Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and {beta}4 integrin function in MDA-MB-231 breast cancer cells

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

Lee, W.-J.; Chen, W.-K.; Wang, C.-J.

2008-01-15

Hepatocyte growth factor (HGF) and its receptor, Met, known to control invasive growth program have recently been shown to play crucial roles in the survival of breast cancer patients. The diet-derived flavonoids have been reported to possess anti-invasion properties; however, knowledge on the pharmacological and molecular mechanisms in suppressing HGF/Met-mediated tumor invasion and metastasis is poorly understood. In our preliminary study, we use HGF as an invasive inducer to investigate the effect of flavonoids including apigenin, naringenin, genistein and kaempferol on HGF-dependent invasive growth of MDA-MB-231 human breast cancer cells. Results show that apigenin presents the most potent anti-migration andmore » anti-invasion properties by Boyden chamber assay. Furthermore, apigenin represses the HGF-induced cell motility and scattering and inhibits the HGF-promoted cell migration and invasion in a dose-dependent manner. The effect of apigenin on HGF-induced signaling activation involving invasive growth was evaluated by immunoblotting analysis, it shows that apigenin blocks the HGF-induced Akt phosphorylation but not Met, ERK, and JNK phosphorylation. In addition to MDA-MB-231 cells, apigenin exhibits inhibitory effect on HGF-induced Akt phosphorylation in hepatoma SK-Hep1 cells and lung carcinoma A549 cells. By indirect immunofluorescence microscopy assay, apigenin inhibits the HGF-induced clustering of {beta}4 integrin at actin-rich adhesive site and lamellipodia through PI3K-dependent manner. Treatment of apigenin inhibited HGF-stimulated integrin {beta}4 function including cell-matrix adhesion and cell-endothelial cells adhesion in MDA-MB-231 cells. By Akt-siRNA transfection analysis, it confirmed that apigenin inhibited HGF-promoted invasive growth involving blocking PI3K/Akt pathway. Finally, we evaluated the effect of apigenin on HGF-promoted metastasis by lung colonization of tumor cells in nude mice and organ metastasis of tumor cells in chick embryo. By histological and gross examination of mouse lung and real-time PCR analysis of human alu in host tissues, it showed that apigenin, wortmannin, as well as anti-{beta}4 antibody all inhibit HGF-promoted metastasis. These data support the inhibitory effect of apigenin on HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and integrin {beta}4 function.« less

Transcriptional and posttranscriptional inhibition of HMGCR and PC biosynthesis by geraniol in 2 Hep-G2 cell proliferation linked pathways.

PubMed

Crespo, Rosana; Montero Villegas, Sandra; Abba, Martín C; de Bravo, Margarita G; Polo, Mónica P

2013-06-01

Geraniol, present in the essential oils of many aromatic plants, has in vitro and in vivo antitumor activity against several cell lines. We investigated the effects of geraniol on lipid metabolic pathways involved in Hep-G2 cell proliferation and found that geraniol inhibits the mevalonate pathway, phosphatidylcholine biosynthesis, cell growth, and cell cycle progression (with an arrest occurring at the G0/G1 interphase) and increases apoptosis. The expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting step in cholesterol synthesis, was inhibited at the transcriptional and posttranscriptional levels, as assessed by real-time RT-PCR, Western blots, and [(14)C]HMG-CoA-conversion radioactivity assays. That geraniol decreased cholesterogenesis but increased the incorporation of [(14)C]acetate into other nonsaponifiable metabolites indicated the existence of a second control point between squalene and cholesterol involved in redirecting the flow of cholesterol-derived carbon toward other metabolites of the mevalonate pathway. That exogenous mevalonate failed to restore growth in geraniol-inhibited cells suggests that, in addition to the inhibition of HMGCR, other dose-dependent actions exist through which geraniol can impact the mevalonate pathway and consequently inhibit cell proliferation. These results suggest that geraniol, a nontoxic compound found in many fruits and herbs, exhibits notable potential as a natural agent for combatting cancer and (or) cardiovascular diseases.

RhoE interferes with Rb inactivation and regulates the proliferation and survival of the U87 human glioblastoma cell line

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

Poch, Enric; Minambres, Rebeca; Mocholi, Enric

2007-02-15

Rho GTPases are important regulators of actin cytoskeleton, but they are also involved in cell proliferation, transformation and oncogenesis. One of this proteins, RhoE, inhibits cell proliferation, however the mechanism that regulates this effect remains poorly understood. Therefore, we undertook the present study to determine the role of RhoE in the regulation of cell proliferation. For this purpose we generated an adenovirus system to overexpress RhoE in U87 glioblastoma cells. Our results show that RhoE disrupts actin cytoskeleton organization and inhibits U87 glioblastoma cell proliferation. Importantly, RhoE expressing cells show a reduction in Rb phosphorylation and in cyclin D1 expression.more » Furthermore, RhoE inhibits ERK activation following serum stimulation of quiescent cells. Based in these findings, we propose that RhoE inhibits ERK activation, thereby decreasing cyclin D1 expression and leading to a reduction in Rb inactivation, and that this mechanism is involved in the RhoE-induced cell growth inhibition. Moreover, we also demonstrate that RhoE induces apoptosis in U87 cells and also in colon carcinoma and melanoma cells. These results indicate that RhoE plays an important role in the regulation of cell proliferation and survival, and suggest that this protein may be considered as an oncosupressor since it is capable to induce apoptosis in several tumor cell lines.« less

Carnosic acid inhibits the proliferation and migration capacity of human colorectal cancer cells

PubMed Central

BARNI, M.V.; CARLINI, M.J.; CAFFERATA, E.G.; PURICELLI, L.; MORENO, S.

2012-01-01

Colorectal cancer (CRC) is the third most common malignant neoplasm worldwide. The objective of this study was to examine whether carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., would inhibit the cell viability of three CRC cell lines: Caco-2, HT29 and LoVo in a dose-dependent manner, with IC50 values in the range of 24–96 μM. CA induced cell death by apoptosis in Caco-2 line after 24 h of treatment and inhibited cell adhesion and migration, possibly by reducing the activity of secreted proteases such as urokinase plasminogen activator (uPA) and metalloproteinases (MMPs). These effects may be associated through a mechanism involving the inhibition of the COX-2 pathway, because we have determined that CA downregulates the expression of COX-2 in Caco-2 cells at both the mRNA and protein levels. Therefore, CA modulates different targets involved in the development of CRC. These findings indicate that carnosic acid may have anticancer activity and may be useful as a novel chemotherapeutic agent. PMID:22246562

In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals.

PubMed Central

Coffey, M D; Cole, R A; Colles, S M; Chisolm, G M

1995-01-01

Mounting evidence supports current theories linking lipoprotein oxidation to atherosclerosis. We sought the cellular biochemical mechanism by which oxidized LDL inflicts cell injury. Inhibitors of candidate pathways of cell death were used to treat human fibroblast target cells exposed to oxidized LDL.. Ebselen, which degrades lipid hydroperoxides, inhibited oxidized LDL toxicity, consistent with our recent report that 7 beta-hydroperoxycholesterol (7 beta-OOH chol) is the major cytotoxin of oxidized LDL. Intracellular chelation of metal ions inhibited, while preloading cells with iron enhanced, toxicity, Inhibition of oxidized LDL and 7 beta-OOH chol toxicity by 2-keto-4-thiolmethyl butyric acid, a putative alkoxyl radical scavenger and by vitamin E, probucol and diphenylphenylenediamine, putative scavengers of peroxyl radicals was consistent with the involvement of these radicals in the lethal sequence. Cell death was thus postulated to occur due to lipid peroxidation via a sequence involving lipid hydroperoxide-induced, iron-mediated formation of alkoxyl, lipid, and peroxyl radicals. Pathways involving other reactive oxygen species, new protein synthesis, or altered cholesterol metabolism were considered less likely, since putative inhibitors failed to lessen toxicity. Understanding the mechanism of cell injury by oxidized LDL and its toxic moiety, 7 beta-OOH chol, may indicate specific interventions in the cell injury believed to accompany vascular lesion development. PMID:7560078

A second target of benzamide riboside: dihydrofolate reductase.

PubMed

Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R

2012-11-01

Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

Mechanisms of inhibition by fluoride of urease activities of cell suspensions and biofilms of Staphylococcus epidermidis, Streptococcus salivarius, Actinomyces naeslundii and of dental plaque.

PubMed

Barboza-Silva, E; Castro, A C D; Marquis, R E

2005-12-01

Fluoride is known to be a potent inhibitor of bacterial ureases and can also act in the form of hydrofluoric acid as a transmembrane proton conductor to acidify the cytoplasm of intact cells with possible indirect, acid inhibition of urease. Our research objectives were to assess the inhibitory potencies of fluoride for three urease-positive bacteria commonly found in the mouth and to determine the relative importance of direct and indirect inhibition of ureases for overall inhibition of intact cells or biofilms. The experimental design involved intact bacteria in suspensions, mono-organism biofilms, cell extracts, and dental plaque. Standard enzymatic assays for ammonia production from urea were used. We found that ureolysis by cells in suspensions or mono-organism biofilms of Staphylococcus epidermidis, Streptococcus salivarius or Actinomyces naeslundii was inhibited by fluoride at plaque levels of 0.1-0.5 mm in a pH-dependent manner. The results of experiments with the organic weak acids indomethacin and capric acid, which do not directly inhibit urease enzyme, indicated that weak-acid effects leading to cytoplasmic acidification are also involved in fluoride inhibition. However, direct fluoride inhibition of urease appeared to be the major mechanism for reduction in ureolytic activity in acid environments. Results of experiments with freshly harvested supragingival dental plaque indicated responses to fluoride similar to those of S. salivarius with pH-dependent fluoride inhibition and both direct and indirect inhibition of urease. Fluoride can act to diminish alkali production from urea by oral bacteria through direct and indirect mechanisms.

Belinostat-induced apoptosis and growth inhibition in pancreatic cancer cells involve activation of TAK1-AMPK signaling axis

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

Wang, Bing, E-mail: wangbin69@yahoo.com; Wang, Xin-bao; Chen, Li-yu

2013-07-19

Highlights: •Belinostat activates AMPK in cultured pancreatic cancer cells. •Activation of AMPK is important for belinostat-induced cytotoxic effects. •ROS and TAK1 are involved in belinostat-induced AMPK activation. •AMPK activation mediates mTOR inhibition by belinostat. -- Abstract: Pancreatic cancer accounts for more than 250,000 deaths worldwide each year. Recent studies have shown that belinostat, a novel pan histone deacetylases inhibitor (HDACi) induces apoptosis and growth inhibition in pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In the current study, we found that AMP-activated protein kinase (AMPK) activation was required for belinostat-induced apoptosis and anti-proliferation in PANC-1 pancreatic cancermore » cells. A significant AMPK activation was induced by belinostat in PANC-1 cells. Inhibition of AMPK by RNAi knockdown or dominant negative (DN) mutation significantly inhibited belinostat-induced apoptosis in PANC-1 cells. Reversely, AMPK activator AICAR and A-769662 exerted strong cytotoxicity in PANC-1 cells. Belinostat promoted reactive oxygen species (ROS) production in PANC-1 cells, increased ROS induced transforming growth factor-β-activating kinase 1 (TAK1)/AMPK association to activate AMPK. Meanwhile, anti-oxidants N-Acetyl-Cysteine (NAC) and MnTBAP as well as TAK1 shRNA knockdown suppressed belinostat-induced AMPK activation and PANC-1 cell apoptosis. In conclusion, we propose that belinostat-induced apoptosis and growth inhibition require the activation of ROS-TAK1-AMPK signaling axis in cultured pancreatic cancer cells.« less

Inhibition of Curcumin on ZAKα Activity Resultant in Apoptosis and Anchorage-Independent Growth in Cancer Cells.

PubMed

Lee, Jin-Sun; Wang, Tsu-Shing; Lin, Ming Cheng; Lin, Wei-Wen; Yang, Jaw-Ji

2017-10-31

Curcumin, a popular yellow pigment of the dietary spice turmeric, has been reported to inhibit cell growth and to induce apoptosis in a wide variety of cancer cells. Although numerous studies have investigated anticancer effects of curcumin, the precise molecular mechanism of action remains unidentified. Whereas curcumin mediates cell survival and apoptosis through mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling cascades, its impact on the upstream regulation of MAPK is unclear. The leucine-zipper and sterile-α motif kinase alpha (ZAKα), a mitogen-activated protein kinase kinase kinase (MAP3K), activates the c-Jun N-terminal kinase (JNK) and NF-κB pathway. This paper investigated the prospective involvement of ZAKα in curcumin-induced effects on cancer cells. Our results suggest that the antitumor activity of curcumin is mediated via a mechanism involving inhibition of ZAKα activity.

Fluoxetine regulates cell growth inhibition of interferon-α.

PubMed

Lin, Yu-Min; Yu, Bu-Chin; Chiu, Wen-Tai; Sun, Hung-Yu; Chien, Yu-Chieh; Su, Hui-Chen; Yen, Shu-Yang; Lai, Hsin-Wen; Bai, Chyi-Huey; Young, Kung-Chia; Tsao, Chiung-Wen

2016-10-01

Fluoxetine, a well-known anti-depression agent, may act as a chemosensitizer to assist and promote cancer therapy. However, how fluoxetine regulates cellular signaling to enhance cellular responses against tumor cell growth remains unclear. In the present study, addition of fluoxetine promoted growth inhibition of interferon-alpha (IFN-α) in human bladder carcinoma cells but not in normal uroepithelial cells through lessening the IFN-α-induced apoptosis but switching to cause G1 arrest, and maintaining the IFN-α-mediated reduction in G2/M phase. Activations and signal transducer and transactivator (STAT)-1 and peroxisome proliferator-activated receptor alpha (PPAR-α) were involved in this process. Chemical inhibitions of STAT-1 or PPAR-α partially rescued bladder carcinoma cells from IFN-α-mediated growth inhibition via blockades of G1 arrest, cyclin D1 reduction, p53 downregulation and p27 upregulation in the presence of fluoxetine. However, the functions of both proteins were not involved in the control of fluoxetine over apoptosis and maintained the declined G2/M phase of IFN-α. These results indicated that activation of PPAR-α and STAT-1 participated, at least in part, in growth inhibition of IFN-α in the presence of fluoxetine.

Dioscin enhances methotrexate absorption by down-regulating MDR1 in vitro and in vivo

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

Wang, Lijuan, E-mail: jlwang1979@163.com; Wang, Changyuan, E-mail: wangcyuan@163.com; Provincial Key Laboratory for Pharmacokinetics and Transport, Liaoning, Dalian Medical University, Dalian, Liaoning

2014-06-01

The purpose of this study was to investigate the enhancing effect of dioscin on the absorption of methotrexate (MTX) and clarify the molecular mechanism involved in vivo and in vitro. Dioscin increased MTX chemosensitivity and transepithelial flux in the absorptive direction, significantly inhibiting multidrug resistance 1 (MDR1) mRNA and protein expression and MDR1 promoter and nuclear factor κ-B (NF-κB) activities in Caco-2 cells. Moreover, inhibitor κB-α (IκB-α) degradation was inhibited by dioscin. Dioscin enhanced the intracellular concentration of MTX by down-regulating MDR1 expression through a mechanism that involves NF-κB signaling pathway inhibition in Caco-2 cells. Dioscin strengthened MTX absorption bymore » inhibiting MDR1 expression in rat intestine. In addition, even though MTX is absorbed into the enterocytes, there was no increase in toxicity observed, and that, in fact, decreased toxicity was seen. - Highlights: • Dioscin raised MTX concentration by inhibiting MDR1 in Caco-2 cells. • Dioscin suppresses MDR1 by inhibiting NF-κB signaling pathway in Caco-2 cells. • Dioscin can enhance MTX absorption via inhibiting MDR1 in vivo and in vitro. • Dioscin did not increase MTX-induced gastrointestinal mucosal toxicity.« less

Chronic inhibition of glycogen synthase kinase-3 protects against rotenone-induced cell death in human neuron-like cells by increasing BDNF secretion.

PubMed

Giménez-Cassina, Alfredo; Lim, Filip; Díaz-Nido, Javier

2012-12-07

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. Likewise, activation of glycogen synthase kinase-3 (GSK-3) has been proposed to play an important role in neurodegeneration. This multifunctional protein kinase is involved in a number of cellular functions and we previously showed that chronic inhibition of GSK-3 protects neuronal cells against mitochondrial dysfunction-elicited cell death, through a mechanism involving increased glucose metabolism and the translocation of hexokinase II (HKII) to mitochondria. Here, we sought to gain deeper insight into the molecular basis of this neuroprotection. We found that chronic inhibition of GSK-3, either genetically or pharmacologically, elicited a marked increase in brain-derived neurotrophic factor (BDNF) secretion, which in turn conferred resistance to mitochondrial dysfunction through subcellular re-distribution of HKII. These results define a molecular pathway through which chronic inhibition of GSK-3 may protect neuronal cells from death. Moreover, they highlight the potential benefits of enhanced neurotrophic factor secretion as a therapeutic approach to treat neurodegenerative diseases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

ME-143 Is Superior to Genistein in Suppression of WNT Signaling in Colon Cancer Cells.

PubMed

Pintova, Sofya; Planutis, Kestutis; Planutiene, Marina; Holcombe, Randall F

2017-04-01

This study tested the effect of the soy isoflavones genistein and ME-143, and two chemotherapeutic agents, 5-fluorouracil (5FU) and oxaliplatin, on WNT signaling. Colon cancer cell lines RKO (hereditary nonpolyposis colorectal cancer type) and DLD1 (most common colorectal cancer type driven by a mutation in WNT pathway) were utilized. WNT throughput was measured using a β-catenin-responsive SuperTopFlash luciferase assay. A stabilized β-catenin construct was employed to test β-catenin involvement in the mechanism of drug activity. ME-143 was a more than 10-fold potent inhibitor of DLD1 proliferation than genistein at 3.125 μM. Genistein alone did not inhibit WNT signaling in either cell line. In RKO cells, oxaliplatin and its combination with 5FU significantly inhibited WNT throughput. Neither 5FU, oxaliplatin nor their combination inhibited WNT signaling in DLD1 cells. In both the RKO and DLD1 cell lines, ME-143 significantly reduced WNT throughput by 65-75%. The introduction of stabilized β-catenin attenuated the ME-143-dependent inhibition of the WNT/β-catenin pathway. ME-143 alone and in combination with 5FU and oxaliplatin effectively inhibits the WNT/β-catenin pathway in colorectal cancer cells of diverse genetic background. β-Catenin is directly involved in the mechanism of inhibition, and clinical studies are warranted. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Nitric Oxide Regulates Lung Carcinoma Cell Anoikis through Inhibition of Ubiquitin-Proteasomal Degradation of Caveolin-1*

PubMed Central

Chanvorachote, Pithi; Nimmannit, Ubonthip; Lu, Yongju; Talbott, Siera; Jiang, Bing-Hua; Rojanasakul, Yon

2009-01-01

Anoikis, a detachment-induced apoptosis, is a principal mechanism of inhibition of tumor cell metastasis. Tumor cells can acquire anoikis resistance which is frequently observed in metastatic lung cancer. This phenomenon becomes an important obstacle of efficient cancer therapy. Recently, signaling mediators such as caveolin-1 (Cav-1) and nitric oxide (NO) have garnered attention in metastasis research; however, their role and the underlying mechanisms of metastasis regulation are largely unknown. Using human lung carcinoma H460 cells, we show that NO impairs the apoptotic function of the cells after detachment. The NO donors sodium nitroprusside and diethylenetriamine NONOate inhibit detachment-induced apoptosis, whereas the NO inhibitors aminoguanidine and 2-(4-carboxyphenyl) tetramethylimidazoline-1-oxyl-3-oxide promote this effect. Resistance to anoikis in H460 cells is mediated by Cav-1, which is significantly down-regulated after cell detachment through a non-transcriptional mechanism involving ubiquitin-proteasomal degradation. NO inhibits this down-regulation by interfering with Cav-1 ubiquitination through a process that involves protein S-nitrosylation, which prevents its proteasomal degradation and induction of anoikis by cell detachment. These findings indicate a novel pathway for NO regulation of Cav-1, which could be a key mechanism of anoikis resistance in tumor cells. PMID:19706615

Nitric oxide regulates lung carcinoma cell anoikis through inhibition of ubiquitin-proteasomal degradation of caveolin-1.

PubMed

Chanvorachote, Pithi; Nimmannit, Ubonthip; Lu, Yongju; Talbott, Siera; Jiang, Bing-Hua; Rojanasakul, Yon

2009-10-09

Anoikis, a detachment-induced apoptosis, is a principal mechanism of inhibition of tumor cell metastasis. Tumor cells can acquire anoikis resistance which is frequently observed in metastatic lung cancer. This phenomenon becomes an important obstacle of efficient cancer therapy. Recently, signaling mediators such as caveolin-1 (Cav-1) and nitric oxide (NO) have garnered attention in metastasis research; however, their role and the underlying mechanisms of metastasis regulation are largely unknown. Using human lung carcinoma H460 cells, we show that NO impairs the apoptotic function of the cells after detachment. The NO donors sodium nitroprusside and diethylenetriamine NONOate inhibit detachment-induced apoptosis, whereas the NO inhibitors aminoguanidine and 2-(4-carboxyphenyl) tetramethylimidazoline-1-oxyl-3-oxide promote this effect. Resistance to anoikis in H460 cells is mediated by Cav-1, which is significantly down-regulated after cell detachment through a non-transcriptional mechanism involving ubiquitin-proteasomal degradation. NO inhibits this down-regulation by interfering with Cav-1 ubiquitination through a process that involves protein S-nitrosylation, which prevents its proteasomal degradation and induction of anoikis by cell detachment. These findings indicate a novel pathway for NO regulation of Cav-1, which could be a key mechanism of anoikis resistance in tumor cells.

Neutral Endopeptidase Inhibits Neuropeptide Mediated Growth of Androgen-Independent Prostate Cancer

DTIC Science & Technology

2000-09-01

The objective of this project is to elucidate the involvement of neutral endopeptidase (NEP), a cell-surface peptidase which inactivates active...pathways. Recombinant NEP and induced NEP can inhibit the bombesin and endothelin-l stimulated FAK phosphorylation and cell migration. These studies will

Inhibition of melanoma cell motility by the snake venom disintegrin eristostatin

PubMed Central

Tian, Jing; Paquette-Straub, Carrie; Sage, E. Helene; Funk, Sarah E.; Patel, Vivek; Galileo, Deni; McLane, Mary Ann

2007-01-01

Eristostatin, an RGD-containing disintegrin isolated from the venom of Eristicophis macmahoni, inhibits lung or liver colonization of melanoma cells in a mouse model. In this study, transwell migration and in vitro wound closure assays were used to determine the effect of eristostatin on the migration of melanoma cells. Eristostatin significantly impaired the migration of 5 human melanoma cell lines. Furthermore, it specifically inhibited cell migration on fibronectin in a concentration-dependent manner, but not that on collagen IV or laminin. In contrast, eristostatin was found to have no effect on cell proliferation or angiogenesis. These results indicate that the interaction between eristostatin and melanoma cells may involve fibronectin-binding integrins that mediate cell migration. Mutations to alanine of seven residues within the RGD loop of eristostatin and four residues outside the RGD loop of eristostatin resulted in significantly less potency in both platelet aggregation and wound closure assays. For six of the mutations, however, decreased activity was found only in the latter assay. We conclude that a different mechanism and/or integrin is involved in these two cell activities. PMID:17316731

Inhibition of selectin binding

DOEpatents

Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

2001-10-09

This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

Inhibition of selectin binding

DOEpatents

Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

1999-01-01

This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

Inhibition of selectin binding

DOEpatents

Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Carolyn

1999-10-05

This invention provides a system for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, this system can be used to palliate certain inflammatory and immunological conditions.

Silencing of karyopherin α2 inhibits cell growth and survival in human hepatocellular carcinoma

PubMed Central

Yang, Yunfeng; Guo, Jian; Hao, Yuxia; Wang, Fuhua; Li, Fengxia; Shuang, Shaomin; Wang, Junping

2017-01-01

Karyopherin α2 (KPNA2), involved in nucleocytoplasmic transport, has been reported to be upregulated in hepatocellular carcinoma and considered as a biomarker for poor prognosis. However, comprehensive studies of KPNA2 functions in hepatocellular carcinogenesis are still lacking. Our study examine the roles and related molecular mechanisms of KPNA2 in hepatocellular carcinoma development. Results show that KPNA2 knockdown inhibited the proliferation and growth of hepatocellular carcinoma cells in vitro and in vivo. KPNA2 knockdown also inhibited colony formation ability, induced cell cycle arrest and cellular apoptosis in two hepatocellular carcinoma cell lines, HepG2 and SMMC-7721. Furthermore, gene expression microarray analysis in HepG2 cells with KPNA2 knockdown revealed that critical signaling pathways involved in cell proliferation and survival were deregulated. In conclusion, this study provided systematic evidence that KPNA2 was an essential factor promoting hepatocellular carcinoma and unraveled potential molecular pathways and networks underlying KPNA2-induced hepatocellular carcinogenesis. PMID:28422734

Silencing of karyopherin α2 inhibits cell growth and survival in human hepatocellular carcinoma.

PubMed

Yang, Yunfeng; Guo, Jian; Hao, Yuxia; Wang, Fuhua; Li, Fengxia; Shuang, Shaomin; Wang, Junping

2017-05-30

Karyopherin α2 (KPNA2), involved in nucleocytoplasmic transport, has been reported to be upregulated in hepatocellular carcinoma and considered as a biomarker for poor prognosis. However, comprehensive studies of KPNA2 functions in hepatocellular carcinogenesis are still lacking. Our study examine the roles and related molecular mechanisms of KPNA2 in hepatocellular carcinoma development. Results show that KPNA2 knockdown inhibited the proliferation and growth of hepatocellular carcinoma cells in vitro and in vivo. KPNA2 knockdown also inhibited colony formation ability, induced cell cycle arrest and cellular apoptosis in two hepatocellular carcinoma cell lines, HepG2 and SMMC-7721. Furthermore, gene expression microarray analysis in HepG2 cells with KPNA2 knockdown revealed that critical signaling pathways involved in cell proliferation and survival were deregulated. In conclusion, this study provided systematic evidence that KPNA2 was an essential factor promoting hepatocellular carcinoma and unraveled potential molecular pathways and networks underlying KPNA2-induced hepatocellular carcinogenesis.

Selective growth inhibition of human breast cancer cells by graviola fruit extract in vitro and in vivo involving downregulation of EGFR expression.

PubMed

Dai, Yumin; Hogan, Shelly; Schmelz, Eva M; Ju, Young H; Canning, Corene; Zhou, Kequan

2011-01-01

The epidermal growth factor receptor (EGFR) is an oncogene frequently overexpressed in breast cancer (BC), and its overexpression has been associated with poor prognosis and drug resistance. EGFR is therefore a rational target for BC therapy development. This study demonstrated that a graviola fruit extract (GFE) significantly downregulated EGFR gene expression and inhibited the growth of BC cells and xenografts. GFE selectively inhibited the growth of EGFR-overexpressing human BC (MDA-MB-468) cells (IC(50) = 4.8 μg/ml) but had no effect on nontumorigenic human breast epithelial cells (MCF-10A). GFE significantly downregulated EGFR mRNA expression, arrested cell cycle in the G0/G1 phase, and induced apoptosis in MDA-MB-468 cells. In the mouse xenograft model, a 5-wk dietary treatment of GFE (200 mg/kg diet) significantly reduced the protein expression of EGFR, p-EGFR, and p-ERK in MDA-MB-468 tumors by 56%, 54%, and 32.5%, respectively. Overall, dietary GFE inhibited tumor growth, as measured by wet weight, by 32% (P < 0.01). These data showed that dietary GFE induced significant growth inhibition of MDA-MB-468 cells in vitro and in vivo through a mechanism involving the EGFR/ERK signaling pathway, suggesting that GFE may have a protective effect for women against EGFR-overexpressing BC.

Effects of G6PD activity inhibition on the viability, ROS generation and mechanical properties of cervical cancer cells.

PubMed

Fang, Zishui; Jiang, Chengrui; Feng, Yi; Chen, Rixin; Lin, Xiaoying; Zhang, Zhiqiang; Han, Luhao; Chen, Xiaodan; Li, Hongyi; Guo, Yibin; Jiang, Weiying

2016-09-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency has been revealed to be involved in the efficacy to anti-cancer therapy but the mechanism remains unclear. We aimed to investigate the anti-cancer mechanism of G6PD deficiency. In our study, dehydroepiandrosterone (DHEA) and shRNA technology were used for inhibiting the activity of G6PD of cervical cancer cells. Peak Force QNM Atomic Force Microscopy was used to assess the changes of topography and biomechanical properties of cells and detect the effects on living cells in a natural aqueous environment. Flow cytometry was used to detect the apoptosis and reactive oxygen species (ROS) generation. Scanning electron microscopy was used to observe cell morphology. Moreover, a laser scanning confocal microscope was used to observe the alterations in cytoskeleton to explore the involved mechanism. When G6PD was inhibited by DHEA or RNA interference, the abnormal Young's modulus and increased roughness of cell membrane were observed in HeLa cells, as well as the idioblasts. Simultaneously, G6PD deficiency resulted in decreased HeLa cells migration and proliferation ability but increased ROS generation inducing apoptosis. What's more, the inhibition of G6PD activity caused the disorganization of microfilaments and microtubules of cytoskeletons and cell shrinkage. Our results indicated the anti-cervix cancer mechanism of G6PD deficiency may be involved with the decreased cancer cells migration and proliferation ability as a result of abnormal reorganization of cell cytoskeleton and abnormal biomechanical properties caused by the increased ROS. Suppression of G6PD may be a promising strategy in developing novel therapeutic methods for cervical cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

Curcumin inhibits tumor epithelial‑mesenchymal transition by downregulating the Wnt signaling pathway and upregulating NKD2 expression in colon cancer cells.

PubMed

Zhang, Zewei; Chen, Haitao; Xu, Chao; Song, Lu; Huang, Lulu; Lai, Yuebiao; Wang, Yuqi; Chen, Hanlu; Gu, Danlin; Ren, Lili; Yao, Qinghua

2016-05-01

Tumor invasion and metastasis are closely associated with epithelial‑mesenchymal transition (EMT). EMT refers to epithelial cells under physiological and pathological conditions that are specific to mesenchymal transition. Curcumin inhibits EMT progression via Wnt signaling. The Wnt signaling pathway is a conservative EMT‑related signaling pathway that is involved in the development of various tumors. In the present study, MTS assays were employed to analyze the proliferation of curcumin‑treated cells. Naked cuticle homolog 2 (NKD2), chemokine receptor 4 (CXCR4) and antibodies associated with EMT were examined in SW620 colorectal cancer cell lines using western blot analysis and real‑time qPCR. NKD2 small‑interfering RNA (siRNA) and CXCR4 expression plasmid was synthesized and transfected into the colorectal cancer cell lines, and NKD2 and CXCR4 expression levels were detected. The results showed that curcumin significantly inhibited the proliferation of colorectal cancer cells and upregulated the expression of NKD2 in SW620 colorectal cancer cells and in the xenograft, resulting in the downregulation of key markers in the Wnt signaling. In addition, the progression of ETM was inhibited due to the overexpression of E‑cadherin as well as the downregulation of vimentin. Curcumin also inhibited tumor metastasis by downregulating the expression of CXCR4 significantly. The results suggested involvement of the NKD2‑Wnt‑CXCR4 signaling pathway in colorectal cancer cells. In addition, curcumin is inhibit this signaling and the development of colorectal cancer.

Curcumin inhibits tumor epithelial-mesenchymal transition by downregulating the Wnt signaling pathway and upregulating NKD2 expression in colon cancer cells

PubMed Central

ZHANG, ZEWEI; CHEN, HAITAO; XU, CHAO; SONG, LU; HUANG, LULU; LAI, YUEBIAO; WANG, YUQI; CHEN, HANLU; GU, DANLIN; REN, LILI; YAO, QINGHUA

2016-01-01

Tumor invasion and metastasis are closely associated with epithelial-mesenchymal transition (EMT). EMT refers to epithelial cells under physiological and pathological conditions that are specific to mesenchymal transition. Curcumin inhibits EMT progression via Wnt signaling. The Wnt signaling pathway is a conservative EMT-related signaling pathway that is involved in the development of various tumors. In the present study, MTS assays were employed to analyze the proliferation of curcumin-treated cells. Naked cuticle homolog 2 (NKD2), chemokine receptor 4 (CXCR4) and antibodies associated with EMT were examined in SW620 colorectal cancer cell lines using western blot analysis and real-time qPCR. NKD2 small-interfering RNA (siRNA) and CXCR4 expression plasmid was synthesized and transfected into the colorectal cancer cell lines, and NKD2 and CXCR4 expression levels were detected. The results showed that curcumin significantly inhibited the proliferation of colorectal cancer cells and upregulated the expression of NKD2 in SW620 colorectal cancer cells and in the xenograft, resulting in the downregulation of key markers in the Wnt signaling. In addition, the progression of ETM was inhibited due to the overexpression of E-cadherin as well as the downregulation of vimentin. Curcumin also inhibited tumor metastasis by downregulating the expression of CXCR4 significantly. The results suggested involvement of the NKD2-Wnt-CXCR4 signaling pathway in colorectal cancer cells. In addition, curcumin is inhibit this signaling and the development of colorectal cancer. PMID:26985708

Decursin prevents TPA-induced invasion through suppression of PKCα/p38/NF-κB-dependent MMP-9 expression in MCF-7 human breast carcinoma cells.

PubMed

Kim, Jeong-Mi; Noh, Eun-Mi; Kim, Mi-Seong; Hwang, Jin-Ki; Hwang, Hong-Yeon; Ryu, Do-Gon; Kim, Hye-Jung; Yu, Hong-Nu; You, Yong-Ouk; Kim, Jong-Suk; Youn, Hyun Jo; Kwon, Kang-Beom; Jung, Sung Hoo; Lee, Young-Rae

2014-05-01

Decursin, a coumarin compound, was first isolated from the roots of Angelica gigas almost four decades ago. It was found to exhibit cytotoxicity against various human cancer cells and to possess anti-amnesic activity in vivo through the inhibition of AChE activity. However, the effect of decursin on breast cancer invasion is unknown. Matrix metalloproteinase-9 (MMP-9) is known to be an important factor for cancer cell invasion. Therefore, in this study, we investigated the inhibitory effect of decursin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion, as well as the molecular mechanisms involved in MCF-7 cells. Our results showed that decursin inhibits TPA-induced MMP-9 expression and cell invasion through the suppression of NF-κB. Furthermore, decursin repressed the TPA-induced phosphorylation of p38 MAPK and inhibited TPA-induced translocation of PKCα from the cytosol to the membrane, but did not affect the translocation of PKCδ. These results indicate that decursin-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the PKCα, MAPK and NF-κB pathways in MCF-7 cells. Thus, decursin may have potential value in restricting breast cancer metastasis.

Knockdown of hTERT and concurrent treatment with interferon-gamma inhibited proliferation and invasion of human glioblastoma cell lines

PubMed Central

George, Joseph; Banik, Naren L.; Ray, Swapan K.

2011-01-01

Human telomerase reverse transcriptase (hTERT) is the catalytic component of telomerase that facilitates tumor cell invasion and proliferation. Telomerase and hTERT are remarkably upregulated in majority of cancers including glioblastoma. Interferon-gamma (IFN-γ) modulates several cellular activities including cell cycle and multiplication through transcriptional regulation. The present investigation was designed to unravel the molecular mechanisms of the inhibition of cell proliferation, migration, and invasion of human glioblastoma SNB-19 and LN-18 cell lines after knockdown of hTERT using a plasmid vector based siRNA and concurrent treatment with IFN-γ. We observed more than 80% inhibition of cell proliferation, migration, and invasion of both cell lines after the treatment with combination of hTERT siRNA and IFN-γ. Our studies also showed accumulation of apoptotic cells in subG1 phase and an increase in cell population in G0/G1 with a reduction in G2/M phase indicating cell cycle arrest in G0/G1 phase for apoptosis. Semiquantitative and real-time RT-PCR analyses demonstrated significant downregulation of c- Myc and upregulation of p21 Waf1 and p27 Kip1. Western blotting confirmed the downregulation of the molecules involved in cell proliferation, migration, and invasion and also showed upregulation of cell cycle inhibitors. In conclusion, our study demonstrated that knockdown of hTERT siRNA and concurrent treatment with IFN-γ effectively inhibited cell proliferation, migration, and invasion in glioblastoma cells through downregulation of the molecules involved in these processes and cell cycle inhibition. Therefore, the combination of hTERT siRNA and IFN-γ offers a potential therapeutic strategy for controlling growth of human glioblastoma cells. PMID:20394835

Prostaglandin E2 inhibition of IL-27 production in murine dendritic cells: a novel mechanism which involves IRF1

PubMed Central

Hooper, Kirsten M; Yen, Jui-Hung; Kong, Weimin; Rahbari, Kate M; Kuo, Ping-Chang; Gamero, Ana M; Ganea, Doina

2016-01-01

IL-27, a multifunctional cytokine produced by antigen-presenting cells, antagonizes inflammation by affecting conventional dendritic cells (cDC), inducing IL-10, and promoting development of regulatory Tr1 cells. Although the mechanisms involved in IL-27 induction are well-studied, much less is known about the factors that negatively impact IL-27 expression. Prostaglandin E2 (PGE2), a major immunomodulatory prostanoid, acts as a pro-inflammatory agent in several models of inflammatory/autoimmune diseases, promoting primarily Th17 development and function. In this study, we report on a novel mechanism which promotes the pro-inflammatory function of PGE2. We showed previously that PGE2 inhibits IL-27 production in murine bone marrow derived DCs. Here, we show that, in addition to BMDCs, PGE2 inhibits IL-27 production in macrophages and in splenic cDC and we identify a novel pathway consisting of signaling through EP2/EP4→induction of cAMP→downregulation of IRF1 expression and binding to the p28 ISRE site. The inhibitory effect of PGE2 on p28 and irf1 expression does not involve endogenous IFNβ, STAT1 or STAT2, and inhibition of IL-27 does not appear to be mediated through PKA, EPAC, PI3K, or MAPKs. We observed similar inhibition of il27p28 expression in vivo in splenic DC following administration of dimethyl PGE2 in conjunction with LPS. Based on the anti-inflammatory role of IL-27 in cDC and through the generation of Tr1 cells, we propose that the PGE2-induced inhibition of IL-27 in activated cDC represents an important additional mechanism for its in vivo pro-inflammatory functions. PMID:28062696

Inhibition of glutathione synthesis eliminates the adaptive response of ascitic hepatoma 22 cells to nedaplatin that targets thioredoxin reductase

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

Wang, Yijun; Lu, Hongjuan; Wang, Dongxu

2012-12-15

Thioredoxin reductase (TrxR) is a target for cancer therapy and the anticancer mechanism of cisplatin involves TrxR inhibition. We hypothesize that the anticancer drug nedaplatin (NDP), an analogue of cisplatin and a second-generation platinum complex, also targets TrxR. Furthermore, we investigate whether the therapeutic efficacy of NDP can be enhanced by simultaneous modulation of 1) TrxR, via NDP, and 2) glutathione (GSH), via the GSH synthesis inhibitor buthionine sulfoximine (BSO). Mice bearing ascitic hepatoma 22 (H22) cells were treated with NDP alone or NDP plus BSO. TrxR activity of H22 cells was inhibited by NDP in a dose-dependent manner. Amore » high correlation between the inhibition of TrxR activity at 6 h and the inhibition of ascitic fluid volume at 72 h was established (r = 0.978, p < 0.01). As an adaptive response, the viable ascitic cancer cells after NDP treatment displayed an enlarged cell phenotype, assembled with several-fold more antioxidant enzymes and GSH-predominant non-protein free thiols. This adaptive response was largely eliminated when BSO was co-administered with NDP, leading to the decimation of the H22 cell population without enhancing renal toxicity, since at this dose, NDP did not inhibit renal TrxR activity. In conclusion, the pharmacological effect of NDP involves TrxR inhibition, and the adaptive response of NDP-treated ascitic H22 cells can be efficiently counteracted by BSO. Simultaneous modulation of TrxR and GSH on ascitic H22 cells using NDP plus BSO greatly enhances therapeutic efficacy as compared with the single modulation of TrxR using NDP alone. -- Highlights: ► Nedaplatin at a pharmacological dose inhibits TrxR in cancer cells but not in kidney. ► The nedaplatin-treated cancer cells exhibit adaptive response. ► Buthionine sulfoximine inhibits glutathione in both cancer cells and kidney. ► Buthionine sulfoximine counteracts the adaptive response to the nedaplatin treatment. ► Buthionine sulfoximine does not aggravate renal toxicity of the nedaplatin treatment.« less

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

Combined inhibition of heat shock proteins 90 and 70 leads to simultaneous degradation of the oncogenic signaling proteins involved in muscle invasive bladder cancer

PubMed Central

Cavanaugh, Alice; Juengst, Brendon; Sheridan, Kathleen; Danella, John F.; Williams, Heinric

2015-01-01

Heat shock protein 90 (HSP90) plays a critical role in the survival of cancer cells including muscle invasive bladder cancer (MIBC). The addiction of tumor cells to HSP90 has promoted the development of numerous HSP90 inhibitors and their use in clinical trials. This study evaluated the role of inhibiting HSP90 using STA9090 (STA) alone or in combination with the HSP70 inhibitor VER155008 (VER) in several human MIBC cell lines. While both STA and VER inhibited MIBC cell growth and migration and promoted apoptosis, combination therapy was more effective. Therefore, the signaling pathways involved in MIBC were systematically interrogated following STA and/or VER treatments. STA and not VER reduced the expression of proteins in the p53/Rb, PI3K and SWI/SWF pathways. Interestingly, STA was not as effective as VER or combination therapy in degrading proteins involved in the histone modification pathway such as KDM6A (demethylase) and EP300 (acetyltransferase) as predicted by The Cancer Genome Atlas (TCGA) data. This data suggests that dual HSP90 and HSP70 inhibition can simultaneously disrupt the key signaling pathways in MIBC. PMID:26556859

Green tea extract and aged garlic extract inhibit anion transport and sickle cell dehydration in vitro.

PubMed

Ohnishi, S T; Ohnishi, T; Ogunmola, G B

2001-01-01

Both green tea extract (GTE or tea polyphenols) and aged garlic extract (AGE) effectively inhibited in vitro dehydration of sickle red blood cells induced by K-Cl cotransport or red cell storage. For K-Cl cotransport induced by 500 mM urea, 0.3 mg/ml EGCg (epigallocatechin gallate; a major component in GTE) almost completely inhibited dehydration, and 6 mg/ml AGE inhibited dehydration to 30% of the control level. Both vitamins E and C had no effect at the level of 2 mM. Different tea extracts had different degrees of inhibition, but the inhibitory activity increased when the number of hydroxyl groups in the compounds increased. With storage of sickle cells at 4 degrees C for 6 days, the cells started to undergo spontaneous dehydration when incubated at 37 degrees C. Neither inhibitors for Ca-induced K efflux nor K-Cl cotransport could inhibit cell dehydration of stored sickle cells, but both GTE and AGE effectively inhibited it. Chloride efflux measurements using a chloride electrode demonstrated that both GTE and AGE inhibited anion transport in red blood cells. The inhibitory mechanism of these compounds may be related to anion transport inhibition, although involvement of their antioxidant activities can not yet be ruled out. Copyright 2001 Academic Press.

The viral protein A238L inhibits cyclooxygenase-2 expression through a nuclear factor of activated T cell-dependent transactivation pathway.

PubMed

Granja, Aitor G; Nogal, Maria L; Hurtado, Carolina; Vila, Virginia; Carrascosa, Angel L; Salas, María L; Fresno, Manuel; Revilla, Yolanda

2004-12-17

Cyclooxygenase-2 is transiently induced upon cell activation or viral infections, resulting in inflammation and modulation of the immune response. Here we report that A238L, an African swine fever virus protein, efficiently inhibits cyclooxygenase-2 gene expression in Jurkat T cells and in virus-infected Vero cells. Transfection of Jurkat cells stably expressing A238L with cyclooxygenase-2 promoter-luciferase constructs containing 5'-terminal deletions or mutations in distal or proximal nuclear factor of activated T cell (NFAT) response elements revealed that these sequences are involved in the inhibition induced by A238L. Overexpression of a constitutively active version of the calcium-dependent phosphatase calcineurin or NFAT reversed the inhibition mediated by A238L on cyclooxygenase-2 promoter activation, whereas overexpression of p65 NFkappaB had no effect. A238L does not modify the nuclear localization of NFAT after phorbol 12-myristate 13-acetate/calcium ionophore stimulation. Moreover, we show that the mechanism by which the viral protein down-regulates cyclooxygenase-2 activity does not involve inhibition of the binding between NFAT and its specific DNA sequences into the cyclooxygenase-2 promoter. Strikingly, A238L dramatically inhibited the transactivation mediated by a GAL4-NFAT fusion protein containing the N-terminal transactivation domain of NFAT1. Taken together, these data indicate that A238L down-regulates cyclooxygenase-2 transcription through the NFAT response elements, being NFAT-dependent transactivation implicated in this down-regulation.

Polypeptide from Chlamys farreri inhibits UVB-induced apoptosis of HaCaT cells via iNOS/NO and HSP90

NASA Astrophysics Data System (ADS)

Zhang, Zhengyang; Liu, Xiaojin; Liu, Tuo; Yan, Lin; Wang, Yuejun; Wang, Chunbo

2009-09-01

Polypeptide from Chlamys farreri (PCF) is a novel marine bioactive product that was isolated from the gonochoric Chinese scallop Chlamys farreri, and was found to be an effective antioxidant in our recent studies. In this study, we investigated the effect of PCF on ultraviolet B (UVB)-induced apoptosis of HaCaT cells and the intracellular signaling pathways involved. Pretreatment with the inducible nitric oxide synthase (iNOS) inhibitor S-methylisothiourea sulfate inhibited UVB-induced apoptosis, indicating that iNOS and NO play important roles in apoptosis. On the other hand, the inhibition of UVB-induced apoptosis in the immortalized keratinocyte (HaCaT) cells by PCF was estimated using a DNA ladder. PCF treatment inhibited UVB-induced iNOS activation, as determined by RT-PCR, NO production, as determined by ESR, and up-regulated heat shock protein (HSP) 90 activation, as determined by Western blotting. Our results indicate that iNOS and NO are involved in UVB-induced apoptosis of HaCaT cells and the protective effect of PCF against UVB irradiation is exerted by suppressing the expression of iNOS, followed by inhibition of NO release and enhanced activation of HSP90.

Effects on Energy Metabolism of Two Guanidine Molecules, (Boc)2 -Creatine and Metformin.

PubMed

Garbati, Patrizia; Ravera, Silvia; Scarfì, Sonia; Salis, Annalisa; Rosano, Camillo; Poggi, Alessandro; Damonte, Gianluca; Millo, Enrico; Balestrino, Maurizio

2017-09-01

Several enzymes are involved in the energy production, becoming a possible target for new anti-cancer drugs. In this paper, we used biochemical and in silico studies to evaluate the effects of two guanidine molecules, (Boc) 2 -creatine and metformin, on creatine kinase, an enzyme involved in the regulation of intracellular energy levels. Our results show that both drugs inhibit creatine kinase activity; however, (Boc) 2 -creatine displays a competitive inhibition, while metformin acts with a non-competitive mechanism. Moreover, (Boc) 2 -creatine is able to inhibit the activity of hexokinase with a non-competitive mechanism. Considering that creatine kinase and hexokinase are involved in energy metabolism, we evaluated the effects of (Boc) 2 -creatine and metformin on the ATP/AMP ratio and on cellular proliferation in healthy fibroblasts, human breast cancer cells (MDA-MB-468), a human neuroblastoma cell line (SH-SY5Y), a human Hodgkin lymphoma cell line (KMH2). We found that healthy fibroblasts were only partially affected by (Boc) 2 -creatine, while both ATP/AMP ratio and viability of the three cancer cell lines were significantly decreased. By inhibiting both creatine kinase and hexokinase, (Boc) 2 -creatine appears as a promising new agent in anticancer treatment. Further research is needed to understand what types of cancer cells are most suitable to treatment by this new compound. J. Cell. Biochem. 118: 2700-2711, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The Inhibitory Effect of Doxycycline on Cisplatin-Sensitive and -Resistant Epithelial Ovarian Cancer

PubMed Central

Ma, Bei; Xu, Ming-juan

2014-01-01

Background Detecting a new effective and hypotoxic anticancer drug is an emerging new strategy for cancer chemotherapy. Doxycycline (DC) is a kind of antibiotics but also inhibits tumorigenesis. Methods MTT and cell invasion assay, flow cytometry, western-blot analysis and nude mice were used to investigate the effects and underlying mechanisms of doxycycline on epithelial ovarian cancer cells. Results Doxycycline inhibited the proliferation and invasion of SKOV3 and SKOV3/DDP; induced moderate apoptosis of SKOV3/DDP. CXCR4 expression at both mRNA and protein levels was downregulated in both cell lines when treated with doxycycline. Akt and ERK1/2 were involved in doxycycline effect on cell proliferation of SKOV3 but not of SKOV3/DDP. Akt and EKR1/2 phosphorylation were activated by SDF-1α, which was then inhibited by doxycycline in SKOV3. Pro-caspase-3 expression was significantly higher in SKOV3 than that in SKOV3/DDP which was upregulated when treated with doxycycline. In vivo, doxycycline inhibited peritoneal tumor xenograft and decreased malignant ascites. Conclusion Doxycycline not only has an inhibitory effect on ovarian cancer, but also can increase sensitivity to cisplatin. SDF-1α/CXCR4-regulated Akt and ERK 1/2 activations are probably involved in the antitumor effect of doxycycline on SKOV3 cells, while upregulation of pro-caspase-3 may be the main mechanism involved in SKOV3/DDP cells. PMID:24598933

Inhibition of cell migration by focal adhesion kinase: Time-dependent difference in integrin-induced signaling between endothelial and hepatoblastoma cells.

PubMed

Yu, Hongchi; Gao, Min; Ma, Yunlong; Wang, Lijuan; Shen, Yang; Liu, Xiaoheng

2018-05-01

angiogenesis plays an important role in the development and progression of tumors, and it involves a series of signaling pathways contributing to the migration of endothelial cells for vascularization and to the invasion of cancer cells for secondary tumor formation. Among these pathways, the focal adhesion kinase (FAK) signaling cascade has been implicated in a variety of human cancers in connection with cell adhesion and migration events leading to tumor angiogenesis, metastasis and invasion. Therefore, the inhibition of FAK in endothelial and/or cancer cells is a potential target for anti‑angiogenic therapy. In the present study, a small‑molecule FAK inhibitor, 1,2,4,5-benzenetetramine tetrahydrochloride (Y15), was used to study the effects of FAK inhibition on the adhesion and migration behaviors of vascular endothelial cells (VECs) and human hepatoblastoma cells. Furthermore, the time-dependent differences in proteins associated with the integrin-mediated FAK/Rho GTPases signaling pathway within 2 h were examined. The results indicated that the inhibition of FAK significantly decreased the migration ability of VECs and human hepatoblastoma cells in a dose-dependent manner. Inhibition of FAK promoted cell detachment by decreasing the expression of focal adhesion components, and blocked cell motility by reducing the level of Rho GTPases. However, the expression of crucial proteins involved in integrin-induced signaling in two cell lines exhibited a time-dependent difference with increased duration of FAK inhibitor treatment, suggesting different mechanisms of FAK-mediated cell migration behavior. These results suggest that the mechanism underlying FAK-mediated adhesion and migration behavior differs among various cells, which is expected to provide evidence for future FAK therapy targeted against tumor angiogenesis.

Disturbance of DKK1 level is partly involved in survival of lung cancer cells via regulation of ROMO1 and γ-radiation sensitivity

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

Kim, In Gyu, E-mail: igkim@kaeri.re.kr; Department of Radiation Biotechnology and Applied Radioisotope, University of Science and Technology; Kim, Seo Yoen

2014-01-03

Highlights: •DKK1 was expressed differently among non-small-cell lung cancer cell lines. •DKK1 negatively regulated ROMO1 gene expression. •Disturbance of DKK1 level induced the imbalance of cellular ROS. •DKK1/ROMO1-induced ROS imbalance is involved in cell survival in NSCLC. -- Abstract: Dickkopf1 (DKK1), a secreted protein involved in embryonic development, is a potent inhibitor of the Wnt signaling pathway and has been postulated to be a tumor suppressor or tumor promoter depending on the tumor type. In this study, we showed that DKK1 was expressed differently among non-small-cell lung cancer cell lines. The DKK1 expression level was much higher in A549 cellsmore » than in H460 cells. We revealed that blockage of DKK1 expression by silencing RNA in A549 cells caused up-regulation of intracellular reactive oxygen species (ROS) modulator (ROMO1) protein, followed by partial cell death, cell growth inhibition, and loss of epithelial–mesenchymal transition property caused by ROS, and it also increased γ-radiation sensitivity. DKK1 overexpression in H460 significantly inhibited cell survival with the decrease of ROMO1 level, which induced the decrease of cellular ROS. Thereafter, exogenous N-acetylcysteine, an antioxidant, or hydrogen peroxide, a pro-oxidant, partially rescued cells from death and growth inhibition. In each cell line, both overexpression and blockage of DKK1 not only elevated p-RB activation, which led to cell growth arrest, but also inactivated AKT/NF-kB, which increased radiation sensitivity and inhibited cell growth. This study is the first to demonstrate that strict modulation of DKK1 expression in different cell types partially maintains cell survival via tight regulation of the ROS-producing ROMO1 and radiation resistance.« less

Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells

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

Thakur, Basant Kumar, E-mail: thakur.basant@mh-hannover.de; Department of Molecular Hematopoiesis, Hannover Medical School, Carl Neuberg Str-1, 30625 Hannover; Dittrich, Tino

2012-08-03

Highlights: Black-Right-Pointing-Pointer In 293T cells, p53 is considered to be inactive due to its interaction with the large T-antigen. Black-Right-Pointing-Pointer Acetylation of p53 at lysine 382 is important for its functional activation. Black-Right-Pointing-Pointer First evidence to document the presence of a functional p53 in 293T cells. Black-Right-Pointing-Pointer Inhibition of NAMPT/SIRT pathway by FK866 in 293T cells increases the functional activity of p53. Black-Right-Pointing-Pointer This activation of p53 involves reversible acetylation of p53 at lysine 382. -- Abstract: Inactivation of p53 protein by endogenous and exogenous carcinogens is involved in the pathogenesis of different human malignancies. In cancer associated with SV-40more » DNA tumor virus, p53 is considered to be non-functional mainly due to its interaction with the large T-antigen. Using the 293T cell line (HEK293 cells transformed with large T antigen) as a model, we provide evidence that p53 is one of the critical downstream targets involved in FK866-mediated killing of 293T cells. A reduced rate of apoptosis and an increased number of cells in S-phase was accompanied after knockdown of p53 in these cells. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway. Additionally, knockdown of p53 attenuated the effect of FK866 on cell proliferation, apoptosis, and cell cycle arrest. The data presented here shed light on two important facts: (1) that p53 in 293T cells is active in the presence of FK866, an inhibitor of NAMPT pathway; (2) the apoptosis induced by FK866 in 293T cells is associated with increased acetylation of p53 at Lys382, which is required for the functional activity of p53.« less

Synergistic Interactions with PI3K Inhibition that Induce Apoptosis. | Office of Cancer Genomics

Cancer.gov

Activating mutations involving the PI3K pathway occur frequently in human cancers. However, PI3K inhibitors primarily induce cell cycle arrest, leaving a significant reservoir of tumor cells that may acquire or exhibit resistance. We searched for genes that are required for the survival of PI3K mutant cancer cells in the presence of PI3K inhibition by conducting a genome scale shRNA-based apoptosis screen in a PIK3CA mutant human breast cancer cell. We identified 5 genes (PIM2, ZAK, TACC1, ZFR, ZNF565) whose suppression induced cell death upon PI3K inhibition.

Induction of apoptosis by plumbagin through reactive oxygen species-mediated inhibition of topoisomerase II

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

Kawiak, Anna; Piosik, Jacek; Stasilojc, Grzegorz

2007-09-15

Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis. Plumbagin, a naphthoquinone exhibiting antitumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topomore » II activity-HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS. In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation. These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II. Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA. The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis. Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin.« less

ROCK Inhibition Extends Passage of Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium

PubMed Central

Croze, Roxanne H.; Buchholz, David E.; Radeke, Monte J.; Thi, William J.; Hu, Qirui; Coffey, Peter J.

2014-01-01

Human embryonic stem cells (hESCs) offer a potentially unlimited supply of cells for emerging cell-based therapies. Unfortunately, the process of deriving distinct cell types can be time consuming and expensive. In the developed world, age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with more than 7.2 million people afflicted in the U.S. alone. Both hESC-derived retinal pigmented epithelium (hESC-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE) are being developed for AMD therapies by multiple groups, but their potential for expansion in culture is limited. To attempt to overcome this passage limitation, we examined the involvement of Rho-associated, coiled-coil protein kinase (ROCK) in hESC-RPE and iPSC-RPE culture. We report that inhibiting ROCK1/2 with Y-27632 allows extended passage of hESC-RPE and iPSC-RPE. Microarray analysis suggests that ROCK inhibition could be suppressing an epithelial-to-mesenchymal transition through various pathways. These include inhibition of key ligands of the transforming growth factor-β pathway (TGFB1 and GDF6) and Wnt signaling. Two important processes are affected, allowing for an increase in hESC-RPE expansion. First, ROCK inhibition promotes proliferation by inducing multiple components that are involved in cell cycle progression. Second, ROCK inhibition affects many pathways that could be converging to suppress RPE-to-mesenchymal transition. This allows hESC-RPE to remain functional for an extended but finite period in culture. PMID:25069775

ROCK Inhibition Extends Passage of Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium.

PubMed

Croze, Roxanne H; Buchholz, David E; Radeke, Monte J; Thi, William J; Hu, Qirui; Coffey, Peter J; Clegg, Dennis O

2014-09-01

Human embryonic stem cells (hESCs) offer a potentially unlimited supply of cells for emerging cell-based therapies. Unfortunately, the process of deriving distinct cell types can be time consuming and expensive. In the developed world, age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with more than 7.2 million people afflicted in the U.S. alone. Both hESC-derived retinal pigmented epithelium (hESC-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE) are being developed for AMD therapies by multiple groups, but their potential for expansion in culture is limited. To attempt to overcome this passage limitation, we examined the involvement of Rho-associated, coiled-coil protein kinase (ROCK) in hESC-RPE and iPSC-RPE culture. We report that inhibiting ROCK1/2 with Y-27632 allows extended passage of hESC-RPE and iPSC-RPE. Microarray analysis suggests that ROCK inhibition could be suppressing an epithelial-to-mesenchymal transition through various pathways. These include inhibition of key ligands of the transforming growth factor-β pathway (TGFB1 and GDF6) and Wnt signaling. Two important processes are affected, allowing for an increase in hESC-RPE expansion. First, ROCK inhibition promotes proliferation by inducing multiple components that are involved in cell cycle progression. Second, ROCK inhibition affects many pathways that could be converging to suppress RPE-to-mesenchymal transition. This allows hESC-RPE to remain functional for an extended but finite period in culture. ©AlphaMed Press.

Curcumin suppresses the TPA-induced invasion through inhibition of PKCα-dependent MMP-expression in MCF-7 human breast cancer cells.

PubMed

Kim, Jeong-Mi; Noh, Eun-Mi; Kwon, Kang-Beam; Kim, Jong-Suk; You, Yong-Ouk; Hwang, Jin-Ki; Hwang, Bo-Mi; Kim, Byeong-Soo; Lee, Sung-Hoo; Lee, Seung Jin; Jung, Sung Hoo; Youn, Hyun Jo; Lee, Young-Rae

2012-09-15

Curcumin (diferuloylmethane) is a polyphenol derived from the plant turmeric (Curcuma longa), which is commonly used as a spice. Although anti-carcinogenic, anti-oxidant, anti-inflammation, and anti-angiogenic properties have been reported, the effect of curcumin on breast cancer metastasis is unknown. Matrix metalloproteinase-9 (MMP-9) is a major component in cancer cell invasion. In this study, we investigated the inhibitory effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion and the molecular mechanisms involved in MCF-7 cells. Our results showed that curcumin inhibits TPA-induced MMP-9 expression and cell invasion through suppressing NF-κB and AP-1 activation. Also, curcumin strongly repressed the TPA-induced phosphorylation of p38 and JNK and inhibited TPA-induced translocation of PKCα from the cytosol to the membrane, but did not affect the translocation of PKCδ. These results indicate that curcumin-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the PKCα, MAPK and NF-κB/AP-1 pathway in MCF-7 cells. Curcumin may have potential value in restricting breast cancer metastasis. Copyright © 2012 Elsevier GmbH. All rights reserved.

Augmentation of Breast Cancer Growth and Metastasis by Chronic Stressor Exposure

DTIC Science & Technology

2012-07-01

good model for examining NE effects on the tumor stromal cells in the absence of direct involvement of β-AR-expressing tumor cells . In that report...SHG+ fiber content also directly inhibit tumor metastasis, perhaps through direct effects on tumor cell motility, and that this inhibition is induced... effects in different cell types within a tumor that contribute to tumor progression. Nevertheless, the overall result is a protumorigenic signal that

Insulin-like growth factor-binding protein-3 inhibits IGF-1-induced proliferation of human hepatocellular carcinoma cells by controlling bFGF and PDGF autocrine/paracrine loops

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

Ma, Yang; Han, Chen-chen; Li, Yifan

Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) produced by hepatocellular carcinoma (HCC) cells are responsible for the growth of HCC cells. Accumulating evidence shows that insulin-like growth factor-binding protein-3 (IGFBP-3) suppresses HCC cell proliferation in both IGF-dependent and independent manners. It's unknown, however, whether treatment with exogenous IGFBP-3 inhibits bFGF and PDGF production in HCC cells. The present study demonstrates that IGFBP-3 suppressed IGF-1-induced bFGF and PDGF expression while it does not affect their expression in the absence of IGF-1. To delineate the underlying mechanism, western-blot and RT-PCR assays confirmed that the transcription factor early growth responsemore » protein 1 (EGR1) is involved in IGFBP-3 regulation of bFGF and PDGF. IGFBP-3 inhibition of type 1 insulin-like growth factor receptor (IGF1R), ERK and AKT activation is IGF-1-dependent. Furthermore, transient transfection with constitutively activated AKT or MEK partially blocks the IGFBP-3 inhibition of EGR1, bFGF and PDGF expression. In conclusion, these findings suggest that IGFBP-3 suppresses transcription of EGR1 and its target genes bFGF and PDGF through inhibiting IGF-1-dependent ERK and AKT activation. It demonstrates the importance of IGFBP-3 in the regulation of HCC cell proliferation, suggesting that IGFBP-3 could be a target for the treatment of HCC. - Highlights: • IGFBP-3 plays an inhibition role in IGF1-induced HCC cell growth. • IGFBP-3 inhibits bFGF and PDGF production in the IGF-dependent manner. • EGR1 is involved in IGFBP-3 regulation of bFGF and PDGF in HCC cells. • IGFBP-3 suppresses EGR1 and its target genes bFGF and PDGF through inhibiting IGF-1-dependent ERK and AKT activation.« less

Silencing of BAG3 inhibits the epithelial-mesenchymal transition in human cervical cancer.

PubMed

Song, Fei; Wang, Geng; Ma, Zhifang; Ma, Yuebing; Wang, Yingying

2017-11-10

Bcl2-associated athanogene 3 (BAG3) has been reported to be involved in aggressive progression of many tumors. In the present study, we examined the expression of BAG3 in human cervical cancer (CC) tissues and investigated the role of BAG3 in SiHa and HeLa cell growth, migration, and invasion. Here, we found that most of CC tissues highly expressed the protein and mRNA of BAG3, while their expression was obviously lower in paired normal tissues (all p<0.001). BAG3 expression was associated with FIGO stage and metastasis (all p<0.05). In-vitro analysis demonstrated that BAG3 siRNAs inhibited SiHa and HeLa cell growth, invasion and migration. Mechanically, BAG3 siRNAs inhibited the expression of EMT-regulating markers, involving MMP2, Slug and N-cadherin, and increased the expression of E-cadherin. In a xenograft nude model, BAG3 siRNAs inhibited tumor growth and the expression of EMT biomarkers. In conclusion, BAG3 is involved in the EMT process, including cell growth, invasion and migration in the development of CC. Thus, BAG3 target might be recommended as a novel therapeutic approach.

Silencing of BAG3 inhibits the epithelial-mesenchymal transition in human cervical cancer

PubMed Central

Song, Fei; Wang, Geng; Ma, Zhifang; Ma, Yuebing; Wang, Yingying

2017-01-01

Bcl2-associated athanogene 3 (BAG3) has been reported to be involved in aggressive progression of many tumors. In the present study, we examined the expression of BAG3 in human cervical cancer (CC) tissues and investigated the role of BAG3 in SiHa and HeLa cell growth, migration, and invasion. Here, we found that most of CC tissues highly expressed the protein and mRNA of BAG3, while their expression was obviously lower in paired normal tissues (all p<0.001). BAG3 expression was associated with FIGO stage and metastasis (all p<0.05). In-vitro analysis demonstrated that BAG3 siRNAs inhibited SiHa and HeLa cell growth, invasion and migration. Mechanically, BAG3 siRNAs inhibited the expression of EMT-regulating markers, involving MMP2, Slug and N-cadherin, and increased the expression of E-cadherin. In a xenograft nude model, BAG3 siRNAs inhibited tumor growth and the expression of EMT biomarkers. In conclusion, BAG3 is involved in the EMT process, including cell growth, invasion and migration in the development of CC. Thus, BAG3 target might be recommended as a novel therapeutic approach. PMID:29221135

Grape seed extract triggers apoptosis in Caco-2 human colon cancer cells through reactive oxygen species and calcium increase: extracellular signal-regulated kinase involvement.

PubMed

Dinicola, Simona; Mariggiò, Maria Addolorata; Morabito, Caterina; Guarnieri, Simone; Cucina, Alessandra; Pasqualato, Alessia; D'Anselmi, Fabrizio; Proietti, Sara; Coluccia, Pierpaolo; Bizzarri, Mariano

2013-09-14

Grape seed extract (GSE) from Italia, Palieri and Red Globe cultivars inhibits cell growth and induces apoptosis in Caco-2 human colon cancer cells in a dose-dependent manner. In order to investigate the mechanism(s) supporting the apoptotic process, we analysed reactive oxygen species (ROS) production, intracellular Ca2+ handling and extracellular signal-regulated kinase (ERK) activation. Upon exposure to GSE, ROS and intracellular Ca2+ levels increased in Caco-2 cells, concomitantly with ERK inactivation. As ERK activity is thought to be essential for promoting survival pathways, inhibition of this kinase is likely to play a relevant role in GSE-mediated anticancer effects. Indeed, pretreatment with N-acetyl cysteine, a ROS scavenger, reversed GSE-induced apoptosis, and promoted ERK phosphorylation. This effect was strengthened by ethylene glycol tetraacetic acid-mediated inhibition of extracellular Ca2+ influx. ROS and Ca2+ influx inhibition, in turn, increased ERK phosphorylation, and hence almost entirely suppressed GSE-mediated apoptosis. These data suggested that GSE triggers a previously unrecognised ERK-based mechanism, involving both ROS production and intracellular Ca2+ increase, eventually leading to apoptosis in cancer cells.

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

Wang, Wei; Department of Infectious Diseases, Peking University Third Hospital, Beijing; Zhang, Yuan

The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a β{sub 2}-adrenergic receptor (β{sub 2}-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of β{sub 2}-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and β-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1βmore » (IL-1β) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of β-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1β (IL-1β) release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1β release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the β{sub 2}-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1β release via β-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases. - Highlights: • β{sub 2}-AR agonist fenoterol exerts its protective effect on LPS-treated THP-1 cells. • Fenoterol inhibits LPS-induced AMPK activation and IL-1β production. • β-arrestin2 mediates fenoterol-inhibited AMPK activation and IL-1β release. • AMPKα1 is involved in LPS-induced NF-κB activation and IL-1β production.« less

Berberine suppresses tumorigenicity and growth of nasopharyngeal carcinoma cells by inhibiting STAT3 activation induced by tumor associated fibroblasts.

PubMed

Tsang, Chi Man; Cheung, Yuk Chun; Lui, Vivian Wai-Yan; Yip, Yim Ling; Zhang, Guitao; Lin, Victor Weitao; Cheung, Kenneth Chat-Pan; Feng, Yibin; Tsao, Sai Wah

2013-12-31

Cortidis rhizoma (Huanglian) and its major therapeutic component, berberine, have drawn extensive attention in recent years for their anti-cancer properties. Growth inhibitory effects of berberine on multiple types of human cancer cells have been reported. Berberine inhibits invasion, induces cell cycle arrest and apoptosis in human cancer cells. The anti-inflammatory property of berberine, involving inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) activation, has also been documented. In this study, we have examined the effects of berberine on tumorigenicity and growth of nasopharyngeal carcinoma (NPC) cells and their relationship to STAT3 signaling using both in vivo and in vitro models. Berberine effectively inhibited the tumorigenicity and growth of an EBV-positive NPC cell line (C666-1) in athymic nude mice. Inhibition of tumorigenic growth of NPC cells in vivo was correlated with effective inhibition of STAT3 activation in NPC cells inside the tumor xenografts grown in nude mice. In vitro, berberine inhibited both constitutive and IL-6-induced STAT3 activation in NPC cells. Inhibition of STAT3 activation by berberine induced growth inhibition and apoptotic response in NPC cells. Tumor-associated fibroblasts were found to secret IL-6 and the conditioned medium harvested from the fibroblasts also induced STAT3 activation in NPC cells. Furthermore, STAT3 activation by conditioned medium of tumor-associated fibroblasts could be blocked by berberine or antibodies against IL-6 and IL-6R. Our observation that berberine effectively inhibited activation of STAT3 induced by tumor-associated fibroblasts suggests a role of berberine in modulating the effects of tumor stroma on the growth of NPC cells. The effective inhibition of STAT3 activation in NPC cells by berberine supports its potential use in the treatment of NPC.

Canonical TGF-β Signaling Pathway Represses Human NK Cell Metabolism.

PubMed

Zaiatz-Bittencourt, Vanessa; Finlay, David K; Gardiner, Clair M

2018-06-15

Cytokines stimulate rapid metabolic changes in human NK cells, including increases in both glycolysis and oxidative phosphorylation pathways. However, how these are subsequently regulated is not known. In this study, we demonstrate that TGF-β can inhibit many of these metabolic changes, including oxidative phosphorylation, glycolytic capacity, and respiratory capacity. TGF-β also inhibited cytokine-induced expression of the transferrin nutrient receptor CD71. In contrast to a recent report on murine NK cells, TGF-β-mediated suppression of these metabolic responses did not involve the inhibition of the metabolic regulator mTORC1. Inhibition of the canonical TGF-β signaling pathway was able to restore almost all metabolic and functional responses that were inhibited by TGF-β. These data suggest that pharmacological inhibition of TGF-β could provide a metabolic advantage to NK cells that is likely to result in improved functional responses. This has important implications for NK cell-based cancer immunotherapies. Copyright © 2018 by The American Association of Immunologists, Inc.

Adherence of Moraxella bovis to cell cultures of bovine origin.

PubMed

Annuar, B O; Wilcox, G E

1985-09-01

The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.

Mechanism of lymphocytic choriomeningitis virus entry into cells.

PubMed

Borrow, P; Oldstone, M B

1994-01-01

The path that the arenavirus lymphocytic choriomeningitis virus (LCMV) uses to enter rodent fibroblastic cell lines was dissected by infectivity and inhibition studies and immunoelectron microscopy. Lysosomotropic weak bases (chloroquine and ammonium chloride) and carboxylic ionophores (monensin and nigericin) inhibited virus entry, assessed as virus nucleoprotein expression at early times post-infection, indicating that the entry process involved a pH-dependent fusion step in intracellular vesicles. That entry occurred in vesicles rather than by direct fusion of virions with the plasma membrane was confirmed by immunoelectron microscopy. The vesicles involved were large (150-300 nm diameter), smooth-walled, and not associated with clathrin. Unlike classical phagocytosis, virus uptake in these vesicles was a microfilament-independent process, as it was not blocked by cytochalasins. LCMV entry into rodent fibroblast cell lines thus involves viropexis in large smooth-walled vesicles, followed by a pH-dependent fusion event inside the cell.

Targeting Metastasis with Snake Toxins: Molecular Mechanisms

PubMed Central

Urra, Félix A.

2017-01-01

Metastasis involves the migration of cancer cells from a primary tumor to invade and establish secondary tumors in distant organs, and it is the main cause for cancer-related deaths. Currently, the conventional cytostatic drugs target the proliferation of malignant cells, being ineffective in metastatic disease. This highlights the need to find new anti-metastatic drugs. Toxins isolated from snake venoms are a natural source of potentially useful molecular scaffolds to obtain agents with anti-migratory and anti-invasive effects in cancer cells. While there is greater evidence concerning the mechanisms of cell death induction of several snake toxin classes on cancer cells; only a reduced number of toxin classes have been reported (i.e., disintegrins/disintegrin-like proteins, C-type lectin-like proteins, C-type lectins, serinproteases, cardiotoxins, snake venom cystatins) as inhibitors of adhesion, migration, and invasion of cancer cells. Here, we discuss the anti-metastatic mechanisms of snake toxins, distinguishing three targets, which involve (1) inhibition of extracellular matrix components-dependent adhesion and migration, (2) inhibition of epithelial-mesenchymal transition, and (3) inhibition of migration by alterations in the actin/cytoskeleton network. PMID:29189742

Mosla dianthera inhibits mast cell-mediated allergic reactions through the inhibition of histamine release and inflammatory cytokine production

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

Lee, Dong-Hee; Kim, Sang-Hyun; Eun, Jae-Soon

2006-11-01

In this study, we investigated the effect of the aqueous extract of Mosla dianthera (Maxim.) (AEMD) on the mast cell-mediated allergy model and studied the possible mechanism of action. Mast cell-mediated allergic disease is involved in many diseases such as asthma, sinusitis and rheumatoid arthritis. The discovery of drugs for the treatment of allergic disease is an important subject in human health. AEMD inhibited compound 48/80-induced systemic reactions in mice. AEMD decreased immunoglobulin E-mediated local allergic reactions, passive cutaneous anaphylaxis. AEMD attenuated intracellular calcium level and release of histamine from rat peritoneal mast cells activated by compound 48/80. Furthermore, AEMDmore » attenuated the phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated TNF-{alpha}, IL-8 and IL-6 secretion in human mast cells. The inhibitory effect of AEMD on the pro-inflammatory cytokines was nuclear factor-{kappa}B (NF-{kappa}B) dependent. AEMD decreased PMA and A23187-induced degradation of I{kappa}B{alpha} and nuclear translocation of NF-{kappa}B. Our findings provide evidence that AEMD inhibits mast cell-derived immediate-type allergic reactions and involvement of pro-inflammatory cytokines and NF-{kappa}B in these effects.« less

Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

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

Li, Jie; Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen; Yang, Xi-fei

2014-10-10

Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we exploremore » the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.« less

Metformin inhibits epithelial–mesenchymal transition in prostate cancer cells: Involvement of the tumor suppressor miR30a and its target gene SOX4

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

Zhang, Jing; Shen, Chengwu; Wang, Lin

2014-09-26

Highlights: • Metformin inhibits TGF-β-induced EMT in prostate cancer (PCa) cells. • Metformin upregulates tumor suppressor miR30a and downregulates SOX4 in PCa cells. • SOX4 is a target gene of miR30a. - Abstract: Tumor metastasis is the leading cause of mortality and morbidity of prostate cancer (PCa) patients. Epithelial–mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. Recent evidence suggested that diabetic patients treated with metformin have lower PCa risk and better prognosis. This study was aimed to investigate the effects of metformin on EMT in PCa cells and the possible microRNA (miRNA)-based mechanisms. MiRNAs have beenmore » shown to regulate various processes of cancer metastasis. We herein showed that metformin significantly inhibits proliferation of Vcap and PC-3 cells, induces G0/G1 cell cycle arrest and inhibits invasiveness and motility capacity of Vcap cells. Metformin could inhibit TGF-β-induced EMT in Vcap cells, as manifested by inhibition of the increase of N-cadherin (p = 0.013), Vimentin (p = 0.002) and the decrease of E-cadherin (p = 0.0023) and β-catenin (p = 0.034) at mRNA and protein levels. Notably, we demonstrated significant upregulation of miR30a levels by metformin (P < 0.05) and further experiments indicated that miR30a significantly inhibits proliferation and EMT process of Vcap cells. Interestingly, we identified that SOX4, a previously reported oncogenic transcriptional factor and modulator of EMT, is a direct target gene of miR30a. Finally, we screened the expression of miR30a and SOX4 in 84 PCa cases with radical prostatectomy. Of note, SOX4 overexpression is significantly associated with decreased levels of miR30a in PCa cases. In all, our study suggested that inhibition of EMT by metformin in PCa cells may involve upregulation of miR30a and downregulation of SOX4.« less

Centchroman inhibits proliferation of head and neck cancer cells through the modulation of PI3K/mTOR Pathway

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

Srivastava, Vikas Kumar; Gara, Rishi Kumar; Bhatt, M.L.B.

Research highlights: {yields} Centchroman (CC) inhibits cellular proliferation in HNSCC cells through the dual inhibition of PI3/mTOR pathway. {yields} CC treatment also inhibits STAT3 activation and alters expression of proteins involved in cell cycle regulation and DNA repair response in HNSCC cells. {yields} CC exhibits anti-proliferative activity in a variety of non-HNSCC cancer cell lines and is devoid of cytotoxicity to normal cell types of diverse origins. -- Abstract: Centchroman (CC; 67/20; INN: Ormeloxifene) is a non-steroidal antiestrogen extensively used as a female contraceptive in India. In the present study, we report the anti-proliferative effect of CC in head andmore » neck squamous cell carcinoma (HNSCC) cells. CC inhibited cell proliferation in a dose dependent manner at 24 h of treatment. Further studies showed that CC treatment induced apoptosis, inhibited Akt/mTOR and signal transducers and activators of transcription protein 3 (STAT3) signaling, altered proteins associated with cell cycle regulation and DNA damage and inhibited colony forming efficiency of HNSCC cells. In addition, CC displayed anti-proliferative activity against a variety of non-HNSCC cell lines of diverse origin. The ability of CC to serve as a dual-inhibitor of Akt/mTOR and STAT3 signaling warrants further studies into its role as a therapeutic strategy against HNSCC.« less

Progressive Inhibition by Water Deficit of Cell Wall Extensibility and Growth along the Elongation Zone of Maize Roots Is Related to Increased Lignin Metabolism and Progressive Stelar Accumulation of Wall Phenolics1

PubMed Central

Fan, Ling; Linker, Raphael; Gepstein, Shimon; Tanimoto, Eiichi; Yamamoto, Ryoichi; Neumann, Peter M.

2006-01-01

Water deficit caused by addition of polyethylene glycol 6000 at −0.5 MPa water potential to well-aerated nutrient solution for 48 h inhibited the elongation of maize (Zea mays) seedling primary roots. Segmental growth rates in the root elongation zone were maintained 0 to 3 mm behind the tip, but in comparison with well-watered control roots, progressive growth inhibition was initiated by water deficit as expanding cells crossed the region 3 to 9 mm behind the tip. The mechanical extensibility of the cell walls was also progressively inhibited. We investigated the possible involvement in root growth inhibition by water deficit of alterations in metabolism and accumulation of wall-linked phenolic substances. Water deficit increased expression in the root elongation zone of transcripts of two genes involved in lignin biosynthesis, cinnamoyl-CoA reductase 1 and 2, after only 1 h, i.e. before decreases in wall extensibility. Further increases in transcript expression and increased lignin staining were detected after 48 h. Progressive stress-induced increases in wall-linked phenolics at 3 to 6 and 6 to 9 mm behind the root tip were detected by comparing Fourier transform infrared spectra and UV-fluorescence images of isolated cell walls from water deficit and control roots. Increased UV fluorescence and lignin staining colocated to vascular tissues in the stele. Longitudinal bisection of the elongation zone resulted in inward curvature, suggesting that inner, stelar tissues were also rate limiting for root growth. We suggest that spatially localized changes in wall-phenolic metabolism are involved in the progressive inhibition of wall extensibility and root growth and may facilitate root acclimation to drying environments. PMID:16384904

Cloning of B cell-specific membrane tetraspanning molecule BTS possessing B cell proliferation-inhibitory function.

