights the therapeutic potentials of SC-CO2 extract of N. sativa in targeting breast cancer. PMID:24328702

Cytotoxic Effects of Ochratoxin A in Neuro-2a Cells: Role of Oxidative Stress Evidenced by N-acetylcysteine.

PubMed

Bhat, Pratiksha V; Pandareesh; Khanum, Farhath; Tamatam, Anand

2016-01-01

Ochratoxin-A (OTA), is toxic secondary metabolite and is found to be a source of vast range of toxic effects like hepatotoxicity, nephrotoxicity. However, the information available currently regarding neurotoxic effects exerted by OTA is scanty. Hence, the present study was aimed to evaluate the neurotoxic effects of OTA and the possible mechanisms of toxicity as well as the role of cytotoxic oxidative stress on neuronal (Neuro-2a) cell line was evaluated in vitro. Results of the MTT and LDH assay showed that, OTA induced dose-dependent cell death in Neuro-2a cells and EC50 value was determined as 500 nM. OTA induced high levels of reactive oxygen species (ROS) and elevated levels of malondialdehyde, also loss of mitochondrial membrane potential was observed in a dose depended manner. Effects of OTA on ROS induced chromosomal DNA damage was assessed by Comet assay and plasmid DNA damage assay in which increase in DNA damage was observed in Neuro-2a cells by increasing the OTA concentration. Further western blotting analysis of OTA treated Neuro-2a cells indicated elevated expression levels of c-Jun, JNK3 and cleaved caspase-3 leading to apoptotic cell death. Other hand realtime-Q-PCR analysis clearly indicates the suppressed expression of neuronal biomarker genes including AChE, BDNF, TH and NOS2. Further N-acetylcysteine (NAC) pretreatment to Neuro-2a cells followed by OTA treatment clearly evidenced that, the significant reversal of toxic effects exerted by OTA on Neuro-2a cells. In the present study, results illustrate that ROS a principle event in oxidative stress was elevated by OTA toxicity in Neuro-2a cells. However, further in vivo, animal studies are in need to conclude the present study reports and the use of NAC as a remedy for OTA induced neuronal stress.

Comparative Therapeutic Effects of Minocycline Treatment and Bone Marrow Mononuclear Cell Transplantation following Striatal Stroke

PubMed Central

Souza, Celice C.; da Silva, Michelle Castro; Lopes, Rosana Telma; Cardoso, Marcelo M.; Santos, Adriano Guimarães; dos Santos, Ijair Rogério

2017-01-01

We explored the comparative effects of minocycline treatment and intrastriatal BMMC transplantation after experimental striatal stroke in adult rats. Male Wistar adult rats were divided as follows: saline-treated (N = 5), minocycline-treated (N = 5), and BMMC-transplanted (N = 5) animals. Animals received intrastriatal microinjections of 80 pmol of endothelin-1 (ET-1). Behavioral tests were performed at 1, 3, and 7 days postischemia. Animals were treated with minocycline (50 mg/kg, i.p.) or intrastriatal transplants of 106 BMMCs at 24 h postischemia. Animals were perfused at 7 days after ischemic induction. Coronal sections were stained with cresyl violet for gross histopathological analysis and immunolabeled for the identification of neuronal bodies (NeuN), activated microglia/macrophages (ED1), and apoptotic cells (active caspase-3). BMMC transplantation and minocycline reduced the number of ED1+ cells (p < 0.05, ANOVA-Tukey), but BMMC afforded better results. Both treatments afforded comparable levels of neuronal preservation compared to control (p > 0.05). BMMC transplantation induced a higher decrease in the number of apoptotic cells compared to control and minocycline treatment. Both therapeutic approaches improved functional recovery in ischemic animals. The results suggest that BMMC transplantation is more effective in modulating microglial activation and reducing apoptotic cell death than minocycline, although both treatments are equally efficacious on improving neuronal preservation. PMID:28713482

Comparative Therapeutic Effects of Minocycline Treatment and Bone Marrow Mononuclear Cell Transplantation following Striatal Stroke.

PubMed

Souza, Celice C; da Silva, Michelle Castro; Lopes, Rosana Telma; Cardoso, Marcelo M; de Souza, Lucas Lacerda; Santos, Adriano Guimarães; Dos Santos, Ijair Rogério; Franco, Edna C S; Gomes-Leal, Walace

2017-01-01

We explored the comparative effects of minocycline treatment and intrastriatal BMMC transplantation after experimental striatal stroke in adult rats. Male Wistar adult rats were divided as follows: saline-treated ( N = 5), minocycline-treated ( N = 5), and BMMC-transplanted ( N = 5) animals. Animals received intrastriatal microinjections of 80 pmol of endothelin-1 (ET-1). Behavioral tests were performed at 1, 3, and 7 days postischemia. Animals were treated with minocycline (50 mg/kg, i.p.) or intrastriatal transplants of 106 BMMCs at 24 h postischemia. Animals were perfused at 7 days after ischemic induction. Coronal sections were stained with cresyl violet for gross histopathological analysis and immunolabeled for the identification of neuronal bodies (NeuN), activated microglia/macrophages (ED1), and apoptotic cells (active caspase-3). BMMC transplantation and minocycline reduced the number of ED1+ cells ( p < 0.05, ANOVA-Tukey), but BMMC afforded better results. Both treatments afforded comparable levels of neuronal preservation compared to control ( p > 0.05). BMMC transplantation induced a higher decrease in the number of apoptotic cells compared to control and minocycline treatment. Both therapeutic approaches improved functional recovery in ischemic animals. The results suggest that BMMC transplantation is more effective in modulating microglial activation and reducing apoptotic cell death than minocycline, although both treatments are equally efficacious on improving neuronal preservation.

Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, M.H.; Neubig, R.R.

1986-03-05

High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonistmore » (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.« less

Measurement of the intracellular ph in human stomach cells: a novel approach to evaluate the gastric acid secretory potential of coffee beverages.

PubMed

Weiss, Carola; Rubach, Malte; Lang, Roman; Seebach, Elisabeth; Blumberg, Simone; Frank, Oliver; Hofmann, Thomas; Somoza, Veronika

2010-02-10

As the consumption of coffee beverages sometimes is reported to cause gastric irritation, for which an increased stomach acid secretion is one of the promoting factors, different processing technologies such as steam-treatment have been developed to reduce putative stomach irritating compounds. There is evidence-based data neither on the effect of detailed processing variations nor on individual coffee components affecting the proton secretory activity (PSA). This work aimed at developing a screening model suitable for investigating the effects of commercial coffee beverages and components thereof on human parietal cells. Human gastric cancer cells (HGT-1) were treated with reconstituted freeze-dried coffee beverages prepared from customary coffee products such as regular coffee (RC, n = 4), mild bean coffee (MBC, n = 5), stomach friendly coffee (SFC, n = 4), and SFC decaffeinated (SFCD, n = 3). PSA was analyzed by flow cytometry using the pH-sensitive dye SNARF-AM. Treatment of the cells with MBC did not result in a PSA different from RC treatment (p

Mesenchymal stromal cell treatment prevents H9N2 avian influenza virus-induced acute lung injury in mice.

PubMed

Li, Yan; Xu, Jun; Shi, Weiqing; Chen, Cheng; Shao, Yan; Zhu, Limei; Lu, Wei; Han, XiaoDong

2016-10-28

The avian influenza virus (AIV) can cross species barriers and expand its host range from birds to mammals, even humans. Avian influenza is characterized by pronounced activation of the proinflammatory cytokine cascade, which perpetuates the inflammatory response, leading to persistent systemic inflammatory response syndrome and pulmonary infection in animals and humans. There are currently no specific treatment strategies for avian influenza. We hypothesized that mesenchymal stromal cells (MSCs) would have beneficial effects in the treatment of H9N2 AIV-induced acute lung injury in mice. Six- to 8-week-old C57BL/6 mice were infected intranasally with 1 × 10 4 MID 50 of A/HONG KONG/2108/2003 [H9N2 (HK)] H9N2 virus to induce acute lung injury. After 30 min, syngeneic MSCs were delivered through the caudal vein. Three days after infection, we measured the survival rate, lung weight, arterial blood gas, and cytokines in both bronchoalveolar lavage fluid (BALF) and serum, and assessed pathological changes to the lungs. MSC administration significantly palliated H9N2 AIV-induced pulmonary inflammation by reducing chemokines and proinflammatory cytokines levels, as well as reducing inflammatory cell recruit into the lungs. Thus, H9N2 AIV-induced lung injury was markedly alleviated in mice treated with MSCs. Lung histopathology and arterial blood gas analysis were improved in mice with H9N2 AIV-induced lung injury following MSC treatment. MSC treatment significantly reduces H9N2 AIV-induced acute lung injury in mice and is associated with reduced pulmonary inflammation. These results indicate a potential role for MSC therapy in the treatment of clinical avian influenza.

Nicotine promotes cell proliferation and induces resistance to cisplatin by α7 nicotinic acetylcholine receptor‑mediated activation in Raw264.7 and El4 cells.

PubMed

Wang, Yan Yan; Liu, Yao; Ni, Xiao Yan; Bai, Zhen Huan; Chen, Qiong Yun; Zhang, Ye; Gao, Feng Guang

2014-03-01

Although nicotine is a risk factor for carcinogenesis and atherosclerosis, epidemiological data indicate that nicotine has therapeutic benefits in treating Alzheimer's disease. Our previous studies also showed that nicotine-treated dendritic cells have potential antitumor effects. Hence, the precise effects of nicotine on the biological characterizations of cells are controversial. The aim of the present study was to assess the roles of α7 nicotinic acetylcholine receptors (nAChRs), Erk1/2-p38-JNK and PI3K-Akt pathway in nicotine-mediated proliferation and anti-apoptosis effects. The results firstly showed that nicotine treatment clearly augmented cell viability and upregulated PCNA expression in both Raw264.7 and El4 cells. Meanwhile, nicotine afforded protection against cisplatin-induced toxicity through inhibiting caspase-3 activation and upregulating anti-apoptotic protein expression. Further exploration demonstrated that nicotine efficiently abolished cisplatin-promoted mitochondria translocation of Bax and the release of cytochrome c. The pretreatment of α-bungarotoxin and tubocurarine chloride significantly attenuated nicotine-augmented cell viability, abolished caspase-3 activation and α7 nAChR upregulation. Both Erk-JNK-p38 and PI3K-Akt signaling pathways could be activated by nicotine treatment in Raw264.7 and El4 cells. Notably, when Erk-JNK and PI3K-Akt activities were inhibited, nicotine-augmented cell proliferation and anti-apoptotic effects were abolished accordingly. The results presented here indicate that nicotine could achieve α7 nAChR-mediated proliferation and anti-apoptotic effects by activating Erk-JNK and PI3K-Akt pathways respectively, providing potential therapeutic molecules to deal with smoking-associated human diseases.

Sustained inhibition of intimal thickening. In vitro and in vivo effects of polymeric beta-cyclodextrin sulfate.

PubMed Central

Bachinsky, W B; Barnathan, E S; Liu, H; Okada, S S; Kuo, A; Raghunath, P N; Muttreja, M; Caron, R J; Tomaszewski, J E; Golden, M A

1995-01-01

Intimal thickening after vascular injury may be modulated in part by heparin binding growth factors. We hypothesized that placement of a therapeutic polymer in the periadventitial space capable of tightly binding growth factors might alter the vascular response to injury. We first demonstrated that incubation of rat aortic smooth muscle cells with an insoluble, sulfated polymer of beta-cyclodextrin (P-CDS) was associated with a dose-dependent inhibition of proliferation induced by fetal calf serum, fibroblast growth factor-2 (FGF-2), platelet-derived growth factor BB, or epidermal growth factor. Preincubation studies of P-CDS with FGF-2 revealed a very rapid removal of mitogenic activity. Using radiolabeled FGF-2 (0.25 microg/ml), we observed a very rapid association rate (0.34 +/- 0.07 min-1, n=4) and a very slow dissociation rate (3.3 +/- 0.2 X 10(-7) min-1) at 37 degrees C, suggesting a high affinity interaction. Using both Transwell and linear under-agarose assays, we demonstrated a significant inhibition of random migration (chemokinesis) by P-CDS. Unsulfated polymeric beta-cyclodextrin (P-CD) had little if any of these effects, suggesting that the high negative charge density of P-CDS was important for the effects. Finally, rats undergoing carotid artery balloon injury were randomized to treatment with periadventitial P-CDS or no treatment, and were killed at 4 (n=20), 14 (n=59), and 88 d (n=14). Morphometric analysis demonstrated significant and sustained inhibition of intimal thickening in P-CDS-treated rats at 14 (P < 0.01) and 88 d (P < 0.05) using absolute intimal area or intima/media area ratios. No inhibition was seen in a group of rats treated with P-CD. In P-CDS-treated rats, bromodeoxyuridine labeling studies revealed fewer labeled smooth muscle cells in the intima at 14 d (P=0.01), while staining with Evans blue revealed enhanced late endothelial cell regrowth. Thus, periadventitially applied sulfated beta-cyclodextrin polymer, which can tightly bind heparin binding growth factors, inhibits intimal thickening in vivo in a sustained fashion without using an additional delivery system. These studies suggest that cellular processes mediated by heparin binding growth factors may be modulated by P-CDS. Images PMID:8675622

Mechanism of enhanced performance on a hybrid direct carbon fuel cell using sawdust biofuels

NASA Astrophysics Data System (ADS)

Li, Shuangbin; Jiang, Cairong; Liu, Juan; Tao, Haoliang; Meng, Xie; Connor, Paul; Hui, Jianing; Wang, Shaorong; Ma, Jianjun; Irvine, John T. S.

2018-04-01

Biomass is expected to play a significant role in power generation in the near future. With the uprising of carbon fuel cells, hybrid direct carbon fuel cells (HDCFCs) show its intrinsic and incomparable advantages in the generation of clean energy with higher efficiency. In this study, two types of biomass treated by physical sieve and pyrolysis from raw sawdust are investigated on an anode-supported HDCFC. The structure and thermal analysis indicate that raw sawdust has well-formed cellulose I phase with very low ash. Electrochemical performance behaviors for sieved and pyrolyzed sawdust combined with various weight ratios of carbonate are compared in N2 and CO2 purge gas. The results show that the power output of sieved sawdust with 789 mWcm-2 is superior to that of pyrolyzed sawdust in CO2 flowing, as well as in N2 flowing. The anode reaction mechanism for the discrepancy of two fuels is explained and the emphasis is also placed on the modified oxygen-reduction cycle mechanism of catalytic effects of Li2CO3 and K2CO3 salts in promoting cell performance.

Ampelopsin-induced reactive oxygen species enhance the apoptosis of colon cancer cells by activating endoplasmic reticulum stress-mediated AMPK/MAPK/XAF1 signaling

PubMed Central

Park, Ga Bin; Jeong, Jee-Yeong; Kim, Daejin

2017-01-01

Ampelopsin (Amp) is bioactive natural product and exerts anti-cancer effects against several cancer types. The present study investigated the anti-colon cancer activity of Amp and explored its mechanism of action. The treatment of colon cancer cells with Amp resulted in the dose- and time-dependent induction of apoptosis via the activation of endoplasmic reticulum (ER) stress, 5′ adenosine monophosphate-activated protein kinase (AMPK), and c-Jun N-terminal protein kinase (JNK)/p38 mitogen-activated protein kinases (MAPKs). Salubrinal, an ER stress inhibitor, prevented the upregulation of ER stress-associated proteins, including phosphorylated protein kinase RNA-like ER kinase, phosphorylated eukaryotic translation initiation factor 2α, glucose-regulated protein 78, and CCAAT/enhancer-binding protein homologous protein, as well as suppressing AMPK activation and the MAPK signaling pathway. Knockdown of AMPK by RNA interference failed to block ER stress. Additionally, SP600125 (a JNK inhibitor) and SB203580 (a p38-MAPK inhibitor) effectively inhibited apoptosis and attenuated the expression of X-linked IAP-associated factor 1 (XAF1) and apoptotic Bcl-2 family proteins (BCL2 antagonist/killer 1 and BCL2-associated X protein) in Amp-treated colon cancer cells. Furthermore, reactive oxygen species (ROS)-mediated ER stress/AMPK apoptotic signaling pathway in Amp-treated colon cancer cells were markedly inhibited by treatment with N-acetyl-L-cysteine, a ROS scavenger. These results demonstrate that treatment with Amp induces the apoptotic death of colon cancer cells through ER stress-initiated AMPK/MAPK/XAF1 signaling. These results also provide experimental information for developing Amp as therapeutic drug against colon cancer. PMID:29250183

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

NASA Technical Reports Server (NTRS)

Chen, N. X.; Ryder, K. D.; Pavalko, F. M.; Turner, C. H.; Burr, D. B.; Qiu, J.; Duncan, R. L.

2000-01-01

Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm(2)). Before application of shear, cells were treated with two Ca(2+) channel inhibitors or various blockers of intracellular Ca(2+) release for 0. 5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca(2+)](i) response, neither gadolinium nor nifedipine, an L-type channel Ca(2+) channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular Ca(2+) chelator, or thapsigargin, which empties intracellular Ca(2+) stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP(3))-induced intracellular Ca(2+) release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP(3)-mediated intracellular Ca(2+) release is required for modulating flow-induced responses in MC3T3-E1 cells.

Escherichia coli Nissle 1917 enhances bioavailability of serotonin in gut tissues through modulation of synthesis and clearance

PubMed Central

Nzakizwanayo, Jonathan; Dedi, Cinzia; Standen, Guy; Macfarlane, Wendy M.; Patel, Bhavik A.; Jones, Brian V.

2015-01-01

Accumulating evidence shows indigenous gut microbes can interact with the human host through modulation of serotonin (5-HT) signaling. Here we investigate the impact of the probiotic Escherichia coli Nissle 1917 (EcN) on 5-HT signalling in gut tissues. Ex-vivo mouse ileal tissue sections were treated with either EcN or the human gut commensal MG1655, and effects on levels of 5-HT, precursors, and metabolites, were evaluated using amperometry and high performance liquid chromatography with electrochemical detection (HPLC-EC). Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT. These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures. In contrast, when supernatant recovered from untreated ileal tissue was pre-incubated with EcN, the derivative cell-free supernatant was able to elevate 5-HT overflow when used to treat fresh ileal tissue. Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA. The former pointed to modulation of tryptophan hydroxylase-1 to enhance 5-HT synthesis, while the latter indicates an impact on clearance into enterocytes through SERT. Taken together, these findings show EcN is able to enhance 5-HT bioavailability in ileal tissues through interaction with compounds secreted from host tissues. PMID:26616662

Anakinra reduces blood pressure and renal fibrosis in one kidney/DOCA/salt-induced hypertension.

PubMed

Ling, Yeong Hann; Krishnan, Shalini M; Chan, Christopher T; Diep, Henry; Ferens, Dorota; Chin-Dusting, Jaye; Kemp-Harper, Barbara K; Samuel, Chrishan S; Hewitson, Timothy D; Latz, Eicke; Mansell, Ashley; Sobey, Christopher G; Drummond, Grant R

2017-02-01

To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal inflammation, structural damage and blood pressure (BP) in mice with established hypertension. Hypertension was induced in male mice by uninephrectomy, deoxycorticosterone acetate (2.4mg/d,s.c.) and replacement of drinking water with saline (1K/DOCA/salt). Control mice received uninephrectomy, a placebo pellet and normal drinking water. 10days post-surgery, mice commenced treatment with anakinra (75mg/kg/d, i.p.) or vehicle (0.9% saline, i.p.) for 11days. Systolic BP was measured by tail cuff while qPCR, immunohistochemistry and flow cytometry were used to measure inflammatory markers, collagen and immune cell infiltration in the kidneys. By 10days post-surgery, 1K/DOCA/salt-treated mice displayed elevated systolic BP (148.3±2.4mmHg) compared to control mice (121.7±2.7mmHg; n=18, P<0.0001). The intervention with anakinra reduced BP in 1K/DOCA/salt-treated mice by ∼20mmHg (n=16, P<0.05), but had no effect in controls. In 1K/DOCA/salt-treated mice, anakinra modestly reduced (∼30%) renal expression of some (CCL5, CCL2; n=7-8; P<0.05) but not all (ICAM-1, IL-6) inflammatory markers, and had no effect on immune cell infiltration (n=7-8, P>0.05). Anakinra reduced renal collagen content (n=6, P<0.01) but paradoxically appeared to exacerbate the renal and glomerular hypertrophy (n=8-9, P<0.001) that accompanied 1K/DOCA/salt-induced hypertension. Despite its anti-hypertensive and renal anti-fibrotic actions, anakinra had minimal effects on inflammation and leukocyte infiltration in mice with 1K/DOCA/salt-induced hypertension. Future studies will assess whether the anti-hypertensive actions of anakinra are mediated by protective actions in other BP-regulating or salt-handling organs such as the arteries, skin and brain. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Clinical outcomes using carbon-ion radiotherapy and dose-volume histogram comparison between carbon-ion radiotherapy and photon therapy for T2b-4N0M0 non-small cell lung cancer-A pilot study.

PubMed

Shirai, Katsuyuki; Kawashima, Motohiro; Saitoh, Jun-Ichi; Abe, Takanori; Fukata, Kyohei; Shigeta, Yuka; Irie, Daisuke; Shiba, Shintaro; Okano, Naoko; Ohno, Tatsuya; Nakano, Takashi

2017-01-01

The safety and efficacy of carbon-ion radiotherapy for advanced non-small cell lung cancer have not been established. We evaluated the clinical outcomes and dose-volume histogram parameters of carbon-ion radiotherapy compared with photon therapy in T2b-4N0M0 non-small cell lung cancer. Twenty-three patients were treated with carbon-ion radiotherapy between May 2011 and December 2015. Seven, 14, and 2 patients had T2b, T3, and T4, respectively. The median age was 78 (range, 53-91) years, with 22 male patients. There were 12 adenocarcinomas, 8 squamous cell carcinomas, 1 non-small cell lung carcinoma, and 2 clinically diagnosed lung cancers. Eleven patients were operable, and 12 patients were inoperable. Most patients (91%) were treated with carbon-ion radiotherapy of 60.0 Gy relative biological effectiveness (RBE) in 4 fractions or 64.0 Gy (RBE) in 16 fractions. Local control and overall survival rates were calculated. Dose-volume histogram parameters of normal lung and tumor coverages were compared between carbon-ion radiotherapy and photon therapies, including three-dimensional conformal radiotherapy (3DCRT) and intensity-modulated radiotherapy (IMRT). The median follow-up of surviving patients was 25 months. Three patients experienced local recurrence, and the 2-year local control rate was 81%. During follow-up, 5 patients died of lung cancer, and 1 died of intercurrent disease. The 2-year overall survival rate was 70%. Operable patients had a better overall survival rate compared with inoperable patients (100% vs. 43%; P = 0.04). There was no grade ≥2 radiation pneumonitis. In dose-volume histogram analysis, carbon-ion radiotherapy had a significantly lower dose to normal lung and greater tumor coverage compared with photon therapies. Carbon-ion radiotherapy was effectively and safely performed for T2b-4N0M0 non-small cell lung cancer, and the dose distribution was superior compared with those for photon therapies. A Japanese multi-institutional study is ongoing to prospectively evaluate these patients and establish the use of carbon-ion radiotherapy.

Geldanamycin Reduces Acute Respiratory Distress Syndrome and Promotes the Survival of Mice Infected with the Highly Virulent H5N1 Influenza Virus.

PubMed

Wang, Chengmin; Liu, Pengpeng; Luo, Jing; Ding, Hua; Gao, Yan; Sun, Lei; Luo, Fubing; Liu, Xiaodong; He, Hongxuan

2017-01-01

Infections with lethal influenza viruses lead to acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), which may be related to the activation of the host's immune system. Here, in our study, male C57BL/6 mice were infected with 10 LD 50 of the H5N1 influenza virus and treated with geldanamycin or oseltamivir 2 h after infection. Lung injury was assessed by histopathology on days 4 and 7. The viral load was quantified by measuring the NP gene expression level on days 2, 4, and 7. Levels of cytokines and chemokines in bronchoalveolar lavage fluids and inflammatory cells were analyzed at different time points. Geldanamycin administration prolonged survival in mice and dramatically reduced lung injury and pulmonary inflammatory compared with other mice. Viral loads in geldanamycin-treated mice also significantly reduced compared with non-treated mice, but not to the extent as the oseltamivir-treated mice. Furthermore, the geldanamycin treatment markedly reduced the production of major proinflammatory cytokines and chemokines and attenuated the infiltration and activation of immune cells, but it did not alter the generation of virus-neutralizing antibodies. In conclusion, geldanamycin plays an important role in attenuating virus infection-induced ALI/ARDS by reducing the host's inflammatory responses and may provide an important reference for clinical treatments.

Atezolizumab in Japanese Patients With Previously Treated Advanced Non-Small-Cell Lung Cancer: A Subgroup Analysis of the Phase 3 OAK Study.

PubMed

Hida, Toyoaki; Kaji, Reiko; Satouchi, Miyako; Ikeda, Norihiko; Horiike, Atsushi; Nokihara, Hiroshi; Seto, Takashi; Kawakami, Tomohisa; Nakagawa, Shintaro; Kubo, Toshio

2018-02-01

Atezolizumab, an anti-programmed death-ligand 1 (PD-L1) agent, is effective and well tolerated in patients with pretreated advanced non-small-cell lung cancer (NSCLC). We assessed its efficacy and safety in Japanese patients through subgroup analyses of the phase 3 OAK study (NCT02008227). Key eligibility criteria of this randomized, controlled, open-label, international study include locally advanced/metastatic NSCLC, ≥ 1 prior platinum-based chemotherapy, age ≥ 18 years, measurable disease (Response Evaluation Criteria in Solid Tumors v1.1), and Eastern Cooperative Oncology Group performance status 0 or 1. Atezolizumab 1200 mg or docetaxel 75 mg/m 2 was provided intravenously every 3 weeks. Co-primary end points were overall survival (OS) in the intention-to-treat (ITT) population and those with ≥ 1% PD-L1 expression on tumor cells (TC) or tumor-infiltrating immune cells (IC; TC1/2/3 or IC1/2/3). Sixty-four ITT patients were Japanese; 19 had TC1/2/3 or IC1/2/3 status. In Japanese ITT patients, median OS in the atezolizumab arm (n = 36) was longer than the docetaxel arm (n = 28; 21.3 months [95% confidence interval (CI), 11.0-not estimable (NE)] versus 17.0 months [95% CI, 12.5-NE], respectively; hazard ratio 0.80 [95% CI, 0.41-1.57]). In the TC1/2/3 or IC1/2/3 population, median OS was 21.3 months (95% CI, 15.0-NE) and NE in the atezolizumab (n = 11) and docetaxel (n = 8) groups, respectively (hazard ratio, 0.81 [95% CI, 0.22-3.05]). Atezolizumab was generally well tolerated, with no treatment-related deaths. Atezolizumab was effective and well tolerated in pretreated Japanese patients with NSCLC. Results are consistent with the primary analysis of OAK. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Are Carotid Stent Fractures Clinically Significant?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia-Toca, Manuel; Rodriguez, Heron E.; Naughton, Peter A.

2012-04-15

Purpose: Late stent fatigue is a known complication after carotid artery stenting (CAS) for cervical carotid occlusive disease. The purpose of this study was to determine the prevalence and clinical significance of carotid stent fractures. Materials and Methods: A single-center retrospective review of 253 carotid bifurcation lesions treated with CAS and mechanical embolic protection from April 2001 to December 2009 was performed. Stent integrity was analyzed by two independent observers using multiplanar cervical plain radiographs with fractures classified into the following types: type I = single strut fracture; type II = multiple strut fractures; type III = transverse fracture; andmore » type IV = transverse fracture with dislocation. Mean follow-up was 32 months. Results: Follow-up imaging was completed on 106 self-expanding nitinol stents (26 closed-cell and 80 open-cell stents). Eight fractures (7.5%) were detected (type I n = 1, type II n = 6, and type III n = 1). Seven fractures were found in open-cell stents (Precise n = 3, ViVEXX n = 2, and Acculink n = 2), and 1 fracture was found in a closed-cell stent (Xact n = 1) (p = 0.67). Only a previous history of external beam neck irradiation was associated with fractures (p = 0.048). No associated clinical sequelae were observed among the patients with fractures, and only 1 patient had an associated significant restenosis ({>=}80%) requiring reintervention. Conclusions: Late stent fatigue after CAS is an uncommon event and rarely clinically relevant. Although cell design does not appear to influence the occurrence of fractures, lesion characteristics may be associated risk factors.« less

Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1).

PubMed

Zhao, Minzhi; Li, Haiyun; Ma, Yan; Gong, He; Yang, Shu; Fang, Qiaojun; Hu, Zhiyuan

2017-01-01

Abraxane (Abr), a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX). To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1), showed significant differential expression ( P <0.05) in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes.

Conditions Inducing Excessive O-GlcNAcylation Inhibit BMP2-Induced Osteogenic Differentiation of C2C12 Cells.

PubMed

Gu, Hanna; Song, Mina; Boonanantanasarn, Kanitsak; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

2018-01-09

Hyperglycemic conditions in diabetic patients can affect various cellular functions, including the modulation of osteogenic differentiation. However, the molecular mechanisms by which hyperglycemia affects osteogenic differentiation are yet to be clarified. This study aimed to investigate whether the aberrant increase in protein O -linked-β- N -acetylglucosamine glycosylation ( O -GlcNAcylation) contributes to the suppression of osteogenic differentiation due to hyperglycemia. To induce osteogenic differentiation, C2C12 cells were cultured in the presence of recombinant human bone morphogenetic protein 2 (BMP2). Excessive protein O -GlcNAcylation was induced by treating C2C12 cells with high glucose, glucosamine, or N -acetylglucosamine concentrations or by O -GlcNAc transferase (OGT) overexpression. The effect of O -GlcNAcylation on osteoblast differentiation was then confirmed by examining the expression levels of osteogenic marker gene mRNAs, activity of alkaline phosphatase, and transcriptional activity of Runx2, a critical transcription factor for osteoblast differentiation and bone formation. Cell treatment with high glucose, glucosamine or N -acetylglucosamine increased O -GlcNAcylation of Runx2 and the total levels of O -GlcNAcylated proteins, which led to a decrease in the transcriptional activity of Runx2, expression levels of osteogenic marker genes (Runx2, osterix, alkaline phosphatase, and type I collagen), and activity of alkaline phosphatase. These inhibitory effects were rescued by lowering protein O -GlcNAcylation levels by adding STO45849, an OGT inhibitor, or by overexpressing β- N -acetylglucosaminidase. Our findings suggest that excessive protein O -GlcNAcylation contributes to high glucose-suppressed osteogenic differentiation.

Phase 1 Results of ZUMA-1: A Multicenter Study of KTE-C19 Anti-CD19 CAR T Cell Therapy in Refractory Aggressive Lymphoma.

PubMed

Locke, Frederick L; Neelapu, Sattva S; Bartlett, Nancy L; Siddiqi, Tanya; Chavez, Julio C; Hosing, Chitra M; Ghobadi, Armin; Budde, Lihua E; Bot, Adrian; Rossi, John M; Jiang, Yizhou; Xue, Allen X; Elias, Meg; Aycock, Jeff; Wiezorek, Jeff; Go, William Y

2017-01-04

Outcomes for patients with refractory diffuse large B cell lymphoma (DLBCL) are poor. In the multicenter ZUMA-1 phase 1 study, we evaluated KTE-C19, an autologous CD3ζ/CD28-based chimeric antigen receptor (CAR) T cell therapy, in patients with refractory DLBCL. Patients received low-dose conditioning chemotherapy with concurrent cyclophosphamide (500 mg/m 2 ) and fludarabine (30 mg/m 2 ) for 3 days followed by KTE-C19 at a target dose of 2 × 10 6 CAR T cells/kg. The incidence of dose-limiting toxicity (DLT) was the primary endpoint. Seven patients were treated with KTE-C19 and one patient experienced a DLT of grade 4 cytokine release syndrome (CRS) and neurotoxicity. Grade ≥3 CRS and neurotoxicity were observed in 14% (n = 1/7) and 57% (n = 4/7) of patients, respectively. All other KTE-C19-related grade ≥3 events resolved within 1 month. The overall response rate was 71% (n = 5/7) and complete response (CR) rate was 57% (n = 4/7). Three patients have ongoing CR (all at 12+ months). CAR T cells demonstrated peak expansion within 2 weeks and continued to be detectable at 12+ months in patients with ongoing CR. This regimen of KTE-C19 was safe for further study in phase 2 and induced durable remissions in patients with refractory DLBCL. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Measuring topology of low-intensity DNA methylation sites for high-throughput assessment of epigenetic drug-induced effects in cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gertych, Arkadiusz, E-mail: gertycha@cshs.org; Bioinformatics, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA; Farkas, Daniel L., E-mail: dlfarkas@gmail.com

2010-11-15

Epigenetic anti-cancer drugs with demethylating effects have shown to alter genome organization in mammalian cell nuclei. The interest in the development of novel epigenetic drugs has increased the demand for cell-based assays to evaluate drug performance in pre-clinical studies. An imaging-based cytometrical approach that can measure demethylation effects as changes in the spatial nuclear distributions of methylated cytosine and global DNA in cancer cells is introduced in this paper. The cells were studied by immunofluorescence with a specific antibody against 5-methylcytosine (MeC), and 4,6-diamidino-2-phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei. In the preprocessing step themore » segmentation of nuclei in three-dimensional images (3-D) is followed by an automated assessment of nuclear DAPI/MeC patterns to exclude dissimilar entities. Next, low-intensity MeC (LIM) and low-intensity DNA (LID) sites of similar nuclei are localized and processed to obtain specific nuclear density profiles. These profiles sampled at half of the total nuclear volume yielded two parameters: LIM{sub 0.5} and LID{sub 0.5}. The analysis shows that zebularine and 5-azacytidine-the two tested epigenetic drugs introduce changes in the spatial distribution of low-intensity DNA and MeC signals. LIM{sub 0.5} and LID{sub 0.5} were significantly different (p < 0.001) in 5-azacytidine treated (n = 660) and zebularine treated (n = 496) vs. untreated (n = 649) DU145 human prostate cancer cells. In the latter case the LIM sites were predominantly found at the nuclear border, whereas treated populations showed different degrees of increase in LIMs towards the interior nuclear space, in which a large portion of heterochromatin is located. The cell-by-cell evaluation of changes in the spatial reorganization of MeC/DAPI signals revealed that zebularine is a more gentle demethylating agent than 5-azacytidine. Measuring changes in the topology of low-intensity sites can potentially be a valuable component in the high-throughput assessment of demethylation and risk of chromatin reorganization in epigenetic-drug screening tasks.« less

Effect of water extracts from edible Myrtaceae plants on uptake of 2-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose in TNF-α-treated FL83B mouse hepatocytes.

PubMed

Chang, Wen-Chang; Shen, Szu-Chuan

2013-02-01

This study investigated the glucose uptake activity of the water extracts from the leaves and fruit of edible Myrtaceae plants, including guava (Psidium guajava Linn.), wax apples [Syzygium samarangense (Blume) Merr. and L.M. Perry], Pu-Tau [Syzygium jambo (L.) Alston], and Kan-Shi Pu-Tau (Syzygium cumini Linn.) in FL83B mouse hepatocytes. The fluorescent dye 2-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose was used to estimate the uptake ability of the cells. Glucose uptake test showed that pink wax apple fruit extract (PWFE) exhibits the highest glucose uptake activity, at an increment of 21% in the insulin-resistant FL83B mouse hepatocytes as compared with the TNF-α-treated control group. Vescalagin was isolated using column chromatography of PWFE. This compound, at the concentration of 6.25 µg/mL, exhibits the same glucose uptake improvement in insulin-resistant cells as PWFE at a 100-µg/mL dose. We postulate that vescalagin is an active component in PWFE that may alleviate the insulin resistance in mouse hepatocytes. Copyright © 2012 John Wiley & Sons, Ltd.

NGAL attenuates renal ischemia/reperfusion injury through autophagy activation and apoptosis inhibition in rats.

PubMed

Zhang, Ya-Li; Qiao, Shu-Kai; Wang, Rong-Ying; Guo, Xiao-Nan

2018-06-01

Ischemia/reperfusion (I/R) injury is a main cause of acute kidney injury (AKI), and currently lacks effective therapies. This study is to investigate the level of Neutrophil gelatinase-associated lipocalin (NGAL) and autophagy status during renal I/R injury, so as to determine whether the exogenous NGAL protein could exert a protective effect for I/R injury and explore the potential mechanisms. Forty male Wistar rats were randomly divided into the following four groups: Sham, I/R, pre-treated with NGAL before I/R (I/R + pre-N), treated with NGAL after I/R (I/R + post-N). All rats were subjected to clamping the left renal pedicle for 45 min after right nephrectomy, followed by 24 h of reperfusion. Serum creatinine (SCr) and blood urea nitrogen (BUN) were used for renal function, tubular cell apoptosis and autophagy were measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method, histological examination and electron microscope, respectively. The tubular cell proliferation was assessed by the protein expression of proliferating cell nuclear antigen (PCNA). Western blotting was used to quantitate the levels of LC3, Beclin-1, Bcl-2 and Bax in kidney tissues. Exogenous NGAL protein intervention significantly improved renal function, reduced tubular cell apoptosis, increased tubular cell proliferation and promoted autophagy activation after renal I/R injury. Further, the efficacy in pre-N was significantly better than post-N. The mechanisms were involved in the regulation of several autophagy and apoptosis-related genes. Our study demonstrated that exogenous NGAL protein play a protective role during I/R injury, which may offer a novel may for prevention and treatment of renal I/R injury. Copyright © 2018. Published by Elsevier B.V.

Melanin content in melanoma metastases affects the outcome of radiotherapy.

PubMed

Brożyna, Anna A; Jóźwicki, Wojciech; Roszkowski, Krzysztof; Filipiak, Jan; Slominski, Andrzej T

2016-04-05

Melanin possess radioprotective and scavenging properties, and its presence can affect the behavior of melanoma cells, its surrounding environment and susceptibility to the therapy, as showed in vitro experiments. To determine whether melanin presence in melanoma affects the efficiency of radiotherapy (RTH) we evaluated the survival time after RTH treatment in metastatic melanoma patients (n = 57). In another cohort of melanoma patients (n = 84), the relationship between melanin level and pT and pN status was determined. A significantly longer survival time was found in patients with amelanotic metastatic melanomas in comparison to the melanotic ones, who were treated with either RTH or chemotherapy (CHTH) and RTH. These differences were more significant in a group of melanoma patients treated only with RTH. A detailed analysis of primary melanomas revealed that melanin levels were significantly higher in melanoma cells invading reticular dermis than the papillary dermis. A significant reduction of melanin pigmentation in pT3 and pT4 melanomas in comparison to pT1 and T2 tumors was observed. However, melanin levels measured in pT3-pT4 melanomas developing metastases (pN1-3, pM1) were higher than in pN0 and pM0 cases. The presence of melanin in metastatic melanoma cells decreases the outcome of radiotherapy, and melanin synthesis is related to higher disease advancement. Based on our previous cell-based and clinical research and present research we also suggest that inhibition of melanogenesis can improve radiotherapy modalities. The mechanism of relationship between melanogenesis and efficacy of RTH requires additional studies, including larger melanoma patients population and orthotopic, imageable mouse models of metastatic melanoma.

Reduction of the potential energy barrier and resistance at wafer-bonded n-GaAs/n-GaAs interfaces by sulfur passivation

NASA Astrophysics Data System (ADS)

Jackson, Michael J.; Jackson, Biyun L.; Goorsky, Mark S.

2011-11-01

Sulfur passivation and subsequent wafer-bonding treatments are demonstrated for III-V semiconductor applications using GaAs-GaAs direct wafer-bonded structures. Two different sulfur passivation processes are addressed. A dry sulfur passivation method that utilizes elemental sulfur vapor activated by ultraviolet light in vacuum is compared with aqueous sulfide and native-oxide-etch treatments. The electrical conductivity across a sulfur-treated 400 - °C-bonded n-GaAs/n-GaAs interface significantly increased with a short anneal (1-2 min) at elevated temperatures (500-600 °C). Interfaces treated with the NH4OH oxide etch, on the other hand, exhibited only mild improvement in accordance with previously published studies in this area. TEM and STEM images revealed similar interfacial microstructure changes with annealing for both sulfur-treated and NH4OH interfaces, whereby some areas have direct semiconductor-semiconductor contact without any interfacial layer. Fitting the observed temperature dependence of zero-bias conductance using a model for tunneling through a grain boundary reveals that the addition of sulfur at the interface lowered the interfacial energy barrier by 0.2 eV. The interface resistance for these sulfur-treated structures is 0.03 Ω.cm at room temperature. These results emphasize that sulfur-passivation techniques reduce interface states that otherwise limit the implementation of wafer bonding for high-efficiency solar cells and other devices.

N-acetyl-L-cysteine increases MnSOD activity and enhances the recruitment of quiescent human fibroblasts to the proliferation cycle during wound healing.

PubMed

Mao, Gaowei; Goswami, Monali; Kalen, Amanda L; Goswami, Prabhat C; Sarsour, Ehab H

2016-01-01

The rebuilding of the connective tissue during wound healing requires the recruitment of fibroblasts to the wound area as well as reentry of quiescent fibroblasts to the proliferative cycle. Whether this process can be modulated by a small molecular weight thiol antioxidant N-acetyl-L-cysteine (NAC) was tested in normal human skin fibroblasts (NHFs) using a uni-directional wound healing assay. NAC treated cells demonstrated a decreased migration rate but increased number of proliferating cells recruited into the wound area post wounding. Fifteen day quiescent control and NAC treated NHFs were re-plated at a lower density and cell numbers counted at different days post-plating. Interestingly, NAC treated cells exhibited increased cellular proliferation indicated by both decreased cell population doubling time and increased S phase cells. NAC treated cells demonstrated decreased steady state levels of reactive oxygen species as well as increased protein and activity levels of manganese superoxide dismutase (MnSOD). NAC treatment failed to induce proliferation in quiescent cells lacking MnSOD expression. These results demonstrate that NAC enhanced the recruitment of quiescent NHFs into proliferation cycle during wound healing. Our results also suggest that the wound healing properties of NAC might be due to its ability to induce and enhance MnSOD expression and activity. Altogether, these findings suggest NAC might be potentially developed as a dietary intervention to improve tissue injury in animals and humans.

Inhibitory effect of N-adamantyl-3,4-dihydroxybenzamide on melanogenesis in melan-a cells and brown guinea pigs.

PubMed

Rho, Ho Sik; Ahn, Soo Mi; Hwang, Jae Sung

2011-04-01

To find novel depigmenting agents, a new synthetic compound, N-adamantyl-3,4-dihydroxybenzamide (NADB) was produced and the effects on melanogenesis were investigated. Our results showed that NADB reduced melanin synthesis in a dose-dependent manner in melan-a cells. Tyrosinase activity was also reduced by NADB treatment in melan-a cells. However, NADB did not inhibit tyrosinase activity directly in a cell-free system. Treatment of melan-a cells with NADB caused a marked decrease in protein and mRNA levels of tyrosinase along with tyrosinase-related protein 1 and dopachrome tautomerase. To determine whether NADB reduces skin pigmentation, the dorsal skin of brown guinea pigs was shaved and irradiated with UV for 3 weeks using a solar simulator. Then NADB (2 or 1% in propylene glycol:ethanol:water = 5:3:2) was applied topically twice daily for 4 weeks. Visual inspection and Fontana-Masson staining both demonstrated that NADB resulted in lower skin pigmentation and total epidermal melanin in comparison to vehicle-treated areas. These findings suggest that NADB is useful in the treatment of hyperpigmentation.

Endothelin-1 Treatment Induces an Experimental Cerebral Malaria-Like Syndrome in C57BL/6 Mice Infected with Plasmodium berghei NK65.

PubMed

Martins, Yuri C; Freeman, Brandi D; Akide Ndunge, Oscar B; Weiss, Louis M; Tanowitz, Herbert B; Desruisseaux, Mahalia S

2016-11-01

Plasmodium berghei ANKA infection of C57BL/6 mice is a widely used model of experimental cerebral malaria (ECM). By contrast, the nonneurotropic P. berghei NK65 (PbN) causes severe malarial disease in C57BL/6 mice but does not cause ECM. Previous studies suggest that endothelin-1 (ET-1) contributes to the pathogenesis of ECM. In this study, we characterize the role of ET-1 on ECM vascular dysfunction. Mice infected with 10 6 PbN-parasitized red blood cells were treated with either ET-1 or saline from 2 to 8 days postinfection (dpi). Plasmodium berghei ANKA-infected mice served as the positive control. ET-1-treated PbN-infected mice exhibited neurological signs, hypothermia, and behavioral alterations characteristic of ECM, dying 4 to 8 dpi. Parasitemia was not affected by ET-1 treatment. Saline-treated PbN-infected mice did not display ECM, surviving until 12 dpi. ET-1-treated PbN-infected mice displayed leukocyte adhesion to the vascular endothelia and petechial hemorrhages throughout the brain at 6 dpi. Intravital microscopic images demonstrated significant brain arteriolar vessel constriction, decreased functional capillary density, and increased blood-brain barrier permeability. These alterations were not present in either ET-1-treated uninfected or saline-treated PbN-infected mice. In summary, ET-1 treatment of PbN-infected mice induced an ECM-like syndrome, causing brain vasoconstriction, adherence of activated leukocytes in the cerebral microvasculature, and blood-brain barrier leakage, indicating that ET-1 is involved in the genesis of brain microvascular alterations that are the hallmark of ECM. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Effects of thyroid hormone on Leydig cell regeneration in the adult rat following ethane dimethane sulphonate treatment.

PubMed

Ariyaratne, H B; Mills, N; Mason, J I; Mendis-Handagama, S M

2000-10-01

We tested the effects of thyroid hormone on Leydig cell (LC) regeneration in the adult rat testis after ethane dimethyl sulphonate (EDS) treatment. Ninety-day-old, thyroid-intact (n = 96) and thyroidectomized (n = 5) male Sprague-Dawley rats were injected intraperitoneally (single injection) with EDS (75 mg/kg) to destroy LC. Thyroid-intact, EDS-treated rats were equally divided into three groups (n = 32 per group) and treated as follows: control (saline-injected), hypothyroid (provided 0.1% propyl thiouracil in drinking water), and hyperthyroid (received daily subcutaneous injections of tri-iodothyronine, 100 microg/kg). Testing was done at Days 2, 7, 14, and 21 for thyroid-intact rats and at Day 21 for thyroidectomized rats after the EDS treatment. Leydig cells were absent in control and hyperthyroid rats at Days 2, 7, and 14; in hypothyroid rats at all ages; and in thyroidectomized rats at Day 21. The LC number per testis in hyperthyroid rats was twice as those of controls at Day 21. 3beta-Hydroxysteroid dehydrogenase (LC marker) immunocytochemistry results agreed with these findings. Mesenchymal cell number per testis was similar in the three treatment groups of thyroid-intact rats on Days 2 and 7, but it was different on Days 14 and 21. The highest number was in the hypothyroid rats, and the lowest was in the hyperthyroid rats. Serum testosterone levels could be measured in control rats only on Day 21, were undetectable in hypothyroid rats at all stages, and were detected in hyperthyroid rats on Days 14 and 21. These levels in hyperthyroid rats were twofold greater than those of controls on Day 21. Serum androstenedione levels could be measured only in the hyperthyroid rats on Day 21. Testosterone and androstenedione levels in the incubation media showed similar patterns to those in serum, but with larger values. These findings indicate that hypothyroidism inhibits LC regeneration and hyperthyroidism results in accelerated differentiation of more mesenchymal cells into LC following the EDS treatment. The observations of the EDS-treated, thyroidectomized rats confirmed that the findings in hypothyroid rats were, indeed, due to the deficiency of thyroid hormone.