PubMed

Suenaga, Tadahiro; Arase, Hisashi; Yamasaki, Sho; Kohno, Masayuki; Yokosuka, Tadashi; Takeuchi, Arata; Hattori, Takamichi; Saito, Takashi

2007-11-01

Lymphocyte proliferation is regulated by signals through antigen receptors, co-stimulatory receptors, and other positive and negative modulators. Several membrane tetraspanning molecules are also involved in the regulation of lymphocyte growth and death. We cloned a new B cell-specific tetraspanning (BTS) membrane molecule, which is similar to CD20 in terms of expression, structure and function. BTS is specifically expressed in the B cell line and its expression is increased after the pre-B cell stage. BTS is expressed in intracellular granules and on the cell surface. Overexpression of BTS in immature B cell lines induces growth retardation through inhibition of cell cycle progression and cell size increase without inducing apoptosis. This inhibitory function is mediated predominantly by the N terminus of BTS. The development of mature B cells is inhibited in transgenic mice expressing BTS, suggesting that BTS is involved in the in vivo regulation of B cells. These results indicate that BTS plays a role in the regulation of cell division and B cell growth.

Human leptin protein activates the growth of HepG2 cells by inhibiting PERK‑mediated ER stress and apoptosis.

PubMed

Xiong, Ying; Zhang, Jie; Liu, Man; An, Mingwei; Lei, Ling; Guo, Wuhua

2014-09-01

Current treatment modalities for various types of hepatic cancer, which has an increasing incidence rate, are inadequate and novel therapies are required. Therefore, identifying targets for liver cancer is becoming increasingly valuable to develop novel methods for therapy. The aim of the present study was to examine the growth activation mechanism of the leptin protein in the liver cancer cell line HepG2. The effects of the leptin protein on cell death were investigated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide analysis. DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling analysis were also performed to detect cell apoptosis. The expression of leptin and three endoplasmic reticulum (ER) stress unfolded protein response (UPR) proteins, including activating transcription factor 6, phosphorylated‑PKR‑like ER kinase (p‑PERK) and inositol requiring protein 1, were investigated for the examination of ER stress. The mRNA UPR proteins were also detected by reverse transcription polymerase chain reaction. The apoptosis‑associated caspase 12 and C/EBP homologous protein (CHOP) was detected by western blot analysis. The expression of or incubation with the leptin protein was able to activate cell growth and inhibit cell death and apoptosis. In cells that expressed leptin or were incubated with leptin protein (pep-LPT), cisplatin‑induced ER stress‑associated mRNA transcription and protein activation were inhibited. Levels of the ER stress UPR pathway protein, PERK, increased significantly in leptin‑silenced cells when treated with cisplatin as compared with those in the leptin‑expressing or pep-LPT cells. Furthermore, caspase 12 activation was inhibited in ex‑LPT, pep‑LPT and HepG2 cells. In conclusion, human leptin protein is involved in promoting the proliferation of HepG2 cells through inhibiting the ER stress‑associated apoptotic pathway. The PERK UPR pathway and the apoptotic factor caspase 12 were found to be involved in the inhibition of apoptosis and enhancement of proliferation.

The adaptor SASH1 acts through NOTCH1 and its inhibitor DLK1 in a 3D model of lumenogenesis involving CEACAM1.

PubMed

Stubblefield, Kandis; Chean, Jennifer; Nguyen, Tung; Chen, Charng-Jui; Shively, John E

2017-10-15

CEACAM1 transfection into breast cancer cells restores lumen formation in a 3D culture model. Among the top up-regulated genes that were associated with restoration of lumen formation, the adaptor protein SASH1 was identified. Furthermore, SASH1 was shown to be critical for lumen formation by RNAi inhibition. Upon analyzing the gene array from CEACAM1/MCF7 cells treated with SASH1 RNAi, DLK1, an inhibitor of NOTCH1 signaling, was found to be down-regulated to the same extent as SASH1. Subsequent treatment of CEACAM1/MCF7 cells with RNAi to DLK1 also inhibited lumen formation, supporting its association with SASH1. In agreement with the role of DLK1 as a NOTCH1 inhibitor, NOTCH1, as well as its regulated genes HES1 and HEY1, were down-regulated in CEACAM1/MCF7 cells by the action of DLK1 RNAi, and up-regulated by SASH1 RNAi. When CEACAM1/MCF7 cells were treated with a γ-secretase inhibitor known to inhibit NOTCH signaling, lumen formation was inhibited. We conclude that restoration of lumen formation by CEACAM1 regulates the NOTCH1 signaling pathway via the adaptor protein SASH1 and the NOTCH1 inhibitor DLK1. These data suggest that the putative involvement of NOTCH1 as a tumor-promoting gene in breast cancer may depend on its lack of regulation in cancer, whereas its involvement in normal lumen formation requires activation of its expression, and subsequently, inhibition of its signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Tetrahydroxystilbene glucoside improves TNF-α-induced endothelial dysfunction: involvement of TGFβ/Smad pathway and inhibition of vimentin expression.

PubMed

Yao, Wenjuan; Gu, Chengjing; Shao, Haoran; Meng, Guoliang; Wang, Huiming; Jing, Xiang; Zhang, Wei

2015-01-01

Endothelial dysfunction plays an important role in the pathogenesis of atherogenesis. 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), an active component of the rhizome extract from Polygonum multiflorum (PM), exhibits significant anti-atherosclerotic activity. Here, we used human umbilical vein endothelial cells (HUVECs) induced by tumor necrosis factor-α (TNF-α) in vitro to investigate the cytoprotective effects of TSG on TNF-α-induced endothelial injury and the related mechanisms. Pretreatment with 50 and 100 μM TSG markedly attenuated TNF-α-induced loss of cell viability and release of lactate dehydrogenase (LDH) and inhibited TNF-α-induced cell apoptosis. The inhibition of vimentin expression was involved in the cytoprotection afforded by TSG. Using inhibitors for PI3K and TGFβ or siRNA for Akt and Smad2, we found that vimentin production in HUVECs is regulated by TGFβ/Smad signaling, but not by PI3K-Akt-mTOR signaling. Meanwhile, TSG inhibited both the expression of TGFβ1 and the phosphorylation of Smad2 and Smad3, and TSG suppressed the nuclear translocation of Smad4 induced by TNF-α. These results suggest that TSG protects HUVECs against TNF-α-induced cell damage by inhibiting vimentin expression via the interruption of the TGFβ/Smad signaling pathway.

Targeting of the BLT2 in chronic myeloid leukemia inhibits leukemia stem/progenitor cell function

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

Xiao, Meifang; Ai, Hongmei; Li, Tao

Imatinib, a tyrosine kinase inhibitor (TKI) has significantly improved clinical outcome for chronic myeloid leukemia (CML) patients. However, patients develop resistance when the disease progresses to the blast phase (BP) and the mechanisms are not well understood. Here we show that BCR-ABL activates BLT2 in hematopoietic stem/progenitor cells to promote leukemogenesis and this involves the p53 signaling pathway. Compared to normal bone marrow (NBM), the mRNA and protein levels of BLT2 are significantly increased in BP-CML CD34{sup +} stem/progenitor cells. This is correlated with increasing BCR-ABL expression. In contrast, knockdown of BCR-ABL or inhibition of its tyrosine kinase activity decreasesmore » Blt2 protein level. BLT2 inhibition induces apoptosis, inhibits proliferation, colony formation and self-renewal capacity of CD34{sup +} cells from TKI-resistant BP-CML patients. Importantly, the inhibitory effects of BCR-ABL TKI on CML stem/progenitor cells are further enhanced upon combination with BLT2 inhibition. We further show that BLT2 activation selectively suppresses p53 but not Wnt or BMP-mediated luciferase activity and transcription. Our results demonstrate that BLT2 is a novel pathway activated by BCR-ABL and critically involved in the resistance of BP-CML CD34{sup +} stem/progenitors to TKIs treatment. Our findings suggest that BLT2 and p53 can serve as therapeutic targets for CML treatment. - Highlights: • BCR-ABL regulates BLT2 expression to promote leukemogenesis. • BLT2 is essential to maintain CML cell function. • Activation of BLT2 suppresses p53 signaling pathway in CML cells. • Inhibition of BLT2 and BCR-ABL synergize in eliminating CML CD34{sup +} stem/progenitors.« less

Zoledronic Acid Inhibits Aromatase Activity and Phosphorylation: Potential Mechanism for Additive Zoledronic Acid and Letrozole Drug Interaction

PubMed Central

Schech, Amanda J.; Nemieboka, Brandon E.; Brodie, Angela H.

2012-01-01

Zoledronic acid (ZA), a bisphosphonate originally indicated for use in osteoporosis, has been reported to exert a direct effect on breast cancer cells, although the mechanism of this effect is currently unknown. Data from the ABCSG-12 and ZO-FAST clinical trials suggest that treatment with the combination of ZA and aromatase inhibitors (AI) result in increased disease free survival in breast cancer patients over AI alone. To determine whether the mechanism of this combination involved inhibition of aromatase, AC-1 cells (MCF-7 human breast cancer cells transfected with an aromatase construct) were treated simultaneously with combinations of ZA and AI letrozole for 72 hours. This combination significantly increased inhibition of aromatase activity of AC-1 cells by compared to letrozole alone. Combination treatment of 1nM letrozole and 1μM and 10μM zoledronic acid resulted in an additive drug interaction on inhibiting cell viability, as measured by MTT assay. Treatment with ZA was found to inhibit phosphorylation of aromatase on serine 473. Zoledronic acid was also shown to be more effective in inhibiting cell viability in aromatase transfected AC-1 cells when compared to inhibition of cell viability observed in non-transfected MCF-7. Estradiol was able to partially rescue the effect of 1μM and 10μM ZA on cell viability following treatment for 72 hours, as shown by a shift to the right in the estradiol dose response curve. In conclusion, these results indicate that the combination of ZA and letrozole results in an additive inhibition of cell viability. Furthermore, ZA alone can inhibit aromatase activity through inhibition of serine phosphorylation events important for aromatase enzymatic activity and contributes to inhibition of cell viability. PMID:22659283

DNA and origin region segregation are not affected by the transition from rod to sphere after inhibition of Escherichia coli MreB by A22.

PubMed

Karczmarek, Aneta; Martínez-Arteaga, Rocío; Baselga, Rocío Martínez-Arteaga; Alexeeva, Svetlana; Hansen, Flemming G; Vicente, Miguel; Nanninga, Nanne; den Blaauwen, Tanneke

2007-07-01

The bacterial actin homologue MreB forms a helix underneath the cytoplasmic membrane and was shown to be essential in the morphogenesis of the rod-shaped cells. Additionally, MreB was implicated to be involved in DNA segregation. However, in our hands the mreBCD deletion strain (PA340-678) grew without apparent DNA segregation defect, suggesting that the reported chromosome segregation inhibition could be caused by a temporarily effect of MreB inhibition or depletion. To assess the involvement of MreB in DNA segregation during the transition from rod to sphere, we compared the effect of A22 and the PBP2 inhibitor mecillinam on the percentage of cells with segregated nucleoids and the number of oriC foci in wild-type Escherichia coli cells. Cells became spherical in the same time window during both treatments and we could not detect any difference in the chromosome or oriC segregation between these two treatments. Additionally, flow cytometric analyses showed that A22 and mecillinam treatment gave essentially the same chromosome segregation pattern. We conclude that MreB is not directly involved in DNA segregation of E. coli.

L-Asparaginase II Produced by Salmonella Typhimurium Inhibits T Cell Responses and Mediates Virulence

PubMed Central

Kullas, Amy L.; McClelland, Michael; Yang, Hee-Jeong; Tam, Jason W.; Torres, AnnMarie; Porwollik, Steffen; Mena, Patricio; McPhee, Joseph B.; Bogomolnaya, Lydia; Andrews-Polymenis, Helene; van der Velden, Adrianus W.M.

2013-01-01

SUMMARY Salmonella enterica serovar Typhimurium avoids clearance by the host immune system by suppressing T cell responses; however, the mechanisms that mediate this immunosuppression remain unknown. We show that S. Typhimurium inhibit T cell responses by producing L-Asparaginase II, which catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia. L-Asparaginase II is necessary and sufficient to suppress T cell blastogenesis, cytokine production, and proliferation and to downmodulate expression of the T cell receptor. Furthermore, S. Typhimurium-induced inhibition of T cells in vitro is prevented upon addition of L-asparagine. S. Typhimurium lacking the L-Asparaginase II gene (STM3106) are unable to inhibit T cell responses and exhibit attenuated virulence in vivo. L-Asparaginases are used to treat acute lymphoblastic leukemia through mechanisms that likely involve amino acid starvation of leukemic cells, and these findings indicate that pathogens similarly use L-asparagine deprivation to limit T cell responses. PMID:23245323

Inhibiting prenylation augments chemotherapy efficacy in renal cell carcinoma through dual inhibition on mitochondrial respiration and glycolysis.

PubMed

Huang, Jiangrong; Yang, Xiaoyu; Peng, Xiaochun; Huang, Wei

2017-11-18

Prenylation is a posttranslational lipid modification required for the proper functions of a number of proteins involved in cell regulation. Here, we show that prenylation inhibition is important for renal cell carcinoma (RCC) growth, survival and response to chemotherapy, and its underlying mechanism may be contributed to mitochondrial dysfunction. We first demonstrated that a HMG-CoA reductase inhibitor pitavastatin inhibited mevalonate pathway and thereby prenylation in RCC cells. In addition, pitavastatin is effective in inhibiting growth and inducing apoptosis in a panel of RCC cell lines. Combination of pitavastatin and paclitaxel is significantly more effective than pitavastatin or paclitaxel alone as shown by both in vitro cell culture system and in vivo RCC xenograft model. Importantly, pitavastatin treatment inhibits mitochondrial respiration via suppressing mitochondrial complex I and II enzyme activities. Interestingly, different from mitochondrial inhibitor phenformin that inhibits mitochondrial respiration but activates glycolytic rate in RCC cells, pitavastatin significantly decreases glycolytic rate. The dual inhibitory action of pitavastatin on mitochondrial respiration and glycolysis results in remarkable energy depletion and oxidative stress in RCC cells. In addition, inhibition of prenylation by depleting Isoprenylcysteine carboxylmethyltransferase (Icmt) also mimics the inhibitory effects of pitavastatin in RCC cells. Our work demonstrates the previously unappreciated association between prenylation inhibition and energy metabolism in RCC, which can be therapeutically exploited, likely in tumors that largely rely on energy metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

Osthole Induces Cell Cycle Arrest and Inhibits Migration and Invasion via PTEN/Akt Pathways in Osteosarcoma.

PubMed

Wang, Lu; Yang, Lei; Lu, Ying; Chen, Yingzhun; Liu, Tianhua; Peng, Yanli; Zhou, Yuhong; Cao, Yang; Bi, Zhenggang; Liu, Tianyi; Liu, Zhenhong; Shan, Hongli

2016-01-01

Osteosarcoma is the second highest cause of cancer-related death in children and adolescents. Majority of osteosarcoma patients (90%) show metastasis. Previous reports revealed that osthole showed antitumor activities via induction of apoptosis and inhibition of proliferation. However, the potential effects and detailed molecular mechanisms involved remained unclear. Cell viability was analyzed by MTT assay in osteosarcoma cell lines MG-63 and SAOS-2. Cell cycle was detected by flow cytometry. The effects of migration and invasion were evaluated by wound healing assay and transwell assays. Moreover, the level of proteins expression was determined by Western blot. The cell viability of MG63 and SAOS-2 were markedly inhibited by osthole in a dose- and time-dependent manner. Cell cycle was arrested and the ability of migration and invasion was obviously reduced when cells were exposed to osthole. Moreover, enzymes involved in PTEN/Akt pathway were regulated such as PTEN and p-Akt proteins. Furthermore, osthole inhibited the tumor growth in vivo. Our study unraveled, for the first time, the ability of osthole to suppress osteosarcoma and elucidated the regulation of PTEN/Akt pathway as a signaling mechanism for the anti-tumor action of osthole. These findings indicate that osthole may represent a novel therapeutic strategy in the treatment of osteosarcoma. © 2016 The Author(s) Published by S. Karger AG, Basel.

Menadione induces G2/M arrest in gastric cancer cells by down-regulation of CDC25C and proteasome mediated degradation of CDK1 and cyclin B1

PubMed Central

Lee, Min Ho; Cho, Yoonjung; Kim, Do Hyun; Woo, Hyun Jun; Yang, Ji Yeong; Kwon, Hye Jin; Yeon, Min Ji; Park, Min; Kim, Sa-Hyun; Moon, Cheol; Tharmalingam, Nagendran; Kim, Tae Ue; Kim, Jong-Bae

2016-01-01

Menadione (vitamin K3) has been reported to induce apoptotic cell death and growth inhibition in various types of cancer cells. However, involvement of menadione in cell cycle control has not been considered in gastric cancer cells yet. In the current study, we have investigated whether menadione is involved in the cell cycle regulation and suppression of growth in gastric cancer cells. In the cell cycle analysis, we found that menadione induced G2/M cell cycle arrest in AGS cells. To elucidate the underlying mechanism, we investigated the cell cycle regulatory molecules involved in the G2/M cell cycle transition. After 24 h of menadione treatment, the protein level of CDK1, CDC25C and cyclin B1 in AGS cells was decreased in a menadione dose-dependent manner. In the time course experiment, the protein level of CDC25C decreased in 6 h, and CDK1and cyclin B1 protein levels began to decrease after 18 h of menadione treatment. We found that mRNA level of CDC25C decreased by menadione treatment in 6 h. Menadione did not have an influence on mRNA level of CDK1 and cyclin B1 though the protein levels were decreased. However, the decreased protein levels of CDK1 and cyclin B1 were recovered by inhibition of proteasome. Collectively, these results suggest that menadione inhibits growth of gastric cancer cells by reducing expression of CDC25C and promoting proteasome mediated degradation of CDK1 and cyclin B1 thereby blocking transition of the cell cycle from G2 phase to M phase. PMID:28077999

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

PubMed

Smith, M Ryan; Vayalil, Praveen K; Zhou, Fen; Benavides, Gloria A; Beggs, Reena R; Golzarian, Hafez; Nijampatnam, Bhavitavya; Oliver, Patsy G; Smith, Robin A J; Murphy, Michael P; Velu, Sadanandan E; Landar, Aimee

2016-08-01

Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

p53 suppresses hyper-recombination by modulating BRCA1 function

PubMed Central

Dong, Chao; Zhang, Fengmei; Luo, Yue; Wang, Hui; Zhao, Xipeng; Guo, Gongshe; Powell, Simon N.; Feng, Zhihui

2015-01-01

Both p53 and BRCA1 are tumor suppressors and are involved in a number of cellular processes including cell cycle arrest, apoptosis, transcriptional regulation, and DNA damage repair. Some studies have suggested that the association of BRCA1 and p53 is required for transcriptional regulation of genes involved in cell replication and DNA repair pathways. However, the relationship between the two proteins in molecular mechanisms of DNA repair is still not clear. Therefore, we sought to determine whether there is a functional link between p53 and BRCA1 in DNA repair. Firstly, using a plasmid recombination substrate, pDR-GFP, integrated into the genome of breast cancer cell line MCF7, we have demonstrated that p53 suppressed Rad51-mediated hyper-recombinational repair by two independent cell models of HPV-E6 induced p53 inactivation and p53 knockdown assay. Our study further indicated that p53 mediated homologous recombination (HR) through inhibiting BRCA1 over-function via mechanism of transcription regulation in response to DNA repair. Since it was found p53 and BRCA1 existed in a protein complex, indicating both proteins may be associated at post-transcriptional level. Moreover, defective p53-induced hyper-recombination was associated with cell radioresistance and chromosomal stability, strongly supporting the involvement of p53 in the inhibition of hyper-recombination, which led to genetic stability and cellular function in response to DNA damage. In addition, it was found that p53 loss rescued BRCA1 deficiency via recovering HR and chromosomal stability, suggesting that p53 is also involved in the HR-inhibition independently of BRCA1. Thus, our data indicated that p53 was involved in inhibiting recombination by both BRCA1-dependent and -independent mechanisms, and there is a functional link between p53-suppression and BRCA1-promotion in regulation of HR activity at transcription level and possible post-transcription level. PMID:26162908

Lysophosphatidylcholine-induced cytotoxicity in osteoblast-like MG-63 cells: involvement of transient receptor potential vanilloid 2 (TRPV2) channels.

PubMed

Fallah, Abdallah; Pierre, Rachel; Abed, Elie; Moreau, Robert

2013-01-01

Epidemiological studies indicate that patients suffering from atherosclerosis are predisposed to develop osteoporosis. Accordingly, atherogenic determinants such as oxidized low density lipoprotein (OxLDL) particles have been shown to alter bone cell functions. In this work, we investigated the cytotoxicity of lysophosphatidylcholine (lysoPC), a major phospholipid component generated upon LDL oxidation, on bone-forming MG-63 osteoblast-like cells. Cell viability was reduced by lysoPC in a concentration-dependent manner with a LC50 of 18.7±0.7 μM. LysoPC-induced cell death was attributed to induction of both apoptosis and necrosis. Since impairment of intracellular calcium homeostasis is often involved in mechanism of cell death, we determined the involvement of calcium in lysoPC-induced cytotoxicity. LysoPC promoted a rapid and transient increase in intracellular calcium attributed to mobilization from calcium stores, followed by a sustained influx. Intracellular calcium mobilization was associated to phospholipase C (PLC)-dependent mobilization of calcium from the endoplasmic reticulum since inhibition of PLC or calcium depletion of reticulum endoplasmic with thapsigargin prevented the calcium mobilization. The calcium influx induced by lysoPC was abolished by inhibition of transient receptor potential vanilloid (TRPV) channels with ruthenium red whereas gadolinium, which inhibits canonical TRP (TRPC) channels, was without effect. Accordingly, expression of TRPV2 and TRPV4 were shown in MG-63 cells. The addition of TRPV2 inhibitor Tranilast in the incubation medium prevent the calcium influx triggered by lysoPC and reduced lysoPC-induced cytotoxicity whereas TRPV4 inhibitor RN 1734 was without effect, which confirms the involvement of TRPV2 activation in lysoPC-induced cell death.

Tumor suppressor NDRG2 inhibits glycolysis and glutaminolysis in colorectal cancer cells by repressing c-Myc expression

PubMed Central

Chu, Dake; Wei, Li; Li, Xia; Yang, Guodong; Liu, Xinping; Yao, Libo; Zhang, Jian; Shen, Lan

2015-01-01

Cancer cells use glucose and glutamine as the major sources of energy and precursor intermediates, and enhanced glycolysis and glutamimolysis are the major hallmarks of metabolic reprogramming in cancer. Oncogene activation and tumor suppressor gene inactivation alter multiple intracellular signaling pathways that affect glycolysis and glutaminolysis. N-Myc downstream regulated gene 2 (NDRG2) is a tumor suppressor gene inhibiting cancer growth, metastasis and invasion. However, the role and molecular mechanism of NDRG2 in cancer metabolism remains unclear. In this study, we discovered the role of the tumor suppressor gene NDRG2 in aerobic glycolysis and glutaminolysis of cancer cells. NDRG2 inhibited glucose consumption and lactate production, glutamine consumption and glutamate production in colorectal cancer cells. Analysis of glucose transporters and the catalytic enzymes involved in glycolysis revealed that glucose transporter 1 (GLUT1), hexokinase 2 (HK2), pyruvate kinase M2 isoform (PKM2) and lactate dehydrogenase A (LDHA) was significantly suppressed by NDRG2. Analysis of glutamine transporter and the catalytic enzymes involved in glutaminolysis revealed that glutamine transporter ASC amino-acid transporter 2 (ASCT2) and glutaminase 1 (GLS1) was also significantly suppressed by NDRG2. Transcription factor c-Myc mediated inhibition of glycolysis and glutaminolysis by NDRG2. More importantly, NDRG2 inhibited the expression of c-Myc by suppressing the expression of β-catenin, which can transcriptionally activate C-MYC gene in nucleus. In addition, the growth and proliferation of colorectal cancer cells were suppressed significantly by NDRG2 through inhibition of glycolysis and glutaminolysis. Taken together, these findings indicate that NDRG2 functions as an essential regulator in glycolysis and glutaminolysis via repression of c-Myc, and acts as a suppressor of carcinogenesis through coordinately targeting glucose and glutamine transporter, multiple catalytic enzymes involved in glycolysis and glutaminolysis, which fuels the bioenergy and biomaterials needed for cancer proliferation and progress. PMID:26317652

The nucleus raphe magnus OFF-cells are involved in diffuse noxious inhibitory controls.

PubMed

Chebbi, R; Boyer, N; Monconduit, L; Artola, A; Luccarini, P; Dallel, R

2014-06-01

Diffuse noxious inhibitory controls (DNIC) are very powerful long-lasting descending inhibitory controls which are pivotal in modulating the activity of spinal and trigeminal nociceptive neurons. DNIC are subserved by a loop involving supraspinal structures such as the lateral parabrachial nucleus and the subnucleus reticularis dorsalis. Surprisingly, though, whether the nucleus raphe magnus (NRM), another supraspinal area which is long known to be important in pain modulation, is involved in DNIC is still a matter of discussion. Here, we reassessed the role of the NRM neurons in DNIC by electrophysiologically recording from wide dynamic range (WDR) neurons in the trigeminal subnucleus oralis and pharmacologically manipulating the NRM OFF- and ON-cells. In control conditions, C-fiber-evoked responses in trigeminal WDR neurons are inhibited by a conditioning noxious heat stimulation applied to the hindpaw. We show that inactivating the NRM by microinjecting the GABAA receptor agonist, muscimol, both facilitates C-fiber-evoked responses of trigeminal WDR neurons and strongly attenuates their inhibition by heat applied to the hindpaw. Interestingly, selective blockade of ON-cells by microinjecting the broad-spectrum excitatory amino acid antagonist, kynurenate, into the NRM neither affects C-fiber-evoked responses nor attenuates DNIC of trigeminal WDR neurons. These results indicate that the NRM tonically inhibits trigeminal nociceptive inputs and is involved in the neuronal network underlying DNIC. Moreover, within NRM, OFF-cells might be more specifically involved in both the tonic and phasic descending inhibitory controls of trigeminal nociception. Copyright © 2014 Elsevier Inc. All rights reserved.

Gallic acid inhibits gastric cancer cells metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity

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

Ho, Hsieh-Hsun; Chang, Chi-Sen; Division of Gastroenterology, Taichung Veterans General Hospital, Taichung 402, Taiwan

2013-01-01

Our previous study demonstrated the therapeutic potential of gallic acid (GA) for controlling tumor metastasis through its inhibitory effect on the motility of AGS cells. A noteworthy finding in our previous experiment was increased RhoB expression in GA-treated cells. The aim of this study was to evaluate the role of RhoB expression on the inhibitory effects of GA on AGS cells. By applying the transfection of RhoB siRNA into AGS cells and an animal model, we tested the effect of GA on inhibition of tumor growth and RhoB expression. The results confirmed that RhoB-siRNA transfection induced GA to inhibit AGSmore » cells’ invasive growth involving blocking the AKT/small GTPase signals pathway and inhibition of NF-κB activity. Finally, we evaluated the effect of GA on AGS cell metastasis by colonization of tumor cells in nude mice. It showed GA inhibited tumor cells growth via the expression of RhoB. These data support the inhibitory effect of GA which was shown to inhibit gastric cancer cell metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity. Thus, GA might be a potential agent in treating gastric cancer. Highlights: ► GA could downregulate AKT signal via increased expression of RhoB. ► GA inhibits metastasis in vitro in gastric carcinoma. ► GA inhibits tumor growth in nude mice model.« less

Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis

USDA-ARS?s Scientific Manuscript database

Shiga toxin 1, exotoxin A, diphtheria toxin and ricin are all AB-type protein toxins that act within the host cytosol to kill the host cell through a pathway involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. In...

Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

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

Shin, Youn Ho; Lee, Seo Jin; Jung, Junyang, E-mail: jjung@khu.ac.kr

Highlights: Black-Right-Pointing-Pointer ATP-treated sciatic explants shows the decreased expression of p75NGFR. Black-Right-Pointing-Pointer Extracellular ATP inhibits the expression of phospho-ERK1/2. Black-Right-Pointing-Pointer Lysosomal exocytosis is involved in Schwann cell dedifferentiation. Black-Right-Pointing-Pointer Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibitsmore » Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.« less

Lactate dehydrogenase downregulation mediates the inhibitory effect of diallyl trisulfide on proliferation, metastasis, and invasion in triple-negative breast cancer.

PubMed

Cheng, Shi-Yann; Yang, Yao-Chih; Ting, Kuan-Lun; Wen, Su-Ying; Viswanadha, Vijaya Padma; Huang, Chih-Yang; Kuo, Wei-Wen

2017-04-01

The Warburg effect plays a critical role in tumorigenesis, suggesting that specific agents targeting Warburg effect key proteins may be a promising strategy for cancer therapy. Previous studies have shown that diallyl trisulfide (DATS) inhibits proliferation of breast cancer cells by inducing apoptosis in vitro and in vivo. However, whether the Warburg effect is involved with the apoptosis-promoting action of DATS is unclear. Here, we show that the action of DATS is associated with downregulation of lactate dehydrogenase A (LDHA), an essential protein of the Warburg effect whose upregulation is closely related to tumorigenesis. Interestingly, inhibition of the Warburg effect by DATS in breast cancer cells did not greatly affect normal cells. Furthermore, DATS inhibited growth of breast cancer cells, particularly in MDA-MB-231, a triple-negative breast cancer (TNBC) cell, and reduced proliferation and migration; invasion was reversed by over-expression of LDHA. These data suggest that DATS inhibits breast cancer growth and aggressiveness through a novel pathway targeting the key enzyme of the Warburg effect. Our study shows that LDHA downregulation is involved in the apoptotic effect of DATS on TNBC. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1390-1398, 2017. © 2016 Wiley Periodicals, Inc.

Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules.

PubMed

Wasylyk, Christine; Zheng, Hong; Castell, Christelle; Debussche, Laurent; Multon, Marie-Christine; Wasylyk, Bohdan

2008-03-01

Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

Inhibition of polyomavirus ori-dependent DNA replication by mSin3B.

PubMed

Xie, An-Yong; Folk, William R

2002-12-01

When tethered in cis to DNA, the transcriptional corepressor mSin3B inhibits polyomavirus (Py) ori-dependent DNA replication in vivo. Histone deacetylases (HDACs) appear not to be involved, since tethering class I and class II HDACs in cis does not inhibit replication and treating the cells with trichostatin A does not specifically relieve inhibition by mSin3B. However, the mSin3B L59P mutation that impairs mSin3B interaction with N-CoR/SMRT abrogates inhibition of replication, suggesting the involvement of N-CoR/SMRT. Py large T antigen interacts with mSin3B, suggesting an HDAC-independent mechanism by which mSin3B inhibits DNA replication.

Regulation of adhesion and growth of fibrosarcoma cells by NF-kappa B RelA involves transforming growth factor beta.

PubMed Central

Perez, J R; Higgins-Sochaski, K A; Maltese, J Y; Narayanan, R

1994-01-01

The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion. Images PMID:8035811

Inhibition of lymphocyte proliferation and antibody production in vitro by silica, talc, bentonite or Corynebacterium parvum: involvement of peroxidative processes.

PubMed

Hoffeld, J T

1983-05-01

This study was undertaken to determine whether and by what means particles which induce granulomata in vivo can affect murine spleen lymphoproliferative and antibody responses in vitro. Particles of silica, talc, Bentonite or C. parvum cells inhibited lipopolysaccharide- or concanavalin A-stimulated proliferation and sheep red blood cell-induced antibody response in vitro. The inhibition required at least 48 hours exposure of the cells to the particles. The late onset of inhibition and its reproducibility at different cell or mitogen concentrations implicated particle-induced injury to both phagocytes and lymphocytes. Either alpha-tocopherol or 2-mercaptoethanol prevented the particle-induced inhibition of spleen cell responses. alpha-Tocopherol and 2-mercaptoethanol have in common the capacity to protect cells against membrane lipid peroxidation. The inhibitory peroxidative process(es) implicated by these studies are most likely attributable to: (a) stimulation of oxidative metabolism of phagocytic cells by particles; and (b) iron-catalyzed peroxidation directly by the particles. These data may be relevant in understanding the pathogenesis of and devising therapeutic approaches toward various granulomatous conditions.

A novel taspine analog, HMQ1611, inhibits growth of non-small cell lung cancer by inhibiting angiogenesis

PubMed Central

LU, WEN; DAI, BINGLING; MA, WEINA; ZHANG, YANMIN

2012-01-01

In the present study, we investigated the antitumor activity of HMQ1611, a novel synthetic taspine derivative, in vivo and evaluated associated potential antiangiogenesis mechanisms. The proliferation of A549 cells was examined by WST-1 assay in vitro. Tube formation and lung tissue vessel models were used to observe the antiangiogenic activity of HMQ1611. In addition, vascular enodthelial growth factor (VEGF) secretion and KDR kinase activities were measured by ELISA and the HTRF®KinEASE™-TK assay. In vivo, the antitumor activity was assessed by implantation of A549 cells in athymic mice. The results showed that HMQ1611 inhibited A549 cell proliferation and VEGF secretion, while it significantly inhibited tube formation and tissue vascularization. Furthermore, HMQ1611 inhibited A549 xenograft tumor growth. In conclusion, the results of our study suggest that HMQ1611 has latent properties for the inhibition of angiogenesis which are involved in its antitumor activity. PMID:23162661

A novel taspine analog, HMQ1611, inhibits growth of non-small cell lung cancer by inhibiting angiogenesis.

PubMed

Lu, Wen; Dai, Bingling; Ma, Weina; Zhang, Yanmin

2012-11-01

In the present study, we investigated the antitumor activity of HMQ1611, a novel synthetic taspine derivative, in vivo and evaluated associated potential antiangiogenesis mechanisms. The proliferation of A549 cells was examined by WST-1 assay in vitro. Tube formation and lung tissue vessel models were used to observe the antiangiogenic activity of HMQ1611. In addition, vascular enodthelial growth factor (VEGF) secretion and KDR kinase activities were measured by ELISA and the HTRF(®)KinEASE(™)-TK assay. In vivo, the antitumor activity was assessed by implantation of A549 cells in athymic mice. The results showed that HMQ1611 inhibited A549 cell proliferation and VEGF secretion, while it significantly inhibited tube formation and tissue vascularization. Furthermore, HMQ1611 inhibited A549 xenograft tumor growth. In conclusion, the results of our study suggest that HMQ1611 has latent properties for the inhibition of angiogenesis which are involved in its antitumor activity.

Interleukin-2-induced survival of natural killer (NK) cells involving phosphatidylinositol-3 kinase-dependent reduction of ceramide through acid sphingomyelinase, sphingomyelin synthase, and glucosylceramide synthase.

PubMed

Taguchi, Yoshimitsu; Kondo, Tadakazu; Watanabe, Mitsumasa; Miyaji, Michihiko; Umehara, Hisanori; Kozutsumi, Yasunori; Okazaki, Toshiro

2004-11-15

Interleukin 2 (IL-2) rescued human natural killer (NK) KHYG-1 cells from apoptosis along with a reduction of ceramide. Conversely, an increase of ceramide inhibited IL-2-rescued survival. IL-2 deprivation-induced activation of acid sphingomyelinase (SMase) and inhibition of glucosylceramide synthase (GCS) and sphingomyelin synthase (SMS) were normalized by IL-2 supplementation. A phosphatidyl inositol-3 (PI-3) kinase inhibitor, LY294002, inhibited IL-2-rescued survival, but a mitogen-activated protein kinase inhibitor, PD98059, and an inhibitor of Janus tyrosine kinase/signal transducer and activator of transcription pathway, AG490, did not. LY294002 inhibited IL-2-induced reduction of ceramide through activation of acid SMase and inhibition of GCS and SMS, suggesting the positive involvement of PI-3 kinase in ceramide reduction through enzymatic regulation. Indeed, a constitutively active PI-3 kinase enhanced growth rate and ceramide reduction through inhibition of acid SMase and activation of GCS and SMS. Further, LY294002 inhibited IL-2-induced changes of transcriptional level as well as mRNA and protein levels in acid SMase and GCS but did not affect the stability of the mRNAs. These results suggest that PI-3 kinase-dependent reduction of ceramide through regulation of acid SMase, GCS, and SMS plays a role in IL-2-rescued survival of NK cells.

The Potential Therapeutic Agent Mepacrine Protects Caco-2 Cells against Clostridium perfringens Enterotoxin Action.

PubMed

Freedman, John C; Hendricks, Matthew R; McClane, Bruce A

2017-01-01

Clostridium perfringens enterotoxin (CPE) causes the diarrhea associated with a common bacterial food poisoning and many antibiotic-associated diarrhea cases. The severity of some CPE-mediated disease cases warrants the development of potential therapeutics. A previous study showed that the presence of mepacrine inhibited CPE-induced electrophysiology effects in artificial lipid bilayers lacking CPE receptors. However, that study did not assess whether mepacrine inactivates CPE or, instead, inhibits a step in CPE action. Furthermore, CPE action in host cells is complex, involving the toxin binding to receptors, receptor-bound CPE oligomerizing into a prepore on the membrane surface, and β-hairpins in the CPE prepore inserting into the membrane to form a pore that induces cell death. Therefore, the current study evaluated the ability of mepacrine to protect cells from CPE. This drug was found to reduce CPE-induced cytotoxicity in Caco-2 cells. This protection did not involve mepacrine inactivation of CPE, indicating that mepacrine affects one or more steps in CPE action. Western blotting then demonstrated that mepacrine decreases CPE pore levels in Caco-2 cells. This mepacrine-induced reduction in CPE pore levels did not involve CPE binding inhibition but rather an increase in CPE monomer dissociation due to mepacrine interactions with Caco-2 membranes. In addition, mepacrine was also shown to inhibit CPE pores when already present in Caco-2 cells. These in vitro studies, which identified two mepacrine-sensitive steps in CPE-induced cytotoxicity, add support to further testing of the therapeutic potential of mepacrine against CPE-mediated disease. IMPORTANCE Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal (GI) symptoms of a common bacterial food poisoning and several nonfoodborne human GI diseases. A previous study showed that, via an undetermined mechanism, the presence of mepacrine blocks CPE-induced electrophysiologic activity in artificial membranes. The current study now demonstrates that mepacrine also inhibits CPE-induced cytotoxicity in human enterocyte-like Caco-2 cells and that mepacrine does not directly inactivate CPE. Instead, this drug reduces both CPE pore formation and CPE pore activity in Caco-2 cells. These results suggest mepacrine as a therapeutic candidate for treating CPE-mediated GI diseases.

Pro-apoptotic activities of polyphenolics from açai (Euterpe oleracea Martius) in human SW-480 colon cancer cells.

PubMed

Dias, Manoela Maciel dos Santos; Noratto, Giuliana; Martino, Hercia Stampini Duarte; Arbizu, Shirley; Peluzio, Maria do Carmo Gouveia; Talcott, Stephen; Ramos, Afonso Mota; Mertens-Talcott, Susanne U

2014-01-01

This study aimed to evaluate the cell growth inhibition activity of açai (Euterpe oleracea Mart.) polyphenolic extract against colon cancer HT-29 and SW-480 cells and the nonmalignant CCD-18Co colon fibroblast cells. Results showed that açai polyphenolic extract (5-20 mg/L) inhibited preferentially the growth of SW-480 cells with no toxicity in CCD-18Co cells, and this was accompanied by reduction of H2O2-induced reactive oxygen species (ROS) generation. The mechanisms involved in SW-480 cell growth-inhibition by açai polyphenolic extract included the downregulation of NF-κB proinflammatory transcription factor and the nuclear factor-kappa B targets intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Furthermore, prooncogenic specificity proteins (Sp) were downregulated as well as Sp-targets Bcl-2, vascular endothelial growth factor, and survivin. This was accompanied by activation of mitochondrial proapoptotic pathway involving increase of cytochrome c, cleavage of caspase-3, and decrease of PARP-1. Results strongly suggest that açai polyphenolic extract has antiinflammatory and cytotoxic activities in colon cancer cells and can be effective as natural colon cancer chemopreventive agents.

Autophagy induced by baicalin involves downregulation of CD147 in SMMC-7721 cells in vitro

PubMed Central

ZHANG, XIANJIAO; TANG, XU; LIU, HANQIANG; LI, LIANXIANG; HOU, QIAN; GAO, JIANMIN

2012-01-01

Baicalin has been demonstrated to exert anticancer effects mainly through induction of tumor cell apoptosis and cell cycle arrest. However, the precise mechanisms underlying its anticancer role remain to be elucidated. In the present study, we investigated whether autophagy was involved in the anticancer activity of baicalin in the human hepatocellular carcinoma (HCC) cell line SMMC-7721 and the possible molecular mechanisms. Our data showed that the viability of SMMC-7721 cells was significantly inhibited by baicalin in a dose- and time-dependent manner. Alongside apoptosis, autophagy was also induced by baicalin dose- and time-dependently with the involvement of the autophagy-associated protein Beclin 1. Moreover, we demonstrated that cell death induced by baicalin was significantly inhibited by the apoptosis inhibitor z-DEVD-fmk or the autophagy inhibitor 3-MA, respectively. In addition, we found that CD147, a key molecule related both to apoptosis and autophagy, was markedly downregulated at the protein level in SMMC-7721 cells treated with baicalin. Collectively, this is the first study to suggest that baicalin induces autophagic cell death in SMMC-7721 cells, which involves the downregulation of CD147. Our study reveals a new mechanism for the anticancer effects of baicalin and puts forward a potential crucial role of CD147 in baicalin-induced cancer cell death. PMID:22200845

Autophagy contributes to gefitinib-induced glioma cell growth inhibition

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

Chang, Cheng-Yi; Graduate Institute of Pharmaceutical Science and Technology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan; Kuan, Yu-Hsiang

Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and geneticmore » silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation. - Highlights: • Gefitinib causes cytotoxic and cytostatic effect on glioma. • Gefitinib induces autophagy. • Gefitinib causes cytostatic effect through autophagy. • Gefitinib induces autophagy involving AMPK.« less

Cellular mechanisms underlying the inhibitory effect of flufenamic acid on chloride secretion in human intestinal epithelial cells.

PubMed

Pongkorpsakol, Pawin; Yimnual, Chantapol; Chatsudthipong, Varanuj; Rukachaisirikul, Vatcharin; Muanprasat, Chatchai

2017-06-01

Intestinal Cl - secretion is involved in the pathogenesis of secretory diarrheas including cholera. We recently demonstrated that flufenamic acid (FFA) suppressed Vibrio cholerae El Tor variant-induced intestinal fluid secretion via mechanisms involving AMPK activation and NF-κB-suppression. The present study aimed to investigate the effect of FFA on transepithelial Cl - secretion in human intestinal epithelial (T84) cells. FFA inhibited cAMP-dependent Cl - secretion in T84 cell monolayers with IC 50 of ∼8 μM. Other fenamate drugs including tolfenamic acid, meclofenamic acid and mefenamic acid exhibited the same effect albeit with lower potency. FFA also inhibited activities of CFTR, a cAMP-activated apical Cl - channel, and KCNQ1/KCNE3, a cAMP-activated basolateral K + channel. Mechanisms of CFTR inhibition by FFA did not involve activation of its negative regulators. Interestingly, FFA inhibited Ca 2+ -dependent Cl - secretion with IC 50 of ∼10 μM. FFA inhibited activities of Ca 2+ -activated Cl - channels and K Ca 3.1, a Ca 2+ -activated basolateral K + channels, but had no effect on activities of Na + -K + -Cl - cotransporters and Na + -K + ATPases. These results indicate that FFA inhibits both cAMP and Ca 2+ -dependent Cl - secretion by suppressing activities of both apical Cl - channels and basolateral K + channels. FFA and other fenamate drugs may be useful in the treatment of secretory diarrheas. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Flavonoids (apigenin, tangeretin) counteract tumor promoter-induced inhibition of intercellular communication of rat liver epithelial cells.

PubMed

Chaumontet, C; Droumaguet, C; Bex, V; Heberden, C; Gaillard-Sanchez, I; Martel, P

1997-03-19

We have shown previously that two flavonoids, apigenin and tangeretin, enhance gap junctional intercellular communication (GJIC) in rat liver epithelial cells, named REL cells. Here, we show that these two flavones also antagonize the inhibition of GJIC induced by tumor promoters like 12-O-tetradecanoyl-phorbol-acetate (TPA) and 3,5,di-tertio-butyl-4-hydroxytoluene (BHT). Their preventive effect is rapid. It does not seem to involve any change of the amount of the connexin expressed in REL cells, connexin 43 (Cx 43), and in its phosphorylation state. Other flavonoids tested including naringenin, myricetin, catechin and chrysin did not enhance GJIC nor counteract TPA-induced inhibition of GJIC.

Silencing heme oxygenase-1 gene expression in retinal pigment epithelial cells inhibits proliferation, migration and tube formation of cocultured endothelial cells

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

Zhang, Wenjie; Zhang, Xiaomei, E-mail: zhangxm667@163.com; Lu, Hong

2013-05-10

Highlights: •HO-1 is highly induced in RPE cells by hypoxia. •Inhibition of HO-1 activity and knockdown of HO-1 expression inhibit VEGF expression in RPE cells under hypoxia. •Knockdown of HO-1 in RPE cells inhibits angiogenesis of endothelial cells in vitro. -- Abstract: Heme oxygenase-1 (HO-1) plays an important role in the vasculature and in the angiogenesis of tumors, wounds and other environments. Retinal pigment epithelial (RPE) cells and choroidal endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), a process in which hypoxia plays an important role. Our aim was to evaluate the role of human RPE-cellmore » HO-1 in the angiogenic activities of cocultured endothelial cells under hypoxia. Small interfering RNA (siRNA) for HO-1 was transfected into human RPE cell line ARPE-19, and zinc protoporphyrin (ZnPP) was used to inhibit HO-1 activity. Knockdown of HO-1 expression and inhibition of HO-1 activity resulted in potent reduction of the expression of vascular endothelial growth factor (VEGF) under hypoxia. Furthermore, knockdown of HO-1 suppressed the proliferation, migration and tube formation of cocultured endothelial cells. These findings indicated that HO-1 might have an angiogenic effect in CNV through modulation of VEGF expression and might be a potential target for treating CNV.« less

Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase.

PubMed

Wang, Yang; Desai, Janish; Zhang, Yonghui; Malwal, Satish R; Shin, Christopher J; Feng, Xinxin; Sun, Hong; Liu, Guizhi; Guo, Rey-Ting; Oldfield, Eric

2016-10-19

We synthesized a series of benzoic acids and phenylphosphonic acids and investigated their effects on the growth of Staphylococcus aureus and Bacillus subtilis. One of the most active compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7, ED 50 ∼0.15 μg mL -1 ) acted synergistically with seven antibiotics known to target bacterial cell-wall biosynthesis (a fractional inhibitory concentration index (FICI) of ∼0.35, on average) but had indifferent effects in combinations with six non-cell-wall biosynthesis inhibitors (average FICI∼1.45). The most active compounds were found to inhibit two enzymes involved in isoprenoid/bacterial cell-wall biosynthesis: undecaprenyl diphosphate synthase (UPPS) and undecaprenyl diphosphate phosphatase (UPPP), but not farnesyl diphosphate synthase, and there were good correlations between bacterial cell growth inhibition, UPPS inhibition, and UPPP inhibition. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Direct evidence that FK506 inhibition of FcepsilonRI-mediated exocytosis from RBL mast cells involves calcineurin.

PubMed

Hultsch, T; Brand, P; Lohmann, S; Saloga, J; Kincaid, R L; Knop, J

1998-05-01

FcepsilonRI-mediated exocytosis of preformed mediators from mast cells and basophils (e.g. histamine, serotonin, beta-hexosaminidase) is sensitive to the immunosuppressants cyclosporin A and FK506 (IC50 200 and 4 nM, respectively) but not rapamycin. The mechanism of inhibition does not appear to involve tyrosine phosphorylation, hydrolysis of inositol phosphates or calcium flux. Here we report experiments using a molecular approach to assess the role of calcineurin, a serine/threonine phosphatase thought to be the primary pharmacological target of these drugs. Calcineurin's activity requires association of its catalytic (A) subunit with an intrinsic regulatory (B) subunit. We hypothesized that calcineurin-sensitive signalling events should be affected by the depletion of calcineurin B subunits, thereby reducing the number of active A:B complexes. We therefore transfected rat basophilic leukemia (RBL) cells with an inhibitory (dominant negative) form of the calcineurin A subunit, which binds the calcineurin B subunit with high affinity but does not possess catalytic activity (B subunit knock-out, BKO). In these transfected cells, the dose-response curve for the inhibition of FcepsilonRI-mediated exocytosis by FK506 was shifted to the left, indicating an increased drug sensitivity of BKO-transfected cells. We conclude that FK506 inhibition of FcepsilonRI-mediated exocytosis in mast cells specifically targets calcineurin activity.

Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control

PubMed Central

Zhao, Bin; Wei, Xiaomu; Li, Weiquan; Udan, Ryan S.; Yang, Qian; Kim, Joungmok; Xie, Joe; Ikenoue, Tsuneo; Yu, Jindan; Li, Li; Zheng, Pan; Ye, Keqiang; Chinnaiyan, Arul; Halder, Georg; Lai, Zhi-Chun; Guan, Kun-Liang

2007-01-01

The Hippo pathway plays a key role in organ size control by regulating cell proliferation and apoptosis in Drosophila. Although recent genetic studies have shown that the Hippo pathway is regulated by the NF2 and Fat tumor suppressors, the physiological regulations of this pathway are unknown. Here we show that in mammalian cells, the transcription coactivator YAP (Yes-associated protein), is inhibited by cell density via the Hippo pathway. Phosphorylation by the Lats tumor suppressor kinase leads to cytoplasmic translocation and inactivation of the YAP oncoprotein. Furthermore, attenuation of this phosphorylation of YAP or Yorkie (Yki), the Drosophila homolog of YAP, potentiates their growth-promoting function in vivo. Moreover, YAP overexpression regulates gene expression in a manner opposite to cell density, and is able to overcome cell contact inhibition. Inhibition of YAP function restores contact inhibition in a human cancer cell line bearing deletion of Salvador (Sav), a Hippo pathway component. Interestingly, we observed that YAP protein is elevated and nuclear localized in some human liver and prostate cancers. Our observations demonstrate that YAP plays a key role in the Hippo pathway to control cell proliferation in response to cell contact. PMID:17974916

Berberine suppresses tumorigenicity and growth of nasopharyngeal carcinoma cells by inhibiting STAT3 activation induced by tumor associated fibroblasts

PubMed Central

2013-01-01

Background Cortidis rhizoma (Huanglian) and its major therapeutic component, berberine, have drawn extensive attention in recent years for their anti-cancer properties. Growth inhibitory effects of berberine on multiple types of human cancer cells have been reported. Berberine inhibits invasion, induces cell cycle arrest and apoptosis in human cancer cells. The anti-inflammatory property of berberine, involving inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) activation, has also been documented. Methods In this study, we have examined the effects of berberine on tumorigenicity and growth of nasopharyngeal carcinoma (NPC) cells and their relationship to STAT3 signaling using both in vivo and in vitro models. Results Berberine effectively inhibited the tumorigenicity and growth of an EBV-positive NPC cell line (C666-1) in athymic nude mice. Inhibition of tumorigenic growth of NPC cells in vivo was correlated with effective inhibition of STAT3 activation in NPC cells inside the tumor xenografts grown in nude mice. In vitro, berberine inhibited both constitutive and IL-6-induced STAT3 activation in NPC cells. Inhibition of STAT3 activation by berberine induced growth inhibition and apoptotic response in NPC cells. Tumor-associated fibroblasts were found to secret IL-6 and the conditioned medium harvested from the fibroblasts also induced STAT3 activation in NPC cells. Furthermore, STAT3 activation by conditioned medium of tumor-associated fibroblasts could be blocked by berberine or antibodies against IL-6 and IL-6R. Conclusions Our observation that berberine effectively inhibited activation of STAT3 induced by tumor-associated fibroblasts suggests a role of berberine in modulating the effects of tumor stroma on the growth of NPC cells. The effective inhibition of STAT3 activation in NPC cells by berberine supports its potential use in the treatment of NPC. PMID:24380387

Evidence for Pipecolate Oxidase in Mediating Protection Against Hydrogen Peroxide Stress.

PubMed

Natarajan, Sathish Kumar; Muthukrishnan, Ezhumalai; Khalimonchuk, Oleh; Mott, Justin L; Becker, Donald F

2017-07-01

Pipecolate, an intermediate of the lysine catabolic pathway, is oxidized to Δ 1 -piperideine-6-carboxylate (P6C) by the flavoenzyme l-pipecolate oxidase (PIPOX). P6C spontaneously hydrolyzes to generate α-aminoadipate semialdehyde, which is then converted into α-aminoadipate acid by α-aminoadipatesemialdehyde dehydrogenase. l-pipecolate was previously reported to protect mammalian cells against oxidative stress. Here, we examined whether PIPOX is involved in the mechanism of pipecolate stress protection. Knockdown of PIPOX by small interference RNA abolished pipecolate protection against hydrogen peroxide-induced cell death in HEK293 cells suggesting a critical role for PIPOX. Subcellular fractionation analysis showed that PIPOX is localized in the mitochondria of HEK293 cells consistent with its role in lysine catabolism. Signaling pathways potentially involved in pipecolate protection were explored by treating cells with small molecule inhibitors. Inhibition of both mTORC1 and mTORC2 kinase complexes or inhibition of Akt kinase alone blocked pipecolate protection suggesting the involvement of these signaling pathways. Phosphorylation of the Akt downstream target, forkhead transcription factor O3 (FoxO3), was also significantly increased in cells treated with pipecolate, further implicating Akt in the protective mechanism and revealing FoxO3 inhibition as a potentially key step. The results presented here demonstrate that pipecolate metabolism can influence cell signaling during oxidative stress to promote cell survival and suggest that the mechanism of pipecolate protection parallels that of proline, which is also metabolized in the mitochondria. J. Cell. Biochem. 118: 1678-1688, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells

PubMed Central

Alotaibi, Moureq; Sharma, Khushboo; Saleh, Tareq; Povirk, Lawrence F.; Hendrickson, Eric A.; Gewirtz, David A.

2016-01-01

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (−/−) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (−/−) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10–20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence. PMID:26934368

Interaction of the Tobacco mosaic virus movement protein with microtubules during the cell cycle in tobacco BY-2 cells.

PubMed

Boutant, Emmanuel; Fitterer, Chantal; Ritzenthaler, Christophe; Heinlein, Manfred

2009-10-01

Cell-to-cell movement of Tobacco mosaic virus (TMV) involves the interaction of virus-encoded 30-kDa movement protein (MP) with microtubules. In cells behind the infection front that accumulate high levels of MP, this activity is reflected by the formation of stabilized MP/microtubule complexes. The ability of MP to bind along and stabilize microtubules is conserved upon expression in mammalian cells. In mammalian cells, the protein also leads to inhibition of mitosis and cell division through a microtubule-independent process correlated with the loss of centrosomal gamma-tubulin and of centrosomal microtubule-nucleation activity. Since MP has the capacity to interact with plant factors involved in microtubule nucleation and dynamics, we used inducible expression in BY-2 cells to test whether MP expression inhibits mitosis and cell division also in plants. We demonstrate that MP:GFP associates with all plant microtubule arrays and, unlike in mammalian cells, does not interfere with mitosis. Thus, MP function and the interaction of MP with factors of the cytoskeleton do not entail an inhibition of mitosis in plants. We also report that the protein targets primary plasmodesmata in BY-2 cells immediately upon or during cytokinesis and that the accumulation of MP in plasmodesmata occurs in the presence of inhibitors of the cytoskeleton and the secretory pathway.

Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

PubMed

Bardon, Clément; Poly, Franck; Piola, Florence; Pancton, Muriel; Comte, Gilles; Meiffren, Guillaume; Haichar, Feth el Zahar

2016-05-01

Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Proton pump inhibitors induce apoptosis of human B-cell tumors through a caspase-independent mechanism involving reactive oxygen species.

PubMed

De Milito, Angelo; Iessi, Elisabetta; Logozzi, Mariantonia; Lozupone, Francesco; Spada, Massimo; Marino, Maria Lucia; Federici, Cristina; Perdicchio, Maurizio; Matarrese, Paola; Lugini, Luana; Nilsson, Anna; Fais, Stefano

2007-06-01

Proton pumps like the vacuolar-type H+ ATPase (V-ATPase) are involved in the control of cellular pH in normal and tumor cells. Treatment with proton pump inhibitors (PPI) induces sensitization of cancer cells to chemotherapeutics via modifications of cellular pH gradients. It is also known that low pH is the most suitable condition for a full PPI activation. Here, we tested whether PPI treatment in unbuffered culture conditions could affect survival and proliferation of human B-cell tumors. First, we showed that PPI treatment increased the sensitivity to vinblastine of a pre-B acute lymphoblastic leukemia (ALL) cell line. PPI, per se, induced a dose-dependent inhibition of proliferation of tumor B cells, which was associated with a dose- and time-dependent apoptotic-like cytotoxicity in B-cell lines and leukemic cells from patients with pre-B ALL. The effect of PPI was mediated by a very early production of reactive oxygen species (ROS), that preceded alkalinization of lysosomal pH, lysosomal membrane permeabilization, and cytosol acidification, suggesting an early destabilization of the acidic vesicular compartment. Lysosomal alterations were followed by mitochondrial membrane depolarization, release of cytochrome c, chromatin condensation, and caspase activation. However, inhibition of caspase activity did not affect PPI-induced cell death, whereas specific inhibition of ROS by an antioxidant (N-acetylcysteine) significantly delayed cell death and protected both lysosomal and mitochondrial membranes. The proapoptotic activity of PPI was consistent with a clear inhibition of tumor growth following PPI treatment of B-cell lymphoma in severe combined immunodeficient mice. This study further supports the importance of acidity and pH gradients in tumor cell homeostasis and suggests new therapeutic approaches for human B-cell tumors based on PPI.

Leptin stimulation of cell cycle and inhibition of apoptosis gene and protein expression in OVCAR-3 ovarian cancer cells.

PubMed

Ptak, Anna; Kolaczkowska, Elzbieta; Gregoraszczuk, Ewa L

2013-04-01

The OVCAR-3 cell line expressing the long (ObRb) and short (ObRt) isoforms of leptin receptor mRNA was used to analyze the effect of leptin on the expression of selected genes and proteins involved in the cell cycle and apoptosis. OVCAR-3 cells were exposed to 2, 20, 40, and 100 ng/ml of leptin. Cell proliferation was determined using the alamarBlue cell viability test and flow cytometry. Apoptosis was measured using a cellular DNA fragmentation ELISA kit. The expression of selected cell cycle and apoptosis genes was evaluated by real-time PCR and confirmed by western blot. The stimulatory action of leptin on cell proliferation was observed as an increase in cells in the S and G2/M phases. Up-regulation of genes responsible for inducing cell proliferation and suppression of genes responsible for inhibition of proliferation were noted. Western blots revealed increased expression of cyclins D and A and inhibition of p21WAF1/CIP1 protein expression by leptin. Inhibition of DNA fragmentation was observed under all leptin doses. Suppression of genes involved in the extrinsic and intrinsic apoptotic pathway was observed. Western blots illustrated decreased Bad, TNFR1, and caspase 6 protein expression in response to leptin treatment. Leptin promotes ovarian cancer cell line growth by up-regulating genes and proteins responsible for inducing cell proliferation as well as down-regulating pro-apoptotic genes and proteins in apoptotic pathways. Results of this study warrant examining the relationship between the risk of ovarian cancer and elevated leptin levels in obese women.

Apoptosis and autophagy induced by pyropheophorbide-α methyl ester-mediated photodynamic therapy in human osteosarcoma MG-63 cells.

PubMed

Huang, Qiu; Ou, Yun-Sheng; Tao, Yong; Yin, Hang; Tu, Ping-Hua

2016-06-01

Pyropheophorbide-α methyl ester (MPPa) was a second-generation photosensitizer with many potential applications. Here, we explored the impact of MPPa-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and autophagy of human osteosarcoma (MG-63) cells as well as the relationships between apoptosis and autophagy of the cells, and investigated the related molecular mechanisms. We found that MPPa-PDT demonstrated the ability to inhibit MG-63 cell viability in an MPPa concentration- and light dose-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, MPPa-PDT could also induce autophagy of MG-63 cell. Meanwhile, the ROS scavenger N-acetyl-L-cysteine (NAC) and the Jnk inhibitor SP600125 were found to inhibit the MPPa-PDT-induced autophagy, and NAC could also inhibit Jnk phosphorylation. Furthermore, pretreatment with the autophagy inhibitor 3-methyladenine or chloroquine showed the potential in reducing the apoptosis rate induced by MPPa-PDT in MG-63 cells. Our results indicated that the mitochondrial pathway was involved in MPPa-PDT-induced apoptosis of MG-63 cells. Meanwhile the ROS-Jnk signaling pathway was involved in MPPa-PDT-induced autophagy, which further promoted the apoptosis in MG-63 cells.

Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: Inactivation of specific transcription factors

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

Fradkin, L.G.; Yoshinaga, S.K.; Berk, A.J.

1987-11-01

The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription ofmore » RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoted, however, was not altered by infection of cells with the virus. The authors conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirtus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.« less

Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

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

Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly, E-mail: anatoly_zhitkovich@brown.edu

2015-07-15

Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effectsmore » of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in normal human cells. • Protective role of proteasomes is linked to repair of DNA–protein crosslinks.« less

Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line

PubMed Central

Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

2017-01-01

The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines. PMID:28810654

Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line.

PubMed

Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

2017-08-01

The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines.

α-Lipoic acid inhibits the migration and invasion of breast cancer cells through inhibition of TGFβ signaling.

PubMed

Tripathy, Joytirmay; Tripathy, Anindita; Thangaraju, Muthusamy; Suar, Mrutyunjay; Elangovan, Selvakumar

2018-05-23

Invasion and metastasis are the main cause of mortality in breast cancer. Hence, novel therapeutic interventions with high specificity toward invasion and metastasis are necessary. α-Lipoic acid showed antiproliferative and cytotoxic effects on several cancers including breast cancer. However, the effect of lipoic acid on breast cancer metastasis remains unclear. In the present study, we examined the effects of lipoic acid on the migration and invasion of MDA-MB-231 and 4 T1 breast cancer cells. Our data showed that lipoic acid effectively inhibited the colony forming ability of highly invasive MDA-MB-231 and 4 T1 cells. Moreover, the nontoxic concentrations of lipoic acid significantly reduced the migration of breast cancer cells. Lipoic acid also inhibited the TGFβ-induced angiopoietin-like 4 (ANGPTL4) expression and reduced the activity of matrix metalloproteinase-9 (MMP-9), an enzyme involved in invasion and metastasis, in both the cell lines. The inhibition of cell migration by lipoic acid is accompanied by the downregulation of FAK, ERK1/2 and AKT phosphorylation, and inhibition of nuclear translocation of β-catenin. Our data demonstrated that lipoic acid inhibited the migration and invasion of metastatic breast cancer cells at least in part through inhibiting ERK1/2 and AKT signaling. Thus, our findings show that the inhibition of TGFβ signaling is a potential mechanism for the anti-invasive effects of lipoic acid. Copyright © 2017. Published by Elsevier Inc.

Tunneling nanotube (TNT) formation is downregulated by cytarabine and NF-κB inhibition in acute myeloid leukemia (AML)

PubMed Central

Omsland, Maria; Bruserud, Øystein; Gjertsen, Bjørn T; Andresen, Vibeke

2017-01-01

Acute myeloid leukemia (AML) is a bone marrow derived blood cancer where intercellular communication in the leukemic bone marrow participates in disease development, progression and chemoresistance. Tunneling nanotubes (TNTs) are intercellular communication structures involved in transport of cellular contents and pathogens, also demonstrated to play a role in both cell death modulation and chemoresistance. Here we investigated the presence of TNTs by live fluorescent microscopy and identified TNT formation between primary AML cells and in AML cell lines. We found that NF-κB activity was involved in TNT regulation and formation. Cytarabine downregulated TNTs and inhibited NF-κB alone and in combination with daunorubicin, providing additional support for involvement of the NF-κB pathway in TNT formation. Interestingly, daunorubicin was found to localize to lysosomes in TNTs connecting AML cells indicating a novel function of TNTs as drug transporting devices. We conclude that TNT communication could reflect important biological features of AML that may be explored in future therapy development. PMID:27974700

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

Lai, Jing; Liu, Gexiu; Yan, Guoyao

By investigating the anti-adipogenic effects of WEHI-3 cells – a murine acute myelomonocytic leukemia cell line – we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPARγ, C/EBPα, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway,more » β-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and β-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and β-catenin are involved in this process. - Highlights: • WEHI-3, an acute myelomonocytic leukemia cell line, inhibited 3T3-L1 preadipocyte from differentiating into adipocyte. • WEHI-3 cells can arrest 3T3-L1 cells in G0/G1 phase by secreting soluble factors and thus inhibit their proliferation. • WEHI-3 cells reduced bone marrow pimelosis in the murine model. • Both ROCKII and β-catenin were involved in the WEHI-3-mediated anti-adipogenic effects.« less

Inhibitory Effects of Leptin on Pancreatic α-Cell Function

PubMed Central

Tudurí, Eva; Marroquí, Laura; Soriano, Sergi; Ropero, Ana B.; Batista, Thiago M.; Piquer, Sandra; López-Boado, Miguel A.; Carneiro, Everardo M.; Gomis, Ramón; Nadal, Angel; Quesada, Ivan

2009-01-01

OBJECTIVE Leptin released from adipocytes plays a key role in the control of food intake, energy balance, and glucose homeostasis. In addition to its central action, leptin directly affects pancreatic β-cells, inhibiting insulin secretion, and, thus, modulating glucose homeostasis. However, despite the importance of glucagon secretion in glucose homeostasis, the role of leptin in α-cell function has not been studied in detail. In the present study, we have investigated this functional interaction. RESEARCH DESIGN AND METHODS The presence of leptin receptors (ObR) was demonstrated by RT-PCR analysis, Western blot, and immunocytochemistry. Electrical activity was analyzed by patch-clamp and Ca2+ signals by confocal microscopy. Exocytosis and glucagon secretion were assessed using fluorescence methods and radioimmunoassay, respectively. RESULTS The expression of several ObR isoforms (a–e) was detected in glucagon-secreting αTC1-9 cells. ObRb, the main isoform involved in leptin signaling, was identified at the protein level in αTC1-9 cells as well as in mouse and human α-cells. The application of leptin (6.25 nmol/l) hyperpolarized the α-cell membrane potential, suppressing the electrical activity induced by 0.5 mmol/l glucose. Additionally, leptin inhibited Ca2+ signaling in αTC1-9 cells and in mouse and human α-cells within intact islets. A similar result occurred with 0.625 nmol/l leptin. These effects were accompanied by a decrease in glucagon secretion from mouse islets and were counteracted by the phosphatidylinositol 3-kinase inhibitor, wortmannin, suggesting the involvement of this pathway in leptin action. CONCLUSIONS These results demonstrate that leptin inhibits α-cell function, and, thus, these cells are involved in the adipoinsular communication. PMID:19401420

Anti-cancer activity of withaferin A in B-cell lymphoma

PubMed Central

McKenna, MK; Gachuki, BW; Alhakeem, SS; Oben, KN; Rangnekar, VM; Gupta, RC; Bondada, S

2015-01-01

Withaferin A (WA), a withanolide from the plant, Ashwagandha (Withania somnifera) used in Ayurvedic medicine, has been found to be valuable in the treatment of several medical ailments. WA has been found to have anticancer activity against various solid tumors, but its effects on hematological malignancies have not been studied in detail. WA strongly inhibited the survival of several human and murine B cell lymphoma cell lines. Additionally, in vivo studies with syngeneic-graft lymphoma cells suggest that WA inhibits the growth of tumor but does not affect other proliferative tissues. We demonstrate that WA inhibits the efficiency of NF-κB nuclear translocation in diffuse large B cell lymphomas and found that WA treatment resulted in a significant decrease in protein levels involved in B cell receptor signaling and cell cycle regulation. WA inhibited the activity of heat shock protein (Hsp) 90 as reflected by a sharp increase in Hsp70 expression levels. Hence, we propose that the anti-cancer effects of WA in lymphomas are likely due to its ability to inhibit Hsp90 function and subsequent reduction of critical kinases and cell cycle regulators that are clients of Hsp90. PMID:26020511

Anti-cancer activity of withaferin A in B-cell lymphoma.

PubMed

McKenna, M K; Gachuki, B W; Alhakeem, S S; Oben, K N; Rangnekar, V M; Gupta, R C; Bondada, S

2015-01-01

Withaferin A (WA), a withanolide from the plant, Ashwagandha (Withania somnifera) used in Ayurvedic medicine, has been found to be valuable in the treatment of several medical ailments. WA has been found to have anticancer activity against various solid tumors, but its effects on hematological malignancies have not been studied in detail. WA strongly inhibited the survival of several human and murine B cell lymphoma cell lines. Additionally, in vivo studies with syngeneic-graft lymphoma cells suggest that WA inhibits the growth of tumor but does not affect other proliferative tissues. We demonstrate that WA inhibits the efficiency of NF-κB nuclear translocation in diffuse large B cell lymphomas and found that WA treatment resulted in a significant decrease in protein levels involved in B cell receptor signaling and cell cycle regulation. WA inhibited the activity of heat shock protein (Hsp) 90 as reflected by a sharp increase in Hsp70 expression levels. Hence, we propose that the anti-cancer effects of WA in lymphomas are likely due to its ability to inhibit Hsp90 function and subsequent reduction of critical kinases and cell cycle regulators that are clients of Hsp90.

Protein kinase C-beta inhibition induces apoptosis and inhibits cell cycle progression in AIDS-related Non-Hodgkin lymphoma cells

PubMed Central

Saba, Nakhle S.; Levy, Laura S.

2011-01-01

AIDS-related Non-Hodgkin Lymphoma (AIDS-NHL) constitutes an aggressive variety of lymphomas characterized by increased extranodal involvement, relapse rate and resistance to chemotherapy. PKCβ targeting showed promising results in preclinical and clinical studies involving a wide variety of cancers, but studies describing the role of PKCβ in AIDS-NHL are primitive if not lacking. In the present study, three AIDS-NHL cell lines were examined: 2F7 (AIDS-Burkitt Lymphoma), BCBL-1 (AIDS-Primary Effusion Lymphoma) and UMCL01-101 (AIDS-Diffuse Large B Cell Lymphoma). Immunoblot analysis demonstrated expression of PKCβ1 and PKCβ2 in 2F7 and UMCL01-101 cells, and PKCβ1 alone in BCBL-1 cells. The viability of 2F7 and BCBL-1 cells decreased significantly in the presence of PKCβ-selective inhibitor at IC50 of 14 μM and 15 μM, respectively, as measured by MTS assay. In contrast, UMCL01-101 cells were relatively resistant. As determined using flow cytometric TUNEL assay with propidium iodide staining, the responsiveness of sensitive cells was associated with apoptotic induction and cell cycle inhibition. PKCβ-selective inhibition was observed not to affect AKT phosphorylation, but to induce a rapid and sustained reduction in the phosphorylation of GSK3β, ribosomal protein S6, and mTOR in sensitive cell lines. The results indicate that PKCβ plays an important role in AIDS-related NHL survival, and suggest that PKCβ targeting should be considered in a broader spectrum of NHL. The observations in BCBL-1 were unexpected in the absence of PKCβ2 expression and implicate PKCβ1 as a regulator in those cells. PMID:21997316

Repurposing the anti-malarial drug dihydroartemisinin suppresses metastasis of non-small-cell lung cancer via inhibiting NF-κB/GLUT1 axis

PubMed Central

Jiang, Jie; Geng, Guojun; Yu, Xiuyi; Liu, Hongming; Gao, Jing; An, Hanxiang; Cai, Chengfu; Li, Ning; Shen, Dongyan; Wu, Xiaoqiang; Zheng, Lisheng; Mi, Yanjun; Yang, Shuyu

2016-01-01

Non-small-cell lung cancer (NSCLC) is an aggressive malignancy and long-term survival remains unsatisfactory for patients with metastatic and recurrent disease. Repurposing the anti-malarial drug dihydroartemisinin (DHA) has been proved to possess potent antitumor effect on various cancers. However, the effects of DHA in preventing the invasion of NSCLC cells have not been studied. In the present study, we determined the inhibitory effects of DHA on invasion and migration and the possible mechanisms involved using A549 and H1975 cells. DHA inhibited in vitro migration and invasion of NSCLC cells even in low concentration with little cytotoxicity. Additionally, low concentration DHA also inhibited Warburg effect in NSCLC cells. Mechanically, DHA negatively regulates NF-κB signaling to inhibit the GLUT1 translocation. Blocking the NF-κB signaling largely abolishes the inhibitory effects of DHA on the translocation of GLUT1 to the plasma membrane and the Warburg effect. Furthermore, GLUT1 knockdown significantly decreased the inhibition of invasion, and migration by DHA. Our results suggested that DHA can inhibit metastasis of NSCLC by targeting glucose metabolism via inhibiting NF-κB signaling pathway and DHA may deserve further investigation in NSCLC treatment. PMID:27895313

DT-13 attenuates human lung cancer metastasis via regulating NMIIA activity under hypoxia condition.

PubMed

Wei, Xiao-Hui; Lin, Sen-Sen; Liu, Yang; Zhao, Ren-Ping; Khan, Ghulam Jilany; Du, Hong-Zhi; Mao, Ting-Ting; Yu, Bo-Yang; Li, Rui-Ming; Yuan, Sheng-Tao; Sun, Li

2016-08-01

Cancer metastasis plays a major role in tumor deterioration. Metastatic processes are known to be regulated by hypoxic microenvironment and non-muscle myosin IIA (NMIIA). DT-13, a bioactive saponin monomer isolated from Ophiopogon japonicus, has been reported to inhibit various cancer metastasis, but whether NMIIA is involved in the anti-metastatic activity of DT-13 under hypoxia remains to be determined. Thus, this study aims to clarify the role of DT-13 in regulating 95D cell metastasis under hypoxic microenvironment and to further investigate whether NMIIA is involved in the anti-metastatic mechanism of DT-13. We found that DT-13 significantly inhibited 95D cells metastasis in vitro and in vivo. Furthermore, hypoxia significantly inhibited the expression of NMIIA and redistributed NMIIA to the cell periphery, whereas DT-13 reversed the hypoxic effects by upregulating the expression of NMIIA. Moreover, DT-13 treatment redistributed NMIIA to the nuclear periphery and reduced the formation of F-actin in 95D cells. In addition, we found that the Raf-ERK1/2 signaling pathway is involved in regulation of NMIIA by DT-13. Collectively, these findings support NMIIA as a target of DT-13 to prevent lung cancer metastasis.

Neuroprotective effects of SB-415286 on hydrogen peroxide-induced cell death in B65 rat neuroblastoma cells and neurons.

PubMed

Pizarro, Javier G; Yeste-Velasco, Marc; Rimbau, Victor; Casadesús, Gemma; Smith, Mark A; Pallàs, Mercè; Folch, Jaume; Camins, Antoni

2008-01-01

Glycogen synthase kinase-3 (GSK-3) is involved in the pathogenesis of several neurodegenerative diseases. In addition, as oxidative stress has been implicated in all neurodegenerative disorders, the inhibition of both pathways offers a potential strategy for preventing or delaying neurodegeneration. We examined the cytoprotective effects of lithium and SB-415286, two inhibitors of GSK-3, using a rat B65 cell line and also in cerebellar granule cells (CGN). H(2)O(2) decreased the inactive form of GSK-3 (phospho-GSK-3 at Ser9), as measured by immunoblot experiments involving an antibody against the inactive form of the enzyme. Moreover, lithium inhibited this effect. While SB-415286 exerted a protective effect, lithium did not attenuate the toxic effects of H(2)O(2) (1mM). We then examined those mechanisms implicated in the protective effects of SB-415286. When we analyzed reactive oxygen species (ROS) production using the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate in B65 cells, as well as in CGN, we found that SB-415286 strongly reduced DCF fluorescence. Lithium, however, did not exhibit any antioxidant properties. We conclude that the GSK-3 inhibitor SB-415286 has antioxidant properties, which may explain the cytoprotective effects against H(2)O(2) damage. Furthermore, inhibition of GSK-3 activity was not involved in this protective effect.

Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through β-arrestin-2 in THP-1 cell line.

PubMed

Wang, Wei; Zhang, Yuan; Xu, Ming; Zhang, You-Yi; He, Bei

2015-06-26

The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a β2-adrenergic receptor (β2-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of β2-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and β-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1β (IL-1β) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of β-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1β (IL-1β) release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1β release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the β2-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1β release via β-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative developmental neurotoxicity of organophosphates in vivo: transcriptional responses of pathways for brain cell development, cell signaling, cytotoxicity and neurotransmitter systems.

PubMed

Slotkin, Theodore A; Seidler, Frederic J

2007-05-30

Organophosphates affect mammalian brain development through a variety of mechanisms beyond their shared property of cholinesterase inhibition. We used microarrays to characterize similarities and differences in transcriptional responses to chlorpyrifos and diazinon, assessing defined gene groupings for the pathways known to be associated with the mechanisms and/or outcomes of chlorpyrifos-induced developmental neurotoxicity. We exposed neonatal rats to daily doses of chlorpyrifos (1mg/kg) or diazinon (1 or 2mg/kg) on postnatal days 1-4 and evaluated gene expression profiles in brainstem and forebrain on day 5; these doses produce little or no cholinesterase inhibition. We evaluated pathways for general neural cell development, cell signaling, cytotoxicity and neurotransmitter systems, and identified significant differences for >60% of 252 genes. Chlorpyrifos elicited major transcriptional changes in genes involved in neural cell growth, development of glia and myelin, transcriptional factors involved in neural cell differentiation, cAMP-related cell signaling, apoptosis, oxidative stress, excitotoxicity, and development of neurotransmitter synthesis, storage and receptors for acetylcholine, serotonin, norepinephrine and dopamine. Diazinon had similar effects on many of the same processes but also showed major differences from chlorpyrifos. Our results buttress the idea that different organophosphates target multiple pathways involved in neural cell development but also that they deviate in key aspects that may contribute to disparate neurodevelopmental outcomes. Equally important, these pathways are compromised at exposures that are unrelated to biologically significant cholinesterase inhibition and its associated signs of systemic toxicity. The approach used here demonstrates how planned comparisons with microarrays can be used to screen for developmental neurotoxicity.

Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4

PubMed Central

Schuette, Verena; Embgenbroich, Maria; Ulas, Thomas; Welz, Meike; Schulte-Schrepping, Jonas; Draffehn, Astrid M.; Quast, Thomas; Koch, Katharina; Nehring, Melanie; König, Jessica; Zweynert, Annegret; Harms, Frederike L.; Steiner, Nancy; Limmer, Andreas; Förster, Irmgard; Berberich-Siebelt, Friederike; Knolle, Percy A.; Wohlleber, Dirk; Kolanus, Waldemar; Beyer, Marc; Schultze, Joachim L.; Burgdorf, Sven

2016-01-01

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8+ T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte–associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality. PMID:27601670

Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4.

PubMed

Schuette, Verena; Embgenbroich, Maria; Ulas, Thomas; Welz, Meike; Schulte-Schrepping, Jonas; Draffehn, Astrid M; Quast, Thomas; Koch, Katharina; Nehring, Melanie; König, Jessica; Zweynert, Annegret; Harms, Frederike L; Steiner, Nancy; Limmer, Andreas; Förster, Irmgard; Berberich-Siebelt, Friederike; Knolle, Percy A; Wohlleber, Dirk; Kolanus, Waldemar; Beyer, Marc; Schultze, Joachim L; Burgdorf, Sven

2016-09-20

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.

C086, a novel analog of curcumin, induces growth inhibition and down-regulation of NFκB in colon cancer cells and xenograft tumors.

PubMed

Chen, Chun; Liu, Yang; Chen, Yuanzhong; Xu, Jianhua

2011-11-01

New analogues of curcumin with improved properties are needed to meet therapeutic requirements. In this study, the effects of C086 on growth inhibition and NFκB pathway regulation were investigated in colon cancer cells and xenograft tumors. C086 exhibited potent antiproliferative activity in all 6 colon cancer cell lines. In a xenograft model of SW480 cells in nude mice, the oral administration of C086 showed significant growth suppression of SW480 tumors, and both Western blot and immunohistochemistry analyses showed decreased NFκB (p65) expression in tumor tissues. Using TNF-α to induce NFκB activation in SW480 cells, it was revealed that C086 inhibited IκBα phosphorylation and its subsequent degradation, and suppressed the nuclear translocation and DNA binding activity of NFκB. C-Myc, cyclin D1, and Bcl-2, NFκB-regulated gene products involving in cellular proliferation and antiapoptosis, were decreased in the C086 treated groups. This effect was accompanied by pro-apoptosis of C086 in colon cancer cells and lower expression of PCNA in C086 treated colon cancer xenografts. Immunostaining for CD31 showed that there were fewer microvessels in C086 treated SW480 tumors, and NFκB-targeted gene products involved in angiogenesis (i.e., vascular endothelial growth factor, matrix metalloproteinase-9) were also downregulated. C086 also inhibited bovine aortic endothelial cell (BAEC) proliferation and tube formation in Matrigel. Overall, our results suggest that C086 is a potent antitumor agent and has promising future in colon cancer. C086 suppressed NFκB activation through inhibition of IκBα phosphorylation. Downregulation of NFκB-regulated gene products contributed to the antiproliferation, pro-apoptosis, and antiangiogenesis effect of C086.

Inhibition of STAT3 and ErbB2 Suppresses Tumor Growth, Enhances Radiosensitivity, and Induces Mitochondria-Dependent Apoptosis in Glioma Cells

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

Gao Ling; Li Fengsheng; Dong Bo

2010-07-15

Purpose: Constitutively activated signal transducer and activator of transcription 3 (STAT3) and ErbB2 are involved in the pathogenesis of many tumors, including astrocytoma. Inactivation of these molecules is reported to result in radiosensitization. The purpose of this study was to investigate whether inhibition of STAT3, ErbB2, or both could enhance radiotherapy in the human glioma model (U251 and U87 cell lines). Methods and Materials: The RNAi plasmids targeting STAT3 or ErbB2 were constructed, and their downregulatory effects on target proteins were examined by immunoblotting. After combination treatment of RNAi with or without irradiation, the cell viability was determined using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliummore » bromide (MTT) and clonogenic assays. The in vivo effect of combined treatment was determined using the U251 xenograft model. The apoptosis caused by the inhibition of STAT3 and ErbB2 was detected, and the mechanism involved in the apoptosis was investigated, including increases in caspase proteins, mitochondrial damage, and the expression of key modulating protein of different apoptosis pathways. Results: Transfection of U251 cells with STAT3 or ErbB2 siRNA plasmids specifically reduced their target gene expressions. Inhibition of STAT3 or ErbB2 greatly decreased glioma cell survival after 2, 4, or 6 Gy irradiation. Inhibition of STAT3 and ErbB2 also enhanced radiation-induced tumor growth inhibition in the U251 xenograft model. Furthermore, the suppression of either STAT3 or ErbB2 could induce U251 cell apoptosis, which was related primarily to the mitochondrial apoptotic pathway. Conclusions: These results indicated that simultaneous inhibition of STAT3 and ErbB2 expression can promote potent antitumor activity and radiosensitizing activity in human glioma.« less

Antimony trichloride induces a loss of cell viability via reactive oxygen species-dependent autophagy in A549 cells.

PubMed

Zhao, Xinyuan; Xing, Fengjun; Cong, Yewen; Zhuang, Yin; Han, Muxi; Wu, Zhiqiang; Yu, Shali; Wei, Haiyan; Wang, Xiaoke; Chen, Gang

2017-12-01

Antimony (Sb) is one of the most prevalent heavy metals and frequently leads to biological toxicity. Although autophagy is believed to be involved in metal-associated cytotoxicity, there is no evidence of its involvement following exposure. Moreover, the underlying mechanism of autophagy remains unclear. In this study, treatment with antimony trichloride caused autophagy in a dose- and time-dependent manner in A549 cells but did not affect the level of Atg5 or Atg7 mRNA expression. Furthermore, Sb enhanced autophagic flux while upregulating p62 gene and protein levels. The classic mechanistic target of rapamycin (mTOR) pathway is not involved in Sb-induced autophagy. However, Sb-induced autophagy and the upregulation of p62 were inhibited by treatment with the antioxidant N-acetylcysteine (NAC). Subsequent analyses demonstrated that the inhibition of autophagy protected A549 cells from a loss of cell viability, while the activation of autophagy by rapamycin had the opposite effect. These data suggest that reactive oxygen species-dependent autophagy mediates Sb-stimulated cell viability loss in A549 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibition of T Cell Protein Tyrosine Phosphatase Enhances Interleukin-18-Dependent Hematopoietic Stem Cell Expansion

PubMed Central

Bourdeau, Annie; Trop, Sébastien; Doody, Karen M; Dumont, Daniel J; Tremblayef, Michel L

2013-01-01

The clinical application of hematopoietic progenitor cell-based therapies for the treatment of hematological diseases is hindered by current protocols, which are cumbersome and have limited efficacy to augment the progenitor cell pool. We report that inhibition of T-cell protein tyrosine phosphatase (TC-PTP), an enzyme involved in the regulation of cytokine signaling, through gene knockout results in a ninefold increase in the number of hematopoietic progenitors in murine bone marrow (BM). This effect could be reproduced using a short (48 hours) treatment with a pharmacological inhibitor of TC-PTP in murine BM, as well as in human BM, peripheral blood, and cord blood. We also demonstrate that the ex vivo use of TC-PTP inhibitor only provides a temporary effect on stem cells and did not alter their capacity to reconstitute all hematopoietic components in vivo. We establish that one of the mechanisms whereby inhibition of TC-PTP mediates its effects involves the interleukin-18 (IL-18) signaling pathway, leading to increased production of IL-12 and interferon-gamma by progenitor cells. Together, our results reveal a previously unrecognized role for IL-18 in contributing to the augmentation of the stem cell pool and provide a novel and simple method to rapidly expand progenitor cells from a variety of sources using a pharmacological compound. Stem Cells 2013;31:293–304 PMID:23135963

Activation of acetyl-coenzyme A carboxylase is involved in Taxol-induced ovarian cancer cell death

PubMed Central

WU, JIANG; JI, FANG; DI, WEN; CHEN, HONGDUO; WAN, YINSHENG

2011-01-01

Acetyl-coenzyme A carboxylase (ACC) is an attractive target for research into the treatment of a variety of human diseases, including diabetes, obesity and cancer. Mounting evidence suggests that the inhibition of ACC induced of cancer cell apoptosis. However, whether the inhibition of ACC regulates apoptosis in CaOV3 cancer cells has yet to be addressed. This study investigated the cytotoxic mechanism of action of ACC inhibition. Results showed that 5-(tetradecyloxy)-2-furoic acid (TOFA), an ACC inhibitor, enhanced Taxol-induced CaOV3 human ovarian cancer cell apoptosis. Notably, when TOFA was administered as a monotherapy, it induced CaOV3 cell apoptosis. Pre-treatment with the EGFR inhibitor PD153035 was found to markedly enhance ACC phosphorylation, whereas AMP-activated protein kinase (AMPK) activator AICAR was found to marginally enhance ACC phosphorylation. Taken together, the data showed ACC is a potential novel molecular target of Taxol. Additionally, ACC inhibition partially contributed to the cytotoxic effect of Taxol in ovarian cancer cells. PMID:22866118

Activation of acetyl-coenzyme A carboxylase is involved in Taxol-induced ovarian cancer cell death.

PubMed

Wu, Jiang; Ji, Fang; DI, Wen; Chen, Hongduo; Wan, Yinsheng

2011-05-01

Acetyl-coenzyme A carboxylase (ACC) is an attractive target for research into the treatment of a variety of human diseases, including diabetes, obesity and cancer. Mounting evidence suggests that the inhibition of ACC induced of cancer cell apoptosis. However, whether the inhibition of ACC regulates apoptosis in CaOV3 cancer cells has yet to be addressed. This study investigated the cytotoxic mechanism of action of ACC inhibition. Results showed that 5-(tetradecyloxy)-2-furoic acid (TOFA), an ACC inhibitor, enhanced Taxol-induced CaOV3 human ovarian cancer cell apoptosis. Notably, when TOFA was administered as a monotherapy, it induced CaOV3 cell apoptosis. Pre-treatment with the EGFR inhibitor PD153035 was found to markedly enhance ACC phosphorylation, whereas AMP-activated protein kinase (AMPK) activator AICAR was found to marginally enhance ACC phosphorylation. Taken together, the data showed ACC is a potential novel molecular target of Taxol. Additionally, ACC inhibition partially contributed to the cytotoxic effect of Taxol in ovarian cancer cells.

D-Phenylalanine inhibits biofilm development of a marine microbe, Pseudoalteromonas sp. SC2014.

PubMed

Li, Ee; Wu, Jiajia; Wang, Peng; Zhang, Dun

2016-09-01

D-Amino acids have been reported to be able to inhibit biofilm formation or disperse existing biofilms of many microbes; in some cases this is due to growth inhibition as an unspecific effect. In this work, six different D-amino acids were tested for their inhibitory effects on biofilm development and bacterial growth of Pseudoalteromonas sp. SC2014, a marine microbe involved in microbiologically influenced corrosion (MIC). Experimental results indicated that D-phenylalanine (D-Phe) inhibited biofilm formation effectively at concentrations that did not affect cell growth, whereas the other D-amino acids either showed little effect or inhibited biofilm formation while inhibiting bacterial growth. Further studies found that D-Phe could inhibit bacterial accumulation on the surface of 316L stainless steel, and prevent bacteria from forming a multilayer biofilm. It was also suggested that D-Phe could promote the disassembly of an established multilayer biofilm but have little effect on the remaining monolayer adherent cells. For the first time, it was found that a D-amino acid could effectively inhibit biofilm formation of an MIC-involved microbe. This might supply a new insight into how MIC could be mitigated. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

PKC signaling is involved in the regulation of progranulin (acrogranin/PC-cell-derived growth factor/granulin-epithelin precursor) protein expression in human ovarian cancer cell lines.

PubMed

Diaz-Cueto, Laura; Arechavaleta-Velasco, Fabian; Diaz-Arizaga, Adriana; Dominguez-Lopez, Pablo; Robles-Flores, Martha

2012-07-01

Overexpression of progranulin (also named acrogranin, PC-cell-derived growth factor, or granulin-epithelin precursor) is associated with ovarian cancer, specifically with cell proliferation, malignancy, chemoresistance, and shortened overall survival. The objective of the current study is to identify the signaling pathways involved in the regulation of progranulin expression in ovarian cancer cell lines. We studied the relation of protein kinase C (PKC), phosphatidylinositol 3-kinase, protein kinase A, P38, extracellular signal-regulated kinase, and Akt pathways on the modulation of progranulin expression levels in NIH-OVCAR-3 and SK-OV-3 ovarian cancer cell lines. The different pathways were examined using pharmacological inhibitors (calphostin C, LY294002, H89, SB203580, PD98059, and Akt Inhibitor), and mRNA and protein progranulin expression were analyzed by reverse transcriptase polymerase chain reaction and Western blot techniques, respectively. Inhibition of PKC signal transduction pathway by calphostin C decreased in a dose-dependent manner protein but not mRNA levels of progranulin in both ovarian cancer cell lines. LY294002 but not wortmannin, which are phosphatidylinositol 3-kinase inhibitors, also diminished the expression of progranulin in both cell lines. In addition, LY294002 treatment produced a significant reduction in cell viability. Inhibition of protein kinase A, P38, extracellular signal-regulated kinase, and Akt did not affect progranulin protein expression. These results suggest that the PKC signaling is involved in the regulation of progranulin protein expression in 2 different ovarian cancer cell lines. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the cellular proliferation and invasion in ovarian cancer produced by progranulin.

Differential ability of formononetin to stimulate proliferation of endothelial cells and breast cancer cells via a feedback loop involving MicroRNA-375, RASD1, and ERα.

PubMed

Chen, Jian; Zhang, Xing; Wang, Yong; Ye, Yu; Huang, Zhaoquan

2018-05-02

For postmenopausal cardiovascular disease, long-term estrogen therapy may increase the risk of breast cancer. To reduce this risk, estrogen may be replaced with the phytoestrogen formononetin, but how formononetin acts on vascular endothelial cells (ECs) and breast cancer cells is unclear. Here, we show that low concentrations of formononetin induced proliferation and inhibited apoptosis more strongly in cultured human umbilical vein endothelial cells (HUVECs) than in breast cancer cells expressing estrogen receptor α (ERα) (MCF-7, BT474) or not (MDA-MB-231), and that this differential stimulation was associated with miR-375 up-regulation in HUVECs. For the first time, we demonstrate the presence of a feedback loop involving miR-375, ras dexamethasone-induced 1 (RASD1), and ERα in normal HUVECs, and we show that formononetin stimulated this feedback loop in HUVECs but not in MCF-7 or BT474 cells. In all three cell lines, formononetin increased Akt phosphorylation and Bcl-2 expression. Inhibiting miR-375 blocked these changes and increased proliferation in HUVECs, but not in MCF-7 or BT474 cells. In ovariectomized rats, formononetin increased uterine weight and caused similar changes in levels of miR-375, RASD1, ERα, and Bcl-2 in aortic ECs as in cultured HUVECs. In mice bearing MCF-7 xenografts, tumor growth was stimulated by 17β-estradiol but not by formononetin. These results suggest selective action of formononetin in ECs (proliferation stimulation and apoptosis inhibition) relative to breast cancer cells, possibly via a feedback loop involving miR-375, RASD1, and ERα. This differential effect may explain why formononetin may not increase the risk of postmenopausal breast cancer. © 2018 Wiley Periodicals, Inc.

Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARα, β/δ, and γ

PubMed Central

Li, Andrew C.; Binder, Christoph J.; Gutierrez, Alejandra; Brown, Kathleen K.; Plotkin, Christine R.; Pattison, Jennifer W.; Valledor, Annabel F.; Davis, Roger A.; Willson, Timothy M.; Witztum, Joseph L.; Palinski, Wulf; Glass, Christopher K.

2004-01-01

PPARα, β/δ, and γ regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor–ABCA1 (LXR-ABCA1) pathway. To investigate the potential importance of these activities in vivo, we performed a systematic analysis of the effects of PPARα, β, and γ agonists on foam-cell formation and atherosclerosis in male LDL receptor–deficient (LDLR–/–) mice. Like the PPARγ agonist, a PPARα-specific agonist strongly inhibited atherosclerosis, whereas a PPARβ-specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPARα and PPARγ agonists, but not the PPARβ agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpectedly, PPARα and PPARγ agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPARα required LXRs, activation of PPARγ reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmacologically to inhibit the development of atherosclerosis. PMID:15578089

Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins.

PubMed

Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Ma, Yue

2016-06-01

Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.

MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells.

PubMed

Ito, Keisuke; Eguchi, Yutaka; Imagawa, Yusuke; Akai, Shuji; Mochizuki, Hideki; Tsujimoto, Yoshihide

2017-01-01

Regulation of cell death is potentially a powerful treatment modality for intractable diseases such as neurodegenerative diseases. Although there have been many reports about the possible involvement of various types of cell death in neurodegenerative diseases, it is still unclear exactly how neurons die in patients with these diseases, thus treatment strategies based on cell death regulation have not been established yet. To obtain some insight into the mechanisms of cell death involved in neurodegenerative diseases, we studied the effect of 1-methyl-4-phenylpyridinium (MPP+) on the human neuroblastoma cell line SH-SY5Y (a widely used model of Parkinson's disease). We found that MPP+ predominantly induced non-apoptotic death of neuronally differentiated SH-SY5Y cells. This cell death was strongly inhibited by necrostatin-1 (Nec-1), a necroptosis inhibitor, and by an indole-containing compound (3,3'-diindolylmethane: DIM). However, it occurred independently of receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3), indicating that this form of cell death was not necroptosis. MPP+-induced cell death was also inhibited by several inhibitors of ferroptosis, including ferrostatin-1 (Fer-1). Although MPP+-induced death and ferroptosis shared some features, such as occurrence of lipid peroxidation and inhibition by Fer-1, MPP+-induced death seemed to be distinct from ferroptosis because MPP+-induced death (but not ferroptosis) was inhibited by Nec-1, was independent of p53, and was accompanied by ATP depletion and mitochondrial swelling. Further investigation of MPP+-induced non-apoptotic cell death may be useful for understanding the mechanisms of neuronal loss and for treatment of neurodegenerative diseases such as Parkinson's disease.

MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells

PubMed Central

Ito, Keisuke; Eguchi, Yutaka; Imagawa, Yusuke; Akai, Shuji; Mochizuki, Hideki; Tsujimoto, Yoshihide

2017-01-01

Regulation of cell death is potentially a powerful treatment modality for intractable diseases such as neurodegenerative diseases. Although there have been many reports about the possible involvement of various types of cell death in neurodegenerative diseases, it is still unclear exactly how neurons die in patients with these diseases, thus treatment strategies based on cell death regulation have not been established yet. To obtain some insight into the mechanisms of cell death involved in neurodegenerative diseases, we studied the effect of 1-methyl-4-phenylpyridinium (MPP+) on the human neuroblastoma cell line SH-SY5Y (a widely used model of Parkinson’s disease). We found that MPP+ predominantly induced non-apoptotic death of neuronally differentiated SH-SY5Y cells. This cell death was strongly inhibited by necrostatin-1 (Nec-1), a necroptosis inhibitor, and by an indole-containing compound (3,3′-diindolylmethane: DIM). However, it occurred independently of receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3), indicating that this form of cell death was not necroptosis. MPP+-induced cell death was also inhibited by several inhibitors of ferroptosis, including ferrostatin-1 (Fer-1). Although MPP+-induced death and ferroptosis shared some features, such as occurrence of lipid peroxidation and inhibition by Fer-1, MPP+-induced death seemed to be distinct from ferroptosis because MPP+-induced death (but not ferroptosis) was inhibited by Nec-1, was independent of p53, and was accompanied by ATP depletion and mitochondrial swelling. Further investigation of MPP+-induced non-apoptotic cell death may be useful for understanding the mechanisms of neuronal loss and for treatment of neurodegenerative diseases such as Parkinson’s disease. PMID:28250973

Estrogen suppresses breast cancer proliferation through GPER / p38 MAPK axis during hypoxia.

PubMed

Sathya, S; Sudhagar, S; Lakshmi, B S

2015-12-05

Breast cancer cells frequently experience hypoxia which is associated with resistance to hormonal therapy and poor clinical prognosis, making it important to understand the function of estrogen under hypoxic condition. Here, we demonstrate that estrogen suppresses breast cancer cell growth under hypoxia, through inhibition at G1/S phase of cell cycle, by elevation of p21 expression. The involvement of GPER in estrogen mediated growth arrest was elucidated using specific ligands and siRNA. Although, estrogen was observed to activate both p44/42 and p38 MAPK signaling, pharmacological inhibition and silencing of p38 MAPK abrogated the induction of p21 expression and growth arrest, during hypoxia. The involvement of estrogen induced ROS in the p38 MAPK mediated p21 expression and cell growth arrest was established by observing that scavenging of ROS by NAC abrogated p38 MAPK activation and p21 expression during hypoxia. In conclusion, Estrogen suppresses breast cancer growth by inhibiting G1/S phase transition through GPER/ROS/p38 MAPK/p21 mediated signaling during hypoxic condition. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Sirtuin 1-dependent resveratrol cytotoxicity and pro-differentiation activity on breast cancer cells.

PubMed

Deus, Cláudia M; Serafim, Teresa L; Magalhães-Novais, Silvia; Vilaça, Andreia; Moreira, Ana C; Sardão, Vilma A; Cardoso, Susana M; Oliveira, Paulo J

2017-03-01

Sirtuins regulate several processes associated with tumor development. Resveratrol was shown to stimulate sirtuin 1 and 3 (SIRT1/3) activities and to result in cytotoxicity for some tumor types. The relationship between modulation of sirtuin activities, cellular metabolic remodeling and resveratrol cytotoxicity mechanism on breast cancer cells is still an open question. Here, we evaluated whether sirtuin 1 and 3 are involved in resveratrol toxicity and whether resveratrol leads to a metabolic remodeling and cell differentiation. Results using the Extracellular Flux Analyzer indicated that resveratrol inhibits mitochondrial respiration in breast cancer cells. We also demonstrated here for the first time that resveratrol cytotoxic effects on breast cancer cells were modulated by SIRT1 and also involved mitochondrial complex I inhibition. Importantly, we also demonstrated that resveratrol reduced the pool of breast cancer cells with stemness markers through a SIRT1-dependent mechanism. Our data highlights the role of SIRT1 in regulating resveratrol induced differentiation and/or toxicity in breast cancer cells.

CedA is a novel Escherichia coli protein that activates the cell division inhibited by chromosomal DNA over-replication.

PubMed

Katayama, T; Takata, M; Sekimizu, K

1997-11-01

We isolated and characterized a new gene related to the control of cell division regulation in Escherichia coli. At 30 degrees C, the dnaAcos mutant causes over-replication of the chromosome, and colony formation is inhibited. We found that, at this temperature, the dnaAcos cells form filaments; therefore, septum formation is inhibited. This inhibition was independent of SfiA, an inhibitor of the septum-forming protein, FtsZ. To identify factors involved in this pathway of inhibition, we isolated seven multicopy suppressors for the cold-sensitive phenotype of the dnaAcos mutant. One of these proved to be a previously unknown gene, which we named cedA. This gene encoded a 12 kDa protein and resided at 38.9min on the E. coli genome map. A multicopy supply of the cedA gene to the dnaAcos cells did not repress over-replication of the chromosome but did stimulate cell division of the host, the result being growth of cells with an abnormally elevated chromosomal copy number. Therefore, the expression level of the cedA gene seems to be important for inhibiting cell division of the dnaAcos mutant at 30 degrees C. We propose that over-replication of the chromosome activates a pathway for inhibiting cell division and that the cedA gene modulates this division control. In the dnaA+ background, cedA also seems to affect cell division.

Gallic acid indanone and mangiferin xanthone are strong determinants of immunosuppressive anti-tumour effects of Mangifera indica L. bark in MDA-MB231 breast cancer cells.

PubMed

García-Rivera, Dagmar; Delgado, René; Bougarne, Nadia; Haegeman, Guy; Berghe, Wim Vanden

2011-06-01

Vimang is a standardized extract derived from Mango bark (Mangifera Indica L.), commonly used as anti-inflammatory phytomedicine, which has recently been used to complement cancer therapies in cancer patients. We have further investigated potential anti-tumour effects of glucosylxanthone mangiferin and indanone gallic acid, which are both present in Vimang extract. We observed significant anti-tumour effects of both Vimang constituents in the highly aggressive and metastatic breast cancer cell type MDA-MB231. At the molecular level, mangiferin and gallic acid both inhibit classical NFκB activation by IKKα/β kinases, which results in impaired IκB degradation, NFκB translocation and NFκB/DNA binding. In contrast to the xanthone mangiferin, gallic acid further inhibits additional NFκB pathways involved in cancer cell survival and therapy resistance, such as MEK1, JNK1/2, MSK1, and p90RSK. This results in combinatorial inhibition of NFκB activity by gallic acid, which results in potent inhibition of NFκB target genes involved in inflammation, metastasis, anti-apoptosis and angiogenesis, such as IL-6, IL-8, COX2, CXCR4, XIAP, bcl2, VEGF. The cumulative NFκB inhibition by gallic acid, but not mangiferin, is also reflected at the level of cell survival, which reveals significant tumour cytotoxic effects in MDA-MB231 cells. Altogether, we identify gallic acid, besides mangiferin, as an essential anti-cancer component in Vimang extract, which demonstrates multifocal inhibition of NFκB activity in the cancer-inflammation network. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

BK Polyomavirus Replication in Renal Tubular Epithelial Cells Is Inhibited by Sirolimus, but Activated by Tacrolimus Through a Pathway Involving FKBP-12.

PubMed

Hirsch, H H; Yakhontova, K; Lu, M; Manzetti, J

2016-03-01

BK polyomavirus (BKPyV) replication causes nephropathy and premature kidney transplant failure. Insufficient BKPyV-specific T cell control is regarded as a key mechanism, but direct effects of immunosuppressive drugs on BKPyV replication might play an additional role. We compared the effects of mammalian target of rapamycin (mTOR)- and calcineurin-inhibitors on BKPyV replication in primary human renal tubular epithelial cells. Sirolimus impaired BKPyV replication with a 90% inhibitory concentration of 4 ng/mL by interfering with mTOR-SP6-kinase activation. Sirolimus inhibition was rapid and effective up to 24 h postinfection during viral early gene expression, but not thereafter, during viral late gene expression. The mTORC-1 kinase inhibitor torin-1 showed a similar inhibition profile, supporting the notion that early steps of BKPyV replication depend on mTOR activity. Cyclosporine A also inhibited BKPyV replication, while tacrolimus activated BKPyV replication and reversed sirolimus inhibition. FK binding protein 12kda (FKBP-12) siRNA knockdown abrogated sirolimus inhibition and increased BKPyV replication similar to adding tacrolimus. Thus, sirolimus and tacrolimus exert opposite effects on BKPyV replication in renal tubular epithelial cells by a mechanism involving FKBP-12 as common target. Immunosuppressive drugs may therefore contribute directly to the risk of BKPyV replication and nephropathy besides suppressing T cell functions. The data provide rationales for clinical trials aiming at reducing the risk of BKPyV replication and disease in kidney transplantation. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

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

Railo, Antti; Nagy, Irina I.; Kilpelaeinen, Pekka

The Wnt family of glycoprotein growth factors controls a number of central cellular processes such as proliferation, differentiation and ageing. All the Wnt proteins analyzed so far either activate or inhibit the canonical {beta}-catenin signaling pathway that regulates transcription of the target genes. In addition, some of them activate noncanonical signaling pathways that involve components such as the JNK, heterotrimeric G proteins, protein kinase C, and calmodulin-dependent protein kinase II, although the precise signaling mechanisms are only just beginning to be revealed. We demonstrate here that Wnt-11 signaling is sufficient to inhibit not only the canonical {beta}-catenin mediated Wnt signalingmore » but also JNK/AP-1 and NF-{kappa}B signaling in the CHO cells, thus serving as a noncanonical Wnt ligand in this system. Inhibition of the JNK/AP-1 pathway is mediated in part by the MAPK kinase MKK4 and Akt. Moreover, protein kinase C is involved in the regulation of JNK/AP-1 by Wnt-11, but not of the NF-{kappa}B pathway. Consistent with the central role of Akt, JNK and NF-{kappa}B in cell survival and stress responses, Wnt-11 signaling promotes cell viability. Hence Wnt-11 is involved in coordination of key signaling pathways.« less

Analysis of surface properties of fixed and live cells using derivatized agarose beads.

PubMed

Navarro, Vanessa M; Walker, Sherri L; Badali, Oliver; Abundis, Maria I; Ngo, Lylla L; Weerasinghe, Gayani; Barajas, Marcela; Zem, Gregory; Oppenheimer, Steven B

2002-01-01

A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

Role of mitochondrial dysfunction in neurotoxicity of MPP+: partial protection of PC12 cells by acetyl-L-carnitine.

PubMed

Virmani, Ashraf; Gaetani, Franco; Binienda, Zbigniew; Xu, Alex; Duhart, Helen; Ali, Syed F

2004-10-01

The damage to the central nervous system that is observed after administration of either methamphetamine (METH) or 1-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is known to be linked to dopamine (DA). The underlying neurotoxicity mechanism for both METH and MPP+ seem to involve free radical formation and impaired mitochondrial function. The MPP+ is thought to selectively kill nigrostriatal dopaminergic neurons by inhibiting mitochondrial complex I, with cell death being attributed to oxidative stress damage to these vulnerable DA neurons. In the present study, MPP+ was shown to significantly inhibit the response to MTT by cultured PC12 cells. This inhibitory action of MPP+ could be partially reversed by the co-incubation of the cells with the acetylated form of carnitine, acetyl-L-carnitine (ALC). Since at least part of the toxic action of MPP+ is related to mitochondrial inhibition, the partial reversal of the inhibition of MTT response by ALC could involve a partial restoration of mitochondrial function. The role carnitine derivatives, such as ALC, play in attenuating MPP+ and METH-evoked toxicity is still under investigation to elucidate the contribution of mitochondrial dysfunction in mechanisms of neurotoxicity.

Rho-Kinase Inhibition During Early Cardiac Development Causes Arrhythmogenic Right Ventricular Cardiomyopathy in Mice.

PubMed

Ellawindy, Alia; Satoh, Kimio; Sunamura, Shinichiro; Kikuchi, Nobuhiro; Suzuki, Kota; Minami, Tatsuro; Ikeda, Shohei; Tanaka, Shinichi; Shimizu, Toru; Enkhjargal, Budbazar; Miyata, Satoshi; Taguchi, Yuhto; Handoh, Tetsuya; Kobayashi, Kenta; Kobayashi, Kazuto; Nakayama, Keiko; Miura, Masahito; Shimokawa, Hiroaki

2015-10-01

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by fibrofatty changes of the right ventricle, ventricular arrhythmias, and sudden death. Though ARVC is currently regarded as a disease of the desmosome, desmosomal gene mutations have been identified only in half of ARVC patients, suggesting the involvement of other associated mechanisms. Rho-kinase signaling is involved in the regulation of intracellular transport and organizes cytoskeletal filaments, which supports desmosomal protein complex at the myocardial cell-cell junctions. Here, we explored whether inhibition of Rho-kinase signaling is involved in the pathogenesis of ARVC. Using 2 novel mouse models with SM22α- or αMHC-restricted overexpression of dominant-negative Rho-kinase, we show that mice with Rho-kinase inhibition in the developing heart (SM22α-restricted) spontaneously develop cardiac dilatation and dysfunction, myocardial fibrofatty changes, and ventricular arrhythmias, resulting in premature sudden death, phenotypes fulfilling the criteria of ARVC in humans. Rho-kinase inhibition in the developing heart results in the development of ARVC phenotypes in dominant-negative Rho-kinase mice through 3 mechanisms: (1) reduction of cardiac cell proliferation and ventricular wall thickness, (2) stimulation of the expression of the proadipogenic noncanonical Wnt ligand, Wnt5b, and the major adipogenic transcription factor, PPARγ (peroxisome proliferator activated receptor-γ), and inhibition of Wnt/β-catenin signaling, and (3) development of desmosomal abnormalities. These mechanisms lead to the development of cardiac dilatation and dysfunction, myocardial fibrofatty changes, and ventricular arrhythmias, ultimately resulting in sudden premature death in this ARVC mouse model. This study demonstrates a novel crucial role of Rho-kinase inhibition during cardiac development in the pathogenesis of ARVC in mice. © 2015 American Heart Association, Inc.

DRAM Triggers Lysosomal Membrane Permeabilization and Cell Death in CD4+ T Cells Infected with HIV

PubMed Central

Laforge, Mireille; Limou, Sophie; Harper, Francis; Casartelli, Nicoletta; Rodrigues, Vasco; Silvestre, Ricardo; Haloui, Houda; Zagury, Jean-Francois; Senik, Anna; Estaquier, Jerome

2013-01-01

Productive HIV infection of CD4+ T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells. PMID:23658518

Atrial natriuretic peptide: a possible mediator involved in dexamethasone's inhibition of cell proliferation in multiple myeloma.

PubMed

Ding, Jiang-Hua; Chang, Yu-Sui

2012-08-01

Atrial natriuretic peptide (ANP) has been recognized for several decades for its role of regulating blood pressure. Recently, cumulating evidences show that ANP plays an anticancer role in various solid tumors via blocking the kinase cascade of Ras-MEK1/2-ERK1/2 with the result of inhibition of DNA synthesis. ANP, as well as its receptors (NPR-A and NPR-C) has been identified present in the embryonic stem cell and a wide range of cancer cells. Various lymphoid organs, such as lymph nodes, have been detected the presence of ANP. Multiple myeloma (MM), though the therapies have evolved significantly, is still an incurable disease as B lymphocyte cell neoplasm. Dexamethasone is the cornerstone in treatment of MM via inactivation of Ras-MEK1/2-ERK1/2 cascade reaction. Coincidently, dexamethasone can increase the expression of ANP markedly. Nevertheless, the role of ANP in MM is unclear. Based on these results above, we raise the hypothesis that ANP is involved in mediating dexamethasone's inhibition of proliferation in MM cells, which suggests that ANP may be a potential agent to treat MM. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Multimodal effects of small molecule ROCK and LIMK inhibitors on mitosis, and their implication as anti-leukemia agents.

PubMed

Oku, Yusuke; Tareyanagi, Chiaki; Takaya, Shinichi; Osaka, Sayaka; Ujiie, Haruki; Yoshida, Kentaro; Nishiya, Naoyuki; Uehara, Yoshimasa

2014-01-01

Accurate chromosome segregation is vital for cell viability. Many cancer cells show chromosome instability (CIN) due to aberrant expression of the genes involved in chromosome segregation. The induction of massive chromosome segregation errors in such cancer cells by small molecule inhibitors is an emerging strategy to kill these cells selectively. Here we screened and characterized small molecule inhibitors which cause mitotic chromosome segregation errors to target cancer cell growth. We screened about 300 chemicals with known targets, and found that Rho-associated coiled-coil kinase (ROCK) inhibitors bypassed the spindle assembly checkpoint (SAC), which delays anaphase onset until proper kinetochore-microtubule interactions are established. We investigated how ROCK inhibitors affect chromosome segregation, and found that they induced microtubule-dependent centrosome fragmentation. Knockdown of ROCK1 and ROCK2 revealed their additive roles in centrosome integrity. Pharmacological inhibition of LIMK also induced centrosome fragmentation similar to that by ROCK inhibitors. Inhibition of ROCK or LIMK hyper-stabilized mitotic spindles and impaired Aurora-A activation. These results suggested that ROCK and LIMK are directly or indirectly involved in microtubule dynamics and activation of Aurora-A. Furthermore, inhibition of ROCK or LIMK suppressed T cell leukemia growth in vitro, but not peripheral blood mononuclear cells. They induced centrosome fragmentation and apoptosis in T cell leukemia cells. These results suggested that ROCK and LIMK can be a potential target for anti-cancer drugs.

p53-independent p21 induction by MELK inhibition.

PubMed

Matsuda, Tatsuo; Kato, Taigo; Kiyotani, Kazuma; Tarhan, Yunus Emre; Saloura, Vassiliki; Chung, Suyoun; Ueda, Koji; Nakamura, Yusuke; Park, Jae-Hyun

2017-08-29

MELK play critical roles in human carcinogenesis through activation of cell proliferation, inhibition of apoptosis and maintenance of stemness. Therefore, MELK is a promising therapeutic target for a wide range of cancers. Although p21 is a well-known p53-downstream gene, we found that treatment with a potent MELK inhibitor, OTS167, could induce p21 protein expression in cancer cell lines harboring loss-of-function TP53 mutations. We also confirmed that MELK knockdown by siRNA induced the p21 expression in p53-deficient cancer cell lines and caused the cell cycle arrest at G1 phase. Further analysis indicated that FOXO1 and FOXO3, two known transcriptional regulators of p21, were phosphorylated by MELK and thus be involved in the induction of p21 after MELK inhibition. Collectively, our herein findings suggest that MELK inhibition may be effective for human cancers even if TP53 is mutated.

p53-independent p21 induction by MELK inhibition

PubMed Central

Matsuda, Tatsuo; Kato, Taigo; Kiyotani, Kazuma; Tarhan, Yunus Emre; Saloura, Vassiliki; Chung, Suyoun; Ueda, Koji; Nakamura, Yusuke; Park, Jae-Hyun

2017-01-01

MELK play critical roles in human carcinogenesis through activation of cell proliferation, inhibition of apoptosis and maintenance of stemness. Therefore, MELK is a promising therapeutic target for a wide range of cancers. Although p21 is a well-known p53-downstream gene, we found that treatment with a potent MELK inhibitor, OTS167, could induce p21 protein expression in cancer cell lines harboring loss-of-function TP53 mutations. We also confirmed that MELK knockdown by siRNA induced the p21 expression in p53-deficient cancer cell lines and caused the cell cycle arrest at G1 phase. Further analysis indicated that FOXO1 and FOXO3, two known transcriptional regulators of p21, were phosphorylated by MELK and thus be involved in the induction of p21 after MELK inhibition. Collectively, our herein findings suggest that MELK inhibition may be effective for human cancers even if TP53 is mutated. PMID:28938528

Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect

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

Ghezali, Lamia; Leger, David Yannick; Limami, Youness

2013-04-15

Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effectmore » on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines. - Highlights: ► Cyclopamine alone but not jervine induces apoptosis in human erythroleukemia cells. ► Cyclopamine and jervine induce COX-2 overexpression. ► COX-2 overexpression is implicated in resistance to cyclopamine-induced apoptosis. ► Apoptotic potential of jervine is restrained by NF-κB pathway activation. ► PKC is involved in cyclopamine-induced apoptosis and COX-2 overexpression.« less

The Wnt5A/Protein Kinase C Pathway Mediates Motility in Melanoma Cells via the Inhibition of Metastasis Suppressors and Initiation of an Epithelial to Mesenchymal Transition*S

PubMed Central

Dissanayake, Samudra K.; Wade, Michael; Johnson, Carrie E.; O’Connell, Michael P.; Leotlela, Poloko D.; French, Amanda D.; Shah, Kavita V.; Hewitt, Kyle J.; Rosenthal, Devin T.; Indig, Fred E.; Jiang, Yuan; Nickoloff, Brian J.; Taub, Dennis D.; Trent, Jeffrey M.; Moon, Randall T.; Bittner, Michael; Weeraratna, Ashani T.

2008-01-01

We have shown that Wnt5A increases the motility of melanoma cells. To explore cellular pathways involving Wnt5A, we compared gain-of-function (WNT5A stable transfectants) versus loss-of-function (siRNA knockdown) of WNT5A by microarray analysis. Increasing WNT5A suppressed the expression of several genes, which were re-expressed after small interference RNA-mediated knockdown of WNT5A. Genes affected by WNT5A include KISS-1, a metastasis suppressor, and CD44, involved in tumor cell homing during metastasis. This could be validated at the protein level using both small interference RNA and recombinant Wnt5A (rWnt5A). Among the genes up-regulated by WNT5A was the gene vimentin, associated with an epithelial to mesenchymal transition (EMT), which involves decreases in E-cadherin, due to up-regulation of the transcriptional repressor, Snail. rWnt5A treatment increases Snail and vimentin expression, and decreases E-cadherin, even in the presence of dominant-negativeTCF4, suggesting that this activation is independent of Wnt/β-catenin signaling. Because Wnt5A can signal via protein kinase C (PKC), the role of PKC in Wnt5A-mediated motility and EMT was also assessed using PKC inhibition and activation studies. Treating cells expressing low levels of Wnt5A with phorbol ester increased Snail expression inhibiting PKC in cells expressing high levels of Wnt5A decreased Snail. Furthermore, inhibition of PKC before Wnt5A treatment blocked Snail expression, implying that Wnt5A can potentiate melanoma metastasis via the induction of EMT in a PKC-dependent manner. PMID:17426020

Hepatic Stellate Cells Inhibit T Cells through Active TGF-β1 from a Cell Surface-Bound Latent TGF-β1/GARP Complex.

PubMed

Li, Yan; Kim, Byung-Gyu; Qian, Shiguang; Letterio, John J; Fung, John J; Lu, Lina; Lin, Feng

2015-09-15

Hepatic stellate cells (HSCs) inhibit T cells, a process that could help the liver to maintain its immunoprivileged status. HSCs secrete latent TGF-β1, but the detailed mechanisms by which latent TGF-β1 is activated and whether it plays any role in HSC-mediated T cell suppression remain unclear. Glycoprotein A repetitions predominant (GARP) is a surface marker of activated regulatory T cells. GARP binds latent TGF-β1 for its activation, which is critical for regulatory T cells to suppress effector T cells; however, it is still unclear whether GARP is present on HSCs and whether it has any impact on HSC function. In this study, we found that TGF-β1(+/-) HSCs, which produce reduced levels of TGF-β1, showed decreased potency in inhibiting T cells. We also found that pharmaceutical or genetic inhibition of the TGF-β1 signaling pathway reduced the T cell-inhibiting activity of HSCs. Additionally, using isolated primary HSCs, we demonstrated that GARP was constitutively expressed on HSCs. Blocking GARP function or knocking down GARP expression significantly impaired the potency of HSCs to suppress the proliferation of and IFN-γ production from activated T cells, suggesting that GARP is important for HSCs to inhibit T cells. These results demonstrate the unexpected presence of GARP on HSCs and its significance in regard to the ability of HSCs to activate latent TGF-β1 and thereby inhibit T cells. Our study reveals a new mechanism for HSC-mediated immune regulation and potentially for other conditions, such as liver fibrosis, that involve HSC-secreted TGF-β1. Copyright © 2015 by The American Association of Immunologists, Inc.

Inhibition of the aquaporin 3 water channel increases the sensitivity of prostate cancer cells to cryotherapy

PubMed Central

Ismail, M; Bokaee, S; Davies, J; Harrington, K J; Pandha, H

2009-01-01

Aquaporins (AQPs) are intrinsic membrane proteins that facilitate selective water and small solute movement across the plasma membrane. In this study, we investigate the role of inhibiting AQPs in sensitising prostate cancer cells to cryotherapy. PC-3 and DU145 prostate cancer cells were cooled to 0, −5 and −10°C. The expression of AQP3 in response to freezing was determined using real-time quantitative polymerase chain reaction (RT–qPCR) and western blot analysis. Aquaporins were inhibited using mercuric chloride (HgCl2) and small interfering RNA (siRNA) duplex, and cell survival was assessed using a colorimetric assay. There was a significant increase in AQP3 expression in response to freezing. Cells treated with AQP3 siRNA were more sensitive to cryoinjury compared with control cells (P<0.001). Inhibition of the AQPs by HgCl2 also increased the sensitivity of both cell lines to cryoinjury and there was a complete loss of cell viability at −10°C (P<0.01). In conclusion, we have shown that AQP3 is involved directly in cryoinjury. Inhibition of AQP3 increases the sensitivity of prostate cancer cells to freezing. This strategy may be exploited in the clinic to improve the efficacy of prostate cryotherapy. PMID:19513079

Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

PubMed Central

Ricci, Alessandro; Pacella, Aurora; Cigliana, Giovanni; Bozzuto, Giuseppina; Podo, Franca; Carpinelli, Giulia

2017-01-01

Background The chemokine receptor CXCR4 plays a crucial role in tumors, including glioblastoma multiforme (GBM), the most aggressive glioma. Phosphatidylcholine-specific phospholipase C (PC-PLC), a catabolic enzyme of PC metabolism, is involved in several aspects of cancer biology and its inhibition down-modulates the expression of growth factor membrane receptors interfering with their signaling pathways. In the present work we investigated the possible interplay between CXCR4 and PC-PLC in GBM cells. Methods Confocal microscopy, immunoprecipitation, western blot analyses, and the evaluation of migration and invasion potential were performed on U87MG cells after PC-PLC inhibition with the xanthate D609. The intracellular metabolome was investigated by magnetic resonance spectroscopy; lactate levels and lactate dehydrogenase (LDH) activity were analyzed by colorimetric assay. Results Our studies demonstrated that CXCR4 and PC-PLC co-localize and are associated on U87MG cell membrane. D609 reduced CXCR4 expression, cell proliferation and invasion, interfering with AKT and EGFR activation and expression. Metabolic analyses showed a decrease in intracellular lactate concentration together with a decrement in LDH activity. Conclusions Our data suggest that inhibition of PC-PLC could represent a new molecular approach in glioma biology not only for its ability in modulating cell metabolism, glioma growth and motility, but also for its inhibitory effect on crucial molecules involved in cancer progression. PMID:28423060

Paracoccidioides brasiliensis interferes on dendritic cells maturation by inhibiting PGE2 production.

PubMed

Fernandes, Reginaldo K; Bachiega, Tatiana F; Rodrigues, Daniela R; Golim, Marjorie de A; Dias-Melicio, Luciane A; Balderramas, Helanderson de A; Kaneno, Ramon; Soares, Ângela M V C

2015-01-01

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil, whose etiologic agent is the thermodimorphic fungus of the genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii. The mechanisms involved in the initial interaction of the fungus with cells of the innate immune response, as dendritic cells (DCs), deserve to be studied. Prostaglandins (PGs) are eicosanoids that play an important role in modulating functions of immune cells including DCs. Here we found that human immature DCs derived from the differentiation of monocytes cultured with GM-CSF and IL-4 release substantial concentrations of PGE2, which, however, were significantly inhibited after challenge with P. brasiliensis. In vitro blocking of pattern recognition receptors (PRRs) by monoclonal antibodies showed the involvement of mannose receptor (MR) in PGE2 inhibition by the fungus. In addition, phenotyping assays showed that after challenge with the fungus, DCs do not change their phenotype of immature cells to mature ones, as well as do not produce IL-12 p70 or adequate concentrations of TNF-α. Assays using exogenous PGE2 confirmed an association between PGE2 inhibition and failure of cells to phenotypically mature in response to P. brasiliensis. We conclude that a P. brasiliensis evasion mechanism exists associated to a dysregulation on DC maturation. These findings may provide novel information for the understanding of the complex interplay between the host and this fungus.

Listeria arpJ gene modifies T helper type 2 subset differentiation.

PubMed

Kanoh, Makoto; Maruyama, Saho; Shen, Hua; Matsumoto, Akira; Shinomiya, Hiroto; Przybilla, Karin; Gouin, Edith; Cossart, Pascale; Goebel, Werner; Asano, Yoshihiro

2015-07-15

Although the T-cell subset differentiation pathway has been characterized extensively from the view of host gene regulation, the effects of genes of the pathogen on T-cell subset differentiation during infection have yet to be elucidated. Especially, the bacterial genes that are responsible for this shift have not yet been determined. Utilizing a single-gene-mutation Listeria panel, we investigated genes involved in the host-pathogen interaction that are required for the initiation of T-cell subset differentiation in the early phase of pathogen infection. We demonstrate that the induction of T helper types 1 and 2 (Th1 and Th2) subsets are separate phenomena and are mediated by distinct Listeria genes. We identified several candidate Listeria genes that appear to be involved in the host-Listeria interaction. Among them, arpJ is the strongest candidate gene for inhibiting Th2 subset induction. Furthermore, the analysis utilizing arpJ-deficient Listeria monocytogenes (Lm) revealed that the tumor necrosis factor (TNF) superfamily (Tnfsf) 9-TNF receptor superfamily (Tnfrsf) 9 interaction inhibits the Th2 response during Lm infection. arpJ is the candidate gene for inhibiting Th2 T-cell subset induction. The arpJ gene product influences the expression of Tnfsf/Tnfrsf on antigen-presenting cells and inhibits the Th2 T-cell subset differentiation during Listeria infection. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

RIP1 Inhibition Rescues from LPS-Induced RIP3-Mediated Programmed Cell Death, Distributed Energy Metabolism and Spatial Memory Impairment.

PubMed

Nikseresht, Sara; Khodagholi, Fariba; Nategh, Mohsen; Dargahi, Leila

2015-10-01

Receptor interacting protein 1 (RIP1) has a critical role in initiation of programmed necrosis or necroptosis. RIP1 in a close collaboration with RIP3 not only mediates necroptosis but also is involved in apoptosis and inflammatory signaling. However, the interpretation of the distinct function of RIP1 and RIP3 is complicated. Herein, we demonstrated that RIP1 inhibition in the context of LPS-induced neuroinflammation decreases RIP3 expression. Concomitant administration of Nec-1, specific inhibitor of RIP1, with LPS also attenuated the activating effect of RIP3 on metabolic enzymes, glutamate-ammonia ligase and glutamate dehydrogenase as bioenergetic determinants, in hippocampal and cortical cells. RIP1 inhibition possessed an anti-inflammatory effect and improved the antioxidant capacity against LPS. Interestingly, and opposed to some reports that necroptosis inhibition sensitizes cells to apoptosis, our results showed that RIP1 inhibition attenuates apoptotic cell death in response to LPS. The survival of neuronal function was also confirmed by measuring spontaneous alternations of rats in Y-maze. In conclusion, effects of RIP1 inhibition on RIP3 and cell death provide new approaches to ameliorate neuroinflammation and relative disorders.

Regulation of CaV2 calcium channels by G protein coupled receptors

PubMed Central

Zamponi, Gerald W.; Currie, Kevin P.M.

2012-01-01

Voltage gated calcium channels (Ca2+ channels) are key mediators of depolarization induced calcium influx into excitable cells, and thereby play pivotal roles in a wide array of physiological responses. This review focuses on the inhibition of CaV2 (N- and P/Q-type) Ca2+-channels by G protein coupled receptors (GPCRs), which exerts important autocrine/paracrine control over synaptic transmission and neuroendocrine secretion. Voltage-dependent inhibition is the most widespread mechanism, and involves direct binding of the G protein βγ dimer (Gβγ) to the α1 subunit of CaV2 channels. GPCRs can also recruit several other distinct mechanisms including phosphorylation, lipid signaling pathways, and channel trafficking that result in voltage-independent inhibition. Current knowledge of Gβγ-mediated inhibition is reviewed, including the molecular interactions involved, determinants of voltage-dependence, and crosstalk with other cell signaling pathways. A summary of recent developments in understanding the voltage-independent mechanisms prominent in sympathetic and sensory neurons is also included. PMID:23063655

LPA Induces Colon Cancer Cell Proliferation through a Cooperation between the ROCK and STAT-3 Pathways.

PubMed

Leve, Fernanda; Peres-Moreira, Rubem J; Binato, Renata; Abdelhay, Eliana; Morgado-Díaz, José A

2015-01-01

Lysophosphatidic acid (LPA) plays a critical role in the proliferation and migration of colon cancer cells; however, the downstream signaling events underlying these processes remain poorly characterized. The aim of this study was to investigate the signaling pathways triggered by LPA to regulate the mechanisms involved in the progression of colorectal cancer (CRC). We have used three cell line models of CRC, and initially analyzed the expression profile of LPA receptors (LPAR). Then, we treated the cells with LPA and events related to their tumorigenic potential, such as migration, invasion, anchorage-independent growth, proliferation as well as apoptosis and cell cycle were evaluated. We used the Chip array technique to analyze the global gene expression profiling that occurs after LPA treatment, and we identified cell signaling pathways related to the cell cycle. The inhibition of these pathways verified the conclusions of the transcriptomic analysis. We found that the cell lines expressed LPAR1, -2 and -3 in a differential manner and that 10 μM LPA did not affect cell migration, invasion and anchorage-independent growth, but it did induce proliferation and cell cycle progression in HCT-116 cells. Although LPA in this concentration did not induce transcriptional activity of β-catenin, it promoted the activation of Rho and STAT-3. Moreover, ROCK and STAT-3 inhibitors prevented LPA-induced proliferation, but ROCK inhibition did not prevent STAT-3 activation. Finally, we observed that LPA regulates the expression of genes related to the cell cycle and that the combined inhibition of ROCK and STAT-3 prevented cell cycle progression and increased the LPA-induced expression of cyclins E1, A2 and B1 to a greater degree than either inhibitor alone. Overall, these results demonstrate that LPA increases the proliferative potential of colon adenocarcinoma HCT-116 cells through a mechanism involving cooperation between the Rho-ROCK and STAT3 pathways involved in cell cycle control.

ERP44 inhibits human lung cancer cell migration mainly via IP3R2.

PubMed

Huang, Xue; Jin, Meng; Chen, Ying-Xiao; Wang, Jun; Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

2016-06-01

Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway.

ERP44 inhibits human lung cancer cell migration mainly via IP3R2

PubMed Central

Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

2016-01-01

Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway. PMID:27347718

Involvement of PPAR-γ in the neuroprotective and anti-inflammatory effects of angiotensin type 1 receptor inhibition: effects of the receptor antagonist telmisartan and receptor deletion in a mouse MPTP model of Parkinson's disease.

PubMed

Garrido-Gil, Pablo; Joglar, Belen; Rodriguez-Perez, Ana I; Guerra, Maria J; Labandeira-Garcia, Jose L

2012-02-22

Several recent studies have shown that angiotensin type 1 receptor (AT1) antagonists such as candesartan inhibit the microglial inflammatory response and dopaminergic cell loss in animal models of Parkinson's disease. However, the mechanisms involved in the neuroprotective and anti-inflammatory effects of AT1 blockers in the brain have not been clarified. A number of studies have reported that AT1 blockers activate peroxisome proliferator-activated receptor gamma (PPAR γ). PPAR-γ activation inhibits inflammation, and may be responsible for neuroprotective effects, independently of AT1 blocking actions. We have investigated whether oral treatment with telmisartan (the most potent PPAR-γ activator among AT1 blockers) provides neuroprotection against dopaminergic cell death and neuroinflammation, and the possible role of PPAR-γ activation in any such neuroprotection. We used a mouse model of parkinsonism induced by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and co-administration of the PPAR-γ antagonist GW9662 to study the role of PPAR-γ activation. In addition, we used AT1a-null mice lesioned with MPTP to study whether deletion of AT1 in the absence of any pharmacological effect of AT1 blockers provides neuroprotection, and investigated whether PPAR-γ activation may also be involved in any such effect of AT1 deletion by co-administration of the PPAR-γ antagonist GW9662. We observed that telmisartan protects mouse dopaminergic neurons and inhibits the microglial response induced by administration of MPTP. The protective effects of telmisartan on dopaminergic cell death and microglial activation were inhibited by co-administration of GW9662. Dopaminergic cell death and microglial activation were significantly lower in AT1a-null mice treated with MPTP than in mice not subjected to AT1a deletion. Interestingly, the protective effects of AT1 deletion were also inhibited by co-administration of GW9662. The results suggest that telmisartan provides effective neuroprotection against dopaminergic cell death and that the neuroprotective effect is mediated by PPAR-γ activation. However, the results in AT1-deficient mice show that blockage of AT1, unrelated to the pharmacological properties of AT1 blockers, also protects against dopaminergic cell death and neuroinflammation. Furthermore, the results show that PPAR-γ activation is involved in the anti-inflammatory and neuroprotective effects of AT1 deletion.

Roxithromycin inhibits VEGF-induced human airway smooth muscle cell proliferation: Opportunities for the treatment of asthma

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

Pei, Qing-Mei, E-mail: 34713316@qq.com; Jiang, Ping, E-mail: jiangping@163.com; Yang, Min, E-mail: YangMin@163.com

Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodelling, which is associated with increased airway smooth muscle (ASM) mass. Roxithromycin (RXM) has been widely used in asthma treatment; however, its mechanism of action is poorly understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodelling in patients with asthma, and shown to promote ASM cell proliferation. Here, we investigated the effect of RXM on VEGF-induced ASM cell proliferation and attempted to elucidate the underlying mechanisms of action. We tested the effect of RXM on proliferationmore » and cell cycle progression, as well as on the expression of phospho-VEGF receptor 2 (VEGFR2), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-Akt, and caveolin-1 in VEGF-stimulated ASM cells. RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. Additionally, VEGF-induced ASM cell proliferation was suppressed by inhibiting the activity of ERK1/2, but not that of Akt. Furthermore, RXM treatment inhibits VEGF-induced activation of VEGFR2 and ERK and downregulation of caveolin-1 in a dose-dependent manner. RXM also inhibited TGF-β-induced VEGF secretion by ASM cells and BEAS-2B cells. Collectively, our findings suggest that RXM inhibits VEGF-induced ASM cell proliferation by suppression of VEGFR2 and ERK1/2 activation and caveolin-1 down-regulation, which may be involved in airway remodelling. Further elucidation of the mechanisms underlying these observations should enable the development of treatments for smooth muscle hyperplasia-associated diseases of the airway such as asthma. - Highlights: • RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. • VEGF-induced cell proliferation was suppressed by inhibiting the activity of ERK1/2. • RXM inhibits activation of VEGFR2 and ERK and downregulation of caveolin-1. • RXM inhibited TGF-β-induced VEGF secretion by ASM cells and BEAS-2B cells. • Our findings expand our knowledge of the role of RXM in airway remodelling.« less

Immune cell inhibition by SLAMF7 is mediated by a mechanism requiring src kinases, CD45, and SHIP-1 that is defective in multiple myeloma cells.

PubMed

Guo, Huaijian; Cruz-Munoz, Mario-Ernesto; Wu, Ning; Robbins, Michael; Veillette, André

2015-01-01

Signaling lymphocytic activation molecule F7 (SLAMF7) is a receptor present on immune cells, including natural killer (NK) cells. It is also expressed on multiple myeloma (MM) cells. This led to development of an anti-SLAMF7 antibody, elotuzumab, showing efficacy against MM. SLAMF7 mediates activating or inhibitory effects in NK cells, depending on whether cells express or do not express the adaptor EAT-2. Since MM cells lack EAT-2, we elucidated the inhibitory effectors of SLAMF7 in EAT-2-negative NK cells and tested whether these effectors were triggered in MM cells. SLAMF7-mediated inhibition in NK cells lacking EAT-2 was mediated by SH2 domain-containing inositol phosphatase 1 (SHIP-1), which was recruited via tyrosine 261 of SLAMF7. Coupling of SLAMF7 to SHIP-1 required Src kinases, which phosphorylated SLAMF7. Although MM cells lack EAT-2, elotuzumab did not induce inhibitory signals in these cells. This was at least partly due to a lack of CD45, a phosphatase required for Src kinase activation. A defect in SLAMF7 function was also observed in CD45-deficient NK cells. Hence, SLAMF7-triggered inhibition is mediated by a mechanism involving Src kinases, CD45, and SHIP-1 that is defective in MM cells. This defect might explain why elotuzumab eliminates MM cells by an indirect mechanism involving the activation of NK cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Pirfenidone inhibits p38-mediated generation of procoagulant microparticles by human alveolar epithelial cells.

PubMed

Neri, Tommaso; Lombardi, Stefania; Faìta, Francesca; Petrini, Silvia; Balìa, Cristina; Scalise, Valentina; Pedrinelli, Roberto; Paggiaro, Pierluigi; Celi, Alessandro

2016-08-01

Pirfenidone is a drug recently approved for idiopathic pulmonary fibrosis but its mechanisms of action are partially unknown. We have previously demonstrated that the airways of patients with idiopathic pulmonary fibrosis contain procoagulant microparticles that activate coagulation factor X to its active form, Xa, a proteinase that signals fibroblast growth and differentiation, thus potentially contributing to the pathogenesis of the disease. We also reported that in vitro exposure of human alveolar cells to H2O2 causes microparticle generation. Since p38 activation is involved in microparticle generation in some cell models and p38 inhibition is one of the mechanisms of action of pirfenidone, we investigated the hypothesis that H2O2-induced generation of microparticles by alveolar cells is dependent on p38 phosphorylation and is inhibited by pirfenidone. H2O2 stimulation of alveolar cells caused p38 phosphorylation that was inhibited by pirfenidone. The drug also inhibited H2O2 induced microparticle generation as assessed by two independent methods (solid phase thrombin generation and flow cytometry). The shedding of microparticle-bound tissue factor activity was also inhibited by pirfenidone. Inhibition of p38-mediated generation of procoagulant microparticle is a previously unrecognized mechanism of action of the antifibrotic drug, pirfenidone. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rosmarinic acid counteracts activation of hepatic stellate cells via inhibiting the ROS-dependent MMP-2 activity: Involvement of Nrf2 antioxidant system

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

Lu, Changfang; Zou, Yu; Liu, Yuzhang

Recently, oxidative stress is involved in hepatofibrogenesis. Matrix metalloproteinase-2 (MMP-2) is required for activation of hepatic stellate cells (HSCs) in response to reactive oxygen species (ROS). This study was designed to explore the hypothesis that the inhibitory effect of rosmarinic acid (RA) on HSCs activation might mainly result from its antioxidant capability by increasing the synthesis of glutathione (GSH) involved in nuclear factor kappa B (NF-κB)-dependent inhibition of MMP-2 activity. Here, we demonstrate that RA reverses activated HSCs to quiescent cells. Concomitantly, RA inhibits MMP-2 activity. RNA interference-imposed knockdown of NF-κB abolished down-regulation of MMP-2 by RA. RA-mediated inactivation ofmore » NF-κB could be blocked by the diphenyleneiodonium chloride (DPI; a ROS inhibitor). Conversely, transfection of dominant-negative (DN) mutant of extracellular signal-regulated kinases 2 (ERK2), c-Jun N-terminal kinase 1 (JNK1), or p38α kinase had no such effect. Simultaneously, RA suppresses ROS generation and lipid peroxidation (LPO) whereas increases cellular GSH in HSC-T6 cells. Furthermore, RA significantly increased antioxidant response element (ARE)-mediated luciferase activity, nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and catalytic subunits from glutamate cysteine ligase (GCLc) expression, but not modulatory subunits from GCL (GCLm). RA-mediated up-regulation of GClc is inhibited by the shRNA-induced Nrf2 knockdown. The knocking down of Nrf2 or buthionine sulfoximine (a GCL inhibitor) abolished RA-mediated inhibition of ROS. Collectively, these results provide novel insights into the mechanisms of RA as an antifibrogenic candidate in the prevention and treatment of liver fibrosis. - Highlights: • RA reverses activated HSCs to quiescent cells. • RA suppresses MMP-2 activity through a NF-κB-dependent pathway. • Inhibition of oxidative stress by RA is dependent on nuclear translocation of Nrf2. • RA-mediated down-regulation of MMP-2 was ROS-dependent.« less

T Lymphocyte Inhibition by Tumor-Infiltrating Dendritic Cells Involves Ectonucleotidase CD39 but Not Arginase-1.

PubMed

Trad, Malika; Gautheron, Alexandrine; Fraszczak, Jennifer; Alizadeh, Darya; Larmonier, Claire; LaCasse, Collin J; Centuori, Sara; Audia, Sylvain; Samson, Maxime; Ciudad, Marion; Bonnefoy, Francis; Lemaire-Ewing, Stéphanie; Katsanis, Emmanuel; Perruche, Sylvain; Saas, Philippe; Bonnotte, Bernard

2015-01-01

T lymphocytes activated by dendritic cells (DC) which present tumor antigens play a key role in the antitumor immune response. However, in patients suffering from active cancer, DC are not efficient at initiating and supporting immune responses as they participate to T lymphocyte inhibition. DC in the tumor environment are functionally defective and exhibit a characteristic of immature phenotype, different to that of DC present in nonpathological conditions. The mechanistic bases underlying DC dysfunction in cancer responsible for the modulation of T-cell responses and tumor immune escape are still being investigated. Using two different mouse tumor models, we showed that tumor-infiltrating DC (TIDC) are constitutively immunosuppressive, exhibit a semimature phenotype, and impair responder T lymphocyte proliferation and activation by a mechanism involving CD39 ectoenzyme.

Alpha fetoprotein antagonises benzyl isothiocyanate inhibition of the malignant behaviors of hepatocellular carcinoma cells.

PubMed

Zhu, Mingyue; Li, Wei; Guo, Junli; Lu, Yan; Dong, Xu; Lin, Bo; Chen, Yi; Zhang, Xueer; Li, Mengsen

2016-11-15

Benzyl isothiocyanate (BITC) is a dietary isothiocyanate derived from cruciferous vegetables. Recent studies showed that BITC inhibited the growth of many cancer cells, including hepatocellular carcinoma (HCC) cells. Alpha-fetoprotein (AFP) is a important molecule for promoting progression of HCC, in the present investigation, we explore the influence of AFP on the role of BITC in the malignant behaviours of HCC cells, and the potential underlying mechanisms. We found thatBITC inhibited viability, migration, invasion and induced apoptosis of human liver cancer cell lines, Bel 7402(AFP producer) and HLE(non-AFP producer) cells in vitro. The role of BITC involve in promoting actived-caspase-3 and PARP-1 expression, and enhancing caspase-3 activity but decreasing MMP-2/9, survivin and CXCR4 expression. AFP antagonized the effect of BITC. This study suggests that BITC induced significant reductions in the viability of HCC cell lines. BITC may activate caspase-3 signal and inhibit the expression of growth- and metastasis-related proteins; AFP is an pivotal molecule for the HCC chemo-resistance of BITC.

Lycium barbarum Polysaccharides Protect Rat Corneal Epithelial Cells against Ultraviolet B-Induced Apoptosis by Attenuating the Mitochondrial Pathway and Inhibiting JNK Phosphorylation.

PubMed

Du, Shaobo; Han, Biao; Li, Kang; Zhang, Xuan; Sha, Xueli; Gao, Lan

2017-01-01

Lycium barbarum polysaccharides (LBPs) have been shown to play a key role in protecting the eyes by reducing the apoptosis induced by certain types of damage. However, it is not known whether LBPs can protect damaged corneal cells from apoptosis. Moreover, no reports have focused on the role of LBPs in guarding against ultraviolet B- (UVB-) induced apoptosis. The present study aimed to investigate the protective effect and underlying mechanism of LBPs against UVB-induced apoptosis in rat corneal epithelial (RCE) cells. The results showed that LBPs significantly prevented the loss of cell viability and inhibited cell apoptosis induced by UVB in RCE cells. LBPs also inhibited UVB-induced loss of mitochondrial membrane potential, downregulation of Bcl-2 , and upregulation of Bax and caspase-3. Finally, LBPs attenuated the phosphorylation of c-Jun NH 2 -terminal kinase (JNK) triggered by UVB. In summary, LBPs protect RCE cells against UVB-induced damage and apoptosis, and the underlying mechanism involves the attenuation of the mitochondrial apoptosis pathway and the inhibition of JNK phosphorylation.

Chemical genetic inhibition of Mps1 in stable human cell lines reveals novel aspects of Mps1 function in mitosis.

PubMed

Sliedrecht, Tale; Zhang, Chao; Shokat, Kevan M; Kops, Geert J P L

2010-04-22

Proper execution of chromosome segregation relies on tight control of attachment of chromosomes to spindle microtubules. This is monitored by the mitotic checkpoint that allows chromosome segregation only when all chromosomes are stably attached. Proper functioning of the attachment and checkpoint processes is thus important to prevent chromosomal instability. Both processes rely on the mitotic kinase Mps1. We present here two cell lines in which endogenous Mps1 has been stably replaced with a mutant kinase (Mps1-as) that is specifically inhibited by bulky PP1 analogs. Mps1 inhibition in these cell lines is highly penetrant and reversible. Timed inhibition during bipolar spindle assembly shows that Mps1 is critical for attachment error-correction and confirms its role in Aurora B regulation. We furthermore show that Mps1 has multiple controls over mitotic checkpoint activity. Mps1 inhibition precludes Mad1 localization to unattached kinetochores but also accelerates mitosis. This acceleration correlates with absence of detectable mitotic checkpoint complex after Mps1 inhibition. Finally, we show that short-term inhibition of Mps1 catalytic activity is sufficient to kill cells. Mps1 is involved in the regulation of multiple key processes that ensure correct chromosome segregation and is a promising target for inhibition in anti-cancer strategies. We report here two cell lines that allow specific and highly penetrant inhibition of Mps1 in a reproducible manner through the use of chemical genetics. Using these cell lines we confirm previously suggested roles for Mps1 activity in mitosis, present evidence for novel functions and examine cell viability after short and prolonged Mps1 inhibition. These cell lines present the best cellular model system to date for investigations into Mps1 biology and the effects of penetrance and duration of Mps1 inhibition on cell viability.

Arsenic trioxide-mediated growth inhibition in gallbladder carcinoma cells via down-regulation of Cyclin D1 transcription mediated by Sp1 transcription factor

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

Ai, Zhilong; Lu, Weiqi; Ton, Saixiong

2007-08-31

Gallbladder carcinoma (GBC), an aggressive and mostly lethal malignancy, is known to be resistant to a number of drug stimuli. Here, we demonstrated that arsenic trioxide inhibited the proliferation of gallbladder carcinoma in vivo and in vitro as well as the transcription of cell cycle-related protein Cyclin D1. And, Cyclin D1 overexpression inhibited the negative role of arsenic trioxide in cell cycle progression. We further explored the mechanisms by which arsenic trioxide affected Cyclin D1 transcription and found that the Sp1 transcription factor was down-regulated by arsenic trioxide, with a corresponding decrease in Cyclin D1 promoter activity. Taken together, thesemore » results suggested that arsenic trioxide inhibited gallbladder carcinoma cell proliferation via down-regulation of Cyclin D1 transcription in a Sp1-dependent manner, which provided a new mechanism of arsenic trioxide-involved cell proliferation and may have important therapeutic implications in gallbladder carcinoma patients.« less

The role of nitric oxide pathway in arginine transport and growth of IPEC-1 cells.

PubMed

Xiao, Hao; Zeng, Liming; Shao, Fangyuan; Huang, Bo; Wu, Miaomiao; Tan, Bie; Yin, Yulong

2017-05-02

L-Arginine itself and its metabolite-nitric oxide play great roles in intestinal physiology. However, the molecular mechanism underlying nitric oxide pathway regulating L-Arginine transport and cell growth is not yet fully understood. We report that inhibition of nitric oxide synthase (NOS) significantly induced cell apoptosis (p < 0.05), and promoted the rate of Arginine uptake and the expressions of protein for CAT-2 and y+LAT-1 (p < 0.05), while reduced protein expression of CAT-1. And NOS inhibition markedly decreased the activation of mammalian target of rapamycin (mTOR) and PI3K-Akt pathways by Arginine in the IPEC-1 cells (p < 0.05). Taken together, these data suggest that inhibition of NO pathway by L-NAME induces a negative feedback increasing of Arginine uptake and CAT-2 and y+LAT-1 protein expression, but promotes cell apoptosis which involved inhibiting the activation of mTOR and PI3K-Akt pathways.

3-bromopyruvate enhanced daunorubicin-induced cytotoxicity involved in monocarboxylate transporter 1 in breast cancer cells.

PubMed

Liu, Zhe; Sun, Yiming; Hong, Haiyu; Zhao, Surong; Zou, Xue; Ma, Renqiang; Jiang, Chenchen; Wang, Zhiwei; Li, Huabin; Liu, Hao

2015-01-01

Increasing evidence demonstrates that the hexokinase inhibitor 3-bromopyruvate (3-BrPA) induces the cell apoptotic death by inhibiting ATP generation in human cancer cells. Interestingly, some tumor cell lines are less sensitive to 3-BrPA-induced apoptosis than others. Moreover, the molecular mechanism of 3-BrPA-trigged apoptosis is unclear. In the present study, we examined the effects of 3-BrPA on the viability of the breast cancer cell lines MDA-MB-231 and MCF-7. We further investigated the potential roles of monocarboxylate transporter 1 (MCT1) in drug accumulation and efflux of breast cancer cells. Finally, we explored whether 3-BrPA enhanced daunorubicin (DNR)-induced cytotoxicity through regulation of MCT1 in breast cancer cells. MTT and colony formation assays were used to measure cell viability. Western blot analysis, flow cytometric analysis and fluorescent microscopy were used to determine the molecular mechanism of actions of MCT1 in different breast cancer cell lines. Whole-body bioluminescence imaging was used to investigate the effect of 3-BrPA in vivo. We found that 3-BrPA significantly inhibited cell growth and induced apoptosis in MCF-7 cell line, but not in MDA-MB-231 cells. Moreover, we observed that 3-BrPA efficiently enhanced DNR-induced cytotoxicity in MCF-7 cells by inhibiting the activity of ATP-dependent efflux pumps. We also found that MCT1 overexpression increased the efficacy of 3-BrPA in MDA-MB-231 cells. 3-BrPA markedly suppressed subcutaneous tumor growth in combination with DNR in nude mice implanted with MCF-7 cells. Lastly, our whole-body bioluminescence imaging data indicated that 3-BrPA promoted DNR accumulation in tumors. These findings collectively suggest that 3-BrPA enhanced DNR antitumor activity in breast cancer cells involved MCT-1, suggesting that inhibition of glycolysis could be an effective therapeutic approach for breast cancer treatment.

CD133: Beyond a Cancer Stem Cell Biomarker.

PubMed

Barzegar Behrooz, Amir; Syahir, Amir; Ahmad, Syahida

2018-06-18

CD133 (prominin-1), a pentaspan membrane glycoprotein, is one of the most well-characterized biomarkers used for the isolation of cancer stem cells (CSCs). The presence of CSCs is one of the main causes of tumor reversal and resilience. Accumulating evidence has shown that CD133 might be responsible for CSCs tumorigenesis, metastasis, and chemoresistance. It is now understood that CD133 interacts with the Wnt/β-catenin and PI3K-Akt signaling pathways. Moreover, CD133 can upregulate the expression of FLICE-like inhibitory protein (FLIP) in CD133-positive cells, inhibiting apoptosis. Additionally, CD133 can increase angiogenesis by activating the Wnt signaling pathway and increasing the expression of vascular endothelial growth factor-A (VEGF-A) and interleukin-8. Therefore, CD133 could be considered to be an "Achilles' heel" for CSCs, because by inhibiting this protein, the signaling pathways that are involved in cell proliferation will also be inhibited. By understanding the molecular biology of CD133, we can not only isolate stem cells, but can also utilize it as a therapeutic strategy. In this review, we summarize new insights into the fundamental cell biology of CD133 and discuss the involvement of CD133 in metastasis, metabolism, tumorigenesis, drug-resistance, apoptosis, and autophagy.

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

PubMed

Zhyvoloup, Alexander; Melamed, Anat; Anderson, Ian; Planas, Delphine; Lee, Chen-Hsuin; Kriston-Vizi, Janos; Ketteler, Robin; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R M; Fassati, Ariberto

2017-07-01

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

Ethylene Inhibits Cell Proliferation of the Arabidopsis Root Meristem1[OPEN

PubMed Central

Street, Ian H.; Aman, Sitwat; Zubo, Yan; Ramzan, Aleena; Wang, Xiaomin; Shakeel, Samina N.; Kieber, Joseph J.; Schaller, G. Eric

2015-01-01

The root system of plants plays a critical role in plant growth and survival, with root growth being dependent on both cell proliferation and cell elongation. Multiple phytohormones interact to control root growth, including ethylene, which is primarily known for its role in controlling root cell elongation. We find that ethylene also negatively regulates cell proliferation at the root meristem of Arabidopsis (Arabidopsis thaliana). Genetic analysis indicates that the inhibition of cell proliferation involves two pathways operating downstream of the ethylene receptors. The major pathway is the canonical ethylene signal transduction pathway that incorporates CONSTITUTIVE TRIPLE RESPONSE1, ETHYLENE INSENSITIVE2, and the ETHYLENE INSENSITIVE3 family of transcription factors. The secondary pathway is a phosphorelay based on genetic analysis of receptor histidine kinase activity and mutants involving the type B response regulators. Analysis of ethylene-dependent gene expression and genetic analysis supports SHORT HYPOCOTYL2, a repressor of auxin signaling, as one mediator of the ethylene response and furthermore, indicates that SHORT HYPOCOTYL2 is a point of convergence for both ethylene and cytokinin in negatively regulating cell proliferation. Additional analysis indicates that ethylene signaling contributes but is not required for cytokinin to inhibit activity of the root meristem. These results identify key elements, along with points of cross talk with cytokinin and auxin, by which ethylene negatively regulates cell proliferation at the root apical meristem. PMID:26149574

Molecular Mechanism of Action of Genistein and Related Phytoestrogens in Estrogen-Receptor Dependent and Independent Growth of Breast Cancer Cells

DTIC Science & Technology

1999-07-01

quercetin inhibited ER-negative MDA-MB-468 breast cancer cell growth with 1050 values of 8.8 and 18.1 Micronmeter, respectively. The other compounds...were less effective. The mechanism of growth inhibition by genistein and quercetin involved G2/M cell cycle arrest, changes in cyclin B1 levels and...apoptosis. Our results indicate that genistein and quercetin may be useful in the treatment of ER-negative tumors. Results of our studies on MDA-MB-468 cells have been documented and submitted for publication.

Inhibition of Human Cytomegalovirus Replication by Artemisinins: Effects Mediated through Cell Cycle Modulation

PubMed Central

Roy, Sujayita; He, Ran; Kapoor, Arun; Forman, Michael; Mazzone, Jennifer R.; Posner, Gary H.

2015-01-01

Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition. PMID:25870074

A single cell observation of staurosporine effect on the Ca2+ signals in rat basophilic leukemia cells.

PubMed

Teshima, R; Ikebuchi, H; Terao, T; Miyagawa, T; Arata, Y; Nakanishi, M

1990-09-17

A digital imaging fluorescence microscope was used to study the effect of a protein kinase inhibitor staurosporine on the antigen-dependent calcium signals in an individual rat basophilic leukemia cell (RBL-2H3). Although dose dependency of staurosporine was different from one cell to another, staurosporine inhibited, at low concentration, the calcium influx from the external medium into RBL-2H3 cells. At high concentration, however, it inhibited both the removal of calcium ion from internal stores and the calcium influx from the external medium. These results indicated that staurosporine is necessary for the inhibition of the calcium influx from the external medium and that a protein kinase (possibly protein kinase C) is involved in the calcium influx from the external medium into the cytoplasm.

Cannabidiol inhibits human glioma cell migration through a cannabinoid receptor-independent mechanism

PubMed Central

Vaccani, Angelo; Massi, Paola; Colombo, Arianna; Rubino, Tiziana; Parolaro, Daniela

2005-01-01

We evaluated the ability of cannabidiol (CBD) to impair the migration of tumor cells stimulated by conditioned medium. CBD caused concentration-dependent inhibition of the migration of U87 glioma cells, quantified in a Boyden chamber. Since these cells express both cannabinoid CB1 and CB2 receptors in the membrane, we also evaluated their engagement in the antimigratory effect of CBD. The inhibition of cell was not antagonized either by the selective cannabinoid receptor antagonists SR141716 (CB1) and SR144528 (CB2) or by pretreatment with pertussis toxin, indicating no involvement of classical cannabinoid receptors and/or receptors coupled to Gi/o proteins. These results reinforce the evidence of antitumoral properties of CBD, demonstrating its ability to limit tumor invasion, although the mechanism of its pharmacological effects remains to be clarified. PMID:15700028

The effect of glutamate on ghrelin release in mice.

PubMed

Chacrabati, Rakhi; Gong, Zhi; Ikenoya, Chika; Kondo, Daisuke; Zigman, Jeffrey M; Sakai, Takafumi; Sakata, Ichiro

2017-03-01

Ghrelin is abundantly produced in the stomach. Here, we found that glutamate decreased ghrelin expression and release in ghrelin-producing cells, and decreased levels of food intake and plasma acyl-ghrelin in mice. Treatment with siRNA of G protein-coupled receptor, family C, group 5, member B (GPRC5B) in ghrelin-producing cell lines completely blocked the effect of glutamate-induced ghrelin suppression. In addition, glutamate inhibited ghrelin release via the extracellular signal-regulated kinase (ERK) activity pathway, and stimulated CREB2 mRNA expression in ghrelin-producing cell lines. These results suggest that glutamate inhibits ghrelin release via ERK-CREB2 pathway. These results suggest that the GPRC5B-ERK-CREB2 pathway is involved in the inhibition of ghrelin expression and secretion in ghrelin cells. © 2017 International Federation for Cell Biology.

Receptor for advanced glycation end products (RAGE) functions as receptor for specific sulfated glycosaminoglycans, and anti-RAGE antibody or sulfated glycosaminoglycans delivered in vivo inhibit pulmonary metastasis of tumor cells.

PubMed

Mizumoto, Shuji; Takahashi, Jun; Sugahara, Kazuyuki

2012-06-01

Altered expression of chondroitin sulfate (CS) and heparan sulfate (HS) at the surfaces of tumor cells plays a key role in malignant transformation and tumor metastasis. Previously we demonstrated that a Lewis lung carcinoma (LLC)-derived tumor cell line with high metastatic potential had a higher proportion of E-disaccharide units, GlcUA-GalNAc(4,6-O-disulfate), in CS chains than low metastatic LLC cells and that such CS chains are involved in the metastatic process. The metastasis was markedly inhibited by the pre-administration of CS-E from squid cartilage rich in E units or by preincubation with a phage display antibody specific for CS-E. However, the molecular mechanism of the inhibition remains to be investigated. In this study the receptor molecule for CS chains containing E-disaccharides expressed on LLC cells was revealed to be receptor for advanced glycation end products (RAGE), which is a member of the immunoglobulin superfamily predominantly expressed in the lung. Interestingly, RAGE bound strongly to not only E-disaccharide, but also HS-expressing LLC cells. Furthermore, the colonization of the lungs by LLC cells was effectively inhibited by the blocking of CS or HS chains at the tumor cell surface with an anti-RAGE antibody through intravenous injections in a dose-dependent manner. These results provide the clear evidence that RAGE is at least one of the critical receptors for CS and HS chains expressed at the tumor cell surface and involved in experimental lung metastasis and that CS/HS and RAGE are potential molecular targets in the treatment of pulmonary metastasis.

ROCK activity affects IL-1-induced signaling possibly through MKK4 and p38 MAPK in Caco-2 cells.