Effect of total hydroalcholic extract of Nigella sativa and its n-hexane and ethyl acetate fractions on ACHN and GP-293 cell lines.

PubMed

Shahraki, Samira; Khajavirad, Abolfazl; Shafei, Mohammad Naser; Mahmoudi, Mahmoud; Tabasi, Nafisa Sadat

2016-01-01

Medicinal plants are noted for their many advantages including the ability to treat diseases such as cancer. In this study, we examined the antitumor effect of the medicinal plant Nigella sativa on the morphology, survival, and apoptosis of ACHN (human renal adenocarcinoma) and GP-293 (normal renal epithelial) cell lines. From a hydroalcoholic extract of N. sativa, n-hexane and ethyl acetate fractions were extracted. Cells were treated with various concentrations of total hydroalcholic extract and n-hexane and ethyl acetate fractions; cell viability, morphological changes, and apoptosis were then determined. Results were presented as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was applied for the statistical analysis of the data. The total extract and the fractions in a dose- and time-dependent manner reduced the cell viability in ACHN with no effect on the GP-293 cell line. In addition, the total extract resulted in more morphological changes in the ACHN cells compared to the GP-293 cells. The effect of the total extract in inducing apoptosis after 48 hours in the ACHN cell line was greater than in GP-293. In addition, the effect of the two fractions was lower than the total extract at all used concentrations. Therefore, the effect of total extract and n-hexane and ethyl acetate fractions of N. sativa on cell viability and apoptosis in the ACHN cell line is greater than in the GP-293 cell line. However, the effect of the total extract is higher than either of the two fractions on their own.

[The efficacy and safety of stereotactic radiotherapy for non-resectable non-small cell lung cancer].

PubMed

Futamura, Yohei; Sawa, Toshiyuki; Horiba, Akane; Ishiguro, Takashi; Yoshida, Tsutomu; Iida, Takayoshi; Marui, Tsutomu

2010-11-01

Stereotactic radiotherapy (SRT) has recently been used for the treatment of small lung tumors. We retrospectively evaluated the efficacy and safety of SRT for non-small cell lung cancer (NSCLC) treated in our hospital. Between October 2007 and December 2009, 31 tumors of 29 patients were treated by SRT (mean age, 75 years: stage IA, n=13; stage IB, n=5; stage IIIA, n=1; stage IIIB, n=1; recurrence, n=11). All of the patients completed the treatment. In one patient who had radiation pneumonitis before SRT, a progression of pulmonary fibrosis was observed, and treated with steroid therapy. In evaluable 29 tumors of 27 patients, the recurrence rates are 11/29 (37.9%). Median progression free survival time was 8 months. The recurrence rate and median progression free survival time of stage IA and IB subgroups were 4/17 (23.5%) and 12 months, respectively. SRT is thought to be a safe and effective treatment for stage I NSCLC. For patients with stage I NSCLC, SRT can be a complemental therapy for surgical resection.

Nuclear quiescence and histone hyper-acetylation jointly improve protamine-mediated nuclear remodeling in sheep fibroblasts

PubMed Central

Palazzese, Luca; Czernik, Marta; Iuso, Domenico; Toschi, Paola

2018-01-01

Recently we have demonstrated the possibility to replace histones with protamine, through the heterologous expression of human protamine 1 (hPrm1) gene in sheep fibroblasts. Here we have optimized protaminization of somatic nucleus by adjusting the best concentration and exposure time to trichostatin A (TSA) in serum-starved fibroblasts (nuclear quiescence), before expressing Prm1 gene. To stop cell proliferation, we starved cells in 0.5% FBS in MEM (“starved”—ST group), whereas in the Control group (CTR) the cells were cultured in 10% FBS in MEM. To find the most effective TSA concentration, we treated the cells with increasing concentrations of TSA in MEM + 10% FBS. Our results show that combination of cell culture conditions in 50 nM TSA, is more effective in terminating cell proliferation than ST and CTR groups (respectively 8%, 17.8% and 90.2% p<0.0001). Moreover, nuclear quiescence marker genes expression (Dicer1, Smarca 2, Ezh1 and Ddx39) confirmed that our culture conditions kept the cells in a nuclear quiescent state. Finally, ST and 50 nM TSA jointly increased the number of spermatid-like cell (39.4%) at higher rate compared to 25 nM TSA (20.4%, p<0.05) and 100 nM TSA (13.7%, p<0.05). To conclude, we have demonstrated that nuclear quiescence in ST cells and the open nuclear structure conferred by TSA resulted in an improved Prm1-mediated conversion of somatic nuclei into spermatid-like structures. This finding might improve nuclear reprogramming of somatic cells following nuclear transfer. PMID:29543876

Neuroprotective Effects of Oleocanthal, A Compound in Virgin Olive Oil, in A Rat Model of Traumatic Brain Injury.

PubMed

Mete, Mesut; Aydemir, Isıl; Unsal, Ulkun Unlu; Collu, Fatih; Vatandas, Gokhan; Gurcu, Beyhan; Duransoy, Yusuf Kurtulus; Taneli, Fatma; Tugrul, Mehmet Ibrahim; Selcuki, Mehmet

2017-11-01

TBI has two distinct phases: primary and secondary injury. Many agents have been used to prevent secondary injury. Oleocanthal (OC) has anti-inflammatory and antioxidant properties similar nonsteroidal anti-inflammatory drug. We evaluated the neuroprotective effects of OC in a rat model of TBI. Twenty-six adult male, Wistar albino rats were used. The rats were divided into 4 groups. group 1, sham (n = 5). group 2, trauma (n = 5): Rats were treated with 10 mg/kg saline intraperitoneally (IP) twice a day. Groups 3 and 4, rats were treated with 10 (group 3, n = 8) or 30 (group 4, n = 8) mg/kg OC IP twice a day. For each group brain samples were collected 72 h after injury. Brain samples and blood were evaluated with histopathological and biochemical methods. Histopathological evaluation revealed a significant difference between group 2 and group 4. Biochemical findings demonstrated that, oxidative stress index was the highest in group 2 and was the lowest in the group 4. Results indicated that OC has a protective effect on neural cells after TBI. This effect is achieved by reducing oxidative stress and apoptosis.

Radiation Therapy-First Strategy After Surgery With or Without Adjuvant Chemotherapy in Stage IIIA-N2 Non-Small Cell Lung Cancer.

PubMed

Lee, Hyun Woo; Noh, O Kyu; Oh, Young-Taek; Choi, Jin-Hyuk; Chun, Mison; Kim, Hwan-Ik; Heo, Jaesung; Ahn, Mi Sun; Park, Seong Yong; Park, Rae Woong; Yoon, Dukyong

2016-03-01

Postoperative radiation therapy (PORT) and postoperative chemotherapy (POCT) can be administered as adjuvant therapies in patients with non-small cell lung cancer (NSCLC). The aim of this study was to present the clinical outcomes in patients treated with PORT-first with or without subsequent POCT in stage IIIA-N2 NSCLC. From January 2002 to November 2014, the conditions of 105 patients with stage IIIA-N2 NSCLC who received PORT-first with or without subsequent POCT were analyzed. PORT was initiated within 4 to 6 weeks after surgical resection. Platinum-based POCT was administered 3 to 4 weeks after the completion of PORT. We analyzed the outcomes and the clinical factors affecting survival. Of 105 patients, 43 (41.0%) received POCT with a median of 4 cycles (range, 2-6 cycles). The follow-up times ranged from 3 to 123 months (median, 30 months), and the 5-year overall survival (OS) was 40.2%. The 5-year OS of patients treated with PORT and POCT was significantly higher than that of patients with PORT (61.3% vs 29.2%, P<.001). The significant prognostic factors affecting OS were the use of POCT (hazard ratio [HR] = 0.453, P=.036) and type of surgery (pneumonectomy/lobectomy; HR = 2.845, P<.001). PORT-first strategy after surgery appeared not to compromise the clinical outcomes in the treatment of stage IIIA-N2 NSCLC. The benefit of POCT on OS was preserved even in the PORT-first setting. Further studies are warranted to compare the sequencing of PORT and POCT, guaranteeing the proper use of POCT. Copyright © 2016 Elsevier Inc. All rights reserved.

Radiation Therapy–First Strategy After Surgery With or Without Adjuvant Chemotherapy in Stage IIIA-N2 Non-Small Cell Lung Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hyun Woo; Noh, O Kyu, E-mail: okyu.noh@gmail.com; Oh, Young-Taek

Purpose: Postoperative radiation therapy (PORT) and postoperative chemotherapy (POCT) can be administered as adjuvant therapies in patients with non-small cell lung cancer (NSCLC). The aim of this study was to present the clinical outcomes in patients treated with PORT-first with or without subsequent POCT in stage IIIA-N2 NSCLC. Methods and Materials: From January 2002 to November 2014, the conditions of 105 patients with stage IIIA-N2 NSCLC who received PORT-first with or without subsequent POCT were analyzed. PORT was initiated within 4 to 6 weeks after surgical resection. Platinum-based POCT was administered 3 to 4 weeks after the completion of PORT. We analyzedmore » the outcomes and the clinical factors affecting survival. Results: Of 105 patients, 43 (41.0%) received POCT with a median of 4 cycles (range, 2-6 cycles). The follow-up times ranged from 3 to 123 months (median, 30 months), and the 5-year overall survival (OS) was 40.2%. The 5-year OS of patients treated with PORT and POCT was significantly higher than that of patients with PORT (61.3% vs 29.2%, P<.001). The significant prognostic factors affecting OS were the use of POCT (hazard ratio [HR] = 0.453, P=.036) and type of surgery (pneumonectomy/lobectomy; HR = 2.845, P<.001). Conclusions: PORT-first strategy after surgery appeared not to compromise the clinical outcomes in the treatment of stage IIIA-N2 NSCLC. The benefit of POCT on OS was preserved even in the PORT-first setting. Further studies are warranted to compare the sequencing of PORT and POCT, guaranteeing the proper use of POCT.« less

Force spectroscopy of membrane hardness of SH-SY5Y neuroblastoma cells before and after differentiation

NASA Astrophysics Data System (ADS)

Kwon, Sangwoo; Yang, Woochul; Choi, Yun Kyong; Park, Jung Keuck

2014-05-01

Atomic force microscopy (AFM) is utilized in many studies for measuring the structure and the physical characteristics of soft and bio materials. In particular, the force spectroscopy function in the AFM system allows us to explore the mechanical properties of bio cells. In this study, we probe the variation in the membrane hardness of human neuroblastoma SH-SY5Y cells (SH-cells) before and after differentiation by using force spectroscopy. The SH-cell, which is usually differentiated by using a chemical treatment with retinoic acid (RA), is a neuronal cell line employed widely as an in-vitro model for neuroscience research. In force spectroscopy, the force-distance curves are obtained from both the original and the RA-treated cells while the AFM tip approaches and pushes on the cell membranes. The slope deduced from linear region in the force-distance curve is the spring constant and corresponds to the hardness of the cell membrane. The spring constant of the RA-treated cells (0.597 ± 0.010 nN/nm) was smaller than that of the original cells (0.794 ± 0.010 nN/nm), reflecting a hardness decrease in the cells differentiated with the RA treatments. The results clearly demonstrated that the differentiated cells are softer than the original cells. The change in the elasticity of the differentiated cells might be caused by morphological modification during differentiation process. We suggest that force spectroscopy can be employed as a novel method to determine the degree of differentiation of stem cells into various functional cells.

Epidermal Growth Factor Receptor Inhibition with Erlotinib Partially Prevents Cisplatin-Induced Nephrotoxicity in Rats

PubMed Central

Matsumoto, Kei; Shindo-Hirai, Yuki; Kuno, Yoshihiro; Yamamoto, Yasutaka; Suzuki, Taihei; Saito, Tomohiro; Iseri, Ken; Shibata, Takanori

2014-01-01

The effects of blocking the epidermal growth factor receptor (EGFR) in acute kidney injury (AKI) are controversial. Here we investigated the renoprotective effect of erlotinib, a selective tyrosine kinase inhibitor that can block EGFR activity, on cisplatin (CP)-induced AKI. Groups of animals were given either erlotinib or vehicle from one day before up to Day 3 following induction of CP- nephrotoxicity (CP-N). In addition, we analyzed the effects of erlotinib on signaling pathways involved in CP-N by using human renal proximal tubular cells (HK-2). Compared to controls, rats treated with erlotinib exhibited significant improvement of renal function and attenuation of tubulointerstitial injury, and reduced the number of apoptotic and proliferating cells. Erlotinib-treated rats had a significant reduction of renal cortical mRNA for profibrogenic genes. The Bax/Bcl-2 mRNA and protein ratios were significantly reduced by erlotinib treatment. In vitro, we observed that erlotinib significantly reduced the phosphorylation of MEK1 and Akt, processes that were induced by CP in HK-2. Taken together, these data indicate that erlotinib has renoprotective properties that are likely mediated through decreases in the apoptosis and proliferation of tubular cells, effects that reflect inhibition of downstream signaling pathways of EGFR. These results suggest that erlotinib may be useful for preventing AKI in patients receiving CP chemotherapy. PMID:25390346

Effects of a traditional Chinese herbal medicine, Kanzo-bushi-to, on the resistance of thermally injured mice infected with herpes simplex virus type 1.

PubMed

Matsuo, R; Ball, M A; Kobayashi, M; Herndon, D N; Pollard, R B; Suzuki, F

1994-10-01

The protective effect of Kanzo-bushi-to (TJS-038) was investigated on the opportunistic infection of herpes simplex virus type 1 (HSV) in thermally injured mice (TI-Mice). We have previously reported that TI-Mice were approximately 100 times more susceptible to HSV infection than normal mice (N-Mice) and that CD8+ suppressor T (ST)-cells induced by burn injury were involved in causing this increased susceptibility of TI-Mice. Increased susceptibility of TI-Mice to the infection was reversed to the levels observed in N-Mice when TI-Mice were treated intraperitoneally with TJS-038 at a dose of 5 mg/kg 1 and 4 days after thermal injury. The activity of ST-cells was greatly decreased in TI-Mice treated with TJS-038. The generation of Vicia villosa lectin-adherent CD4+ CD28+ TCR-alpha/beta+ contrasuppressor T (Contra-ST)-cells associated with the appearance of ST-cells was expanded and occurred earlier in spleens of TJS-038-treated TI-Mice as compared with that of untreated TI-Mice. The improved resistance of TJS-038-treated TI-Mice to the infection was transferred to untreated TI-Mice by adoptive transfer of Contra-ST-cells prepared from TJS-038-treated TI-Mice. These results suggest that TJS-038 may restore the resistance of TI-Mice to the HSV infection through the expanded generation of Contra-ST-cells.

Pilot study of nelarabine in combination with intensive chemotherapy in high-risk T-cell acute lymphoblastic leukemia: a report from the Children's Oncology Group.

PubMed

Dunsmore, Kimberly P; Devidas, Meenakshi; Linda, Stephen B; Borowitz, Michael J; Winick, Naomi; Hunger, Stephen P; Carroll, William L; Camitta, Bruce M

2012-08-01

Children's Oncology Group study AALL00P2 was designed to assess the feasibility and safety of adding nelarabine to a BFM 86-based chemotherapy regimen in children with newly diagnosed T-cell acute lymphoblastic leukemia (T-ALL). In stage one of the study, eight patients with a slow early response (SER) by prednisone poor response (PPR; ≥ 1,000 peripheral blood blasts on day 8 of prednisone prephase) received chemotherapy plus six courses of nelarabine 400 mg/m(2) once per day; four patients with SER by high minimal residual disease (MRD; ≥ 1% at day 36 of induction) received chemotherapy plus five courses of nelarabine; 16 patients with a rapid early response (RER) received chemotherapy without nelarabine. In stage two, all patients received six 5-day courses of nelarabine at 650 mg/m(2) once per day (10 SER patients [one by MRD, nine by PPR]) or 400 mg/m(2) once per day (38 RER patients; 12 SER patients [three by MRD, nine by PPR]). The only significant difference in toxicities was decreased neutropenic infections in patients treated with nelarabine (42% with v 81% without nelarabine). Five-year event-free survival (EFS) rates were 73% for 11 stage one SER patients and 67% for 22 stage two SER patients treated with nelarabine versus 69% for 16 stage one RER patients treated without nelarabine and 74% for 38 stage two RER patients treated with nelarabine. Five-year EFS for all patients receiving nelarabine (n = 70) was 73% versus 69% for those treated without nelarabine (n = 16). Addition of nelarabine to a BFM 86-based chemotherapy regimen was well tolerated and produced encouraging results in pediatric patients with T-ALL, particularly those with a SER, who have historically fared poorly.

Pilot Study of Nelarabine in Combination With Intensive Chemotherapy in High-Risk T-Cell Acute Lymphoblastic Leukemia: A Report From the Children's Oncology Group

PubMed Central

Dunsmore, Kimberly P.; Devidas, Meenakshi; Linda, Stephen B.; Borowitz, Michael J.; Winick, Naomi; Hunger, Stephen P.; Carroll, William L.; Camitta, Bruce M.

2012-01-01

Purpose Children's Oncology Group study AALL00P2 was designed to assess the feasibility and safety of adding nelarabine to a BFM 86–based chemotherapy regimen in children with newly diagnosed T-cell acute lymphoblastic leukemia (T-ALL). Patients and Methods In stage one of the study, eight patients with a slow early response (SER) by prednisone poor response (PPR; ≥ 1,000 peripheral blood blasts on day 8 of prednisone prephase) received chemotherapy plus six courses of nelarabine 400 mg/m2 once per day; four patients with SER by high minimal residual disease (MRD; ≥ 1% at day 36 of induction) received chemotherapy plus five courses of nelarabine; 16 patients with a rapid early response (RER) received chemotherapy without nelarabine. In stage two, all patients received six 5-day courses of nelarabine at 650 mg/m2 once per day (10 SER patients [one by MRD, nine by PPR]) or 400 mg/m2 once per day (38 RER patients; 12 SER patients [three by MRD, nine by PPR]). Results The only significant difference in toxicities was decreased neutropenic infections in patients treated with nelarabine (42% with v 81% without nelarabine). Five-year event-free survival (EFS) rates were 73% for 11 stage one SER patients and 67% for 22 stage two SER patients treated with nelarabine versus 69% for 16 stage one RER patients treated without nelarabine and 74% for 38 stage two RER patients treated with nelarabine. Five-year EFS for all patients receiving nelarabine (n = 70) was 73% versus 69% for those treated without nelarabine (n = 16). Conclusion Addition of nelarabine to a BFM 86–based chemotherapy regimen was well tolerated and produced encouraging results in pediatric patients with T-ALL, particularly those with a SER, who have historically fared poorly. PMID:22734022

Cross-Species Analysis of Nicotine-Induced Proteomic Alterations in Pancreatic Cells

PubMed Central

Paulo, Joao A.; Urrutia, Raul; Kadiyala, Vivek; Banks, Peter

2014-01-01

Background Toxic compounds in tobacco, such as nicotine, may have adversely affect pancreatic function. We aim to determine nicotine-induced protein alterations in pancreatic cells, which may reveal a link between nicotine exposure and pancreatic disease. Methods We compared the proteomic alterations induced by nicotine treatment in cultured pancreatic cells (mouse, rat and human stellate cells and human duct cells) using mass spectrometry-based techniques, specifically GeLC-MS/MS and spectral counting. Results We identified thousands of proteins in pancreatic cells, hundreds of which were identified exclusively or in higher abundance in either nicotine-treated or untreated cells. Inter-species comparisons of stellate cell proteins revealed several differentially-abundant proteins (in nicotine treated versus untreated cells) common among the 3 species. Proteins appearing in all nicotine-treated stellate cells include amyloid beta (A4), procollagen type VI alpha 1, integral membrane protein 2B,and Toll interacting protein. Conclusions Proteins which were differentially expressed upon nicotine treatment across cell lines, were enriched in certain pathways, including nAChR, cytokine, and integrin signaling. At this analytical depth, we conclude that similar pathways are affected by nicotine, but alterations at the protein level among stellate cells of different species vary. Further interrogation of such pathways will lead to insights into the potential effect of nicotine on pancreatic cells at the biomolecular level and the extension of this concept to the effect of nicotine on pancreatic disease. PMID:23456891

Targeting insulin resistance in type 2 diabetes via immune modulation of cord blood-derived multipotent stem cells (CB-SCs) in stem cell educator therapy: phase I/II clinical trial.

PubMed

Zhao, Yong; Jiang, Zhaoshun; Zhao, Tingbao; Ye, Mingliang; Hu, Chengjin; Zhou, Huimin; Yin, Zhaohui; Chen, Yana; Zhang, Ye; Wang, Shanfeng; Shen, Jie; Thaker, Hatim; Jain, Summit; Li, Yunxiang; Diao, Yalin; Chen, Yingjian; Sun, Xiaoming; Fisk, Mary Beth; Li, Heng

2013-07-09

The prevalence of type 2 diabetes (T2D) is increasing worldwide and creating a significant burden on health systems, highlighting the need for the development of innovative therapeutic approaches to overcome immune dysfunction, which is likely a key factor in the development of insulin resistance in T2D. It suggests that immune modulation may be a useful tool in treating the disease. In an open-label, phase 1/phase 2 study, patients (N=36) with long-standing T2D were divided into three groups (Group A, oral medications, n=18; Group B, oral medications+insulin injections, n=11; Group C having impaired β-cell function with oral medications+insulin injections, n=7). All patients received one treatment with the Stem Cell Educator therapy in which a patient's blood is circulated through a closed-loop system that separates mononuclear cells from the whole blood, briefly co-cultures them with adherent cord blood-derived multipotent stem cells (CB-SCs), and returns the educated autologous cells to the patient's circulation. Clinical findings indicate that T2D patients achieve improved metabolic control and reduced inflammation markers after receiving Stem Cell Educator therapy. Median glycated hemoglobin (HbA1C) in Group A and B was significantly reduced from 8.61%±1.12 at baseline to 7.25%±0.58 at 12 weeks (P=2.62E-06), and 7.33%±1.02 at one year post-treatment (P=0.0002). Homeostasis model assessment (HOMA) of insulin resistance (HOMA-IR) demonstrated that insulin sensitivity was improved post-treatment. Notably, the islet beta-cell function in Group C subjects was markedly recovered, as demonstrated by the restoration of C-peptide levels. Mechanistic studies revealed that Stem Cell Educator therapy reverses immune dysfunctions through immune modulation on monocytes and balancing Th1/Th2/Th3 cytokine production. Clinical data from the current phase 1/phase 2 study demonstrate that Stem Cell Educator therapy is a safe approach that produces lasting improvement in metabolic control for individuals with moderate or severe T2D who receive a single treatment. In addition, this approach does not appear to have the safety and ethical concerns associated with conventional stem cell-based approaches. ClinicalTrials.gov number, NCT01415726.

Enhancement of DNA ligase I level by gemcitabine in human cancer cells.