PubMed

Banerjee, Sayantan; McGee, Dennis W

2016-09-01

Elevated levels of interleukin-1 (IL-1) accompany inflammatory bowel disease. IL-1-stimulated intestinal epithelial cells can secrete potent chemokines like CXCL8 to exacerbate inflammation. Previously, we found that inhibiting the Rho-associated kinase (ROCK) could inhibit IL-1- or TNF-α-induced CXCL8 secretion by the Caco-2 colonic epithelial cell line. This ROCK inhibition did not affect IκBα phosphorylation and degradation, but suppressed the phosphorylation of c-Jun N-terminal kinase (JNK). Therefore, ROCK must play an important role in epithelial cell CXCL8 responses through an effect on the JNK signaling pathway. Here, we extend these studies by showing that inhibiting ROCK suppressed the IL-1-induced phosphorylation of MKK4, a known activator of JNK, but not MKK7. Yet, ROCK inhibition had no significant effect on the IL-1-induced phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2. Inhibiting ROCK also suppressed the phosphorylation of p38 MAPK after IL-1 stimulation, but this inhibition had no significant effect on the stability of CXCL8 messenger RNA (mRNA) after IL-1 stimulation. These results suggest that ROCK may be important in IL-1-induced signaling through MKK4 to JNK and the activation of p38 MAPK. Finally, inhibiting ROCK in IL-1 and TNF-α co-stimulated Caco-2 cells also resulted in a significant suppression of CXCL8 secretion and mRNA levels suggesting that inhibiting ROCK may be a mechanism to inhibit the overall response of epithelial cells to both cytokines. These studies indicate a novel signaling event, which could provide a target for suppressing intestinal epithelial cells (IEC) chemokine responses involved in mucosal inflammation.

Fluoroquinolones inhibit human polyomavirus BK (BKV) replication in primary human kidney cells.

PubMed

Sharma, Biswa Nath; Li, Ruomei; Bernhoff, Eva; Gutteberg, Tore Jarl; Rinaldo, Christine Hanssen

2011-10-01

Reactivation of human polyomavirus BK (BKV) may cause polyomavirus-associated nephropathy or polyomavirus-associated hemorrhagic cystitis in renal- or bone marrow-transplant patients, respectively. Lack of treatment options has led to exploration of fluoroquinolones that inhibit topoisomerase II and IV in prokaryotes and possibly large T-antigen (LT-ag) helicase activity in polyomavirus. We characterized the effects of ofloxacin and levofloxacin on BKV replication in the natural host cells - primary human renal proximal tubular epithelial cells (RPTECs). Ofloxacin and levofloxacin inhibited BKV load in a dose-dependent manner yielding a ∼90% inhibition at 150 μg/ml. Ofloxacin at 150 μg/ml inhibited LT-ag mRNA and protein expression from 24h post infection (hpi). BKV genome replication was 77% reduced at 48 hpi and a similar reduction was found in VP1 and agnoprotein expression. At 72 hpi, the reduction in genome replication and protein expression was less pronounced. A dose-dependent cytostatic effect was noted. In infected cells, 150 μg/ml ofloxacin led to a 26% and 6% inhibition of cellular DNA replication and total metabolic activity, respectively while 150 μg/ml levofloxacin affected this slightly more, particularly in uninfected cells. Cell counting and xCELLigence results revealed that cell numbers were not reduced. In conclusion, ofloxacin and levofloxacin inhibit but do not eradicate BKV replication in RPTECs. At a concentration of ofloxacin giving ∼90% inhibition in BKV load, no significant cytotoxicity was observed. This concentration can be achieved in urine and possibly in the kidneys. Our results support a mechanism involving inhibition of LT-ag expression or functions but also suggest inhibition of cellular enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.

Involvement of Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Advanced Glycation End Products-Induced Glomerular Mesangial Cell Injury

PubMed Central

Chiang, Chih-Kang; Wang, Ching-Chia; Lu, Tien-Fong; Huang, Kuo-How; Sheu, Meei-Ling; Liu, Shing-Hwa; Hung, Kuan-Yu

2016-01-01

Advanced glycation end-products (AGEs)-induced mesangial cell death is one of major causes of glomerulus dysfunction in diabetic nephropathy. Both endoplasmic reticulum (ER) stress and autophagy are adaptive responses in cells under environmental stress and participate in the renal diseases. The role of ER stress and autophagy in AGEs-induced mesangial cell death is still unclear. Here, we investigated the effect and mechanism of AGEs on glomerular mesangial cells. AGEs dose-dependently decreased mesangial cell viability and induced cell apoptosis. AGEs also induced ER stress signals in a time- and dose-dependent manner. Inhibition of ER stress with 4-phenylbutyric acid effectively inhibited the activation of eIF2α and CHOP signals and reversed AGEs-induced cell apoptosis. AGEs also activated LC-3 cleavage, increased Atg5 expression, and decreased p62 expression, which indicated the autophagy induction in mesangial cells. Inhibition of autophagy by Atg5 siRNAs transfection aggravated AGEs-induced mesangial cell apoptosis. Moreover, ER stress inhibition by 4-phenylbutyric acid significantly reversed AGEs-induced autophagy, but autophagy inhibition did not influence the AGEs-induced ER stress-related signals activation. These results suggest that AGEs induce mesangial cell apoptosis via an ER stress-triggered signaling pathway. Atg5-dependent autophagy plays a protective role. These findings may offer a new strategy against AGEs toxicity in the kidney. PMID:27665710

Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis.

PubMed

Cherubin, Patrick; Quiñones, Beatriz; Teter, Ken

2018-02-06

Ricin, Shiga toxin, exotoxin A, and diphtheria toxin are AB-type protein toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. Intoxication is therefore viewed as an irreversible process. Using flow cytometry and a fluorescent reporter system to monitor protein synthesis, we show a single molecule of cytosolic toxin is not sufficient for complete inhibition of protein synthesis or cell death. Furthermore, cells can recover from intoxication: cells with a partial loss of protein synthesis will, upon removal of the toxin, increase the level of protein production and survive the toxin challenge. Thus, in contrast to the prevailing model, ongoing toxin delivery to the cytosol appears to be required for the death of cells exposed to sub-optimal toxin concentrations.

PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

PubMed

Marklein, Diana; Graab, Ulrike; Naumann, Ivonne; Yan, Tiandong; Ridzewski, Rosalie; Nitzki, Frauke; Rosenberger, Albert; Dittmann, Kai; Wienands, Jürgen; Wojnowski, Leszek; Fulda, Simone; Hahn, Heidi

2012-01-01

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.

PI3K Inhibition Enhances Doxorubicin-Induced Apoptosis in Sarcoma Cells

PubMed Central

Marklein, Diana; Graab, Ulrike; Naumann, Ivonne; Yan, Tiandong; Ridzewski, Rosalie; Nitzki, Frauke; Rosenberger, Albert; Dittmann, Kai; Wienands, Jürgen; Wojnowski, Leszek; Fulda, Simone; Hahn, Heidi

2012-01-01

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines. PMID:23300809

Osthole exhibits anti-cancer property in rat glioma cells through inhibiting PI3K/Akt and MAPK signaling pathways.

PubMed

Ding, Daofang; Wei, Songpu; Song, Yi; Li, Linghui; Du, Guoqing; Zhan, Hongsheng; Cao, Yuelong

2013-01-01

The purpose of this study was to investigate how Osthole affects glioma cell proliferation, apoptosis, invasion and migration. Rat glioma cells were treated with different concentrations of Osthole (0 µM, 25 µM, 50 µM, and 100 µM). Cell proliferation was assessed by measuring PCNA expression and CCK8 assay at different time points. Apoptosis was evaluated by measuring the expression of pro-apoptotic protein including Bax, Bcl2, PARP, and cleaved Caspase3, and of anti-apoptotic protein Survivin. Cell migration and invasion were assessed using different methods. Signaling pathways such as PI3K/Akt and MAPK, which are involved in the development of glioma cells, were also investigated in this study. Treatment with Osthole markedly inhibits glioma cell proliferation, as assessed by western blot with the PCNA antibody. Osthole also induces cell apoptosis by upregulating the expression of pro-apoptotic proteins, and by reducing the expression of anti-apoptotic factors. Moreover, C6 cell migration and invasion were efficiently inhibited in groups treated with Osthole, compared to the control group. Additionally, inhibition of PI3K/Akt and MAPK signaling pathway was also observed in C6 cells treated with Osthole. Our findings showed an anti-cancer effect of Osthole on glioma cells, including the proliferation inhibition, apoptosis induction, and migration/invasion inhibition. Further investigation in C6 glioma cells implicated the role of Osthole in essential pathways controlling glioma cell progression. Taken together, our data suggested that Osthole may have a potential application in glioma therapy. © 2014 S. Karger AG, Basel.

Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A

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

Siu, W.P.; Pun, Pamela Boon Li; Latchoumycandane, Calivarathan

2008-03-15

Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24-48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (> 500 {mu}M) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 {mu}M) fully inhibited diclofenac-induced cell injury, suggesting thatmore » mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca{sup 2+} chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca{sup 2+}-Bid-Bax-MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury.« less

Comparative Developmental Neurotoxicity of Organophosphates In Vivo: Transcriptional Responses of Pathways for Brain Cell Development, Cell Signaling, Cytotoxicity and Neurotransmitter Systems

PubMed Central

Slotkin, Theodore A.; Seidler, Frederic J.

2007-01-01

Organophosphates affect mammalian brain development through a variety of mechanisms beyond their shared property of cholinesterase inhibition. We used microarrays to characterize similarities and differences in transcriptional responses to chlorpyrifos and diazinon, assessing defined gene groupings for the pathways known to be associated with the mechanisms and/or outcomes of chlorpyrifos-induced developmental neurotoxicity. We exposed neonatal rats to daily doses of chlorpyrifos (1 mg/kg) or diazinon (1 or 2 mg/kg) on postnatal days 1-4 and evaluated gene expression profiles in brainstem and forebrain on day 5; these doses produce little or no cholinesterase inhibition. We evaluated pathways for general neural cell development, cell signaling, cytotoxicity and neurotransmitter systems, and identified significant differences for >60% of 252 genes. Chlorpyrifos elicited major transcriptional changes in genes involved in neural cell growth, development of glia and myelin, transcriptional factors involved in neural cell differentiation, cAMP-related cell signaling, apoptosis, oxidative stress, excitotoxicity, and development of neurotransmitter synthesis, storage and receptors for acetylcholine, serotonin, norepinephrine and dopamine. Diazinon had similar effects on many of the same processes but also showed major differences from chlorpyrifos. Our results buttress the idea that different organophosphates target multiple pathways involved in neural cell development but also that they deviate in key aspects that may contribute to disparate neurodevelopmental outcomes. Equally important, these pathways are compromised at exposures that are unrelated to biologically significant cholinesterase inhibition and its associated signs of systemic toxicity. The approach used here demonstrates how planned comparisons with microarrays can be used to screen for developmental neurotoxicity. PMID:17452286

Brucella and Osteoarticular Cell Activation: Partners in Crime

PubMed Central

Giambartolomei, Guillermo H.; Arriola Benitez, Paula C.; Delpino, M. Victoria

2017-01-01

Osteoarticular brucellosis is the most common presentation of human active disease although its prevalence varies widely. The three most common forms of osteoarticular involvement are sacroiliitis, spondylitis, and peripheral arthritis. The molecular mechanisms implicated in bone damage have been recently elucidated. B. abortus induces bone damage through diverse mechanisms in which TNF-α and the receptor activator of nuclear factor kappa-B ligand (RANKL)-the natural modulator of bone homeostasis are involved. These processes are driven by inflammatory cells, like monocytes/macrophages, neutrophils, Th17 CD4+ T, and B cells. In addition, Brucella abortus has a direct effect on osteoarticular cells and tilts homeostatic bone remodeling. These bacteria inhibit bone matrix deposition by osteoblasts (the only bone cells involved in bone deposition), and modify the phenotype of these cells to produce matrix metalloproteinases (MMPs) and cytokine secretion, contributing to bone matrix degradation. B. abortus also affects osteoclasts (cells naturally involved in bone resorption) by inducing an increase in osteoclastogenesis and osteoclast activation; thus, increasing mineral and organic bone matrix resorption, contributing to bone damage. Given that the pathology induced by Brucella species involved joint tissue, experiments conducted on synoviocytes revealed that besides inducing the activation of these cells to secrete chemokines, proinflammatory cytokines and MMPS, the infection also inhibits synoviocyte apoptosis. Brucella is an intracellular bacterium that replicates preferentially in the endoplasmic reticulum of macrophages. The analysis of B. abortus-infected synoviocytes indicated that bacteria also replicate in their reticulum suggesting that they could use this cell type for intracellular replication during the osteoarticular localization of the disease. Finally, the molecular mechanisms of osteoarticular brucellosis discovered recently shed light on how the interaction between B. abortus and immune and osteoarticular cells may play an important role in producing damage in joint and bone. PMID:28265268

Inhibitory effect of green tea on injury to a cultured renal epithelial cell line, LLC-PK1.

PubMed

Yokozawa, T; Dong, E; Chung, H Y; Oura, H; Nakagawa, H

1997-01-01

When cells from a cultured renal epithelial cell line, LLC-PK1, were cultured under hypoxic conditions (oxygen concentration of 2% or less) before reoxygenation was applied (95% air, 5% CO2), the leakage of lactate dehydrogenase (LDH) into the medium increased. This phenomenon was inhibited in the presence of dimethyl sulfoxide, a hydroxyl radical scavenger, suggesting the involvement of free radicals. Such oxidative stress was significantly inhibited by a green tea extract, and more potently by a tannin mixture. On the other hand, under ordinary culture conditions (95%, air, 5% CO2), there was cell injury, although the LDH leakage was less than that under hypoxia/reoxygenation, and such injury was inhibited by the green tea extract and the tannin mixture.

Involvement of up-regulated Necl-5/Tage4/PVR/CD155 in the loss of contact inhibition in transformed NIH3T3 cells

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

Minami, Yukiko; Department of Surgery and Clinical Oncology, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Osaka; Ikeda, Wataru

2007-01-26

Normal cells show contact inhibition of cell movement and proliferation, but this is lost following transformation. We found that Necl-5, originally identified as a poliovirus receptor and up-regulated in many cancer cells, enhances growth factor-induced cell movement and proliferation. We showed that when cells contact other cells, Necl-5 interacts in trans with nectin-3 and is removed by endocytosis from the cell surface, resulting in a reduction of cell movement and proliferation. We show here that up-regulation of the gene encoding Necl-5 by the oncogene V12-Ki-Ras causes enhanced cell movement and proliferation. Upon cell-cell contact, de novo synthesis of Necl-5 exceedsmore » the rate of Necl-5 endocytosis, eventually resulting in a net increase in the amount of Necl-5 at the cell surface. In addition, expression of the gene encoding nectin-3 is markedly reduced in transformed cells. Thus, up-regulation of Necl-5 following transformation contributes to the loss of contact inhibition in transformed cells.« less

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

PubMed

Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

2010-02-01

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

Mycobacterium tuberculosis surface protein Rv0227c contains high activity binding peptides which inhibit cell invasion.

PubMed

Rodríguez, Diana Marcela; Ocampo, Marisol; Curtidor, Hernando; Vanegas, Magnolia; Patarroyo, Manuel Elkin; Patarroyo, Manuel Alfonso

2012-12-01

Mycobacterium tuberculosis surface proteins involved in target cell invasion may be identified as a strategy for developing subunit-based, chemically-synthesized vaccines. The Rv0227c protein was thus selected to assess its role in the invasion and infection of Mycobacterium tuberculosis target cells. Results revealed Rv0227c localization on mycobacterial surface by immunoelectron microscopy and Western blot. Receptor-ligand assays using 20-mer, non-overlapping peptides covering the complete Rv0227c protein sequence revealed three high activity binding peptides for U937 phagocytic cells and seven for A549 cells. Peptide 16944 significantly inhibited mycobacterial entry to both cell lines while 16943 and 16949 only managed to inhibit entrance to U937 cells and 16951 to A549 cells. The Jnet bioinformatics tool predicted secondary structure elements for the complete protein, agreeing with elements determined for such chemically-synthesized peptides. It was thus concluded that high activity binding peptides which were able to inhibit mycobacterial entry to target cells are of great importance when selecting peptide candidates for inclusion in an anti-tuberculosis vaccine. Copyright © 2012 Elsevier Inc. All rights reserved.

Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

NASA Technical Reports Server (NTRS)

Betz, N. A.; Westhoff, B. A.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

1995-01-01

Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells results in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression.

Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor

NASA Technical Reports Server (NTRS)

Morre, D. James

2002-01-01

The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

Ethylene Upregulates Auxin Biosynthesis in Arabidopsis Seedlings to Enhance Inhibition of Root Cell Elongation[W

PubMed Central

Swarup, Ranjan; Perry, Paula; Hagenbeek, Dik; Van Der Straeten, Dominique; Beemster, Gerrit T.S.; Sandberg, Göran; Bhalerao, Rishikesh; Ljung, Karin; Bennett, Malcolm J.

2007-01-01

Ethylene represents an important regulatory signal for root development. Genetic studies in Arabidopsis thaliana have demonstrated that ethylene inhibition of root growth involves another hormone signal, auxin. This study investigated why auxin was required by ethylene to regulate root growth. We initially observed that ethylene positively controls auxin biosynthesis in the root apex. We subsequently demonstrated that ethylene-regulated root growth is dependent on (1) the transport of auxin from the root apex via the lateral root cap and (2) auxin responses occurring in multiple elongation zone tissues. Detailed growth studies revealed that the ability of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid to inhibit root cell elongation was significantly enhanced in the presence of auxin. We conclude that by upregulating auxin biosynthesis, ethylene facilitates its ability to inhibit root cell expansion. PMID:17630275

Dexamethasone inhibits IL-12p40 production in lipopolysaccharide-stimulated human monocytic cells by down-regulating the activity of c-Jun N-terminal kinase, the activation protein-1, and NF-kappa B transcription factors.

PubMed

Ma, Wei; Gee, Katrina; Lim, Wilfred; Chambers, Kelly; Angel, Jonathan B; Kozlowski, Maya; Kumar, Ashok

2004-01-01

IL-12 plays a critical role in the development of cell-mediated immune responses and in the pathogenesis of inflammatory and autoimmune disorders. Dexamethasone (DXM), an anti-inflammatory glucocorticoid, has been shown to inhibit IL-12p40 production in LPS-stimulated monocytic cells. In this study, we investigated the molecular mechanism by which DXM inhibits IL-12p40 production by studying the role of the mitogen-activated protein kinases (MAPKs), and the key transcription factors involved in human IL-12p40 production in LPS-stimulated monocytic cells. A role for c-Jun N-terminal kinase (JNK) MAPK in LPS-induced IL-12p40 regulation in a promonocytic THP-1/CD14 cell line was demonstrated by using specific inhibitors of JNK activation, SP600125 and a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase-1 mutant. To identify transcription factors regulating IL-12p40 gene transcription, extensive deletion analyses of the IL-12p40 promoter was performed. The results revealed the involvement of a sequence encompassing the AP-1-binding site, in addition to that of NF-kappaB. The role of AP-1 in IL-12p40 transcription was confirmed by using antisense c-fos and c-jun oligonucleotides. Studies conducted to understand the regulation of AP-1 and NF-kappaB activation by JNK MAPK revealed that both DXM and SP600125 inhibited IL-12p40 gene transcription by inhibiting the activation of AP-1 and NF-kappaB transcription factors as revealed by luciferase reporter and gel mobility shift assays. Taken together, our results suggest that DXM may inhibit IL-12p40 production in LPS-stimulated human monocytic cells by down-regulating the activation of JNK MAPK, the AP-1, and NF-kappaB transcription factors.

Involvement of DNA methylation in the control of cell growth during heat stress in tobacco BY-2 cells.

PubMed

Centomani, Isabella; Sgobba, Alessandra; D'Addabbo, Pietro; Dipierro, Nunzio; Paradiso, Annalisa; De Gara, Laura; Dipierro, Silvio; Viggiano, Luigi; de Pinto, Maria Concetta

2015-11-01

The alteration of growth patterns, through the adjustment of cell division and expansion, is a characteristic response of plants to environmental stress. In order to study this response in more depth, the effect of heat stress on growth was investigated in tobacco BY-2 cells. The results indicate that heat stress inhibited cell division, by slowing cell cycle progression. Cells were stopped in the pre-mitotic phases, as shown by the increased expression of CycD3-1 and by the decrease in the NtCycA13, NtCyc29 and CDKB1-1 transcripts. The decrease in cell length and the reduced expression of Nt-EXPA5 indicated that cell expansion was also inhibited. Since DNA methylation plays a key role in controlling gene expression, the possibility that the altered expression of genes involved in the control of cell growth, observed during heat stress, could be due to changes in the methylation state of their promoters was investigated. The results show that the altered expression of CycD3-1 and Nt-EXPA5 was consistent with changes in the methylation state of the upstream region of these genes. These results suggest that DNA methylation, controlling the expression of genes involved in plant development, contributes to growth alteration occurring in response to environmental changes.

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

Felipe, K.B.; Benites, J.; Glorieux, C.

Highlights: •Phenylaminonaphthoquinones are redox cyclers able to form ROS. •Phenylaminonaphthoquinones plus ascorbate inhibit T24 cell growth. •Phenylaminonaphthoquinones plus ascorbate lead to necrotic-like cell death. •Phenylaminonaphthoquinones plus ascorbate impair cell cycle and affect MAPKs. •Phenylaminonaphthoquinones plus ascorbate induce a senescent cancer cell phenotype. -- Abstract: Quinone-containing molecules have been developed against cancer mainly for their redox cycling ability leading to reactive oxygen species (ROS) formation. We have previously shown that donor-acceptor phenylaminonaphthoquinones are biologically active against a panel of cancer cells. In this report, we explored the mechanisms involved in cancer cell growth inhibition caused by two phenylaminonaphthoquinones, namely Q7 andmore » Q9, with or without ascorbate (ASC). The results show that Q7 and Q9 are both redox cyclers able to form ROS, which strongly inhibit the proliferation of T24 cells. Q9 was a better redox cycler than Q7 because of marked stabilization of the semiquinone radical species arising from its reduction by ascorbate. Indeed, ASC dramatically enhances the inhibitory effect of Q9 on cell proliferation. Q9 plus ASC impairs the cell cycle, causing a decrease in the number of cells in the G2/M phase without involving other cell cycle regulating key proteins. Moreover, Q9 plus ASC influences the MAPK signaling pathways, provoking the appearance of a senescent cancer cell phenotype and ultimately leading to necrotic-like cell death. Because cellular senescence limits the replicative capacity of cells, our results suggest that induction of senescence may be exploited as a basis for new approaches to cancer therapy.« less

Adhesion inhibition of Mycoplasma iowae to chicken lymphoma DT40 cells by monoclonal antibodies reacting with a 65-kD polypeptide.

PubMed

Fiorentin, L; Panangala, V S; Zhang, Y; Toivio-Kinnucan, M

1998-01-01

Tissue- and cell-specific attachment of mycoplasmas is a key aspect of the host-parasite relationship. In this study, monoclonal antibodies (MAbs) recognizing surface membrane polypeptides with molecular masses of 46 kD (p46) and 65 kD (p65), respectively, were examined in a microtiter cell attachment (agglutination) inhibition assay. MAbs MI3, MI6, and MI12 reacting with p65 polypeptide of Mycoplasma iowae inhibited attachment of the organisms to chicken lymphoma (DT 40) cells. One MAb (MI2) that reacted with p65 in immunoblots did not inhibit cell attachment, possibly because of the intrinsic native conformation of the epitope(s) in intact mycoplasmas as opposed to the linear state (sodium dodecyl sulfate denatured) in immunoblots. More pronounced M. iowae adherence inhibition was demonstrated by polyclonal turkey and mouse anti-M. iowae antisera compared with MAbs. Immunogold labelling followed by electron microscopy allowed us to localize the MAb-recognized epitopes on the membrane surface of M. iowae. On the basis of the cell attachment inhibition of M. iowae by specific MAbs (MI3, MI6, and MI12), we propose that the p65 polypeptide plays a role in cytadherence. The ability of polyclonal antisera to inhibit attachment of M. iowae more efficiently than the MAbs suggests that additional epitopes within p65 and/or other proteins are involved in cell attachment.

Antitumor and antimetastatic activities of grape skin polyphenols in a murine model of breast cancer.

PubMed

Sun, T; Chen, Q Y; Wu, L J; Yao, X M; Sun, X J

2012-10-01

Treatment modalities are not effective once breast cancer metastasis has occurred. Dietary botanicals may have a better protective effect. We therefore investigated the effects of grape skin polyphenols on a highly metastatic mouse mammary carcinoma cell line. In vitro treatment of 4T1 cells, with grape skin polyphenols resulted in inhibition of the migration and viability in a dose-dependent manner. The migration of 4T1 cells was significantly inhibited by grape skin polyphenols, even at a very low concentration (5 μg/ml), and was totally inhibited when the concentration was 20 μg/ml. However, 20 μg/ml of grape skin polyphenols inhibited cell viability by only 11.4%. The inhibition of migration is independent of decreased cell viability or apoptosis induction. Further analysis indicated that the inhibition of migration by grape skin polyphenols is involved in blocking the PI3k/Akt and MAPK pathways. The effects of dietary grape skin polyphenols were then examined using an in vivo model in which 4T1 cells were implanted subcutaneously in Balb/c mice. The metastasis of tumor cells to the lungs was inhibited significantly by dietary grape skin extracts (0.5 and 1.0 mg/ml in drinking water) and the survival of the mice enhanced. These data suggest that grape skin polyphenols possess chemotherapeutic efficacy against breast cancer with metastases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Silibinin induces hepatic stellate cell cycle arrest via enhancing p53/p27 and inhibiting Akt downstream signaling protein expression.

PubMed

Ezhilarasan, Devaraj; Evraerts, Jonathan; Sid, Brice; Calderon, Pedro Buc; Karthikeyan, Sivanesan; Sokal, Etienne; Najimi, Mustapha

2017-02-01

Proliferation of hepatic stellate cells (HSCs) plays a pivotal role in the progression of liver fibrosis consequent to chronic liver injury. Silibinin, a flavonoid compound, has been shown to possess anti-fibrogenic effects in animal models of liver fibrosis. This was attributed to an inhibition of cell proliferation of activated HSCs. The present study was to gain insight into the molecular pathways involved in silibinin anti-fibrogenic effect. The study was conducted on LX-2 human stellate cells treated with three concentrations of silibinin (10, 50 and 100 μmol/L) for 24 and 96 hours. At the end of the treatment cell viability and proliferation were evaluated. Protein expression of p27, p21, p53, Akt and phosphorylated-Akt was evaluated by Western blotting analysis and Ki-67 protein expression was by immunocytochemistry. Sirtuin activity was evaluated by chemiluminescence based assay. Silibinin inhibits LX-2 cell proliferation in dose- and time-dependent manner; we showed that silibinin upregulated the protein expressions of p27 and p53. Such regulation was correlated to an inhibition of both downstream Akt and phosphorylated-Akt protein signaling and Ki-67 protein expression. Sirtuin activity also was correlated to silibinin-inhibited proliferation of LX-2 cells. The anti-proliferative effect of silibinin on LX-2 human stellate cells is via the inhibition of the expressions of various cell cycle targets including p27, Akt and sirtuin signaling.

The hepatocyte growth factor antagonist NK4 inhibits indoleamine-2,3-dioxygenase expression via the c-Met-phosphatidylinositol 3-kinase-AKT signaling pathway

PubMed Central

WANG, DONGDONG; SAGA, YASUSHI; SATO, NAOTO; NAKAMURA, TOSHIKAZU; TAKIKAWA, OSAMU; MIZUKAMI, HIROAKI; MATSUBARA, SHIGEKI; FUJIWARA, HIROYUKI

2016-01-01

Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme involved in tumor malignancy. However, the regulatory mechanism underlying its involvement remains largely uncharacterized. The present study aimed to investigate the hypothesis that NK4, an antagonist of hepatocyte growth factor (HGF), can regulate IDO and to characterize the signaling mechanism involved. Following successful transfection of the human ovarian cancer cell line SKOV-3 (which constitutively expresses IDO) with an NK4 expression vector, we observed that NK4 expression suppressed IDO expression; furthermore, NK4 expression did not suppress cancer cell growth in vitro [in the absence of natural killer (NK) cells], but did influence tumor growth in vivo. In addition, NK4 enhanced the sensitivity of cancer cells to NK cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. In an effort to clarify the mechanisms by which NK4 interacts with IDO, we performed investigations utilizing various biochemical inhibitors. The results of these investigations were as follows. First, c-Met (a receptor of HGF) tyrosine kinase inhibitor PHA-665752, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 both suppress IDO expression. Second, enhanced expression of PTEN (a known tumor suppressor) via negative regulation within a PI3K-AKT pathway, inhibits IDO expression. Conversely, neither the MEK1/2 inhibitor U0126 nor the STAT3 inhibitor WP1066 affects IDO expression. These results suggest that NK4 inhibits IDO expression via a c-Met-PI3K-AKT signaling pathway. PMID:27082119

The 5'-untranslated region of p23 mRNA from the Ehrlich ascites tumor is involved in translation control of the growth related protein p23.

PubMed

Böhm, H; Gross, B; Gaestel, M; Bommer, U A; Ryffel, G; Bielka, H

1991-01-01

The growth-related protein p23 of the Ehrlich ascites tumor (EAT) is preferentially expressed in the exponentially growing tumor; its synthesis is translationally controlled. p23 mRNA is efficiently translated in the wheat germ cell-free lysate. In contrast, p23 mRNA present in poly(A)+RNA isolated from EAT is not translated in cell-free systems of EAT and reticulocytes. Moreover, translation of a p23 transcript is inhibited in the presence of total poly(A)+RNA. This inhibition is abolished by the removal of the 5'-UTR of the p23 transcript. Solution hybridization/RNase protection experiments point to the presence of a nucleotide sequence complementary to the 5'-UTR of p23 mRNA which might be involved in p23 mRNA inhibition.

Subinhibitory concentrations of phloretin repress the virulence of Salmonella typhimurium and protect against Salmonella typhimurium infection.

PubMed

Shuai-Cheng, Wu; Ben-Dong, Fu; Xiu-Ling, Chu; Jian-Qing, Su; Yun-Xing, Fu; Zhen-Qiang, Cui; Dao-Xiu, Xu; Zong-Mei, Wu

2016-11-01

Phloretin, a natural component of many fruits, exhibits anti-virulence effects and provides a new alternative to counter bacterial infection. The aim of this study was to determine the effect of subinhibitory concentrations of phloretin on the virulence of Salmonella typhimurium. At concentrations where growth of Salmonella was not inhibited, phloretin significantly inhibited bacteria biofilm formation and motility. Subinhibitory concentrations of phloretin repressed eight genes involved in the Salmonella pathogenicity island 1 and 3 genes involved in flagella production. Furthermore, subinhibitory concentrations of phloretin inhibited the adhesion and invasion of Salmonella in IEC-6 cells and reduced the LDH levels of S. typhimurium-infected IEC-6 cells. Additionally, phloretin significantly decreased the cecum bacterial loads of the mice infected with live S. typhimurium containing subinhibitory concentrations of phloretin by gavage. These results suggested that subinhibitory concentrations of phloretin attenuate the virulence of S. typhimurium and protect against S. typhimurium infection.

ERβ up-regulation was involved in silibinin-induced growth inhibition of human breast cancer MCF-7 cells.

PubMed

Zheng, Nan; Liu, Lu; Liu, Weiwei; Zhang, Ping; Huang, Huai; Zang, Linghe; Hayashi, Toshihiko; Tashiro, Shin-ichi; Onodera, Satoshi; Xia, Mingyu; Ikejima, Takashi

2016-02-01

We previously reported that silibinin induced a loss of cell viability in breast cancer (MCF-7) cells by ERα down-regulation. But whether this cytotoxicity depends on another estrogen receptor, ERβ, has yet to be elucidated. Therefore, we sought to explore the effects of ERβ modulation on cell viability by using an ERβ-selective agonist (Diarylprepionitrile, DPN) and an antagonist (PHTPP). Our data demonstrated that ERβ served as a growth suppressor in MCF-7 cells, and the incubation of silibinin, elevated ERβ expression, resulting in the tumor growth inhibition. The cytotoxic effect of silibinin was diminished by PHTPP and enhanced by DPN. Silencing of ERβ by siRNA confirmed these results. Apoptotic cascades, including the sequential activation of caspase-9 and -6, and finally the cleavage of caspase substrates, PARP and ICAD, caused by treatment with silibinin, were all repressed by PHTPP pre-treatment but exacerbated by DPN. Unlike ERα, ERβ did not involve autophagic process in the regulation, since neither autophagic inhibitor (3-MA) nor the inducer (rapamycin) affected the cell survival rates regardless ERβ activity. Taken together, silibinin induced apoptosis through mitochondrial pathway by up-regulating ERβ pathways in MCF-7 cells without the involvement of autophagy. Copyright © 2016. Published by Elsevier Inc.

Aldolase sequesters WASP and affects WASP/Arp2/3-stimulated actin dynamics.

PubMed

Ritterson Lew, Carolyn; Tolan, Dean R

2013-08-01

In addition to its roles in sugar metabolism, fructose-1,6-bisphosphate aldolase (aldolase) has been implicated in cellular functions independent from these roles, termed "moonlighting functions." These moonlighting functions likely involve the known aldolase-actin interaction, as many proteins with which aldolase interacts are involved in actin-dependent processes. Specifically, aldolase interacts both in vitro and in cells with Wiskott-Aldrich Syndrome Protein (WASP), a protein involved in controlling actin dynamics, yet the function of this interaction remains unknown. Here, the effect of aldolase on WASP-dependent processes in vitro and in cells is investigated. Aldolase inhibits WASP/Arp2/3-dependent actin polymerization in vitro. In cells, knockdown of aldolase results in a decreased rate of cell motility and cell spreading, two WASP-dependent processes. Expression of exogenous aldolase rescues these defects. Whether these effects of aldolase on WASP-dependent processes were due to aldolase catalysis or moonlighting functions is tested using aldolase variants defective in either catalytic or actin-binding activity. While the actin-binding deficient aldolase variant is unable to inhibit actin polymerization in vitro and is unable to rescue cell motility defects in cells, the catalytically inactive aldolase is able to perform these functions, providing evidence that aldolase moonlighting plays a role in WASP-mediated processes. Copyright © 2013 Wiley Periodicals, Inc.

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

PubMed

Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

2017-06-15

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. Copyright © 2017 American Society for Microbiology.

Inhibition of canonical WNT signaling pathway by β-catenin/CBP inhibitor ICG-001 ameliorates liver fibrosis in vivo through suppression of stromal CXCL12.

PubMed

Akcora, Büsra Öztürk; Storm, Gert; Bansal, Ruchi

2018-03-01

Quiescent hepatic stellate cells (HSCs), in response to liver injury, undergo characteristic morphological transformation into proliferative, contractile and ECM-producing myofibroblasts. In this study, we investigated the implication of canonical Wnt signaling pathway in HSCs and liver fibrogenesis. Canonical Wnt signaling pathway activation and inhibition using β-catenin/CBP inhibitor ICG001 was examined in-vitro in TGFβ-activated 3T3, LX2, primary human HSCs, and in-vivo in CCl 4 -induced acute liver injury mouse model. Fibroblasts-conditioned medium studies were performed to assess the Wnt-regulated paracrine factors involved in crosstalk between HSCs-macrophages and HSCs-endothelial cells. Canonical Wnt signaling pathway components were significantly up-regulated in-vitro and in-vivo. In-vitro, ICG-001 significantly inhibited fibrotic parameters, 3D-collagen contractility and wound healing. Conditioned medium induced fibroblasts-mediated macrophage and endothelial cells activation was significantly inhibited by ICG-001. In-vivo, ICG-001 significantly attenuated collagen accumulation and HSC activation. Interestingly, ICG-001 drastically inhibited macrophage infiltration, intrahepatic inflammation and angiogenesis. We further analyzed the paracrine factors involved in Wnt-mediated effects and found CXCL12 was significantly suppressed both in-vitro and in-vivo following Wnt inhibition. Wnt-regulated CXCL12 secretion from activated HSCs potentiated macrophage infiltration and activation, and angiogenesis. Pharmacological inhibition of canonical Wnt signaling pathway via suppression of stromal CXCL12 suggests a potential therapeutic approach targeting activated HSCs in liver fibrosis. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Therapeutic Efficacy of Suppressing the JAK/STAT Pathway in Multiple Models of EAE1

PubMed Central

Liu, Yudong; Holdbrooks, Andrew T.; De Sarno, Patrizia; Rowse, Amber L.; Yanagisawa, Lora L.; McFarland, Braden C.; Harrington, Laurie E.; Raman, Chander; Sabbaj, Steffanie; Benveniste, Etty N.; Qin, Hongwei

2014-01-01

Pathogenic T helper cells and myeloid cells are involved in the pathogenesis of Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE), an animal model of MS. The JAK/STAT pathway is utilized by numerous cytokines for signaling, and is critical for development, regulation and termination of immune responses. Dysregulation of the JAK/STAT pathway has pathological implications in autoimmune and neuroinflammatory diseases. Many of the cytokines involved in MS/EAE, including IL-6, IL-12, IL-23, IFN-γ and GM-CSF, use the JAK/STAT pathway to induce biological responses. Thus, targeting JAKs has implications for treating autoimmune inflammation of the brain. We have utilized AZD1480, a JAK1/2 inhibitor, to investigate the therapeutic potential of inhibiting the JAK/STAT pathway in models of EAE. AZD1480 treatment inhibits disease severity in MOG-induced classical and atypical EAE models by preventing entry of immune cells into the brain, suppressing differentiation of Th1 and Th17 cells, deactivating myeloid cells, inhibiting STAT activation in the brain, and reducing expression of pro-inflammatory cytokines and chemokines. Treatment of SJL/J mice with AZD1480 delays disease onset of PLP-induced relapsing-remitting disease, reduces relapses and diminishes clinical severity. AZD1480 treatment was also effective in reducing ongoing paralysis induced by adoptive transfer of either pathogenic Th1 or Th17 cells. In vivo AZD1480 treatment impairs both the priming and expansion of T-cells, and attenuates antigen-presentation functions of myeloid cells. Inhibition of the JAK/STAT pathway has clinical efficacy in multiple pre-clinical models of MS, suggesting the feasibility of the JAK/STAT pathway as a target for neuroinflammatory diseases. PMID:24323580

The silencing of Pokemon attenuates the proliferation of hepatocellular carcinoma cells in vitro and in vivo by inhibiting the PI3K/Akt pathway.

PubMed

Lin, Chan-Chan; Zhou, Jing-Ping; Liu, Yun-Peng; Liu, Jing-Jing; Yang, Xiao-Ning; Jazag, Amarsanaa; Zhang, Zhi-Ping; Guleng, Bayasi; Ren, Jian-Lin

2012-01-01

Pokemon (POK erythroid myeloid ontogenic factor), which belongs to the POK protein family, is also called LRF, OCZF and FBI-1. As a transcriptional repressor, Pokemon assumes a critical function in cellular differentiation and oncogenesis. Our study identified an oncogenic role for Pokemon in human hepatocellular carcinoma (HCC). We successfully established human HepG2 and Huh-7 cell lines in which Pokemon was stably knocked down. We demonstrated that Pokemon silencing inhibited cell proliferation and migration. Pokemon knockdown inhibited the PI3K/Akt and c-Raf/MEK/ERK pathways and modulated the expression of various cell cycle regulators in HepG2 and Huh-7 cells. Therefore, Pokemon may also be involved in cell cycle progression in these cells. We confirmed that Pokemon silencing suppresses hepatocellular carcinoma growth in tumor xenograft mice. These results suggest that Pokemon promotes cell proliferation and migration in hepatocellular carcinoma and accelerates tumor development in an Akt- and ERK-signaling-dependent manner.

Inhibition of cell proliferation by nobiletin, a dietary phytochemical, associated with apoptosis and characteristic gene expression, but lack of effect on early rat hepatocarcinogenesis in vivo.

PubMed

Ohnishi, Hiroyuki; Asamoto, Makoto; Tujimura, Kazunari; Hokaiwado, Naomi; Takahashi, Satoru; Ogawa, Kumiko; Kuribayashi, Masanori; Ogiso, Tadashi; Okuyama, Harumi; Shirai, Tomoyuki

2004-12-01

Dietary phytochemicals can inhibit the development of certain types of tumors. We here investigated the effects of nobiletin (Nob), garcinol (Gar), auraptene (Aur), beta-cryptoxanthin- and hesperidine-rich pulp (CHRP) and 1,1'-acetoxychavicol acetate (ACA) on hepatocarcinogenesis in a rat medium-term liver bioassay, and also examined their influence on cell proliferation, cell cycle kinetics, apoptosis and cell invasion of rat and human hepatocellular carcinoma (HCC) cells, MH1C1 and HepG2, respectively. While there were no obvious suppressive effects on the development of putative preneoplastic liver lesions, inhibition of hepatocarcinoma cell proliferation was evident in the Nob group. Nob also caused G2/M cell cycle arrest and apoptosis. Microarray analysis identified a set of genes specifically regulated by Nob, and these are likely to be involved in the observed growth suppression of HCC cells. These results suggest that phytochemicals might have chemopreventive potential in late stages of hepatocarcinogenesis.

Protective effects of red wine flavonols on 4-hydroxynonenal-induced apoptosis in PC12 cells.

PubMed

Jang, Young Jin; Kang, Nam Joo; Lee, Ki Won; Lee, Hyong Joo

2009-08-01

There is accumulating evidence that a moderate consumption of red wine has health benefits, such as the inhibition of neurodegenerative diseases. Although this is generally attributed to resveratrol, the protective mechanisms and the active substance(s) remain unclear. We examined whether and how red wine extract (RWE) and red wine flavonols quercetin and myricetin inhibited 4-hydroxynonenal (HNE)-induced apoptosis of rat pheochromocytoma PC12 cells. RWE attenuated HNE-induced PC12 cell death in a dose-dependent manner. HNE induced cleavage of poly(ADP-ribose) polymerase, which is involved in DNA repair in the nucleus, and this was inhibited by RWE treatment. Treatment with RWE also inhibited HNE-induced nuclear condensation in PC12 cells. Data of 2',7'-dichlorofluorescin diacetate showed that RWE protected against apoptosis of PC12 cells by attenuating intracellular reactive oxygen species. The cytoprotective effects on HNE-induced cell death were stronger for quercetin and myricetin than for resveratrol. HNE-induced nuclear condensation was attenuated by quercetin and myricetin. These results suggest that the neuroprotective potential of red wine is attributable to flavonols rather than to resveratrol.

Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

PubMed

Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

2001-04-01

The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

Inhibition of cancer cell growth by exposure to a specific time-varying electromagnetic field involves T-type calcium channels.

PubMed

Buckner, Carly A; Buckner, Alison L; Koren, Stan A; Persinger, Michael A; Lafrenie, Robert M

2015-01-01

Electromagnetic field (EMF) exposures affect many biological systems. The reproducibility of these effects is related to the intensity, duration, frequency, and pattern of the EMF. We have shown that exposure to a specific time-varying EMF can inhibit the growth of malignant cells. Thomas-EMF is a low-intensity, frequency-modulated (25-6 Hz) EMF pattern. Daily, 1 h, exposures to Thomas-EMF inhibited the growth of malignant cell lines including B16-BL6, MDA-MB-231, MCF-7, and HeLa cells but did not affect the growth of non-malignant cells. Thomas-EMF also inhibited B16-BL6 cell proliferation in vivo. B16-BL6 cells implanted in syngeneic C57b mice and exposed daily to Thomas-EMF produced smaller tumours than in sham-treated controls. In vitro studies showed that exposure of malignant cells to Thomas-EMF for > 15 min promoted Ca(2+) influx which could be blocked by inhibitors of voltage-gated T-type Ca(2+) channels. Blocking Ca(2+) uptake also blocked Thomas-EMF-dependent inhibition of cell proliferation. Exposure to Thomas-EMF delayed cell cycle progression and altered cyclin expression consistent with the decrease in cell proliferation. Non-malignant cells did not show any EMF-dependent changes in Ca(2+) influx or cell growth. These data confirm that exposure to a specific EMF pattern can affect cellular processes and that exposure to Thomas-EMF may provide a potential anti-cancer therapy.

Inhibition of Cancer Cell Growth by Exposure to a Specific Time-Varying Electromagnetic Field Involves T-Type Calcium Channels

PubMed Central

Buckner, Carly A.; Buckner, Alison L.; Koren, Stan A.; Persinger, Michael A.; Lafrenie, Robert M.

2015-01-01

Electromagnetic field (EMF) exposures affect many biological systems. The reproducibility of these effects is related to the intensity, duration, frequency, and pattern of the EMF. We have shown that exposure to a specific time-varying EMF can inhibit the growth of malignant cells. Thomas-EMF is a low-intensity, frequency-modulated (25-6 Hz) EMF pattern. Daily, 1 h, exposures to Thomas-EMF inhibited the growth of malignant cell lines including B16-BL6, MDA-MB-231, MCF-7, and HeLa cells but did not affect the growth of non-malignant cells. Thomas-EMF also inhibited B16-BL6 cell proliferation in vivo. B16-BL6 cells implanted in syngeneic C57b mice and exposed daily to Thomas-EMF produced smaller tumours than in sham-treated controls. In vitro studies showed that exposure of malignant cells to Thomas-EMF for > 15 min promoted Ca2+ influx which could be blocked by inhibitors of voltage-gated T-type Ca2+ channels. Blocking Ca2+ uptake also blocked Thomas-EMF-dependent inhibition of cell proliferation. Exposure to Thomas-EMF delayed cell cycle progression and altered cyclin expression consistent with the decrease in cell proliferation. Non-malignant cells did not show any EMF-dependent changes in Ca2+ influx or cell growth. These data confirm that exposure to a specific EMF pattern can affect cellular processes and that exposure to Thomas-EMF may provide a potential anti-cancer therapy. PMID:25875081

Myeloid-derived suppressor cells as a potential therapy for experimental autoimmune myasthenia gravis

PubMed Central

Li, Yan; Tu, Zhidan; Qian, Shiguang; Fung, John J.; Markowitz, Sanford D.; Kusner, Linda L.; Kaminski, Henry J.; Lu, Lina; Lin, Feng

2016-01-01

We recently demonstrated that hepatic stellate cells induce the differentiation of myeloid-derived suppressor cells (MDSCs) from myeloid progenitors. In this study, we found that adoptive transfer of these MDSCs effectively reversed disease progression in experimental autoimmune myasthenia gravis (EAMG), a T-cell-dependent and B-cell-mediated model for myasthenia gravis. In addition to ameliorated disease severity, MDSC-treated EAMG mice showed suppressed acetylcholine receptors (AChR)-specific T-cell responses, decreased levels of serum anti-AChR IgGs, and reduced complement activation at the neuromuscular junctions. Incubating MDSCs with B cells activated by anti-IgM or anti-CD40 antibodies inhibited the proliferation of these in vitro activated B cells. Administering MDSCs into mice immunized with a T-cell-independent antigen inhibited the antigen-specific antibody production in vivo. MDSCs directly inhibit B cells through multiple mechanisms including prostaglandin E2, inducible nitric oxide synthase and arginase. Interestingly, MDSC treatment in EMAG mice does not appear to significantly inhibit their immune response to a non-relevant antigen, ovalbumin. These results demonstrated that hepatic stellate cell-induced MDSCs concurrently suppress both T- and B- cell autoimmunity, leading to effective treatment of established EAMG; and that the MDSCs inhibit AChR-specific immune responses at least partially in an antigen-specific manner. These data suggest that MDSCs could be further developed as a novel approach to treating myasthenia gravis and, even more broadly, other diseases in which T and B cells are involved in pathogenesis. PMID:25057008

Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.

PubMed

Frémin, Christophe; Bessard, Anne; Ezan, Frédéric; Gailhouste, Luc; Régeard, Morgane; Le Seyec, Jacques; Gilot, David; Pagès, Gilles; Pouysségur, Jacques; Langouët, Sophie; Baffet, Georges

2009-03-01

We investigated the specific role of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)-dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF-dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2-dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long-term survival and cell cycle regulation of hepatocytes.

Circadian rhythm genes mediate fenvalerate-induced inhibition of testosterone synthesis in mouse Leydig cells.

PubMed

Guo, Yichen; Shen, Ouxi; Han, Jingjing; Duan, Hongyu; Yang, Siyuan; Zhu, Zhenghong; Tong, Jian; Zhang, Jie

2017-01-01

Fenvalerate (Fen), a widely used pesticide, is known to impair male reproductive functions by mechanisms that remain to be elucidated. Recent studies indicated that circadian clock genes may play an important role in successful male reproduction. The aim of this study was to determine the effects of Fen on circadian clock genes involved in the biosynthesis of testosterone using TM3 cells derived from mouse Leydig cells. Data demonstrated that the circadian rhythm of testosterone synthesis in TM3 cells was disturbed following Fen treatment as evidenced by changes in the circadian rhythmicity of core clock genes (Bmal1, Rev-erbα, Rorα). Further, the observed altered rhythms were accompanied by increased intracellular Ca 2+ levels and modified steroidogenic acute regulatory (StAR) mRNA expression. Thus, data suggested that Fen inhibits testosterone synthesis via pathways involving intracellular Ca 2+ and clock genes (Bmal1, Rev-Erbα, Rorα) as well as StAR mRNA expression in TM3 cells.

Decreased miR-106a inhibits glioma cell glucose uptake and proliferation by targeting SLC2A3 in GBM.

PubMed

Dai, Dong-Wei; Lu, Qiong; Wang, Lai-Xing; Zhao, Wen-Yuan; Cao, Yi-Qun; Li, Ya-Nan; Han, Guo-Sheng; Liu, Jian-Min; Yue, Zhi-Jian

2013-10-14

MiR-106a is frequently down-regulated in various types of human cancer. However the underlying mechanism of miR-106a involved in glioma remains elusive. The association of miR-106a with glioma grade and patient survival was analyzed. The biological function and target of miR-106a were determined by bioinformatic analysis and cell experiments (Western blot, luciferase reporter, cell cycle, ntracellular ATP production and glucose uptake assay). Finally, rescue expression of its target SLC2A3 was used to test the role of SLC2A3 in miR-106a-mediated cell glycolysis and proliferation. Here we showed that miR-106a was a tumor suppressor miRNA was involved in GBM cell glucose uptake and proliferation. Decreased miR-106a in GBM tissues and conferred a poor survival of GBM patients. SLC2A3 was identified as a core target of miR-106a in GBM cells. Inhibition of SLC2A3 by miR-106a attenuated cell proliferation and inhibited glucose uptake. In addition, for each biological process we identified ontology-associated transcripts that significantly correlated with SLC2A3 expression. Finally, the expression of SLC2A3 largely abrogated miR-106a-mediated cell proliferation and glucose uptake in GBM cells. Taken together, miR-106a and SLC2A3 could be potential therapeutic approaches for GBM.

Oncostatic action of melatonin: facts and question marks.

PubMed

Pawlikowski, Marek; Winczyk, Katarzyna; Karasek, Michal

2002-04-01

The paper presents the data concerning the in vivo effects of melatonin on experimentally-induced tumors in animals and the in vitro effects on animal and human tumor cells. The majority of experimental tumors responded to the melatonin treatment with growth inhibition. However, some negative or opposite results (i.e. stimulation of tumor instead of inhibition) were also reported. Some of the negative results can be attributed to the improper timing of melatonin administration. Melatonin was also shown to inhibit the growth of several animal and human tumor cell lines in vitro. On the basis of these experiments, a hypothesis of the oncostatic action of melatonin was put forward. The mechanism of the postulated action is complex and probably includes: 1) modulation of the endocrine system; 2) modulation of the immune system; 3) the direct oncostatic action of melatonin on tumor cells. The latter includes the recently discovered anti-oxidative action which probably plays an important role in the countering the DNA damage during the radiation challenge or the exposure to chemical carcinogens. It also includes the antiproliferative and pro-apoptotic effects exerted via melatonin receptors expressed by tumor cells. The involvement of the membrane melatonin receptors is mainly assumed. However, the recent data from our and other laboratories suggest also the involvement of RZR/ROR receptors (the putative melatonin nuclear receptors) in both melatonin-induced proliferation inhibition and apoptosis.

Astaxanthin Inhibits JAK/STAT-3 Signaling to Abrogate Cell Proliferation, Invasion and Angiogenesis in a Hamster Model of Oral Cancer

PubMed Central

Kowshik, J.; Baba, Abdul Basit; Giri, Hemant; Deepak Reddy, G.; Dixit, Madhulika; Nagini, Siddavaram

2014-01-01

Identifying agents that inhibit STAT-3, a cytosolic transcription factor involved in the activation of various genes implicated in tumour progression is a promising strategy for cancer chemoprevention. In the present study, we investigated the effect of dietary astaxanthin on JAK-2/STAT-3 signaling in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by examining the mRNA and protein expression of JAK/STAT-3 and its target genes. Quantitative RT-PCR, immunoblotting and immunohistochemical analyses revealed that astaxanthin supplementation inhibits key events in JAK/STAT signaling especially STAT-3 phosphorylation and subsequent nuclear translocation of STAT-3. Furthermore, astaxanthin downregulated the expression of STAT-3 target genes involved in cell proliferation, invasion and angiogenesis, and reduced microvascular density, thereby preventing tumour progression. Molecular docking analysis confirmed inhibitory effects of astaxanthin on STAT signaling and angiogenesis. Cell culture experiments with the endothelial cell line ECV304 substantiated the role of astaxanthin in suppressing angiogenesis. Taken together, our data provide substantial evidence that dietary astaxanthin prevents the development and progression of HBP carcinomas through the inhibition of JAK-2/STAT-3 signaling and its downstream events. Thus, astaxanthin that functions as a potent inhibitor of tumour development and progression by targeting JAK/STAT signaling may be an ideal candidate for cancer chemoprevention. PMID:25296162

Strain-specific inhibition of the adherence of uropathogenic bacteria to bladder cells by probiotic Lactobacillus spp.

PubMed

de Llano, Dolores González; Arroyo, Amalia; Cárdenas, Nivia; Rodríguez, Juan Miguel; Moreno-Arribas, M Victoria; Bartolomé, Begoña

2017-06-01

Urinary tract infections (UTIs), one of most common infections worldwide, face high recurrence rates and increasing antimicrobial resistance. Probiotic bacteria, especially of the genus Lactobacillus, are considered a promising preventive and/or treatment therapy against UTIs. In order to elucidate the mechanisms involved in these beneficial effects, we studied the impact of different Lactobacillus strains (Lactobacillus salivarius UCM572, L. plantarum CLC17 and L. acidophilus 01) in the adherence of reference and clinical uropathogenic strains (Escherichia coli ATCC® 53503, E. coli 10791, Enterococcus faecalis 04-1, En. faecalis 08-1 and Staphylococcus epidermidis 08-3) to T24 epithelial bladder cells. In general, the Lactobacillus strains with previous in vivo evidence of beneficial effects against UTIs (L. salivarius UCM572 and L. acidophilus 01) significantly inhibited the adherence of the five uropathogens to T24 cells, displaying percentages of inhibition ranging between 22.2% and 43.9%, and between 16.5% and 53.7%, respectively. On the other hand, L. plantarum CLC17, a strain with no expected effects on UTIs, showed almost negligible anti-adherence effects.Therefore, these in vitro results suggest that inhibition of the adherence of uropathogens to epithelial bladder cells may be one of the mechanisms involved in the potential beneficial effects of probiotics against UTIs in vivo. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

KH-type splicing regulatory protein is involved in esophageal squamous cell carcinoma progression.

PubMed

Fujita, Yuji; Masuda, Kiyoshi; Hamada, Junichi; Shoda, Katsutoshi; Naruto, Takuya; Hamada, Satoshi; Miyakami, Yuko; Kohmoto, Tomohiro; Watanabe, Miki; Takahashi, Rizu; Tange, Shoichiro; Saito, Masako; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Tangoku, Akira; Otsuji, Eigo; Imoto, Issei

2017-11-24

KH-type splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein, which is involved in several post-transcriptional aspects of RNA metabolism, including microRNA (miRNA) biogenesis. It affects distinct cell functions in different tissues and can have an impact on various pathological conditions. In the present study, we investigated the oncogenic functions of KHSRP and their underlying mechanisms in the pathogenesis of esophageal squamous cell carcinoma (ESCC). KHSRP expression levels were elevated in ESCC tumors when compared with those in non-tumorous tissues by immunohistochemistry, and cytoplasmic KHSRP overexpression was found to be an independent prognosticator for worse overall survival in a cohort of 104 patients with ESCC. KHSRP knockdown inhibited growth, migration, and invasion of ESCC cells. KHSRP knockdown also inhibited the maturation of cancer-associated miRNAs, such as miR-21, miR-130b, and miR-301, and induced the expression of their target mRNAs, such as BMP6, PDCD4, and TIMP3, resulting in the inhibition of epithelial-to-mesenchymal transition. Our findings uncover a novel oncogenic function of KHSRP in esophageal tumorigenesis and implicate its use as a marker for prognostic evaluation and as a putative therapeutic target in ESCC.

Inhibition of murine T-cell responses by anti-oxidants: the targets of lipo-oxygenase pathway inhibitors.

PubMed Central

Dornand, J; Gerber, M

1989-01-01

We have previously established that oxidative phenomena are involved in human T-cell activation (Sekkat, Dornand & Gerber, 1988). In the present work we have studied the effect of different anti-oxidants (scavengers of O2-, .OH and lipo-oxygenase inhibitors) on the stimulation of murine T cells. We report here that all the anti-oxidants used suppressed T-lymphocyte proliferation and IL-2 synthesis, the former effect resulting very likely from the latter. This inhibition was concomitant with the triggering of activation. We also demonstrate that the various anti-oxidants have different biochemical targets. Unlike the other compounds, the phenolic drugs nordihydroguaiaretic acid (NDGA) and butylated hydroxyanisole (BHA), which block lipid peroxidation, affect both signals triggered by the binding of lectin to its receptors: they suppress the rise of intracellular free calcium concentration and inhibit some of the events, depending on the sole protein kinase C activation, namely IL-2 receptor expression and phorbol myristate acetate (PMA)-induced pH change. Our results are discussed within the framework of a possible involvement of reactive oxygen species and of arachidonic acid derivative(s) in T-cell activation and IL-2 production. PMID:2512249

Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation.

PubMed

Choi, Yong-Min; Kim, Han-Kyul; Shim, Wooyoung; Anwar, Muhammad Ayaz; Kwon, Ji-Woong; Kwon, Hyuk-Kwon; Kim, Hyung Joong; Jeong, Hyobin; Kim, Hwan Myung; Hwang, Daehee; Kim, Hyung Sik; Choi, Sangdun

2015-01-01

The chemotherapeutic use of cisplatin is limited by its severe side effects. In this study, by conducting different omics data analyses, we demonstrated that cisplatin induces cell death in a proximal tubular cell line by suppressing glycolysis- and tricarboxylic acid (TCA)/mitochondria-related genes. Furthermore, analysis of the urine from cisplatin-treated rats revealed the lower expression levels of enzymes involved in glycolysis, TCA cycle, and genes related to mitochondrial stability and confirmed the cisplatin-related metabolic abnormalities. Additionally, an increase in the level of p53, which directly inhibits glycolysis, has been observed. Inhibition of p53 restored glycolysis and significantly reduced the rate of cell death at 24 h and 48 h due to p53 inhibition. The foremost reason of cisplatin-related cytotoxicity has been correlated to the generation of mitochondrial reactive oxygen species (ROS) that influence multiple pathways. Abnormalities in these pathways resulted in the collapse of mitochondrial energy production, which in turn sensitized the cells to death. The quenching of ROS led to the amelioration of the affected pathways. Considering these observations, it can be concluded that there is a significant correlation between cisplatin and metabolic dysfunctions involving mROS as the major player.

Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

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

Xu, Yang; Pang, Xiaoyan; Dong, Mei

Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesitymore » has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.« less

Isorhapontigenin induced cell growth inhibition and apoptosis by targeting EGFR-related pathways in prostate cancer.

PubMed

Zhu, Cuicui; Zhu, Qingyi; Wu, Zhaomeng; Yin, Yingying; Kang, Dan; Lu, Shan; Liu, Ping

2018-02-01

Isorhapontigenin (ISO), a naturally phytopolyphenol compound existing in Chinese herb, apples, and various vegetables, has attracted extensive interest in recent years for its diverse pharmacological characteristics. Increasing evidences reveal that ISO can inhibit cancer cell growth by induced apoptosis, however, the molecular mechanisms is not fully understood. In this study, we found for the first time that ISO apparently induced cell growth inhibition and apoptosis by targeting EGFR and its downstream signal pathways in prostate cancer (PCa) cells both in vitro and in vivo, whereas no obviously effect on normal prostate cells. From the results, we found that ISO competitively targeted EGFR with EGF and inhibited EGFR auto-phosphorylation, and then decreased the levels of p-Erk1/2, p-PI3 K, and p-AKT, and further induced down-regulation of p-FOXO1 and promoted FOXO1 nuclear translocation; and finally resulted in a significantly up-regulation of Bim/p21/27/Bax/cleaved Caspase-3/cleaved PARP-1 and a markedly down-regulation of Sp1/Bcl-2/XIAP/Cyclin D1. Moreover, our experimental data demonstrated that treatment of ISO decreased protein level of AR via both inhibiting the expression of AR gene and promoting the ubiquitination/degradation of AR proteins in proteasome. In vivo, we also found that ISO inhibited the growth of subcutaneous xenotransplanted tumor in nude mice by inducing PCa cell growth inhibition and apoptosis. Taken together, all findings here clearly implicated that EGFR-related signal pathways, including EGFR-PI3K-Akt and EGFR-Erk1/2 pathways, were involved in ISO-induced cell growth inhibition and apoptosis in PCa cells, providing a more solid theoretical basis for the application of ISO to treat patients with prostate cancer in clinic. © 2017 Wiley Periodicals, Inc.

Trehalose protects against cadmium-induced cytotoxicity in primary rat proximal tubular cells via inhibiting apoptosis and restoring autophagic flux

PubMed Central

Wang, Xin-Yu; Yang, Heng; Wang, Min-Ge; Yang, Du-Bao; Wang, Zhen-Yong; Wang, Lin

2017-01-01

Autophagy has an important renoprotective function and we recently found that autophagy inhibition is involved in cadmium (Cd)-induced nephrotoxicity. Here, we aimed to investigate the protective effect of trehalose (Tre), a novel autophagy activator, against Cd-induced cytotoxicity in primary rat proximal tubular (rPT) cells. First, data showed that Tre treatment significantly decreased Cd-induced apoptotic cell death of rPT cells via inhibiting caspase-dependent apoptotic pathway, evidenced by morphological analysis, flow cytometric and immunoblot assays. Also, administration with Tre protected rPT cells against Cd-induced lipid peroxidation. Inhibition of autophagic flux in Cd-exposed rPT cells was markedly restored by Tre administration, demonstrated by immunoblot analysis of autophagy marker proteins and GFP and RFP tandemly tagged LC3 method. Resultantly, Cd-induced autophagosome accumulation was obviously alleviated by Tre treatment. Meanwhile, blockage of autophagosome–lysosome fusion by Cd exposure was noticeably restored by Tre, which promoted the autophagic degradation in Cd-exposed rPT cells. Moreover, Tre treatment markedly recovered Cd-induced lysosomal alkalinization and impairment of lysosomal degradation capacity in rPT cells, demonstrating that Tre has the ability to restore Cd-impaired lysosomal function. Collectively, these findings demonstrate that Tre treatment alleviates Cd-induced cytotoxicity in rPT cells by inhibiting apoptosis and restoring autophagic flux. PMID:29022917

Statins and cancer.

PubMed

Vallianou, Natalia G; Kostantinou, Alexandra; Kougias, Marios; Kazazis, Christos

2014-06-01

Statins have pleiotropic properties and might exert an effect even in the field of cancer. Statins competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme (HMG-CoA) reductase, the major rate-limiting enzyme that controls the conversion of HMG-CoA to mevalonic acid. Specifically, inhibition of HMG-CoA reductase by statins has been proved to prevent the synthesis of mevalonic acid, a precursor of non-steroidal isoprenoids, which are lipid attachment molecules for small G proteins, such as Ras, Rho and Rac. Thus, statins may inhibit the synthesis of isoprenoids and thereby suppress the activation of small G proteins. In addition, statins exert pro-apoptotic, anti-angiogenic, and immunomodulatory effects, which may prevent cancer growth. Statins may inhibit the growth of a variety of cancer cell types, including breast, gastric, pancreatic, and prostate carcinoma, neuroblastoma, melanoma, mesothelioma and acute myeloid leukemia cells. They exert pro-apoptotic effects in a wide range of cancer cell lines, but with many differences in the sensitivity to statin-induced cell death among different cancer cell types. Regarding anti-angiogenic effects, multiple statin effects on blood vessel formation by inhibition of angiogenesis through down-regulation of pro-angiogenic factors, such as vascular endothelial growth factor, inhibition of endothelial cell proliferation and inhibition of adhesion to extracellular matrix by blocking intercellular adhesion molecules have been suggested. The molecular mechanisms of statin immunomodulation often implicate multiple pathways, regarding the regulation of genes encoding key molecules, which are involved in antigen presentation and subsequent immunomodulation. Another mechanism involves the down-regulation of the nuclear factor-kappa-B, which is responsible for the transcription of many genes involved in immunologic mechanisms, such as interferon-inducible protein-10, monocyte chemo-attractant protein 1 and cyclooxygenase-2. Statins have been associated with a significantly lower risk of breast, colorectal, ovarian, pancreatic, lung cancers and lymphoma in several observational studies. On the other hand, other studies, including meta-analyses have raised concerns about the safety of statins among elderly patients. A recent study upon the relationship between statin use (prior to cancer diagnosis) and cancer-related mortality in the entire Danish population from 1995-2009 in adults > 40 years of age has been conducted. As compared to statin non-users, patients using statins prior to cancer diagnosis were 15% less likely to die from any cause or cancer specifically. Further investigation is needed to elaborate on their mode of action as well as their true significance on cancer prevention and perhaps as an adjuvant to cancer chemotherapy.

Curcumin inhibits epigen and amphiregulin upregulated by 2,4,6-trinitrochlorobenzene associated with attenuation of skin swelling.

PubMed

Sakai, Hiroyasu; Sato, Ken; Sato, Fumiaki; Kai, Yuki; Mandokoro, Kazutaka; Matsumoto, Kenjiro; Kato, Shinichi; Yumoto, Tetsuro; Narita, Minoru; Chiba, Yoshihiko

2017-08-01

Contact dermatitis model involving repeated application of hapten is used as a tool to assess dermatitis, as characterized by thickening. Involvement of cell proliferation, elicited by repeated hapten-stimulation, in this swelling has been unclear. Curcumin is reported to reduce inflammation. We examined involvement of cell proliferation and the role of extracellular regulated kinase (ERK) in 2,4,6-trinitrochlorobenzene (TNCB) challenge-induced ear swelling. We also examined the effects of curcumin in this model. Mice were sensitized with TNCB to the abdominal skin. Then, they were challenged with TNCB to the ear three times. The ERK activation inhibitor U0126 or curcumin was applied 30 min before each TNCB challenge. TNCB challenge-induced increased epidermal cell number and dermal thickening. Gene expressions of epithelial mitogen (EPGN), amphiregulin (AREG) and heparin-binding-epidermal growth factor (HB-EGF) were increased in the ears after the last TNCB challenge. Ki-67 immunoreactivity was increased in the dermis in TNCB-challenged ears. TNCB-induced swelling was inhibited by U0126 and curcumin. Curcumin also attenuated TNCB-induced ERK phosphorylation and expression of EPGN and AREG genes. Ear swelling induced by TNCB challenge might be mediated, in part, by the EPGN- and AREG-ERK proliferation pathway and was inhibited by curcumin.

Essential roles of caspases and their upstream regulators in rotenone-induced apoptosis

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

Lee Jihjong; Huang, M.-S.; Yang, I-C.

2008-06-20

In the present study, we examined whether caspases and their upstream regulators are involved in rotenone-induced cytotoxicity. Rotenone significantly inhibited the proliferation of oral cancer cell lines in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that rotenone treatment induced apoptosis following G2/M arrest. Western blotting showed activation of both the caspase-8 and caspase-9 pathways, which differed from previous studies conducted in other cell types. Furthermore, p53 protein and its downstream pro-apoptotic target, Bax, were induced in SAS cells after treatment with rotenone. Rotenone-induced apoptosis was inhibited by antioxidants (glutathione, N-acetylcysteine, andmore » tiron). In conclusion, our results demonstrate significant involvement of caspases and their upstream regulators in rotenone-induced cytotoxicity.« less

T Lymphocyte Inhibition by Tumor-Infiltrating Dendritic Cells Involves Ectonucleotidase CD39 but Not Arginase-1

PubMed Central

Trad, Malika; Gautheron, Alexandrine; Fraszczak, Jennifer; Larmonier, Claire; LaCasse, Collin J.; Centuori, Sara; Audia, Sylvain; Samson, Maxime; Ciudad, Marion; Bonnefoy, Francis; Lemaire-Ewing, Stéphanie; Katsanis, Emmanuel; Perruche, Sylvain; Saas, Philippe; Bonnotte, Bernard

2015-01-01

T lymphocytes activated by dendritic cells (DC) which present tumor antigens play a key role in the antitumor immune response. However, in patients suffering from active cancer, DC are not efficient at initiating and supporting immune responses as they participate to T lymphocyte inhibition. DC in the tumor environment are functionally defective and exhibit a characteristic of immature phenotype, different to that of DC present in nonpathological conditions. The mechanistic bases underlying DC dysfunction in cancer responsible for the modulation of T-cell responses and tumor immune escape are still being investigated. Using two different mouse tumor models, we showed that tumor-infiltrating DC (TIDC) are constitutively immunosuppressive, exhibit a semimature phenotype, and impair responder T lymphocyte proliferation and activation by a mechanism involving CD39 ectoenzyme. PMID:26491691

Pre-clinical assessment of A-674563 as an anti-melanoma agent

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

Zou, Ying; Fan, Guobiao; Wang, Xuemin, E-mail: wangxuemeidr@yeah.net

The present study aims to investigate the anti-melanoma activity by an Akt1 specific inhibitor A-674563. We showed that A-674563 was anti-proliferative and cytotoxic when added to human melanoma cells (A375, WM-115 and SK-Mel-2 lines). A-674563 induced caspase-dependent apoptotic death of human melanoma cells, and its cytotoxicity was inhibited with pre-treatment of caspase inhibitors. Further, A-674563 treatment blocked Akt and its downstream S6 Kinase 1 (S6K1) activation in A375 melanoma cells. Significantly, restoring Akt-S6K1 activation via introduction of constitutively-active Akt1 (ca-Akt1) only partially attenuated A-674563's cytotoxicity against A375 cells. Further, A-674563 induced pro-apoptotic ceramide production in A375 cells. Significantly, sphingosine-1-phosphate (S1P) inhibited A-674563-inducedmore » ceramide production and subsequent A375 cell apoptosis. On the other hand, co-treatment with the glucosylceramide synthase (GCS) inhibitor PDMP or the cell permeable short-chain ceramide (C6) potentiated A-674563's cytotoxicity against A375 cells. In vivo, A-674563 oral gavage inhibited A375 xenograft growth in severe combined immunodeficiency (scid) mice. Akt inactivation, caspase-3 activation and ceramide production were also observed in A-674563-treated A375 xenografts. Together, these results suggest that A-674563 exerts potent anti-melanoma activity, involving Akt-dependent and Akt-independent mechanisms. - Highlights: • A-674563 inhibits human melanoma cell survival and proliferation. • A-674563 induces melanoma cell apoptotic death, inhibited by caspase inhibitors. • A-674563 inhibits melanoma cells via Akt-dependent and -independent mechanisms. • A-674563 induces ceramide production in melanoma cells, independent of Akt inhibition. • A-674563 oral administration potently inhibits A375 xenograft growth in mice.« less

Edaravone protected PC12 cells against MPP(+)-cytoxicity via inhibiting oxidative stress and up-regulating heme oxygenase-1 expression.

PubMed

Cheng, Baohua; Guo, Yunliang; Li, Chuangang; Ji, Bingyuan; Pan, Yanyou; Chen, Jing; Bai, Bo

2014-08-15

Oxidative stress is involved in the pathogenesis of Parkinson's disease (PD). Edaravone has been shown to have a neuroprotective effect. In the present work, we investigated the effect of edaravone on 1-methyl-4-phenylpyridinium (MPP(+))-treated PC12 cells. Edaravone inhibited the decrease of cell viability and apoptosis induced by MPP(+) in PC12 cells. In addition, edaravone alleviated intracellular reactive oxygen species (ROS) production. MPP(+) induced heme oxygenase-1 (HO-1) expression, which was further enhanced by edaravone. The inhibitor of HO-1 zinc protoporphyrin-IX attenuated the neuroprotection of edaravone. So edaravone protected PC12 cells against MPP(+)-cytoxicity via inhibiting oxidative stress and up-regulating HO-1 expression. The data showed that edaravone was neuroprotective and could be potentially therapeutics for PD in future. Copyright © 2014 Elsevier B.V. All rights reserved.

Silibinin, a novel chemokine receptor type 4 antagonist, inhibits chemokine ligand 12-induced migration in breast cancer cells.

PubMed

Wang, Yan; Liang, Wei-Cheng; Pan, Wen-Liang; Law, Wai-Kit; Hu, Jian-Shu; Ip, Denis Tsz-Ming; Waye, Mary Miu-Yee; Ng, Tzi-Bun; Wan, David Chi-Cheong

2014-09-25

C-X-C chemokine receptor type 4 (CXCR4) signaling has been demonstrated to be involved in cancer invasion and migration; therefore, CXCR4 antagonist can serve as an anti-cancer drug by preventing tumor metastasis. This study aimed to identify the CXCR4 antagonists that could reduce and/or inhibit tumor metastasis from natural products. According to the molecular docking screening, we reported here silibinin as a novel CXCR4 antagonist. Biochemical characterization showed that silibinin blocked chemokine ligand 12 (CXCL12)-induced CXCR4 internalization by competitive binding to CXCR4, therefore inhibiting downstream intracellular signaling. In human breast cancer cells MDA-MB-231, which expresses high levels of CXCR4, inhibition of CXCL12-induced chemomigration can be found under silibinin treatment. Overexpression of CXCL12 sensitized MDA-MB-231 cells to the inhibition of silibinin, which was abolished by CXCR4 knockdown. The inhibition of silibinin was also observed in MCF-7/CXCR4 cells rather than MCF-7 cells that express low level of CXCR4. Our work demonstrated that silibinin is a novel CXCR4 antagonist that may have potential therapeutic use for prevention of tumor metastasis. Copyright © 2014 Elsevier GmbH. All rights reserved.

Feedback inhibition of nitric oxide synthase activity by nitric oxide.

PubMed Central

Assreuy, J.; Cunha, F. Q.; Liew, F. Y.; Moncada, S.

1993-01-01

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly. PMID:7682140

Protein kinase C-beta inhibition induces apoptosis and inhibits cell cycle progression in acquired immunodeficiency syndrome-related non-hodgkin lymphoma cells.

PubMed

Saba, Nakhle S; Levy, Laura S

2012-01-01

Acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin lymphoma (NHL) constitutes an aggressive variety of lymphomas characterized by increased extranodal involvement, relapse rate, and resistance to chemotherapy. Protein kinase C-beta (PKCβ) targeting showed promising results in preclinical and clinical studies involving a wide variety of cancers, but studies describing the role of PKCβ in AIDS-NHL are primitive if not lacking. In the present study, 3 AIDS-NHL cell lines were examined: 2F7 (AIDS-Burkitt lymphoma), BCBL-1 (AIDS-primary effusion lymphoma), and UMCL01-101 (AIDS-diffuse large B-cell lymphoma). Immunoblot analysis demonstrated expression of PKCβ1 and PKCβ2 in 2F7 and UMCL01-101 cells, and PKCβ1 alone in BCBL-1 cells. The viability of 2F7 and BCBL-1 cells decreased significantly in the presence of PKCβ-selective inhibitor at half-maximal inhibitory concentration of 14 and 15 μmol/L, respectively, as measured by tetrazolium dye reduction assay. In contrast, UMCL01-101 cells were relatively resistant. As determined using flow cytometric deoxynucleotidyl transferase dUTP nick-end labeling assay with propidium iodide staining, the responsiveness of sensitive cells was associated with apoptotic induction and cell cycle inhibition. Protein kinase C-beta-selective inhibition was observed not to affect AKT phosphorylation but to induce a rapid and sustained reduction in the phosphorylation of glycogen synthase kinase-3 beta, ribosomal protein S6, and mammalian target of rapamycin in sensitive cell lines. The results indicate that PKCβ plays an important role in AIDS-related NHL survival and suggest that PKCβ targeting should be considered in a broader spectrum of NHL. The observations in BCBL-1 were unexpected in the absence of PKCβ2 expression and implicate PKCβ1 as a regulator in those cells.

Pleiotrophin-induced endothelial cell migration is regulated by xanthine oxidase-mediated generation of reactive oxygen species.

PubMed

Tsirmoula, Sotiria; Lamprou, Margarita; Hatziapostolou, Maria; Kieffer, Nelly; Papadimitriou, Evangelia

2015-03-01

Pleiotrophin (PTN) is a heparin-binding growth factor that induces cell migration through binding to its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and integrin alpha v beta 3 (ανβ3). In the present work, we studied the effect of PTN on the generation of reactive oxygen species (ROS) in human endothelial cells and the involvement of ROS in PTN-induced cell migration. Exogenous PTN significantly increased ROS levels in a concentration and time-dependent manner in both human endothelial and prostate cancer cells, while knockdown of endogenous PTN expression in prostate cancer cells significantly down-regulated ROS production. Suppression of RPTPβ/ζ through genetic and pharmacological approaches, or inhibition of c-src kinase activity abolished PTN-induced ROS generation. A synthetic peptide that blocks PTN-ανβ3 interaction abolished PTN-induced ROS generation, suggesting that ανβ3 is also involved. The latter was confirmed in CHO cells that do not express β3 or over-express wild-type β3 or mutant β3Y773F/Y785F. PTN increased ROS generation in cells expressing wild-type β3 but not in cells not expressing or expressing mutant β3. Phosphoinositide 3-kinase (PI3K) or Erk1/2 inhibition suppressed PTN-induced ROS production, suggesting that ROS production lays down-stream of PI3K or Erk1/2 activation by PTN. Finally, ROS scavenging and xanthine oxidase inhibition completely abolished both PTN-induced ROS generation and cell migration, while NADPH oxidase inhibition had no effect. Collectively, these data suggest that xanthine oxidase-mediated ROS production is required for PTN-induced cell migration through the cell membrane functional complex of ανβ3 and RPTPβ/ζ and activation of c-src, PI3K and ERK1/2 kinases. Copyright © 2015 Elsevier Inc. All rights reserved.

Fermented guava leaf extract inhibits LPS-induced COX-2 and iNOS expression in Mouse macrophage cells by inhibition of transcription factor NF-kappaB.

PubMed

Choi, Soo-Youn; Hwang, Joon-Ho; Park, Soo-Young; Jin, Yeong-Jun; Ko, Hee-Chul; Moon, Sang-Wook; Kim, Se-Jae

2008-08-01

The goal of this study was to elucidate the antiinflammatory activities of Psidium guajava L. (guava) leaf. To improve the functionality of guava leaf, it was fermented with Phellinus linteus mycelia, Lactobacillus plantarum and Saccharomyces cerevisiae. The ethanol extract from fermented guava leaf inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production. Western blot analysis showed that fermented guava leaf extract decreased LPS-induced inducible nitric oxide synthase (iNOS) and the cyclooxygenase-2 (COX-2) protein level in RAW 264.7 cells. To investigate the mechanism involved, the study examined the effect of fermented guava leaf extract on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. Fermented guava leaf extract significantly inhibited LPS-induced NF-kappaB transcriptional activity. Immunochemical analysis revealed that fermented guava leaf extract suppressed LPS-induced degradation of I-kappaBalpha. Taken together, the data indicate that fermented guava leaf extract is involved in the inhibition of iNOS and COX-2 via the down-regulation of NF-kappaB pathway, revealing a partial molecular basis for the antiinflammatory properties of fermented guava leaf extract.

Novel Midkine Inhibitor iMDK Inhibits Tumor Growth and Angiogenesis in Oral Squamous Cell Carcinoma.

PubMed

Masui, Masanori; Okui, Tatsuo; Shimo, Tsuyoshi; Takabatake, Kiyofumi; Fukazawa, Takuya; Matsumoto, Kenichi; Kurio, Naito; Ibaragi, Soichiro; Naomoto, Yoshio; Nagatsuka, Hitoshi; Sasaki, Akira

2016-06-01

Midkine is a heparin-binding growth factor highly expressed in various human malignant tumors. However, its role in the growth of oral squamous cell carcinoma is not well understood. In this study, we analyzed the antitumor effect of a novel midkine inhibitor (iMDK) against oral squamous cell carcinoma. Administration of iMDK induced a robust antitumor response and suppressed cluster of differentiation 31 (CD31) expression in oral squamous cell carcinoma HSC-2 cells and SAS cells xenograft models. iMDK inhibited the proliferation of these cells dose-dependently, as well as the expression of midkine and phospho-extracellular signal-regulated kinase in HSC-2 and SAS cells. Moreover, iMDK significantly inhibited vascular endothelial growth factor and induced tube growth of human umbilical vein endothelial cells in a dose-dependent fashion. These findings suggest that midkine is critically involved in oral squamous cell carcinoma and iMDK can be effectively used for the treatment of oral squamous cell carcinoma. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Chrna2-Martinotti Cells Synchronize Layer 5 Type A Pyramidal Cells via Rebound Excitation

PubMed Central

Leão, Richardson N.; Edwards, Steven J.

2017-01-01

Martinotti cells are the most prominent distal dendrite–targeting interneurons in the cortex, but their role in controlling pyramidal cell (PC) activity is largely unknown. Here, we show that the nicotinic acetylcholine receptor α2 subunit (Chrna2) specifically marks layer 5 (L5) Martinotti cells projecting to layer 1. Furthermore, we confirm that Chrna2-expressing Martinotti cells selectively target L5 thick-tufted type A PCs but not thin-tufted type B PCs. Using optogenetic activation and inhibition, we demonstrate how Chrna2-Martinotti cells robustly reset and synchronize type A PCs via slow rhythmic burst activity and rebound excitation. Moreover, using optical feedback inhibition, in which PC spikes controlled the firing of surrounding Chrna2-Martinotti cells, we found that neighboring PC spike trains became synchronized by Martinotti cell inhibition. Together, our results show that L5 Martinotti cells participate in defined cortical circuits and can synchronize PCs in a frequency-dependent manner. These findings suggest that Martinotti cells are pivotal for coordinated PC activity, which is involved in cortical information processing and cognitive control. PMID:28182735

Ascomycin macrolactam derivative SDZ ASM 981 inhibits the release of granule-associated mediators and of newly synthesized cytokines in RBL 2H3 mast cells in an immunophilin-dependent manner.

PubMed

Hultsch, T; Müller, K D; Meingassner, J G; Grassberger, M; Schopf, R E; Knop, J

1998-09-01

Mast cells play an important role in the pathological development of many inflammatory and allergic diseases and inhibition of mast cell activation is a potential target for therapeutic intervention. Therefore, the effect of the novel ascomycin macrolactam derivative SDZ ASM 981 on Fc epsilonRI-mediated activation of rat basophilic leukemia (RBL) cells, as a model for mast cell activation, was investigated. First, the ability to inhibit different mast cell immunophilins in vitro was tested. Using recombinant macrophilin-12 (FKBP-12), inhibition of rotamase activity with an IC50 of approximately 6 nM was observed. The rotamase activity of cyclophilin A (18 kDa) was not affected. Secondly, the effect of SDZ ASM 981 on Fc epsilonRI-mediated mast cell activation was investigated in the RBL cell model. SDZ ASM 981 inhibited exocytosis of preformed mediators (e.g. serotonin) with an IC50 of approximately 30 nM. Transcription and release of newly synthesized mediators (e.g. TNF-alpha) was inhibited with an IC50 of approximately 100 nM. The inhibitory effect of SDZ ASM 981 was antagonized by rapamycin. We conclude that SDZ ASM 981 is a potent inhibitor of Fc epsilonRI-mediated activation of mast cells in vitro. The mechanism of action involves formation of (calcineurin) inhibitory complexes with macrophilins. We suggest that this inhibitory action on mast cells might contribute to the antiinflammatory effect of SDZ ASM 981 observed in vivo (e.g. in aptopic dermatitis and psoriasis).

Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells.

PubMed

Silva, Mariana C C; de Paula, Cláudia A A; Ferreira, Joana G; Paredes-Gamero, Edgar J; Vaz, Angela M S F; Sampaio, Misako U; Correia, Maria Tereza S; Oliva, Maria Luiza V

2014-07-01

Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity. MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting. BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α1, α6 and β1 integrin subunit expression, and increases α5 subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21. BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells. Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Abrogation of TNF-mediated cytotoxicity by space flight involves protein kinase C

NASA Technical Reports Server (NTRS)

Woods, K. M.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

Experiments conducted on STS-50 indicated that space flight significantly inhibited tumor necrosis factor (TNF)-mediated killing of LM929 cells compared to ground controls. In ground-based studies, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also inhibited TNF-mediated killing of LM929 cells. Therefore, we used PKC inhibitors to determine if the inhibitory effects of spaceflight on TNF-mediated cytotoxicity involved the activation of PKC. In experiments conducted onboard space shuttle mission STS-54, we saw that in the presence of the protein kinase C inhibitors H7 and H8, TNF-mediated cytotoxicity was restored to levels of those observed in the ground controls. Subsequent experiments done during the STS-57 mission tested the dose response of two protein kinase inhibitors, H7 and HA1004. We again saw that killing was restored in a dose-dependent manner, with inhibitor concentrations known to inhibit PKC being most effective. These data suggest that space flight ameliorates the action of TNF by affecting PKC in target cells.

Phosphatidic acid inhibits ceramide 1-phosphate-stimulated macrophage migration.

PubMed

Ouro, Alberto; Arana, Lide; Rivera, Io-Guané; Ordoñez, Marta; Gomez-Larrauri, Ana; Presa, Natalia; Simón, Jorge; Trueba, Miguel; Gangoiti, Patricia; Bittman, Robert; Gomez-Muñoz, Antonio

2014-12-15

Ceramide 1-phosphate (C1P) was recently demonstrated to potently induce cell migration. This action could only be observed when C1P was applied exogenously to cells in culture, and was inhibited by pertussis toxin. However, the mechanisms involved in this process are poorly understood. In this work, we found that phosphatidic acid (PA), which is structurally related to C1P, displaced radiolabeled C1P from its membrane-binding site and inhibited C1P-stimulated macrophage migration. This effect was independent of the saturated fatty acid chain length or the presence of a double bond in each of the fatty acyl chains of PA. Treatment of RAW264.7 macrophages with exogenous phospholipase D (PLD), an enzyme that produces PA from membrane phospholipids, also inhibited C1P-stimulated cell migration. Likewise, PA or exogenous PLD inhibited C1P-stimulated extracellularly regulated kinases (ERK) 1 and 2 phosphorylation, leading to inhibition of cell migration. However, PA did not inhibit C1P-stimulated Akt phosphorylation. It is concluded that PA is a physiological regulator of C1P-stimulated macrophage migration. These actions of PA may have important implications in the control of pathophysiological functions that are regulated by C1P, including inflammation and various cellular processes associated with cell migration such as organogenesis or tumor metastasis. Copyright © 2014 Elsevier Inc. All rights reserved.

Inhibition of neurotensin-stimulated mast cell secretion and carboxypeptidase A activity by the peptide inhibitor of carboxypeptidase A and neurotensin-receptor antagonist SR 48692.

PubMed

Miller, L A; Cochrane, D E; Feldberg, R S; Carraway, R E

1998-06-01

Neurotensin (NT), a peptide found in brain and several peripheral tissues, is a potent stimulus for mast cell secretion and its actions are blocked by the specific NT receptor antagonist, SR 48692. Subsequent to stimulation, NT is rapidly degraded by mast cell carboxypeptidase A (CPA). In the experiments described here, we tested for the involvement of CPA activity in the activation of mast cell secretion by the peptide, NT. Mast cells were isolated from the peritoneal and pleural cavities of rats, purified over metrizamide gradients and incubated at 37 degrees C in Locke solution or Locke containing the appropriate inhibitors. For some experiments, media derived from mast cells stimulated by compound 48/80 were used as a source of mast cell CPA activity. Treatment of mast cells with the highly specific peptide inhibitor of CPA derived from potato (PCI) inhibited histamine release in response to NT and NT8-13 (the biologically active region of NT). This inhibition required some 20 min to develop and was only partially reversed by a 20-min wash period. PCI (10 microM) did not inhibit histamine release in response to NT1-12, bradykinin, compound 48/80, the calcium ionophore, A23187, or anti-IgE serum. PCI also inhibited mast cell CPA activity. SR 48692, a highly selective antagonist of the brain NT receptor and of NT-stimulated mast cell secretion, also inhibited mast cell CPA activity as well as bovine pancreatic CPA activity in a concentration-dependent manner. It is suggested that the mast cell binding site for NT and the active site for CPA may share similar characteristics. The results are discussed in terms of NT mechanism of action on the mast cell.

Excess thyroid hormone inhibits embryonic neural stem/progenitor cells proliferation and maintenance through STAT3 signalling pathway.

PubMed

Chen, Chunhai; Zhou, Zhou; Zhong, Min; Li, Maoquan; Yang, Xuesen; Zhang, Yanwen; Wang, Yuan; Wei, Aimin; Qu, Mingyue; Zhang, Lei; Xu, Shangcheng; Chen, Shude; Yu, Zhengping

2011-07-01

Hyperthyroidism is prevalent during pregnancy, but little is known about the effects of excess thyroid hormone on the development of embryonic neural stem/progenitor cells (NSCs), and the mechanisms underlying these effects. Previous studies indicate that STAT3 plays a crucial role in determining NSC fate during neurodevelopment. In this study, we investigated the effects of a supraphysiological dose of 3,5,3'-L-triiodothyronine (T3) on the proliferation and maintenance of NSCs derived from embryonic day 13.5 mouse neocortex, and the involvement of STAT3 in this process. Our results suggest that excess T3 treatment inhibits NSC proliferation and maintenance. T3 decreased tyrosine phosphorylation of JAK1, JAK2 and STAT3, and subsequently inhibited STAT3-DNA binding activity. Furthermore, proliferation and maintenance of NSCs were decreased by inhibitors of JAKs and STAT3, indicating that the STAT3 signalling pathway is involved in the process of NSC proliferation and maintenance. Taken together, these results suggest that the STAT3 signalling pathway is involved in the process of T3-induced inhibition of embryonic NSC proliferation and maintenance. These findings provide data for understanding the effects of hyperthyroidism during pregnancy on fetal brain development, and the mechanisms underlying these effects.

ALG2 regulates glioblastoma cell proliferation, migration and tumorigenicity

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

Zhang, Dunke; Wang, Feng; Pang, Yi

Apoptosis-linked gene-2 (ALG-2), also known as programmed cell death 6 (PDCD6), has recently been reported to be aberrantly expressed in various tumors and required for tumor cell viability. The aim of the present study was to investigate whether ALG-2 plays a crucial role in tumor cell proliferation, migration and tumorigenicity. In this study, we examined the expression of PDCD6 in glioblastoma cell lines and found that ALG-2 was generally expressed in glioblastoma cell lines. We also performed an analysis of an online database and found that high expression of ALG-2 was associated with poor prognosis (p = 0.039). We found that over-expressionmore » of ALG2 in glioblastoma could inhibit cell proliferation and, conversely, that down-regulation of ALG2 could promote cell proliferation. Further studies showed that over-expression of ALG2 inhibited the migration of tumor cells, whereas down-regulation of ALG2 promoted tumor cell migration. Finally, in vitro and in vivo studies showed that over-expression of ALG2 inhibited the tumorigenic ability of tumor cells, while down-regulation of ALG2 promoted tumor cell tumorigenic ability. In conclusion, ALG2 has a tumor suppressive role in glioblastoma and might be a potential target for the treatment of glioblastoma. - Highlights: • Low ALG2 expression is indicative of poor prognosis in glioblastoma patients. • ALG2 is required for cell proliferation in GBM cells. • ALG2 is involved in GBM cell migration. • ALG2 is involved in GBM cell self-renewal and tumorigenesis in vitro and in vivo.« less

Differential routes of Ca2+ influx in Swiss 3T3 fibroblasts in response to receptor stimulation.

PubMed Central

Miyakawa, T; Kojima, M; Ui, M

1998-01-01

Ca2+ influx into cells in response to stimulation of various receptors was studied with Swiss 3T3 fibroblasts. The mechanisms involved were found to be so diverse that they were classified into four groups, Type I to IV. Type-I influx occurred, via pertussis toxin-susceptible G-proteins, immediately after internal Ca2+ mobilization by bradykinin, thrombin, endothelin, vasopressin or angiotensin II. Type-II influx induced by bombesin differed from Type I in its insusceptibility to pertussis toxin treatment. Ca2+ influx induced by prostaglandin E1, referred to as Type-III influx, was unique in that phospholipase C was apparently not activated without extracellular Ca2+, strongly suggesting that the Ca2+ influx preceded and was responsible for InsP3 generation and internal Ca2+ mobilization. More Ca2+ entered the cells more slowly via the Type-IV route opened by platelet-derived and other growth factors. These types of Ca2+ influx could be differentiated by their different susceptibilities to protein kinase C maximally activated by 1 h of exposure of cells to PMA, which inhibited phospholipase Cbeta coupled to receptors involved in Type-I and -II influx but did not inhibit growth-factor-receptor-coupled phospholipase Cgamma. Type-I and -II Ca2+ influxes, together with store-operated influx induced by thapsigargin, were not directly inhibited by exposure of cells to PMA, but Type-III and -IV influxes were completely inhibited. In addition, stimulation of receptors involved in Type-I and -IV Ca2+ influx, but not Type-II and -III influx, led to phospholipase A2 activation in the presence of extracellular Ca2+. Inhibition of Type-I and -IV Ca2+ influxes by their respective inhibitors, diltiazem and nifedipine, resulted in abolition of phospholipase A2 activation induced by the respective receptor agonists, in agreement with the notion that Ca2+ influx via these routes is responsible for receptor-mediated phospholipase A2 activation. PMID:9405282

Differential routes of Ca2+ influx in Swiss 3T3 fibroblasts in response to receptor stimulation.

PubMed

Miyakawa, T; Kojima, M; Ui, M

1998-01-01

Ca2+ influx into cells in response to stimulation of various receptors was studied with Swiss 3T3 fibroblasts. The mechanisms involved were found to be so diverse that they were classified into four groups, Type I to IV. Type-I influx occurred, via pertussis toxin-susceptible G-proteins, immediately after internal Ca2+ mobilization by bradykinin, thrombin, endothelin, vasopressin or angiotensin II. Type-II influx induced by bombesin differed from Type I in its insusceptibility to pertussis toxin treatment. Ca2+ influx induced by prostaglandin E1, referred to as Type-III influx, was unique in that phospholipase C was apparently not activated without extracellular Ca2+, strongly suggesting that the Ca2+ influx preceded and was responsible for InsP3 generation and internal Ca2+ mobilization. More Ca2+ entered the cells more slowly via the Type-IV route opened by platelet-derived and other growth factors. These types of Ca2+ influx could be differentiated by their different susceptibilities to protein kinase C maximally activated by 1 h of exposure of cells to PMA, which inhibited phospholipase Cbeta coupled to receptors involved in Type-I and -II influx but did not inhibit growth-factor-receptor-coupled phospholipase Cgamma. Type-I and -II Ca2+ influxes, together with store-operated influx induced by thapsigargin, were not directly inhibited by exposure of cells to PMA, but Type-III and -IV influxes were completely inhibited. In addition, stimulation of receptors involved in Type-I and -IV Ca2+ influx, but not Type-II and -III influx, led to phospholipase A2 activation in the presence of extracellular Ca2+. Inhibition of Type-I and -IV Ca2+ influxes by their respective inhibitors, diltiazem and nifedipine, resulted in abolition of phospholipase A2 activation induced by the respective receptor agonists, in agreement with the notion that Ca2+ influx via these routes is responsible for receptor-mediated phospholipase A2 activation.

Effects of advanced glycation end products on ezrin-dependent functions in LLC-PK1 proximal tubule cells.

PubMed

Bach, Leon A; Gallicchio, Marisa A; McRobert, E Anne; Tikoo, Anjali; Cooper, Mark E

2005-06-01

We have recently shown that advanced glycation products (AGEs) bind to the ERM (ezrin, radixin, moesin) family of proteins. ERM proteins act as cross-linkers between cell membrane proteins and the actin cytoskeleton. They are also involved in signal transduction pathways. They therefore have a critical role in normal cell processes, including modulation of cell shape, adhesion, and motility. We postulate that AGEs may contribute to diabetic complications by disrupting ERM function. In support of this hypothesis, AGEs inhibit ezrin-dependent tubulogenesis of proximal tubule cells. Phosphorylation is an important activating mechanism for ERM proteins, and AGEs inhibit ezrin phosphorylation mediated by the epidermal growth factor receptor.

Excess Iodide Induces an Acute Inhibition of the Sodium/Iodide Symporter in Thyroid Male Rat Cells by Increasing Reactive Oxygen Species

PubMed Central

Arriagada, Alejandro A.; Albornoz, Eduardo; Opazo, Ma. Cecilia; Becerra, Alvaro; Vidal, Gonzalo; Fardella, Carlos; Michea, Luis; Carrasco, Nancy; Simon, Felipe; Elorza, Alvaro A.; Bueno, Susan M.; Kalergis, Alexis M.

2015-01-01

Na+/I− symporter (NIS) mediates iodide (I−) uptake in the thyroid gland, the first and rate-limiting step in the biosynthesis of the thyroid hormones. The expression and function of NIS in thyroid cells is mainly regulated by TSH and by the intracellular concentration of I−. High doses of I− for 1 or 2 days inhibit the synthesis of thyroid hormones, a process known as the Wolff-Chaikoff effect. The cellular mechanisms responsible for this physiological response are mediated in part by the inhibition of I− uptake through a reduction of NIS expression. Here we show that inhibition of I− uptake occurs as early as 2 hours or 5 hours after exposure to excess I− in FRTL-5 cells and the rat thyroid gland, respectively. Inhibition of I− uptake was not due to reduced NIS expression or altered localization in thyroid cells. We observed that incubation of FRTL-5 cells with excess I− for 2 hours increased H2O2 generation. Furthermore, the inhibitory effect of excess I− on NIS-mediated I− transport could be recapitulated by H2O2 and reverted by reactive derived oxygen species scavengers. The data shown here support the notion that excess I− inhibits NIS at the cell surface at early times by means of a posttranslational mechanism that involves reactive derived oxygen species. PMID:25594695

PARP activity and inhibition in fetal and adult oligodendrocyte precursor cells: Effect on cell survival and differentiation.

PubMed

Baldassarro, Vito A; Marchesini, Alessandra; Giardino, Luciana; Calzà, Laura

2017-07-01

Poly (ADP-ribose) polymerase (PARP) family members are ubiquitously expressed and play a key role in cellular processes, including DNA repair and cell death/survival balance. Accordingly, PARP inhibition is an emerging pharmacological strategy for cancer and neurodegenerative diseases. Consistent evidences support the critical involvement of PARP family members in cell differentiation and phenotype maturation. In this study we used an oligodendrocyte precursor cells (OPCs) enriched system derived from fetal and adult brain to investigate the role of PARP in OPCs proliferation, survival, and differentiation. The PARP inhibitors PJ34, TIQ-A and Olaparib were used as pharmacological tools. The main results of the study are: (i) PARP mRNA expression and PARP activity are much higher in fetal than in adult-derived OPCs; (ii) the culture treatment with PARP inhibitors is cytotoxic for OPCs derived from fetal, but not from adult, brain; (iii) PARP inhibition reduces cell number, according to the inhibitory potency of the compounds; (iv) PARP inhibition effect on fetal OPCs is a slow process; (v) PARP inhibition impairs OPCs maturation into myelinating OL in fetal, but not in adult cultures, according to the inhibitory potency of the compounds. These results have implications for PARP-inhibition therapies for diseases and lesions of the central nervous system, in particular for neonatal hypoxic/ischemic encephalopathy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

c-MYC inhibition impairs hypoxia response in glioblastoma multiforme

PubMed Central

Falchetti, Maria Laura; Illi, Barbara; Bozzo, Francesca; Valle, Cristiana; Helmer-Citterich, Manuela; Ferrè, Fabrizio; Nasi, Sergio; Levi, Andrea

2016-01-01

The c-MYC oncoprotein is a DNA binding transcription factor that enhances the expression of many active genes. c-MYC transcriptional signatures vary according to the transcriptional program defined in each cell type during differentiation. Little is known on the involvement of c-MYC in regulation of gene expression programs that are induced by extracellular cues such as a changing microenvironment. Here we demonstrate that inhibition of c-MYC in glioblastoma multiforme cells blunts hypoxia-dependent glycolytic reprogramming and mitochondria fragmentation in hypoxia. This happens because c-MYC inhibition alters the cell transcriptional response to hypoxia and finely tunes the expression of a subset of Hypoxia Inducible Factor 1-regulated genes. We also show that genes whose expression in hypoxia is affected by c-MYC inhibition are able to distinguish the Proneural subtype of glioblastoma multiforme, thus potentially providing a molecular signature for this class of tumors that are the least tractable among glioblastomas. PMID:27119353

SOX7 Suppresses Wnt Signaling by Disrupting β-Catenin/BCL9 Interaction.

PubMed

Fan, Rong; He, HaiYan; Yao, Wang; Zhu, YanFeng; Zhou, XunJie; Gui, MingTai; Lu, Jing; Xi, Hao; Deng, ZhongLong; Fan, Min

2018-02-01

The Wnt signaling is involved in angiogenesis and tumor development. β-catenin is the core component of the Wnt pathway, which mediates oncogenic transcription and regulated by a series of proteins. Sex-determining region Y-box 7 (SOX7) is a member of high-mobility-group transcription factor family, which inhibits oncogenic Wnt signaling in lots of tumor cells with unknown mechanism. By coimmunoprecipitation (co-IP) and super Topflash reporter assay, SOX7 can bind β-catenin and inhibit β-catenin/T cell factor (TCF)-mediated transcription. Meanwhile, B cell lymphoma 9 (BCL9) drives Wnt signaling path through direct binding-mediated β-catenin. Finally, we found that SOX7 inhibits oncogenic β-catenin-mediated transcription by disrupting the β-catenin/BCL9 interaction. Mechanistically, SOX7 compete with BCL9 to bind β-catenin. Our results show SOX7 inhibited Wnt signaling as suppressor and could be an important target for anticancer therapy.

Parallel processing by cortical inhibition enables context-dependent behavior.

PubMed

Kuchibhotla, Kishore V; Gill, Jonathan V; Lindsay, Grace W; Papadoyannis, Eleni S; Field, Rachel E; Sten, Tom A Hindmarsh; Miller, Kenneth D; Froemke, Robert C

2017-01-01

Physical features of sensory stimuli are fixed, but sensory perception is context dependent. The precise mechanisms that govern contextual modulation remain unknown. Here, we trained mice to switch between two contexts: passively listening to pure tones and performing a recognition task for the same stimuli. Two-photon imaging showed that many excitatory neurons in auditory cortex were suppressed during behavior, while some cells became more active. Whole-cell recordings showed that excitatory inputs were affected only modestly by context, but inhibition was more sensitive, with PV + , SOM + , and VIP + interneurons balancing inhibition and disinhibition within the network. Cholinergic modulation was involved in context switching, with cholinergic axons increasing activity during behavior and directly depolarizing inhibitory cells. Network modeling captured these findings, but only when modulation coincidently drove all three interneuron subtypes, ruling out either inhibition or disinhibition alone as sole mechanism for active engagement. Parallel processing of cholinergic modulation by cortical interneurons therefore enables context-dependent behavior.

Targeting the mevalonate cascade as a new therapeutic approach in heart disease, cancer and pulmonary disease.

PubMed

Yeganeh, Behzad; Wiechec, Emilia; Ande, Sudharsana R; Sharma, Pawan; Moghadam, Adel Rezaei; Post, Martin; Freed, Darren H; Hashemi, Mohammad; Shojaei, Shahla; Zeki, Amir A; Ghavami, Saeid

2014-07-01

The cholesterol biosynthesis pathway, also known as the mevalonate (MVA) pathway, is an essential cellular pathway that is involved in diverse cell functions. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) is the rate-limiting step in cholesterol biosynthesis and catalyzes the conversion of HMG-CoA to MVA. Given its role in cholesterol and isoprenoid biosynthesis, the regulation of HMGCR has been intensely investigated. Because all cells require a steady supply of MVA, both the sterol (i.e. cholesterol) and non-sterol (i.e. isoprenoid) products of MVA metabolism exert coordinated feedback regulation on HMGCR through different mechanisms. The proper functioning of HMGCR as the proximal enzyme in the MVA pathway is essential under both normal physiologic conditions and in many diseases given its role in cell cycle pathways and cell proliferation, cholesterol biosynthesis and metabolism, cell cytoskeletal dynamics and stability, cell membrane structure and fluidity, mitochondrial function, proliferation, and cell fate. The blockbuster statin drugs ('statins') directly bind to and inhibit HMGCR, and their use for the past thirty years has revolutionized the treatment of hypercholesterolemia and cardiovascular diseases, in particular coronary heart disease. Initially thought to exert their effects through cholesterol reduction, recent evidence indicates that statins also have pleiotropic immunomodulatory properties independent of cholesterol lowering. In this review we will focus on the therapeutic applications and mechanisms involved in the MVA cascade including Rho GTPase and Rho kinase (ROCK) signaling, statin inhibition of HMGCR, geranylgeranyltransferase (GGTase) inhibition, and farnesyltransferase (FTase) inhibition in cardiovascular disease, pulmonary diseases (e.g. asthma and chronic obstructive pulmonary disease (COPD)), and cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Binding and inhibition of Cdc25 phosphatases by vitamin K analogues.

PubMed

Kar, Siddhartha; Lefterov, Iliya M; Wang, Meifang; Lazo, John S; Scott, Colleen N; Wilcox, Craig S; Carr, Brian I

2003-09-09

A synthetic K vitamin analogue, 2-(2-mercaptothenol)-3-methyl-1,4-naphthoquinone or Cpd 5, was previously found to be a potent inhibitor of cell growth [Nishikawa et al., (1995) J. Biol. Chem. 270, 28304-28310]. The mechanisms of cell growth were hypothesized to include the inactivation of cellular protein tyrosine phosphatases, especially the Cdc25 family [Tamura et al. (2000) Cancer Res. 60, 1317-1325]. In this study, we synthesized PD 49, a new biotin containing Cpd 5 derivative, to search for evidence of direct interaction of these arylating analogues with Cdc25A, Cdc25B, and Cdc25C phosphatases. PD 49 was shown to directly bind to GST-Cdc25A, GST-Cdc25B, their catalytic fragments, and GST-Cdc25C. The binding could be competed with excess glutathione or Cpd 5, and a cysteine-to-serine mutation of the catalytic cysteine abolished binding. This was consistent with an involvement in binding of cysteine in the catalytic domain. This interaction between PD 49 and Cdc25 also occurred in lysates of treated cells. PD 49 also bound to protein phosphatases other than Cdc25. We found that the new analogue also inhibited Hep3B human hepatoma cell growth. This growth inhibition involved ERK1/2 phosphorylation and was inhibited by a MEK antagonist. The results demonstrate a direct interaction and binding between this growth-inhibiting K vitamin derivative with both purified as well as with cellular Cdc25A, Cdc25B, and Cdc25C.

Targeting GLI by GANT61 involves mechanisms dependent on inhibition of both transcription and DNA licensing.

PubMed

Zhang, Ruowen; Wu, Jiahui; Ferrandon, Sylvain; Glowacki, Katie J; Houghton, Janet A

2016-12-06

The GLI genes are transcription factors and in cancers are oncogenes, aberrantly and constitutively activated. GANT61, a specific GLI inhibitor, has induced extensive cytotoxicity in human models of colon cancer. The FOXM1 promoter was determined to be a transcriptional target of GLI1. In HT29 cells, inhibition of GLI1 binding at the GLI consensus sequence by GANT61 led to inhibited binding of Pol II, the pause-release factors DSIF, NELF and p-TEFb. The formation of R-loops (RNA:DNA hybrids, ssDNA), were reduced by GANT61 at the FOXM1 promoter. Pretreatment of HT29 cells with α-amanitin reduced GANT61-induced γH2AX foci. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication to be linked. By co-immunoprecipitation and confocal microscopy, GLI1 co-localized with the DNA licensing factors ORC4, CDT1, and MCM2. Significant co-localization of GLI1 and ORC4 was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. CDT1 was found to be a transcription target of GLI1. Overexpression of CDT1 in HT29 and SW480 cells reduced GANT61-induced cell death, gH2AX foci, and cleavage of caspase-3. Data demonstrate involvement of transcription and of DNA replication licensing factors by non-transcriptional and transcriptional mechanisms in the GLI-dependent mechanism of action of GANT61.

Dimethyl phenyl piperazine iodide (DMPP) induces glioma regression by inhibiting angiogenesis

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

He, Yan-qing; Li, Yan; Wang, Xiao-yu

1,1-Dimethyl-4-phenyl piperazine iodide (DMPP) is a synthetic nicotinic acetylcholine receptor (nAChR) agonist that could reduce airway inflammation. In this study, we demonstrated that DMPP could dramatically inhibit glioma size maintained on the chick embryonic chorioallantoic membrane (CAM). We first performed MTT and BrdU incorporation experiments on U87 glioma cells in vitro to understand the mechanism involved. We established that DMPP did not significantly affect U87 cell proliferation and survival. We speculated that DMPP directly caused the tumor to regress by affecting the vasculature in and around the implanted tumor on our chick CAM model. Hence, we conducted detailed analysis ofmore » DMPP's inhibitory effects on angiogenesis. Three vasculogenesis and angiogenesis in vivo models were used in the study which included (1) early chick blood islands formation, (2) chick yolk-sac membrane (YSW) and (3) CAM models. The results revealed that DMPP directly suppressed all developmental stages involved in vasculogenesis and angiogenesis – possibly by acting through Ang-1 and HIF-2α signaling. In sum, our results show that DMPP could induce glioma regression grown on CAM by inhibiting vasculogenesis and angiogenesis. - Highlights: ●We demonstrated that DMPP inhibited the growth of glioma cells on chick CAM. ●DMPP did not significantly affect the proliferation and survival of U87 cells. ●We revealed that DMPP suppressed vasculogenesis and angiogenesis in chick embryo. ●Angiogenesis in chick CAM was inhibited by DMPP via most probably Ang-1 and HIF-2α. ●DMPP could be potentially developed as an anti-tumor drug in the future.« less

Follistatin induces muscle hypertrophy through satellite cell proliferation and inhibition of both myostatin and activin.

PubMed

Gilson, Hélène; Schakman, Olivier; Kalista, Stéphanie; Lause, Pascale; Tsuchida, Kunihiro; Thissen, Jean-Paul

2009-07-01

Follistatin (FS) inhibits several members of the TGF-beta superfamily, including myostatin (Mstn), a negative regulator of muscle growth. Mstn inhibition by FS represents a potential therapeutic approach of muscle atrophy. The aim of our study was to investigate the mechanisms of the FS-induced muscle hypertrophy. To test the role of satellite cells in the FS effect, we used irradiation to destroy their proliferative capacity. FS overexpression increased the muscle weight by about 37% in control animals, but the increase reached only 20% in irradiated muscle, supporting the role of cell proliferation in the FS-induced hypertrophy. Surprisingly, the muscle hypertrophy caused by FS reached the same magnitude in Mstn-KO as in WT mice, suggesting that Mstn might not be the only ligand of FS involved in the regulation of muscle mass. To assess the role of activin (Act), another FS ligand, in the FS-induced hypertrophy, we electroporated FSI-I, a FS mutant that does not bind Act with high affinity. Whereas FS electroporation increased muscle weight by 32%, the muscle weight gain induced by FSI-I reached only 14%. Furthermore, in Mstn-KO mice, FSI-I overexpression failed to induce hypertrophy, in contrast to FS. Therefore, these results suggest that Act inhibition may contribute to FS-induced hypertrophy. Finally, the role of Act as a regulator of muscle mass was supported by the observation that ActA overexpression induced muscle weight loss (-15%). In conclusion, our results show that satellite cell proliferation and both Mstn and Act inhibition are involved in the FS-induced muscle hypertrophy.

Lipotoxicity Mediated Cell Dysfunction and Death Involves Lysosomal Membrane Permeabilization and Cathepsin L Activity

PubMed Central

Almaguel, Frankis G.; Liu, Jo-Wen; Pacheco, Fabio J.; De Leon, Daisy; Casiano, Carlos A.; De Leon, Marino

2010-01-01

Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis, and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity, and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity. PMID:20043885

Withaferin A inhibits experimental epithelial-mesenchymal transition in MCF-10A cells and suppresses vimentin protein level in vivo in breast tumors.

PubMed

Lee, Joomin; Hahm, Eun-Ryeong; Marcus, Adam I; Singh, Shivendra V

2015-06-01

We have shown previously that withaferin A (WA), a bioactive component of the medicinal plant Withania somnifera, inhibits growth of cultured and xenografted human breast cancer cells and prevents breast cancer development and pulmonary metastasis incidence in a transgenic mouse model. The present study was undertaken to determine if the anticancer effect of WA involved inhibition of epithelial-mesenchymal transition (EMT). Experimental EMT induced by exposure of MCF-10A cells to tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β) was partially reversed by treatment with WA but not by its structural analogs withanone or withanolide A. Combined TNF-α and TGF-β treatments conferred partial protection against MCF-10A cell migration inhibition by WA. Inhibition of TNF-α and TGF-β-induced MCF-10A cell migration by WA exposure was modestly attenuated by knockdown of E-cadherin protein. MCF-7 and MDA-MB-231 cells exposed to WA exhibited sustained (MCF-7) or transient (MDA-MB-231) induction of E-cadherin protein. On the other hand, the level of vimentin protein was increased markedly after 24 h treatment of MDA-MB-231 cells with WA. WA-induced apoptosis was not affected by vimentin protein knockdown in MDA-MB-231 cells. Protein level of vimentin was significantly lower in the MDA-MB-231 xenografts as well as in MMTV-neu tumors from WA-treated mice compared with controls. The major conclusions of the present study are that (a) WA treatment inhibits experimental EMT in MCF-10A cells, and (b) mammary cancer growth inhibition by WA administration is associated with suppression of vimentin protein expression in vivo. © 2013 Wiley Periodicals, Inc.

Irisin suppresses the migration, proliferation, and invasion of lung cancer cells via inhibition of epithelial-to-mesenchymal transition

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

Shao, Lei; Jinan Central Hospital Affiliated to Shandong University, Jinan, 250012; Li, Huanjie

Irisin is involved in promoting metabolism, immune regulation, and affects chronic inflammation in many systemic diseases, including gastric cancer. However, the role of irisin in lung cancer is not well characterized. To determine whether irisin has a protective effect against lung cancer, we cultured A549 and NCI-H446 lung cancer cells and treated them with irisin. We detected the proliferation by MTT assay, and assessed the migration and invasion of the cells by scratch wound healing assay and Tran-swell assay. The expression levels of epithelial-to-mesenchymal transition (EMT) markers and the related signaling pathways were detected by western blot analysis. Meanwhile, anmore » inhibitor of PI3K was used to investigate the effect of irsin. Finally, the expression of Snail was detected. We demonstrated that irisin inhibits the proliferation, migration, and invasion of lung cancer cells, and has a novel role in mediating the PI3K/AKT pathway in the cells. Irisin can reverse the activity of EMT and inhibit the expression of Snail via mediating the PI3K/AKT pathway, which is a key regulator of Snail. These results revealed that irisin inhibited EMT and reduced the invasion of lung cancer cells via the PI3K/AKT/Snail pathway. - Highlights: • Irisin inhibits the proliferation of lung cancer cells. • Irisin inhibits the migration and invasion of lung cancer cells. • Irisin affects the expression of EMT markers via inhibiting the PI3K/AKT pathway in lung cancer cells. • Irisin induces Snail downregulation via PI3K/AKT pathway activation.« less

Inhibition of GRP78 abrogates radioresistance in oropharyngeal carcinoma cells after EGFR inhibition by cetuximab.

PubMed

Sun, Chaonan; Han, Chuyang; Jiang, Yuanjun; Han, Ning; Zhang, Miao; Li, Guang; Qiao, Qiao

2017-01-01

The EGFR-specific mAb cetuximab is one of the most effective treatments for oropharyngeal carcinoma, while patient responses to EGFR inhibitors given alone are modest. Combination treatment with radiation can improve the efficacy of treatment through increasing radiosensitivity, while resistance to radiation after administration of cetuximab limits its efficiency. Radiation and drugs can damage the endoplasmic reticulum (ER) homeostatic state and result in ER stress (ERS), subsequently causing resistance to radiation and drugs. Whether the ERS pathway is involved in radioresistance after administration of cetuximab has not been reported. Herein, we show that cetuximab could increase the radiosensitivity of FaDu cells but not Detroit562 cells. In addition, cetuximab inhibited the radiation-induced activation of the ERS signalling pathway IRE1α/ATF6-GRP78 in FaDu cells, while this effect was absent in Detroit562 cells. Silencing GRP78 increased the radiosensitivity of oropharyngeal carcinoma cells and inhibited radiation-induced DNA double-strand-break (DSB) repair and autophagy. More interestingly, silencing GRP78 abrogated resistance to cetuximab and radiation in Detroit562 cells and had a synergistic effect with cetuximab in increasing the radiosensitivity of FaDu cells. Immunohistochemistry showed that overexpression of both GRP78 and EGFR was associated with a poor prognosis in oropharyngeal carcinoma patients (P<0.05). Overall, the results of this study show that radioresistance after EGFR inhibition by cetuximab is mediated by the ERS signalling pathway IRE1α/ATF6-GRP78. This suppression was consequently unable to inhibit radiation-induced DSB repair and autophagy in oropharyngeal carcinoma cells, which conferred resistance to radiotherapy and cetuximab. These results suggest that the cooperative effects of radiotherapy and cetuximab could be further improved by inhibiting GRP78 in non-responsive oropharyngeal carcinoma patients.

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation

PubMed Central

Planas, Delphine; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R. M.

2017-01-01

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation. PMID:28727807

Asparagine deprivation mediated by Salmonella asparaginase causes suppression of activation-induced T cell metabolic reprogramming.

PubMed

Torres, AnnMarie; Luke, Joanna D; Kullas, Amy L; Kapilashrami, Kanishk; Botbol, Yair; Koller, Antonius; Tonge, Peter J; Chen, Emily I; Macian, Fernando; van der Velden, Adrianus W M

2016-02-01

Salmonellae are pathogenic bacteria that induce immunosuppression by mechanisms that remain largely unknown. Previously, we showed that a putative type II l-asparaginase produced by Salmonella Typhimurium inhibits T cell responses and mediates virulence in a murine model of infection. Here, we report that this putative L-asparaginase exhibits L-asparagine hydrolase activity required for Salmonella Typhimurium to inhibit T cells. We show that L-asparagine is a nutrient important for T cell activation and that L-asparagine deprivation, such as that mediated by the Salmonella Typhimurium L-asparaginase, causes suppression of activation-induced mammalian target of rapamycin signaling, autophagy, Myc expression, and L-lactate secretion. We also show that L-asparagine deprivation mediated by the Salmonella Typhimurium L-asparaginase causes suppression of cellular processes and pathways involved in protein synthesis, metabolism, and immune response. Our results advance knowledge of a mechanism used by Salmonella Typhimurium to inhibit T cell responses and mediate virulence, and provide new insights into the prerequisites of T cell activation. We propose a model in which l-asparagine deprivation inhibits T cell exit from quiescence by causing suppression of activation-induced metabolic reprogramming. © Society for Leukocyte Biology.

Selected Phytochemicals and Culinary Plant Extracts Inhibit Fructose Uptake in Caco-2 Cells.

PubMed

Lee, Yurim; Lim, Yeni; Kwon, Oran

2015-09-18

This study compared the ability of nine culinary plant extracts containing a wide array of phytochemicals to inhibit fructose uptake and then explored the involvement of intestinal fructose transporters and phytochemicals for selected samples. The chemical signature was characterized by high performance liquid chromatography with mass spectrometry. Inhibition of [(14)C]-fructose uptake was tested by using human intestinal Caco-2 cells. Then, the relative contribution of the two apical-facing intestinal fructose transporters, GLUT2 and GLUT5, and the signature components for fructose uptake inhibition was confirmed in naive, phloretin-treated and forskolin-treated Caco-2 cells. HPLC/MS analysis of the chemical signature revealed that guava leaf contained quercetin and catechin, and turmeric contained curcumin, bisdemethoxycurcumin and dimethoxycurcumin. Similar inhibition of fructose uptake (by ~50%) was observed with guava leaf and turmeric in Caco-2 cells, but with a higher contribution of GLUT2 for turmeric and that of GLUT5 for guava leaf. The data suggested that, in turmeric, demethoxycurcumin specifically contributed to GLUT2-mediated fructose uptake inhibition, and curcumin did the same to GLUT5-mediated fructose uptake inhibition, but GLUT2 inhibition was more potent. By contrast, in guava leaf, catechin specifically contributed to GLUT5-mediated fructose uptake inhibition, and quercetin affected both GLUT5- and GLUT2-mediated fructose uptake inhibition, resulting in the higher contribution of GLUT5. These results suggest that demethoxycurcumin is an important contributor to GLUT2-mediated fructose uptake inhibition for turmeric extract, and catechin is the same to GLUT5-mediated fructose uptake inhibition for guava leaf extract. Quercetin, curcumin and bisdemethoxycurcumin contributed to both GLUT5- and GLUT2-mediated fructose uptake inhibition, but the contribution to GLUT5 inhibition was higher than the contribution to GLUT2 inhibition.

Role of ERK1/2 kinase in the expression of iNOS by NDMA in human neutrophils.

PubMed

Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Garley, Marzena; Jablonski, Jakub; Radziwon, Piotr

2013-01-01

Potential role of ERK1/2 kinase in conjunction with p38 in the regulation of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and superoxide anion generation by human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was determined. Increased synthesis of NO due to the involvement of iNOS in neutrophils exposed to NDMA was observed. In addition, intensified activation of ERK1/2 and p38 kinases was determined in these cells. Inhibition of kinase regulated by extracellular signals (ERK1/2) pathway, in contrast to p38 pathway, led to an increased production of NO and expression of iNOS in PMNs. Moreover, as a result of inhibition of ERK1/2 pathway, a decreased activation of p38 kinase was observed in neutrophils, while inhibition of p38 kinase did not affect activation of ERK1/2 pathway in these cells. An increased ability to release superoxide anion by the studied PMNs was observed, which decreased after ERK1/2 pathway inhibition. In conclusion, in human neutrophils, ERK1/2 kinase is not directly involved in the regulation of iNOS and NO production induced by NDMA; however, the kinase participates in superoxide anion production in these cells.

Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-γ.

PubMed

Pahl, Jens H W; Kwappenberg, Kitty M C; Varypataki, Eleni M; Santos, Susy J; Kuijjer, Marieke L; Mohamed, Susan; Wijnen, Juul T; van Tol, Maarten J D; Cleton-Jansen, Anne-Marie; Egeler, R Maarten; Jiskoot, Wim; Lankester, Arjan C; Schilham, Marco W

2014-03-10

In osteosarcoma, the presence of tumor-infiltrating macrophages positively correlates with patient survival in contrast to the negative effect of tumor-associated macrophages in patients with other tumors. Liposome-encapsulated muramyl tripeptide (L-MTP-PE) has been introduced in the treatment of osteosarcoma patients, which may enhance the potential anti-tumor activity of macrophages. Direct anti-tumor activity of human macrophages against human osteosarcoma cells has not been described so far. Hence, we assessed osteosarcoma cell growth after co-culture with human macrophages. Monocyte-derived M1-like and M2-like macrophages were polarized with LPS + IFN-γ, L-MTP-PE +/- IFN-γ or IL-10 and incubated with osteosarcoma cells. Two days later, viable tumor cell numbers were analyzed. Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab. M1-like macrophages inhibited osteosarcoma cell growth when activated with LPS + IFN-γ. Likewise, stimulation of M1-like macrophages with liposomal muramyl tripeptide (L-MTP-PE) inhibited tumor growth, but only when combined with IFN-γ. Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages. The inhibition was mediated by supernatants of activated M1-like macrophages, containing TNF-α and IL-1β. However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation. While LPS + IFN-γ-activated M2-like macrophages had low anti-tumor activity, IL-10-polarized M2-like macrophages were able to reduce osteosarcoma cell growth in the presence of the anti-EGFR cetuximab involving antibody-dependent tumor cell phagocytosis. This study demonstrates that human macrophages can be induced to exert direct anti-tumor activity against osteosarcoma cells. Our observation that the induction of macrophage anti-tumor activity by L-MTP-PE required IFN-γ may be of relevance for the optimization of L-MTP-PE therapy in osteosarcoma patients.

Characterization of putative toxin/antitoxin systems in Vibrio parahaemolyticus.

PubMed

Hino, M; Zhang, J; Takagi, H; Miyoshi, T; Uchiumi, T; Nakashima, T; Kakuta, Y; Kimura, M

2014-07-01

To obtain more information about the toxin/antitoxin (TA) systems in the Vibrio genus and also to examine their involvement in the induction of a viable but nonculturable (VBNC) state, we searched homologues of the Escherichia coli TA systems in the Vibrio parahaemolyticus genome. We found that a gene cluster, vp1842/vp1843, in the V. parahaemolyticus genome database has homology to that encoding the E. coli TA proteins, DinJ/YafQ. Expression of the putative toxin gene vp1843 in E. coli cells strongly inhibited the cell growth, while coexpression with the putative antitoxin gene vp1842 neutralized this effect. Mutational analysis identified Lys37 and Pro45 in the gene product VP1843 of vp1843 as crucial residues for the growth retardation of E. coli cells. VP1843, unlike the E. coli toxin YafQ, has no protein synthesis inhibitory activity, and that instead the expression of vp1843 in E. coli caused morphological change of the cells. The gene cluster vp1842/vp1843 encodes the V. parahaemolyticus TA system; VP1843 inhibits cell growth, whereas VP1842 serves as an antitoxin by forming a stable complex with VP1843. The putative toxin, VP1843, may be involved in the induction of the VBNC state in V. parahaemolyticus by inhibiting cell division. © 2014 The Society for Applied Microbiology.

Detection of Necroptosis in Ligand-Mediated and Hypoxia-Induced Injury of Hepatocytes Using a Novel Optic Probe Detecting Receptor-Interacting Protein (RIP)1/RIP3 Binding.

PubMed

Haga, Sanae; Kanno, Akira; Ozawa, Takeaki; Morita, Naoki; Asano, Mami; Ozaki, Michitaka

2017-07-21

Liver injury is often observed in various pathological conditions including posthepatectomy state and cancer chemotherapy. It occurs mainly as a consequence of the combined necrotic and apoptotic types of cell death. In order to study liver/hepatocyte injury by necrotic type of cell death, we studied signal-regulated necrosis (necroptosis) by newly developing an optic probe detecting receptor-interacting protein (RIP)1/RIP3 binding, an essential process for necroptosis induction. In the mouse hepatocyte cell line, TIB-73 cells, TNF-a/cycloheximide (T/C) induced RIP1/3 binding only when caspase activity was suppressed by z-VAD-fmk (zVAD), a caspase-specific inhibitor. T/C/zVADinduced RIP1/3-binding was inhibited by necrostatin-1 (Nec-1), an allosteric inhibitor of RIP1. The reduced cell survival by T/C/zVAD was improved by Nec-1. These facts indicate that T/C induces necroptosis of hepatocytes when apoptotic pathway is inhibited/unavailable. FasL also induced cell death which was only partially inhibited by zVAD, indicating the possible involvement of necroptosis other than apoptosis. FasL activated caspase-3 and, similarly, induced RIP1/3-binding when caspases were inactivated. Interestingly, FasL-induced RIP1/3 binding was significantly suppressed by the antioxidants, Trolox and N-acetyl cysteine (NAC), suggesting the involvement of reactive oxygen species (ROS) in FasL-induced necroptotic cellular processes. H₂O₂, by itself, induced RIP1/3 binding that was suppressed by Nec-1, but not by zVAD. Hypoxia induced RIP1/3 binding after reoxygenation, which was suppressed by Nec-1 or by the antioxidants. Cell death induced by hypoxia/reoxygenation (H/R) was also improved by Nec-1. Similar to H₂O₂, H/R did not require caspase inhibition for RIP1/3 binding, suggesting the involvement of a caspase-independent mechanism for non-ligand induced and/or redox-mediated necroptosis. These data indicate that ROS induce necroptosis, and mediate the FasL- and hypoxia-induced necroptosis via a molecular mechanism that differs from a conventional caspase-dependent pathway. In conclusion, necroptosis is potentially involved in liver/hepatocyte injury induced by oxidative stress and FasL, other than apoptosis.

Insights into the etiology-associated gene regulatory networks in hepatocellular carcinoma from The Cancer Genome Atlas.

PubMed

Seshachalam, Veerabrahma Pratap; Sekar, Karthik; Hui, Kam M

2018-04-19

Hepatitis B virus, hepatitis C virus, alcoholic consumption and non-alcoholic fatty liver are the major known risk factors for Hepatocellular carcinoma (HCC). There have been very few studies comparing the underlying biological mechanisms associated with the different etiologies of HCC. In this study, we hypothesized the existence of different regulatory networks associated with different liver disease etiologies involved in hepatocarcinogenesis. Using upstream regulatory analysis tool in ingenuity pathway analysis software, URs were predicted using differential expressed genes for HCC to facilitate the interrogation of global gene regulation. Analysis of regulatory networks for HBV HCC revealed E2F1 as activated UR, regulating genes involved in cell cycle and DNA replication and HNF4A and HNF1A as inhibited UR. In HCV HCC, IFNG, involved in cellular movement and signaling was activated while IL1RN, MAPK1 involved in IL-22 signaling and immune response was inhibited. In Alcoholic-consumption HCC, ERBB2 involved in inflammatory response and cellular movement was activated, whereas HNF4A, NUPR1 were inhibited. For HCC derived from Non-alcoholic fatty liver disease, miR-1249-5p was activated and NUPR1 involved in cell cycle and apoptosis was inhibited. The prognostic value of representative genes identified in the regulatory networks for HBV HCC can be further validated by an independent HBV HCC dataset established in our laboratory with survival data. Our study identified functionally distinct candidate URs for HCC developed from different etiologic risk factors. Further functional validation studies of these regulatory networks could facilitate the management of HCC towards personalized medicine. This article is protected by copyright. All rights reserved.

Intravenous transplantation of mesenchymal stromal cells has therapeutic effects in a sepsis mouse model through inhibition of septic natural killer cells.

PubMed

Liu, Wenhua; Gao, Yang; Li, Haibo; Wang, Hongliang; Ye, Ming; Jiang, Guihua; Chen, Yongsheng; Liu, Yang; Kong, Junying; Liu, Wei; Sun, Meng; Hou, Meng; Yu, Kaijiang

2016-10-01

Transplantation of mesenchymal stromal cells is a promising strategy for treating sepsis. Natural killer cells are important in the development of sepsis, and their functions can be inhibited by mesenchymal stromal cells, we asked whether mesenchymal stromal cells exert their therapeutic effects through inhibiting the functions of natural killer cells in a septic mouse model generated with cecal ligation puncture method. Using co-cultures of cells, small interfering RNA, enzyme-linked immnuosorbent assays, fluorescence assays, western blotting, and pathological examination, we investigated the levels of inflammatory cytokines, proliferation of natural killer cells, inflammatory infiltration of important organs in mice, and activity of the Janus kinase/signal transducer and activator of transcription signaling pathway and found that mesenchymal stromal cells inhibited the function and proliferation of septic natural killer cells, increased interleukin-10 levels and increased the expression of components, such as Janus kinase 1, Janus kinase 2, and signal transducer and activator of transcription 3 in the Janus kinase/signal transducer and activator of transcription pathway both in vitro and in vivo. We conclude that mesenchymal stromal cells have their therapeutic effect in the septic mouse model through inhibiting the function and proliferation of septic natural killer cells. This biological process may involve interleukin-10 and suppressor of cytokine signaling 3 as well as other pathway components in the Janus kinase/signal transducer and activator of transcription pathway. Transplantation of mesenchymal stromal cells is an effective strategy to treat sepsis. Copyright © 2016. Published by Elsevier Ltd.

Nitric oxide-mediated inhibition of taurocholate uptake involves S-nitrosylation of NTCP.

PubMed

Schonhoff, Christopher M; Ramasamy, Umadevi; Anwer, M Sawkat

2011-02-01

The sodium-taurocholate (TC) cotransporting polypeptide (NTCP) facilitates bile formation by mediating sinusoidal Na(+)-TC cotransport. During sepsis-induced cholestasis, there is a decrease in NTCP-dependent uptake of bile acids and an increase in nitric oxide (NO) levels in hepatocytes. In rat hepatocytes NO inhibits Na(+)-dependent uptake of taurocholate. The aim of this study was to extend these findings to human NTCP and to further investigate the mechanism by which NO inhibits TC uptake. Using a human hepatoma cell line stably expressing NTCP (HuH-NTCP), we performed experiments with the NO donors sodium nitroprusside and S-nitrosocysteine and demonstrated that NO inhibits TC uptake in these cells. Kinetic analyses revealed that NO significantly decreased the V(max) but not the K(m) of TC uptake by NTCP, indicating noncompetitive inhibition. NO decreased the amount of NTCP in the plasma membrane, providing a molecular mechanism for the noncompetitive inhibition of TC uptake. One way that NO can modify protein function is through a posttranslational modification known as S-nitrosylation: the binding of NO to cysteine thiols. Using a biotin switch technique we observed that NTCP is S-nitrosylated under conditions in which NO inhibits TC uptake. Moreover, dithiothreitol reversed NO-mediated inhibition of TC uptake and S-nitrosylation of NTCP, indicating that NO inhibits TC uptake via modification of cysteine thiols. In addition, NO treatment led to a decrease in Ntcp phosphorylation. Taken together these results indicate that the inhibition of TC uptake by NO involves S-nitrosylation of NTCP.

Photodynamic treatment of herpes simplex virus during its replicative cycle.

PubMed Central

Khan, N C; Melnick, J L; Biswal, N

1977-01-01

Photodynamic treatment of herpes simplex virus type 1-infected hamster embryo fibroblasts (LSH strain) with a low concentration of proflavine (0.08 mug/10(5) cells per ml), a 3-9-diamine acridine dye, inhibited production not only of infectious progeny but also of virion particles. However, there was no appreciable inhibition of viral or cellular DNA synthesis, even when the infected cells were repeatedly exposed to this low concentration of dye and light during the replication cycle of the virus. It thus appears that photodynamic treatment of infected cells interferes with the processes involved in virus maturation. PMID:189063

AMPK inhibits MTDH expression via GSK3β and SIRT1 activation: potential role in triple negative breast cancer cell proliferation.

PubMed

Gollavilli, Paradesi Naidu; Kanugula, Anantha Koteswararao; Koyyada, Rajeswari; Karnewar, Santosh; Neeli, Praveen Kumar; Kotamraju, Srigiridhar

2015-10-01

Recent studies have highlighted the involvement of metadherin (MTDH), an oncogenic protein, in promoting cancer progression, metastasis and chemoresistance in many cancers including mammary carcinomas. However, the molecular regulation of MTDH is still not completely understood. In this study we document that AMP activated protein kinase (AMPK) activation-induced anti-proliferative effects are, in part, mediated by inhibiting MTDH expression in MDA-MB-231 and BT-549 triple negative breast cancer (TNBC) cells. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, caused growth arrest, inhibition of migration and invasion of TNBC cells. Intriguingly, AICAR or metformin treatment resulted in significant downregulation of MTDH expression via inhibiting c-Myc expression. In contrast, treatment of cells with compound C, an inhibitor of AMPK, increased both c-Myc and MTDH expressions in TNBC cells. Also, AMPK activation caused increased glycogen synthase kinase 3β (GSK3β) activity by inhibiting the inactive phosphorylation at Ser9, on the one hand, and activation of sirtuin1 (SIRT1) by inhibiting Ser47 phosphorylation, as evidenced by deacetylation of p53, on the other hand. Moreover, AMPK-induced GSK3β and SIRT1 activities were found to be responsible for inhibiting c-Myc-mediated upregulation of MTDH, as LiCl (an inhibitor of GSK3β) and EX-527 (an inhibitor of SIRT1) reversed AICAR-mediated downregulation of c-Myc and MTDH expressions. Similar results were observed with siSIRT1 treatment. Furthermore, AICAR and EX-527 treatments caused increased cell death under MTDH-depleted conditions. Finally, we uncovered a novel regulation of MTDH expression and showed that AMPK activation by inducing GSK3β and SIRT1 downregulates MTDH expression via inhibiting c-Myc in TNBC cells. © 2015 FEBS.

FXR blocks the growth of liver cancer cells through inhibiting mTOR-s6K pathway

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

Huang, Xiongfei, E-mail: xiongfeihuang@hotmail.com; Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, Fuzhou 350108, Fujian; Zeng, Yeting

The nuclear receptor Farnesoid X Receptor (FXR) is likely a tumor suppressor in liver tissue but its molecular mechanism of suppression is not well understood. In this study, the gene expression profile of human liver cancer cells was investigated by microarray. Bioinformatics analysis of these data revealed that FXR might regulate the mTOR/S6K signaling pathway. This was confirmed by altering the expression level of FXR in liver cancer cells. Overexpression of FXR prevented the growth of cells and induced cell cycle arrest, which was enhanced by the mTOR/S6K inhibitor rapamycin. FXR upregulation also intensified the inhibition of cell growth bymore » rapamycin. Downregulation of FXR produced the opposite effect. Finally, we found that ectopic expression of FXR in SK-Hep-1 xenografts inhibits tumor growth and reduces expression of the phosphorylated protein S6K. Taken together, our data provide the first evidence that FXR suppresses proliferation of human liver cancer cells via the inhibition of the mTOR/S6K signaling pathway. FXR expression can be used as a biomarker of personalized mTOR inhibitor treatment assessment for liver cancer patients. -- Highlights: •FXR inhibits the proliferation of liver cancer cells by prolonging G0/G1 phase. •Microarray results indicate that mTOR-S6k signaling is involved in cellular processes in which FXR plays an important role. •FXR blocks the growth of liver cancer cells via the inhibition of the mTOR/S6K signaling pathway in vitro and in vivo.« less

FOXO/TXNIP pathway is involved in the suppression of hepatocellular carcinoma growth by glutamate antagonist MK-801

PubMed Central

2013-01-01

Background Accumulating evidence has suggested the importance of glutamate signaling in cancer growth, yet the signaling pathway has not been fully elucidated. N-methyl-D-aspartic acid (NMDA) receptor activates intracellular signaling pathways such as the extracellular-signal-regulated kinase (ERK) and forkhead box, class O (FOXO). Suppression of lung carcinoma growth by NMDA receptor antagonists via the ERK pathway has been reported. However, series of evidences suggested the importance of FOXO pathways for the regulation of normal and cancer cell growth. In the liver, FOXO1 play important roles for the cell proliferation such as hepatic stellate cells as well as liver metabolism. Our aim was to investigate the involvement of the FOXO pathway and the target genes in the growth inhibitory effects of NMDA receptor antagonist MK-801 in human hepatocellular carcinoma. Methods Expression of NMDAR1 in cancer cell lines from different tissues was examined by Western blot. NMDA receptor subunits in HepG2, HuH-7, and HLF were examined by reverse transcriptase polymerase chain reaction (RT-PCR), and growth inhibition by MK-801 and NBQX was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of MK-801 on the cell cycle were examined by flow cytometry and Western blot analysis. Expression of thioredoxin-interacting protein (TXNIP) and p27 was determined by real-time PCR and Western blotting. Activation of the FOXO pathway and TXNIP induction were examined by Western blotting, fluorescence microscopy, Chromatin immunoprecipitation (ChIP) assay, and reporter gene assay. The effects of TXNIP on growth inhibition were examined using the gene silencing technique. Results NMDA receptor subunits were expressed in all cell lines examined, and MK-801, but not NBQX, inhibited cell growth of hepatocellular carcinomas. Cell cycle analysis showed that MK-801 induced G1 cell cycle arrest by down-regulating cyclin D1 and up-regulating p27. MK-801 dephosphorylated Thr24 in FOXO1 and induced its nuclear translocation, thus increasing transcription of TXNIP, a tumor suppressor gene. Knock-down of TXNIP ameliorated the growth inhibitory effects of MK-801. Conclusions Our results indicate that functional NMDA receptors are expressed in hepatocellular carcinomas and that the FOXO pathway is involved in the growth inhibitory effects of MK-801. This mechanism could be common in hepatocellular carcinomas examined, but other mechanisms such as ERK pathway could exist in other cancer cells as reported in lung carcinoma cells. Altered expression levels of FOXO target genes including cyclin D1 and p27 may contribute to the inhibition of G1/S cell cycle transition. Induction of the tumor suppressor gene TXNIP plays an important role in the growth inhibition by MK-801. Our report provides new evidence that FOXO-TXNIP pathway play a role in the inhibition of the hepatocellular carcinoma growth by MK-801. PMID:24112473

Brefeldin A Inhibits Cell-Free, De Novo Synthesis of Poliovirus

PubMed Central

Cuconati, Andrea; Molla, Akhteruzzaman; Wimmer, Eckard

1998-01-01

Brefeldin A (BFA), an inhibitor of intracellular vesicle-dependent secretory transport, is a potent inhibitor of poliovirus RNA replication in infected cells. We have determined that the unknown mechanism of BFA inhibition of replication is reproduced in the cell-free poliovirus translation, replication, and encapsidation system. Furthermore, we provide evidence suggesting that the cellular mechanism targeted by BFA, the GTP-dependent synthesis of secretory transport vesicles, may be involved in viral RNA replication in the system via a soluble cellular GTP-binding and -hydrolyzing activity. This activity is related to the ARF (ADP-ribosylation factor) family of GTP-binding proteins. ARFs are required for the formation of several classes of secretory vesicles, and some family members are indirectly inactivated by BFA. Peptides that function as competitive inhibitors of ARF activity in cell-free transport systems also inhibit poliovirus RNA replication, and this inhibitory effect can be countered by the addition of exogenous ARF. We suggest that BFA inhibition of replication is diagnostic of a requirement for ARF activity in the cell-free system. PMID:9658088

Phase I Trial: Cirmtuzumab Inhibits ROR1 Signaling and Stemness Signatures in Patients with Chronic Lymphocytic Leukemia.

PubMed

Choi, Michael Y; Widhopf, George F; Ghia, Emanuela M; Kidwell, Reilly L; Hasan, Md Kamrul; Yu, Jian; Rassenti, Laura Z; Chen, Liguang; Chen, Yun; Pittman, Emily; Pu, Minya; Messer, Karen; Prussak, Charles E; Castro, Januario E; Jamieson, Catriona; Kipps, Thomas J

2018-06-01

Cirmtuzumab is a humanized monoclonal antibody (mAb) that targets ROR1, an oncoembryonic orphan receptor for Wnt5a found on cancer stem cells (CSCs). Aberrant expression of ROR1 is seen in many malignancies and has been linked to Rho-GTPase activation and cancer stem cell self-renewal. For patients with chronic lymphocytic leukemia (CLL), self-renewing, neoplastic B cells express ROR1 in 95% of cases. High-level leukemia cell expression of ROR1 is associated with an unfavorable prognosis. We conducted a phase 1 study involving 26 patients with progressive, relapsed, or refractory CLL. Patients received four biweekly infusions, with doses ranging from 0.015 to 20 mg/kg. Cirmtuzumab had a long plasma half-life and did not have dose-limiting toxicity. Inhibition of ROR1 signaling was observed, including decreased activation of RhoA and HS1. Transcriptome analyses showed that therapy inhibited CLL stemness gene expression signatures in vivo. Cirmtuzumab is safe and effective at inhibiting tumor cell ROR1 signaling in patients with CLL. Copyright © 2018. Published by Elsevier Inc.

Human mesenchymal stem cells target adhesion molecules and receptors involved in T cell extravasation.

PubMed

Benvenuto, Federica; Voci, Adriana; Carminati, Enrico; Gualandi, Francesca; Mancardi, Gianluigi; Uccelli, Antonio; Vergani, Laura

2015-12-10

Systemic delivery of bone marrow-derived mesenchymal stem cells (MSC) seems to be of benefit in the treatment of multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS) sustained by migration of T cells across the brain blood barrier (BBB) and subsequent induction of inflammatory lesions into CNS. MSC have been found to modulate several effector functions of T cells. In this study, we investigated the effects of MSC on adhesion molecules and receptors on T cell surface that sustain their transendothelial migration. We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate the expression both at the mRNA and at the plasma-membrane level of α4 integrin, β2 integrin, ICAM-1 and CXCR3. In parallel, we assessed if MSC are able to modulate expression of adhesion molecules on the endothelial cells that interact with T cells during their transendothelial migration. Our in vitro analyses revealed that MSC: (i) inhibit proliferation and activation of both peripheral blood mononuclear cells (PBMC) and CD3(+)-selected lymphocytes through the release of soluble factors; (ii) exert suppressive effects on those surface molecules highly expressed by activated lymphocytes and involved in transendothelial migration; (iii) inhibit CXCL10-driven chemotaxis of CD3(+) cells; (iv) down-regulated expression of adhesion molecules on endothelial cells. Taken together, these data demonstrate that the immunosuppressive effect of MSC does not exclusively depends on their anti-proliferative activity on T cells, but also on the impairment of leukocyte migratory potential through the inhibition of the adhesion molecules and receptors that are responsible for T cell trafficking across BBB. This could suggest a new mechanism through which MSC modulate T cell responses.

TSA-induced cell death in prostate cancer cell lines is caspase-2 dependent and involves the PIDDosome.

PubMed

Taghiyev, Agshin F; Guseva, Natalya V; Glover, Rebecca A; Rokhlin, Oskar W; Cohen, Michael B

2006-09-01

The histone deacetylase inhibitor Trichostatin A (TSA) has previously been found to induce caspase activity in the human prostate cancer cell lines DU145 and LNCaP. TSA treatment resulted in the release of cytochrome c and Smac/DIABLO from mitochondria in DU145, and activation of caspase-9 in both cell lines. We concluded that TSA mediated its effect via the mitochondrial pathway. The aim of the current study was to determine how TSA initiated the caspase cascade. The results revealed that caspase-2 plays an important role in TSA-induced apoptosis. Inhibition of caspase-2 by siRNA or expression of caspase-2dn substantially decreased caspase activity after TSA treatment in both cell lines, siRNA caspase-2 also inhibited TSA-induced cell death. Caspase-2 acts upstream of caspase-8 and -9 and mediates mitochondrial cytochrome c release. Coimmunoprecipitation experiments show that caspase-2 formed protein complexes with RADD/RAIDD and PIDD. Together, these data indicate that caspase-2 initiates caspase cascade after TSA treatment and involves the formation of the PIDDosome.

The role of MAPK signaling pathway in the Her-2-positive meningiomas

PubMed Central

Wang, Zhaoyin; Wang, Weijia; Xu, Shan; Wang, Shanshan; Tu, Yi; Xiong, Yifeng; Mei, Jinhong; Wang, Chunliang

2016-01-01

Meningiomas are common types of adult nerve system tumors. Although most cases are considered benign, due to its high rate of recurrence and easy malignant progression to anaplastic meningioma they present a puzzle for the current treatment. The HER-2 oncogene has important value for meningioma cells development and progression. So far, little is known about the effect on the exact underlying signal pathway and molecular mechanisms of HER-2-positive meningioma cells. The goal of the present study was to determine the effects of HER-2 gene and possible involvement of MAPK signal pathway in human malignant meningioma. We applied q-PCR analysis, immunofluorescence (IF) staining, western blot analysis, animal model, MAPK inhibition, MTT assay and cell invasion analysis for the investigation. The results demonstrated that the downregulation of the expression of HER-2 significantly inhibited cell motility and proliferation of human meningioma cells in vivo. Accordingly, in the HER-2-overexpression meningioma cells with the inhibition of ERK1/2, ERK5, JNK, in the cells with the ERK1/2, ERK5 inhibition, protein expression was markedly suppressed as well as the cell proliferation resistance. No difference was observed in the HER-2-overexpression meningioma cells with the inhibition of JNK. These findings suggest that HER-2 gene can affect the proliferation ability of human meningioma cells in vivo and MAPK signal pathway may contribute to the carcinogenesis and development of human meningiomas combinating with HER-2. PMID:27279438

Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator

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

Fitzpatrick, L.A.; Yasumoto, T.; Aurbach, G.D.

1989-01-01

Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivatesmore » a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release.« less

Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells

PubMed Central

Son, Yonghae; Kim, Bo-Young; Eo, Seong-Kug; Park, Young Chul; Kim, Koanhoi

2016-01-01

Oxysterol like 27-hydroxycholesterol (27OHChol) has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx) affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells. PMID:27340507

Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells.

PubMed

Son, Yonghae; Kim, Bo-Young; Eo, Seong-Kug; Park, Young Chul; Kim, Koanhoi

2016-01-01

Oxysterol like 27-hydroxycholesterol (27OHChol) has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx) affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells.

Escin suppresses migration and invasion involving the alteration of CXCL16/CXCR6 axis in human gastric adenocarcinoma AGS cells.

PubMed

Lee, Hyun Sook; Hong, Ji Eun; Kim, Eun Ji; Kim, Sun Hyo

2014-01-01

Escin, a natural mixture of triterpene saponins isolated from horse chestnut, has been reported to possess anticancer activity in many human cancer cells. However, the effect of escin on the metastasis has not been studied. The present study examined the effect of escin on the migration and invasion of AGS human gastric cancer cells. To examine the effects of escin on metastatic capacities of gastric cancer cells, AGS cells were cultured in the presence of 0-4 μmol/L escin. Escin inhibited cell migration and invasion in AGS cells. However, escin did not affect the viability of these cells at these concentrations. The chemokine receptor and its ligands play an important role in cancer metastasis. Escin decreased the production of soluble C-X-C motif chemokine (CXCL)16 but increased the expression of trans-membranous CXCL16. The expression of C-X-C chemokine receptor (CXCR)6 was not affected by escin treatment. Exogenous CXCL16 reversed escin-induced migration inhibition. In addition, escin inhibited the phosphorylation of focal adhesion kinase and Akt. These results demonstrate that escin inhibited the migration and invasion of AGS cells, which is associated with altered CXCL16/CXCR6 axis. These findings suggest that escin has potential as an antimetastatic agent in gastric cancer.

Inhibition of Notch-1 pathway is involved in rottlerin-induced tumor suppressive function in nasopharyngeal carcinoma cells

PubMed Central

Hou, Yingying; Feng, Shaoyan; Wang, Lixia; Zhao, Zhe; Su, Jingna; Yin, Xuyuan; Zheng, Nana; Zhou, Xiuxia; Xia, Jun; Wang, Zhiwei

2017-01-01

Recent studies have revealed that rottlerin is a natural chemical drug to exert its anti-cancer activity. However, the molecular mechanisms of rottlerin-induced tumor suppressive function have not been fully elucidated. Notch signaling pathway has been characterized to play a crucial role in tumorigenesis. Therefore, regulation of Notch pathway could be beneficial for the treatment of human cancer. The aims of our current study were to explore whether rottlerin could suppress Notch-1 expression, which leads to inhibition of cell proliferation, migration and invasion in nasopharyngeal carcinoma cells. We performed several approaches, such as CTG, Flow cytometry, scratch healing assay, transwell and Western blotting. Our results showed that rottlerin treatment inhibited cell growth, migration and invasion, and triggered apoptosis, and arrested cell cycle to G1 phase. Moreover, the expression of Notch-1 was obvious decreased in nasopharyngeal carcinoma cells after rottlerin treatment. Importantly, overexpression of Notch-1 promoted cell growth and invasion, whereas down-regulation of Notch-1 inhibited cell growth and invasion in nasopharyngeal carcinoma cells. Notably, we found the over-expression of Notch-1 could abrogate the anti-cancer function induced by rottlerin. Strikingly, our study implied that Notch-1 could be a useful target of rottlerin for the prevention and treatment of human nasopharyngeal carcinoma. PMID:28977931

MicroRNA expression profile and functional analysis reveal their roles in contact inhibition and its disruption switch of rat vascular smooth muscle cells.

PubMed

Sun, Ye-Ying; Qin, Shan-Shan; Cheng, Yun-Hui; Wang, Chao-Yun; Liu, Xiao-Jun; Liu, Ying; Zhang, Xiu-Li; Zhang, Wendy; Zhan, Jia-Xin; Shao, Shuai; Bian, Wei-Hua; Luo, Bi-Hui; Lu, Dong-Feng; Yang, Jian; Wang, Chun-Hua; Zhang, Chun-Xiang

2018-05-01

Contact inhibition and its disruption of vascular smooth muscle cells (VSMCs) are important cellular events in vascular diseases. But the underlying molecular mechanisms are unclear. In this study we investigated the roles of microRNAs (miRNAs) in the contact inhibition and its disruption of VSMCs and the molecular mechanisms involved. Rat VSMCs were seeded at 30% or 90% confluence. MiRNA expression profiles in contact-inhibited confluent VSMCs (90% confluence) and non-contact-inhibited low-density VSMCs (30% confluence) were determined. We found that multiple miRNAs were differentially expressed between the two groups. Among them, miR-145 was significantly increased in contact-inhibited VSMCs. Serum could disrupt the contact inhibition as shown by the elicited proliferation of confluent VSMCs. The contact inhibition disruption accompanied with a down-regulation of miR-145. Serum-induced contact inhibition disruption of VSMCs was blocked by overexpression of miR-145. Moreover, downregulation of miR-145 was sufficient to disrupt the contact inhibition of VSMCs. The downregulation of miR-145 in serum-induced contact inhibition disruption was related to the activation PI3-kinase/Akt pathway, which was blocked by the PI3-kinase inhibitor LY294002. KLF5, a target gene of miR-145, was identified to be involved in miR-145-mediated effect on VSMC contact inhibition disruption, as it could be inhibited by knockdown of KLF5. In summary, our results show that multiple miRNAs are differentially expressed in contact-inhibited VSMCs and in non-contact-inhibited VSMCs. Among them, miR-145 is a critical gene in contact inhibition and its disruption of VSMCs. PI3-kinase/Akt/miR-145/KLF5 is a critical signaling pathway in serum-induced contact inhibition disruption. Targeting of miRNAs related to the contact inhibition of VSMCs may represent a novel therapeutic approach for vascular diseases.

Ferroptosis is Involved in Acetaminophen Induced Cell Death.

PubMed

Lőrincz, Tamás; Jemnitz, Katalin; Kardon, Tamás; Mandl, József; Szarka, András

2015-09-01

The recently described form of programmed cell death, ferroptosis can be induced by agents causing GSH depletion or the inhibition of GPX4. Ferroptosis clearly shows distinct morphologic, biochemical and genetic features from apoptosis, necrosis and autophagy. Since NAPQI the highly reactive metabolite of the widely applied analgesic and antipyretic, acetaminophen induces a cell death which can be characterized by GSH depletion, GPX inhibition and caspase independency the involvement of ferroptosis in acetaminophen induced cell death has been investigated. The specific ferroptosis inhibitor ferrostatin-1 failed to elevate the viability of acetaminophen treated HepG2 cells. It should be noticed that these cells do not form NAPQI due to the lack of phase I enzyme expression therefore GSH depletion cannot be observed. However in the case of acetaminophen treated primary mouse hepatocytes the significant elevation of cell viability could be observed upon ferrostatin-1 treatment. Similar to ferrostatin-1 treatment, the addition of the RIP1 kinase inhibitor necrostatin-1 could also elevate the viability of acetaminophen treated primary hepatocytes. Ferrostatin-1 has no influence on the expression of CYP2E1 or on the cellular GSH level which suggest that the protective effect of ferrostatin-1 in APAP induced cell death is not based on the reduced metabolism of APAP to NAPQI or on altered NAPQI conjugation by cellular GSH. Our results suggest that beyond necroptosis and apoptosis a third programmed cell death, ferroptosis is also involved in acetaminophen induced cell death in primary hepatocytes.

Nicotine induces cell proliferation in association with cyclin D1 up-regulation and inhibits cell differentiation in association with p53 regulation in a murine pre-osteoblastic cell line

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

Sato, Tsuyoshi; Abe, Takahiro; Nakamoto, Norimichi

Recent studies have suggested that nicotine critically affects bone metabolism. Many studies have examined the effects of nicotine on proliferation and differentiation, but the underlying molecular mechanisms remain unclear. We examined cell cycle regulators involved in the proliferation and differentiation of MC3T3-E1 cells. Nicotine induced cell proliferation in association with p53 down-regulation and cyclin D1 up-regulation. In differentiated cells, nicotine reduced alkaline phosphatase activity and mineralized nodule formation in dose-dependent manners. Furthermore, p53 expression was sustained in nicotine-treated cells during differentiation. These findings indicate that nicotine promotes the cell cycle and inhibits differentiation in association with p53 regulation in pre-osteoblasticmore » cells.« less

Inhibition of Herpes Simplex Virus gD and Lymphotoxin-α Binding to HveA by Peptide Antagonists

PubMed Central

Sarrias, Maria Rosa; Whitbeck, J. Charles; Rooney, Isabelle; Spruce, Lynn; Kay, Brian K.; Montgomery, Rebecca I.; Spear, Patricia G.; Ware, Carl F.; Eisenberg, Roselyn J.; Cohen, Gary H.; Lambris, John D.

1999-01-01

The herpesvirus entry mediator A (HveA) is a recently characterized member of the tumor necrosis factor receptor family that mediates the entry of most herpes simplex virus type 1 (HSV-1) strains into mammalian cells. Studies on the interaction of HSV-1 with HveA have shown that of all the viral proteins involved in uptake, only gD has been shown to bind directly to HveA, and this binding mediates viral entry into cells. In addition to gD binding to HveA, the latter has been shown to interact with proteins of tumor necrosis factor receptor-associated factor family, lymphotoxin-α (LT-α), and a membrane-associated protein referred to as LIGHT. To study the relationship between HveA, its natural ligands, and the viral proteins involved in HSV entry into cells, we have screened two phage-displayed combinatorial peptide libraries for peptide ligands of a recombinant form of HveA. Affinity selection experiments yielded two peptide ligands, BP-1 and BP-2, which could block the interaction between gD and HveA. Of the two peptides, only BP-2 inhibited HSV entry into CHO cells transfected with an HveA-expressing plasmid. When we analyzed these peptides for the ability to interfere with HveA binding to its natural ligand LT-α, we found that BP-1 inhibited the interaction of cellular LT-α with HveA. Thus, we have dissected the sites of interaction between the cell receptor, its natural ligand LT-α and gD, the virus-specific protein involved in HSV entry into cells. PMID:10364318

Tannic acid and chromic chloride-induced binding of protein to red cells: a preliminary study of possible binding sites and reaction mechanisms.

PubMed

Hunt, A F; Reed, M I

1990-07-01

The binding mechanisms and binding sites involved in the tannic acid and chromic chloride-induced binding of protein to red cells were investigated using the binding of IgA paraprotein to red cells as model systems. Inhibition studies of these model systems using amino acid homopolymers and compounds (common as red cell membrane constituents) suggest that the mechanisms involved are similar to those proposed for the conversion of hide or skin collagen to leather, as in commercial tanning. These studies also suggest that tannic acid-induced binding of IgA paraprotein to red cells involves the amino acid residues of L-arginine, L-lysine, L-histidine, and L-proline analogous to tanning with phenolic plant extracts. The amino acid residues of L-aspartate, L-glutamate and L-asparagine are involved in a similar manner in chronic chloride-induced binding of protein to red cells.

Silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway.

PubMed

Li, Yumei; Zhang, Chunmei; Cai, Danfeng; Chen, Congde; Mu, Dongmei

2017-12-01

Rhabdoid tumors, which tend to occur prior to the age of 2 years, are one of the most aggressive malignancies and have a poor prognosis due to the frequency of metastasis. Silibinin, a natural extract, has been approved as a potential tumor suppressor in various studies, however, whether or not it also exerts its antitumor capacity in rhabdoid tumors, particularly with regards to tumor migration and invasion, is unclear. The rhabdoid tumor G401 cell line was used in the present in vitro study. An MTT assay was used to assess the cytotoxicity of silibinin on G401 cells, cell migration was studied using a wound healing assay and a Transwell migration assay, and cell invasion was determined using a Transwell invasion assay. The underlying mechanism in silibinin inhibited cell migration and invasion was investigated by western blot analysis and further confirmed using a specific inhibitor. Experimental results demonstrated that high doses of silibinin suppressed cell viability, and that low doses of silibinin inhibited cell migration and invasion without affecting cell proliferation. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway was involved in the silibinin-induced inhibition of metastasis. Silibinin inactivated the PI3K/Akt pathway, and inhibited cell migration and invasion, an effect that was further enhanced when LY294002, a classic PI3K inhibitor, was used concurrently. In general, silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway and may be a potential chemotherapeutic drug to combat rhabdoid tumors in the future.