PubMed

Sun, Daekyu; Urrabaz, Rheanna; Kelly, Susan; Nguyen, Myhanh; Weitman, Steve

2002-04-01

DNA ligase I is an essential enzyme for completing DNA replication and DNA repair by ligating Okazaki fragments and by joining single-strand breaks formed either directly by DNA-damaging agents or indirectly by DNA repair enzymes, respectively. In this study, we examined whether the DNA ligase I level could be modulated in human tumor cell lines by treatment with gemcitabine (2', 2'-difluoro-2'-deoxycytidine), which is a nucleoside analogue of cytidine with proven antitumor activity against a broad spectrum of human cancers in clinical studies. To determine the effect of gemcitabine on DNA ligase I expression, Western blot analysis was used to measure the DNA ligase I levels in MiaPaCa, NGP, and SK-N-BE cells treated with different concentrations of gemcitabine and harvested at different time intervals. Cell cycle analysis was also performed to determine the underlying mechanism of DNA ligase I level enhancement in response to gemcitabine. In addition, other agents that share the same mechanism of action with gemcitabine were used to elucidate further details. When different types of tumor cell lines, including MiaPaCa, NGP, and SK-N-BE, were treated with gemcitabine, the level of DNA ligase I increased severalfold despite significant cell growth inhibition. In contrast, other DNA ligases (III and IV) either remained unchanged or decreased with treatment. Cell cycle analysis showed that arrest in S-phase corresponded to an increase of DNA ligase I levels in gemcitabine treated cells. Other agents, such as 1-beta-D-arabinofuranosylcytosine and hydroxyurea, which partly share mechanisms of action with gemcitabine by targeting DNA polymerases and ribonucleotide reductase, respectively, also caused an increase of DNA ligase I levels. However, 5-fluorouracil, which predominantly targets thymidylate synthase, did not cause an increase of DNA ligase I level. Our results suggest that an arrest of DNA replication caused by gemcitabine treatment through incorporation of gemcitabine triphosphate into replicating DNA and inhibition of ribonucleotide reductase would trigger an increase in DNA ligase I levels in cancer cells. The elevated presence of DNA ligase I in S-phase-arrested cells leads us to speculate that DNA ligase I might have an important role in repairing DNA damage caused by stalled replication forks.

Improvement over the years of long-term survival in high-risk lymphoma patients treated with hematopoietic stem cell transplantation as consolidation or salvage therapy.

PubMed

Calderón-Cabrera, C; Márquez-Malaver, F J; de la Cruz-Vicente, F; Falantes, F; Carrillo, E; Parody, R; Montero, I; González Campos, J; Martino, M L; Carmona, M; Pérez-Simón, J A; Espigado, I

2013-01-01

The role of hemopoietic stem cell transplantation (HSCT) is not well established in certain types of lymphoma, such as those with a high relapse risk or relapsing after initial therapy. New chemotherapeutic schemes and immunotherapy have improved survival of these patients. Nevertheless, there is not enough evidence regarding whether transplantation is the best therapeutic approach. Moreover, published data on long-term follow-up of high-risk lymphoma patients treated with HSCT are scarce. We analyzed 177 consecutive patients diagnosed with a high risk of relapse or with relapsed lymphoma who underwent HSCT after induction with standard chemotherapy in a tertiary academic center from 1989 to 2013. The median age was 40 years. Diagnoses were Hodgkin disease (n = 56), diffuse large B-cell lymphoma (n = 44), follicular lymphoma (n = 29), mantle cell lymphoma (n = 15), T-cell lymphoma (n = 18), and others (n = 15). Patients received either an autologous graft (n = 154) in first complete remission (1CR; n = 59) or more advanced stages (AS; n = 95), or an allogeneic graft (n = 23) in 1CR (n = 4) or AS (n = 19). In the autologous group, overall survival (OS) at 5 years was 57% and 75% in the periods 1989-2001 and 2002-2013, respectively (P = .05). Patients receiving an allogeneic graft presented an OS of 25% and 43% in the 2 periods. With a mean follow-up of 5 years (95% confidence interval 3.5-6.6), for patients receiving a transplant in 1CR, OS at 5 years was 80%, and for those receiving a transplant in AS it was 59% (P = .003). Nonrelapse mortality (NRM) at 5 years was 3.1% in the autologous group and 27.9% in the allogeneic group (P < .001). The main cause of NRM was infection (44%) in the whole cohort. All this leads to the conclusion that transplantation, as a therapeutic strategy, has shown a high long-term OS in this subgroup of patients with such a poor prognosis. OS improved over the years and reaching 1CR was a good prognostic feature. Infections were the main cause of NRM. Copyright © 2013 Elsevier Inc. All rights reserved.

Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

NASA Technical Reports Server (NTRS)

Schatten, H.; Ripple, M.; Balczon, R.; Weindruch, R.; Chakrabarti, A.; Taylor, M.; Hueser, C. N.

2000-01-01

We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well. Copyright 2000 Wiley-Liss, Inc.

Broad-spectrum neutralization of avian influenza viruses by sialylated human milk oligosaccharides: in vivo assessment of 3'-sialyllactose against H9N2 in chickens.

PubMed

Pandey, Ramesh Prasad; Kim, Dae Hee; Woo, Jinsuk; Song, Jaeyoung; Jang, Sang Ho; Kim, Joon Bae; Cheong, Kwang Myun; Oh, Jin Sik; Sohng, Jae Kyung

2018-02-07

Two sialylated human milk oligosaccharides (SHMOs) 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) were accessed for their possible antiviral activity against six different subtypes of thirteen avian influenza (AI) viruses in vitro. 3'-SL exhibited promising antiviral activity against almost all subtypes of tested AI viruses in hemagglutination inhibition assay, whereas 6'-SL showed activity against few selected H1N1, H1N2, and H3N2 subtype strains. 3'-SL has minimum inhibitory concentration values of 15.62 mM or less in more than half of the viruses examined. 3'-SL also showed effective inactivation of H9N2 Korea isolate (A/Chicken/Korea/MS96/1996) at 12.5 mM concentration in Madin Darby Canine Kidney (MDCK) cell line. Thus, 3'-SL was further studied for in vivo study against H9N2 virus in pathogen free chicken experiment models. In vivo study exhibited improved clinical symptoms on H9N2 infected chickens when treated with 3'-SL. Moreover, treating chickens with 3'-SL resulted in complete elimination of H9N2 viruses within 24 h of virus infection (0.8 HAU of H9N2). Indirect ELISA assay confirmed complete wash-out of H9N2 viruses from the colon after neutralization by 3'-SL without entering the blood stream. These in vivo results open up possible applications of 3'-SL for the prevention of AI virus infections in birds by a simple cleansing mechanism.

Can vitamin d suppress endothelial cells apoptosis in multiple sclerosis patients?

PubMed

Dehghani, Leila; Meamar, Rokhsareh; Etemadifar, Masoud; Sheshde, Zahra Dehghani; Shaygannejad, Vahid; Sharifkhah, Mostafa; Tahani, Soheil

2013-05-01

Multiple sclerosis (MS) is an autoimmune disease of central nerves system, in which neurological disabilities occur in young adults. Despite increasing number of studies on MS, some aspects of this disorder are still unclear. In the previous studies, it has been proven that there is direct relation between MS incidence and vitamin D deficiency. Thereby, strong evidence in MS pathogenesis suggests that endothelial cells (EC) could be harmed in MS. In addition, functional changes in EC and macrovascular injuries lead blood-brain barrier disruption in MS. Current study is the first investigation to elucidate positive influences of vitamin D against EC apoptosis in MS. Human umbilical vein endothelial cells (HUVECs) were cultured and then treated with sera from patients with active MS (in relapse) and sera from healthy volunteer participants as control group (each group n=15). 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay for cell surveillance and cell-death detection kit for evaluating apoptosis were used in this study. There was a significant decrease in apoptosis rate by the serum of patients, just when 1,25(OH)2D3 applied before treating HUVECs with sera from active MS (in relapse). Furthermore, the cells surveillance increased markedly with the presence of 1,25(OH)(2)D(3) in culture, too. Withregard to increment in EC apoptosis rate, which treated by the sera from MS patients and decrement in apoptosis rate by the presence of vitamin D in culture media, it could be proposed that vitamin D pre-treatment can be used for MS patients, due to its beneficial effects on protecting EC apoptosis.

EDC Cross-linking of Decellularized Tissue: A Promising Approach?

PubMed

Lehmann, Nadine; Christ, Torsten; Daugs, Aila; Bloch, Oliver; Holinski, Sebastian

2017-07-01

Decellularization of xenogenous cardiovascular structures is a promising approach to create scaffolds for tissue engineering. Unfortunately, handling and pliability of the unfixed tissue is challenging. N-(3-dimethylaminopropyl)-N9-ethylcarbodiimide (EDC) is an alternative cross-linking agent to glutaraldehyde (GA). Applied in native tissue, it provides biocompatibility and shows no potential for calcification. In addition, EDC can be used to link growth factors (GFs) to tissue scaffolds after decellularization. EDC cross-linking could thereby help to improve decellularized tissue without the toxicity of GA. Porcine aortic wall tissue specimens (TS) were decellularized, treated with EDC, and coated with fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF). Afterward, TS were subcutaneously implanted in 36 Lewis rats along with one decellularized TS without EDC treatment. After 2, 4, and 6 weeks TS were explanted from 12 rats, respectively. Vital cells were evaluated by RNA quantification, general cellular infiltration by hematoxylin and eosin staining (H&E), macrophage infiltration by CD68 staining, calcification by Von-Kossa staining, and tissue degradation by measurement of TS thickness. Quantification of vital cells showed reduced reseeding of EDC-treated TS compared to noncross-linked TS after 2 (p < 0.05) and 4 weeks (p < 0.05), while after 6 weeks only EDC+VEGF showed fewer viable cells (p < 0.01). Histological evaluation confirmed a reduced infiltration of EDC-treated TS. Macrophage infiltration decreased in all groups from 2 to 6 weeks, with the smallest population in EDC+VEGF-treated TS (p > 0.05). In EDC+FGF-treated TS, macrophages were reduced after 2 weeks compared to noncross-linked TS (p < 0.05), while after 4 and 6 weeks no significant difference was found (p > 0.05). Von-Kossa staining revealed no calcification in any of the specimens. Thickness of noncross-linked and EDC+FGF-treated TS was not different at the respective times of explantation, but decreased in both groups toward 6 weeks. EDC cross-linking combined with GF coating of decellularized aortic wall tissue showed encouraging results. The treatment did not impair the advantages of decellularized tissue such as long-term recellularization, absence of calcification, and tissue integrity. Based on the low macrophage infiltration and minimal tissue degradation, treatment with EDC and VEGF could be useful after decellularization. However, further research is necessary to verify these findings in models, including mechanical stress.

Antitumor effects of the flavone chalcone: inhibition of invasion and migration through the FAK/JNK signaling pathway in human gastric adenocarcinoma AGS cells.

PubMed

Lin, Su-Hsuan; Shih, Yuan-Wei

2014-06-01

Chalcones (benzylideneacetophenone) are cancer-preventive food components found in a human diet rich in fruits and vegetables. In this study, we first report the chemopreventive effect of chalcone in human gastric adenocarcinoma cell lines: AGS. The results showed that chalcone could inhibit the abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay, Boyden chamber invasion/migration assay, and wound-healing assay. Molecular data showed that the effect of chalcone in AGS cells might be mediated via sustained inactivation of the phosphorylation of focal adhesion kinase (FAK) and c-Jun N-terminal kinase 1 and 2 (JNK1/2) signal involved in the downregulation of the expressions of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Next, chalcone-treated AGS cells showed tremendous decrease in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, treating FAK small interfering RNA (FAK siRNA) and specific inhibitor for JNK (SP600125) to AGS cells could reduce the phosphorylation of JNK1/2 and the activity of MMP-2 and MMP-9. Our results revealed that chalcone significantly inhibited the metastatic ability of AGS cells by reducing MMP-2 and MMP-9 expressions concomitantly with a marked reduction on cell invasion and migration through suppressing and JNK signaling pathways. We suggest that chalcone may offer the application in clinical medicine.

A phase 2 study of weekly temsirolimus and bortezomib for relapsed or refractory B-cell non-Hodgkin lymphoma: A Wisconsin Oncology Network study.

PubMed

Fenske, Timothy S; Shah, Namrata M; Kim, Kyung Mann; Saha, Sandeep; Zhang, Chong; Baim, Arielle E; Farnen, John P; Onitilo, Adedayo A; Blank, Jules H; Ahuja, Harish; Wassenaar, Tim; Qamar, Rubina; Mansky, Patrick; Traynor, Anne M; Mattison, Ryan J; Kahl, Brad S

2015-10-01

Proteasome inhibitors and mammalian target of rapamycin inhibitors each have activity in various B-cell malignancies and affect distinct cellular pathways. Their combination has demonstrated synergy in vitro and in mouse models. The authors conducted a single-arm, phase 2 trial of combined temsirolimus and bortezomib in patients with relapsed and refractory B-cell non-Hodgkin lymphoma (NHL) using a dosing scheme that was previously tested in multiple myeloma. The patients received bortezomib and temsirolimus weekly on days 1, 8, 15, and 22 of a 35-day cycle. Of 39 patients who received treatment, 3 achieved a complete response (7.7%; 95% confidence interval [CI], 1.6%-21%), and 9 had a partial response (PR) (23%; 95% CI, 11%-39%). Thus, the overall response rate (12 of 39 patients) was 31% (95% CI, 17%-48%), and the median progression-free survival was 4.7 months (95% CI, 2.1-7.8 months; 2 months for patients with diffuse large B-cell lymphoma [n = 18], 7.5 months for those with mantle cell lymphoma [n = 7], and 16.5 months for those with follicular lymphoma [n = 9]). Two extensively treated patients with diffuse large B-cell lymphoma achieved a complete response. There were no unexpected toxicities from the combination. The current results demonstrate that the combination of a mammalian target of rapamycin inhibitor and a proteasome inhibitor is safe and has activity in patients with heavily pretreated B-cell NHL. Further studies with this combination are warranted in specific subtypes of NHL. © 2015 American Cancer Society.

The effects of diazinon and cypermethrin on the differentiation of neuronal and glial cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flaskos, J.; Harris, W.; Sachana, M.

2007-03-15

Diazinon and cypermethrin are pesticides extensively used in sheep dipping. Diazinon is a known anti-cholinesterase, but there is limited information regarding its molecular mechanism of action. This paper describes the effects of diazinon and cypermethrin at a morphological and molecular level on differentiating mouse N2a neuroblastoma and rat C6 glioma cell lines. Concentrations up to 10 {mu}M of both compounds and their mixture had no effect on the viability of either cell line, as determined by methyl blue tetrazolium reduction and total protein assays. Microscopic analysis revealed that 1 {mu}M and 10 {mu}M diazinon but not cypermethrin inhibited the outgrowthmore » of axon-like processes in N2a cells after a 24-h exposure but neither compound affected process outgrowth by differentiating C6 cells at these concentrations. Under these conditions, 10 {mu}M diazinon inhibited AChE slightly compared to the control after a 4-h exposure but not after 24 h. Western blotting analysis showed that morphological changes were associated with reduced cross-reactivity with antibodies that recognize the neurofilament heavy chain (NFH), microtubule associated protein MAP 1B and HSP-70 compared to control cell extracts, whereas reactivity with anti-{alpha}-tubulin antibodies was unchanged. Aggregation of NFH was observed in cell bodies of diazinon-treated N2a cells, as determined by indirect immunofluorescence staining. These data demonstrate that diazinon specifically targets neurite outgrowth in neuronal cells and that this effect is associated with disruption of axonal cytoskeleton proteins, whereas cypermethrin has no effect on the same parameters.« less

Inorganic mercury causes pancreatic beta-cell death via the oxidative stress-induced apoptotic and necrotic pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Yawen; Huang Chunfa; Yang Chingyao

2010-03-15

Mercury is a well-known highly toxic metal. In this study, we characterize and investigate the cytotoxicity and its possible mechanisms of inorganic mercury in pancreatic beta-cells. Mercury chloride (HgCl{sub 2}) dose-dependently decreased the function of insulin secretion and cell viability in pancreatic beta-cell-derived HIT-T15 cells and isolated mouse pancreatic islets. HgCl{sub 2} significantly increased ROS formation in HIT-T15 cells. Antioxidant N-acetylcysteine effectively reversed HgCl{sub 2}-induced insulin secretion dysfunction in HIT-T15 cells and isolated mouse pancreatic islets. Moreover, HgCl{sub 2} increased sub-G1 hypodiploids and annexin-V binding in HIT-T15 cells, indicating that HgCl{sub 2} possessed ability in apoptosis induction. HgCl{sub 2} alsomore » displayed several features of mitochondria-dependent apoptotic signals including disruption of the mitochondrial membrane potential, increase of mitochondrial cytochrome c release and activations of poly (ADP-ribose) polymerase (PARP) and caspase 3. Exposure of HIT-T15 cells to HgCl{sub 2} could significantly increase both apoptotic and necrotic cell populations by acridine orange/ethidium bromide dual staining. Meanwhile, HgCl{sub 2} could also trigger the depletion of intracellular ATP levels and increase the LDH release from HIT-T15 cells. These HgCl{sub 2}-induced cell death-related signals could be significantly reversed by N-acetylcysteine. The intracellular mercury levels were markedly elevated in HgCl{sub 2}-treated HIT-T15 cells. Taken together, these results suggest that HgCl{sub 2}-induced oxidative stress causes pancreatic beta-cell dysfunction and cytotoxicity involved the co-existence of apoptotic and necrotic cell death.« less

In vitro and in vivo immunomodulatory activity of sulfated polysaccharides from red seaweed Nemalion helminthoides.

PubMed

Pérez-Recalde, Mercedes; Matulewicz, María C; Pujol, Carlos A; Carlucci, María J

2014-02-01

Water-soluble sulfated polysaccharides from the red seaweed Nemalion helminthoides: two xylomannan fractions (N3 and N4) and a mannan fraction (N6) were investigated to determine their in vitro and in vivo immunomodulatory activities. N3 and N4 induced in vitro proliferation of macrophages of the murine cell line RAW 264.7 and significantly stimulated the production of nitric oxide (NO) and cytokines (IL-6 and TNF-α) in the same cells, whereas this response was not observed with the mannan N6. The cytokine production was also stimulated by sulfated xylomannans in vivo in BALB/c mice inoculated intravenously with these polysaccharides. Remarkably, when mice were treated with N3 and N4 for 1 h before being infected with Herpes simplex virus type 2, they remained asymptomatic with no signs of disease. The in vitro and in vivo results suggest that sulfated xylomannans could be strong immunomodulators. Copyright © 2013 Elsevier B.V. All rights reserved.

Alterations to the protein profile of bladder carcinoma cell lines induced by plant extract MINA-05 in vitro.

PubMed

Nguyen-Khuong, Terry; White, Melanie Y; Hung, Tzong-Tyng; Seeto, Shona; Thomas, Melissa L; Fitzgerald, Anna M; Martucci, Carlos E; Luk, Sharon; Pang, Shiu-Fu; Russell, Pamela J; Walsh, Bradley J

2009-04-01

Bladder cancer (BLCa) is a severe urological cancer of both men and women that commonly recurs and once invasive, is difficult to treat. MINA-05 (CK Life Sciences Int'l, Hong Kong) is a derivative of complex botanical extracts, shown to reduce cellular proliferation of bladder and prostate carcinomas. We tested the effects of MINA-05 against human BLCa cell sublines, B8, B8-RSP-GCK, B8-RSP-LN and C3, from a transitional cell carcinoma, grade IV, to determine the molecular targets of treatment by observing the cellular protein profile. Cells were acclimatised for 48 h then treated for 72 h with concentrations of MINA-05 reflecting 1/2 IC(50), IC(50) and 2 x IC(50) (n = 3) or with vehicle, (0.5% DMSO). Dose-dependant changes in protein abundance were detected and characterised using 2-dimensional electrophoresis and MS. We identified 10 proteins that underwent changes in abundance, pI and/or molecular mass in response to treatment. MINA-05 was shown to influence proteins across numerous functional classes including cytoskeletal proteins, energy metabolism proteins, protein degradation proteins and tumour suppressors, suggesting a global impact on these cell lines. This study implies that the ability of MINA-05 to retard cellular proliferation is attributed to its ability to alter cell cycling, metabolism, protein degradation and the cancer cell environment.

Escherichia coli Nissle 1917 protects gnotobiotic pigs against human rotavirus by modulating pDC and NK-cell responses.

PubMed

Vlasova, Anastasia N; Shao, Lulu; Kandasamy, Sukumar; Fischer, David D; Rauf, Abdul; Langel, Stephanie N; Chattha, Kuldeep S; Kumar, Anand; Huang, Huang-Chi; Rajashekara, Gireesh; Saif, Linda J

2016-10-01

Lactobacillus rhamnosus GG (LGG), a gram-positive lactic acid bacterium, is one of the most widely used probiotics; while fewer gram-negative probiotics including Escherichia coli Nissle 1917 (EcN) are characterized. A mechanistic understanding of their individual and interactive effects on human rotavirus (HRV) and immunity is lacking. In this study, noncolonized, EcN-, LGG-, and EcN + LGG-colonized neonatal gnotobiotic (Gn) pigs were challenged with HRV. EcN colonization is associated with a greater protection against HRV, and induces the highest frequencies of plasmacytoid dendritic cells (pDCs), significantly increased NK-cell function and decreased frequencies of apoptotic and TLR4 + mononuclear cells (MNCs). Consistent with the highest NK-cell activity, splenic CD172 + MNCs (DC enriched fraction) of EcN-colonized pigs produced the highest levels of IL-12 in vitro. LGG colonization has little effect on the above parameters, which are intermediate in EcN + LGG-colonized pigs, suggesting that probiotics modulate each other's effects. Additionally, in vitro EcN-treated splenic or intestinal MNCs produce higher levels of innate, immunoregulatory and immunostimulatory cytokines, IFN-α, IL-12, and IL-10, compared to MNCs of pigs treated with LGG. These results indicate that the EcN-mediated greater protection against HRV is associated with potent stimulation of the innate immune system and activation of the DC-IL-12-NK immune axis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synergistic inhibition of glioma cell proliferation by Withaferin A and tumor treating fields.

PubMed

Chang, Edwin; Pohling, Christoph; Beygui, Nooshin; Patel, Chirag B; Rosenberg, Jarrett; Ha, Dong Ho; Gambhir, Sanjiv S

2017-09-01

Glioblastoma (GBM) is the most aggressive and lethal form of brain cancer. Standard therapies are non-specific and often of limited effectiveness; thus, efforts are underway to uncover novel, unorthodox therapies against GBM. In previous studies, we investigated Withaferin A, a steroidal lactone from Ayurvedic medicine that inhibits proliferation in cancers including GBM. Another novel approach, tumor treating fields (TTFields), is thought to disrupt mitotic spindle formation and stymie proliferation of actively dividing cells. We hypothesized that combining TTFields with Withaferin A would synergistically inhibit proliferation in glioblastoma. Human glioblastoma cells (GBM2, GBM39, U87-MG) and human breast adenocarcinoma cells (MDA-MB-231) were isolated from primary tumors. The glioma cell lines were genetically engineered to express firefly luciferase. Proliferative potential was assessed either by bioluminescence imaging or cell counting via hemocytometer. TTFields (4 V/cm) significantly inhibited growth of the four cancer cell lines tested (n = 3 experiments per time point, four measurements per sample, p < 0.02 at least; 2-way ANOVA, control vs. treatment). The combination of Withaferin A (10-100 nM) with TTFields significantly inhibited the growth of the glioma cells to a degree beyond that of Withaferin A or TTFields alone. The interaction of the Withaferin A and TTFields on glioma cells was found to be synergistic in nature (p < 0.01, n = 3 experiments). These findings were validated by both bioluminescence and hemocytometric measurements. The combination of Withaferin A with TTFields represents a novel approach to treat GBM in a manner that is likely better than either treatment alone and that is synergistic.

Neuroblastoma: treatment outcome after incomplete resection of primary tumors.