Quercetin Is More Effective than Cromolyn in Blocking Human Mast Cell Cytokine Release and Inhibits Contact Dermatitis and Photosensitivity in Humans

PubMed Central

Asadi, Shahrzad; Sismanopoulos, Nikolaos; Butcher, Alan; Fu, Xueyan; Katsarou-Katsari, Alexandra; Antoniou, Christina; Theoharides, Theoharis C.

2012-01-01

Mast cells are immune cells critical in the pathogenesis of allergic, but also inflammatory and autoimmune diseases through release of many pro-inflammatory cytokines such as IL-8 and TNF. Contact dermatitis and photosensitivity are skin conditions that involve non-immune triggers such as substance P (SP), and do not respond to conventional treatment. Inhibition of mast cell cytokine release could be effective therapy for such diseases. Unfortunately, disodium cromoglycate (cromolyn), the only compound marketed as a mast cell “stabilizer”, is not particularly effective in blocking human mast cells. Instead, flavonoids are potent anti-oxidant and anti-inflammatory compounds with mast cell inhibitory actions. Here, we first compared the flavonoid quercetin (Que) and cromolyn on cultured human mast cells. Que and cromolyn (100 µM) can effectively inhibit secretion of histamine and PGD2. Que and cromolyn also inhibit histamine, leukotrienes and PGD2 from primary human cord blood-derived cultured mast cells (hCBMCs) stimulated by IgE/Anti-IgE. However, Que is more effective than cromolyn in inhibiting IL-8 and TNF release from LAD2 mast cells stimulated by SP. Moreover, Que reduces IL-6 release from hCBMCs in a dose-dependent manner. Que inhibits cytosolic calcium level increase and NF-kappa B activation. Interestingly, Que is effective prophylactically, while cromolyn must be added together with the trigger or it rapidly loses its effect. In two pilot, open-label, clinical trials, Que significantly decreased contact dermatitis and photosensitivity, skin conditions that do not respond to conventional treatment. In summary, Que is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases, especially in formulations that permit more sufficient oral absorption. PMID:22470478

Myeloid-derived suppressor cells as a potential therapy for experimental autoimmune myasthenia gravis.

PubMed

Li, Yan; Tu, Zhidan; Qian, Shiguang; Fung, John J; Markowitz, Sanford D; Kusner, Linda L; Kaminski, Henry J; Lu, Lina; Lin, Feng

2014-09-01

We recently demonstrated that hepatic stellate cells induce the differentiation of myeloid-derived suppressor cells (MDSCs) from myeloid progenitors. In this study, we found that adoptive transfer of these MDSCs effectively reversed disease progression in experimental autoimmune myasthenia gravis (EAMG), a T cell-dependent and B cell-mediated model for myasthenia gravis. In addition to ameliorated disease severity, MDSC-treated EAMG mice showed suppressed acetylcholine receptor (AChR)-specific T cell responses, decreased levels of serum anti-AChR IgGs, and reduced complement activation at the neuromuscular junctions. Incubating MDSCs with B cells activated by anti-IgM or anti-CD40 Abs inhibited the proliferation of these in vitro-activated B cells. Administering MDSCs into mice immunized with a T cell-independent Ag inhibited the Ag-specific Ab production in vivo. MDSCs directly inhibit B cells through multiple mechanisms, including PGE2, inducible NO synthase, and arginase. Interestingly, MDSC treatment in EAMG mice does not appear to significantly inhibit their immune response to a nonrelevant Ag, OVA. These results demonstrated that hepatic stellate cell-induced MDSCs concurrently suppress both T and B cell autoimmunity, leading to effective treatment of established EAMG, and that the MDSCs inhibit AChR-specific immune responses at least partially in an Ag-specific manner. These data suggest that MDSCs could be further developed as a novel approach to treating myasthenia gravis and, even more broadly, other diseases in which T and B cells are involved in pathogenesis. Copyright © 2014 by The American Association of Immunologists, Inc.

Ovarian cancer therapeutic potential of glutamine depletion based on GS expression.

PubMed

Furusawa, Akiko; Miyamoto, Morikazu; Takano, Masashi; Tsuda, Hitoshi; Song, Yong Sang; Aoki, Daisuke; Miyasaka, Naoyuki; Inazawa, Johji; Inoue, Jun

2018-05-28

Amino acids (AAs) are biologically important nutrient compounds necessary for the survival of any cell. Of the 20 AAs, cancer cells depend on the uptake of several extracellular AAs for survival. However, which extracellular AA is indispensable for the survival of cancer cells and the molecular mechanism involved have not been fully defined. In this study, we found that the reduction of cell survival caused by glutamine (Gln) depletion is inversely correlated with the expression level of glutamine synthetase (GS) in ovarian cancer (OVC) cells. GS expression was downregulated in 45 of 316 OVC cases (14.2%). The depletion of extracellular Gln by treatment with l-asparaginase, in addition to inhibiting Gln uptake via the knockdown of a Gln transporter, led to the inhibition of cell growth in OVC cells with low expression of GS (GSlow-OVC cells). Furthermore, the re-expression of GS in GSlow-OVC cells induced the inhibition of tumor growth in vitro and in vivo. Thus, these findings provide novel insight into the development of an OVC therapy based on the requirement of Gln.

The mechanism of retrovirus suppression of human T cell proliferation in vitro.

PubMed

Copelan, E A; Rinehart, J J; Lewis, M; Mathes, L; Olsen, R; Sagone, A

1983-10-01

Immunosuppression is commonly associated with retrovirus-induced animal tumors. Studies in the murine and feline retrovirus systems suggest that the 15,000-dalton envelope protein (p15E) of the virion may contribute to immunosuppression by interfering with normal lymphocyte function. We examined the effect of inactivated feline leukemia virus (UV-FeLV) and p15E derived from this virus on concanavalin A (Con A) driven human T cell proliferation. Virus and p15E markedly suppressed mononuclear cell proliferative response to Con A. Suppression was not due to inhibition of monocyte accessory cell function, or interleukin 1 (IL 1) secretion. In fact, the presence of monocytes partially protected T cells from UV-FeLV suppression. UV-FeLV, however, suppressed T cell secretion of and response to interleukin 2 (IL 2). We conclude that UV-FeLV and derived p15E inhibit T cell proliferation by direct inhibition of T cell function. These findings, extended to the in vivo situations, suggest that retrovirus-associated suppression of the immune response involves the induction of T cell but not monocyte dysfunction.

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

Nakayama, Hironao; Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115; Division of Cell Growth and Tumor Regulation, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295

Class 3 semaphorins were discovered as a family of axon guidance molecules, but are now known to be involved in diverse biologic processes. In this study, we investigated the anti-angiogenic potential of SEMA3E and SEMA3F (SEMA3E&F) in infantile hemangioma (IH). IH is a common vascular tumor that involves both vasculogenesis and angiogenesis. Our lab has identified and isolated hemangioma stem cells (HemSC), glucose transporter 1 positive (GLUT1{sup +}) endothelial cells (designated as GLUT1{sup sel} cells) based on anti-GLUT1 magnetic beads selection and GLUT1-negative endothelial cells (named HemEC). We have shown that these types of cells play important roles in hemangiogenesis.more » We report here that SEMA3E inhibited HemEC migration and proliferation while SEMA3F was able to suppress the migration and proliferation in all three types of cells. Confocal microscopy showed that stress fibers in HemEC were reduced by SEMA3E&F and that stress fibers in HemSC were decreased by SEMA3F, which led to cytoskeletal collapse and loss of cell motility in both cell types. Additionally, SEMA3E&F were able to inhibit vascular endothelial growth factor (VEGF)-induced sprouts in all three types of cells. Further, SEMA3E&F reduced the level of p-VEGFR2 and its downstream p-ERK in HemEC. These results demonstrate that SEMA3E&F inhibit IH cell proliferation and suppress the angiogenic activities of migration and sprout formation. SEMA3E&F may have therapeutic potential to treat or prevent growth of highly proliferative IH. - Highlights: • SEMA3E&F reduce actin stress fibers and induce cytoskeletal collapse in HemEC. • SEMA3E&F inhibit angiogenic activities of HemEC. • SEMA3E&F can interrupt the VEGF-A-VEGFR2-ERK signaling pathway in HemEC. • Plexin D1 and NRP2 are induced during HemSC/GLUT1{sup sel}-to-EC differentiation.« less

Involvement of FFA1 and FFA4 in the regulation of cellular functions during tumor progression in colon cancer cells.

PubMed

Takahashi, Kaede; Fukushima, Kaori; Onishi, Yuka; Minami, Kanako; Otagaki, Shiho; Ishimoto, Kaichi; Fukushima, Nobuyuki; Honoki, Kanya; Tsujiuchi, Toshifumi

2018-08-01

Free fatty acid receptor 1 (FFA1) and FFA4 mediate a variety of biological responses through binding of medium- and long-chain free fatty acids. The aim of this study was to investigate an involvement of FFA1 and FFA4 in the regulation of cellular functions during tumor progression in colon cancer cells. The long-term fluorouracil (5-FU) and cisplatin (CDDP) treated cells were generated from DLD1 cells (DLD-5FU and DLD-CDDP cells, respectively). FFAR1 expressions were lower in DLD-5FU and DLD-CDDP cells than in DLD1 cells. In contrast, DLD-5FU and DLD-CDDP cells showed the high FFAR4 expressions, compared with DLD1 cells. The cell motile activities of DLD-5FU and DLD-CDDP cells were reduced by GW9508 which is an agonist of FFA1 and FFA4. Moreover, GW1100, an antagonist of FFA1, inhibited the cell motile activities of DLD-5FU and DLD-CDDP cells. To evaluate whether FFA1 and FFA4 regulate the enhancement of cell motility, invasion and colony formation, highly migratory (hmDLD1) cells were established from DLD1 cells. FFAR1 expression was significantly higher in hmDLD1 cells than in DLD1 cells, but no change of FFAR4 expression was observed. The elevated cell motile and invasive activities and colony formation of hmDLD1 cells were suppressed by FFA1 inhibition. These results suggest that FFA1 and FFA4 are involved in the regulation of cellular functions during tumor progression in colon cancer DLD1 cells. Copyright © 2018 Elsevier Inc. All rights reserved.

NHERF1 inhibits proliferation of triple-negative breast cancer cells by suppressing GPER signaling.

PubMed

Wang, Yan; Peng, Zhiqiang; Meng, Ran; Tao, Tao; Wang, Qiqi; Zhao, Chunjuan; Liu, Hua; Song, Ran; Zheng, Junfang; Qin, Qiong; He, Junqi

2017-07-01

G protein-coupled estrogen receptor (GPER) signaling is activated in triple-negative breast cancer (TNBC); however, the detailed mechanisms of its regulation remain unclear. The present study aimed to elucidate the molecular mechanisms involved in GPER activation in TNBC. In MDA-MB-231 cells, a TNBC cell line, NHERF1 interaction with GPER was verified by co-immunoprecipitation and immunofluorescent staining assays. Overexpression of NHERF1 in MDA-MB-231 cells inhibited GPER-mediated proliferation and phosphorylation of ERK1/2 and Akt. Furthermore, NHERF1 expression levels were negatively correlated with the gene signatures of GPER activation, ERK1/2 and Akt signaling, and cell proliferation in early stage of TNBC tumors from the TCGA data set. Taken together, NHERF1 inhibited the activation of GPER-mediated signaling and suppressed the proliferation of triple-negative breast cancer cells. Loss of NHERF1 expression may play a pivotal role in the early stage of TNBC carcinogenesis.

The neural cell adhesion molecule is involved in the metastatic capacity in a murine model of lung cancer.

PubMed

Campodónico, Paola B; de Kier Joffé, Elisa D Bal; Urtreger, Alejandro J; Lauria, Lilia S; Lastiri, José M; Puricelli, Lydia I; Todaro, Laura B

2010-04-01

Neural cell adhesion molecule (NCAM) is involved in cell growth, migration, and differentiation. Its expression and/or polysialylation appear to be deregulated in many different cancer types. We employed the lung tumor cell line LP07, syngeneic in BALB/c mice to investigate the role of NCAM in malignant progression. LP07 cells express the three main NCAM isoforms, all of them polysialylated. This cells line, pretreated with an anti-NCAM antibody and inoculated intravenously (i.v.) into syngeneic mice, developed less and smaller lung metastases. In vitro studies showed that NCAM bound antibody inhibited cell growth, mainly due to an increase in apoptosis, associated with a decrease of cyclin D1 and enhanced expression of active caspase 3 and caspase 9. Anti-NCAM-treated LP07 cells showed impairment in their ability to migrate and adhere to several extracellular matrix components. Secreted uPA activity was also reduced. NCAM-140 knocked-down by siRNA in LP07 cells pretreated or not with anti-NCAM showed an impaired metastasizing ability upon i.v. inoculation into mice. These results suggest that anti-NCAM treatment could be mimicking homophilic trans-interactions and NCAM-140 knocked-down impairs heterophilic interactions, both leading to inhibition of metastatic dissemination. The involvement of NCAM in lung tumor progression was confirmed in human NSCLC tumors. Sixty percent of the cases expressed NCAM at tumor cell level. A multivariate analysis indicated that NCAM expression was associated with a shorter overall survival in this homogeneous series of Stages I and II NSCLC patients. NCAM may be able to modulate mechanisms involved in lung carcinoma progression and represents an attractive target to control metastatic progression.

Heat shock protein 90 inhibitor NVP-AUY922 exerts potent activity against adult T-cell leukemia-lymphoma cells.

PubMed

Taniguchi, Hiroaki; Hasegawa, Hiroo; Sasaki, Daisuke; Ando, Koji; Sawayama, Yasushi; Imanishi, Daisuke; Taguchi, Jun; Imaizumi, Yoshitaka; Hata, Tomoko; Tsukasaki, Kunihiro; Uno, Naoki; Morinaga, Yoshitomo; Yanagihara, Katsunori; Miyazaki, Yasushi

2014-12-01

Adult T-cell leukemia-lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL-related cell lines. Analysis using FACS revealed that AUY922 induced cell-cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho-Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM-1, -2, -3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922-induced death of ATL cells. In fact, SGI-1776, a pan-PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL-related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Heat shock protein 90 inhibitor NVP-AUY922 exerts potent activity against adult T-cell leukemia–lymphoma cells

PubMed Central

Taniguchi, Hiroaki; Hasegawa, Hiroo; Sasaki, Daisuke; Ando, Koji; Sawayama, Yasushi; Imanishi, Daisuke; Taguchi, Jun; Imaizumi, Yoshitaka; Hata, Tomoko; Tsukasaki, Kunihiro; Uno, Naoki; Morinaga, Yoshitomo; Yanagihara, Katsunori; Miyazaki, Yasushi

2014-01-01

Adult T-cell leukemia–lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL-related cell lines. Analysis using FACS revealed that AUY922 induced cell-cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho-Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM-1, -2, -3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922-induced death of ATL cells. In fact, SGI-1776, a pan-PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL-related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target. PMID:25263741

Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway.

PubMed

Lakhan, Ram; Said, Hamid M

2017-04-01

Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr 78 Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway.

Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway

PubMed Central

Lakhan, Ram

2017-01-01

Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr78Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway. PMID:28052864

17β-Estradiol inhibits TNF-α-induced proliferation and migration of vascular smooth muscle cells via suppression of TRAIL.

PubMed

Li, Hengchang; Cheng, Yang; Simoncini, Tommaso; Xu, Shiyuan

2016-07-01

Atherosclerosis is an inflammatory disease and involves migration of vascular smooth muscle cells (VSMCs). Estrogen inhibits VSMCs migration, while the underlying mechanism remains to be revealed. Recent years, there is emerging evidence showing that TNF-related apoptosis-inducing ligand (TRAIL) increases proliferation and migration of VSMCs. In this study, we investigated the regulatory effect of estrogen on TRAIL expression in VSMCs. TNF-α greatly enhanced TRAIL protein expression and stimulated VSMCs proliferation and migration. This effect was partially inhibited by the addition of TRAIL neutralizing antibody, suggesting that TRAIL is important in TNF-α-induced migration. 17β-estradiol (E2) inhibited TRAIL expression under TNF-α stimulation in a time- and concentration-dependent manner. This effect was was mimicked by ERα agonist 4',4″,4‴-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), but not ERβ agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN), indicating that ERα is involved in this action. TNF-α led to nuclear factor kappa B (NF-κB) p65 phosphorylation and the inhibitor pyrrolidine dithiocarbama (PDTC) inhibited TRAIL expression, suggesting that NF-κB signaling is crucial for TARIL production. E2 suppressed p65 phosphorylation in VSMCs and the overexpression of p65 subunit reversed the inhibitory effect of E2 on TRAIL expression and cell proliferation and migration. Taken together, our results indicate that E2 inhibits VSMCs proliferation and migration by downregulation of TRAIL expression via suppression of NF-κB pathway.

Thrombin enhances herpes simplex virus infection of cells involving protease-activated receptor 1.

PubMed

Sutherland, M R; Friedman, H M; Pryzdial, E L G

2007-05-01

We have previously shown that the surface of purified herpes family viruses can initiate thrombin production by expressing host-encoded and virus-encoded procoagulant factors. These enable the virus to bypass the normal cell-regulated mechanisms for initiating coagulation, and provide a link between infection and vascular disease. In the current study we investigated why these viruses may have evolved to generate thrombin. Using cytolytic viral plaque assays, the current study examines the effect of thrombin on human umbilical vein endothelial cell (HUVEC) or human foreskin fibroblast (HFF) infection by purified herpes simplex virus type 1 (HSV1) and type 2 (HSV2). Demonstrating that the availability of thrombin is an advantage to the virus, purified thrombin added to serum-free inoculation media resulted in up to a 3-fold enhancement of infection depending on the virus strain and cell type. The effect of thrombin on HUVEC infection was generally greater than its effect on HFF. To illustrate the involvement of thrombin produced during inoculation, hirudin was shown to inhibit the infection of each HSV strain, but only when serum containing clotting factors for thrombin production was present in media. The involvement of protease-activated receptor 1 (PAR1) was supported using PAR1-activating peptides in place of thrombin and PAR1-specific antibodies to inhibit the effects of thrombin. These data show that HSV1 and HSV2 initiate thrombin production to increase the susceptibility of cells to infection through a mechanism involving PAR1-mediated cell modulation.

Induction of apoptosis in hepatocellular carcinoma Smmc-7721 cells by vitamin K(2) is associated with p53 and independent of the intrinsic apoptotic pathway.

PubMed

Li, Lu; Qi, Zhiling; Qian, Jin; Bi, Fuyong; Lv, Jun; Xu, Lei; Zhang, Ling; Chen, Hongyu; Jia, Renbing

2010-09-01

Vitamin K(2) (VK(2)) can exert cell growth inhibitory effects in various human cancer cells. In this study, we investigated the cell growth inhibitory effects of VK(2) in hepatocellular carcinoma Smmc-7721 cells and the mechanisms involved. We found that VK(2)-inhibited cell proliferation in Smmc-7721 cells in a dose-dependent manner, and the IC50 of VK(2) in Smmc-7721 cells was 9.73 microM at 24 h. The data from flow cytometric analyses, DNA fragmentation assays, and caspase 3 activity assays revealed that apoptosis was the determining factor in VK(2) activity. Furthermore, a significant increase in p53 phosphorylation and protein level was exhibited in apoptotic cells treated with VK(2), although there were no changes in p53 mRNA expression. Bax expression was unaffected by VK(2) in Smmc-7721 cells. In addition, our study showed that caspase 3 was activated by caspase 8, not caspase 9, in Smmc-7721 cells treated with VK(2). In summary, these data suggested that VK(2) can inhibit the growth of Smmc-7721 cells by induction of apoptosis involving caspase 8 activation and p53. This apoptotic process was not mediated by the intrinsic apoptotic pathway.

Saussurea lappa extract suppresses TPA-induced cell invasion via inhibition of NF-κB-dependent MMP-9 expression in MCF-7 breast cancer cells

PubMed Central

2014-01-01

Background Saussurea lappa (SL) has been used as a traditional herbal medicine to treat abdominal pain and tenesmus, and has been suggested to possess various biological activities, including anti-tumor, anti-ulcer, anti-inflammatory, anti-viral, and cardiotonic activities. The effect of SL on breast cancer metastasis, however, is unknown. Cell migration and invasion are crucial in neoplastic metastasis. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix, is a major component in cancer cell invasion. Methods Cell viability was examined by MTT assay, whereas cell motility was measured by invasion assay. Western blot, Real-time PCR, and Zymography assays were used to investigate the inhibitory effects of ESL on matrix metalloproteinase-9 (MMP-9) expression level in MCF-7 cells. EMSA confirmed the inhibitory effects of ESL on DNA binding of NF- κB in MCF-7 cells. Results Cells threated with various concentrations of Saussurea lappa (ESL) for 24 h. Concentrations of 2 or 4 μM did not lead to a significant change in cell viability or morphology. Therefore, subsequent experiments utilized the optimal non-toxic concentration (2 or 4 μM) of ESL. In this study, we investigated the inhibitory effect of ethanol extract of ESL on MMP-9 expression and cell invasion in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MCF-7 cells. ESL inhibited the TPA-induced transcriptional activation of nuclear factor-kappa B (NF-κB). However, this result obtained that ESL did not block the TPA-induced phosphorylation of the kinases: p38, ERK, and JNK. Therefore, ELS-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of NF-kB pathway in MCF-7 cells. Conclusions These results indicate that ELS-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of NF-kB pathway in MCF-7 cells. Thus, ESL has potential for controlling breast cancer invasiveness in vitro. PMID:24885456

Fluid shear stress induces upregulation of COX-2 and PGI2 release in endothelial cells via a pathway involving PECAM-1, PI3K, FAK, and p38.

PubMed

Russell-Puleri, Sparkle; Dela Paz, Nathaniel G; Adams, Diana; Chattopadhyay, Mitali; Cancel, Limary; Ebong, Eno; Orr, A Wayne; Frangos, John A; Tarbell, John M

2017-03-01

Vascular endothelial cells play an important role in the regulation of vascular function in response to mechanical stimuli in both healthy and diseased states. Prostaglandin I 2 (PGI 2 ) is an important antiatherogenic prostanoid and vasodilator produced in endothelial cells through the action of the cyclooxygenase (COX) isoenzymes COX-1 and COX-2. However, the mechanisms involved in sustained, shear-induced production of COX-2 and PGI 2 have not been elucidated but are determined in the present study. We used cultured endothelial cells exposed to steady fluid shear stress (FSS) of 10 dyn/cm 2 for 5 h to examine shear stress-induced induction of COX-2/PGI 2 Our results demonstrate the relationship between the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1) and the intracellular mechanoresponsive molecules phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and mitogen-activated protein kinase p38 in the FSS induction of COX-2 expression and PGI 2 release. Knockdown of PECAM-1 (small interference RNA) expression inhibited FSS-induced activation of α 5 β 1 -integrin, upregulation of COX-2, and release of PGI 2 in both bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). Furthermore, inhibition of the PI3K pathway (LY294002) substantially inhibited FSS activation of α 5 β 1 -integrin, upregulation of COX-2 gene and protein expression, and release of PGI 2 in BAECs. Inhibition of integrin-associated FAK (PF573228) and MAPK p38 (SB203580) also inhibited the shear-induced upregulation of COX-2. Finally, a PECAM-1 -/- mouse model was characterized by reduced COX-2 immunostaining in the aorta and reduced plasma PGI 2 levels compared with wild-type mice, as well as complete inhibition of acute flow-induced PGI 2 release compared with wild-type animals. NEW & NOTEWORTHY In this study we determined the major mechanotransduction pathway by which blood flow-driven shear stress activates cyclooxygenase-2 (COX-2) and prostaglandin I 2 (PGI 2 ) release in endothelial cells. Our work has demonstrated for the first time that COX-2/PGI 2 mechanotransduction is mediated by the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1). Copyright © 2017 the American Physiological Society.

Fluid shear stress induces upregulation of COX-2 and PGI2 release in endothelial cells via a pathway involving PECAM-1, PI3K, FAK, and p38

PubMed Central

Russell-Puleri, Sparkle; dela Paz, Nathaniel G.; Adams, Diana; Chattopadhyay, Mitali; Cancel, Limary; Ebong, Eno; Orr, A. Wayne; Frangos, John A.

2017-01-01

Vascular endothelial cells play an important role in the regulation of vascular function in response to mechanical stimuli in both healthy and diseased states. Prostaglandin I2 (PGI2) is an important antiatherogenic prostanoid and vasodilator produced in endothelial cells through the action of the cyclooxygenase (COX) isoenzymes COX-1 and COX-2. However, the mechanisms involved in sustained, shear-induced production of COX-2 and PGI2 have not been elucidated but are determined in the present study. We used cultured endothelial cells exposed to steady fluid shear stress (FSS) of 10 dyn/cm2 for 5 h to examine shear stress-induced induction of COX-2/PGI2. Our results demonstrate the relationship between the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1) and the intracellular mechanoresponsive molecules phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and mitogen-activated protein kinase p38 in the FSS induction of COX-2 expression and PGI2 release. Knockdown of PECAM-1 (small interference RNA) expression inhibited FSS-induced activation of α5β1-integrin, upregulation of COX-2, and release of PGI2 in both bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). Furthermore, inhibition of the PI3K pathway (LY294002) substantially inhibited FSS activation of α5β1-integrin, upregulation of COX-2 gene and protein expression, and release of PGI2 in BAECs. Inhibition of integrin-associated FAK (PF573228) and MAPK p38 (SB203580) also inhibited the shear-induced upregulation of COX-2. Finally, a PECAM-1−/− mouse model was characterized by reduced COX-2 immunostaining in the aorta and reduced plasma PGI2 levels compared with wild-type mice, as well as complete inhibition of acute flow-induced PGI2 release compared with wild-type animals. NEW & NOTEWORTHY In this study we determined the major mechanotransduction pathway by which blood flow-driven shear stress activates cyclooxygenase-2 (COX-2) and prostaglandin I2 (PGI2) release in endothelial cells. Our work has demonstrated for the first time that COX-2/PGI2 mechanotransduction is mediated by the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1). PMID:28011582

Inhibition of PTP1B disrupts cell-cell adhesion and induces anoikis in breast epithelial cells.

PubMed

Hilmarsdottir, Bylgja; Briem, Eirikur; Halldorsson, Skarphedinn; Kricker, Jennifer; Ingthorsson, Sævar; Gustafsdottir, Sigrun; Mælandsmo, Gunhild M; Magnusson, Magnus K; Gudjonsson, Thorarinn

2017-05-11

Protein tyrosine phosphatase 1B (PTP1B) is a well-known inhibitor of insulin signaling pathways and inhibitors against PTP1B are being developed as promising drug candidates for treatment of obesity. PTP1B has also been linked to breast cancer both as a tumor suppressor and as an oncogene. Furthermore, PTP1B has been shown to be a regulator of cell adhesion and migration in normal and cancer cells. In this study, we analyzed the PTP1B expression in normal breast tissue, primary breast cells and the breast epithelial cell line D492. In normal breast tissue and primary breast cells, PTP1B is widely expressed in both epithelial and stromal cells, with highest expression in myoepithelial cells and fibroblasts. PTP1B is widely expressed in branching structures generated by D492 when cultured in 3D reconstituted basement membrane (3D rBM). Inhibition of PTP1B in D492 and another mammary epithelial cell line HMLE resulted in reduced cell proliferation and induction of anoikis. These changes were seen when cells were cultured both in monolayer and in 3D rBM. PTP1B inhibition affected cell attachment, expression of cell adhesion proteins and actin polymerization. Moreover, epithelial to mesenchymal transition (EMT) sensitized cells to PTP1B inhibition. A mesenchymal sublines of D492 and HMLE (D492M and HMLEmes) were more sensitive to PTP1B inhibition than D492 and HMLE. Reversion of D492M to an epithelial state using miR-200c-141 restored resistance to detachment induced by PTP1B inhibition. In conclusion, we have shown that PTP1B is widely expressed in the human breast gland with highest expression in myoepithelial cells and fibroblasts. Inhibition of PTP1B in D492 and HMLE affects cell-cell adhesion and induces anoikis-like effects. Finally, cells with an EMT phenotype are more sensitive to PTP1B inhibitors making PTP1B a potential candidate for further studies as a target for drug development in cancer involving the EMT phenotype.

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells.

PubMed

Chen, Lisa S; Redkar, Sanjeev; Bearss, David; Wierda, William G; Gandhi, Varsha

2009-11-05

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-X(L), XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction.

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells

PubMed Central

Chen, Lisa S.; Redkar, Sanjeev; Bearss, David; Wierda, William G.

2009-01-01

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-XL, XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction. PMID:19734450

Proinflammatory Cytokines IL-6 and TNF-α Increased Telomerase Activity through NF-κB/STAT1/STAT3 Activation, and Withaferin A Inhibited the Signaling in Colorectal Cancer Cells.

PubMed

Chung, Seyung S; Wu, Yong; Okobi, Quincy; Adekoya, Debbie; Atefi, Mohammad; Clarke, Orette; Dutta, Pranabananda; Vadgama, Jaydutt V

2017-01-01

There are increasing evidences of proinflammatory cytokine involvement in cancer development. Here, we found that two cytokines, IL-6 and TNF- α , activated colorectal cancer cells to be more invasive and stem-like. Combined treatment of IL-6 and TNF- α phosphorylated transcription factors STAT3 in a synergistic manner. STAT3, STAT1, and NF- κ B physically interacted upon the cytokine stimulation. STAT3 was bound to the promoter region of human telomerase reverse transcriptase (hTERT). IL-6 and TNF- α stimulation further enhanced STAT3 binding affinity. Stem cell marker Oct-4 was upregulated in colorectal cancer cells upon IL-6 and TNF- α stimulation. Withaferin A, an anti-inflammatory steroidal lactone, inhibited the IL-6- and TNF- α -induced cancer cell invasion and decreased colonosphere formation. Notably, withaferin A inhibited STAT3 phosphorylation and abolished the STAT3, STAT1, and NF- κ B interactions. Oct-4 expression was also downregulated by withaferin A inhibition. The binding of STAT3 to the hTERT promoter region and telomerase activity showed reduction with withaferin A treatments. Proinflammatory cytokine-induced cancer cell invasiveness is mediated by a STAT3-regulated mechanism in colorectal cancer cells. Our data suggest that withaferin A could be a promising anticancer agent that effectively inhibits the progression of colorectal cancer.

SOCS1 and SOCS3 Are Targeted by Hepatitis C Virus Core/gC1qR Ligation To Inhibit T-Cell Function

PubMed Central

Yao, Zhi Qiang; Waggoner, Stephen N.; Cruise, Michael W.; Hall, Caroline; Xie, Xuefang; Oldach, David W.; Hahn, Young S.

2005-01-01

T cells play an important role in the control of hepatitis C virus (HCV) infection. We have previously demonstrated that the HCV core inhibits T-cell responses through interaction with gC1qR. We show here that core proteins from chronic and resolved HCV patients differ in sequence, gC1qR-binding ability, and T-cell inhibition. Specifically, chronic core isolates bind to gC1qR more efficiently and inhibit T-cell proliferation as well as gamma interferon (IFN-γ) production more profoundly than resolved core isolates. This inhibition is mediated by the disruption of STAT phosphorylation through the induction of SOCS molecules. Silencing either SOCS1 or SOCS3 by small interfering RNA dramatically augments the production of IFN-γ in T cells, thereby abrogating the inhibitory effect of core. Additionally, the ability of core proteins from patients with chronic infections to induce SOCS proteins and suppress STAT activation greatly exceeds that of core proteins from patients with resolved infections. These results suggest that the HCV core/gC1qR-induced T-cell dysfunction involves the induction of SOCS, a powerful inhibitor of cytokine signaling, which represents a novel mechanism by which a virus usurps the host machinery for persistence. PMID:16306613

Reprogramming of leukemic cell metabolism through the naphthoquinonic compound Quambalarine B

PubMed Central

Vališ, Karel; Grobárová, Valéria; Hernychová, Lucie; Bugáňová, Martina; Kavan, Daniel; Kalous, Martin; Černý, Jiří; Stodůlková, Eva; Kuzma, Marek; Flieger, Miroslav; Černý, Jan; Novák, Petr

2017-01-01

Abnormalities in cancer metabolism represent potential targets for cancer therapy. We have recently identified a natural compound Quambalarine B (QB), which inhibits proliferation of several leukemic cell lines followed by cell death. We have predicted ubiquinone binding sites of mitochondrial respiratory complexes as potential molecular targets of QB in leukemia cells. Hence, we tracked the effect of QB on leukemia metabolism by applying several omics and biochemical techniques. We have confirmed the inhibition of respiratory complexes by QB and found an increase in the intracellular AMP levels together with respiratory substrates. Inhibition of mitochondrial respiration by QB triggered reprogramming of leukemic cell metabolism involving disproportions in glycolytic flux, inhibition of proteins O-glycosylation, stimulation of glycine synthesis pathway, and pyruvate kinase activity, followed by an increase in pyruvate and a decrease in lactate levels. Inhibition of mitochondrial complex I by QB suppressed folate metabolism as determined by a decrease in formate production. We have also observed an increase in cellular levels of several amino acids except for aspartate, indicating the dependence of Jurkat (T-ALL) cells on aspartate synthesis. These results indicate blockade of mitochondrial complex I and II activity by QB and reduction in aspartate and folate metabolism as therapeutic targets in T-ALL cells. Anti-cancer activity of QB was also confirmed during in vivo studies, suggesting the therapeutic potential of this natural compound. PMID:29262552

LRD-22, a novel dual dithiocarbamatic acid ester, inhibits Aurora-A kinase and induces apoptosis and cell cycle arrest in HepG2 cells

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

Wang, Huiling; Li, Ridong; Li, Li

In this study we investigated the antitumor activity of the novel dual dithiocarbamatic acid ester LRD-22 in vitro and in vivo. Several cancer cell lines were employed to determine the effect of LRD-22 on cell growth, and the MTT assay showed there was a significant decrease in viable tumor cell numbers in the presence of LRD-22, especially in the HepG2 cell line. Colony formation assay also showed LRD-22 strongly inhibits HepG2 cell growth. Evaluation of the mechanism involved showed that inhibitory effects of LRD-22 on cell growth are due to induction of apoptosis and G2/M arrest. LRD-22 inhibited Aurora-A phosphorylation at Thr{submore » 288} and subsequently impaired p53 phosphorylation at Ser{sub 315} which was associated with the proteasome degradation pathway. Tumor suppressor protein p53 is stabilized by this mechanism and accumulates through inhibition of Aurora-A kinase activity via treatment with LRD-22. In vivo study of HepG2 xenograft in nude mice also shows LRD-22 suppresses tumor growth at a concentration of 5 mg/kg without animals suffering loss of body weight. In conclusion, our results demonstrate LRD-22 acts as an Aurora-A kinase inhibitor to induce apoptosis and inhibit proliferation in HepG2 cells, and should be considered as a promising targeting agent for HCC therapy. - Highlights: • LRD-22 significantly inhibits cancer cell growth, especially in the HepG2 cell line. • The inhibitory effect of LRD-22 is due to induction of apoptosis and cell cycle arrest. • LRD-22 inhibits Aurora-A phosphorylation which results in subsequent impairment of the p53 pathway. • LRD-22 suppresses tumor growth in xenograft mice without body weight loss.« less

Involvement of PI3K/Akt and p38 MAPK in the induction of COX-2 expression by bacterial lipopolysaccharide in murine adrenocortical cells.

PubMed

Mercau, M E; Astort, F; Giordanino, E F; Martinez Calejman, C; Sanchez, R; Caldareri, L; Repetto, E M; Coso, O A; Cymeryng, C B

2014-03-25

Previous studies from our laboratory demonstrated the involvement of COX-2 in the stimulation of steroid production by LPS in murine adrenocortical Y1 cells, as well as in the adrenal cortex of male Wistar rats. In this paper we analyzed signaling pathways involved in the induction of this key regulatory enzyme in adrenocortical cells and demonstrated that LPS triggers an increase in COX-2 mRNA levels by mechanisms involving the stimulation of reactive oxygen species (ROS) generation and the activation of p38 MAPK and Akt, in addition to the previously demonstrated increase in NFκB activity. In this sense we showed that: (1) inhibition of p38 MAPK or PI3K/Akt (pharmacological or molecular) prevented the increase in COX-2 protein levels by LPS, (2) LPS induced p38 MAPK and Akt phosphorylation, (3) antioxidant treatment blocked the effect of LPS on p38 MAPK phosphorylation and in COX-2 protein levels, (4) PI3K inhibition with LY294002 prevented p38 MAPK phosphorylation and, (5) the activity of an NFκB reporter was decreased by p38 MAPK or PI3K inhibition. These results suggest that activation of both p38 MAPK and PI3K/Akt pathways promote the stimulation of NFκB activity and that PI3K/Akt activity might regulate both p38 MAPK and NFκB signaling pathways. In summary, in this study we showed that in adrenal cells, LPS induces COX-2 expression by activating p38 MAPK and PI3K/Akt signaling pathways and that both pathways converge in the modulation of NFκB transcriptional activity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Pannexin channels mediate the acquisition of myogenic commitment in C2C12 reserve cells promoted by P2 receptor activation

PubMed Central

Riquelme, Manuel A.; Cea, Luis A.; Vega, José L.; Puebla, Carlos; Vargas, Aníbal A.; Shoji, Kenji F.; Subiabre, Mario; Sáez, Juan C.

2015-01-01

The acquisition of myoblast commitment to the myogenic linage requires rises in intracellular free Ca2+ concentration ([Ca2+]i). Putative cell membrane pathways involved in these [Ca2+]i increments are P2 receptors (P2Rs) as well as connexin (Cx) and/or pannexin (Panx) hemichannels and channels (Cx HChs and Panx Chs), respectively, which are known to permeate Ca2+. Reserve cells (RCs) are uncommitted myoblasts obtained from differentiated C2C12 cell cultures, which acquire commitment upon replating. Regarding these cells, we found that extracellular ATP increases the [Ca2+]i via P2Rs. Moreover, ATP increases the plasma membrane permeability to small molecules and a non-selective membrane current, both of which were inhibited by Cx HCh/Panx1Ch blockers. However, RCs exposed to divalent cation-free saline solution, which is known to activate Cx HChs (but not Panx Chs), did not enhance membrane permeability, thus ruling out the possible involvement of Cx HChs. Moreover, ATP-induced membrane permeability was inhibited with blockers of P2Rs that activate Panx Chs. In addition, exogenous ATP induced the expression of myogenic commitment and increased MyoD levels, which was prevented by the inhibition of P2Rs or knockdown of Panx1 Chs. Similarly, increases in MyoD levels induced by ATP released by RCs were inhibited by Panx Ch/Cx HCh blockers. Myogenic commitment acquisition thus requires a feed-forward mechanism mediated by extracellular ATP, P2Rs, and Panx Chs. PMID:26000275

Prevention of Escherichia coli K1 penetration of the blood-brain barrier by counteracting the host cell receptor and signaling molecule involved in E. coli invasion of human brain microvascular endothelial cells.

PubMed

Zhu, Longkun; Pearce, Donna; Kim, Kwang Sik

2010-08-01

Escherichia coli meningitis is an important cause of mortality and morbidity, and a key contributing factor is our incomplete understanding of the pathogenesis of E. coli meningitis. We have shown that E. coli penetration into the brain requires E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. E. coli invasion of HBMEC involves its interaction with HBMEC receptors, such as E. coli cytotoxic necrotizing factor 1 (CNF1) interaction with its receptor, the 67-kDa laminin receptor (67LR), and host signaling molecules including cytosolic phospholipase A(2)alpha (cPLA(2)alpha). In the present study, we showed that treatment with etoposide resulted in decreased expression of 67LR on HBMEC and inhibited E. coli invasion of HBMEC. Pharmacological inhibition of cysteinyl leukotrienes, lipoxygenated products of arachidonic acid released by cPLA(2)alpha, using montelukast (an antagonist of the type 1 cysteinyl leukotriene receptor) also inhibited E. coli invasion of HBMEC. E. coli penetration into the brain was significantly decreased by etoposide as well as by montelukast, and a combination of etoposide and montelukast was significantly more effective in inhibiting E. coli K1 invasion of HBMEC than single agents alone. These findings demonstrate for the first time that counteracting the HBMEC receptor and signaling molecule involved in E. coli invasion of HBMEC provides a novel approach for prevention of E. coli penetration into the brain, the essential step required for development of E. coli meningitis.

Flavonoid silybin improves the response to radiotherapy in invasive bladder cancer.

PubMed

Prack Mc Cormick, Barbara; Langle, Yanina; Belgorosky, Denise; Vanzulli, Silvia; Balarino, Natalia; Sandes, Eduardo; Eiján, Ana M

2018-01-24

Conservative treatment for invasive bladder cancer (BC) involves a complete transurethral tumor resection combined with chemotherapy (CT) and radiotherapy (RT). The major obstacles of chemo-radiotherapy are the addition of the toxicities of RT and CT, and the recurrence due to RT and CT resistances. The flavonoid Silybin (Sb) inhibits pathways involved in cell survival and resistance mechanisms, therefore the purpose of this paper was to study in vitro and in vivo, the ability of Sb to improve the response to RT, in two murine BC cell lines, with different levels of invasiveness, placing emphasis on radio-sensitivity, and pathways involved in radio-resistance and survival. In vitro, Sb radio-sensitized murine invasive cells through the inhibition of RT-induced NF-κB and PI3K pathways, and the increase of oxidative stress, while non-invasive cells did not show to be sensitized. In vivo, Sb improved RT-response and overall survival in invasive murine tumors. As Sb is already being tested in clinical trials for other urological cancers and it improves RT-response in invasive BC, these results could have translational relevance, supporting further research. © 2018 Wiley Periodicals, Inc.

The flavonoid, fisetin, inhibits UV radiation-induced oxidative stress and the activation of NF-kappaB and MAPK signaling in human lens epithelial cells.

PubMed

Yao, Ke; Zhang, Li; Zhang, Yidong; Ye, PanPan; Zhu, Ning

2008-01-01

Ultraviolet (UV) radiation-induced oxidative stress plays a significant role in the progression of cataracts. This study investigated the photoprotective effect of fisetin on UV radiation-induced oxidative stress in human lens epithelial cells and the possible molecular mechanism involved. SRA01/04 cells exposed to different doses of ultraviolet B (UVB) were cultured with various concentrations of fisetin and subsequently monitored for cell viability by the 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of fisetin on the generation of reactive oxygen species (ROS) of SRA01/04 cells was determined by flow cytometry. Translocation of nuclear factor kappa-B (NF-kappaB) was examined by immunocytochemistry. Expression of NF-kappaB/P65, inhibiter kappa B (IkappaB), and mitogen activated protein kinase (MAPK) proteins were measured by western blot. Treatment of SRA01/04 cells with fisetin inhibited UVB-induced cell death and the generation of ROS. Fisetin inhibited UVB-induced activation and translocation of NF-kappaB/p65, which was mediated through an inhibition of the degradation and activation of IkappaB. Fisetin also inhibited UVB-induced phosphorylation of the p38 and c-Jun N-terminal kinase (JNK) proteins of the MAPK family at various time points studied. The flavonoid, fisetin, could be useful in attenuation of UV radiation-induced oxidative stress and the activation of NF-kappaB and MAPK signaling in human lens epithelial cells, which suggests that fisetin has a potential protective effect against cataractogenesis.

Adeno-associated virus type 2 rep gene-mediated inhibition of basal gene expression of human immunodeficiency virus type 1 involves its negative regulatory functions.

PubMed Central

Oelze, I; Rittner, K; Sczakiel, G

1994-01-01

Adeno-associated virus type 2 (AAV-2), a human parvovirus which is apathogenic in adults, inhibits replication and gene expression of human immunodeficiency virus type 1 (HIV-1) in human cells. The rep gene of AAV-2, which was shown earlier to be sufficient for this negative interference, also down-regulated the expression of heterologous sequences driven by the long terminal repeat (LTR) of HIV-1. This effect was observed in the absence of the HIV-1 transactivator Tat, i.e., at basal levels of LTR-driven transcription. In this work, we studied the involvement of functional subsequences of the HIV-1 LTR in rep-mediated inhibition in the absence of Tat. Mutated LTRs driving an indicator gene (cat) were cointroduced into human SW480 cells together with rep alone or with double-stranded DNA fragments or RNA containing sequences of the HIV-1 LTR. The results indicate that rep strongly enhances the function of negative regulatory elements of the LTR. In addition, the experiments revealed a transcribed sequence element located within the TAR-coding sequence termed AHHH (AAV-HIV homology element derived from HIV-1) which is involved in rep-mediated inhibition. The AHHH element is also involved in down-regulation of basal expression levels in the absence of rep, suggesting that AHHH also contributes to negative regulatory functions of the LTR of HIV-1. In contrast, positive regulatory elements of the HIV-1 LTR such as the NF kappa B and SP1 binding sites have no significant influence on the rep-mediated inhibition. Images PMID:8289357

Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process

PubMed Central

Ghosal, Abhisek; Sekar, Thillai V.

2014-01-01

Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na+-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na+-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS. PMID:24904078

Secretory function of ovarian cells and myometrial contractions in cow are affected by chlorinated insecticides (chlordane, heptachlor, mirex) in vitro

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

Wrobel, Michael Hubert, E-mail: m.wrobel@pan.olszt

The aim of the study was to investigate the effect of chlordane, heptachlor and mirex, on hormonal regulation of the force of myometrial contractions. Myometrial, endometrial, granulosa and luteal cells as well as strips of myometrium from non-pregnant cows were incubated with three insecticides at environmentally relevant doses (0.1, 1 or 10 ng/ml). None of the insecticides affected the viability of studied cells. Chlordane stimulated, while heptachlor and mirex inhibited, secretion of testosterone and estradiol from granulosa cells as well as secretion of progesterone from luteal cells, respectively. Secretion of oxytocin (OT) from granulosa cells was increased after incubation withmore » all studied insecticides. Only mirex stimulated OT secretion from luteal cells, while heptachlor inhibited this effect. None of them affected synthesis of OT in luteal cells and prostaglandins (PGF2 and PGE2) secretion from uterine cells, except PGE2 secretion from endometrial cells was decreased when the cells were incubated with 0.1 ng/ml of chlordane. Basal and OT-stimulated myometrial contractions were increased by mirex and decreased by heptachlor. The data show that the insecticides altered secretory function of ovarian cells. Heptachlor and mirex affected also myometrial contractions in vitro, but uterine secretion of prostaglandins were not involved in the mechanism of that adverse effect of insecticides. The data indicate on potential of these insecticides to disturb fertilisation, blastocyst implantation or even the length of gestation. - Highlights: • The studied insecticides affected steroids and oxytocin secretion from ovaries. • Mirex stimulated bovine myometrial contractions. • Heptachlor inhibited bovine myometrial contractions. • Prostaglandins are not involved in adverse effect of the insecticides on uterine contractions.« less

Inhibition of Protein Ubiquitination by Paraquat and 1-Methyl-4-Phenylpyridinium Impairs Ubiquitin-Dependent Protein Degradation Pathways.

PubMed

Navarro-Yepes, Juliana; Anandhan, Annadurai; Bradley, Erin; Bohovych, Iryna; Yarabe, Bo; de Jong, Annemieke; Ovaa, Huib; Zhou, You; Khalimonchuk, Oleh; Quintanilla-Vega, Betzabet; Franco, Rodrigo

2016-10-01

Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson's disease (PD). Ubiquitin (Ub), alpha (α)-synuclein, p62/sequestosome 1, and oxidized proteins are the major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effects of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP(+)) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells (SK-N-SH) treated with PQ or MPP(+), or in mice chronically exposed to PQ. PQ decreased Ub protein content, but not its mRNA transcription. Protein synthesis inhibition with cycloheximide depleted Ub levels and potentiated PQ-induced cell death. The inhibition of proteasomal activity by PQ was found to be a late event in cell death progression and had neither effect on the toxicity of either MPP(+) or PQ, nor on the accumulation of oxidized sulfenylated, sulfonylated (DJ-1/PARK7 and peroxiredoxins), and carbonylated proteins induced by PQ. PQ- and MPP(+)-induced Ub protein depletion prompted the dimerization/inactivation of the Ub-binding protein p62 that regulates the clearance of ubiquitinated proteins by autophagy. We confirmed that PQ and MPP(+) impaired autophagy flux and that the blockage of autophagy by the overexpression of a dominant-negative form of the autophagy protein 5 (dnAtg5) stimulated their toxicity, but there was no additional effect upon inhibition of the proteasome. PQ induced an increase in the accumulation of α-synuclein in dopaminergic cells and membrane-associated foci in yeast cells. Our results demonstrate that the inhibition of protein ubiquitination by PQ and MPP(+) is involved in the dysfunction of Ub-dependent protein degradation pathways.

Inhibition of protein ubiquitination by paraquat and 1-methyl-4-phenylpyridinium impairs ubiquitin-dependent protein degradation pathways

PubMed Central

Navarro-Yepes, Juliana; Anandhan, Annadurai; Bradley, Erin; Bohovych, Iryna; Yarabe, Bo; de Jong, Annemieke; Ovaa, Huib; Zhou, You; Khalimonchuk, Oleh; Quintanilla-Vega, Betzabet; Franco, Rodrigo

2016-01-01

Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson’s disease (PD). Ubiquitin (Ub), alpha [α]-synuclein, p62/sequestosome 1 and oxidized proteins are major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effect of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells (SK-N-SH) treated with PQ or MPP+, or in mice chronically exposed to PQ. PQ decreased Ub protein content, but not its mRNA transcription. Protein synthesis inhibition with cycloheximide depleted Ub levels and potentiated PQ–induced cell death. Inhibition of proteasomal activity by PQ was found to be a late event in cell death progression, and had no effect on either the toxicity of MPP+ or PQ, or the accumulation of oxidized sulfenylated, sulfonylated (DJ-1/PARK7 and peroxiredoxins) and carbonylated proteins induced by PQ. PQ- and MPP+-induced Ub protein depletion prompted the dimerization/inactivation of the Ub-binding protein p62 that regulates the clearance of ubiquitinated proteins by autophagic. We confirmed that PQ and MPP+ impaired autophagy flux, and that the blockage of autophagy by the overexpression of a dominant-negative form of the autophagy protein 5 (dnAtg5) stimulated their toxicity, but there was no additional effect upon inhibition of the proteasome. PQ induced an increase in the accumulation of α-synuclein in dopaminergic cells and membrane associated foci in yeast cells. Our results demonstrate that inhibition of protein ubiquitination by PQ and MPP+ is involved in the dysfunction of Ub-dependent protein degradation pathways. PMID:26409479

Benzene and its metabolite decreases cell proliferation via LncRNA-OBFC2A-mediated anti-proliferation effect involving NOTCH1 and KLF15

PubMed Central

Sun, Pengling; Wang, Jing; Guo, Xiaoli; Chen, Yujiao; Xing, Caihong; Gao, Ai

2017-01-01

LncRNA has been considered to play a crucial role in the progression of several diseases by affecting cell proliferation. However, its role in benzene toxicity remains unclear. Our study showed that the expression of lncRNA-OBFC2A increased accompanied with the change of cell proliferation related-genes in benzene-exposed workers. In vitro experiments, 1,4-Benzoquinone dose-dependently inhibited cell proliferation and simultaneously caused the decrease of NOTCH1 expression and the increase of KLF15 in AHH-1 cell lines. Meanwhile, 1, 4-Benzoquinone obviously increased the expression of lncRNA-OBFC2A, which was consistent with our previous population results. Therefore, we propose that lncRNA-OBFC2A is involved in benzene toxicity by regulating cell proliferation. Further, we successfully constructed a lentivirus model of interfering the expression of lncRNA-OBFC2A. After interfering lncRNA-OBFC2A, the cell proliferation inhibition and the expression of NOTCH1 and KLF15 induced by 1, 4-Benzoquinone were reversed. Subsequently, RNA fluorescence in situ Hybridization assay showed that lncRNA-OBFC2A was located in cell nuclei. These results suggest that benzene and its metabolite decreases cell proliferation via LncRNA-OBFC2A-mediated anti-proliferation effect involving NOTCH1 and KLF15. LncRNA-OBFC2A can be a potential biomarker for benzene toxicity. PMID:28388563

Benzene and its metabolite decreases cell proliferation via LncRNA-OBFC2A-mediated anti-proliferation effect involving NOTCH1 and KLF15.

PubMed

Sun, Pengling; Wang, Jing; Guo, Xiaoli; Chen, Yujiao; Xing, Caihong; Gao, Ai

2017-06-20

LncRNA has been considered to play a crucial role in the progression of several diseases by affecting cell proliferation. However, its role in benzene toxicity remains unclear. Our study showed that the expression of lncRNA-OBFC2A increased accompanied with the change of cell proliferation related-genes in benzene-exposed workers. In vitro experiments, 1,4-Benzoquinone dose-dependently inhibited cell proliferation and simultaneously caused the decrease of NOTCH1 expression and the increase of KLF15 in AHH-1 cell lines. Meanwhile, 1, 4-Benzoquinone obviously increased the expression of lncRNA-OBFC2A, which was consistent with our previous population results. Therefore, we propose that lncRNA-OBFC2A is involved in benzene toxicity by regulating cell proliferation. Further, we successfully constructed a lentivirus model of interfering the expression of lncRNA-OBFC2A. After interfering lncRNA-OBFC2A, the cell proliferation inhibition and the expression of NOTCH1 and KLF15 induced by 1, 4-Benzoquinone were reversed. Subsequently, RNA fluorescence in situ Hybridization assay showed that lncRNA-OBFC2A was located in cell nuclei. These results suggest that benzene and its metabolite decreases cell proliferation via LncRNA-OBFC2A-mediated anti-proliferation effect involving NOTCH1 and KLF15. LncRNA-OBFC2A can be a potential biomarker for benzene toxicity.

The inhibition of macrophage foam cell formation by tetrahydroxystilbene glucoside is driven by suppressing vimentin cytoskeleton.

PubMed

Yao, Wenjuan; Huang, Lei; Sun, Qinju; Yang, Lifeng; Tang, Lian; Meng, Guoliang; Xu, Xiaole; Zhang, Wei

2016-10-01

Macrophage foam cell formation triggered by oxLDL is an important event that occurs during the development of atherosclerosis. 2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside (TSG) exhibits significant anti-atherosclerotic activity. Herein we used U937 cells induced by PMA and oxLDL in vitro to investigate the inhibitory effects of TSG on U937 differentiation and macrophage foam cell formation. TSG pretreatment markedly inhibited cell differentiation induced by PMA, macrophage apoptosis and foam cell formation induced by oxLDL. The inhibition of vimentin expression and cleavage was involved in these inhibitory effects of TSG. The suppression of vimentin by siRNA in U937 significantly inhibited cell differentiation, apoptosis and foam cell formation. Using inhibitors for TGFβR1 and PI3K, we found that vimentin production in U937 cells is regulated by TGFβ/Smad signaling, but not by PI3K-Akt-mTOR signaling. Meanwhile, TSG pretreatment inhibited both the expression of TGFβ1 and the phosphorylation of Smad2 and Smad3, and TSG suppressed the nuclear translocation of Smad4 induced by PMA and oxLDL. Furthermore, TSG attenuated the induced caspase-3 activation and adhesion molecules levels by PMA and oxLDL. PMA and oxLDL increased the co-localization of vimentin with ICAM-1, which was attenuated by pretreatment with TSG. These results suggest that TSG inhibits macrophage foam cell formation through suppressing vimentin expression and cleavage, adhesion molecules expression and vimentin-ICAM-1 co-localization. The interruption of TGFβ/Smad pathway and caspase-3 activation is responsible for the downregulation of TSG on vimentin expression and degradation, respectively. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Influence of sulfur oxyanions on reductive dehalogenation activities in Desulfomonile tiedjei.

PubMed Central

Townsend, G T; Suflita, J M

1997-01-01

The inhibition of aryl reductive dehalogenation reactions by sulfur oxyanions has been demonstrated in environmental samples, dehalogenating enrichments, and the sulfate-reducing bacterium Desulfomonile tiedjei; however, this phenomenon is not well understood. We examined the effects of sulfate, sulfite, and thiosulfate on reductive dehalogenation in the model microorganism D. tiedjei and found separate mechanisms of inhibition due to these oxyanions under growth versus nongrowth conditions. Dehalogenation activity was greatly reduced in extracts of cells grown in the presence of both 3-chlorobenzoate, the substrate or inducer for the aryl dehalogenation activity, and either sulfate, sulfite, or thiosulfate, indicating that sulfur oxyanions repress the requisite enzymes. In extracts of fully induced cells, thiosulfate and sulfite, but not sulfate, were potent inhibitors of aryl dehalogenation activity even in membrane fractions lacking the cytoplasmically located sulfur oxyanion reductase. These results suggest that under growth conditions, sulfur oxyanions serve as preferred electron acceptors and negatively influence dehalogenation activity in D. tiedjei by regulating the amount of active aryl dehalogenase in cells. Additionally, in vitro inhibition by sulfur oxyanions is due to the interaction of the reactive species with enzymes involved in dehalogenation and need not involve competition between two respiratory processes for reducing equivalents. Sulfur oxyanions also inhibited tetrachloroethylene dehalogenation by the same mechanisms, further indicating that chloroethylenes are fortuitously dehalogenated by the aryl dehalogenase. The commonly observed inhibition of reductive dehalogenation reactions under sulfate-reducing conditions may be due to similar regulation mechanisms in other dehalogenating microorganisms that contain multiple respiratory activities. PMID:9293011

Nitric-oxide synthase trafficking inducer is a pleiotropic regulator of endothelial cell function and signaling

PubMed Central

2017-01-01

Endothelial nitric-oxide synthase (eNOS) and its bioactive product, nitric oxide (NO), mediate many endothelial cell functions, including angiogenesis and vascular permeability. For example, vascular endothelial growth factor (VEGF)-mediated angiogenesis is inhibited upon reduction of NO bioactivity both in vitro and in vivo. Moreover, genetic disruption or pharmacological inhibition of eNOS attenuates angiogenesis during tissue repair, resulting in delayed wound closure. These observations emphasize that eNOS-derived NO can promote angiogenesis. Intriguingly, eNOS activity is regulated by nitric-oxide synthase trafficking inducer (NOSTRIN), which sequesters eNOS, thereby attenuating NO production. This has prompted significant interest in NOSTRIN's function in endothelial cells. We show here that NOSTRIN affects the functional transcriptome of endothelial cells by down-regulating several genes important for invasion and angiogenesis. Interestingly, the effects of NOSTRIN on endothelial gene expression were independent of eNOS activity. NOSTRIN also affected the expression of secreted cytokines involved in inflammatory responses, and ectopic NOSTRIN overexpression functionally restricted endothelial cell proliferation, invasion, adhesion, and VEGF-induced capillary tube formation. Furthermore, NOSTRIN interacted directly with TNF receptor-associated factor 6 (TRAF6), leading to the suppression of NFκB activity and inhibition of AKT activation via phosphorylation. Interestingly, TNF-α-induced NFκB pathway activation was reversed by NOSTRIN. We found that the SH3 domain of NOSTRIN is involved in the NOSTRIN-TRAF6 interaction and is required for NOSTRIN-induced down-regulation of endothelial cell proteins. These results have broad biological implications, as aberrant NOSTRIN expression leading to deactivation of the NFκB pathway, in turn triggering an anti-angiogenic cascade, might inhibit tumorigenesis and cancer progression. PMID:28235804

Inhibition of EphB4-ephrin-B2 signaling enhances response to Cetuximab-radiation therapy in head and neck cancers.

PubMed

Bhatia, Shilpa; Sharma, Jaspreet; Bukkapatnam, Sanjana; Oweida, Ayman; Lennon, Shelby; Phan, Andy V; Milner, Dallin; Uyanga, Nomin; Jimeno, Antonio; Raben, David; Somerset, Hilary; Heasley, Lynn; Karam, Sana D

2018-05-30

The clinical success of targeted therapies such as cetuximab and radiation (RT) is hampered by the low response rates and development of therapeutic resistance. In the current study, we investigated the involvement of EphB4-ephrin-B2 pro-tumorigenic signaling in mediating resistance to EGFR inhibition and radiation therapy in head and neck cancers. We used patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma (HNSCC) and HNSCC cell lines to test our hypothesis. Tumor tissues were subjected to PhosphoRTK array, and western blotting to detect changes in EphB4-ephrin-B2 targets. mRNA sequencing and microarray data analysis was performed on PDX tumors and HNSCC cell lines respectively to determine differences in gene expression of molecules involved in tumor cell growth, proliferation, and survival pathways. Effects on cell growth were determined by MTT assay on HNSCC cells downregulated for EphB4/ephrin-B2 expression, with and without EGFR inhibitor and radiation. Our data from locally-advanced HNSCC patients treated with standard of care definitive chemo-RT show elevated EphB4 and ephrin-B2 levels after failure of treatment. We observed significant response towards cetuximab and radiation therapy following EphB4-ephrin-B2 inhibition resulting in improved survival in tumor-bearing mice. Tumor growth inhibition was accompanied by decrease in the levels of proliferation and pro-survival molecules and increased apoptosis. Our findings underscore the importance of adopting rational drug combinations to enhance therapeutic effect. Our study documenting enhanced response of HNSCC to cetuximab-RT therapy with EphB4-ephrin-B2 blockade has the potential to translate into clinic to benefit this patient population. Copyright ©2018, American Association for Cancer Research.

Inhibition of protein phosphorylation in MIA pancreatic cancer cells: Confluence of metabolic and signaling pathways

PubMed Central

Zhang, Hengwei; Cao, Rui; Lee, Wai-Nang Paul; Deng, Caishu; Zhao, Yingchun; Lappe, Joan; Recker, Robert; Yen, Yun; Wang, Qi; Tsai, Ming-Ying; Go, Vay Liang; Xiao, Gary Guishan

2010-01-01

Oxythiamine (OT), a transketolase inhibitor, is known to inhibit pancreatic cancer cell proliferation. In this study, we investigated the effect of inhibition of the transketolase pathway on signaling pathways in MIA PaCa cancer cells using in-house proteomic techniques. We hypothesized that OT alter protein phosphorylation thus affecting cell cycle arrest and cell proliferation. MIA PaCa-2 cells were cultured in media containing an algal 15N amino acid mixture at 50% enrichment, with and without OT, to determine protein expression and synthesis. Analysis of cell lysates using two-dimensional gel electrophoresis matrix assisted laser desorption and ionization time-of-flight and time-of-flight mass spectrometry (2-DE-MALDI-TOF/TOF MS) identified 12 phosphor proteins that were significantly suppressed by OT treatment. Many of these proteins are involved in regulation of cycle activities and apoptosis. Among the proteins identified, expression of the phosphor heat shock protein 27 (Hsp27) was dramatically inhibited by OT treatment while the level of its total protein remained unchanged. Hsp27 expression and phoshporylation is known to be associated with drug resistance and cancer cell survival. The changes in phosphorylation of key proteins of cancer proliferation and survival suggest that protein phosphorylation is the confluence of the effects of OT on metabolic and signaling pathways. PMID:20035555

Inhibitory crosstalk between ERK and AMPK in the growth and proliferation of cardiac fibroblasts

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

Du Jianhai; Guan Tongju; Zhang Hui

2008-04-04

Extracellular signal-regulated kinase (ERK) is one of the key protein kinases that regulate the growth and proliferation in cardiac fibroblasts (CFs). As an energy sensor of cellular metabolism, AMP-activated protein kinase (AMPK) is found recently to be involved in myocardial remodeling. In this study, we investigated the crosstalk between ERK and AMPK in the growth and proliferation of CFs. In neonatal rat cardiac fibroblasts (NRCFs), we found that serum significantly inhibited basal AMPK phosphorylation between 10 min and 24 h and also partially inhibited AMPK phosphorylation by AMPK activator, 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR). Furthermore, ERK inhibitor could greatly reverse the inhibition ofmore » AMPK by serum. Conversely, activation of AMPK by AICAR also showed a significant inhibition of basal and serum-induced ERK phosphorylation but it showed a delayed and steadfast inhibition which appeared after 60 min and lasted until 12 h. Moreover, inhibition of ERK could repress the activation of p70S6K, an important kinase in cardiac proliferation, and AICAR could also inhibit p70S6K phosphorylation. In addition, under both serum and serum-free medium, AICAR significantly inhibited the DNA synthesis and cell numbers, and reduced cells at S phase. In conclusion, AMPK activation with AICAR inhibited growth and proliferation in cardiac fibroblasts, which involved inhibitory interactions between ERK and AMPK. This is the first report that AMPK could be a target of ERK in growth factors-induced proliferation, which may give a new mechanism that growth factors utilize in their promotion of proliferation in cardiac fibroblasts.« less

The Silencing of Pokemon Attenuates the Proliferation of Hepatocellular Carcinoma Cells In Vitro and In Vivo by Inhibiting the PI3K/Akt Pathway

PubMed Central

Liu, Yun-Peng; Liu, Jing-Jing; Yang, Xiao-Ning; Jazag, Amarsanaa; Zhang, Zhi-Ping; Guleng, Bayasi; Ren, Jian-Lin

2012-01-01

Pokemon (POK erythroid myeloid ontogenic factor), which belongs to the POK protein family, is also called LRF, OCZF and FBI-1. As a transcriptional repressor, Pokemon assumes a critical function in cellular differentiation and oncogenesis. Our study identified an oncogenic role for Pokemon in human hepatocellular carcinoma (HCC). We successfully established human HepG2 and Huh-7 cell lines in which Pokemon was stably knocked down. We demonstrated that Pokemon silencing inhibited cell proliferation and migration. Pokemon knockdown inhibited the PI3K/Akt and c-Raf/MEK/ERK pathways and modulated the expression of various cell cycle regulators in HepG2 and Huh-7 cells. Therefore, Pokemon may also be involved in cell cycle progression in these cells. We confirmed that Pokemon silencing suppresses hepatocellular carcinoma growth in tumor xenograft mice. These results suggest that Pokemon promotes cell proliferation and migration in hepatocellular carcinoma and accelerates tumor development in an Akt- and ERK-signaling-dependent manner. PMID:23300578

p110α Inhibition Overcomes Stromal Cell-Mediated Ibrutinib Resistance in Mantle Cell Lymphoma.

PubMed

Guan, Jiyu; Huang, Dan; Yakimchuk, Konstantin; Okret, Sam

2018-05-01

Acquired resistance to cancer drugs is common, also for modern targeted drugs like the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, a new drug approved for the treatment of the highly aggressive and relapsing mantle cell lymphoma (MCL). The tumor microenvironment often impacts negatively on drug response. Here, we demonstrate that stromal cells protect MCL cells from ibrutinib-induced apoptosis and support MCL cell regrowth after drug removal by impairing ibrutinib-mediated downregulation of PI3K/AKT signaling. Importantly, the stromal cell-mediated ibrutinib resistance was overcome in vitro by inhibiting AKT activity using the PI3K catalytic p110α subunit-specific inhibitor BYL719. This was seen both for MCL cell lines and primary MCL cells. Furthermore, inhibition of p110α activity by BYL719 potentiated the ability of ibrutinib to inhibit MCL tumor growth in vivo in a mouse xenograft model. The stromal cell-mediated ibrutinib resistance was found to be due to a direct interaction with MCL cells and involves the integrin VLA-4, as disrupting stromal cell-MCL cell interaction using a VLA-4 blocking antibody abrogated the ibrutinib resistance. This suggests that combined treatment with ibrutinib and a p110α inhibitor, alternatively by disrupting stromal cell-MCL cell interaction, may be a promising therapeutic strategy to overcome stromal cell-mediated ibrutinib resistance in MCL. Mol Cancer Ther; 17(5); 1090-100. ©2018 AACR . ©2018 American Association for Cancer Research.

Combination Treatment with Apricoxib and IL-27 Enhances Inhibition of Epithelial-Mesenchymal Transition in Human Lung Cancer Cells through a STAT1 Dominant Pathway

PubMed Central

Lee, Mi-Heon; Kachroo, Puja; Pagano, Paul C; Yanagawa, Jane; Wang, Gerald; Walser, Tonya C; Krysan, Kostyantyn; Sharma, Sherven; John, Maie St.; Dubinett, Steven M; Lee, Jay M

2015-01-01

Background The cyclooxygenase 2 (COX-2) pathway has been implicated in the molecular pathogenesis of many malignancies, including lung cancer. Apricoxib, a selective COX-2 inhibitor, has been described to inhibit epithelial-mesenchymal transition (EMT) in human malignancies. The mechanism by which apricoxib may alter the tumor microenvironment by affecting EMT through other important signaling pathways is poorly defined. IL-27 has been shown to have anti-tumor activity and our recent study showed that IL-27 inhibited EMT through a STAT1 dominant pathway. Objective The purpose of this study is to investigate the role of apricoxib combined with IL-27 in inhibiting lung carcinogenesis by modulation of EMT through STAT signaling. Methods and Results Western blot analysis revealed that IL-27 stimulation of human non-small cell lung cancer (NSCLC) cell lines results in STAT1 and STAT3 activation, decreased Snail protein and mesenchymal markers (N-cadherin and vimentin) and a concomitant increase in expression of epithelial markers (E-cadherin, β-and γ-catenins), and inhibition of cell migration. The combination of apricoxib and IL-27 resulted in augmentation of STAT1 activation. However, IL-27 mediated STAT3 activation was decreased by the addition of apricoxib. STAT1 siRNA was used to determine the involvement of STAT1 pathway in the enhanced inhibition of EMT and cell migration by the combined IL-27 and apricoxib treatment. Pretreatment of cells with STAT1 siRNA inhibited the effect of combined IL-27 and apricoxib in the activation of STAT1 and STAT3. In addition, the augmented expression of epithelial markers, decreased expression mesenchymal markers, and inhibited cell migration by the combination treatment were also inhibited by STAT1 siRNA, suggesting that the STAT1 pathway is important in the enhanced effect from the combination treatment. Conclusion Combined apricoxib and IL-27 has an enhanced effect in inhibition of epithelial-mesenchymal transition and cell migration in human lung cancer cells through a STAT1 dominant pathway. PMID:26523208

mda-7/IL-24 induces cell death in neuroblastoma through a novel mechanism involving AIF and ATM

PubMed Central

Bhoopathi, Praveen; Lee, Nathaniel; Pradhan, Anjan K.; Shen, Xue-Ning; Das, Swadesh K.; Sarkar, Devanand; Emdad, Luni; Fisher, Paul B.

2016-01-01

Advanced stages of neuroblastoma, the most common extracranial malignant solid tumor of the central nervous system in infants and children, are refractive to therapy. Ectopic expression of melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24) promotes broad-spectrum antitumor activity in vitro, in vivo in pre-clinical animal models and in a Phase I clinical trial in patients with advanced cancers, without harming normal cells. mda-7/IL-24 exerts cancer-specific toxicity (apoptosis or toxic autophagy) by promoting ER stress and modulating multiple signal transduction pathways regulating cancer cell growth, invasion, metastasis, survival and angiogenesis. To enhance cancer-selective expression and targeted anti-cancer activity of mda-7/IL-24 we created a tropism-modified Cancer Terminator Virus (Ad.5/3-CTV), which selectively replicates in cancer cells producing robust expression of mda-7/IL-24. We now show that Ad.5/3-CTV induces profound neuroblastoma anti-proliferative activity and apoptosis in a caspase 3/9-independent manner both in vitro and in vivo in a tumor xenograft model. Ad.5/3-CTV promotes these effects through a unique pathway involving apoptosis inducing factor (AIF) translocation into the nucleus. Inhibiting AIF rescued neuroblastoma cells from Ad.5/3-CTV-induced cell death, whereas pan-caspase inhibition failed to promote survival. Ad.5/3-CTV infection of neuroblastoma cells increased ATM phosphorylation instigating nuclear translocation and increased γ–H2AX, triggering nuclear translocation and intensified expression of AIF. These results were validated further using two ATM small molecule inhibitors that attenuated PARP cleavage by inhibiting γ–H2AX, which in turn inhibited AIF changes in Ad.5/3-CTV-infected neuroblastoma cells. Taken together, we elucidate a novel pathway for mda-7/IL-24-induced caspase-independent apoptosis in neuroblastoma cells mediated through modulation of AIF, ATM and γ–H2AX. PMID:27197168

Potentiation of in vitro and in vivo antitumor efficacy of doxorubicin by cyclin-dependent kinase inhibitor P276-00 in human non-small cell lung cancer cells.

PubMed

Rathos, Maggie J; Khanwalkar, Harshal; Joshi, Kavita; Manohar, Sonal M; Joshi, Kalpana S

2013-01-23

In the present study, we show that the combination of doxorubicin with the cyclin-dependent kinase inhibitor P276-00 was synergistic at suboptimal doses in the non-small cell lung carcinoma (NSCLC) cell lines and induces extensive apoptosis than either drug alone in H-460 human NSCLC cells. Synergistic effects of P276-00 and doxorubicin on growth inhibition was studied using the Propidium Iodide (PI) assay. The doses showing the best synergistic effect was determined and these doses were used for further mechanistic studies such as western blotting, cell cycle analysis and RT-PCR. The in vivo efficacy of the combination was evaluated using the H-460 xenograft model. The combination of 100 nM doxorubicin followed by 1200 nM P276-00 showed synergistic effect in the p53-positive and p53-mutated cell lines H-460 and H23 respectively as compared to the p53-null cell line H1299. Abrogation of doxorubicin-induced G2/M arrest and induction of apoptosis was observed in the combination treatment. This was associated with induction of tumor suppressor protein p53 and reduction of anti-apoptotic protein Bcl-2. Furthermore, doxorubicin alone greatly induced COX-2, a NF-κB target and Cdk-1, a target of P276-00, which was downregulated by P276-00 in the combination. Doxorubicin when combined with P276-00 in a sequence-specific manner significantly inhibited tumor growth, compared with either doxorubicin or P276-00 alone in H-460 xenograft model. These findings suggest that this combination may increase the therapeutic index over doxorubicin alone and reduce systemic toxicity of doxorubicin most likely via an inhibition of doxorubicin-induced chemoresistance involving NF-κB signaling and inhibition of Cdk-1 which is involved in cell cycle progression.

Potentiation of in vitro and in vivo antitumor efficacy of doxorubicin by cyclin-dependent kinase inhibitor P276-00 in human non-small cell lung cancer cells

PubMed Central

2013-01-01

Background In the present study, we show that the combination of doxorubicin with the cyclin-dependent kinase inhibitor P276-00 was synergistic at suboptimal doses in the non-small cell lung carcinoma (NSCLC) cell lines and induces extensive apoptosis than either drug alone in H-460 human NSCLC cells. Methods Synergistic effects of P276-00 and doxorubicin on growth inhibition was studied using the Propidium Iodide (PI) assay. The doses showing the best synergistic effect was determined and these doses were used for further mechanistic studies such as western blotting, cell cycle analysis and RT-PCR. The in vivo efficacy of the combination was evaluated using the H-460 xenograft model. Results The combination of 100 nM doxorubicin followed by 1200 nM P276-00 showed synergistic effect in the p53-positive and p53-mutated cell lines H-460 and H23 respectively as compared to the p53-null cell line H1299. Abrogation of doxorubicin-induced G2/M arrest and induction of apoptosis was observed in the combination treatment. This was associated with induction of tumor suppressor protein p53 and reduction of anti-apoptotic protein Bcl-2. Furthermore, doxorubicin alone greatly induced COX-2, a NF-κB target and Cdk-1, a target of P276-00, which was downregulated by P276-00 in the combination. Doxorubicin when combined with P276-00 in a sequence-specific manner significantly inhibited tumor growth, compared with either doxorubicin or P276-00 alone in H-460 xenograft model. Conclusion These findings suggest that this combination may increase the therapeutic index over doxorubicin alone and reduce systemic toxicity of doxorubicin most likely via an inhibition of doxorubicin-induced chemoresistance involving NF-κB signaling and inhibition of Cdk-1 which is involved in cell cycle progression. PMID:23343191

Inhibition of PTP1B disrupts cell–cell adhesion and induces anoikis in breast epithelial cells

PubMed Central

Hilmarsdottir, Bylgja; Briem, Eirikur; Halldorsson, Skarphedinn; Kricker, Jennifer; Ingthorsson, Sævar; Gustafsdottir, Sigrun; Mælandsmo, Gunhild M; Magnusson, Magnus K; Gudjonsson, Thorarinn

2017-01-01

Protein tyrosine phosphatase 1B (PTP1B) is a well-known inhibitor of insulin signaling pathways and inhibitors against PTP1B are being developed as promising drug candidates for treatment of obesity. PTP1B has also been linked to breast cancer both as a tumor suppressor and as an oncogene. Furthermore, PTP1B has been shown to be a regulator of cell adhesion and migration in normal and cancer cells. In this study, we analyzed the PTP1B expression in normal breast tissue, primary breast cells and the breast epithelial cell line D492. In normal breast tissue and primary breast cells, PTP1B is widely expressed in both epithelial and stromal cells, with highest expression in myoepithelial cells and fibroblasts. PTP1B is widely expressed in branching structures generated by D492 when cultured in 3D reconstituted basement membrane (3D rBM). Inhibition of PTP1B in D492 and another mammary epithelial cell line HMLE resulted in reduced cell proliferation and induction of anoikis. These changes were seen when cells were cultured both in monolayer and in 3D rBM. PTP1B inhibition affected cell attachment, expression of cell adhesion proteins and actin polymerization. Moreover, epithelial to mesenchymal transition (EMT) sensitized cells to PTP1B inhibition. A mesenchymal sublines of D492 and HMLE (D492M and HMLEmes) were more sensitive to PTP1B inhibition than D492 and HMLE. Reversion of D492M to an epithelial state using miR-200c-141 restored resistance to detachment induced by PTP1B inhibition. In conclusion, we have shown that PTP1B is widely expressed in the human breast gland with highest expression in myoepithelial cells and fibroblasts. Inhibition of PTP1B in D492 and HMLE affects cell–cell adhesion and induces anoikis-like effects. Finally, cells with an EMT phenotype are more sensitive to PTP1B inhibitors making PTP1B a potential candidate for further studies as a target for drug development in cancer involving the EMT phenotype. PMID:28492548

3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway.

PubMed

Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan; Xian, Shulin; Lu, Yunfei

2016-06-17

Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway

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

Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan, E-mail: moonsonlife@yahoo.com

Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generationmore » and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. -- Highlights: •Blockage of glycolysis might be a novel way to anticancer. •Both 3-bromopyruvate and sodium citrate could inhibit glycolysis and regulate mitochondrial pathway in cancer cells. •Both 3-bromopyruvate and sodium citrate would be the novel agents on treatment of gastric cancer.« less

Class IA PI3K inhibition inhibits cell growth and proliferation in mantle cell lymphoma.

PubMed

Tabe, Yoko; Jin, Linhua; Konopleva, Marina; Shikami, Masato; Kimura, Shinya; Andreeff, Michael; Raffeld, Mark; Miida, Takashi

2014-01-01

Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway preferentially occurs in aggressive blastoid variants of mantle cell lymphoma (MCL) and is implicated in the pathogenesis of this disease. In this study, we investigated the role of PI3K isoforms on proliferation of aggressive MCL cells. The changes in cell viability, cell cycle distribution and apoptosis induction by the PI3K isoform-selective inhibitors were evaluated. The molecular basis underlying the effects of the specific inhibition of PI3K isoforms was investigated by Western blot analysis. Our results demonstrated that a class IA PI3K isoform is most commonly involved in the constitutive activation of Akt in aggressive MCL. Treatment with a p110α isoform-specific inhibitor induced prominent cell cycle arrest followed by apoptosis through complete abolishment of phosphorylated (p)-Akt and its downstream targets. An inhibitor of isoform p110δ induced moderate cell cycle arrest with downregulation of p-Akt and p-S6K. A dual inhibitor of p110α and p110δ GDC-0941 caused more prominent cell growth inhibition compared to selective p110α or p110δ inhibitors. Inhibition of the class IB PI3K isoform p110γ did not cause cell cycle arrest or induce apoptosis in MCL cells. These findings suggest that the therapeutic ablation of class IA PI3K may be a promising strategy for the treatment of refractory, aggressive MCL. Copyright © 2013 S. Karger AG, Basel.

Fluorescein transport properties across artificial lipid membranes, Caco-2 cell monolayers and rat jejunum.

PubMed

Berginc, Katja; Zakelj, Simon; Levstik, Lea; Ursic, Darko; Kristl, Albin

2007-05-01

Membrane transport characteristics of a paracellular permeability marker fluorescein were evaluated using artificial membrane, Caco-2 cell monolayers and rat jejunum, all mounted in side-by-side diffusion cells. Modified Ringer buffers with varied pH values were applied as incubation salines on both sides of artificial membrane, cell culture monolayers or rat jejunum. Passive transport according to pH partition theory was determined using all three permeability models. In addition to that, active transport of fluorescein in the M-S (mucosal-to-serosal) direction through rat jejunum was observed. The highest M-S P(app) values regarding the active transport through the rat jejunum were observed in incubation saline with pH 6.5. Fluorescein transport through the rat jejunum was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and alpha-CHC (alpha-cyano-4-hydroxycinnamic acid). Thus, we assume that two pH-dependent influx transporters could be involved in the fluorescein membrane transport through the intestinal (jejunal) epithelium. One is very likely an MCT (monocarboxylic acid cotransporter) isoform, inhibited by specific MCT inhibitor alpha-CHC, while the involvement of the second one with overlapping substrate/inhibitor specificities (most probably a member of the organic anion-transporting polypeptide family, inhibited at least partially by DIDS) could not be excluded.

KH-type splicing regulatory protein is involved in esophageal squamous cell carcinoma progression

PubMed Central

Shoda, Katsutoshi; Naruto, Takuya; Hamada, Satoshi; Miyakami, Yuko; Kohmoto, Tomohiro; Watanabe, Miki; Takahashi, Rizu; Tange, Shoichiro; Saito, Masako; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Tangoku, Akira; Otsuji, Eigo; Imoto, Issei

2017-01-01

KH-type splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein, which is involved in several post-transcriptional aspects of RNA metabolism, including microRNA (miRNA) biogenesis. It affects distinct cell functions in different tissues and can have an impact on various pathological conditions. In the present study, we investigated the oncogenic functions of KHSRP and their underlying mechanisms in the pathogenesis of esophageal squamous cell carcinoma (ESCC). KHSRP expression levels were elevated in ESCC tumors when compared with those in non-tumorous tissues by immunohistochemistry, and cytoplasmic KHSRP overexpression was found to be an independent prognosticator for worse overall survival in a cohort of 104 patients with ESCC. KHSRP knockdown inhibited growth, migration, and invasion of ESCC cells. KHSRP knockdown also inhibited the maturation of cancer-associated miRNAs, such as miR-21, miR-130b, and miR-301, and induced the expression of their target mRNAs, such as BMP6, PDCD4, and TIMP3, resulting in the inhibition of epithelial-to-mesenchymal transition. Our findings uncover a novel oncogenic function of KHSRP in esophageal tumorigenesis and implicate its use as a marker for prognostic evaluation and as a putative therapeutic target in ESCC. PMID:29254151

3,5,6,7,8,3',4'-Heptamethoxyflavone, a Citrus Flavonoid, Inhibits Collagenase Activity and Induces Type I Procollagen Synthesis in HDFn Cells.

PubMed

Kim, Hong-Il; Jeong, Yong-Un; Kim, Jong-Hyeon; Park, Young-Jin

2018-02-22

Citrus fruits contain various types of flavonoids with powerful anti-aging and photoprotective effects on the skin, and have thus been attracting attention as potential, efficacious skincare agents. Here, we aimed to investigate the chemical composition of Citrus unshiu and its protective effects on photoaging. We isolated and identified a bioactive compound, 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF), from C. unshiu peels using ethanol extraction and hexane fractionation. HMF inhibited collagenase activity and increased type I procollagen content in UV-induced human dermal fibroblast neonatal (HDFn) cells. HMF also suppressed the expression of matrix metalloproteinases 1 (MMP-1) and induced the expression of type I procollagen protein in UV-induced HDFn cells. Additionally, HMF inhibited ultraviolet B (UVB)-induced phosphorylation of the mitogen-activated protein kinases (MAPK) cascade signaling components-ERK, JNK, and c-Jun-which are involved in the induction of MMP-1 expression. Furthermore, HMF affected the TGF-β/Smad signaling pathway, which is involved in the regulation of type I procollagen expression. In particular, HMF induced Smad3 protein expression and suppressed Smad7 protein expression in UV-induced HDFn cells in a dose-dependent manner. These findings suggest a role for Citrus unshiu in the preparation of skincare products in future.

Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation

PubMed Central

Shim, Wooyoung; Anwar, Muhammad Ayaz; Kwon, Ji-Woong; Kwon, Hyuk-Kwon; Kim, Hyung Joong; Jeong, Hyobin; Kim, Hwan Myung; Hwang, Daehee; Kim, Hyung Sik; Choi, Sangdun

2015-01-01

The chemotherapeutic use of cisplatin is limited by its severe side effects. In this study, by conducting different omics data analyses, we demonstrated that cisplatin induces cell death in a proximal tubular cell line by suppressing glycolysis- and tricarboxylic acid (TCA)/mitochondria-related genes. Furthermore, analysis of the urine from cisplatin-treated rats revealed the lower expression levels of enzymes involved in glycolysis, TCA cycle, and genes related to mitochondrial stability and confirmed the cisplatin-related metabolic abnormalities. Additionally, an increase in the level of p53, which directly inhibits glycolysis, has been observed. Inhibition of p53 restored glycolysis and significantly reduced the rate of cell death at 24 h and 48 h due to p53 inhibition. The foremost reason of cisplatin-related cytotoxicity has been correlated to the generation of mitochondrial reactive oxygen species (ROS) that influence multiple pathways. Abnormalities in these pathways resulted in the collapse of mitochondrial energy production, which in turn sensitized the cells to death. The quenching of ROS led to the amelioration of the affected pathways. Considering these observations, it can be concluded that there is a significant correlation between cisplatin and metabolic dysfunctions involving mROS as the major player. PMID:26247588

Improving the outcome of adoptive cell transfer by targeting tumor escape

PubMed Central

Kaluza, Karen M.; Vile, Richard

2013-01-01

Adoptive T-cell transfer is among the most promising immunotherapies against cancer. To continue increasing the potential of this therapy, our studies focus on the inhibition of tumor recurrence. Recently, we have demonstrated several ways in which combination therapies involving multiple T-cell populations and immunostimulatory chemotherapy can enhance long-term survival. PMID:23483796

Vaticaffinol, a resveratrol tetramer, exerts more preferable immunosuppressive activity than its precursor in vitro and in vivo through multiple aspects against activated T lymphocytes

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

Feng, Li-Li; Wu, Xue-Feng; Liu, Hai-Liang

2013-03-01

In the present study, we aimed to investigate the immunosuppressive activity of vaticaffinol, a resveratrol tetramer isolated from Vatica mangachapoi, on T lymphocytes both in vitro and in vivo, and further explored its potential molecular mechanism. Resveratrol had a wide spectrum of healthy beneficial effects with multiple targets. Interestingly, its tetramer, vaticaffinol, exerted more intensive immunosuppressive activity than resveratrol. Vaticaffinol significantly inhibited T cells proliferation activated by concanavalin A (Con A) or anti-CD3 plus anti-CD28 in a dose- and time-dependent manner. It also induced Con A-activated T cells undergoing apoptosis through mitochondrial pathway. Moreover, this compound prevented cells from enteringmore » S phase and G2/M phase during T cells activation. In addition, vaticaffinol inhibited ERK and AKT signaling pathways in Con A-activated T cells. Furthermore, vaticaffinol significantly ameliorated ear swelling in a mouse model of picryl chloride-induced ear contact dermatitis in vivo. In most of the aforementioned experiments, however, resveratrol had only slight effects on the inhibition of T lymphocytes compared with vaticaffinol. Taken together, our findings suggest that vaticaffinol exerts more preferable immunosuppressive activity than its precursor resveratrol both in vitro and in vivo by affecting multiple targets against activated T cells. - Graphical abstract: Vaticaffinol, a resveratrol tetramer isolated from Vatica mangachapoi, exerts more intensive immunosuppressive activity than its precursor resveratrol does in vitro and in vivo. Its mechanism may involve multiple effects against activated T cells: regulation of signalings involved in cell proliferation, G0/G1 arrest of T cells, as well as an apoptosis induction in activated effector T cells. Highlights: ► Vaticaffinol, a resveratrol tetramer, exerts more potent activity than its precursor. ► It inhibited T cells proliferation and prevented them from entering cell cycles. ► It led to apoptosis of activated T cells through mitochondrial pathway. ► It down-regulated ERK and AKT signaling pathways in Con A-activated T cells. ► It significantly ameliorated picryl chloride-induced ear swelling.« less

Apoptotic cell death through inhibition of protein kinase CKII activity by 3,4-dihydroxybenzaldehyde purified from Xanthium strumarium.

PubMed

Lee, Bang Hyo; Yoon, Soo-Hyun; Kim, Yun-Sook; Kim, Sang Kook; Moon, Byong Jo; Bae, Young-Seuk

2008-01-01

The CKII inhibitory compound was purified from the fruit of Xanthium strumarium by organic solvent extraction and silica gel chromatography. The inhibitory compound was identified as 3,4-dihydroxybenzaldehyde by analysis with FT-IR, FAB-Mass, EI-Mass, (1)H-NMR and (13)C-NMR. 3,4-dihydroxybenzaldehyde inhibited the phosphotransferase activity of CKII with IC(50) of about 783 microM. Steady-state studies revealed that the inhibitor acts as a competitive inhibitor with respect to the substrate ATP. A value of 138.6 microM was obtained for the apparent K(i). Concentration of 300 microM 3,4-dihydroxybenzaldehyde caused 50% growth inhibition of human cancer cell U937. 3,4-dihydroxybenzaldehyde-induced cell death was characterised with the cleavage of poly(ADP-ribose) polymerase and procaspase-3. Furthermore, the inhibitor induced the fragmentation of DNA into multiples of 180 bp, indicating that it triggered apoptosis. This induction of apoptosis by 3,4-dihydroxybenzaldehyde was also confirmed by using flow cytometry analysis. Since CKII is involved in cell proliferation and oncogenesis, these results suggest that 3,4-dihydroxybenzaldehyde may function by inhibiting oncogenic disease, at least in part, through the inhibition of CKII activity.

Sustained ERK inhibition maximizes responses of BrafV600E thyroid cancers to radioiodine

PubMed Central

Nagarajah, James; Le, Mina; Montero-Conde, Cristina; Pillarsetty, Nagavarakishore; Bolaender, Alexander; Irwin, Christopher; Krishnamoorthy, Gnana Prakasam; Larson, Steven M.; Ho, Alan L.; Seshan, Venkatraman; Ishii, Nobuya; Carrasco, Nancy; Rosen, Neal; Weber, Wolfgang A.; Fagin, James A.

2016-01-01

Radioiodide (RAI) therapy of thyroid cancer exploits the relatively selective ability of thyroid cells to transport and accumulate iodide. Iodide uptake requires expression of critical genes that are involved in various steps of thyroid hormone biosynthesis. ERK signaling, which is markedly increased in thyroid cancer cells driven by oncogenic BRAF, represses the genetic program that enables iodide transport. Here, we determined that a critical threshold for inhibition of MAPK signaling is required to optimally restore expression of thyroid differentiation genes in thyroid cells and in mice with BrafV600E-induced thyroid cancer. Although the MEK inhibitor selumetinib transiently inhibited ERK signaling, which subsequently rebounded, the MEK inhibitor CKI suppressed ERK signaling in a sustained manner by preventing RAF reactivation. A small increase in ERK inhibition markedly increased the expression of thyroid differentiation genes, increased iodide accumulation in cancer cells, and thereby improved responses to RAI therapy. Only a short exposure to the drug was necessary to obtain a maximal response to RAI. These data suggest that potent inhibition of ERK signaling is required to adequately induce iodide uptake and indicate that this is a promising strategy for the treatment of BRAF-mutant thyroid cancer. PMID:27669459

Sustained ERK inhibition maximizes responses of BrafV600E thyroid cancers to radioiodine.

PubMed

Nagarajah, James; Le, Mina; Knauf, Jeffrey A; Ferrandino, Giuseppe; Montero-Conde, Cristina; Pillarsetty, Nagavarakishore; Bolaender, Alexander; Irwin, Christopher; Krishnamoorthy, Gnana Prakasam; Saqcena, Mahesh; Larson, Steven M; Ho, Alan L; Seshan, Venkatraman; Ishii, Nobuya; Carrasco, Nancy; Rosen, Neal; Weber, Wolfgang A; Fagin, James A

2016-11-01

Radioiodide (RAI) therapy of thyroid cancer exploits the relatively selective ability of thyroid cells to transport and accumulate iodide. Iodide uptake requires expression of critical genes that are involved in various steps of thyroid hormone biosynthesis. ERK signaling, which is markedly increased in thyroid cancer cells driven by oncogenic BRAF, represses the genetic program that enables iodide transport. Here, we determined that a critical threshold for inhibition of MAPK signaling is required to optimally restore expression of thyroid differentiation genes in thyroid cells and in mice with BrafV600E-induced thyroid cancer. Although the MEK inhibitor selumetinib transiently inhibited ERK signaling, which subsequently rebounded, the MEK inhibitor CKI suppressed ERK signaling in a sustained manner by preventing RAF reactivation. A small increase in ERK inhibition markedly increased the expression of thyroid differentiation genes, increased iodide accumulation in cancer cells, and thereby improved responses to RAI therapy. Only a short exposure to the drug was necessary to obtain a maximal response to RAI. These data suggest that potent inhibition of ERK signaling is required to adequately induce iodide uptake and indicate that this is a promising strategy for the treatment of BRAF-mutant thyroid cancer.

Biochemical Characterization of Ferulic Acid and Caffeic Acid Which Effectively Inhibit Melanin Synthesis via Different Mechanisms in B16 Melanoma Cells.

PubMed

Maruyama, Hiroko; Kawakami, Fumitaka; Lwin, Thet-Thet; Imai, Motoki; Shamsa, Fazel

2018-01-01

In this study, we examined the inhibitory effects of ferulic acid and caffeic acid on melanin production using a murine B16 melanoma cell line. The mechanisms by which the two acids inhibit melanin production were investigated by evaluating their effects on the activity of tyrosinase, which is involved is the first step of melanin biosynthesis. Ferulic acid showed no toxicity against the melanoma cells at any dose, whereas caffeic acid exerted cellular toxicity at concentrations higher than 0.35 mM. Both ferulic and caffeic acids effectively inhibited melanin production in the B16 melanoma cells. Ferulic acid reduced tyrosinase activity by directly binding to the enzyme, whereas no binding was observed between caffeic acid and tyrosinase. Both ferulic acid and caffeic acid inhibited casein kinase 2 (CK2)-induced phosphorylation of tyrosinase in a dose-dependent manner in vitro. Ferulic acid was found to be a more effective inhibitor of melanin production than caffeic acid; this difference in the inhibitory efficacy between the two substances could be attributable to the difference in their tyrosine-binding activity. Our analysis revealed that both substances also inhibited the CK2-mediated phosphorylation of tyrosinase.

Voltage-Gated Na+ Channels are Modulated by Glucose and Involved in Regulating Cellular Insulin Content of INS-1 Cells.

PubMed

Chen, Chong; Wang, Songhua; Hu, Qingjuan; Zeng, Lvming; Peng, Hailong; Liu, Chao; Huang, Li-Ping; Song, Hao; Li, Yuping; Yao, Li-Hua; Meng, Wei

2018-01-01

Islet beta cells (β-cells) are unique cells that play a critical role in glucose homeostasis by secreting insulin in response to increased glucose levels. Voltage-gated ion channels in β-cells, such as K+ and Ca2+ channels, contribute to insulin secretion. The response of voltage-gated Na+ channels (VGSCs) in β-cells to the changes in glucose levels remains unknown. This work aims to determine the role of extracellular glucose on the regulation of VGSC. The effect of glucose on VGSC currents (INa) was investigated in insulin-secreting β-cell line (INS-1) cells of rats using whole-cell patch clamp techniques, and the effects of glucose on insulin content and cell viability were determined using Enzyme-Linked Immunosorbent Assay (ELISA) and Methylthiazolyldiphenyl-tetrazolium Bromide (MTT) assay methods respectively. Our results show that extracellular glucose application can inhibit the peak of INa in a concentration-dependent manner. Glucose concentration of 18 mM reduced the amplitude of INa, suppressed the INa of steady-state activation, shifted the steady-state inactivation curves of INa to negative potentials, and prolonged the time course of INa recovery from inactivation. Glucose also enhanced the activity-dependent attenuation of INa and reduced the fraction of activated channels. Furthermore, 18 mM glucose or low concentration of tetrodotoxin (TTX, a VGSC-specific blocker) partially inhibited the activity of VGSC and also improved insulin synthesis. These results revealed that extracellular glucose application enhances the insulin synthesis in INS-1 cells and the mechanism through the partial inhibition on INa channel is involved. Our results innovatively suggest that VGSC plays a vital role in modulating glucose homeostasis. © 2018 The Author(s). Published by S. Karger AG, Basel.

Rotenone-induced death of RGC-5 cells is caspase independent, involves the JNK and p38 pathways and is attenuated by specific green tea flavonoids.

PubMed

Kamalden, T A; Ji, D; Osborne, N N

2012-05-01

The aim of the present studies was to characterise cell death following inhibition of mitochondrial complex I with rotenone in a transformed cell line (RGC-5 cells) and to examine the neuroprotective properties of the flavonoids genistein, epigallocatechin gallate (EGCG), epicatechin (EC) and baicalin. Rotenone-induced cell death of RGC-5 cells results in a generation of reactive oxygen species, a breakdown of DNA, the translocation of membrane phosphatidylserine, an up-regulation of haemoxygenase-1 and is unaffected by necrostatin-1 (inhibitor of necroptosis), z-VAD-fmk (pan caspase inhibitor) or NU1025 (PARP inhibitor) but attenuated with SP600125 (JNK inhibitor). Rotenone-induced toxicity of RGC-5 cells also caused an activation of mitogen-activated kinases indicated by an up-regulation and translocation into mitochondria of p-c-Jun, pJNK and pp38. Exposure of RGC-5 cells to rotenone does not affect apoptosis inducing factor or significantly stimulate caspase-3 activity. EGCG and EC both significantly blunt rotenone toxicity of RGC-5 cells at concentrations of 50 μM while genistein and baicalin were without effect. Significantly, genistein is approximately 20 times less efficacious than EGCG (IC(50) 2.5 μM) and EC (IC(50) 1.5 μM) at inhibiting sodium nitroprusside-induced lipid peroxidation. These studies show that rotenone toxicity of RGC-5 cells is neither necroptosis nor caspase-dependent apoptosis but involves the activation of mitogen-activated kinases and is inhibited by a JNK inhibitor, EGCG and EC. Genistein attenuates lipid peroxidation less efficaciously than EC and EGCG and does not affect rotenone toxicity of RGC-5 cells.

Connexin43 synthesis, phosphorylation, and degradation in regulation of transient inhibition of gap junction intercellular communication by the phorbol ester TPA in rat liver epithelial cells.

PubMed

Rivedal, Edgar; Leithe, Edward

2005-01-15

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces transient inhibition of gap junction intercellular communication (GJIC) in several cell types. The initial block in GJIC has been attributed to protein kinase C (PKC) mediated phosphorylation of connexin gap junction proteins, including connexin43 (Cx43). Restoration of GJIC, associated with normalization of the Cx43 phosphorylation status, has been ascribed to different events, including dephosphorylation of Cx43 and de novo synthesis of Cx43 or other, non-gap junctional, proteins. The data presented suggest that restoration of GJIC during continuous TPA exposure in normal and transformed rat liver epithelial cells is dependent on synthesis of Cx43 protein, as well as the transport of already synthesized Cx43 from intracellular pools to the plasma membrane. Reactivation of inactivated Cx43 by dephosphorylation does not appear to be involved in the recovery of GJIC. Both PKC and MAP kinase is involved in TPA-induced degradation of Cx43 and inhibition of GJIC. We show that coincubation of TPA with the protein synthesis inhibitor cycloheximide or the transcription inhibitor actinomycin D results in synergistic enhancement of the level of activated ERK1/2. Together, the present data highlight Cx43 degradation and synthesis as critical determinants in TPA-induced modifications of cell-cell communication via gap junctions.

Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVβ3 and Inhibits Osteoclast Differentiation.

PubMed

Miyazaki, Tatsuya; Miyauchi, Satoshi; Anada, Takahisa; Tawada, Akira; Suzuki, Osamu

2015-10-01

Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVβ3, and inhibited the direct binding of OA to integrin αVβ3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVβ3 and HSPG. © 2015 Wiley Periodicals, Inc.

Parallel regulation of feedforward inhibition and excitation during whisker map plasticity

PubMed Central

House, David RC; Elstrott, Justin; Koh, Eileen; Chung, Jason; Feldman, Daniel E.

2011-01-01

Sensory experience drives robust plasticity of sensory maps in cerebral cortex, but the role of inhibitory circuits in this process is not fully understood. We show that classical deprivation-induced whisker map plasticity in layer 2/3 (L2/3) of rat somatosensory (S1) cortex involves robust weakening of L4-L2/3 feedforward inhibition. This weakening was caused by reduced L4 excitation onto L2/3 fast-spiking (FS) interneurons, which mediate sensitive feedforward inhibition, and was partially offset by strengthening of unitary FS to L2/3 pyramidal cell synapses. Weakening of feedforward inhibition paralleled the known weakening of feedforward excitation, so that mean excitatory-inhibitory balance and timing onto L2/3 pyramidal cells were preserved. Thus, reduced feedforward inhibition is a covert compensatory process that can maintain excitatory-inhibitory balance during classical deprivation-induced Hebbian map plasticity. PMID:22153377

Kif18A is involved in human breast carcinogenesis.

PubMed

Zhang, Chunpeng; Zhu, Changjun; Chen, Hongyan; Li, Linwei; Guo, Liping; Jiang, Wei; Lu, Shih Hsin

2010-09-01

Microtubule (MT) kinesin motor proteins orchestrate various cellular processes (e.g. mitosis, motility and organelle transportation) and have been implicated in human carcinogenesis. Kif18A, a plus-end directed MT depolymerase kinesin, regulates MT dynamics, chromosome congression and cell division. In this study, we report that Kif18A is overexpressed in human breast cancers and Kif18A overexpression is associated with tumor grade, metastasis and poor survival. Functional analyses reveal that ectopic overexpression of Kif18A results in cell multinucleation, whereas ablation of Kif18A expression significantly inhibits the proliferative capability of breast cancer cells in vitro and in vivo. Inhibition of Kif18A not only affects the critical mitotic function of Kif18A but also decreases cancer cell migration by stabilizing MTs at leading edges and ultimately induces anoikis of cells with inactivation of the phosphatidylinositol 3-kinase-Akt signaling pathway. Together, our results indicate that Kif18A is involved in human breast carcinogenesis and may serve as a potential therapeutic target for human breast cancer.

Parkin induces G2/M cell cycle arrest in TNF-α-treated HeLa cells.

PubMed

Lee, Min Ho; Cho, Yoonjung; Jung, Byung Chul; Kim, Sung Hoon; Kang, Yeo Wool; Pan, Cheol-Ho; Rhee, Ki-Jong; Kim, Yoon Suk

2015-08-14

Parkin is a known tumor suppressor. However, the mechanism by which parkin acts as a tumor suppressor remains to be fully elucidated. Previously, we reported that parkin expression induces caspase-dependent apoptotic cell death in TNF-α-treated HeLa cells. However, at that time, we did not consider the involvement of parkin in cell cycle control. In the current study, we investigated whether parkin is involved in cell cycle regulation and suppression of cancer cell growth. In our cell cycle analyses, parkin expression induced G2/M cell cycle arrest in TNF-α-treated HeLa cells. To elucidate the mechanism(s) by which parkin induces this G2/M arrest, we analyzed cell cycle regulatory molecules involved in the G2/M transition. Parkin expression induced CDC2 phosphorylation which is known to inhibit CDC2 activity and cause G2/M arrest. Cyclin B1, which is degraded during the mitotic transition, accumulated in response to parkin expression, thereby indicating parkin-induced G2/M arrest. Next, we established that Myt1, which is known to phosphorylate and inhibit CDC2, increased following parkin expression. In addition, we found that parkin also induces increased Myt1 expression, G2/M arrest, and reduced cell viability in TNF-α-treated HCT15 cells. Furthermore, knockdown of parkin expression by parkin-specific siRNA decreased Myt1 expression and phosphorylation of CDC2 and resulted in recovered cell viability. These results suggest that parkin acts as a crucial molecule causing cell cycle arrest in G2/M, thereby suppressing tumor cell growth. Copyright © 2015 Elsevier Inc. All rights reserved.

Fisetin inhibits human melanoma cell growth through direct binding to p70S6K and mTOR: findings from 3-D melanoma skin equivalents and computational modeling

PubMed Central

Syed, Deeba N.; Chamcheu, Jean-Christopher; Khan, Mohammad Imran; Sechi, Mario; Lall, Rahul K.; Adhami, Vaqar M.; Mukhtar, Hasan

2014-01-01

The incidence of melanoma continues to rise. Inspite of treatment advances, the prognosis remains grim once the disease has metastasized, emphasizing the need to explore additional therapeutic strategies. One such approach is through the use of mechanism-based dietary intervention. We previously showed that the flavonoid fisetin inhibits melanoma cell proliferation, in vitro and in vivo. Here, we studied fisetin-mediated regulation of kinases involved in melanoma growth and progression. Time-course analysis in 3-D melanoma constructs that transitioned from radial to vertical growth showed that fisetin treatment resulted in significant decrease in melanocytic lesions in contrast to untreated controls that showed large tumor nests and invading disseminated cells. Further studies in melanoma cultures and mouse xenografts showed that fisetin-mediated growth inhibition was associated with dephosphorylation of AKT, mTOR and p70S6K proteins. In silico modeling indicated direct interaction of fisetin with mTOR and p70S6K with favorable free energy values. These findings were validated by cell-free competition assays that established binding of fisetin to p70S6K and mTOR while little affinity was detected with AKT. Kinase activity studies reflected similar trend with % inhibition observed for p70S6K and mTOR at lower doses than AKT. Our studies characterized, for the first time, the differential interactions of any botanical agent with kinases involved in melanoma growth and demonstrate that fisetin inhibits mTOR and p70S6K through direct binding while the observed inhibitory effect of fisetin on AKT is mediated indirectly, through targeting interrelated pathways. PMID:24675012

Fisetin inhibits human melanoma cell growth through direct binding to p70S6K and mTOR: findings from 3-D melanoma skin equivalents and computational modeling.

PubMed

Syed, Deeba N; Chamcheu, Jean-Christopher; Khan, Mohammad Imran; Sechi, Mario; Lall, Rahul K; Adhami, Vaqar M; Mukhtar, Hasan

2014-06-01

The incidence of melanoma continues to rise. Inspite of treatment advances, the prognosis remains grim once the disease has metastasized, emphasizing the need to explore additional therapeutic strategies. One such approach is through the use of mechanism-based dietary intervention. We previously showed that the flavonoid fisetin inhibits melanoma cell proliferation, in vitro and in vivo. Here, we studied fisetin-mediated regulation of kinases involved in melanoma growth and progression. Time-course analysis in 3-D melanoma constructs that transitioned from radial to vertical growth showed that fisetin treatment resulted in significant decrease in melanocytic lesions in contrast to untreated controls that showed large tumor nests and invading disseminated cells. Further studies in melanoma cultures and mouse xenografts showed that fisetin-mediated growth inhibition was associated with dephosphorylation of AKT, mTOR and p70S6K proteins. In silico modeling indicated direct interaction of fisetin with mTOR and p70S6K with favorable free energy values. These findings were validated by cell-free competition assays that established binding of fisetin to p70S6K and mTOR while little affinity was detected with AKT. Kinase activity studies reflected similar trend with % inhibition observed for p70S6K and mTOR at lower doses than AKT. Our studies characterized, for the first time, the differential interactions of any botanical agent with kinases involved in melanoma growth and demonstrate that fisetin inhibits mTOR and p70S6K through direct binding while the observed inhibitory effect of fisetin on AKT is mediated indirectly, through targeting interrelated pathways. Published by Elsevier Inc.

Escin Ia suppresses the metastasis of triple-negative breast cancer by inhibiting epithelial-mesenchymal transition via down-regulating LOXL2 expression.

PubMed

Wang, Yuhui; Xu, Xiaotian; Zhao, Peng; Tong, Bei; Wei, Zhifeng; Dai, Yue

2016-04-26

The saponin fraction of Aesculus chinensis Bunge fruits (SFAC) could inhibit the invasion and migration of MDA-MB-231 cells. Among which, escin Ia showed more potent inhibition of the invasion than other five main saponin constituents. It selectively reduced the expression of LOXL2 mRNA and promoted the expression of E-cadherin mRNA, and prevented the EMT process of MDA-MB-231 cells and TNF-α/TGF-β-stimulated MCF-7 cells. Moreover, it reduced the LOXL2 level in MDA-MB-231 cells but not in MCF-7 cells. When MCF-7 cells were stimulated with TNF-α/TGF-β, transfected with LOXL2 or treated with hypoxia, escin Ia down-regulated the level of LOXL2 in MCF-7 cells. Meanwhile, escin Ia suppressed the EMT process in LOXL2-transfected or hypoxia-treated MCF-7 cells. Of interest, escin Ia did not alter the level of HIF-1α in hypoxia-induced MCF-7 cells. In TNBC xenograft mice, the metastasis and EMT of MDA-MB-231 cells were suppressed by escin Ia. In conclusion, escin Ia was the main active ingredient of SFAC for the anti-TNBC metastasis activity, and its action mechanisms involved inhibition of EMT process by down-regulating LOXL2 expression.

Escin Ia suppresses the metastasis of triple-negative breast cancer by inhibiting epithelial-mesenchymal transition via down-regulating LOXL2 expression

PubMed Central

Zhao, Peng; Tong, Bei; Wei, Zhifeng; Dai, Yue

2016-01-01

The saponin fraction of Aesculus chinensis Bunge fruits (SFAC) could inhibit the invasion and migration of MDA-MB-231 cells. Among which, escin Ia showed more potent inhibition of the invasion than other five main saponin constituents. It selectively reduced the expression of LOXL2 mRNA and promoted the expression of E-cadherin mRNA, and prevented the EMT process of MDA-MB-231 cells and TNF-α/TGF-β-stimulated MCF-7 cells. Moreover, it reduced the LOXL2 level in MDA-MB-231 cells but not in MCF-7 cells. When MCF-7 cells were stimulated with TNF-α/TGF-β, transfected with LOXL2 or treated with hypoxia, escin Ia down-regulated the level of LOXL2 in MCF-7 cells. Meanwhile, escin Ia suppressed the EMT process in LOXL2-transfected or hypoxia-treated MCF-7 cells. Of interest, escin Ia did not alter the level of HIF-1α in hypoxia-induced MCF-7 cells. In TNBC xenograft mice, the metastasis and EMT of MDA-MB-231 cells were suppressed by escin Ia. In conclusion, escin Ia was the main active ingredient of SFAC for the anti-TNBC metastasis activity, and its action mechanisms involved inhibition of EMT process by down-regulating LOXL2 expression. PMID:27008697

[Mechanism of apoptosis of nasopharyngeal carcinoma cells induced by polysaccharides extracts from Hedyotic diffusa].

PubMed

Jing Yan; Kang Min; Liu Jin; Li Jingyu; Tang Anzhou

2015-04-01

To explore the proliferation inhibition and apoptosis of polysaccharides extracts from polysaccharides extracts from Hedyotic diffusa (PEHD) on Human Nasopharyngeal Carcinoma (NPC)cell line CNE2 cells in vitro. CNE2 cells treated with various concentrations of PEHD were detected by MTT assay at 24 h, 48 h, and 72 h. The apoptotic cells were analyzed by flow cytometry with Annexin V/PI staining. The expression levels of Bax, Bcl-2 and caspase-3 protein were examined by Western blotting method. The growth of CNE2 cells were suppressed after treatment with PEHD (P < 0.05), MTT assay showed that the highest cell inhibition rate reached to 76.5%, the inhibition in the doses from 2 to 6 mg/ml showed dose-and-time-dependent. The percent of apoptosis in 4 and 6 mg/ml PEHD treatment groups for 48 h were 31.32%, 46.28%, respectively, and significantly higher than that in control groups, 4.86% (P < 0.01). After the cells being treated with PEHD for 48 h, the expression of Bax and caspase-3 protein increased, and the expression of Bcl-2 protein decreased gradually. PEHD could inhibited the growth of CNE2 cells and was dose-and-time-dependent, the mechanism may involve induction of cell apoptosis, which was associated with the activation of Bax and caspase-3 protein and the down-regulation of Bcl-2 protein expression.

Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation.

PubMed

Yan, Dan; He, Yujuan; Dai, Jun; Yang, Lili; Wang, Xiaoyan; Ruan, Qiurong

2017-06-30

Macrophages are largely involved in the whole process of atherosclerosis from an initiation lesion to an advanced lesion. Endothelial disruption is the initial step and macrophage-derived foam cells are the hallmark of atherosclerosis. Promotion of vascular integrity and inhibition of foam cell formation are two important strategies for preventing atherosclerosis. How can we inhibit even the reverse negative role of macrophages in atherosclerosis? The present study was performed to investigate if overexpressing endogenous human vascular endothelial growth factor (VEGF) could facilitate transdifferentiation of macrophages into endothelial-like cells (ELCs) and inhibit foam cell formation. We demonstrated that VEGF-modified macrophages which stably overexpressed human VEGF (hVEGF 165 ) displayed a high capability to alter their phenotype and function into ELCs in vitro Exogenous VEGF could not replace endogenous VEGF to induce the transdifferentiation of macrophages into ELCs in vitro We further showed that VEGF-modified macrophages significantly decreased cytoplasmic lipid accumulation after treatment with oxidized LDL (ox-LDL). Moreover, down-regulation of CD36 expression in these cells was probably one of the mechanisms of reduction in foam cell formation. Our results provided the in vitro proof of VEGF-modified macrophages as atheroprotective therapeutic cells by both promotion of vascular repair and inhibition of foam cell formation. © 2017 The Author(s).

Inhibition of IGF-1-Mediated Cellular Migration and Invasion by Migracin A in Ovarian Clear Cell Carcinoma Cells.

PubMed

Ukaji, Tamami; Lin, Yinzhi; Banno, Kouji; Okada, Shoshiro; Umezawa, Kazuo

2015-01-01

Previously we isolated migracin A from a Streptomyces culture filtrate as an inhibitor of cancer cell migration. In the present research, we found that migracin A inhibited migration and invasion of ovarian clear cell carcinoma ES-2 cells. In the course of our mechanistic study, migracin A was shown to enhance vasohibin-1 expression in an angiogenesis array. We also confirmed that it increased the mRNA expression of this protein. Moreover, overexpression of vasohibin-1 lowered the migration but not the invasion of ES-2 cells. Then, we looked for another target protein employing a motility array, and found that migracin A lowered the IGF-1 expression. Knockdown of IGF-1 by siRNA decreased the migration and invasion of ES-2 cells. Migracin A also decreased Akt phosphorylation involved in the downstream signaling. Crosstalk analysis indicated that overexpression of vasohibin-1 decreased the IGF-1 expression. On the other hand, it showed no direct anticancer activity in terms of the ES-2 growth in agar. Migracin A inhibited the migration and IGF-1 expression in not only ES-2 but also another ovarian clear cell carcinoma JHOC-5 cells. In addition, it also inhibited capillary tube formation of human umbilical vein endothelial cells. Since its cytotoxicity is very low, migracin A may be a candidate for an anti-metastasis agent not exhibiting prominent toxicity.

MK-2206, an AKT Inhibitor, Promotes Caspase-Independent Cell Death and Inhibits Leiomyoma Growth

PubMed Central

Sefton, Elizabeth C.; Qiang, Wenan; Serna, Vanida; Kurita, Takeshi; Wei, Jian-Jun; Chakravarti, Debabrata

2013-01-01

Uterine leiomyomas (ULs), benign tumors of the myometrium, are the number one indication for hysterectomies in the United States due to a lack of an effective alternative therapy. ULs show activation of the pro-survival AKT pathway compared with normal myometrium; however, substantial data directly linking AKT to UL cell survival are lacking. We hypothesized that AKT promotes UL cell survival and that it is a viable target for inhibiting UL growth. We used the investigational AKT inhibitor MK-2206, currently in phase II trials, on cultured primary human UL and myometrial cells, immortalized leiomyoma cells, and in leiomyoma grafts grown under the kidney capsule in mice. MK-2206 inhibited AKT and PRAS40 phosphorylation but did not regulate serum- and glucocorticoid-induced kinase and ERK1/2, demonstrating its specificity for AKT. MK-2206 reduced UL cell viability and decreased UL tumor volumes. UL cells exhibited disruption of mitochondrial structures and underwent cell death that was independent of caspases. Additionally, mammalian target of rapamycin and p70S6K phosphorylation were reduced, indicating that mammalian target of rapamycin complex 1 signaling was compromised by AKT inhibition in UL cells. MK-2206 also induced autophagy in UL cells. Pretreatment of primary UL cells with 3-methyladenine enhanced MK-2206-mediated UL cell death, whereas knockdown of ATG5 and/or ATG7 did not significantly influence UL cell viability in the presence of MK-2206. Our data provide molecular evidence for the involvement of AKT in UL cell survival and suggest that AKT inhibition by MK-2206 may be a viable option to consider for the treatment of ULs. PMID:24002033

Nrf2 mediates redox adaptation in NOX4-overexpressed non-small cell lung cancer cells

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

Wu, Qipeng; Yao, Bei; Li, Ning

The redox adaptation mechanisms in cancer cells are very complex and remain largely unclear. Our previous studies have confirmed that NADPH oxidase 4 (NOX4) is abundantly expressed in non-small cell lung cancer (NSCLC) and confers apoptosis resistance on NSCLC cells. However, the comprehensive mechanisms for NOX4-mediated oxidative resistance of cancer cells remain still undentified. The present study found that NOX4-derived H{sub 2}O{sub 2} enhanced the nuclear factor erythroid 2-related factor 2 (Nrf2) stability via disruption of redox-dependent proteasomal degradation and stimulated its activity through activation of PI3K signaling. Specifically, the results showed that ectopic NOX4 expression did not induce apoptosismore » of A549 cells; however, inhibition of Nrf2 resulted in obvious apoptotic death of NOX4-overexpressed A549 cells, accompanied by a significant increase in H{sub 2}O{sub 2} level and decrease in GSH content. Besides, inhibition of Nrf2 could suppress cell growth and efficiently reverse the enhancement effect of NOX4 on cell growth. The in vivo data confirmed that inhibition of Nrf2 could interfere apoptosis resistance in NOX4-overexpressed A549 tumors and led to cell growth inhibition. In conclusion, these results reveal that Nrf2 is critically involved in redox adaptation regulation in NOX4-overexpressed NSCLC cells. Therefore, NOX4 and Nrf2 may be promising combination targets against malignant progression of NSCLC. - Highlights: • NOX4-derived H{sub 2}O{sub 2} upregulates Nrf2 expression and activity in NSCLC. • Nrf2 confers apoptosis resistance in NOX4-overexpressed NSCLC cells. • Inhibition of Nrf2 reverses the enhancement effect of NOX4 on cell growth.« less

Resveratrol suppresses TPA-induced matrix metalloproteinase-9 expression through the inhibition of MAPK pathways in oral cancer cells.

PubMed

Lin, Feng-Yan; Hsieh, Yi-Hsien; Yang, Shun-Fa; Chen, Chang-Tai; Tang, Chih-Hsin; Chou, Ming-Yung; Chuang, Yi-Ting; Lin, Chiao-Wen; Chen, Mu-Kuan

2015-10-01

Naturally occurring agents, such as resveratrol, have been determined to benefit health. Numerous studies have demonstrated that resveratrol has antioxidative, cardioprotective, and neuroprotective properties. However, the effect of resveratrol exerts on the metastasis of oral cancer cells remains unclear. In this study, we investigated the effect the anti-invasive activity of resveratrol on a human oral cancer cell line (SCC-9) in vitro and the underlying mechanisms. Cell viability was examined by MTT assay, whereas cell motility was measured by migration and wound-healing assays. Zymography, reverse-transcriptase polymerase chain reaction (PCR), and promoter assays confirmed the inhibitory effects of resveratrol on matrix metalloproteinase-9 (MMP-9) expression in oral cancer cells. We established that various concentrations (0-100 μM) of resveratrol inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced migration capacities of SCC-9 cells and caused no cytotoxic effects. Zymography and Western blot analyses suggested that resveratrol inhibited TPA-induced MMP-9 gelatinolytic activity and protein expression. In addition, the results indicated that resveratrol inhibited the phosphorylation of c-Jun N-terminal kinase (JNK)1/2 and extracellular-signal-regulated kinase (ERK)1/2 involved in downregulating protein expression and the transcription of MMP-9. In summary, resveratrol inhibited MMP-9 expression and oral cancer cell metastasis by downregulating JNK1/2 and ERK1/2 signals pathways and, thus, exerts beneficial effects in chemoprevention. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Silymarin suppresses the PGE2 -induced cell migration through inhibition of EP2 activation; G protein-dependent PKA-CREB and G protein-independent Src-STAT3 signal pathways.

PubMed

Woo, Seon Min; Min, Kyoung-Jin; Chae, In Gyeong; Chun, Kyung-Soo; Kwon, Taeg Kyu

2015-03-01

Silymarin has been known as a chemopreventive agent, and possesses multiple anti-cancer activities including induction of apoptosis, inhibition of proliferation and growth, and blockade of migration and invasion. However, whether silymarin could inhibit prostaglandin (PG) E2 -induced renal cell carcinoma (RCC) migration and what are the underlying mechanisms are not well elucidated. Here, we found that silymarin markedly inhibited PGE2 -stimulated migration. PGE2 induced G protein-dependent CREB phosphorylation via protein kinase A (PKA) signaling, and PKA inhibitor (H89) inhibited PGE2 -mediated migration. Silymarin reduced PGE2 -induced CREB phosphorylation and CRE-promoter activity. PGE2 also activated G protien-independent signaling pathways (Src and STAT3) and silymarin reduced PGE2 -induced phosphorylation of Src and STAT3. Inhibitor of Src (Saracatinib) markedly reduced PGE2 -mediated migration. We found that EP2, a PGE2 receptor, is involved in PGE2 -mediated cell migration. Down regulation of EP2 by EP2 siRNA and EP2 antagonist (AH6809) reduced PGE2 -inudced migration. In contrast, EP2 agonist (Butaprost) increased cell migration and silymarin effectively reduced butaprost-mediated cell migration. Moreover, PGE2 increased EP2 expression through activation of positive feedback mechanism, and PGE2 -induced EP2 expression, as well as basal EP2 levels, were reduced in silymarin-treated cells. Taken together, our study demonstrates that silymarin inhibited PGE2 -induced cell migration through inhibition of EP2 signaling pathways (G protein dependent PKA-CREB and G protein-independent Src-STAT3). © 2013 Wiley Periodicals, Inc.

Bax/Tubulin/Epithelial-Mesenchymal Pathways Determine the Efficacy of Silybin Analog HM015k in Colorectal Cancer Cell Growth and Metastasis.

PubMed

Amawi, Haneen; Hussein, Noor A; Ashby, Charles R; Alnafisah, Rawan; Sanglard, Leticia M; Manivannan, Elangovan; Karthikeyan, Chandrabose; Trivedi, Piyush; Eisenmann, Kathryn M; Robey, Robert W; Tiwari, Amit K

2018-01-01

The inhibition of apoptosis, disruption of cellular microtubule dynamics, and over-activation of the epithelial mesenchymal transition (EMT), are involved in the progression, metastasis, and resistance of colorectal cancer (CRC) to chemotherapy. Therefore, the design of a molecule that can target these pathways could be an effective strategy to reverse CRC progression and metastasis. In this study, twelve novel silybin derivatives, HM015a-HM015k (15a-15k) and compound 17, were screened for cytotoxicity in CRC cell lines. Compounds HM015j and HM015k (15k and 15j) significantly decreased cell proliferation, inhibited colony formation, and produced cell cycle arrest in CRC cells. Furthermore, 15k significantly induced the formation of reactive oxygen species and apoptosis. It induced the cleavage of the intrinsic apoptotic protein (Bax p21) to its more efficacious fragment, p18. Compound 15k also inhibited tubulin expression and disrupted its structure. Compound 15k significantly decreased metastatic LOVO cell migration and invasion. Furthermore, 15k reversed mesenchymal morphology in HCT116 and LOVO cells. Additionally, 15k significantly inhibited the expression of the mesenchymal marker N-cadherin and upregulated the expression of the epithelial marker, E-cadherin. Compound 15k inhibited the expression of key proteins known to induce EMT (i.e., DVL3, β-catenin, c-Myc) and upregulated the anti-metastatic protein, cyclin B1. Overall, in vitro , 15k significantly inhibited CRC progression and metastasis by inhibiting apoptosis, tubulin activity and the EMT pathways. Overall, these data suggest that compound 15k should be tested in vivo in a CRC animal model for further development.

Methylselenol, a selenium metabolite, induces cell cycle arrest in G1 phase and apoptosis via the extracellular-regulated kinase 1/2 pathway and other cancer signaling genes.

PubMed

Zeng, Huawei; Wu, Min; Botnen, James H

2009-09-01

Methylselenol has been hypothesized to be a critical selenium (Se) metabolite for anticancer activity in vivo, and our previous study demonstrated that submicromolar methylselenol generated by incubating methionase with seleno-l-methionine inhibits the migration and invasive potential of HT1080 tumor cells. However, little is known about the association between cancer signal pathways and methylselenol's inhibition of tumor cell invasion. In this study, we demonstrated that methylselenol exposure inhibited cell growth and we used a cancer signal pathway-specific array containing 15 different signal transduction pathways involved in oncogenesis to study the effect of methylselenol on cellular signaling. Using real-time RT-PCR, we confirmed that cellular mRNA levels of cyclin-dependent kinase inhibitor 1C (CDKN1C), heme oxygenase 1, platelet/endothelial cell adhesion molecule, and PPARgamma genes were upregulated to 2.8- to 5.7-fold of the control. BCL2-related protein A1, hedgehog interacting protein, and p53 target zinc finger protein genes were downregulated to 26-52% of the control, because of methylselenol exposure. These genes are directly related to the regulation of cell cycle and apoptosis. Methylselenol increased apoptotic cells up to 3.4-fold of the control and inhibited the extracellular-regulated kinase 1/2 (ERK1/2) signaling and cellular myelocytomatosis oncogene (c-Myc) expression. Taken together, our studies identify 7 novel methylselenol responsive genes and demonstrate that methylselenol inhibits ERK1/2 pathway activation and c-Myc expression. The regulation of these genes is likely to play a key role in G1 cell cycle arrest and apoptosis, which may contribute to the inhibition of tumor cell invasion.

Antiproliferative and Apoptotic Effect of Dendrosomal Curcumin Nanoformulation in P53 Mutant and Wide-Type Cancer Cell Lines.

PubMed

Montazeri, Maryam; Pilehvar-Soltanahmadi, Younes; Mohaghegh, Mina; Panahi, Alireza; Khodi, Samaneh; Zarghami, Nosratollah; Sadeghizadeh, Majid

2017-01-01

The aim of this paper is to investigate the effect of dendrosomal curcumin (DNC) on the expression of p53 in both p53 mutant cell lines SKBR3/SW480 and p53 wild-type MCF7/HCT116 in both RNA and protein levels. Curcumin, derived from Curcumin longa, is recently considered in cancer related researches for its cell growth inhibition properties. p53 is a common tumor-suppressor gene involved in cancers and its mutation not only inhibits tumor suppressor activity but also promotes oncogenic activity. Here, p53 mutant/Wild-type cells were employed to study the toxicity of DNC using MTT assay, Flow cytometry and Annexin-V, Real-time PCR and Western blot were used to analyze p53, BAX, Bcl-2, p21 and Noxa changes after treatment. During the time, DNC increased the SubG1 cells and decreased G1, S and G2/M cells, early apoptosis also indicated the inhibition of cell growth in early phase. Real-Time PCR assay showed an increased mRNA of BAX, Noxa and p21 during the time with decreased Bcl-2. The expression of p53 mutant decreased in SKBR3/SW480, and the expression of p53 wild-type increased in MCF7/HCT116. Consequently, p53 plays an important role in mediating the survival by DNC, which can prevent tumor cell growth by modulating the expression of genes involved in apoptosis and proliferation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Analysis of repair and PCNA complex formation induced by ionizing radiation in human fibroblast cell lines.

PubMed

Karmakar, P; Balajee, A S; Natarajan, A T

2001-05-01

Proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerase delta and epsilon, is involved in both DNA replication and repair. Previous studies in vitro have demonstrated the requirement of PCNA in the resynthesis step of nucleotide excision repair (NER) and base excision repair (BER). Using a native chromatin template isolated under near physiological conditions, we have analysed the involvement of PCNA in the BER pathway in different NER defective human cell lines. The repair sites and PCNA were visualized by indirect immunolabelling followed by fluorescence microscopy. The results indicate that exposure to X-rays triggers the induction of PCNA in all the three human fibroblast cell lines studied, namely normal, xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). In all the cell lines, induction of PCNA and repair patches occurred in a dose- and time-dependent fashion. Induction of repair patches in NER-deficient XP-A cells suggests that the X-ray-induced lesions are largely repaired via the BER pathway involving PCNA as one of the key components of this pathway. X-ray-induced repair synthesis was greatly inhibited by treatment of cells with DNA polymerase inhibitors aphidicolin and cytosine arabinoside. Interestingly, inhibition of repair resynthesis did not affect the intensity of PCNA staining in X-irradiated cells indicating that the PCNA may be required for the BER pathway at a step preceding the resynthesis step.

Effects of tumour necrosis factor α upon the metabolism of the endocannabinoid anandamide in prostate cancer cells.

PubMed

Karlsson, Jessica; Gouveia-Figueira, Sandra; Alhouayek, Mireille; Fowler, Christopher J

2017-01-01

Tumour necrosis factor α (TNFα) is involved in the pathogenesis of prostate cancer, a disease where disturbances in the endocannabinoid system are seen. In the present study we have investigated whether treatment of DU145 human prostate cancer cells affects anandamide (AEA) catabolic pathways. Additionally, we have investigated whether cyclooxygenase-2 (COX-2) can regulate the uptake of AEA into cells. Levels of AEA synthetic and catabolic enzymes were determined by qPCR. AEA uptake and hydrolysis in DU145 and RAW264.7 macrophage cells were assayed using AEA labeled in the arachidonic and ethanolamine portions of the molecule, respectively. Levels of AEA, related N-acylethanolamines (NAEs), prostaglandins (PG) and PG-ethanolamines (PG-EA) in DU145 cells and medium were quantitated by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. TNFα treatment of DU145 cells increased mRNA levels of PTSG2 (gene of COX-2) and decreased the mRNA of the AEA synthetic enzyme N-acyl-phosphatidylethanolamine selective phospholipase D. mRNA levels of the AEA hydrolytic enzymes fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase were not changed. AEA uptake in both DU145 and RAW264.7 cells was inhibited by FAAH inhibition, but not by COX-2 inhibition, even in RAW264.7 cells where the expression of this enzyme had greatly been induced by lipopolysaccharide + interferon γ treatment. AEA and related NAEs were detected in DU145 cells, but PGs and PGE2-EA were only detected when the cells had been preincubated with 100 nM AEA. The data demonstrate that in DU145 cells, TNFα treatment changes the relative expression of the enzymes involved in the hydrolytic and oxygenation catabolic pathways for AEA. In RAW264.7 cells, COX-2, in contrast to FAAH, does not regulate the cellular accumulation of AEA. Further studies are necessary to determine the extent to which inflammatory mediators are involved in the abnormal endocannabinoid signalling system in prostate cancer.

The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells.

PubMed

Jakab, Martin; Ketterl, Nina; Fürst, Johannes; Beyreis, Marlena; Kittl, Michael; Kiesslich, Tobias; Hauser-Kronberger, Cornelia; Gaisberger, Martin; Ritter, Markus

2017-01-01

Glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem), activation of voltage-activated Ca2+ currents (ICav) and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi) or cell volume, which also affect β-cell viability, can elicit or modify insulin release. In β-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi) recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume (MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. We found that the α-subunit of the non-gastric H+/K+ ATPase (HKα2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release was unaffected. The L-type ICav blocker nifedipine caused a full inhibition of GSIS at 10 and 20 µM. At 20 µM, SCH-28080 inhibited ICav comparable to 20 µM nifedipine and in addition augmented IKATP recorded at -60 mV and hyperpolarized Vmem by ∼15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC50 values of 22.9 µM and 15.3 µM, respectively. At 20 µM the percentages of Annexin-V+, caspase+ and propidium iodide+ cells were significantly increased after 24 and 48 h. Concurrently, the MCV was significantly decreased (apoptotic volume decrease, AVD) and the SSC signal was increased. At concentrations >40-50 µM, SCH-28080 became progressively cytotoxic causing a steep increase in necrotic cells already 2 h post treatment and a breakdown of ΔΨm within 4 h under 50 and 100 µM while 10 and 20 µM had no effect on ΔΨm within 24 h. We demonstrate expression of HKα2 in rat INS-1E cells. However, the pump is apparently non-functional under the given conditions. Nonetheless the H+/K+ ATPase blocker SCH-28080 inhibits insulin secretion and induces cell death. Importantly, we show that SCH-28080 inhibits ICav - and activates KATP channels identifying them as novel "off-targets" of the inhibitor, causing hyperpolarization of Vmem and inhibition of insulin secretion. © 2017 The Author(s). Published by S. Karger AG, Basel.

In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

PubMed

Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M

2014-04-01

A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

Antioxidative Dietary Compounds Modulate Gene Expression Associated with Apoptosis, DNA Repair, Inhibition of Cell Proliferation and Migration

PubMed Central

Wang, Likui; Gao, Shijuan; Jiang, Wei; Luo, Cheng; Xu, Maonian; Bohlin, Lars; Rosendahl, Markus; Huang, Wenlin

2014-01-01

Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2) called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE), which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix) polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen) and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair. PMID:25226533

Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in primary porcine endothelial cells.

PubMed

Autheman, Delphine; Wyder, Marianne; Popoff, Michel; D'Herde, Katharina; Christen, Stephan; Posthaus, Horst

2013-01-01

Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis").

Muscle FBPase binds to cardiomyocyte mitochondria under glycogen synthase kinase-3 inhibition or elevation of cellular Ca2+ level.

PubMed

Gizak, Agnieszka; Pirog, Michal; Rakus, Dariusz

2012-01-02

A growing body of research suggests that fructose 1,6-bisphosphatase (FBPase) might be involved in regulation of cell mortality/survival. However, the precise role of FBPase in the process remains unknown. Here, we show for the first time that in HL-1 cardiomyocytes, inhibition of glycogen synthase kinase-3 results in translocation of FBPase to mitochondria. In vitro experiments demonstrate that FBPase reduces the rate of calcium-induced mitochondrial swelling, affects ATP synthesis and interacts with mitochondrial proteins involved in regulation of volume and energy homeostasis. We suggest that FBPase might be engaged in a regulation of cell survival by influencing mitochondrial function. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Cbl-b-regulated extracellular signal-regulated kinase signaling is involved in the shikonin-induced apoptosis of lung cancer cells in vitro

PubMed Central

QU, DAN; CHEN, YU; XU, XIAO-MAN; ZHANG, MENG; ZHANG, YI; LI, SHENG-QI

2015-01-01

Shikonin (SK), a naturally occurring naphthoquinone, exhibits antitumor activity. However, its precise mechanisms of action are unknown. In the present study, the effects of SK on NCI-H460 human lung cancer cells were investigated. It was found that SK reduced cell viability and induced apoptosis in the NCI-H460 cells. Additionally, SK inhibited extracellular signal-regulated kinase (ERK) activity, which indicates that inhibition of the ERK pathway is probably one of the mechanisms by which SK induced NCI-H460 cell apoptosis. The expression of Cbl-b was significantly increased by treatment with SK for 4 h, and gradually increased to a maximal level at 24 h; the time taken for the upregulation of Cbl-b protein was in accordance to that required for the downregulation of phospho (p)-ERK protein. The Cbl inhibitor Ps341 reversed the SK-induced downregulation of p-ERK and apoptosis of NCI-H460 cells. These results indicate that Cbl-b potentiates the apoptotic action of SK by inhibiting the ERK pathway in lung cancer cells. PMID:25780420

Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity

PubMed Central

Kim, Won Sam; Kim, Mi Jeong; Kim, Dong Oh; Byun, Jae-Eun; Huy, Hangsak; Song, Hae Young; Park, Young-Jun; Kim, Tae-Don; Yoon, Suk Ran; Choi, Eun-Ji; Jung, Haiyoung; Choi, Inpyo

2017-01-01

Suppressor of cytokine signaling (SOCS) proteins are negative regulators of cytokine responses. Although recent reports have shown regulatory roles for SOCS proteins in innate and adaptive immunity, their roles in natural killer (NK) cell development are largely unknown. Here, we show that SOCS2 is involved in NK cell development. SOCS2−/− mice showed a high frequency of NK cells in the bone marrow and spleen. Knockdown of SOCS2 was associated with enhanced differentiation of NK cells in vitro, and the transplantation of hematopoietic stem cells (HSCs) into congenic mice resulted in enhanced differentiation in SOCS2−/− HSCs. We found that SOCS2 could inhibit Janus kinase 2 (JAK2) activity and JAK2-STAT5 signaling pathways via direct interaction with JAK2. Furthermore, SOCS2−/− mice showed a reduction in lung metastases and an increase in survival following melanoma challenge. Overall, our findings suggest that SOCS2 negatively regulates the development of NK cells by inhibiting JAK2 activity via direct interaction. PMID:28383049

Inhibition by TNF-alpha and IL-4 of cationic lipid mediated gene transfer in cystic fibrosis tracheal gland cells.

PubMed

Bastonero, Sonia; Gargouri, Myriem; Ortiou, Sandrine; Guéant, Jean-Louis; Merten, Marc D

2005-11-01

In vivo, tracheal gland serous cells highly express the cystic fibrosis transmembrane conductance regulator (cftr) gene. This gene is mutated in the lethal monogenic disease cystic fibrosis (CF). Clinical trials in which the human CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in weak and transient gene expression. As CF is characterized by mucus inspissation, airway infection, and severe inflammation, we tested the hypothesis that inflammation and especially two cytokines involved in the Th1/Th2 inflammatory response, interleukin 4 (IL-4) and TNFalpha, could inhibit gene transfer efficiency using a model of human CF tracheal gland cells (CF-KM4) and Lipofectamine reagent as a transfection reagent. The specific secretory defects of CF-KM4 cells were corrected by Lipofectamine-mediated human CFTR gene transfer. However, this was altered when cells were pre-treated with IL-4 and TNFalpha. Inhibition of luciferase reporter gene expression by IL-4 and TNFalpha pre-treated CF-KM4 cells was measured by activity and real-time RT-PCR. Both cytokines induced similar and synergistic inhibition of transgene expression and activity. This cytokine-mediated inhibition could be prevented by anti-inflammatory agents such as glucocorticoids but not by non-steroidal (NSAI) agents. This data suggests that an inflammatory context generated by IL-4 and TNFalpha can inhibit human CFTR gene transfer in CF tracheal gland cells and that glucocorticoids may have a protecting action. Copyright (c) 2005 John Wiley & Sons, Ltd.

Inhibition of H3K27me3 Histone Demethylase Activity Prevents the Proliferative Regeneration of Zebrafish Lateral Line Neuromasts

PubMed Central

Bao, Beier; He, Yingzi; Tang, Dongmei; Li, Wenyan; Li, Huawei

2017-01-01

The H3K27 demethylases are involved in a variety of biological processes, including cell differentiation, proliferation, and cell death by regulating transcriptional activity. However, the function of H3K27 demethylation in the field of hearing research is poorly understood. Here, we investigated the role of H3K27me3 histone demethylase activity in hair cell regeneration using an in vivo animal model. Our data showed that pharmacologic inhibition of H3K27 demethylase activity with the specific small-molecule inhibitor GSK-J4 decreased the number of regenerated hair cells in response to neomycin damage. Furthermore, inhibition of H3K27me3 histone demethylase activity dramatically suppressed cell proliferation and activated caspase-3 levels in the regenerating neuromasts of the zebrafish lateral line. GSK-J4 administration also increased the expression of p21 and p27 in neuromast cells and inhibited the ERK signaling pathway. Collectively, our findings indicate that H3K27me3 demethylation is a key epigenetic regulator in the process of hair cell regeneration in zebrafish and suggest that H3K27me3 histone demethylase activity might be a novel therapeutic target for the treatment of hearing loss. PMID:28348517

MiR224-3p inhibits hypoxia-induced autophagy by targeting autophagy-related genes in human glioblastoma cells.

PubMed

Guo, Xing; Xue, Hao; Guo, Xiaofan; Gao, Xiao; Xu, Shugang; Yan, Shaofeng; Han, Xiao; Li, Tong; Shen, Jie; Li, Gang

2015-12-08

Human glioblastoma multiforme (GBM) is a malignant solid tumor characterized by severe hypoxia. Autophagy plays a protective role in cancer cells under hypoxia. However, the microRNA (miRNA)-related molecular mechanisms underlying hypoxia-reduced autophagy remain poorly understood in GBM. In this study, we performed a miRNA microarray analysis on GBM cells and found that numerous miRNAs were differentially expressed under hypoxic conditions. Further research showed that miR224-3p, one of the significantly down-regulated miRNAs, was involved in regulating hypoxia-induced autophagy in GBM cells. Overexpression of miR224-3p abolished hypoxia-induced autophagy, whereas knocking down endogenous miR224-3p increased autophagic activity under normoxia. In addition, we demonstrated that miR224-3p inhibited autophagy by directly suppressing the expression of two autophagy-related genes (ATGs), ATG5 and FAK family-interacting protein of 200 kDa (FIP200). Furthermore, in vitro, miR224-3p attenuated cell proliferation and promoted hypoxia-induced apoptosis, and in vivo, overexpression of miR224-3p inhibited tumorigenesis of GBM cells. Collectively, our study identified a novel hypoxia-down-regulated miRNA, miR224-3p, as a key modulator of autophagy by inhibiting ATGs in GBM cells.

Effects of modulators of AMP-activated protein kinase on TASK-1/3 and intracellular Ca(2+) concentration in rat carotid body glomus cells.

PubMed

Kim, Donghee; Kang, Dawon; Martin, Elizabeth A; Kim, Insook; Carroll, John L

2014-05-01

Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca(2+) concentration ([Ca(2+)]i). Recent studies suggest that AMP-activated protein kinase (AMPK) mediates these effects of hypoxia by inhibiting the background K(+) channels such as TASK. Here we studied the effects of modulators of AMPK on TASK activity in cell-attached patches. Activators of AMPK (1mM AICAR and 0.1-0.5mM A769662) did not inhibit TASK activity or cause depolarization during acute (10min) or prolonged (2-3h) exposure. Hypoxia inhibited TASK activity by ∼70% in cells pretreated with AICAR or A769662. Both AICAR and A769662 (15-40min) failed to increase [Ca(2+)]i in glomus cells. Compound C (40μM), an inhibitor of AMPK, showed no effect on hypoxia-induced inhibition of TASK. AICAR and A769662 phosphorylated AMPKα in PC12 cells, and Compound C blocked the phosphorylation. Our results suggest that AMPK does not affect TASK activity and is not involved in hypoxia-induced elevation of intracellular [Ca(2+)] in isolated rat carotid body glomus cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture.

PubMed

Gangalum, Rajendra K; Bhat, Ankur M; Kohan, Sirus A; Bhat, Suraj P

2016-06-17

Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Tangeretin and its metabolite 4'-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K.

PubMed

Cheng, Z; Surichan, S; Ruparelia, K; Arroo, R; Boarder, M R

2011-04-01

The mechanisms by which the dietary compound tangeretin has anticancer effects may include acting as a prodrug, forming an antiproliferative product in cancer cells. Here we show that tangeretin also inhibits cell cycle progression in hepatocytes and investigate the role of its primary metabolite 4'-hydroxy-5,6,7,8-tetramethoxyflavone (4'-OH-TMF) in this effect. We used epidermal growth factor (EGF)-stimulated rat hepatocytes, with [(3)H]-thymidine incorporation into DNA as an index of progression to S-phase of the cell cycle, and Western blots for phospho-proteins involved in the cell signalling cascade. Incubation of tangeretin with microsomes expressing CYP1A, or with hepatocytes, generated a primary product we identified as 4'-OH-TMF. Low micromolar concentrations of tangeretin or 4'-OH-TMF gave a concentration-dependent inhibition of EGF-stimulated progression to S-phase while having little effect on cell viability. To determine whether time for conversion of tangeretin to an active metabolite would enhance the inhibitory effect we used long pre-incubations; this reduced the inhibitory effect, in parallel with a reduction in the concentration of tangeretin. The EGF-stimulation of hepatocyte cell cycle progression requires signalling through Akt/mTOR/p70S6K kinase cascades. The tangeretin metabolite 4'-OH-TMF selectively inhibited S6K phosphorylation in the absence of significant inhibition of upstream Akt activity, suggesting an effect at the level of mTOR. Tangeretin and 4'-OH-TMF both inhibit cell cycle progression in primary hepatocytes. The inhibition of p70S6K phosphorylation by 4'-OH-TMF raises the possibility that inhibition of the mTOR pathway may contribute to the anticancer influence of a flavonoid-rich diet. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

NF-κB RelB Negatively Regulates Osteoblast Differentiation and Bone Formation

PubMed Central

Yao, Zhenqiang; Li, Yanyun; Yin, Xiaoxiang; Dong, Yufeng; Xing, Lianping; Boyce, Brendan F.

2013-01-01

RelA-mediated NF-κB canonical signaling promotes mesenchymal progenitor cell (MPC) proliferation, but inhibits differentiation of mature osteoblasts (OBs) and thus negatively regulates bone formation. Previous studies suggest that NF-κB RelB may also negatively regulate bone formation through non-canonical signaling, but they involved a complex knockout mouse model and the molecular mechanisms involved were not investigated. Here, we report that RelB−/− mice develop age-related increased trabecular bone mass associated with increased bone formation. RelB−/− bone marrow stromal cells expanded faster in vitro and have enhanced OB differentiation associated with increased expression of the osteoblastogenic transcription factor, Runx2. In addition, RelB directly targeted the Runx2 promoter to inhibit its activation. Importantly, RelB−/− bone-derived MPCs formed bone more rapidly than wild-type cells after they were injected into a murine tibial bone defect model. Our findings indicate that RelB negatively regulates bone mass as mice age and limits bone formation in healing bone defects, suggesting that inhibition of RelB could reduce age-related bone loss and enhance bone repair. PMID:24115294

PTENp1, a natural sponge of miR-21, mediates PTEN expression to inhibit the proliferation of oral squamous cell carcinoma.

PubMed

Gao, Ling; Ren, Wenhao; Zhang, Linmei; Li, Shaoming; Kong, Xinjuan; Zhang, Hao; Dong, Jianwei; Cai, Guangfeng; Jin, Changxiong; Zheng, Danqing; Zhi, Keqian

2017-04-01

PTENp1, non-coding RNA (ncRNA) pseudogene, is involved in oral squamous cell carcinoma (OSCC). The precise effects mediated by PTENp1 transcripts within intricate regulatory networks involving molecular interactions with ancestral gene PTEN and tumorigenicity in OSCC remain unclear. Here, we found that PTENp1 was aberrantly expressed in OSCC. There was a positive correlation between the expression levels of PTENp1 and PTEN. Further, we showed that PTENp1 acted as a competing endogenous RNA that protects PTEN transcripts from being inhibited by miR-21, and consequently inhibited proliferation and colony formation and triggered S-G2/M cell cycle arrest through the AKT pathway. Also, the homogeneous relationship between expression of PTENp1 and PTEN was confirmed in OSCC tumor xenografts. Finally, low expression of PTENp1 and PTEN was negatively associated with histological differentiation and OSCC prognosis. The present work provided the first evidence for the extraordinary crosstalk among PTENp1, PTEN, and miR-21, and rendered a new light on the treatment of OSCC. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Hyperforin inhibits Akt1 kinase activity and promotes caspase-mediated apoptosis involving Bad and Noxa activation in human myeloid tumor cells.

PubMed

Merhi, Faten; Tang, Ruoping; Piedfer, Marion; Mathieu, Julie; Bombarda, Isabelle; Zaher, Murhaf; Kolb, Jean-Pierre; Billard, Christian; Bauvois, Brigitte

2011-01-01

The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo. HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser(473)) and Akt1 substrate Bad (at Ser(136)) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells. Our data provide new evidence that HF's pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment.

Hyperforin Inhibits Akt1 Kinase Activity and Promotes Caspase-Mediated Apoptosis Involving Bad and Noxa Activation in Human Myeloid Tumor Cells

PubMed Central

Merhi, Faten; Tang, Ruoping; Piedfer, Marion; Mathieu, Julie; Bombarda, Isabelle; Zaher, Murhaf; Kolb, Jean-Pierre; Billard, Christian; Bauvois, Brigitte

2011-01-01

Background The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo. Methodology and Results HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser473) and Akt1 substrate Bad (at Ser136) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells. Significance Our data provide new evidence that HF's pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment. PMID:21998731

Jaceosidin, isolated from dietary mugwort (Artemisia princeps), induces G2/M cell cycle arrest by inactivating cdc25C-cdc2 via ATM-Chk1/2 activation.

PubMed

Lee, Jong-Gyu; Kim, Ji-Hyun; Ahn, Ji-Hye; Lee, Kyung-Tae; Baek, Nam-In; Choi, Jung-Hye

2013-05-01

Jaceosidin, a flavonoid derived from Artemisia princeps (Japanese mugwort), has been shown to inhibit the growth of several human cancer cells, However, the exact mechanism for the cytotoxic effect of jaceosidin is not completely understood. In this study, we investigated the molecular mechanism involved in the antiproliferative effect of jaceosidin in human endometrial cancer cells. We demonstrated that jaceosidin is a more potent inhibitor of cell growth than cisplatin in human endometrial cancer cells. In contrast, jaceosidin-induced cytotoxicity in normal endometrial cells was lower than that observed for cisplatin. Jaceosidin induced G2/M phase cell cycle arrest and modulated the levels of cyclin B and p-Cdc2 in Hec1A cells. Knockdown of p21 using specific siRNAs partially abrogated jaceosidin-induced cell growth inhibition. Additional mechanistic studies revealed that jaceosidin treatment resulted in an increase in phosphorylation of Cdc25C and ATM-Chk1/2. Ku55933, an ATM inhibitor, reversed jaceosidin-induced cell growth inhibition, in part. Moreover, jaceosidin treatment resulted in phosphorylation of ERK, and pretreatment with the ERK inhibitor, PD98059, attenuated cell growth inhibition by jaceosidin. These data suggest that jaceosidin, isolated from Japanese mugwort, modulates the ERK/ATM/Chk1/2 pathway, leading to inactivation of the Cdc2-cyclin B1 complex, followed by G2/M cell cycle arrest in endometrial cancer cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Proteomic analysis of the molecular response of Raji cells to maslinic acid treatment.

PubMed

Yap, W H; Khoo, K S; Lim, S H; Yeo, C C; Lim, Y M

2012-01-15

Maslinic acid, a natural pentacyclic triterpene has been shown to inhibit growth and induce apoptosis in some tumour cell lines. We studied the molecular response of Raji cells towards maslinic acid treatment. A proteomics approach was employed to identify the target proteins. Seventeen differentially expressed proteins including those involved in DNA replication, microtubule filament assembly, nucleo-cytoplasmic trafficking, cell signaling, energy metabolism and cytoskeletal organization were identified by MALDI TOF-TOF MS. The down-regulation of stathmin, Ran GTPase activating protein-1 (RanBP1), and microtubule associated protein RP/EB family member 1 (EB1) were confirmed by Western blotting. The study of the effect of maslinic acid on Raji cell cycle regulation showed that it induced a G1 cell cycle arrest. The differential proteomic changes in maslinic acid-treated Raji cells demonstrated that it also inhibited expression of dUTPase and stathmin which are known to induce early S and G2 cell cycle arrests. The mechanism of maslinic acid-induced cell cycle arrest may be mediated by inhibiting cyclin D1 expression and enhancing the levels of cell cycle-dependent kinase (CDK) inhibitor p21 protein. Maslinic acid suppressed nuclear factor-kappa B (NF-κB) activity which is known to stimulate expression of anti-apoptotic and cell cycle regulatory gene products. These results suggest that maslinic acid affects multiple signaling molecules and inhibits fundamental pathways regulating cell growth and survival in Raji cells. Copyright © 2011 Elsevier GmbH. All rights reserved.

Molecular role of TGF-beta, secreted from a new type of CD4+ suppressor T cell, NY4.2, in the prevention of autoimmune IDDM in NOD mice.

PubMed

Han, H S; Jun, H S; Utsugi, T; Yoon, J W

1997-06-01

A new type of CD4+ T cell clone (NY4.2) isolated from pancreatic islet-infiltrated lymphocytes of acutely diabetic non-obese diabetic (NOD) mice prevents the development of insulin-dependent diabetes mellitus (IDDM) in NOD mice, as well as the recurrence of autoimmune diabetes in syngeneic islet-transplanted NOD mice. It has been demonstrated that the cytokine TGF-beta, secreted from the cells of this clone, is the substance which prevents autoimmune IDDM. This investigation was initiated to determine the molecular role TGF-beta plays in the prevention of autoimmune IDDM by determining its effect on IL-2-induced signal transduction in Con A-activated NOD mouse splenocytes and HT-2 cells. First, we determined whether TGF-beta, secreted from NY4.2 T cells, inhibits IL-2-dependent T cell proliferation in HT-2 cells (IL-2-dependent T cell line) and NOD splenocytes. We found that TGF-beta suppresses IL-2-dependent T cell proliferation. Second, we determined whether TGF-beta inhibits the activation of Janus kinases (JAKs), as well as signal transducers and activators of transcription (STAT) proteins, involved in an IL-2-induced signalling pathway that normally leads to the proliferation of T cells. We found that TGF-beta inhibited tyrosine phosphorylation of JAK1, JAK3, STAT3 and STAT5 in Con A blasts from NOD splenocytes and HT-2 cells. Third, we examined whether TGF-beta inhibits the cooperation between STAT proteins and mitogen-activated protein kinase (MAPK), especially extracellular signal-regulated kinase 2 (ERK2). We found that TGF-beta inhibited the association of STAT3 and STAT5 with ERK2 in Con A blasts from NOD splenocytes and HT-2 cells. On the basis of these observations, we conclude that TGF-beta may interfere with signal transduction via inhibition of the IL-2-induced JAK/STAT pathway and inhibition of the association of STAT proteins with ERK2 in T cells from NOD splenocytes, resulting in the inhibition of IL-2-dependent T cell proliferation. TGF-beta-mediated suppression of T cell activation may be responsible for the prevention of effector T cell-mediated autoimmune IDDM in NOD mice by TGF-beta-producing CD4+ suppressor T cells.

Sirt2 Deacetylase Is a Novel AKT Binding Partner Critical for AKT Activation by Insulin*

PubMed Central

Ramakrishnan, Gopalakrishnan; Davaakhuu, Gantulga; Kaplun, Ludmila; Chung, Wen-Cheng; Rana, Ajay; Atfi, Azeddine; Miele, Lucio; Tzivion, Guri

2014-01-01

AKT/PKB kinases transmit insulin and growth factor signals downstream of phosphatidylinositol 3-kinase (PI3K). AKT activation involves phosphorylation at two residues, Thr308 and Ser473, mediated by PDK1 and the mammalian target of rapamycin complex 2 (mTORC2), respectively. Impaired AKT activation is a key factor in metabolic disorders involving insulin resistance, whereas hyperactivation of AKT is linked to cancer pathogenesis. Here, we identify the cytoplasmic NAD+-dependent deacetylase, Sirt2, as a novel AKT interactor, required for optimal AKT activation. Pharmacological inhibition or genetic down-regulation of Sirt2 diminished AKT activation in insulin and growth factor-responsive cells, whereas Sirt2 overexpression enhanced the activation of AKT and its downstream targets. AKT was prebound with Sirt2 in serum or glucose-deprived cells, and the complex dissociated following insulin treatment. The binding was mediated by the pleckstrin homology and the kinase domains of AKT and was dependent on AMP-activated kinase. This regulation involved a novel AMP-activated kinase-dependent Sirt2 phosphorylation at Thr101. In cells with constitutive PI3K activation, we found that AKT also associated with a nuclear sirtuin, Sirt1; however, inhibition of PI3K resulted in dissociation from Sirt1 and increased association with Sirt2. Sirt1 and Sirt2 inhibitors additively inhibited the constitutive AKT activity in these cells. Our results suggest potential usefulness of Sirt1 and Sirt2 inhibitors in the treatment of cancer cells with up-regulated PI3K activity and of Sirt2 activators in the treatment of insulin-resistant metabolic disorders. PMID:24446434

The natural compound magnolol inhibits invasion and exhibits potential in human breast cancer therapy

PubMed Central

Liu, Ying; Cao, Wei; Zhang, Bo; Liu, Yong-qiang; Wang, Zhong-yuan; Wu, Yan-ping; Yu, Xian-jun; Zhang, Xu-dong; Ming, Ping-hong; Zhou, Guang-biao; Huang, Laiqiang

2013-01-01

Invasion and metastasis are the main causes of treatment failure and death in breast cancer. Thus, novel invasion-based therapies such as those involving natural agents are urgently required. In this study, we examined the effects of magnolol (Mag), a compound extracted from medicinal herbs, on breast cancer cells in vitro and in vivo. Highly invasive cancer cells were found to be highly sensitive to treatment. Mag markedly inhibited the activity of highly invasive MDA-MB-231 cells. Furthermore, Mag significantly downregulated matrix metalloproteinase-9 (MMP-9) expression, an enzyme critical to tumor invasion. Mag also inhibited nuclear factor-κB (NF-κB) transcriptional activity and the DNA binding of NF-κB to MMP-9 promoter. These results indicate that Mag suppresses tumor invasion by inhibiting MMP-9 through the NF-κB pathway. Moreover, Mag overcame the promoting effects of phorbol 12-myristate 13-acetate (PMA) on the invasion of MDA-MB-231 cells. Our findings reveal the therapeutic potential and mechanism of Mag against cancer. PMID:24226295

The Tyrosine Kinase c-Met Contributes to the Pro-tumorigenic Function of the p38 Kinase in Human Bile Duct Cholangiocarcinoma Cells*

PubMed Central

Dai, Rongyang; Li, Juanjuan; Fu, Jing; Chen, Yao; Wang, Ruoyu; Zhao, Xiaofang; Luo, Tao; Zhu, Junjie; Ren, Yibin; Cao, Jie; Qian, Youwen; Li, Ning; Wang, Hongyang

2012-01-01

Pro-tumorigenic function of the p38 kinase plays a critical role in human cholangiocarcinogenesis. However, the underlying mechanism remains incompletely understood. Here, we report that c-Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), contributes to the pro-tumorigenic ability of p38 in human cholangiocarcinoma cells. Both p38 and c-Met promote the proliferation and invasion of human cholangiocarcinoma cells. Importantly, inhibition or knockdown of p38 decreased the basal activation of c-Met. Tyrosine phosphatase inhibitor studies revealed that p38 promotes the activity of c-Met, at least in part, by inhibiting dephosphorylation of the receptor. Moreover, density enhanced phosphatase-1 (DEP-1) is involved in p38-mediated inhibiting dephosphorylation of c-Met. Furthermore, p38 inhibits the degradation of c-Met. Taken together, these data provide a potential mechanism to explain how p38 promotes human cholangiocarcinoma cell proliferation and invasion. We propose that the link between p38 and c-Met is implicated in the progression of human cholangiocarcinoma. PMID:23024367

Involvement of estrogen receptor β5 in the progression of glioma.

PubMed

Li, Wenjun; Winters, Ali; Poteet, Ethan; Ryou, Myoung-Gwi; Lin, Song; Hao, Shuyu; Wu, Zhen; Yuan, Fang; Hatanpaa, Kimmo J; Simpkins, James W; Yang, Shao-Hua

2013-03-29

Emerging evidence suggests a decline of ERβ expression in various peripheral cancers. ERβ has been proposed as a cancer brake that inhibits tumor proliferation. In the current study, we have identified ERβ5 as the predominant isoform of ERβ in human glioma and its expression was significantly increased in human glioma as compared with non-neoplastic brain tissue. Hypoxia and activation of hypoxia inducible factor (HIF) increased ERβ transcription in U87 cells, suggesting elevated ERβ expression in glioma might be induced by the hypoxic stress in the tumor. Over-expression of either ERβ1 or ERβ5 increased PTEN expression and inhibited activation of the PI3K/AKT/mTOR pathway. In addition, ERβ5 inhibited the MAPK/ERK pathway. In U87 cells, ERβ1 and ERβ5 inhibit cell proliferation and reduced cells in the S+G2/M phase. Our findings suggest hypoxia induced ERβ5 expression in glioma as a self-protective mechanism against tumor proliferation and that ERβ5 might serve as a therapeutic target for the treatment of glioma. Copyright © 2013 Elsevier B.V. All rights reserved.