PubMed

Moon, Suk-Bae; Park, Kwi-Won; Jung, Sung-Eun; Youn, Woong-Jae

2009-09-01

For International Neuroblastoma Staging System (INSS) stages III or IV neuroblastoma (intermediate or high risk), complete excision of the primary tumor is not always feasible. Most current studies on the treatment outcome of these patients have reported on the complete excision status. The aim of this study is to review the treatment outcome after the incomplete resection. The medical records of 37 patients that underwent incomplete resection between January 1986 and December 2005 were reviewed retrospectively. Incomplete resection was assessed by review of the operative notes and postoperative computerized tomography. Age, gender, tumor location, INSS stage, N-myc gene copy number, pre- and postoperative therapy, and treatment outcome were reviewed. The treatment outcome was evaluated according to the postoperative treatment protocol in the high-risk group. Intermediate-risk patients were treated with conventional chemotherapy, isotretinoin (ITT) and interleukin-2 (IL-2). High-risk patients were treated with peripheral blood stem cell transplantation (PBSCT), ITT, and IL-2 (N = 11). Before the introduction of PBSCT, the high-risk patients were also treated with the conventional chemotherapy (N = 19). Intermediate-risk patients (N = 5) currently have no evidence of disease (NED). For the high-risk patients (N = 32), 19 patients were treated with chemotherapy alone; 15 patients died of their disease while four patients currently have an NED status. Eight of 11 patients that underwent PBSCT are currently alive. For intermediate risk, conventional chemotherapy appears to be acceptable treatment. However, for high-risk patients, every effort should be made to control residual disease including the use of myeloablative chemotherapy, differentiating agents and immune-modulating agents.

Differentiation of V2a interneurons from human pluripotent stem cells

PubMed Central

Butts, Jessica C.; McCreedy, Dylan A.; Martinez-Vargas, Jorge Alexis; Mendoza-Camacho, Frederico N.; Hookway, Tracy A.; Gifford, Casey A.; Taneja, Praveen; Noble-Haeusslein, Linda; McDevitt, Todd C.

2017-01-01

The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however, the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here, we report the directed differentiation of CHX10+ V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid, sonic hedgehog, and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ∼25% CHX10+ cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time, CHX10+ cells expressed neuronal markers [neurofilament, NeuN, and vesicular glutamate transporter 2 (VGlut2)], and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10+ cells within the differentiated population, which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice, hPSC-derived V2a cultures survived at the site of injection, coexpressed NeuN and VGlut2, extended neurites >5 mm, and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI. PMID:28438991

Noninvasive positron emission tomography imaging of cell death using a novel small-molecule probe, (18)F labeled bis(zinc(II)-dipicolylamine) complex.

PubMed

Wang, Hongliang; Tang, Xiaolan; Tang, Ganghua; Huang, Tingting; Liang, Xiang; Hu, Kongzhen; Deng, Huaifu; Yi, Chang; Shi, Xinchong; Wu, Kening

2013-08-01

The synthetic bis(zinc(II)-dipicolylamine) (DPAZn2) coordination complexes are known to have a high specific and selective affinity to target the exposed phosphatidylserine (PS) on the surface of dead and dying cells. An (18)F-labeled DPAZn2 complex (4-(18)F-Fluoro-benzoyl-bis(zinc(II)-dipicolylamine), (18)F-FB-DPAZn2) as positron emission tomography (PET) tracer was developed and evaluated for in vivo imaging of tumor treated with a chemical agent. The in vitro cell stain studies revealed that fluorescent DPAZn2 complexes (Dansyl-DPAZn2) stained the same cells (apoptotic and necrotic cells) as fluorescein isothiocyanate (FITC) labeled Annexin V (FITC-Annexin V). The radiosynthesis of (18)F-FB-DPAZn2 was achieved through the amidation the precursor bis(2,2'-dipicolylamine) derivative (DPA2) with the prosthetic group N-succinimidyl-4-[(18)F]-fluorobenzoate ((18)F-SFB) and chelation with zinc nitrate. In the biodistribution study, the fast clearance of (18)F-FB-DPAZn2 from blood and kidney was observed and high uptake in liver and intestine within 90 min postinjection was also found. For the PET imaging, significantly higher tumor uptake of (18)F-FB-DPAZn2 was observed in the adriamycin (ADM)-treated Hepa1-6 hepatocellular carcinoma-bearing mice than that in the untreated tumor-model mice, while a slightly decreased tumor uptake of (18)F-FDG was found in the ADM-treated tumor-bearing mice. The results indicate that (18)F-FB-DPAZn2 has the similar capability of apoptosis detection as FITC-Annexin V and seems to be a potential PET tracer for noninvasive evaluation and monitoring of anti-tumor chemotherapy. The high uptake of (18)F-FB-DPAZn2 in the abdomen needs to optimize the structure for improving its pharmacokinetics characteristics in the future work.

Effects of cholesterol oxides on cell death induction and calcium increase in human neuronal cells (SK-N-BE) and evaluation of the protective effects of docosahexaenoic acid (DHA; C22:6 n-3).

PubMed

Zarrouk, Amira; Nury, Thomas; Samadi, Mohammad; O'Callaghan, Yvonne; Hammami, Mohamed; O'Brien, Nora M; Lizard, Gérard; Mackrill, John J

2015-07-01

Some oxysterols are associated with neurodegenerative diseases. Their lipotoxicity is characterized by an oxidative stress and induction of apoptosis. To evaluate the capacity of these molecules to trigger cellular modifications involved in neurodegeneration, human neuronal cells SK-N-BE were treated with 7-ketocholesterol, 7α- and 7β-hydroxycholesterol, 6α- and 6β-hydroxycholesterol, 4α- and 4β-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol (50-100μM, 24h) without or with docosahexaenoic acid (50μM). The effects of these compounds on mitochondrial activity, cell growth, production of reactive oxygen species (ROS) and superoxide anions (O2(-)), catalase and superoxide dismutase activities were determined. The ability of the oxysterols to induce increases in Ca(2+) was measured after 10min and 24h of treatment using fura-2 videomicroscopy and Von Kossa staining, respectively. Cholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, and 24(S)-hydroxycholesterol (100μM) induced mitochondrial dysfunction, cell growth inhibition, ROS overproduction and cell death. A slight increase in the percentage of cells with condensed and/or fragmented nuclei, characteristic of apoptotic cells, was detected. With 27-hydroxycholesterol, a marked increase of O2(-) was observed. Increases in intracellular Ca(2+) were only found with 7-ketocholesterol, 7β-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol. Pre-treatment with docosahexaenoic acid showed some protective effects depending on the oxysterol considered. According to the present data, 7-ketocholesterol, 7β-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol could favor neurodegeneration by their abilities to induce mitochondrial dysfunctions, oxidative stress and/or cell death associated or not with increases in cytosolic calcium levels. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of selective inhibitors of Aurora kinases on anaplastic thyroid carcinoma cell lines.

PubMed

Baldini, Enke; Tuccilli, Chiara; Prinzi, Natalie; Sorrenti, Salvatore; Antonelli, Alessandro; Gnessi, Lucio; Morrone, Stefania; Moretti, Costanzo; Bononi, Marco; Arlot-Bonnemains, Yannick; D'Armiento, Massimino; Ulisse, Salvatore

2014-10-01

Aurora kinases are serine/threonine kinases that play an essential role in cell division. Their aberrant expression and/or function induce severe mitotic abnormalities, resulting in either cell death or aneuploidy. Overexpression of Aurora kinases is often found in several malignancies, among which is anaplastic thyroid carcinoma (ATC). We have previously demonstrated the in vitro efficacy of Aurora kinase inhibitors in restraining cell growth and survival of different ATC cell lines. In this study, we sought to establish which Aurora might represent the preferential drug target for ATC. To this end, the effects of two selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) on four human ATC cell lines (CAL-62, BHT-101, 8305C, and 8505C) were analysed. Both inhibitors reduced cell proliferation in a time- and dose-dependent manner, with IC50 ranges of 44.3-134.2 nM for MLN8237 and of 9.2-461.3 nM for AZD1152. Immunofluorescence experiments and time-lapse videomicroscopy yielded evidence that each inhibitor induced distinct mitotic phenotypes, but both of them prevented the completion of cytokinesis. As a result, poliploidy increased in all AZD1152-treated cells, and in two out of four cell lines treated with MLN8237. Apoptosis was induced in all the cells by MLN8237, and in BHT-101, 8305C, and 8505C by AZD1152, while CAL-62 exposed to AZD1152 died through necrosis after multiple rounds of endoreplication. Both inhibitors were capable of blocking anchorage-independent cell growth. In conclusion, we demonstrated that either Aurora-A or Aurora-B might represent therapeutic targets for the ATC treatment, but inhibition of Aurora-A appears more effective for suppressing ATC cell proliferation and for inducing the apoptotic pathway. © 2014 Society for Endocrinology.

Coagulation profile of Sudanese children with homozygous sickle cell disease and the effect of treatment with omega-3 fatty acid on the coagulation parameters.

PubMed

Awoda, Shiekh; Daak, Ahmed A; Husain, Nazik Elmalaika; Ghebremeskel, Kebreab; Elbashir, Mustafa I

2017-01-01

It has been reported that patients with SCD do have an abnormal coagulation profile. Coagulopathy is thought to be one of the key factors that contribute to the vaso-occlusive crisis that characterises sickle cell disease (SCD). In this study, we investigated whether Sudanese sickle cell patients have an abnormal coagulation profile. In addition, the effect of treatment with either omega-3 fatty acids or hydroxyurea on coagulation profile was assessed. Homozygous SCD patients untreated ( n  = 52), omega-3 treated ( n  = 44), hydroxyurea (HU) treated ( n  = 8) and healthy (HbAA) controls ( n  = 52) matched for age (4-20 years), gender and socioeconomic status were enrolled. Patients on omega-3 fatty acids, according to age, received one to four capsules containing 277.8 mg DHA and 39.0 mg eicosapentnoic. Patients on Hydroxyurea were in on dosage more than 20 mg/kg/day. The steady state levels of the coagulation parameters and the effect of the treatments with either HU or omega-3 fatty acids on markers of coagulation were investigated. Compared to the healthy controls, treated and untreated HbSS patients had lower hemoglobin, plasma Protein C, proteins S and higher white blood cell count (WBC), platelets count (PLTs) and plasma D-dimer levels,( p  < 0.05). In comparison to untreated HbSS, treatment with neither omega-3 nor HU had effect on the WBC, plasma proteins C and S, ( p  > 0.05). HU treated group had a lower PLTs count compared to HbSS untreated group ( p  < 0.5). The prothrombin and activated partial thromboplastin times and international normalized ratio (INR) of untreated patients are significantly higher than n-3 treated, HU-treated patients and health controls, ( p  < 0.05). Patients treated with omega-3 had lowered D-dimer levels in comparison to HU-treated and untreated HbSS patients, ( p  < 0.001). This study provides evidence that Sudanes patients have abnormal coagulation profile and treatment with either HU or omega-3 fatty acids might partially ameliorate SCD-associated chronic coagulopathic state.

Anti-fibrotic effects of thalidomide on hepatic stellate cells and dimethylnitrosamine-intoxicated rats.

PubMed

Chong, Lee-Won; Hsu, Yi-Chao; Chiu, Yung-Tsung; Yang, Kuo-Ching; Huang, Yi-Tsau

2006-05-01

Tumor necrosis factor-alpha (TNF-alpha) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide, alpha-N-phthalidoglutarimide, (C(13)H(10)N(2))(4), has been shown to have immunomodulatory and anti-inflammatory properties, possibly mediated through its anti-TNF-alpha effect. In this study, we investigated the in vitro and in vivo effects of thalidomide on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1) or TNF-alpha. The inhibitory effects of thalidomide on the NFkappaB signaling cascade and fibrosis markers including alpha-smooth muscle actin (alpha-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats, which were randomly assigned to 1 of 4 groups: vehicle (0.7% carboxyl methyl cellulose, CMC), thalidomide (40 mg/kg), thalidomide (200 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. Thalidomide (100-800 nM) concentration-dependently inhibited NFkappaB transcriptional activity induced by TNF-alpha, including IKKalpha expression and IkappaBalpha phosphorylation in HSC-T6 cells. In addition, thalidomide also suppressed TGF-beta1-induced alpha-SMA expression and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats receiving high dose of thalidomide (0.89 +/- 0.20) were significantly reduced in comparison with those of DMN-treated rats receiving vehicle (1.56 +/- 0.18). Hepatic collagen contents of DMN rats were also significantly reduced by either thalidomide or silymarin treatment. Immunohistochemical double staining results showed that alpha-SMA- and NFkappaB-positive cells were decreased in the livers from DMN rats receiving either thalidomide or silymarin treatment. In addition, real-time PCR analysis indicated that hepatic mRNA expressions of TGF-beta1, alpha-SMA, collagen 1alpha2, TNF-alpha and iNOS genes were attenuated by thalidomide treatment. In conclusion, our results showed that thalidomide inhibited activation of HSC-T6 cells by TNF-alpha and ameliorated liver fibrosis in DMN-intoxicated rats.

Fluorescent Photo-conversion: A second chance to label unique cells.

PubMed

Mellott, Adam J; Shinogle, Heather E; Moore, David S; Detamore, Michael S

2015-03-01

Not all cells behave uniformly after treatment in tissue engineering studies. In fact, some treated cells display no signs of treatment or show unique characteristics not consistent with other treated cells. What if the "unique" cells could be isolated from a treated population, and further studied? Photo-convertible reporter proteins, such as Dendra2 , allow for the ability to selectively identify unique cells with a secondary label within a primary labeled treated population. In the current study, select cells were identified and labeled through photo-conversion of Dendra2 -transfected human Wharton's Jelly cells (hWJCs) for the first time. Robust photo-conversion of green-to-red fluorescence was achieved consistently in arbitrarily selected cells, allowing for precise cell identification of select hWJCs. The current study demonstrates a method that offers investigators the opportunity to selectively label and identify unique cells within a treated population for further study or isolation from the treatment population. Photo-convertible reporter proteins, such as Dendra2 , offer the ability over non-photo-convertible reporter proteins, such as green fluorescent protein, to analyze unique individual cells within a treated population, which allows investigators to gain more meaningful information on how a treatment affects all cells within a target population.

A retrospective analysis of RET translocation, gene copy number gain and expression in NSCLC patients treated with vandetanib in four randomized Phase III studies.

PubMed

Platt, Adam; Morten, John; Ji, Qunsheng; Elvin, Paul; Womack, Chris; Su, Xinying; Donald, Emma; Gray, Neil; Read, Jessica; Bigley, Graham; Blockley, Laura; Cresswell, Carl; Dale, Angela; Davies, Amanda; Zhang, Tianwei; Fan, Shuqiong; Fu, Haihua; Gladwin, Amanda; Harrod, Grace; Stevens, James; Williams, Victoria; Ye, Qingqing; Zheng, Li; de Boer, Richard; Herbst, Roy S; Lee, Jin-Soo; Vasselli, James

2015-03-23

To determine the prevalence of RET rearrangement genes, RET copy number gains and expression in tumor samples from four Phase III non-small-cell lung cancer (NSCLC) trials of vandetanib, a selective inhibitor of VEGFR, RET and EGFR signaling, and to determine any association with outcome to vandetanib treatment. Archival tumor samples from the ZODIAC ( NCT00312377 , vandetanib ± docetaxel), ZEAL ( NCT00418886 , vandetanib ± pemetrexed), ZEPHYR ( NCT00404924 , vandetanib vs placebo) and ZEST ( NCT00364351 , vandetanib vs erlotinib) studies were evaluated by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in 944 and 1102 patients. The prevalence of RET rearrangements by FISH was 0.7% (95% CI 0.3-1.5%) among patients with a known result. Seven tumor samples were positive for RET rearrangements (vandetanib, n = 3; comparator, n = 4). 2.8% (n = 26) of samples had RET amplification (innumerable RET clusters, or ≥7 copies in > 10% of tumor cells), 8.1% (n = 76) had low RET gene copy number gain (4-6 copies in ≥40% of tumor cells) and 8.3% (n = 92) were RET expression positive (signal intensity ++ or +++ in >10% of tumor cells). Of RET-rearrangement-positive patients, none had an objective response in the vandetanib arm and one patient responded in the comparator arm. Radiologic evidence of tumor shrinkage was observed in two patients treated with vandetanib and one treated with comparator drug. The objective response rate was similar in the vandetanib and comparator arms for patients positive for RET copy number gains or RET protein expression. We have identified prevalence for three RET biomarkers in a population predominated by non-Asians and smokers. RET rearrangement prevalence was lower than previously reported. We found no evidence of a differential benefit for efficacy by IHC and RET gene copy number gains. The low prevalence of RET rearrangements (0.7%) prevents firm conclusions regarding association of vandetanib treatment with efficacy in the RET rearrangement NSCLC subpopulation. Randomized Phase III clinical trials ( NCT00312377 , ZODIAC; NCT00418886 , ZEAL; NCT00364351 , ZEST; NCT00404924 , ZEPHYR).

Regulatory and activated effector T cells in chronic hepatitis C virus: Relation to autoimmunity

PubMed Central

Fouad, Hanan; El Raziky, Maissa; Hassan, Eman Medhat; Aziz, Ghada Mahmoud Abdel; Darweesh, Samar K; Sayed, Ahmed Reda

2016-01-01

AIM To investigate how Tregs are regulated in chronic hepatitis C virus (HCV) patients via assessment of Tregs markers (granzyme 2, CD69 and FoxP3), Teffs markers [TNFRSF4 (OX40), INFG] and CD4, CD25 genes. METHODS A prospective study was conducted on 120 subjects divided into 4 groups: Group I (n = 30) treatment naïve chronic HCV patients; Group II (n = 30) chronic HCV treated with Peg/Riba; Group III (n = 30) chronic HCV associated with non-organ specific autoantibody and Group IV (n = 30) healthy persons as a control group. Tregs and Teffs markers were assessed in peripheral blood mononuclear cells by quantitative real time reverse transcriptase-polymerase chain reaction. RESULTS Chronic HCV patients exhibited significant higher levels of both Teffs and Tregs in comparison to healthy control group. Tregs markers were significantly decreased in Peg/Riba treated HCV patients in comparison to treatment naïve HCV group. In HCV patients with antinuclear antibody (ANA) +ve, Tregs markers were significantly decreased in comparison to all other studied groups. Teffs markers were significantly elevated in all HCV groups in comparison to control and in HCV group with ANA +ve in comparison to treatment naïve HCV group. CONCLUSION Elevated Tregs cells in chronic HCV patients dampen both CD4+ and CD8+ autologous T cell immune response. Interferon-α and ribavirin therapy suppress proliferation of Tregs. More significant suppression of Tregs was observed in HCV patients with autoantibodies favoring pathological autoimmune response. PMID:27843539

SU-E-T-146: Effects of Uncertainties of Radiation Sensitivity of Biological Modelling for Treatment Planning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oita, M; Department of Life System, Institute of Technology and Science, Graduate School, The Tokushima University; Uto, Y

Purpose: The aim of this study was to evaluate the distribution of uncertainty of cell survival by radiation, and assesses the usefulness of stochastic biological model applying for gaussian distribution. Methods: For single cell experiments, exponentially growing cells were harvested from the standard cell culture dishes by trypsinization, and suspended in test tubes containing 1 ml of MEM(2x10{sup 6} cells/ml). The hypoxic cultures were treated with 95% N{sub 2}−5% CO{sub 2} gas for 30 minutes. In vitro radiosensitization was also measured in EMT6/KU single cells to add radiosensitizer under hypoxic conditions. X-ray irradiation was carried out by using an Xraymore » unit (Hitachi X-ray unit, model MBR-1505R3) with 0.5 mm Al/1.0 mm Cu filter, 150 kV, 4 Gy/min). In vitro assay, cells on the dish were irradiated with 1 Gy to 24 Gy, respectively. After irradiation, colony formation assays were performed. Variations of biological parameters were investigated at standard cell culture(n=16), hypoxic cell culture(n=45) and hypoxic cell culture(n=21) with radiosensitizers, respectively. The data were obtained by separate schedule to take account for the variation of radiation sensitivity of cell cycle. Results: At standard cell culture, hypoxic cell culture and hypoxic cell culture with radiosensitizers, median and standard deviation of alpha/beta ratio were 37.1±73.4 Gy, 9.8±23.7 Gy, 20.7±21.9 Gy, respectively. Average and standard deviation of D{sub 50} were 2.5±2.5 Gy, 6.1±2.2 Gy, 3.6±1.3 Gy, respectively. Conclusion: In this study, we have challenged to apply these uncertainties of parameters for the biological model. The variation of alpha values, beta values, D{sub 50} as well as cell culture might have highly affected by probability of cell death. Further research is in progress for precise prediction of the cell death as well as tumor control probability for treatment planning.« less

Chlorpyrifos- and chlorpyrifos oxon-induced neurite retraction in pre-differentiated N2a cells is associated with transient hyperphosphorylation of neurofilament heavy chain and ERK 1/2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sindi, Ramya A., E-mail: ramya.sindi2010@my.ntu.ac

Chlorpyrifos (CPF) and CPF-oxon (CPO) are known to inhibit neurite outgrowth but little is known about their ability to induce neurite retraction in differentiating neuronal cells. The aims of this study were to determine the ability of these compounds to destabilize neurites and to identify the key molecular events involved. N2a cells were induced to differentiate for 20 h before exposure to CPF or CPO for 2–8 h. Fixed cell monolayers labeled with carboxyfluorescein succinimidyl ester or immunofluorescently stained with antibodies to tubulin (B512) or phosphorylated neurofilament heavy chain (Ta51) showed time- and concentration-dependent reductions in numbers and length ofmore » axon-like processes compared to the control, respectively, retraction of neurites being observed within 2 h of exposure by live cell imaging. Neurofilament disruption was also observed in treated cells stained by indirect immunofluorescence with anti-phosphorylated neurofilament heavy chain (NFH) monoclonal antibody SMI34, while the microtubule network was unaffected. Western blotting analysis revealed transiently increased levels of reactivity of Ta51 after 2 h exposure and reduced levels of reactivity of the same antibody following 8 h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2 h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8 h exposure. These findings suggest for the first time that organophosphorothionate pesticide-induced neurite retraction in N2a cells is associated with transient increases in NFH phosphorylation and ERK1/2 activation. - Highlights: • Chlorpyrifos and chlorpyrifos oxon induced rapid neurite retraction in N2a cells. • This occurred following transient hyperphosphorylation of ERK 1/2. • It was concomitant with hyperphosphorylation of neurofilament heavy chain (NFH). • Subsequent NFH dephosphorylation was associated with increased phosphatase activity. • Thus, the above changes reflect key events in chlorpyrifos induced neurodegeneration.« less

The effect of cisplatin pretreatment on the accumulation of MIBG by neuroblastoma cells in vitro.

PubMed

Armour, A; Cunningham, S H; Gaze, M N; Wheldon, T E; Mairs, R J

1997-01-01

[131I]meta-iodobenzylguanidine ([131I]MIBG) provides a means of selectively delivering radiation to neuroblastoma cells and is a promising addition to the range of agents used to treat neuroblastoma. As MIBG is now being incorporated into multimodal approaches to therapy, important questions arise about the appropriate scheduling and sequencing of the various agents employed. As the ability of neuroblastoma cells to actively accumulate MIBG is crucial to the success of this therapy, the effect of chemotherapeutic agents on this uptake capacity needs to be investigated. We report here our initial findings on the effect of cisplatin pretreatment on the neuroblastoma cell line SK-N-BE (2c). After treating these cells with therapeutically relevant concentrations of cisplatin (2 microM and 20 microM), a stimulation in uptake of [131I]MIBG was observed. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that this effect was due to increased expression of the noradrenaline transporter. These results suggest that appropriate scheduling of cisplatin and [131I]MIBG may lead to an increase in tumour uptake of this radiopharmaceutical with consequent increases in radiation dose to the tumour.

Single-wall carbon nanohorns inhibited activation of microglia induced by lipopolysaccharide through blocking of Sirt3

NASA Astrophysics Data System (ADS)

Li, Lihong; Zhang, Jinqian; Yang, Yang; Wang, Qiang; Gao, Li; Yang, Yanlong; Chang, Tao; Zhang, Xingye; Xiang, Guoan; Cao, Yongmei; Shi, Zujin; Zhao, Ming; Gao, Guodong

2013-02-01

Single-wall carbon nanohorns (SWNHs) have been demonstrated to accumulate in cytotoxic levels within organs of various animal models and cell types, which emerge as a wide range of promising biomedical imaging. Septic encephalopathy (SE) is an early sign of sepsis and associated with an increased rate of morbidity and mortality. Microglia activation plays an important role in neuroinflammation, which contributes to neuronal damage. Inhibition of microglia activation may have therapeutic benefits, which can alleviate the progression of neurodegeneration. Therefore, we investigated the functional changes of mice microglia cell lines pre-treated with or without lipopolysaccharide (LPS) induced by SWNHs. To address this question, the research about direct role of SWNHs on the growth, proliferation, and apoptosis of microglia cell lines in mice (N9 and BV2) pre-treated with or without LPS had been performed. Our results indicate that the particle diameter of SWNHs in water is between 342 to 712 nm. The images in scanning electron microscope showed that SWNHs on polystyrene surface are individual particles. LPS induced activation of mice microglia, promoted its growth and proliferation, and inhibited its apoptosis. SWNHs inhibited proliferation, delayed mitotic entry, and promoted apoptosis of mice microglia cells. The effects followed gradually increasing cultured time and concentrations of SWNHs, especially in cells pre-treated with LPS. SWNHs induced a significantly increase in G1 phase and inhibition of S phase of mice microglia cells in a dose-manner dependent of SWNHs, especially in cells pre-treated with LPS. The transmission electron microscope images showed that individual spherical SWNH particles smaller than 100 nm in diameters were localized inside lysosomes of mice microglia cells. SWNHs inhibited mitotic entry, growth and proliferation of mice microglia cells, and promoted its apoptosis, especially in cells pre-treated with LPS. SWNHs inhibited expression of Sirt3 and energy metabolism related with Sirt3 in mice microglia cells in a dose-dependent manner, especially in cells pre-treated with LPS. The role of SWNHs on mice microglia was implicating Sirt3 and energy metabolism associated with it.

Significant Prognostic Factors for Completely Resected pN2 Non-small Cell Lung Cancer without Neoadjuvant Therapy

PubMed Central

Nakao, Masayuki; Mun, Mingyon; Nakagawa, Ken; Nishio, Makoto; Ishikawa, Yuichi; Okumura, Sakae

2015-01-01

Purpose: To identify prognostic factors for pathologic N2 (pN2) non-small cell lung cancer (NSCLC) treated by surgical resection. Methods: Between 1990 and 2009, 287 patients with pN2 NSCLC underwent curative resection at the Cancer Institute Hospital without preoperative treatment. Results: The 5-year overall survival (OS), cancer-specific survival (CSS), and recurrence-free survival (RFS) rates were 46%, 55% and 24%, respectively. The median follow-up time was 80 months. Multivariate analysis identified four independent predictors for poor OS: multiple-zone mediastinal lymph node metastasis (hazard ratio [HR], 1.616; p = 0.003); ipsilateral intrapulmonary metastasis (HR, 1.042; p = 0.002); tumor size >30 mm (HR, 1.013; p = 0.002); and clinical stage N1 or N2 (HR, 1.051; p = 0.030). Multivariate analysis identified three independent predictors for poor RFS: multiple-zone mediastinal lymph node metastasis (HR, 1.457; p = 0.011); ipsilateral intrapulmonary metastasis (HR, 1.040; p = 0.002); and tumor size >30 mm (HR, 1.008; p = 0.032). Conclusion: Multiple-zone mediastinal lymph node metastasis, ipsilateral intrapulmonary metastasis, and tumor size >30 mm were common independent prognostic factors of OS, CSS, and RFS in pN2 NSCLC. PMID:25740454

Effects of guaifenesin, N-acetylcysteine, and ambroxol on MUC5AC and mucociliary transport in primary differentiated human tracheal-bronchial cells.

PubMed

Seagrave, Jeanclare; Albrecht, Helmut H; Hill, David B; Rogers, Duncan F; Solomon, Gail

2012-10-31

Therapeutic intervention in the pathophysiology of airway mucus hypersecretion is clinically important. Several types of drugs are available with different possible modes of action. We examined the effects of guaifenesin (GGE), N-acetylcysteine (NAC) and ambroxol (Amb) on differentiated human airway epithelial cells stimulated with IL-13 to produce additional MUC5AC. After IL-13 pre-treatment (3 days), the cultures were treated with GGE, NAC or Amb (10-300 μM) in the continued presence of IL-13. Cellular and secreted MUC5AC, mucociliary transport rates (MTR), mucus rheology at several time points, and the antioxidant capacity of the drugs were assessed. IL-13 increased MUC5AC content (~25%) and secretion (~2-fold) and decreased MTR, but only slightly affected the G' (elastic) or G" (viscous) moduli of the secretions. GGE significantly inhibited MUC5AC secretion and content in the IL-13-treated cells in a concentration-dependent manner (IC50s at 24 hr ~100 and 150 μM, respectively). NAC or Amb were less effective. All drugs increased MTR and decreased G' and G" relative to IL-13 alone. Cell viability was not affected and only NAC exhibited antioxidant capacity. Thus, GGE effectively reduces cellular content and secretion of MUC5AC, increases MTR, and alters mucus rheology, and may therefore be useful in treating airway mucus hypersecretion and mucostasis in airway diseases.

Effects of guaifenesin, N-acetylcysteine, and ambroxol on MUC5AC and mucociliary transport in primary differentiated human tracheal-bronchial cells

PubMed Central

2012-01-01

Background Therapeutic intervention in the pathophysiology of airway mucus hypersecretion is clinically important. Several types of drugs are available with different possible modes of action. We examined the effects of guaifenesin (GGE), N-acetylcysteine (NAC) and ambroxol (Amb) on differentiated human airway epithelial cells stimulated with IL-13 to produce additional MUC5AC. Methods After IL-13 pre-treatment (3 days), the cultures were treated with GGE, NAC or Amb (10–300 μM) in the continued presence of IL-13. Cellular and secreted MUC5AC, mucociliary transport rates (MTR), mucus rheology at several time points, and the antioxidant capacity of the drugs were assessed. Results IL-13 increased MUC5AC content (~25%) and secretion (~2-fold) and decreased MTR, but only slightly affected the G’ (elastic) or G” (viscous) moduli of the secretions. GGE significantly inhibited MUC5AC secretion and content in the IL-13-treated cells in a concentration-dependent manner (IC50s at 24 hr ~100 and 150 μM, respectively). NAC or Amb were less effective. All drugs increased MTR and decreased G’ and G” relative to IL-13 alone. Cell viability was not affected and only NAC exhibited antioxidant capacity. Conclusions Thus, GGE effectively reduces cellular content and secretion of MUC5AC, increases MTR, and alters mucus rheology, and may therefore be useful in treating airway mucus hypersecretion and mucostasis in airway diseases. PMID:23113953

Suppression of meiosis of male germ cells by an antagonist of luteinizing hormone-releasing hormone.

PubMed Central

Szende, B; Redding, T W; Schally, A V

1990-01-01

Male nude mice were implanted with osmotic minipumps releasing 50 micrograms of a potent antagonist of luteinizing hormone-releasing hormone (LH-RH) per day [N-Ac-[D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH] (SB-75) [Nal(2), 3-(2-naphthyl)alanine; Phe(pCl), 4-chlorophenylalanine; Pal(3), 3-(3-pyridyl)alanine; Cit, citrulline], or they were treated with s.c. injections of SB-75 (25 micrograms twice a day). Another group of nude mice received an injection of microcapsules of the agonist [D-Trp6]LH-RH liberating 25 micrograms/day. One month after the initiation of treatment, the testicular weights were significantly reduced and the blood testosterone values were at castration levels in all treated groups. Histologically, only the testicles of the mice treated with SB-75 released from minipumps showed a significant decrease of meiosis. The most advanced forms of germ cells were spermatogonia in 26%, spermatocytes in 17%, and round spermatids in 35% of the seminiferous tubules. Only 22% of the tubules contained elongated spermatids. The suppression of meiotic activity by this modern LH-RH antagonist can possibly be used for the development of methods for male contraception and for the protection of germ cells against the damage caused by cytotoxic drugs and x-radiation. Images PMID:2405399

Histone Deacetylase Inhibitor Improves the Development and Acetylation Levels of Cat–Cow Interspecies Cloned Embryos

PubMed Central

Wittayarat, Manita; Sato, Yoko; Do, Lanh Thi Kim; Morita, Yasuhiro; Chatdarong, Kaywalee; Techakumphu, Mongkol; Taniguchi, Masayasu

2013-01-01

Abstract Abnormal epigenetic reprogramming, such as histone acetylation, might cause low efficiency of interspecies somatic cell nuclear transfer (iSCNT). This study was conducted to evaluate the effects of trichostatin A (TSA) on the developmental competence and histone acetylation of iSCNT embryos reconstructed from cat somatic cells and bovine cytoplasm. The iSCNT cat and parthenogenetic bovine embryos were treated with various concentrations of TSA (0, 25, 50, or 100 nM) for 24 h, respectively, following fusion and activation. Treatment with 50 nM TSA produced significantly higher rates of cleavage and blastocyst formation (84.3% and 4.6%, respectively) of iSCNT embryos than the rates of non-TSA–treated iSCNT embryos (63.8% and 0%, respectively). Similarly, the treatment of 50 nM TSA increased the blastocyst formation rate of parthenogenetic bovine embryos. The acetylation levels of histone H3 lysine 9 (H3K9) in the iSCNT embryos with the treatment of 50 nM TSA were similar to those of in vitro–fertilized embryos and significantly higher (p<0.05) than those of non-TSA–treated iSCNT embryos (control), irrespective of the embryonic development stage (two-cell, four-cell, and eight-cell stages). These results indicated that the treatment of 50 nM TSA postfusion was beneficial for development to the blastocyst stage of iSCNT cat embryos and correlated with the increasing levels of acetylation at H3K9. PMID:23790014

Novel Small Molecule Inhibitor of Tyk2: Lucrative Therapeutic Target in Lupus

DTIC Science & Technology

2017-09-01

collected and analyzed by ELISA . mg/kg of SAR20351 (n=12) or vehicle respectively (n=8) for each group. The animals were treated for a total of six...populations in either of the mouse strains. We also performed ELISA on serum collected from these animals at the end of the 6-week study and found...was collected and analyzed by ELISA . MRL/lpr mice is genetically deficient in Fas receptor where immune cells fail to undergo apoptosis and results

Silibinin Inhibits Platelet-Derived Growth Factor-Driven Cell Proliferation via Downregulation of N-Glycosylation in Human Tenon's Fibroblasts in a Proteasome-Dependent Manner.

PubMed

Chen, Yi-Hao; Chen, Ching-Long; Lu, Da-Wen; Liang, Chang-Min; Tai, Ming-Cheng; Chen, Jiann-Torng

2016-01-01

The objective of this study was to evaluate the effects of silibinin on cell proliferation in platelet-derived growth factor (PDGF)-treated human Tenon's fibroblasts (HTFs). The effect of silibinin on cell proliferation in PDGF-treated HTFs was determined by examining the expression of proliferating cell nuclear antigen (PCNA) and performing WST-1 assays. Cell cycle progression was evaluated using flow cytometry. The related cyclins and cyclin-dependent kinases (CDKs) were also analyzed using western blot. A modified rat trabeculectomy model was established to evaluate the effect of silibinin on cell proliferation in vivo. Western blot analysis was carried out to determine the effect of silibinin on the expression of PDGF receptor and on the downstream signaling pathways regulated by PDGF receptor. PDGF elevated the expression of PCNA in HTFs, and this elevation was inhibited by silibinin. The inhibitory effect of silibinin on cell proliferation was also confirmed via WST-1 assay. PDGF-stimulated cell cycle in HTFs was delayed by silibinin, and the related cyclin D1 and CDK4 were also suppressed by silibinin. In the rat model of trabeculectomy, silibinin reduced the expression of PCNA at the site of blebs in vivo. The effects of silibinin on PDGF-stimulated HTFs were mediated via the downregulation of PDGF receptor-regulated signaling pathways, such as ERKs and STATs, which may be partially caused by the downregulation of N-glycosylation of PDGF receptor beta (PDGFRβ). The effect of silibinin on modulation of N-glycosylation of PDGFRβ was mediated in a proteasome-dependent manner. Silibinin inhibited cell proliferation and delayed cell cycle progression in PDGF-treated HTFs in vitro. PDGF also modulated the process of N-glycosylation of the PDGFRβ in a proteasome-dependent manner. Our findings suggest that silibinin has potential therapeutic applications in glaucoma filtering surgery.

Silibinin Inhibits Platelet-Derived Growth Factor-Driven Cell Proliferation via Downregulation of N-Glycosylation in Human Tenon's Fibroblasts in a Proteasome-Dependent Manner

PubMed Central

Chen, Yi-Hao; Chen, Ching-Long; Lu, Da-Wen; Liang, Chang-Min; Tai, Ming-Cheng; Chen, Jiann-Torng

2016-01-01

The objective of this study was to evaluate the effects of silibinin on cell proliferation in platelet-derived growth factor (PDGF)-treated human Tenon's fibroblasts (HTFs). The effect of silibinin on cell proliferation in PDGF-treated HTFs was determined by examining the expression of proliferating cell nuclear antigen (PCNA) and performing WST-1 assays. Cell cycle progression was evaluated using flow cytometry. The related cyclins and cyclin-dependent kinases (CDKs) were also analyzed using western blot. A modified rat trabeculectomy model was established to evaluate the effect of silibinin on cell proliferation in vivo. Western blot analysis was carried out to determine the effect of silibinin on the expression of PDGF receptor and on the downstream signaling pathways regulated by PDGF receptor. PDGF elevated the expression of PCNA in HTFs, and this elevation was inhibited by silibinin. The inhibitory effect of silibinin on cell proliferation was also confirmed via WST-1 assay. PDGF-stimulated cell cycle in HTFs was delayed by silibinin, and the related cyclin D1 and CDK4 were also suppressed by silibinin. In the rat model of trabeculectomy, silibinin reduced the expression of PCNA at the site of blebs in vivo. The effects of silibinin on PDGF-stimulated HTFs were mediated via the downregulation of PDGF receptor-regulated signaling pathways, such as ERKs and STATs, which may be partially caused by the downregulation of N-glycosylation of PDGF receptor beta (PDGFRβ). The effect of silibinin on modulation of N-glycosylation of PDGFRβ was mediated in a proteasome-dependent manner. Silibinin inhibited cell proliferation and delayed cell cycle progression in PDGF-treated HTFs in vitro. PDGF also modulated the process of N-glycosylation of the PDGFRβ in a proteasome-dependent manner. Our findings suggest that silibinin has potential therapeutic applications in glaucoma filtering surgery. PMID:28030611

Regulation of GnRH I receptor gene expression by the GnRH agonist triptorelin, estradiol, and progesterone in the gonadotroph-derived cell line alphaT3-1.

PubMed

Weiss, J M; Polack, S; Treeck, O; Diedrich, K; Ortmann, O

2006-08-01

The secretion of luteinizing hormone (LH) and the GnRH receptor (GnRH-R) concentration are modulated by ovarian steroids and GnRH. To elucidate whether this regulation is due to alterations at the transcriptional level, we examined the GnRH I-R mRNA expression in the gonadotroph-derived cell line alphaT3-1 treated with different estradiol and progesterone paradigms and the GnRH I agonist triptorelin. alphaT3-1 cells were treated with different steroid paradigms: 1 nM estradiol or 100 nM progesterone for 48 h alone or in combination. Cells were exposed to 10 nM or 100 pM triptorelin for 30 min, 3 h, 9 h, or, in pulsatile way, with a 5-min pulse per hour. The GnRH I-R mRNA was determined by Northern blot analysis. GnRH I-R mRNA from cells treated with continuous triptorelin decreased in a time- and concentration-dependent manner. Pulsatile triptorelin increased GnRH I-R gene expression. Progesterone alone further enhanced this effect, whereas estradiol and its combination with progesterone diminished it. Continuous combined treatment with estradiol and progesterone lead to a significant decrease of GnRH I-R mRNA by 30% and by 35% for estradiol alone. The addition of 10 nM triptorelin for 30 min or 3 h could not influence that steroid effect. In conclusion, estradiol and progesterone exclusively decreased GnRH I-R mRNA in alphaT3-1 cells no matter whether they are treated additionally with the GnRH I agonist triptorelin. The enhanced sensitivity of gonadotrophs and GnRH I-R upregulation by estradiol is not due to increased GnRH I gene expression because GnRH I-R mRNA is downregulated by estradiol and progesterone. Other pathways of the GnRH I-R signal transduction might be involved.

27-hydroxycholesterol and the expression of three estrogen-sensitive proteins in MCF7 cells.

PubMed

Cruz, Pamela; Epuñán, María José; Ramírez, María Eugenia; Torres, Cristian G; Valladares, Luis E; Sierralta, Walter D

2012-09-01

The principal aim of this study was to analyze in estrogen receptor-positive MCF7 cells the response of three estrogen-dependent proteins to 27-hydroxycholesterol (27OHC), a major circulating cholesterol metabolite. Immunofluorescence, immunoblotting and immunogold labelling analyses of MCF7 cells exposed for up to 72 h to 2 nM estradiol (E2) or to 2 µM 27OHC demonstrated similar responses in the expression of MnSOD and ERβ compared to the non-stimulated cells. Thus, the results confirm 27OHC's function as a novel selective estrogen receptor modulator (SERM). The epithelial to mesenchymal transition (EMT), observed in MCF7 cells stimulated for longer than 48 h with 2 µM 27OHC, was accompanied by lower immunoreactive levels of nuclear FOXM1 in comparison to E2-treated cells. The results presented in this study are discussed taking into consideration the relationship of hypercholesterolemia, 27OHC production, ROS synthesis and macrophage infiltration, potentially occurring in obese patients with ERα-positive, infiltrated mammary tumors.

Theaflavin-3,3'-digallate, a component of black tea: an inducer of oxidative stress and apoptosis.

PubMed

Schuck, Alyssa G; Ausubel, Miriam B; Zuckerbraun, Harriet L; Babich, Harvey

2008-04-01

Treatment of human oral squamous carcinoma HSC-2 cells and normal GN46 fibroblasts with theaflavin-3,3'-digallate (TF-3), a polyphenol in black tea, showed a concentration and time dependent inhibition of growth, with the tumor cells more sensitive than the fibroblasts. In buffer and in cell culture medium, TF-3 generated reactive oxygen species, with lower levels detected in buffer amended with catalase and superoxide dismutase, indicating the generation of hydrogen peroxide and superoxide, respectively, and suggesting that TF-3 may be an inducer of oxidative stress. The toxicity of TF-3 was decreased in the presence of catalase, pyruvate, and divalent cobalt, all scavengers of reactive oxygen species, but was potentiated in the presence of diethyldithiocarbamate, an inhibitor of superoxide dismutase. The intracellular level of glutathione in HSC-2 cells was lessened after a 4-h exposure to 250 and 500 microM TF-3. However, for GN46 fibroblasts, a 4-h exposure to 250 microM TF-3 stimulated, but to 500 microM TF-3 lessened, intracellular glutathione. Treatment of the cells with the glutathione depleters, 1,3-bis(2-chloroethyl)-N-nitrosourea, 1-chloro-2,4-dinitrobenzene, and d,l-buthionine-[S,R]-sulfoximine potentiated the toxicity of TF-3. Induction of apoptotic cell death in HSC-2 cells treated with TF-3 was noted by apoptotic cell morphologies, by TUNEL staining, by PARP cleavage, and by elevated activity of caspase-3. Apoptosis was not noted in GN46 fibroblasts treated with TF-3.

Safety and Efficacy of the Intravenous Infusion of Umbilical Cord Mesenchymal Stem Cells in Patients With Heart Failure: A Phase 1/2 Randomized Controlled Trial (RIMECARD Trial [Randomized Clinical Trial of Intravenous Infusion Umbilical Cord Mesenchymal Stem Cells on Cardiopathy]).

PubMed

Bartolucci, Jorge; Verdugo, Fernando J; González, Paz L; Larrea, Ricardo E; Abarzua, Ema; Goset, Carlos; Rojo, Pamela; Palma, Ivan; Lamich, Ruben; Pedreros, Pablo A; Valdivia, Gloria; Lopez, Valentina M; Nazzal, Carolina; Alcayaga-Miranda, Francisca; Cuenca, Jimena; Brobeck, Matthew J; Patel, Amit N; Figueroa, Fernando E; Khoury, Maroun

2017-10-27

Umbilical cord-derived mesenchymal stem cells (UC-MSC) are easily accessible and expanded in vitro, possess distinct properties, and improve myocardial remodeling and function in experimental models of cardiovascular disease. Although bone marrow-derived mesenchymal stem cells have been previously assessed for their therapeutic potential in individuals with heart failure and reduced ejection fraction, no clinical trial has evaluated intravenous infusion of UC-MSCs in these patients. Evaluate the safety and efficacy of the intravenous infusion of UC-MSC in patients with chronic stable heart failure and reduced ejection fraction. Patients with heart failure and reduced ejection fraction under optimal medical treatment were randomized to intravenous infusion of allogenic UC-MSCs (Cellistem, Cells for Cells S.A., Santiago, Chile; 1×10 6 cells/kg) or placebo (n=15 per group). UC-MSCs in vitro, compared with bone marrow-derived mesenchymal stem cells, displayed a 55-fold increase in the expression of hepatocyte growth factor, known to be involved in myogenesis, cell migration, and immunoregulation. UC-MSC-treated patients presented no adverse events related to the cell infusion, and none of the patients tested at 0, 15, and 90 days presented alloantibodies to the UC-MSCs (n=7). Only the UC-MSC-treated group exhibited significant improvements in left ventricular ejection fraction at 3, 6, and 12 months of follow-up assessed both through transthoracic echocardiography ( P =0.0167 versus baseline) and cardiac MRI ( P =0.025 versus baseline). Echocardiographic left ventricular ejection fraction change from baseline to month 12 differed significantly between groups (+7.07±6.22% versus +1.85±5.60%; P =0.028). In addition, at all follow-up time points, UC-MSC-treated patients displayed improvements of New York Heart Association functional class ( P =0.0167 versus baseline) and Minnesota Living with Heart Failure Questionnaire ( P <0.05 versus baseline). At study completion, groups did not differ in mortality, heart failure admissions, arrhythmias, or incident malignancy. Intravenous infusion of UC-MSC was safe in this group of patients with stable heart failure and reduced ejection fraction under optimal medical treatment. Improvements in left ventricular function, functional status, and quality of life were observed in patients treated with UC-MSCs. URL: https://www.clinicaltrials.gov/ct2/show/NCT01739777. Unique identifier: NCT01739777. © 2017 The Authors.

Improving the photovoltaic performance of the all-solid-state TiO2 NR/CuInS2 solar cell by hydrogen plasma treatment.

PubMed

Chen, Bingfeng; Niu, Wenzhe; Lou, Zirui; Ye, Zhizhen; Zhu, Liping

2018-07-06

The interfacial properties of the heterojunction between p-type and n-type materials play an important role in the performance of the solar cell. In this paper, a p-type CuInS 2 film was deposited on TiO 2 nanorod arrays by spin coating to fabricate an all-solid-state solar cell and the TiO 2 nanorod arrays were treated with hydrogen plasma(H:TiO 2 ) to ameliorate the interfacial properties. The influence of the hydrogen plasma treatment on the performance of the solar cell was investigated. The short-circuit current density was obviously raised and the power conversion efficiency of the solar cell improved to 0.30%, which is three times that of solar cells without hydrogen plasma treatment. The enhancement of the performance is attributed to not only the enhancement of carrier separation and transport, but the reduction of the recombination of electrons and holes, which is caused by hydrogen plasma treatment.

Improving the photovoltaic performance of the all-solid-state TiO2 NR/CuInS2 solar cell by hydrogen plasma treatment

NASA Astrophysics Data System (ADS)

Chen, Bingfeng; Niu, Wenzhe; Lou, Zirui; Ye, Zhizhen; Zhu, Liping

2018-07-01

The interfacial properties of the heterojunction between p-type and n-type materials play an important role in the performance of the solar cell. In this paper, a p-type CuInS2 film was deposited on TiO2 nanorod arrays by spin coating to fabricate an all-solid-state solar cell and the TiO2 nanorod arrays were treated with hydrogen plasma(H:TiO2) to ameliorate the interfacial properties. The influence of the hydrogen plasma treatment on the performance of the solar cell was investigated. The short-circuit current density was obviously raised and the power conversion efficiency of the solar cell improved to 0.30%, which is three times that of solar cells without hydrogen plasma treatment. The enhancement of the performance is attributed to not only the enhancement of carrier separation and transport, but the reduction of the recombination of electrons and holes, which is caused by hydrogen plasma treatment.

Patterns of care and outcomes for stage IIIB non-small cell lung cancer in the TNM-7 era: Results from the Netherlands Cancer Registry.

PubMed

Dickhoff, C; Dahele, M; Smit, E F; Paul, M A; Senan, S; Hartemink, K J; Damhuis, R A

2017-08-01

There is limited data on the pattern of care for locally advanced, clinical (c) IIIB non-small cell lung cancer (NSCLC) in the TNM-7 staging era. The primary aim of this study was to investigate national patterns of care and outcomes in the Netherlands, with a secondary focus on the use of surgery. Data from patients treated for TNM-7 cIIIB NSCLC between 2010 and 2014, was extracted from the Netherlands Cancer Registry (NCR). Survival data was obtained from the automated Civil Registry. 43.762 patients with NSCLC were recorded in the NCR during this 5-year period, with cIIIB accounting for 10% (n=4.401). Clinical N2 (37%) and N3 (63%) nodal involvement was pathologically confirmed in 50.8%. The use of endobronchial ultrasound (EBUS) increased with time from 9% to 29% (p<0.001), while the rate of pathological confirmation of N2 or N3 nodes increased from 44% to 54% (p<0.001). 48% of patients received chemoradiotherapy (CRT), 19% chemotherapy (CT), RT in 10% and surgery in 2.2%. 22% received best supportive care (BSC). The percentage of patients treated with CRT decreased from 65% for patients aged <60 years to 13% for patients aged 80 years or older. Overall survival for surgery was 28 months, followed by CRT (19mths), CT (9mths), RT (8mths) and BSC (3mths). In the Netherlands, CRT is the most frequent treatment for cIIIB NSCLC in the TNM-7 era. The use of surgery is limited. Accurate staging requires specific attention and the scarce use of radical treatment in elderly patients merits further evaluation. Copyright © 2017 Elsevier B.V. All rights reserved.

Gravity, an Regulation Factor in BMSCs Differentiation to osteoblasts

NASA Astrophysics Data System (ADS)

Yan, Huang; Yinghui, Li; Fen, Yang; Zhongquan, Dai

PURPOSE Most studies of regulatory mechanisms of adult stem cell differentiation are concentrated in chemical factors but few efforts are put into physical factors Recent space life science studies indicate mechanical factors participate in the differentiation of cells The aim of this study is to investigate the effects of simulated microgravity or hypergravity on the osteogenic differentiation of rat bone marrow mesenchymal stem cells BMSCs METHODOLOGY The BMSCs at day 7 were added osteogenic inducer 10nM dexamethasone 10mM beta -glycerophosphate and 50 mu M asorbic acid-2-phosphate for 7 days and cultured under simulated microgravity or hypergravity 2g for 1 day 3 days 5 days or 7 days RESULTS After treating BMSCs with osteogenic inducer and hypergravity the cells expressed more ColIA1 Cbfa1 and ALP than in single steogenic inducer treatment Reversely the cells treated with osteogenic inducer and simulated microgravity expressed less ColIA1 Cbfa1 and ALP CONCLUSIONS Our study suggests that hypergravity promotes the osteogenic differentiation of BMSCs and simulated microgravity inhibits this process Gravity is an important regulation factor in BMSCs differentiation to osteoblasts

Exclusive radiotherapy for stage T1-T2N0M0 lanryngeal cancer: retrospective study of 59 patients at CFB and CHU de Caen.

PubMed

Cuny, F; Géry, B; Florescu, C; Clarisse, B; Blanchard, D; Rame, J-P; Babin, E; De Raucourt, D

2013-11-01

Study of patients with stage T1N0M0 or T2N0M0 glottic cancer treated by exclusive radiotherapy and comparison of the survival and functional results of this series with those of the literature. Retrospective study of stage T1N0M0 or T2N0M0 glottic cancers diagnosed between 1st January 2000 and 31st December 2010 and treated by exclusive radiotherapy. Evaluation of survival, recurrence and larynx preservation rates. CLCC François-Baclesse and CHU de Caen. Fifty-nine patients (53 men and sixwomen) treated for glottic cancer (57 squamous cell carcinomas, two verrucous carcinomas) comprising 51 T1N0M0 and eight T2N0M0 tumours. Treatment with exclusive radiotherapy (mean dose of 70 Grays limited to the thyroid cartilage for 57 patients, with lymph node irradiation for two patients). In this series, five (9.8%) patients with stage T1N0M0 glottic cancer and three patients (37.5%) with stage T2N0M0 glottic cancer relapsed, corresponding to a global recurrence rate of 13.6%. Three of the eight recurrences involved lymph nodes exclusively (N), two patients relapsed exclusively at the primary tumour site (T) and three patients presented local and lymph node recurrence (T and N). Treatment consisted of salvage total laryngectomy with bilateral cervical lymph node dissection in three cases, bilateral cervical lymph node dissection and sensitized radiotherapy in two cases, exclusive chemotherapy in one case, cervical lymph node dissection and cervical radiotherapy in one case. The last patient with recurrence died prior to salvage therapy. The larynx preservation rate was 94.9%. In comparison with the literature, treatment of stage T1-T2N0M0 glottic cancer by exclusive radiotherapy gives very good results, with a larynx preservation rate of 95%. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Single cell kinase signaling assay using pinched flow coupled droplet microfluidics.

PubMed

Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S; Tan Shao Weng, Daniel; Thakor, Nitish V; Teck Lim, Chwee; Chen, Chia-Hung

2014-05-01

Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib.

Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS.

PubMed

Zhang, Hong-Hai; Lechuga, Thomas J; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

2016-05-01

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-(12)C6 (14)N4-Arg and L-(12)C6 (14)N2-Lys) or "heavy" (L-(13)C6 (15)N4-Arg and L-(13)C6 (15)N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. © 2016 by the Society for the Study of Reproduction, Inc.

Efficacy of icotinib in lung squamous-cell cancer: A real-world experience from single institution.

PubMed

Xu, Jianping; Liu, Xiaoyan; Yang, Sheng; Zhang, Xiangru; Shi, Yuankai

2017-12-01

Squamous cell carcinoma is a less common type of nonsmall cell lung cancer (NSCLC) which associates with a poor clinical prognosis and lacks specific therapy. This study aimed to evaluate the efficacy and safety of icotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has proven to be effective in EGFR-mutated NSCLC, in patients with lung squamous-cell cancer. Retrospective analysis was conducted in patients who had advanced lung squamous-cell cancer confirmed by cytology or histology. Patients were treated orally with icotinib (125 mg, three times daily) until event of unacceptable toxicity, disease progression or death. The primary endpoint was overall survival. The secondary endpoints were progression-free survival, overall response rate and disease control rate. Between January 2014 and May 2016, 20 patients were enrolled and evaluated for the efficacy and safety of icotinib. Overall, the median overall survival and progression-free survival were 9.93 months (95% confidence interval (CI): 3.46-16.40) and 3.0 months (95% CI: 0.00-8.35), respectively. The overall response rate and disease control rate were 20% and 70%, respectively. For treatment-naive patients (n = 11), the overall survival and progression-free survival were 9.93 months (95% CI: 0.00-23.49) and 6.27 months (95% CI: 0.00-12.61); the response rate and disease control rate were 27.3% and 54.5%, respectively. The overall survival and progression-free survival of patients treated with second- or multiple-line icotinib treatment (n = 9) were 6.5 months (95% CI: 0.80-12.20) and 1.2 months (95% CI: 1.10-1.30). A total of 11 patients experienced at least one treatment-related adverse event, most of which were mild to moderate. The most common manifestations were rash (n = 6, 30%) followed by diarrhea (n = 2, 10%). Icotinib has demonstrated a favorable efficacy and safety profile in patients with advanced lung squamous-cell cancer. © 2017 John Wiley & Sons Australia, Ltd.

Salt Sensitive Tet-Off-Like Systems to Knockdown Primordial Germ Cell Genes for Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus.

PubMed

Li, Hanbo; Su, Baofeng; Qin, Guyu; Ye, Zhi; Alsaqufi, Ahmed; Perera, Dayan A; Shang, Mei; Odin, Ramjie; Vo, Khoi; Drescher, David; Robinson, Dalton; Zhang, Dan; Abass, Nermeen; Dunham, Rex A

2017-05-31

Repressible knockdown approaches were investigated for transgenic sterilization in channel catfish, Ictalurus punctatus . Two primordial germ cell (PGC) marker genes, nanos and dead end , were targeted for knockdown, and an off-target gene, vasa , was monitored. Two potentially salt sensitive repressible promoters, zebrafish adenylosuccinate synthase 2 (ADSS) and zebrafish racemase (Rm), were each coupled with four knockdown strategies: ds-sh RNA targeting the 5' end (N1) or 3' end (N2) of channel catfish nanos , full-length cDNA sequence of channel catfish nanos for overexpression (cDNA) and ds-sh RNA targeting channel catfish dead end (DND). Each construct had an untreated group and treated group with sodium chloride as the repressor compound. Spawning rates of full-sibling P₁ fish exposed or not exposed to the constructs as treated and untreated embryos were 93% and 59%, respectively, indicating potential sterilization of fish and repression of the constructs. Although the mRNA expression data of PGC marker genes were inconsistent in P₁ fish, most F₁ individuals were able to downregulate the target genes in untreated groups and repress the knockdown process in treated groups. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but more data from F₂ or F₃ are needed for evaluation.

Patterns of Disease Recurrence Following Treatment of Oropharyngeal Cancer With Intensity Modulated Radiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garden, Adam S., E-mail: agarden@mdanderson.org; Dong, Lei; Morrison, William H.

Purpose: To report mature results of a large cohort of patients diagnosed with squamous cell carcinoma of the oropharynx who were treated with intensity modulated radiation therapy (IMRT). Methods and Materials: The database of patients irradiated at The University of Texas, M.D. Anderson Cancer Center was searched for patients diagnosed with oropharyngeal cancer and treated with IMRT between 2000 and 2007. A retrospective review of outcome data was performed. Results: The cohort consisted of 776 patients. One hundred fifty-nine patients (21%) were current smokers, 279 (36%) former smokers, and 337 (43%) never smokers. T and N categories and American Jointmore » Committee on Cancer group stages were distributed as follows: T1/x, 288 (37%); T2, 288 (37%); T3, 113 (15%); T4, 87 (11%); N0, 88(12%); N1/x, 140 (18%); N2a, 101 (13%); N2b, 269 (35%); N2c, 122 (16%); and N3, 56 (7%); stage I, 18(2%); stage II, 40(5%); stage III, 150(19%); and stage IV, 568(74%). Seventy-one patients (10%) presented with nodes in level IV. Median follow-up was 54 months. The 5-year overall survival, locoregional control, and overall recurrence-free survival rates were 84%, 90%, and 82%, respectively. Primary site recurrence developed in 7% of patients, and neck recurrence with primary site control in 3%. We could only identify 12 patients (2%) who had locoregional recurrence outside the high-dose target volumes. Poorer survival rates were observed in current smokers, patients with larger primary (T) tumors and lower neck disease. Conclusions: Patients with oropharyngeal cancer treated with IMRT have excellent disease control. Locoregional recurrence was uncommon, and most often occurred in the high dose volumes. Parotid sparing was accomplished in nearly all patients without compromising tumor coverage.« less

FGF-1-induced matrix metalloproteinase-9 expression in breast cancer cells is mediated by increased activities of NF-kappaB and activating protein-1.

PubMed

Lungu, Gina; Covaleda, Lina; Mendes, Odete; Martini-Stoica, Heidi; Stoica, George

2008-06-01

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor-1 (FGF-1) on the expression of MMP-9 in ENU1564, an ethyl-N-nitrosourea-induced rat mammary adenocarcinoma cell line. We observed that FGF-1 induces a dose-dependent increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP-9 regulation by FGF-1, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system since NF-kappaB and AP-1 transcription factor binding sites have been characterized in the upstream region of the MMP-9 gene. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-kappaBp65, and enhances degradation of cytoplasmic IkappaBalpha. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data show that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-kappaB and AP-1 in FGF-1-treated cells and that mutation of either NF-kappaB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1. Taken together, these results demonstrated that FGF-1-induced MMP-9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF-kappaB and AP-1 and involve activation of a dual signaling pathway, PI3K-Akt and MEK1/2-ERK. (c) 2007 Wiley-Liss, Inc.

CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery

PubMed Central

2012-01-01

Background Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model. Methods CD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery. Results The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs. Conclusion CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms. PMID:22647574

Salt Sensitive Tet-Off-Like Systems to Knockdown Primordial Germ Cell Genes for Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus

PubMed Central

Li, Hanbo; Su, Baofeng; Qin, Guyu; Ye, Zhi; Alsaqufi, Ahmed; Perera, Dayan A.; Shang, Mei; Odin, Ramjie; Vo, Khoi; Drescher, David; Robinson, Dalton; Zhang, Dan; Abass, Nermeen; Dunham, Rex A.

2017-01-01

Repressible knockdown approaches were investigated for transgenic sterilization in channel catfish, Ictalurus punctatus. Two primordial germ cell (PGC) marker genes, nanos and dead end, were targeted for knockdown, and an off-target gene, vasa, was monitored. Two potentially salt sensitive repressible promoters, zebrafish adenylosuccinate synthase 2 (ADSS) and zebrafish racemase (Rm), were each coupled with four knockdown strategies: ds-sh RNA targeting the 5′ end (N1) or 3′ end (N2) of channel catfish nanos, full-length cDNA sequence of channel catfish nanos for overexpression (cDNA) and ds-sh RNA targeting channel catfish dead end (DND). Each construct had an untreated group and treated group with sodium chloride as the repressor compound. Spawning rates of full-sibling P1 fish exposed or not exposed to the constructs as treated and untreated embryos were 93% and 59%, respectively, indicating potential sterilization of fish and repression of the constructs. Although the mRNA expression data of PGC marker genes were inconsistent in P1 fish, most F1 individuals were able to downregulate the target genes in untreated groups and repress the knockdown process in treated groups. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but more data from F2 or F3 are needed for evaluation. PMID:28561774

Therapy with granulocyte colony-stimulating factor in the chronic stage, but not in the acute stage, improves experimental autoimmune myocarditis in rats via nitric oxide.

PubMed

Shimada, Kana; Okabe, Taka-aki; Mikami, Yu; Hattori, Miki; Fujita, Masatoshi; Kishimoto, Chiharu

2010-09-01

We systematically investigated serial efficacy of granulocyte colony-stimulating factor (G-CSF) therapy upon experimental autoimmune myocarditis (EAM) in rats treated with and without the inhibition of nitric oxide (NO) with the analyses of tissue regeneration. G-CSF could mobilize multipotent progenitor cells of bone marrow into the peripheral blood and may improve ventricular function. A rat model of porcine myosin-induced EAM was used. After the immunization of myosin, G-CSF (10 microg/kg/day) or saline was injected intraperitoneally on days 0-21 in experiment 1 and on days 21-42 in experiment 2. Additional myosin-immunized rats were orally given 25 mg/kg/day of N(G)-nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide synthase (NOS), in each experiment (each group; n=8-21). Serum cytokines and peripheral blood cell counts were measured in each group. In experiment 1, G-CSF treatment aggravated cardiac pathology associated with increased macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) levels and enhanced superoxide production. In experiment 2, G-CSF treatment reduced the severity of myocarditis with increased capillary density and improved left ventricular ejection fraction. In the rats with EAM treated with G-CSF associated with oral L-NAME treatment in experiment 2, the severity of myocarditis was not reduced. Myocardial c-kit(+) cells were demonstrated only in G-CSF-treated group in experiment 2 but not in other groups. G-CSF has differential effects on EAM in rats associated with the modulation of cytokine network. The overwhelming superoxide production by G-CSF administration in the acute stage may worsen the disease. G-CSF therapy improved cardiac function via NO system in a rat model of myocarditis in the chronic stage, but not in the acute stage, possibly through the myocardial regeneration and acceleration of healing process. Copyright 2010 Elsevier Ltd. All rights reserved.

Intravenous adipose-derived mesenchymal stem cell therapy for the treatment of feline asthma: a pilot study.

PubMed

Trzil, Julie E; Masseau, Isabelle; Webb, Tracy L; Chang, Chee-Hoon; Dodam, John R; Liu, Hong; Quimby, Jessica M; Dow, Steven W; Reinero, Carol R

2016-12-01

The aim of this study was to evaluate the feasibility and efficacy of serially administered adipose-derived mesenchymal stem cells (MSCs) in an experimental feline asthma model. Allergic asthma was acutely induced with Bermuda grass allergen in six purpose-bred cats. Five intravenous infusions of allogeneic MSCs (n = 4; MSC-treated) or saline (n = 2; placebo-treated) were administered over the first 130 days after asthma induction. Infusions contained 2 × 10 6 , 4 × 10 6 , 4.7 × 10 6 , 1 × 10 7 and 1 × 10 7 cryopreserved MSCs/cat. For thoracic imaging additional cats were enrolled as control groups: four untreated, experimentally asthmatic cats (combined with placebo-treated cats), and six healthy, non-asthmatic cats. Outcome measures included airway eosinophilia, pulmonary mechanics, thoracic computed tomography and several immunologic assays. Cats were assessed for 9 months after treatment. At early points, airway eosinophil percentage was not affected by MSC administration (post-treatment average of days 12, 26, 47, 108 and 133 in MSC-treated cats was 41 ± 15% and in placebo-treated cats it was 34 ± 16%). By month 9, eosinophil percentages in all MSC-treated cats decreased to normal reference intervals (MSC-treated 6%; placebo-treated 20%; normal <17%). Diminished airway hyper-responsiveness was noted in all MSC-treated compared with placebo-treated cats at day 133 (dose of methacholine to double baseline airway resistance: MSC-treated median 22.9 mg/ml [range 6.4-64.0]; individual placebo-treated cats 1.1 and 5.0 mg/ml). Lung attenuation (mean ± SEM MSC-treated -865 ± 12 Hounsfield units [HU]; untreated asthmatics -820 ± 11 HU; P = 0.004) and bronchial wall thickening scores (median [interquartile range] MSC-treated 0 [0-1.5]; untreated asthmatic 11.6 [7.3-27.3]; P = 0.010) were significantly reduced in MSC-treated vs untreated asthmatic cats, consistent with decreased airway remodeling at month 9. No clear immunologic mechanisms by which MSCs act were determined. MSCs may have a delayed effect in reducing airway inflammation, airway hyper-responsiveness and remodeling in experimentally induced asthmatic cats. Results warrant additional investigation of MSC therapy for asthma in cats. © The Author(s) 2015.

Soybean agglutinin binding to corneal endothelial cell surfaces disrupts in situ monolayer integrity and actin organization and interferes with wound repair.

PubMed

Gordon, Sheldon R; Wood, Meredith

2009-03-01

Rat corneal endothelium demonstrates cell-surface soybean agglutinin (SBA) binding during organ-culture or injury. When organ-cultured in medium containing SBA, the endothelial monolayer is disrupted because of cell-cell and cell-matrix alterations. SBA binding disorganizes the circumferential microfilament bundles (CMBs), an effect that is partially prevented by phallacidin preincubation. This disruption is reversible if tissues are returned to standard culture medium. Serum heightens SBA binding, whereas puromycin prevents it. Neither actinomycin D nor alpha-amanitin inhibits SBA binding, suggesting that SBA-binding protein(s) may be post-transcriptionally regulated. During injury-induced cell migration in the presence of SBA, cellular processes are blunted and fail to extend significantly outward. By 72 h post-injury, cells of SBA-treated tissues repopulate the wound but demonstrate little association with neighboring cells. Cells migrating in the presence of N-acetylgalactosamine appear normal but also fail to reassociate with other cells in the jury zone. Immunofluorescent staining for ZO-1 reveals punctuate patterns in cells of control tissues, whereas neither SBA- nor N-acetylgalactosamine-treated tissues exhibit ZO-1 staining. Terminal N-acetylgalactosamine removal fails to affect cell morphology, actin organization, or migration but prevents lectin binding. Our results suggest that SBA binding reflects the synthesis of a stress-induced protein(s) that may play a role in reestablishing cell-cell relationships during monolayer reorganization following injury.

Treatment with sodium nitroprusside improves the endothelial function in aortic rings with endothelial dysfunction.

PubMed

Buzinari, Tereza Cristina; Oishi, Jorge Camargo; De Moraes, Thiago Francisco; Vatanabe, Izabela Pereira; Selistre-de-Araújo, Heloisa Sobreiro; Pestana, Cezar Rangel; Rodrigues, Gerson Jhonatan

2017-07-15

Verify if sodium nitroprusside (SNP) is able to improve endothelial function and if this effect is independent of nitric oxide (NO) release of the compound. Normotensive (2K) and hypertensive (2K-1C) wistar rats were used. Intact endothelium aortas were placed in a myograph and incubated with SNP: 0.1nM; 1nM or 10nM during 30min. Cumulative concentration-effect curves for acetylcholine (Ach) were realized to measure the relaxing capacity. Intracellular NO were measured (by DAF-2DA probe) in HUVEC treated with SNP 0.1nM or DETA/NO 0.1μM. The detection of intracellular superoxide radical (O 2 •- ) was obtained by using DHE probe. Treatment of 2K-1C aortic rings with SNP (0.1; 1.0 and 10nM) improved endothelium dependent relaxation induced by acetylcholine. This improvement induced by SNP was verified at the concentration of 0.1nM, which does not release NO, suggesting that this effect was not induced due to NO release by SNP compound. Besides, we show that the cell treatment with 0.1nM of SNP decreased the fluorescence intensity to DHE in cells stimulated with angiotensin II. These results indicate that SNP decreases the concentration of O 2 •- in HUVEC cells. The SNP at a concentration that does not release NO inside the cells is able to attenuate endothelial dysfunction. Acetylcholine (Ach) (PubChem CID:6060); angiotensin II human (Ang II) (PubChem CID: 16211177); diethylenetriamine/nitric oxide (DETA-NO) (PubChem CID 4518); dihydroethidium (DHE) (PubChem CID: 128682); phenylephrine (Phe) (PubChem CID: 5284443); sodium nitroprusside (SNP) (PubChem CID: 11963579); Thiazolyl Blue Tetrazolium Bromide (MTT) (PubChem CID: 64965); 4,5-diaminofluorescein diacetate (DAF-2DA); 4-hidroxy-Tempo (Tempol) (PubChem CID: 137994), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elgqvist, Joergen; Andersson, Hakan; Bernhardt, Peter

Purpose: To elucidate the therapeutic efficacy of {alpha}-radioimmunotherapy of ovarian cancer in mice. This study: (i) estimated the minimum required activity (MRA), giving a reasonable high therapeutic efficacy; and (ii) calculated the specific energy to tumor cell nuclei and the metastatic cure probability (MCP) using various assumptions regarding monoclonal-antibody (mAb) distribution in measured tumors. The study was performed using the {alpha}-particle emitter Astatine-211 ({sup 211}At) labeled to the mAb MX35 F(ab'){sub 2}. Methods and Materials: Animals were inoculated intraperitoneally with {approx}1 x 10{sup 7} cells of the cell line NIH:OVCAR-3. Four weeks later animals were treated with 25, 50, 100,more » or 200 kBq {sup 211}At-MX35 F(ab'){sub 2} (n = 74). Another group of animals was treated with a nonspecific mAb: 100 kBq {sup 211}At-Rituximab F(ab'){sub 2} (n = 18). Eight weeks after treatment the animals were sacrificed and presence of macro- and microscopic tumors and ascites was determined. An MCP model was developed and compared with the experimentally determined tumor-free fraction (TFF). Results: When treatment was given 4 weeks after cell inoculation, the TFFs were 25%, 22%, 50%, and 61% after treatment with 25, 50, 100, or 200 kBq {sup 211}At-MX35 F(ab'){sub 2}, respectively, the specific energy to irradiated cell nuclei varying between {approx}2 and {approx}400 Gy. Conclusion: As a significant increase in the therapeutic efficacy was observed between the activity levels of 50 and 100 kBq (TFF increase from 22% to 50%), the conclusion was that the MRA is {approx}100 kBq {sup 211}At-MX35 F(ab'){sub 2}. MCP was most consistent with the TFF when assuming a diffusion depth of 30 {mu}m of the mAbs in the tumors.« less

[Mechanisms of (2-methyl-n-butyl) shikonin induced apoptosis of gastric cancer SGC-7901 cells].

PubMed

Wang, Hai-Bing; Ma, Xiao-Qiong

2012-06-01

This study is to investigate the effect of (2-methyl-n-butyl) shikonin (MBS) on inducing apoptosis of human gastric cancer cell line SGC-7901 and the role of ERK1/2 signal pathway in the apoptosis. MTT assay was used to detect SGC-7901 cell proliferation. DNA condensation was measured by DAPI stain. Cell apoptosis was analyzed by flow cytometry. Mitochondrial membrane potential (MMP) was analyzed by JC-1 staining. The protein expressions of Bcl-2, Bax, Survivin, cleaved caspase-9, cleaved caspase-3, cleaved PARP, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38 and p38 were detected by Western blotting. The results showed that MBS reduced the cell viability of SGC-7901 cells in a dose- and time-dependent manner. The IC50 at 24 h and 48 h for SGC-7901 cells was 10.113 and 4.196 micromolL(-1), respectively. After being treated with MBS, the typical nuclear condensation was observed in SGC-7901 cells by DAPI stain. Apoptosis in SGC-7901 cells was induced by MBS in a dose dependent manner. The protein expression of Bcl-2 was down-regulated, while the protein expressions of cleaved caspase-9, cleaved caspase-3, cleaved PARP, p-ERK1/2 and p-JNK were up-regulated after MBS treatment. U0126, a specific MAP kinase (MEK1/2) inhibitor, blocked the ERK1/2 activation by MBS. MMP was decreased by MBS treatment. It can be concluded that MBS could inhibit SGC-7901 cell proliferation and induce apoptosis. Mitochondrial apoptosis pathway, ERK1/2 signal pathway and JNK signal pathway might be involved in this process.

Ammonia and greenhouse gas emissions from a subtropical wheat field under different nitrogen fertilization strategies.

PubMed

Liu, Shuai; Wang, Jim J; Tian, Zhou; Wang, Xudong; Harrison, Stephen

2017-07-01

Minimizing soil ammonia (NH 3 ) and nitrous oxide (N 2 O) emission factors (EFs) has significant implications in regional air quality and greenhouse gas (GHG) emissions besides nitrogen (N) nutrient loss. The aim of this study was to investigate the impacts of different N fertilizer treatments of conventional urea, polymer-coated urea, ammonia sulfate, urease inhibitor (NBPT, N-(n-butyl) thiophosphoric triamide)-treated urea, and nitrification inhibitor (DCD, dicyandiamide)-treated urea on emissions of NH 3 and GHGs from subtropical wheat cultivation. A field study was established in a Cancienne silt loam soil. During growth season, NH 3 emission following N fertilization was characterized using active chamber method whereas GHG emissions of N 2 O, carbon dioxide (CO 2 ), and methane (CH 4 ) were by passive chamber method. The results showed that coated urea exhibited the largest reduction (49%) in the EF of NH 3 -N followed by NBPT-treated urea (39%) and DCD-treated urea (24%) over conventional urea, whereas DCD-treated urea had the greatest suppression on N 2 O-N (87%) followed by coated urea (76%) and NBPT-treated urea (69%). Split fertilization of ammonium sulfate-urea significantly lowered both NH 3 -N and N 2 O-N EF values but split urea treatment had no impact over one-time application of urea. Both NBPT and DCD-treated urea treatments lowered CO 2 -C flux but had no effect on CH 4 -C flux. Overall, application of coated urea or urea with NPBT or DCD could be used as a mitigation strategy for reducing NH 3 and N 2 O emissions in subtropical wheat production in Southern USA. Copyright © 2017. Published by Elsevier B.V.

[Evaluation of selected endocrine complications in patients treated with auto- and allo-haematopoietic stem cell transplantation].

PubMed

Niedzielska, Ewa; Wójcik, Dorota; Barg, Ewa; Pietras, Wojciech; Sega-Pondel, Dorota; Doroszko, Adrian; Niedzielska, Małgorzata; Skarzyńska, Małgorzata; Chybicka, Alicja

2008-01-01

The aim of this study was to evaluate the endocrine complications, in particular disorders of growth and thyroid function and glucose metabolism dysfunctions in patients treated with allo- and auto-haematopoietic stem cell transplantation (HSCT). The investigated group consisted of: I. 16 patients after auto-HSCT (6 girls, 10 boys) aged 3-20 years (average 10,8+/-) because of acute myelogenous leukaemia (n=5), non Hodgkin lymphoma (n=3), neuroblastoma (n=3), embryonal cancer (n=2), medulloblastoma (n=1), Ewing's sarcoma/PNET (n=1), hyper eosinophilic syndrome (n=1). High dose chemiotherapy (HDC/T) included: BU/MEL (busulfan/melfalan) (n=7), BEAM (carmustine, eteposide, cytosine arabinose, melfalan) (n=3). II. 30 patients after allo-HSCT (20 girls, 10 boys) aged 3-17 years (average 9,56). Indication for HSCT was acute lymphoblastic leukaemia (n=11), acute myelogenous leukaemia (n=5), chronic myeloid leukaemia-CML (n=6), myelodysplastic syndromes (n=2), non Hodgkin lymphoma (n=1), juvenile myelomonocytic leukemia (n=1), severe aplastic anaemia (n=1), Blackfan-Diamond anaemia (n=1), severe combined immune deficiency (n=1), rhabdomyosarcoma (n=1). The patients underwent the following types of transplantation: HSCT of matched sibling donor (n=13), HSCT of matched unrelated donor (n=11) and HLA-mismatched related donor (n=6). The preparative regimens consisted of HDC/T usually BU/MEL (n=3); BU/CY/VP (busulfan, cyclophosphamide, etoposide) (6); BU/CY/ATG (anti-thymocyte globulin) (n=5), VP/ATG/TBI (total body irradiation) (n=3). 19 children received CI (cranial irradiation) prior to grafting: auto-HSCT (n=6) and allo-HSCT (n=13) and 6 patients underwent TBI. 18 children received high steroid doses at least 28 days before transplant, 4 patients in the auto-HSCT group, and in the allo-HSCT group 14 patients before and 20 after HSCT procedure. The analysis of thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), free thyroxine (fT4), insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein 3 (IGFBP-3), hemoglobin A1c (HbA1c), prolactine (PRL), oral glucose tolerance test, growth hormone (GH test) and thyrotropin releasing hormone (TRH) test was performed in each case. Hypothyroidism was found in 5 patients (3 after allo-HSCT, 2 after auto-HSCT). Thyroid hormone substitution was applied. No case of hyperthyroidism was diagnosed. Growth deficit was found in 8 patients (6 girls, 2 boys) between 13 to 70 months after allo-transplantation (average 36 months). Three children from the above group received CI. Growth hormone substitution was applied in 1 girl (ALL, HLA MM REL, CI). An impaired excretion of GH after stimulation was diagnosed in 14 pts (10 after allo-HSCT, 4 after auto-HSCT). The growth process should still be observed in this subgroup. Glucose intolerance was found in 7 patients: in 4 treated with auto-HSCT and in 3 after allo-HSCT. Diabetes mellitus was diagnosed in none of them. An impaired glucose tolerance curve with increased excretion of insulin was diagnosed in 12 children. Early endocrinological care is necessary in patients treated both with auto-HSCT and allo-HSCT due to high risk of hormonal disorders.

Induction chemoradiation in stage IIIA/N2 non-small-cell lung cancer: a phase 3 randomised trial.

PubMed

Pless, Miklos; Stupp, Roger; Ris, Hans-Beat; Stahel, Rolf A; Weder, Walter; Thierstein, Sandra; Gerard, Marie-Aline; Xyrafas, Alexandros; Früh, Martin; Cathomas, Richard; Zippelius, Alfred; Roth, Arnaud; Bijelovic, Milorad; Ochsenbein, Adrian; Meier, Urs R; Mamot, Christoph; Rauch, Daniel; Gautschi, Oliver; Betticher, Daniel C; Mirimanoff, René-Olivier; Peters, Solange

2015-09-12

One of the standard options in the treatment of stage IIIA/N2 non-small-cell lung cancer is neoadjuvant chemotherapy and surgery. We did a randomised trial to investigate whether the addition of neoadjuvant radiotherapy improves outcomes. We enrolled patients in 23 centres in Switzerland, Germany and Serbia. Eligible patients had pathologically proven, stage IIIA/N2 non-small-cell lung cancer and were randomly assigned to treatment groups in a 1:1 ratio. Those in the chemoradiotherapy group received three cycles of neoadjuvant chemotherapy (100 mg/m(2) cisplatin and 85 mg/m(2) docetaxel) followed by radiotherapy with 44 Gy in 22 fractions over 3 weeks, and those in the control group received neoadjuvant chemotherapy alone. All patients were scheduled to undergo surgery. Randomisation was stratified by centre, mediastinal bulk (less than 5 cm vs 5 cm or more), and weight loss (5% or more vs less than 5% in the previous 6 months). The primary endpoint was event-free survival. Analyses were done by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00030771. From 2001 to 2012, 232 patients were enrolled, of whom 117 were allocated to the chemoradiotherapy group and 115 to the chemotherapy group. Median event-free survival was similar in the two groups at 12·8 months (95% CI 9·7-22·9) in the chemoradiotherapy group and 11·6 months (8·4-15·2) in the chemotherapy group (p=0·67). Median overall survival was 37·1 months (95% CI 22·6-50·0) with radiotherapy, compared with 26·2 months (19·9-52·1) in the control group. Chemotherapy-related toxic effects were reported in most patients, but 91% of patients completed three cycles of chemotherapy. Radiotherapy-induced grade 3 dysphagia was seen in seven (7%) patients. Three patients died in the control group within 30 days after surgery. Radiotherapy did not add any benefit to induction chemotherapy followed by surgery. We suggest that one definitive local treatment modality combined with neoadjuvant chemotherapy is adequate to treat resectable stage IIIA/N2 non-small-cell lung cancer. Swiss State Secretariat for Education, Research and Innovation (SERI), Swiss Cancer League, and Sanofi. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of Thermally Activated Metalloenediyne Cytotoxicity in Human Melanoma Cells.

PubMed

Keller, Eric J; Porter, Meghan; Garrett, Joy E; Varie, Meredith; Wang, Haiyan; Pollok, Karen E; Turchi, John J; Zaleski, Jeffrey M; Dynlacht, Joseph R

2018-05-15

Enediynes are a highly cytotoxic class of compounds. However, metallation of these compounds may modulate their activation, and thus their cytotoxicity. We previously demonstrated that cytotoxicity of two different metalloenediynes, including (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine] (PyED), is potentiated when the compounds are administered to HeLa cells during hyperthermia treatment at concentrations that are minimally or not cytotoxic at 37°C. In this study, we further characterized the concentration, time and temperature dependence of cytotoxicity of PyED on human U-1 melanoma cells. We also investigated the potential mechanisms by which PyED cytotoxicity is enhanced during hyperthermia treatment. Cell killing with PyED was dependent on concentration, temperature during treatment and time of exposure. Potentiation of cytotoxicity was observed when cells were treated with PyED at temperatures ≥39.5°C, and enhancement of cell killing increased with temperature and with increasing time at a given temperature. All cells treated with PyED were shown to have DNA damage, but substantially more damage was observed in cells treated with PyED during heating. DNA repair was also inhibited in cells treated with the drug during hyperthermia. Thus, potentiation of PyED cytotoxicity by hyperthermia may be due to enhancement of drug-induced DNA lesions, and/or the inhibition of repair of sublethal DNA damage. While the selective thermal activation of PyED supports the potential clinical utility of metalloenediynes as cancer thermochemotherapeutic agents, therapeutic gain could be optimized by identifying compounds that produce minimal toxicity at 37°C but which become activated and show enhancement of cytotoxicity within a tumor subjected to localized hyperthermic or thermal ablative treatment, or which might act as bifunctional agents. We thus also describe the development and initial characterization of a novel cofactor complex of PyED, platinated PyED (Pt-PyED). Pt-PyED binds to DNA-like cisplatin, and much like PyED, cytotoxicity is greatly enhanced after treatment with the drug at elevated temperatures. However, in contrast to PyED, Pt-PyED is only minimally cytotoxic at 37°C, at concentrations at which cytotoxicity is enhanced by hyperthermia. Further development of cisplatin-based enediynes may result in compounds which, when activated, will possess multiple DNA binding modalities similar to cisplatin, but produce less side effects in tissues at normothermic temperatures.

Apigenin reduce lipoteichoic acid-induced inflammatory response in rat cardiomyoblast cells.

PubMed

Gutiérrez-Venegas, Gloria; González-Rosas, Zeltzin

2017-02-01

Infective endocarditis is caused by Streptococcus sanguinis present in dental plaque, which can induce inflammatory responses in the endocardium. The present study depicts research on the properties of apigenin in embryonic mouse heart cells (H9c2) treated with lipoteichoic acid (LTA) obtained from S. sanguinis. Interleukin-1β and cyclooxygenase (COX)-2 expression were detected by reverse transcriptase polymerase chain reaction. In addition, western blot assays and immuno-fluorescence staining were used to assess translocation of nuclear factor kappa beta (NF-κB), degradation of IκB, as well as activity of the mitogen activated protein kinases: extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). Effect of apigenin on cell viability was equally assessed in other experimental series. Our results showed that apigenin blocked activation of ERK, JNK, and p38 in cardiomyocytes treated with LTA in a dose-dependent fashion. Moreover, apigenin showed no cytotoxic effects; it blocked NF-κB translocation and IκB degradation. Our findings suggested that apigenin possessed potential value in the treatment of infectious endocarditis.

In vitro synergistic efficacy of conjugated linoleic acid, oleic acid, safflower oil and taxol cytotoxicity on PC3 cells.

PubMed

Kızılşahin, Sadi; Nalbantsoy, Ayşe; Yavaşoğlu, N Ülkü Karabay

2015-01-01

The aim of this study was to determine in vitro synergistic efficacy of conjugated linoleic acid (CLA), oleic acid (OLA), safflower oil and taxol (Tax) cytotoxicity on human prostate cancer (PC3) cell line. To determine synergistic efficacy of oil combinations, PC3 treated with different doses of compounds alone and combined with 10 μg/mL Tax. The MTT results indicated that OLA-Tax combinations exhibited cytotoxicity against PC3 at doses of 30 nM+10 μg-Tax, 15 nM+5 μg-Tax and 7.5 nM+2.5 μg-Tax. The treatment of OLA or Tax did not show significant inhibition on PC3, while OLA-Tax combinations showed effective cytotoxicity at treated doses. CLA-Tax combinations demonstrated the same effect on PC3 as combined form with 45.72% versus the alone form as 74.51% viability. Cytotoxic synergy between Tax, OLA and CLA shows enhanced cytotoxicity on PC3 which might be used in the therapy of prostate cancer.

Electrochemical impedance spectroscopy study of carbon electrodes prepared from date pits and fibers of oil palm empty fruit bunches

NASA Astrophysics Data System (ADS)

Hamdan, E.; Deraman, M.; Suleman, M.; Nor, N. S. M.; Basri, N. H.; Hanappi, M. F. Y. M.; Sazali, N. E. S.; Tajuddin, N. S. M.; Omar, R.; Othman, M. A. R.; Shamsudin, S. A.

2016-11-01

In this study, we produced pre-carbonized date pits (PDP) and self-adhesive carbon grains (SACGs) from oil palm empty fruit bunches (EFB) by a low temperature (200°C for DP and 280°C for SACGs, respectively) carbonization method followed by KOH treatment to obtain KOH treated PDP (T-PDP) and KOH treated SACGs (T-SACGs). Four sets of green monolith (GMs) denoted as GM-A, GM-B, GM-C and GM-D were prepared respectively from SACGs (100 wt. %), mixture of PDP and SACGs (50:50 wt. %), T-SACGs (100 wt. %), and mixture of T-SACGs and T-PDP (50:50 wt. %), respectively. From these GMs the respective activated carbon monolith (ACMs) electrodes namely ACM-A, ACM-B, ACM-C and ACM-D were prepared via carbonization (N2 carbonization) and activation (CO2 environment). These ACMs electrodes were used to fabricate the corresponding EDLC cells: Cell-A, Cell-B, Cell-C and Cell-D, respectively. The electrochemical impedance spectroscopy tests conducted on the cells found that the Cell-D showed the maximum value of specific capacitance, Csp (˜ 135 F g-1) whereas the Cell-A showed the minimum values of ESR and characteristic response time, respectively, ˜ 2.14 Ω and ˜ 46 s. Therefore, it can be concluded that the KOH treatment can improve the capacitance but caused the increase in the ESR and response time.

Radiation Therapy Overcomes Adverse Prognostic Role of Cyclooxygenase-2 Expression on Reed-Sternberg Cells in Early Hodgkin Lymphoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mestre, Francisco; Gutiérrez, Antonio, E-mail: antoniom.gutierrez@ssib.es; Rodriguez, Jose

Purpose: To analyze the role of radiation therapy (RT) on the adverse prognostic influence of cyclooxygenase-2 (COX-2) expression on Reed-Sternberg (RS) cells, in the setting of early Hodgkin lymphoma (HL) treated with ABVD (adriamycin, vinblastine, bleomycin, dacarbazine). Methods and Materials: In the present study we retrospectively investigated the prognostic value of COX-2 expression in a large (n=143), uniformly treated early HL population from the Spanish Network of HL using tissue microarrays. Univariate and multivariate analyses were done, including the most recognized clinical variables and the potential role of administration of adjuvant RT. Results: Median age was 31 years; the expression of COX-2more » defined a subgroup with significantly worse prognosis. Considering COX-2{sup +} patients, those who received RT had significantly better 5-year progression-free survival (PFS) (80% vs 54% if no RT; P=.008). In contrast, COX-2{sup −} patients only had a modest, nonsignificant benefit from RT in terms of 5-year PFS (90% vs 79%; P=.13). When we compared the outcome of patients receiving RT considering the expression of COX-2 on RS cells, we found a nonsignificant 10% difference in terms of PFS between COX-2{sup +} and COX-2{sup −} patients (P=.09), whereas the difference between the 2 groups was important (25%) in patients not receiving RT (P=.04). Conclusions: Cyclooxygenase-2 RS cell expression is an adverse independent prognostic factor in early HL. Radiation therapy overcomes the worse prognosis associated with COX-2 expression on RS cells, acting in a chemotherapy-independent way. Cyclooxygenase-2 RS cell expression may be useful for determining patient candidates with early HL to receive consolidation with RT.« less

The synergistic effect of chemical carcinogens enhances Epstein-Barr virus reactivation and tumor progression of nasopharyngeal carcinoma cells.

PubMed

Fang, Chih-Yeu; Huang, Sheng-Yen; Wu, Chung-Chun; Hsu, Hui-Yu; Chou, Sheng-Ping; Tsai, Ching-Hwa; Chang, Yao; Takada, Kenzo; Chen, Jen-Yang

2012-01-01

Seroepidemiological studies imply a correlation between Epstein-Barr virus (EBV) reactivation and the development of nasopharyngeal carcinoma (NPC). N-nitroso compounds, phorbols, and butyrates are chemicals found in food and herb samples collected from NPC high-risk areas. These chemicals have been reported to be risk factors contributing to the development of NPC, however, the underlying mechanism is not fully understood. We have demonstrated previously that low dose N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.1 µg/ml) had a synergistic effect with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate (SB) in enhancing EBV reactivation and genome instability in NPC cells harboring EBV. Considering that residents in NPC high-risk areas may contact regularly with these chemical carcinogens, it is vital to elucidate the relation between chemicals and EBV and their contributions to the carcinogenesis of NPC. In this study, we constructed a cell culture model to show that genome instability, alterations of cancer hallmark gene expression, and tumorigenicity were increased after recurrent EBV reactivation in NPC cells following combined treatment of TPA/SB and MNNG. NPC cells latently infected with EBV, NA, and the corresponding EBV-negative cell, NPC-TW01, were periodically treated with MNNG, TPA/SB, or TPA/SB combined with MNNG. With chemically-induced recurrent reactivation of EBV, the degree of genome instability was significantly enhanced in NA cells treated with a combination of TPA/SB and MNNG than those treated individually. The Matrigel invasiveness, as well as the tumorigenicity in mouse, was also enhanced in NA cells after recurrent EBV reactivation. Expression profile analysis by microarray indicates that many carcinogenesis-related genes were altered after recurrent EBV reactivation, and several aberrations observed in cell lines correspond to alterations in NPC lesions. These results indicate that cooperation between chemical carcinogens can enhance the reactivation of EBV and, over recurrent reactivations, lead to alteration of cancer hallmark gene expression with resultant enhancement of tumorigenesis in NPC.

Transfer of minimally manipulated CMV-specific T cells from stem cell or third-party donors to treat CMV infection after allo-HSCT.

PubMed

Neuenhahn, M; Albrecht, J; Odendahl, M; Schlott, F; Dössinger, G; Schiemann, M; Lakshmipathi, S; Martin, K; Bunjes, D; Harsdorf, S; Weissinger, E M; Menzel, H; Verbeek, M; Uharek, L; Kröger, N; Wagner, E; Kobbe, G; Schroeder, T; Schmitt, M; Held, G; Herr, W; Germeroth, L; Bonig, H; Tonn, T; Einsele, H; Busch, D H; Grigoleit, G U

2017-10-01

Cytomegalovirus (CMV) infection is a common, potentially life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We assessed prospectively the safety and efficacy of stem cell-donor- or third-party-donor-derived CMV-specific T cells for the treatment of persistent CMV infections after allo-HSCT in a phase I/IIa trial. Allo-HSCT patients with drug-refractory CMV infection and lacking virus-specific T cells were treated with a single dose of ex vivo major histocompatibility complex-Streptamer-isolated CMV epitope-specific donor T cells. Forty-four allo-HSCT patients receiving a T-cell-replete (D + repl; n=28) or T-cell-depleted (D + depl; n=16) graft from a CMV-seropositive donor were screened for CMV-specific T-cell immunity. Eight D + depl recipients received adoptive T-cell therapy from their stem cell donor. CMV epitope-specific T cells were well supported and became detectable in all treated patients. Complete and partial virological response rates were 62.5% and 25%, respectively. Owing to longsome third-party donor (TPD) identification, only 8 of the 57 CMV patients transplanted from CMV-seronegative donors (D - ) received antigen-specific T cells from partially human leukocyte antigen (HLA)-matched TPDs. In all but one, TPD-derived CMV-specific T cells remained undetectable. In summary, adoptive transfer correlated with functional virus-specific T-cell reconstitution in D + depl patients. Suboptimal HLA match may counteract expansion of TPD-derived virus-specific T cells in D - patients.