Terbinafine inhibits gap junctional intercellular communication

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

Lee, Ju Yeun, E-mail: whitewndus@naver.com

Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca{sup 2+} concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibitsmore » GJIC with a so far unknown mechanism of action. - Highlights: • In vitro pharmacological studies were performed on FRT-Cx43 and LN215 cells. • Terbinafine inhibits gap junctional intercellular communication in both cell lines. • The inhibitory effect of terbinafine is reversible and dose-dependent. • Treatment of terbinafine does not alter Cx43 phosphorylation or cytosolic Ca{sup 2+} concentration. • Inhibition of squalene epoxidase is not involved in this new effect of terbinafine.« less

NSs protein of Schmallenberg virus counteracts the antiviral response of the cell by inhibiting its transcriptional machinery.

PubMed

Barry, Gerald; Varela, Mariana; Ratinier, Maxime; Blomström, Anne-Lie; Caporale, Marco; Seehusen, Frauke; Hahn, Kerstin; Schnettler, Esther; Baumgärtner, Wolfgang; Kohl, Alain; Palmarini, Massimo

2014-08-01

Bunyaviruses have evolved a variety of strategies to counteract the antiviral defence systems of mammalian cells. Here we show that the NSs protein of Schmallenberg virus (SBV) induces the degradation of the RPB1 subunit of RNA polymerase II and consequently inhibits global cellular protein synthesis and the antiviral response. In addition, we show that the SBV NSs protein enhances apoptosis in vitro and possibly in vivo, suggesting that this protein could be involved in SBV pathogenesis in different ways. © 2014 The Authors.

TGFβ Pathway Inhibition Redifferentiates Human Pancreatic Islet β Cells Expanded In Vitro

PubMed Central

Toren-Haritan, Ginat; Efrat, Shimon

2015-01-01

In-vitro expansion of insulin-producing cells from adult human pancreatic islets could provide an abundant cell source for diabetes therapy. However, proliferation of β-cell-derived (BCD) cells is associated with loss of phenotype and epithelial-mesenchymal transition (EMT). Nevertheless, BCD cells maintain open chromatin structure at β-cell genes, suggesting that they could be readily redifferentiated. The transforming growth factor β (TGFβ) pathway has been implicated in EMT in a range of cell types. Here we show that human islet cell expansion in vitro involves upregulation of the TGFβ pathway. Blocking TGFβ pathway activation using short hairpin RNA (shRNA) against TGFβ Receptor 1 (TGFBR1, ALK5) transcripts inhibits BCD cell proliferation and dedifferentiation. Treatment of expanded BCD cells with ALK5 shRNA results in their redifferentiation, as judged by expression of β-cell genes and decreased cell proliferation. These effects, which are reproducible in cells from multiple human donors, are mediated, at least in part, by AKT-FOXO1 signaling. ALK5 inhibition synergizes with a soluble factor cocktail to promote BCD cell redifferentiation. The combined treatment may offer a therapeutically applicable way for generating an abundant source of functional insulin-producing cells following ex-vivo expansion. PMID:26418361

Novel therapeutic applications of nitric oxide donors in cancer: roles in chemo- and immunosensitization to apoptosis and inhibition of metastases.

PubMed

Bonavida, Benjamin; Baritaki, Stavroula; Huerta-Yepez, Sara; Vega, Mario I; Chatterjee, Devasis; Yeung, Kam

2008-09-01

The treatment of primary tumors results in an initial response to approved conventional therapeutics. However, recurrences and malignancies develop as a result of tumors' acquisition of anti-apoptotic mechanisms of resistance. Hence, there is an urgent need of novel therapeutics that can reverse resistance. One approach of interest is the inhibition of cell survival and anti-apoptotic pathways by sensitizing agents that can render resistant tumor cells sensitive to respond to various cytotoxic therapies. We have found that nitric oxide donors, similar to DETANONOate, inhibit cell survival anti-apoptotic pathways, such as the constitutively activated NF-kappaB and sensitize drug-resistant tumor cells to apoptosis by both chemotherapy and immunotherapy. Sensitization by DETANONOate was shown to inhibit the transcription repressor Yin Yang1 (YY1) shown to regulate resistance to both Fas ligand and TRAIL. In addition, DETANONOate-induced inhibition of NF-kappaB results downstream in the inhibition of several anti-apoptotic gene products, thus facilitating the activation of the apoptotic pathways with both chemotherapy and immunotherapy. In addition, DETANONOate induces the expression of the metastatic tumor suppressor gene product, Raf-1 Kinase Inhibitor Protein (RKIP), which inhibits the survival pathways induced by NF-kappaB and Raf-1/MEK which also contributes to the sensitizing activity. This indicates a novel finding that RKIP may also play an important role in the prevention of metastasis. Inhibition of NF-kappaB activation by DETANONOate results downstream in the inhibition of the RKIP transcription repressor Snail, resulting in upregulation of RKIP. Inhibition of Snail results in downstream inhibition of the metastatic cascade initiated by the epithelial-mesenchymal transition (EMT). Thus, nitric oxide donors have the dual functions of both sensitizing tumor cells to chemotherapy and immunotherapy and are also involved in the regulation and inhibition of metastasis.

MCLR-induced PP2A inhibition and subsequent Rac1 inactivation and hyperphosphorylation of cytoskeleton-associated proteins are involved in cytoskeleton rearrangement in SMMC-7721 human liver cancer cell line.

PubMed

Wang, Hao; Liu, Jinghui; Lin, Shuyan; Wang, Beilei; Xing, Mingluan; Guo, Zonglou; Xu, Lihong

2014-10-01

Cyanobacteria-derived toxin microcystin-LR (MCLR) has been widely investigated in its effects on normal cells, there is little information concerning its effects on cancer cells. In the present study, the SMMC-7721 human liver cancer cell line treated with MCLR was used to investigate the change of PP2A, cytoskeleton rearrangement, phosphorylation levels of PP2A substrates that related with cytoskeleton stability and explored underlying mechanisms. Here, we confirmed that MCLR entered into SMMC-7721 cells, bound to PP2A/C subunit and inhibited the activity of PP2A. The upregulation of phosphorylation of the PP2A/C subunit and PP2A regulation protein α4, as well as the change in the association of PP2A/C with α4, were responsible for the decrease in PP2A activity. Another novel finding is that the rearrangement of filamentous actin and microtubules led by MCLR may attribute to the increased phosphorylation of HSP27, VASP and cofilin due to PP2A inhibition. As a result of weakened interactions with PP2A and alterations in its subcellular localization, Rac1 may contribute to the cytoskeletal rearrangement induced by MCLR in SMMC-7721 cells. The current paper presents the first report demonstrating the characteristic of PP2A in MCLR exposed cancer cells, which were more susceptible to MCLR compared with the normal cell lines we previously found, which may be owing to the absence of some type of compensatory mechanisms. The hyperphosphorylation of cytoskeleton-associated proteins and Rac1 inactivation which were induced by inhibition of PP2A are shown to be involved in cytoskeleton rearrangement. Copyright © 2014 Elsevier Ltd. All rights reserved.

miR-4725-3p targeting Stim1 signaling is involved in xanthohumol inhibition of glioma cell invasion.

PubMed

Ho, Kuo-Hao; Chang, Cheng-Kuei; Chen, Peng-Hsu; Wang, Yu-Jia; Chang, Wei-Chiao; Chen, Ku-Chung

2018-05-10

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. Due to its highly invasive nature, it is not easy to treat, resulting in high mortality rates. Stromal interacting molecule 1 (Stim1) plays important roles in regulating store-operated Ca 2+ entry (SOCE), and controls invasion by cancer cells. However, the mechanisms and functions of Stim1 in glioma progression are still unclear. In this study, we investigated the effects of targeting Stim1 expression on glioma cell invasion. By analyzing profiles of GBM patients from RNA-sequencing data in The Cancer Genome Atlas (TCGA), higher expression levels of STIM1 were correlated with the poor survival. Furthermore, signaling pathways associated with tumor malignancy, including the epithelial-to-mesenchymal transition (EMT), were activated in patients with high STIM1 expression according to gene set enrichment analyses. Higher Stim1 levels were found in glioma cells compared to human astrocytes, and these higher levels enhanced glioma cell invasion. Xanthohumol (XN), a prenylated flavonoid extracted from the hop plant Humulus lupulus L. (Cannabaceae), significantly reduced cell invasion through inhibiting Stim1 expression. From an micro(mi)RNA array analysis, miR-4725-3p was upregulated by XN treatment. Overexpression of miR-4725-3p inhibited glioma cell invasion via directly targeting the 3'-untranslated region of STIM1. The extracellular signal-regulated kinase/c-Fos pathway was also validated to participate in XN-upregulated miR-4725-3p expression according to promoter and chromatin immunoprecipitation assays. These results emphasize that miR-4725-3p-inhibited STIM1 signaling is involved in XN-attenuated glioma cell invasion. These findings may provide insights into novel therapeutic strategies for future glioblastoma therapy and drug development. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Second Generation Amphiphilic Poly-Lysine Dendrons Inhibit Glioblastoma Cell Proliferation without Toxicity for Neurons or Astrocytes

PubMed Central

Janiszewska, Jolanta; Posadas, Inmaculada; Játiva, Pablo; Bugaj-Zarebska, Marta; Urbanczyk-Lipkowska, Zofia; Ceña, Valentín

2016-01-01

Glioblastomas are the most common malignant primary brain tumours in adults and one of the most aggressive and difficult-to-treat cancers. No effective treatment exits actually for this tumour and new therapeutic approaches are needed for this disease. One possible innovative approach involves the nanoparticle-mediated specific delivery of drugs and/or genetic material to glioblastoma cells where they can provide therapeutic benefits. In the present work, we have synthesised and characterised several second generation amphiphilic polylysine dendrons to be used as siRNA carriers. We have found that, in addition to their siRNA binding properties, these new compounds inhibit the proliferation of two glioblastoma cell lines while being nontoxic for non-tumoural central nervous system cells like neurons and glia, cell types that share the anatomical space with glioblastoma cells during the course of the disease. The selective toxicity of these nanoparticles to glioblastoma cells, as compared to neurons and glial cells, involves mitochondrial depolarisation and reactive oxygen species production. This selective toxicity, together with the ability to complex and release siRNA, suggests that these new polylysine dendrons might offer a scaffold in the development of future nanoparticles designed to restrict the proliferation of glioblastoma cells. PMID:27832093

Ras/ERK signaling pathway is involved in curcumin-induced cell cycle arrest and apoptosis in human gastric carcinoma AGS cells.

PubMed

Cao, Ai-Li; Tang, Qing-Feng; Zhou, Wen-Chao; Qiu, Yan-Yan; Hu, Song-Jiao; Yin, Pei-Hao

2015-01-01

Curcumin, the biologically active compound from the rhizome of Curcuma longa, could inhibit cell growth and induce apoptosis in gastric carcinoma. However, the underlying mechanism of curcumin on gastric carcinoma cells still needs further investigation. In this study, morphological observation indicated that curcumin inhibited the proliferation of AGS cells in a dose-dependent manner. According to the flow cytometric analysis, curcumin treatment resulted in G2/M arrest in AGS cells, accompanied with an increased expression of cyclin B1 and a decreased expression of cyclin D1. In addition, DNA ladders were observed by gel electrophoresis. Meanwhile, the activities of caspase-3, -8, and -9 were also enhanced in curcumin-treated AGS cells. Nevertheless, the increased activities could be inhibited by benzyloxycarbonyl-Val-Ala-Asp (OME)-fluoromethylketone (z-VAD-fmk), which suggested that the apoptosis was caspase-dependent. Furthermore, downregulation of rat sarcoma (Ras) and upregulation of extracellular-signal-regulated kinase (ERK) were also observed in AGS cells treated with curcumin by Western blot. U0126, an ERK inhibitor, blocked curcumin-induced apoptosis. The results suggested that curcumin inhibited the growth of the AGS cells and induced apoptosis through the activation of Ras/ERK signaling pathway and downstream caspase cascade, and curcumin might be a potential target for the treatment of gastric carcinoma.

NF-κB is involved in the LPS-mediated proliferation and apoptosis of MAC-T epithelial cells as part of the subacute ruminal acidosis response in cows.

PubMed

Fan, Wen-Jie; Li, He-Ping; Zhu, He-Shui; Sui, Shi-Ping; Chen, Pei-Ge; Deng, Yue; Sui, Tong-Ming; Wang, Yue-Ying

2016-11-01

To determine the effect of NF-κB on cell proliferation and apoptosis, we investigate the expression of inflammation and apoptosis-related factors in the bovine mammary epithelial cell line, MAC-T. MAC-T cells were cultured in vitro and MTT and LDH assays used to determine the effects of lipopolysaccharide (LPS) on proliferation and cytotoxicity respectively. RT-PCR and western blotting were used to evaluate the effect of LPS and NF-κB inhibition [pyrrolidine dithiocarbamate (PDTC) treatment] on the expression of inflammation and apoptosis-related factors. LPS significantly inhibited MAC-T cell proliferation in a dose- and time-dependent manner. Furthermore, LPS promoted apoptosis while the NF-кB inhibitor PDTC attenuated this effect. After LPS treatment, the NF-кB signaling pathway was activated, and the expression of inflammation and apoptosis-related factors increased. When PDTC blocked NF-кB signaling, the expression of inflammation and apoptosis-related factors were decreased in MAC-T cells. LPS activates the TLR4/NF-κB signaling pathway, inhibits proliferation and promotes apoptosis in MAC-T cells. NF-кB inhibition attenuates MAC-T cell apoptosis and TLR4/NF-κB signaling pathway. NF-кB inhibitor alleviating MAC-T cell apoptosis is presumably modulated by NF-кB.

Myeloid-derived suppressor cells in breast cancer.

PubMed

Markowitz, Joseph; Wesolowski, Robert; Papenfuss, Tracey; Brooks, Taylor R; Carson, William E

2013-07-01

Myeloid-derived suppressor cells (MDSCs) are a population of immature myeloid cells defined by their suppressive actions on immune cells such as T cells, dendritic cells, and natural killer cells. MDSCs typically are positive for the markers CD33 and CD11b but express low levels of HLADR in humans. In mice, MDSCs are typically positive for both CD11b and Gr1. These cells exert their suppressive activity on the immune system via the production of reactive oxygen species, arginase, and cytokines. These factors subsequently inhibit the activity of multiple protein targets such as the T cell receptor, STAT1, and indoleamine-pyrrole 2,3-dioxygenase. The numbers of MDSCs tend to increase with cancer burden while inhibiting MDSCs improves disease outcome in murine models. MDSCs also inhibit immune cancer therapeutics. In light of the poor prognosis of metastatic breast cancer in women and the correlation of increasing levels of MDSCs with increasing disease burden, the purposes of this review are to (1) discuss why MDSCs may be important in breast cancer, (2) describe model systems used to study MDSCs in vitro and in vivo, (3) discuss mechanisms involved in MDSC induction/function in breast cancer, and (4) present pre-clinical and clinical studies that explore modulation of the MDSC-immune system interaction in breast cancer. MDSCs inhibit the host immune response in breast cancer patients and diminishing MDSC actions may improve therapeutic outcomes.

Arctigenin inhibits lipopolysaccharide-induced iNOS expression in RAW264.7 cells through suppressing JAK-STAT signal pathway.

PubMed

Kou, Xianjuan; Qi, Shimei; Dai, Wuxing; Luo, Lan; Yin, Zhimin

2011-08-01

Arctigenin has been demonstrated to have an anti-inflammatory function, but the precise mechanisms of its action remain to be fully defined. In the present study, we determined the effects of arctigenin on lipopolysaccharide (LPS)-induced production of proinflammatory mediators and the underlying mechanisms involved in RAW264.7 cells. Our results indicated that arctigenin exerted its anti-inflammatory effect by inhibiting ROS-dependent STAT signaling through its antioxidant activity. Arctigenin also significantly reduced the phosphorylation of STAT1 and STAT 3 as well as JAK2 in LPS-stimulated RAW264.7 cells. The inhibitions of STAT1 and STAT 3 by arctigenin prevented their translocation to the nucleus and consequently inhibited expression of iNOS, thereby suppressing the expression of inflammation-associated genes, such as IL-1β, IL-6 and MCP-1, whose promoters contain STAT-binding elements. However, COX-2 expression was slightly inhibited at higher drug concentrations (50 μM). Our data demonstrate that arctigenin inhibits iNOS expression via suppressing JAK-STAT signaling pathway in macrophages. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Inhibition by Agrobacterium tumefaciens and Pseudomonas savastanoi of development of the hypersensitive response elicited by Pseudomonas syringae pv. phaseolicola.

PubMed Central

Robinette, D; Matthysse, A G

1990-01-01

Injection into tobacco leaves of biotype 1 Agrobacterium tumefaciens or of Pseudomonas savastanoi inhibited the development of a visible hypersensitive response to the subsequent injection at the same site of Pseudomonas syringae pv. phaseolicola. This interference with the hypersensitive response was not seen with injection of bacterial growth medium or Escherichia coli cells. Live A. tumefaciens cells were required for the inhibitory effect. Various mutants and strains of A. tumefaciens were examined to determine the genes involved. Known chromosomal mutations generally had no effect on the ability of A. tumefaciens to inhibit the hypersensitive response, except for chvB mutants which showed a reduced (but still significant) inhibition of the hypersensitive response. Ti plasmid genes appeared to be required for the inhibition of the hypersensitive response. The bacteria did not need to be virulent in order to inhibit the hypersensitive response. Deletion of the vir region from pTi had no effect on the inhibition. However, the T region of the Ti plasmid was required for inhibition. Studies of transposon mutants suggested that the tms but not tmr or ocs genes were required. These genes were not acting after transfer to plant cells since they were effective in strains lacking vir genes and thus unable to transfer DNA to plant cells. The results suggest that the expression of the tms genes in the bacteria may inhibit the development of the hypersensitive response by the plant. An examination of the genes required in P. savastanoi for the inhibition of the hypersensitive response suggested that bacterial production of auxin was also required for the inhibition of the hypersensitive response by these bacteria. Images PMID:2211508

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

Yuan, Bo; Cui, Jinquan, E-mail: jinquancuijqc@163.com; Wang, Wuliang

Several reports have indicated a role for the members of the G12 family of heterotrimeric G proteins (Gα12 and Gα13) in oncogenesis and tumor cell growth. The aims of the present study were to evaluate the role of G12 signaling in cervical cancer. We demonstrated that expression of the G12 proteins was highly upregulated in cervical cancer cells. Additionally, expression of the activated forms of Gα12/Gα13 but not expression of activated Gαq induced cell invasion through the activation of the RhoA family of G proteins, but had no effect on cell proliferation in the cervical cancer cells. Inhibition of G12more » signaling by expression of the RGS domain of the p115-Rho-specific guanine nucleotide exchange factor (p115-RGS) blocked thrombin-stimulated cell invasion, but did not inhibit cell proliferation in cervical cells, whereas the inhibition of Gαq (RGS2) had no effect. Furthermore, G12 signaling was able to activate Rho proteins, and this stimulation was inhibited by p115-RGS, and Gα12-induced invasion was blocked by an inhibitor of RhoA/B/C (C3 toxin). Pharmacological inhibition of JNK remarkably decreased G12-induced JNK activation. Both a JNK inhibitor (SP600125) and a ROCK inhibitor (Y27632) reduced G12-induced JNK and c-Jun activation, and markedly inhibited G12-induced cellular invasion. Collectively, these findings demonstrate that stimulation of G12 proteins is capable of promoting invasion through RhoA/ROCK-JNK activation. -- Highlights: •Gα12/Gα13 is upregulated in cervical cancer cell lines. •Gα12/Gα13 is not involved in cervical cancer cell proliferation. •Gα12/Gα13 promotes cervical cancer cell invasion. •The role of Rho G proteins in G12-promoted cervical cancer cell invasion. •G12 promotes cell invasion through activation of the ROCK-JNK signaling axis.« less

Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

PubMed

Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

1984-09-01

In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the therapeutic use of IgG1 monoclonal antibodies.

Identification of genes involved in the ACC-mediated control of root cell elongation in Arabidopsis thaliana

PubMed Central

2012-01-01

Background Along the root axis of Arabidopsis thaliana, cells pass through different developmental stages. In the apical meristem repeated cycles of division increase the numbers of cells. Upon leaving the meristem, these cells pass the transition zone where they are physiologically and mechanically prepared to undergo subsequent rapid elongation. During the process of elongation epidermal cells increase their length by 300% in a couple of hours. When elongation ceases, the cells acquire their final size, shape and functions (in the differentiation zone). Ethylene administered as its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is capable of inhibiting elongation in a concentration-dependent way. Using a microarray analysis, genes and/or processes involved in this elongation arrest are identified. Results Using a CATMA-microarray analysis performed on control and 3h ACC-treated roots, 240 differentially expressed genes were identified. Quantitative Real-Time RT-PCR analysis of the 10 most up and down regulated genes combined with literature search confirmed the accurateness of the analysis. This revealed that inhibition of cell elongation is, at least partly, caused by restricting the events that under normal growth conditions initiate elongation and by increasing the processes that normally stop cellular elongation at the end of the elongation/onset of differentiation zone. Conclusions ACC interferes with cell elongation in the Arabidopsis thaliana roots by inhibiting cells from entering the elongation process and by immediately stimulating the formation of cross-links in cell wall components, diminishing the remaining elongation capacity. From the analysis of the differentially expressed genes, it becomes clear that many genes identified in this response, are also involved in several other kind of stress responses. This suggests that many responses originate from individual elicitors, but that somewhere in the downstream signaling cascade, these are converged to a ’common pathway’. Furthermore, several potential keyplayers, such as transcription factors and auxin-responsive genes, were identified by the microarray analysis. They await further analysis to reveal their exact role in the control of cell elongation. PMID:23134674

Importance of inverse correlation between ALDH3A1 and PPARγ in tumor cells and tissue regeneration.

PubMed

Oraldi, M; Saracino, S; Maggiora, M; Chiaravalloti, A; Buemi, C; Martinasso, G; Paiuzzi, E; Thompson, D; Vasiliou, V; Canuto, R A

2011-05-30

Aldehyde dehydrogenase (ALDH) enzymes are involved in maintaining cellular homeostasis by metabolizing both endogenous and exogenous reactive aldehydes. They modulate several cell functions including proliferation, differentiation, survival as well as cellular response to oxidative stress. We previously reported that ALDH3A1 expression is inversely correlated with the activation of PPARs (Peroxisome Proliferators-Activated Receptors), a category of orphan nuclear hormone receptors, in both rat and human cells. PPARγ is involved in cell proliferation. In this study, we have used PPARγ transfection and inhibition to examine the relationship between ALDH3A1 and PPARγ and their role as regulators of cell proliferation. Induction of PPARγ in A549 and NCTC 2544 cells by transfection caused a decrease in ALDH3A1 and inhibition of cell proliferation, a result we obtained previously using ligands that induce PPARγ. A reduction of PPARγ expression using siRNA increased ALDH3A1 expression and cell proliferation. In cells induced to proliferate in a model of tissue regeneration, ALDH3A1 expression increased during the period of proliferation, whereas PPARγ expression decreased. In conclusion, through modulation of PPARγ or ALDH3A1, it may be possible to reduce cell proliferation in tumor cells or stimulate cell proliferation in normal cells during tissue regeneration. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Endothelial microparticle uptake in target cells is annexin I/phosphatidylserine receptor dependent and prevents apoptosis.

PubMed

Jansen, Felix; Yang, Xiaoyan; Hoyer, Friedrich Felix; Paul, Kathrin; Heiermann, Nadine; Becher, Marc Ulrich; Abu Hussein, Nebal; Kebschull, Moritz; Bedorf, Jörg; Franklin, Bernardo S; Latz, Eicke; Nickenig, Georg; Werner, Nikos

2012-08-01

Endothelial microparticles (EMP) are released from activated or apoptotic cells, but their effect on target cells and the exact way of incorporation are largely unknown. We sought to determine the uptake mechanism and the biological effect of EMP on endothelial and endothelial-regenerating cells. EMP were generated from starved endothelial cells and isolated by ultracentrifugation. Caspase 3 activity assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that EMP protect target endothelial cells against apoptosis in a dose-dependent manner. Proteomic analysis was performed to identify molecules contained in EMP, which might be involved in EMP uptake. Expression of annexin I in EMP was found and confirmed by Western blot, whereas the corresponding receptor phosphatidylserine receptor was present on endothelial target cells. Silencing either annexin I on EMP or phosphatidylserine receptor on target cells using small interfering RNA showed that the uptake of EMP by human coronary artery endothelial cells is annexin I/phosphatidylserine receptor dependent. Annexin I-downregulated EMP abrogated the EMP-mediated protection against apoptosis of endothelial target cells. p38 activation was found to mediate camptothecin-induced apoptosis. Finally, human coronary artery endothelial cells pretreated with EMP inhibited camptothecin-induced p38 activation. EMP are incorporated by endothelial cells in an annexin I/phosphatidylserine receptor-dependent manner and protect target cells against apoptosis. Inhibition of p38 activity is involved in EMP-mediated protection against apoptosis.

Celecoxib enhances the anti-inflammatory effects of farnesylthiosalicylic acid on T cells independent of prostaglandin E(2) production.

PubMed

Mor, Adam; Aizman, Elizabeta; Kloog, Yoel

2012-10-01

Celecoxib (Celebrex(®)), a non-steroidal anti-inflammatory drug and selective cyclooxygenase-2 inhibitor, is widely used to treat arthritis and other inflammatory disorders. Awareness of its anti-proliferative properties has prompted another indication for its use, in preventing colon polyps in high-risk populations. Farnesylthiosalicylic acid (FTS; Salirasib(®)), designed to inhibit oncogenic Ras and currently under evaluation in phase I/II and II clinical trials, was recently shown by our group to exert anti-inflammatory effects on both lymphocytes and mast cells. Here we examined whether celecoxib combined with FTS would enhance this anti-inflammatory activity. While each drug separately inhibited Ras activation in these cells, their combination yielded more marked inhibition as well as further inhibition of ERK phosphorylation, lymphocyte adhesion, and interleukin-2 secretion. The inhibitory effects, moreover, were independent of prostaglandin E(2) secretion. These data point to the promising potential of combined treatment with celecoxib and FTS for inflammatory disorders involving lymphocytes.

The N-terminal neurotensin fragment, NT1-11, inhibits cortisol secretion by human adrenocortical cells.

PubMed

Sicard, Flavie; Contesse, Vincent; Lefebvre, Hervé; Ait-Ali, Djida; Gras, Marjorie; Cartier, Dorthe; Decker, Annick; Chartrel, Nicolas; Anouar, Youssef; Vaudry, Hubert; Delarue, Catherine

2006-08-01

Neurotensin (NT) modulates corticosteroid secretion from the mammalian adrenal gland. The objective of this study was to investigate the possible involvement of NT in the control of cortisol secretion in the human adrenal gland. In vitro studies were conducted on cultured human adrenocortical cells. This study was conducted in a university research laboratory. Adrenal explants from patients undergoing expanded nephrectomy for kidney cancer were studied. Cortisol secretion from cultured adrenocortical cells was measured. NT1-11, the N-terminal fragment of NT, dose-dependently inhibited basal and ACTH-stimulated cortisol production by human adrenocortical cells in primary culture. In contrast, NT had no influence on cortisol output at concentrations up to 10(-6) m. HPLC and RT-PCR analyses failed to detect any significant amounts of NT and NT mRNA, respectively, in adrenal extracts. Molecular and pharmacological studies were performed to determine the type of NT receptor involved in the corticostatic effect of NT1-11. RT-PCR analysis revealed the expression of NT receptor type (NTR) 3 mRNA but not NTR1 and NTR2 mRNAs in the human adrenal tissue. However, the pharmacological profile of the adrenal NT1-11 receptor was different from that of NTR3, indicating that this receptor type is not involved in the action of NT1-11 on corticosteroidogenesis. Our results indicate that NT1-11 may act as an endocrine factor to inhibit cortisol secretion through activation of a receptor distinct from the classical NTR1, NTR2, and NTR3.

Procyanidins from evening primrose (Oenothera paradoxa) defatted seeds inhibit invasiveness of breast cancer cells and modulate the expression of selected genes involved in angiogenesis, metastasis, and apoptosis.

PubMed

Lewandowska, Urszula; Szewczyk, Karolina; Owczarek, Katarzyna; Hrabec, Zbigniew; Podsędek, Anna; Sosnowska, Dorota; Hrabec, Elżbieta

2013-01-01

There is a growing interest in plant polyphenols (including flavanols) that exhibit pleiotropic biological activities such as antiinflammatory, antioxidant, and anticancer effects. Here, we report for the first time the inhibition of MDA-MB-231 breast cancer cell viability and invasiveness by an evening primrose flavanol preparation (EPFP). We observed a decrease in MDA-MB-231 viability of 50% vs. a control after 72 h of incubation with EPFP at a concentration of 58 μM gallic acid equivalents (GAE) and an inhibition of their invasiveness of 65% vs. a control at 75 μM GAE after 48 h of incubation. EPFP caused a 10-fold reduction in matrix metalloproteinase-9 (MMP-9) activity at 100 μM GAE. Furthermore, through modulation of mRNA expression, EPFP reduced the expression levels of the following proteins: antiapoptotic Bcl-2, angiogenic vascular endothelial growth factor (VEGF), and 2 transcription factors (c-Jun, c-Fos). Moreover, analysis by flow cytometry revealed that EPFP induced apoptosis in MDA-MB-231 cells. In conclusion, our data shows that EPFP inhibits cell viability by increasing apoptosis and decreases cell invasiveness by decreasing angiogenesis.

Characterization of γδ regulatory T cells from peripheral blood in patients with multiple myeloma

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

Ma, Yongyong; Department of Hematology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000; Lei, Huyi

γδ regulatory T cells are able to inhibit the activation and function of T cells involved in antigen-specific immune responses. This study aimed to investigate the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in patients diagnosed as multiple myeloma (MM). We measured the levels of γδ T cells, the distribution and clonally amplified TCR Vγ and VδT cells in peripheral blood of healthy donors, patients recently diagnosed with MM, and MM patients in remission cohorts. In addition, we evaluated the ability of γδ regulatory T cells to inhibit the proliferation of CD4+CD25- T cells andmore » detected the expression of immunoregulatory-associated molecules. We found that the levels of γδ regulatory T cells from the peripheral blood in patients of MM were significantly higher than those in healthy donors. Comparison of γδT regulatory cells function in MM and healthy donors showed similarly inhibitory effects on the proliferation of T cells. Additionally, TLR8 expression level increased significantly in MM patients compared to healthy donors, while the expression levels of Foxp3, CD25, CTLA4, GITR, GATA3 and Tbet in MM patients and healthy donors showed no significant difference. Taken together, our study reveals the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in MM patients.« less

Involvement of histone methylation in macrophage apoptosis and unstable plaque formation in methionine-induced hyperhomocysteinemic ApoE-/- mice.

PubMed

Cong, Guangzhi; Yan, Ru; Huang, Hui; Wang, Kai; Yan, Ning; Jin, Ping; Zhang, Na; Hou, Jianjun; Chen, Dapeng; Jia, Shaobin

2017-03-15

Hyperhomocysteinemia (Hhcy) is an independent risk factor of atherosclerosis and promotes unstable plaque formation. Epigenetic mechanisms play an important role in the pathogenesis of atherosclerosis induced by Hhcy. However, the exact mechanism is still undefined. Lesional apoptotic cells and necrotic core formation contribute greatly to the progression of plaque. The present study sought to determine whether modification of histone methylation is involved in macrophage apoptosis and unstable plaque formation in the condition of Hhcy. The unstable plaque formation, lesional apoptotic cells and status of histone methylation were monitored in the aortas of Hhcy ApoE -/- mice induced by a high-methionine (HM) diet for 20weeks. Involvement of histone methylation in macrophage apoptosis and foam cell formation were assessed in macrophage Raw 264.7 cells after being challenged with homocysteine alone or in combination with the histone methylation inhibitor BIX 01294. The unstable plaque formation and lesion apoptotic cells are increased in ApoE -/ - mice supplemented with high-methionine (HM), accompanied with a decreased expression of histone H3 lysine 9 dimethylation. Hhcy increases the apoptosis of macrophages and inhibits the histone H3 lysine 9 dimethylation, as well as the expression of histone methyltransferase G9a in vitro. Inhibition of histone methylation by BIX01294 enhances macrophage apoptosis and foam cell formation in vitro. Our data suggest that Hhcy promotes the progression of atherosclerosis via macrophage apoptosis. Histone methylation might be involved in macrophage apoptosis and unstable plaque formation in methionine induced hyperhomocysteinemic ApoE -/- mice. Copyright © 2017 Elsevier Inc. All rights reserved.

On the pharmacology of ascending, descending and recurrent postsynatic inhibition of the cuneothalamic relay cells in the cat

PubMed Central

Kelly, J. S.; Renaud, L. P.

1973-01-01

1. In cats decerebrated or anaesthetized with pentobarbitone, cells of the middle third of the cuneate nucleus that were excited by tactile stimulation of the ipsilateral forelimb (responding to displacement of hairs, skin or joints) and inhibited by electrical stimulation of the contralateral pyramid, were invariably inhibited by electrical stimulation of the ipsilateral forepaw and the contralateral forelimb nerves. 2. In 50% of the cats, the cells were more fully identified by placing electrodes stereotaxically in the contralateral medial lemniscus. Recurrent inhibition was always a concomitant of the antidromic action potential. 3. The intensity and the duration of inhibition evoked by all of these pathways was totally resistant to iontophoretic and intravenous strychnine in doses at least 5 times that required to block completely the response of the same cells to iontophoretic glycine and was extremely sensitive to either iontophoretic bicuculline or picrotoxin. 4. Although the inhibition was invariably sensitive to intravenous picrotoxin, no significant change occurred in the duration or intensity of the inhibition when bicuculline was administered intravenously (5 or 6 times) as repeated doses of 0·2 mg/kg. 5. Postsynaptic inhibition in the cuneate may be mediated by γ-aminobutyric acid released from the nerve terminals of a common pool of interneurones shared by ascending, descending and recurrent pathways. Since the receptors involved in this pathway are resistant to intravenous bicuculline, they may well be distinct from those responsible for changes in the primary afferent terminal excitability, usually believed to be associated with presynaptic inhibition. PMID:4357959

Effects of murine natural killer cells on Cryptococcus neoformans

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

Nabavi Nouri, N.

Previous data generated by Murphy and McDaniel indicate that normal murine nylon wool nonadherent splenic cells, with the characteristics of natural killer (NK) cells, effectively inhibit the in vitro growth of Cryptococcus neoformans, a yeast-like pathogen. Nylon wood nonadherent cells from spleens of 7-8 week old mice were further fractionated on discontinuous Percoll gradients. The enrichment of NK cells in Percoll fractions 1 and 2 was confirmed by morphological examination, immunofluorescent staining, and by assessing the cytolytic activity of each Percoll cell fraction against YAC-1 targets in the 4 h /sup 51/Cr release assay. Cells isolated from each Percoll fractionmore » were tested for growth inhibitory activity against C neoformans, using an in vitro 18 h growth inhibition assay. The results showed that NK cell enrichment was concomitant with the enrichment of anti-cryptococcal activity the Percoll fractions 1 and 2. An immunolabeling method combined with scanning electron microscopy was used to demonstrate that the effector cells attached to C. neoformans were asialo GM/sub 1/ positive and, therefore, had NK cell characteristics. NK cells have Fc receptors on their surfaces , and are capable of antibody-dependent cell-mediated cytotoxicity (ADCC) against IgG-coated target cells. The author examined the effects of the IgG fraction of rabbit anti-cryptococcal antibody on the NK cell-mediated growth inhibition of C. neoformans. The data indicated that the effector cells involved in antibody-dependent growth inhibition of cryptococci are either NK cells or copurify and coexist in the same population with NK cells.« less

Rapamycin has suppressive and stimulatory effects on human plasmacytoid dendritic cell functions

PubMed Central

Boor, P P C; Metselaar, H J; Mancham, S; van der Laan, L J W; Kwekkeboom, J

2013-01-01

Plasmacytoid dendritic cells (PDC) are involved in innate immunity by interferon (IFN)-α production, and in adaptive immunity by stimulating T cells and inducing generation of regulatory T cells (Treg). In this study we studied the effects of mammalian target of rapamycin (mTOR) inhibition by rapamycin, a commonly used immunosuppressive and anti-cancer drug, on innate and adaptive immune functions of human PDC. A clinically relevant concentration of rapamycin inhibited Toll-like receptor (TLR)-7-induced IFN-α secretion potently (−64%) but TLR-9-induced IFN-α secretion only slightly (−20%), while the same concentration suppressed proinflammatory cytokine production by TLR-7-activated and TLR-9-activated PDC with similar efficacy. Rapamycin inhibited the ability of both TLR-7-activated and TLR-9-activated PDC to stimulate production of IFN-γ and interleukin (IL)-10 by allogeneic T cells. Surprisingly, mTOR-inhibition enhanced the capacity of TLR-7-activated PDC to stimulate naive and memory T helper cell proliferation, which was caused by rapamycin-induced up-regulation of CD80 expression on PDC. Finally, rapamycin treatment of TLR-7-activated PDC enhanced their capacity to induce CD4+forkhead box protein 3 (FoxP3)+ regulatory T cells, but did not affect the generation of suppressive CD8+CD38+lymphocyte activation gene (LAG)-3+ Treg. In general, rapamycin inhibits innate and adaptive immune functions of TLR-stimulated human PDC, but enhances the ability of TLR-7-stimulated PDC to stimulate CD4+ T cell proliferation and induce CD4+FoxP3+ regulatory T cell generation. PMID:23968562

Retinol induces morphological alterations and proliferative focus formation through free radical-mediated activation of multiple signaling pathways.

PubMed

Gelain, Daniel Pens; Pasquali, Matheus Augusto de Bittencourt; Caregnato, Fernanda Freitas; Castro, Mauro Antonio Alves; Moreira, José Claudio Fonseca

2012-04-01

Toxicity of retinol (vitamin A) has been previously associated with apoptosis and/or cell malignant transformation. Thus, we investigated the pathways involved in the induction of proliferation, deformation and proliferative focus formation by retinol in cultured Sertoli cells of rats. Sertoli cells were isolated from immature rats and cultured. The cells were subjected to a 24-h treatment with different concentrations of retinol. Parameters of oxidative stress and cytotoxicity were analyzed. The effects of the p38 inhibitor SB203580 (10 μmol/L), the JNK inhibitor SP600125 (10 μmol/L), the Akt inhibitor LY294002 (10 μmol/L), the ERK inhibitor U0126 (10 μmol/L) the pan-PKC inhibitor Gö6983 (10 μmol/L) and the PKA inhibitor H89 (1 μmol/L) on morphological and proliferative/transformation-associated modifications were studied. Retinol (7 and 14 μmol/L) significantly increases the reactive species production in Sertoli cells. Inhibition of p38, JNK, ERK1/2, Akt, and PKA suppressed retinol-induced [(3)H]dT incorporation into the cells, while PKC inhibition had no effect. ERK1/2 and p38 inhibition also blocked retinol-induced proliferative focus formation in the cells, while Akt and JNK inhibition partially decreased focus formation. ERK1/2 and p38 inhibition hindered transformation-associated deformation in retinol-treated cells, while other treatments had no effect. Our results suggest that activation of multiple kinases is responsible for morphological and proliferative changes associated to malignancy development in Sertoli cells by retinol at the concentrations higher than physiological level.

Mitochondrial fission proteins regulate programmed cell death in yeast.

PubMed

Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

2004-11-15

The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

Proinflammatory Cytokines IL-6 and TNF-α Increased Telomerase Activity through NF-κB/STAT1/STAT3 Activation, and Withaferin A Inhibited the Signaling in Colorectal Cancer Cells

PubMed Central

Okobi, Quincy; Adekoya, Debbie; Atefi, Mohammad; Clarke, Orette; Dutta, Pranabananda; Vadgama, Jaydutt V.

2017-01-01

There are increasing evidences of proinflammatory cytokine involvement in cancer development. Here, we found that two cytokines, IL-6 and TNF-α, activated colorectal cancer cells to be more invasive and stem-like. Combined treatment of IL-6 and TNF-α phosphorylated transcription factors STAT3 in a synergistic manner. STAT3, STAT1, and NF-κB physically interacted upon the cytokine stimulation. STAT3 was bound to the promoter region of human telomerase reverse transcriptase (hTERT). IL-6 and TNF-α stimulation further enhanced STAT3 binding affinity. Stem cell marker Oct-4 was upregulated in colorectal cancer cells upon IL-6 and TNF-α stimulation. Withaferin A, an anti-inflammatory steroidal lactone, inhibited the IL-6- and TNF-α-induced cancer cell invasion and decreased colonosphere formation. Notably, withaferin A inhibited STAT3 phosphorylation and abolished the STAT3, STAT1, and NF-κB interactions. Oct-4 expression was also downregulated by withaferin A inhibition. The binding of STAT3 to the hTERT promoter region and telomerase activity showed reduction with withaferin A treatments. Proinflammatory cytokine-induced cancer cell invasiveness is mediated by a STAT3-regulated mechanism in colorectal cancer cells. Our data suggest that withaferin A could be a promising anticancer agent that effectively inhibits the progression of colorectal cancer. PMID:28676732

Caffeine Inhibits the Activation of Hepatic Stellate Cells Induced by Acetaldehyde via Adenosine A2A Receptor Mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK Signal Pathway

PubMed Central

Yang, Wanzhi; Wang, Qi; Zhao, Han; Yang, Feng; Lv, Xiongwen; Li, Jun

2014-01-01

Hepatic stellate cell (HSC) activation is an essential event during alcoholic liver fibrosis. Evidence suggests that adenosine aggravates liver fibrosis via the adenosine A2A receptor (A2AR). Caffeine, which is being widely consumed during daily life, inhibits the action of adenosine. In this study, we attempted to validate the hypothesis that caffeine influences acetaldehyde-induced HSC activation by acting on A2AR. Acetaldehyde at 50, 100, 200, and 400 μM significantly increased HSC-T6 cells proliferation, and cell proliferation reached a maximum at 48 h after exposure to 200 μM acetaldehyde. Caffeine and the A2AR antagonist ZM241385 decreased the cell viability and inhibited the expression of procollagen type I and type III in acetaldehyde-induced HSC-T6 cells. In addition, the inhibitory effect of caffeine on the expression of procollagen type I was regulated by A2AR-mediated signal pathway involving cAMP, PKA, SRC, and ERK1/2. Interestingly, caffeine’s inhibitory effect on the expression of procollagen type III may depend upon the A2AR-mediated P38 MAPK-dependent pathway. Conclusions: Caffeine significantly inhibited acetaldehyde-induced HSC-T6 cells activation by distinct A2AR mediated signal pathway via inhibition of cAMP-PKA-SRC-ERK1/2 for procollagen type I and via P38 MAPK for procollagen type III. PMID:24682220

Cytotoxic Effects of 24-Methylenecyloartanyl Ferulate on A549 Nonsmall Cell Lung Cancer Cells through MYBBP1A Up-Regulation and AKT and Aurora B Kinase Inhibition.

PubMed

Doello, Sofia; Liang, Zhibin; Cho, Il Kyu; Kim, Jung Bong; Li, Qing X

2018-04-11

Lung cancer is the second most prevalent cancer. Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer. The low efficacy in current chemotherapies impels us to find new alternatives to prevent or treat NSCLC. Rice bran oil is cytotoxic to A549 cells, a NSCLC cell line. Here, we identified 24-methylenecyloartanyl ferulate (24-mCAF) as the main component responsible for the cytotoxicity in A549 cells. An iTRAQ-based quantitative proteomics analysis revealed that 24-mCAF inhibits cell proliferation and activates cell death and apoptosis. 24-mCAF induces up-regulation of Myb binding protein 1A (MYBBP1A), a tumor suppressor that halts cancer progression. 24-mCAF inhibits the activity of AKT and Aurora B kinase, two Ser/Thr kinases involved in MYBBP1A regulation and that represent important targets in NSCLC. This study provides the first insight of the effect of 24-mCAF, the main component of rice bran oil, on A459 cells at the cellular and molecular levels.

Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential

PubMed Central

Sonke, Eric; Verrydt, Megan; Postenka, Carl O.; Pardhan, Siddika; Willie, Chantalle J.; Mazzola, Clarisse R.; Hammers, Matthew D.; Pluth, Michael D.; Lobb, Ian; Power, Nicholas E.; Chambers, Ann F.; Leong, Hon S.; Sener, Alp

2016-01-01

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel–Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H2S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H2S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism and angiogenesis. Using the H2S-specific probe MeRhoAz, it was found that H2S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H2S-producing enzymes could reduce the proliferation, metabolism and survival of ccRCC cell lines, as determined by live-cell imaging, XTT/ATP assay, and flow cytometry respectively. Using the chorioallantoic membrane angiogenesis model, it was found that systemic inhibition of endogenous H2S production was able to decrease vascularization of VHL-deficient ccRCC xenografts. Endogenous H2S production is an attractive new target in ccRCC due to its involvement in multiple aspects of disease. PMID:26068241

Metformin inhibits 7,12-dimethylbenz[a]anthracene-induced breast carcinogenesis and adduct formation in human breast cells by inhibiting the cytochrome P4501A1/aryl hydrocarbon receptor signaling pathway

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

Maayah, Zaid H.; Ghebeh, Hazem; Alhaider, Abdulqader A.

2015-04-15

Recent studies have established that metformin (MET), an oral anti-diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen-induced animal models and cell lines. However, whether MET can protect against breast cancer has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET in non-cancerous human breast MCF10A cells and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1 (CYP1A1)/aryl hydrocarbon receptor (AhR) pathway. Transformation of the MCF10A cells into initiated breast cancer cells with DNA adduct formation was conducted usingmore » 7,12-dimethylbenz[a]anthracene (DMBA), an AhR ligand. The chemopreventive effect of MET against DMBA-induced breast carcinogenesis was evidenced by the capability of MET to restore the induction of the mRNA levels of basic excision repair genes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1), and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). Interestingly, the inhibition of DMBA-induced DNA adduct formation was associated with proportional decrease in CYP1A1 and in NAD(P)H:quinone oxidoreductase 1 (NQO1) gene expression. Mechanistically, the involvements of AhR and nuclear factor erythroid 2-related factor-2 (Nrf2) in the MET-mediated inhibition of DMBA-induced CYP1A1 and NQO1 gene expression were evidenced by the ability of MET to inhibit DMBA-induced xenobiotic responsive element and antioxidant responsive element luciferase reporter gene expression which suggests an AhR- and Nrf2-dependent transcriptional control. However, the inability of MET to bind to AhR suggests that MET is not an AhR ligand. In conclusion, the present work shows a strong evidence that MET inhibits the DMBA-mediated carcinogenicity and adduct formation by inhibiting the expression of CYP1A1 through an AhR ligand-independent mechanism. - Highlights: • Metformin (MET) protects against DMBA-induced DNA adduct and oxidative stress in human breast MCF10A cells. • MET inhibits DMBA-induced CYP1A1 gene expression through XRE-dependent mechanism. • MET inhibits DMBA-induced NQO1 gene expression through ARE-dependent mechanism. • MET did not displace [{sup 3}H]-TCDD from its binding site while successfully inhibited the nuclear translocation of AhR. • MET is not an AhR ligand.« less

Delivering the lateral inhibition punchline: it's all about the timing.

PubMed

Axelrod, Jeffrey D

2010-10-26

Experimental and theoretical biologists have long been fascinated with the emergence of self-organizing patterns in developing organisms, and much attention has focused on Notch-mediated lateral inhibition. Within sheets of cells that may adopt either of two possible cell fates, lateral inhibition establishes patterns through the activity of a negative intercellular feedback loop involving the receptor, Notch, and its ligands Delta or Serrate. Despite a long history of intensive study in Drosophila, where the mechanism was first described, as well as in other organisms, new work continues to yield important insights. Mathematical modeling, combined with biological analyses, has now shed light on two features of the process: how antagonistic and activating ligand-receptor interactions work together to accelerate inhibition and ensure fidelity, and how filopodial dynamics contribute to the observed pattern refinement and spacing.

Characterizing the Anti-HIV Activity of Papuamide A

PubMed Central

Andjelic, Cynthia D; Planelles, Vicente; Barrows, Louis R

2008-01-01

Papuamide A is representative of a class of marine derived cyclic depsipeptides, reported to have cytoprotective activity against HIV-1 in vitro. We show here that papuamide A acts as an entry inhibitor, preventing human immunodeficiency virus infection of host cells and that this inhibition is not specific to R5 or X4 tropic virus. This inhibition of viral entry was determined to not be due to papuamide A binding to CD4 or HIV gp120, the two proteins involved in the cell-virus recognition and binding. Furthermore, papuamide A was able to inhibit HIV pseudotype viruses expressing envelope glycoproteins from vesicular stomatitis virus or amphotropic murine leukemia virus indicating the mechanism of viral entry inhibition is not HIV-1 envelope glycoprotein specific. Time delayed addition studies with the pseudotyped viruses show that papuamide A inhibits viral infection only at the initial stage of the viral life cycle. Additionally, pretreatment studies revealed that the virus, and not the cell, is the target of papuamide A’s action. Together, these results suggest a direct virucidal mechanism of HIV-1 inhibition by papuamide A. We also demonstrate here that the other papuamides (B-D) are able to inhibit viral entry indicating that the free amino moiety of 2,3-diaminobutanoic acid residue is not required for the virucidal activity. PMID:19172193

The roles of MCP-1 and protein kinase C delta activation in human eosinophilic leukemia EoL-1 cells.

PubMed

Lee, Ji-Sook; Yang, Eun Ju; Kim, In Sik

2009-12-01

Idiopathic hypereosinophilc syndrome is a disorder associated with clonally eosinophilic proliferation. The importance of FIP1-like-1-platelet-derived growth factor receptor-alpha (FIP1L1-PDGFRA) in the pathogenesis and classification of HES has been recently reported. In this study, we investigated the contribution of monocyte chemoattractant protein-1 (MCP-1)/CCL2 to chemotactic activity and protein kinase C delta (PKC delta in the human eosinophilic leukemia cell line EoL-1. These cells express CCR2 protein among the CC chemokine receptors (CCR1-5). MCP-1 induces strong migration of EoL-1 cells and the chemotaxis signal in response to MCP-1 involves a G(i)/G(o) protein, phospholipase C (PLC), PKC delta, p38 MAPK and NF-kappaB. MCP-1 activates p38 MAPK via G(i)/G(o) protein, PLC and PKC delta cascade. MCP-1 also induces NF-kappaB translocation and the activation is inhibited by PKC delta activation. The increase in the basal expression and activity of PKC delta in EoL-1 cells, compared to normal eosinophils, inhibits apoptosis in EoL-1 cells. Anti-apoptotic mechanism of PKC delta is related to inhibition of caspase 3 and caspase 9, but not to FIP1L1-PDGFRA. PKC delta functions as an anti-apoptotic molecule, and is involved in EoL-1 cell movement stimulated by MCP-1. This study contributes to an understanding of MCP-1 in eosinophil biology and pathogenic mechanism of eosinophilic disorders.

Caspase-8 regulates the expression of pro- and anti-inflammatory cytokines in human bone marrow-derived mesenchymal stromal cells.

PubMed

Moen, Siv H; Westhrin, Marita; Zahoor, Muhammad; Nørgaard, Nikolai N; Hella, Hanne; Størdal, Berit; Sundan, Anders; Nilsen, Nadra J; Sponaas, Anne-Marit; Standal, Therese

2016-09-01

Mesenchymal stem cells, also called mesenchymal stromal cells, MSCs, have great potential in stem cell therapy partly due to their immunosuppressive properties. How these cells respond to chronic inflammatory stimuli is therefore of importance. Toll-like receptors (TLR)s are innate immune receptors that mediate inflammatory signals in response to infection, stress, and damage. Caspase-8 is involved in activation of NF-kB downstream of TLRs in immune cells. Here we investigated the role of caspase-8 in regulating TLR-induced cytokine production from human bone marrow-derived mesenchymal stromal cells (hBMSCs). Cytokine expression in hBMCs in response to poly(I:C) and LPS was evaluated by PCR, multiplex cytokine assay, and ELISA. TLR3, TRIF, and caspase-8 were silenced using siRNA. Caspase-8 was also inhibited using a caspase-8 inhibitor, z-IEDT. We found that TLR3 agonist poly(I:C) and TLR4 agonist LPS induced secretion of several pro-inflammatory cytokines in a TLR-dependent manner which required the TLR signaling adaptor molecule TRIF. Further, poly(I:C) reduced the expression of anti-inflammatory cytokines HGF and TGFβ whereas LPS reduced HGF expression only. Notably, caspase-8 was involved in the induction of IL- IL-1β, IL-6, CXCL10, and in the inhibition of HGF and TGFβ. Caspase-8 appears to modulate hBMSCs into gaining a pro-inflammatory phenotype. Therefore, inhibiting caspase-8 in hBMSCs might promote an immunosuppressive phenotype which could be useful in clinical applications to treat inflammatory disorders.

Withaferin A Inhibits the Proteasome Activity in Mesothelioma In Vitro and In Vivo

PubMed Central

Cheryan, Vino T.; Wu, Wenjuan; Cui, Cindy Qiuzhi; Polin, Lisa A.; Pass, Harvey I.; Dou, Q. Ping; Rishi, Arun K.; Wali, Anil

2012-01-01

The medicinal plant Withania somnifera has been used for over centuries in Indian Ayurvedic Medicine to treat a wide spectrum of disorders. Withaferin A (WA), a bioactive compound that is isolated from this plant, has anti-inflammatory, immuno-modulatory, anti-angiogenic, and anti-cancer properties. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of WA and the molecular mechanisms involved. WA inhibited growth of the murine as well as patient-derived MPM cells in part by decreasing the chymotryptic activity of the proteasome that resulted in increased levels of ubiquitinated proteins and pro-apoptotic proteasome target proteins (p21, Bax, IκBα). WA suppression of MPM growth also involved elevated apoptosis as evidenced by activation of pro-apoptotic p38 stress activated protein kinase (SAPK) and caspase-3, elevated levels of pro-apoptotic Bax protein and cleavage of poly-(ADP-ribose)-polymerase (PARP). Our studies including gene-array based analyses further revealed that WA suppressed a number of cell growth and metastasis-promoting genes including c-myc. WA treatments also stimulated expression of the cell cycle and apoptosis regulatory protein (CARP)-1/CCAR1, a novel transducer of cell growth signaling. Knock-down of CARP-1, on the other hand, interfered with MPM growth inhibitory effects of WA. Intra-peritoneal administration of 5 mg/kg WA daily inhibited growth of murine MPM cell-derived tumors in vivo in part by inhibiting proteasome activity and stimulating apoptosis. Together our in vitro and in vivo studies suggest that WA suppresses MPM growth by targeting multiple pathways that include blockage of proteasome activity and stimulation of apoptosis, and thus holds promise as an anti-MPM agent. PMID:22912669

Cajanol, a novel anticancer agent from Pigeonpea [Cajanus cajan (L.) Millsp.] roots, induces apoptosis in human breast cancer cells through a ROS-mediated mitochondrial pathway.

PubMed

Luo, Meng; Liu, Xia; Zu, Yuangang; Fu, Yujie; Zhang, Su; Yao, Liping; Efferth, Thomas

2010-10-06

Cajanol (5-hydroxy-3-(4-hydroxy-2-methoxyphenyl)-7-methoxychroman-4-one) is an isoflavanone from Pigeonpea [Cajanus cajan (L.) Millsp.] roots. As the most effective phytoalexin in pigeonpea, the cytotoxic activity of cajanol towards cancer cells has not been report as yet. In the present study, the anticancer activity of cajanol towards MCF-7 human breast cancer cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of cajanol, cell cycle distribution, DNA fragmentation assay and morphological assessment of nuclear change, ROS generation, mitochondrial membrane potential (DeltaPsim) disruption, and expression of caspase-3 and caspase-9, Bax, Bcl-2, PARP and cytochrome c were measured in MCF-7 cells. Cajanol inhibited the growth of MCF-7 cells in a time and dose-dependent manner. The IC(50) value was 54.05 microM after 72 h treatment, 58.32 microM after 48 h; and 83.42 microM after 24h. Cajanol arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of cajanol towards cancer cells in vitro. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A

PubMed Central

Austin, Pamela; Heller, Markus; Williams, David E.; McIntosh, Lawrence P.; Vogl, A. Wayne; Foster, Leonard J.; Andersen, Raymond J.; Roberge, Michel; Roskelley, Calvin D.

2010-01-01

Background Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. Methodology/Principal Findings We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of β1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: β1 integrin and numerous α integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD) proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that β1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. Conclusions/Significance NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface. PMID:20520768

Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

PubMed

Wang, Zheng; Cheng, Xingan; Meng, Qianqian; Wang, Peidan; Shu, Benshui; Hu, Qiongbo; Hu, Meiying; Zhong, Guohua

2015-07-01

Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tauroursodeoxycholic acid inhibits endoplasmic reticulum stress, blocks mitochondrial permeability transition pore opening, and suppresses reperfusion injury through GSK-3ß in cardiac H9c2 cells.

PubMed

Xie, Yuxi; He, Yonggui; Cai, Zhiliang; Cai, Jianhang; Xi, Mengyao; Zhang, Yidong; Xi, Jinkun

2016-01-01

This study investigates whether inhibition of endoplasmic reticulum (ER) stress prevents opening of the mitochondrial permeability transition pore (mPTP) and evaluates the corresponding signaling pathways involved in this process. Exposure of cardiac H9c2 cells to 800 µM H 2 O 2 for 20 min opened mPTP in response to oxidative stress, as demonstrated by quenching of tetramethylrhodamine ethyl ester (TMRE) fluorescence. Oxidative stress-induced mPTP opening was rescued by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) in a dose-dependent manner at low concentrations. The PI3K and PKG inhibitors LY294002 and KT5823 inhibited the effect of TUDCA on mPTP opening, suggesting the involvement of PI3K/Akt and PKG signaling pathways. TUDCA significantly increased glycogen synthase kinase 3 (GSK-3β) phosphorylation at Ser-9, with peak effect at 30 µM TUDCA. The level of GRP78 (ER chaperone) expression was significantly upregulated by 30 µM TUDCA. TUDCA-induced increases in Akt and GSK-3β phosphorylation were inhibited by LY294002, whereas KT5823 suppressed TUDCA-induced increases in VASP and GSK-3β phosphorylation. Oxidative stress severely affected cell morphology and ultrastructure. TUDCA prevented H 2 O 2 -induced ER swelling and mitochondrial damage. TUDCA boosted the viability of cells disrupted by ischemia/reperfusion (I/R), indicating that TUDCA eased reperfusion injury. However, TUDCA did not improve the viability of cells expressing the constitutively active GSK-3β mutant (GSK-3β-S9A-HA) that were subjected to I/R, suggesting an essential role of GSK-3β inactivation in TUDCA-mediated cardioprotection against reperfusion damage. These data indicate that ER stress inhibition prevents mPTP opening and attenuates reperfusion injury through GSK-3β inactivation. The PI3K/Akt and PKG pathways may mediate GSK-3β inactivation.

MicroRNA-21-Mediated Inhibition of Mast Cell Degranulation Involved in the Protective Effect of Berberine on 2,4-Dinitrofluorobenzene-Induced Allergic Contact Dermatitis in Rats via p38 Pathway.

PubMed

Li, Weihua; Liu, Fanxiu; Wang, Jun; Long, Man; Wang, Zhigang

2018-03-01

The study aimed to investigate the effect of berberine on allergic contact dermatitis (ACD) in rats and explore its underlying mechanisms. Firstly, ACD model was established by sensitizing and challenging with 2,4-dinitrofluorobenzene (DNFB) topically, and the rats were treated with berberine. Ear swelling was assessed, and cytokine, IgE, and histamine productions were measured. The ear biopsies were obtained for histology analysis. Additionally, rat peritoneal mast cells (RPMCs) were isolated for detection of microRNA-21 (miR-21) expression, mitogen-activated protein kinase (MAPK) signaling, and MC degranulation. Lastly, RPMCs were transfected with miR-21 mimic or miR-21 inhibitor to investigate the relationship between miR-21 and p38 pathway in MC. Our results showed that berberine significantly attenuated ear swelling in DNFB-induced ACD (ACD vs high dose of berberine 0.48 ± 0.03 vs. 0.33 ± 0.03 mm, P < 0.01), inhibited inflammatory cell infiltration (86 ± 5.16 vs. 58 ± 4.32 cells/mm 2 , P < 0.01), reduced MC recruitment (61 ± 4.07 vs. 39 ± 3.42 mast cells/mm 2 , P < 0.01), as well as decreased inflammatory cytokine, IgE, and histamine productions (all P < 0.05). Berberine treatment inhibited miR-21 expression, suppressed β-hexosaminidase and histamine release, and prevented p38 phosphorylation (all P < 0.05), which was abrogated by pretreatment with miR-21 overexpression. These findings indicate that miR-21-mediated inhibition of MC degranulation is involved in the anti-ACD effect of berberine via inhibiting p38 pathway, which provide a new insight into the immunopharmacological role of berberine and suggest its potential application for the treatment of allergic inflammation, such as ACD.

Inhibition of transglutaminase 2 reduces efferocytosis in human macrophages: Role of CD14 and SR-AI receptors.

PubMed

Eligini, S; Fiorelli, S; Tremoli, E; Colli, S

2016-10-01

Transglutaminase 2 (TGM2), a member of the transglutaminase family of enzymes, is a multifunctional protein involved in numerous events spanning from cell differentiation, to signal transduction, apoptosis, and wound healing. It is expressed in a variety of cells, macrophages included. Macrophage TGM2 promotes the clearance of apoptotic cells (efferocytosis) and emerging evidence suggests that defective efferocytosis contributes to the consequences of inflammation-associated diseases, including atherosclerotic lesion progression and its sequelae. Of interest, active TGM2 identified in human atherosclerotic lesions plays critical roles in plaque stability through effects on matrix cross-linking and TGFβ activity. This study explores the mechanisms by which TGM2 controls efferocytosis in human macrophages. Herein we show that TGM2 increases progressively during monocyte differentiation towards macrophages and controls their efferocytic potential as well as morphology and viability. Two experimental approaches that took advantage of the inhibition of TGM2 activity and protein silencing give proof that TGM2 reduction significantly impairs macrophage efferocytosis. Among the mechanisms involved we highlighted a role of the receptors CD14 and SR-AI whose levels were markedly reduced by TGM2 inhibition. Conversely, CD36 receptor and αvβ3 integrin levels were not influenced. Of note, lipid accumulation and IL-10 secretion were reduced in macrophages displaying defective efferocytosis. Overall, our data define a crucial role of TGM2 activity during macrophage differentiation via mechanisms involving CD14 and SR-AI receptors and show that TGM2 inhibition triggers a pro-inflammatory phenotype. Copyright © 2016 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.

Epigallocatechin 3-gallate inhibits 7-ketocholesterol-induced monocyte-endothelial cell adhesion.

PubMed

Yamagata, Kazuo; Tanaka, Noriko; Suzuki, Koichi

2013-07-01

7-Ketocholesterol (7KC) induces monocytic adhesion to endothelial cells, and induces arteriosclerosis while high-density lipoprotein (HDL) inhibits monocytic adhesion to the endothelium. Epigallocatechin 3-gallate (EGCG) was found to have a protective effect against arteriosclerosis. Therefore, the purpose of this study was to examine the possible HDL-like mechanisms of EGCG in endothelial cells by investigating whether EGCG inhibits 7KC-induced monocyte-endothelial cell adhesion by activating HDL-dependent signal transduction pathways. 7KC and/or EGCG were added to human endothelial cells (ISO-HAS), and the adhesion of pro-monocytic U937 cells was examined. The expression of genes associated with HDL effects such as Ca(2+)/calmodulin-dependent kinase II (CaMKKII), liver kinase B (LKD1), PSD-95/Dlg/ZO-1 kinase 1 (PDZK1), phosphatidylinositol 3-kinase (PI3K), intercellular adhesion molecule-1 (ICAM-1), monocyte chemotactic protein-1 (MCP-1), and endothelial nitric oxide synthase (eNOS) was examined by RT-PCR, and ICAM-1 protein expression was evaluated by western blot (WB). Production of reactive oxygen species (ROS) was examined with H2DCFDA. 7KC significantly induced adhesion of U937 cells to human endothelial cells while significantly increasing gene expressions of ICAM-1 and MCP-1 and decreasing eNOS and CaMKKII gene expressions. EGCG inhibited 7KC-induced monocytic adhesion to endothelial cells, and induced expression of eNOS and several genes involved in the CaMKKII pathway. Stimulation of endothelial cells with EGCG produced intracellular ROS, whereas treatment with N-acetylcysteine (NAC) blocked EGCG-induced expression of eNOS and CaMKKII. These results suggest that inhibition of monocyte-endothelial cell adhesion by EGCG is associated with CaMKKII pathway activation by ROS. Inhibition of 7KC-induced monocyte-endothelial cell adhesion induced by EGCG may function similarly to HDL. Copyright © 2013 Elsevier Inc. All rights reserved.

Inhibition of the Rac1-WAVE2-Arp2/3 signaling pathway promotes radiosensitivity via downregulation of cofilin-1 in U251 human glioma cells.

PubMed

Zhou, Tao; Wang, Chen-Han; Yan, Hua; Zhang, Rui; Zhao, Jin-Bing; Qian, Chun-Fa; Xiao, Hong; Liu, Hong-Yi

2016-05-01

The Ras-related C3 botulinum toxin substrate 1 (Rac1)-WASP-family verprolin-homologous protein-2 (WAVE2)-actin-related protein 2/3 (Arp2/3) signaling pathway has been identified to be involved in cell migration and invasion in various types of cancer cell. Cofilin‑1 (CFL‑1), which is regulated by the Rac1‑WAVE2‑Arp2/3 signaling pathway, may promote radioresistance in glioma. Therefore, the present study aimed to investigate the potential role of the Rac1‑WAVE2‑Arp2/3 signaling pathway in radioresistance in U251 human glioma cells and elucidate its affect on CFL‑1 expression. Western blot analysis was performed to evaluate the protein expression of CFL‑1. In the present study, Rac1 was inhibited by NSC 23766, WAVE2 was inhibited by transfection with short hairpin (sh)RNA‑WAVE2 using Lipofectamine™ 2000 and Arp2/3 was inhibited by CK‑666. Cell viability was measured using the 3‑(4,5‑dimethylthiazol‑2‑yl)-2,5‑diphenyltetrazolium bromide assay, the cell migration ability was examined by a wound‑healing assay, and the cell invasion ability was assessed using a Transwell culture chamber system. The results showed that inhibition of the Rac1‑WAVE2‑Arp2/3 signaling pathway using NSC 23766, shRNA‑WAVE2 or CK‑666 reduced the cell viability, migration and invasion abilities in U251 human glioma cells, concordant with a reduced expression of CFL‑1. Furthermore, the expression of CFL‑1 was significantly increased in radioresistant U251 glioma cells when compared with normal U251 human glioma cells. These findings indicate that inhibition of the Rac1‑WAVE2‑Arp2/3 signaling pathway may promote radiosensitivity, which may partially result from the downregulation of CFL‑1 in U251 human glioma cells.

Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: Involvement of the effector proteins in the regulatory mechanism

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

Saxena, Sunil K.; Kaur, Simarna

Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) andmore » Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.« less

STAT3 signaling pathway is necessary for cell survival and tumorsphere forming capacity in ALDH{sup +}/CD133{sup +} stem cell-like human colon cancer cells

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

Lin, Li, E-mail: lin.796@osu.edu; Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030; Fuchs, James

2011-12-16

Highlights: Black-Right-Pointing-Pointer The phosphorylated or activated form of STAT3 was expressed in colon cancer stem-like cells. Black-Right-Pointing-Pointer STAT3 inhibitor, FLLL32 inhibits P-STAT3 and STAT3 target genes in colon cancer stem-like cells. Black-Right-Pointing-Pointer Inhibition of STAT3 resulted in decreased cell viability and reduced numbers of tumorspheres. Black-Right-Pointing-Pointer STAT3 is required for survival and tumorsphere forming capacity in colon cancer stem-like cells. Black-Right-Pointing-Pointer Targeting STAT3 in cancer stem-like cells may offer a novel treatment approach for colon cancer. -- Abstract: Persistent activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequently detected in colon cancer. Increasing evidence suggests the existencemore » of a small population of colon cancer stem or cancer-initiating cells may be responsible for tumor initiation, metastasis, and resistance to chemotherapy and radiation. Whether STAT3 plays a role in colon cancer-initiating cells and the effect of STAT3 inhibition is still unknown. Flow cytometry was used to isolate colon cancer stem-like cells from three independent human colon cancer cell lines characterized by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulation (ALDH{sup +}/CD133{sup +}). The effects of STAT3 inhibition in colon cancer stem-like cells were examined. The phosphorylated or activated form of STAT3 was expressed in colon cancer stem-like cells and was reduced by a STAT3-selective small molecular inhibitor, FLLL32. FLLL32 also inhibited the expression of potential STAT3 downstream target genes in colon cancer stem-like cells including survivin, Bcl-XL, as well as Notch-1, -3, and -4, which may be involved in stem cell function. Furthermore, FLLL32 inhibited cell viability and tumorsphere formation as well as induced cleaved caspase-3 in colon cancer stem-like cells. FLLL32 is more potent than curcumin as evidenced with lower IC50 in colon cancer stem-like cells. In summary, our results indicate that STAT3 is a novel therapeutic target in colon cancer stem-like cells and inhibition of STAT3 in cancer stem-like cells may offer a potential treatment for colorectal cancer.« less

Dihydroavenanthramide D inhibits human breast cancer cell invasion through suppression of MMP-9 expression

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

Lee, Young-Rae; Noh, Eun-Mi; Oh, Hyun Ju

2011-02-25

Research highlights: {yields} MMP-9 plays a pivotal role in the invasion of MCF-7 breast cancer cells. {yields} TPA stimulates MMP-9 expression through activation of MAPK/NF-{kappa}B and MAPK/AP-1 pathways. {yields} Dihydroavenanthramide D suppresses MMP-9 expression via inhibition of TPA-induced MAPK/NF-{kappa}B and MAPK/AP-1 activations. {yields} Dihydroavenanthramide D blocks cell invasion of MCF-7 breast cancer cells. -- Abstract: Dihydroavenanthramide D (DHAvD) is a synthetic analog to naturally occurring avenanthramide, which is the active component of oat. Previous study demonstrates that DHAvD strongly inhibits activation of nuclear factor-kappa B (NF-{kappa}B), which is a major component in cancer cell invasion. The present study investigated whethermore » DHAvD can modulate MMP-9 expression and cell invasion in MCF-7 human breast cancer cells. MMP-9 expression and cell invasion in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) was increased, whereas these inductions were muted by DHAvD. DHAvD also suppressed activation of mitogen-activated protein kinase (MAPK), and MAPK-mediated nuclear factor-kappa B (NF-{kappa}B) and activator protein-1 (AP-1) activations in TPA-treated MCF-7 cells. The results indicate that DHAvD-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the MAPK/NF-{kappa}B and MAPK/AP-1 pathways in MCF-7 cells. DHAvD may have potential value in breast cancer metastasis.« less

Nectin-like molecule 1 inhibits the migration and invasion of U251 glioma cells by regulating the expression of an extracellular matrix protein osteopontin.

PubMed

Yin, Bin; Li, Ke-han; An, Tai; Chen, Tao; Peng, Xiao-zhong

2010-06-01

To investigate the molecular mechanism of nectin-like molecule 1 (NECL1) inhibiting the migration and invasion of U251 glioma cells. We infected U251 glioma cells with adeno-nectin-like molecule 1 (Ad-NECL1) or empty adenovirus (Ad). Transwell and wound healing assays were performed to observe the migration of U251 cells incubated with the cell supernatant from Ad-NECL1 or Ad infected U251 cells. DNA microarray was applied to screen the gene expression profile after the restoration of NECL1 in U251 glioma cell lines. The differential expression of osteopontin (OPN), a gene related to migration and invasion, was further analyzed with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. The restoration of NECL1 inhibited migration of U251 cells significantly (P<0.05). Altogether 195 genes were found differentially expressed by microarray, in which 175 were up-regulated and 20 down-regulated, including 9 extracellular matrix proteins involved in the migration of cells. Both mRNA and protein expressions of OPN, the most markedly reduced extracellular matrix protein, were found decreased in U251 cells after restoration of NECL1. Immunohistochemical assay also detected an increase of OPN in glioma tissues, related with the progressing of malignant grade. A link might exist between NECL1 and the extracellular matrix protein OPN in inhibiting the migration and invasion of U251 glioma cells.

Terrestrosin D, a steroidal saponin from Tribulus terrestris L., inhibits growth and angiogenesis of human prostate cancer in vitro and in vivo.

PubMed

Wei, Shihu; Fukuhara, Hideo; Chen, Guang; Kawada, Chiaki; Kurabayashi, Atsushi; Furihata, Mutsuo; Inoue, Keiji; Shuin, Taro

2014-01-01

The aim of this study was to investigate whether terrestrosin D (TED) inhibits the progression of castration-resistant prostate cancer and consider its mechanism. Cell cycle, mitochondrial membrane potential (ΔΨm) and apoptosis were determined by flow cytometry. Caspase-3 activity and vascular endothelial growth factor secretion were detected by a caspase-3 assay and human vascular endothelial growth factor kit, respectively. A PC-3 xenograft mouse model was used to evaluate the anticancer effect of TED in vivo. In vitro, TED strongly suppressed the growth of prostate cancer cells and endothelial cells in a dose-dependent manner. TED induced cell cycle arrest and apoptosis in PC-3 cells and human umbilical vascular endothelial cells (HUVECs). TED-induced apoptosis did not involve the caspase pathway. TED also decreased ΔΨm in PC-3 cells and HUVECs. In vivo, TED significantly suppressed tumor growth in nude mice bearing PC-3 cells, without any overt toxicity. Immunohistochemical analysis showed TED induced apoptotic cell death and inhibited angiogenesis in xenograft tumor cells. Cell cycle arrest and induction of apoptosis in cancer cells and endothelial cells might be plausible mechanisms of actions for the observed antitumor and antiangiogenic activities of TED. © 2014 S. Karger AG, Basel.

Transport characteristics of L-citrulline in renal apical membrane of proximal tubular cells.

PubMed

Mitsuoka, Keisuke; Shirasaka, Yoshiyuki; Fukushi, Akimasa; Sato, Masanobu; Nakamura, Toshimichi; Nakanishi, Takeo; Tamai, Ikumi

2009-04-01

L-Citrulline has diagnostic potential for renal function, because its plasma concentration increases with the progression of renal failure. Although L-citrulline extracted by glomerular filtration in kidney is mostly reabsorbed, the mechanism involved is not clearly understood. The present study was designed to characterize L-citrulline transport across the apical membranes of renal epithelial tubular cells, using primary-cultured rat renal proximal tubular cells, as well as the human kidney proximal tubular cell line HK-2. L-Citrulline was transported in a Na(+)-dependent manner from the apical side of both cell types cultured on permeable supports with a microporous membrane. Kinetic analysis indicated that the transport involves two distinct Na(+)-dependent saturable systems and one Na(+)-independent saturable system in HK-2 cells. The uptake was competitively inhibited by neutral and cationic, but not anionic amino acids. Relatively large cationic and anionic compounds inhibited the uptake, but smaller ones did not. In HK-2 cells, mRNA expression of SLC6A19 and SLC7A9, which encode B(0)AT1 and b(0,+)AT, respectively, was detected by RT-PCR. In addition, L-citrulline transport was significantly decreased in HK-2 cells in which either SLC6A19 or SLC7A9 was silenced. Hence, these results suggest that amino acid transporters B(0)AT1 and b(0,+)AT are involved in the reabsorption of L-citrulline in the kidney, at least in part, by mediating the apical membrane transport of L-citrulline in renal tubule cells.

Deficiency in methionine, tryptophan, isoleucine, or choline induces apoptosis in cultured cells.

PubMed

Yen, Chi-Liang E; Mar, Mei-Heng; Craciunescu, Corneliu N; Edwards, Lloyd J; Zeisel, Steven H

2002-07-01

Cells in culture die by apoptosis when deprived of the essential nutrient choline. We now report that cells (both proliferating PC12 cells and postmitotic neurons isolated from fetal rat brains) undergo apoptosis when deprived of other individual essential nutrients (methionine, tryptophan or isoleucine). In PC12 cells, deficiencies of each nutrient independently led to ceramide accumulation and to caspase activation, both recognized signals of several apoptotic pathways. A similar profile of caspases was activated in PC12 cells deprived of choline, methionine, tryptophan or isoleucine. More than one caspase was involved and these caspases appeared to transmit parallel signals for apoptosis induction because only broad-spectrum caspase inhibitors, but not inhibitors for specific individual caspases inhibited apoptosis in choline- or methionine-deprived cells. The induction of these caspase-dependent apoptosis pathways likely did not involve the same upstream signals. Choline deficiency perturbed choline metabolism but did not affect protein synthesis, whereas amino acid deficiencies inhibited protein synthesis but did not perturb choline metabolism. In addition, a subclone of PC12 cells that was resistant to choline deficiency-induced apoptosis was not resistant to tryptophan deficiency-induced apoptosis. These observations suggest that deficiency of each studied nutrient activates different pathways for signaling apoptosis that ultimately converge on a common execution pathway.

Pleiotrophin Exerts Its Migration and Invasion Effect through the Neuropilin-1 Pathway

PubMed Central

Elahouel, Rania; Blanc, Charly; Carpentier, Gilles; Frechault, Sophie; Cascone, Ilaria; Destouches, Damien; Delbé, Jean; Courty, José; Hamma-Kourbali, Yamina

2015-01-01

Pleiotrophin (PTN) is a pleiotropic growth factor that exhibits angiogenic properties and is involved in tumor growth and metastasis. Although it has been shown that PTN is expressed in tumor cells, few studies have investigated its receptors and their involvement in cell migration and invasion. Neuropilin-1 (NRP-1) is a receptor for multiple growth factors that mediates cell motility and plays an important role in angiogenesis and tumor progression. Here we provide evidence for the first time that NRP-1 is crucial for biological activities of PTN. We found that PTN interacted directly with NRP-1 through its thrombospondin type-I repeat domains. Importantly, binding of PTN to NRP-1 stimulated the internalization and recycling of NRP-1 at the cell surface. Invalidation of NRP-1 by RNA interference in human carcinoma cells inhibited PTN-induced intracellular signaling of the serine-threonine kinase, mitogen-activated protein MAP kinase, and focal adhesion kinase pathways. Accordingly, NRP-1 silencing or blocking by antibody inhibited PTN-induced human umbilical vein endothelial cell migration and tumor cell invasion. These results suggest that NRP-1/PTN interaction provides a novel mechanism for controlling the response of endothelial and tumoral cells to PTN and may explain, at least in part, how PTN contributes to tumor angiogenesis and cancer progression. PMID:26408254

α-Lipoic acid inhibits human lung cancer cell proliferation through Grb2-mediated EGFR downregulation.

PubMed

Yang, Lan; Wen, Ya; Lv, Guoqing; Lin, Yuntao; Tang, Junlong; Lu, Jingxiao; Zhang, Manqiao; Liu, Wen; Sun, Xiaojuan

2017-12-09

Alpha lipoic acid (α -LA) is a naturally occurring antioxidant and metabolic enzyme co-factor. Recently, α -LA has been reported to inhibit the growth of various cancer cells, but the precise signaling pathways that mediate the effects of α -LA on non-small cell lung cancer (NSCLC) development remain unclear. The CCK-8 assay was used to assess cell proliferation in NSCLC cell lines after α -LA treatment. The expression of growth factor receptor-bound protein 2 (Grb2), cyclin-dependent kinase (CDK)-2, CDK4, CDK6, Cyclin D3, Cyclin E1, Ras, c-Raf, epidermal growth factor receptor (EGFR), ERK1/2 and activated EGFR and ERK1/2 was evaluated by western blotting. Grb2 levels were restored in α-LA-treated cells by transfection of a plasmid carrying Grb2 and were reduced in NSCLC cells via specific siRNA-mediated knockdown. α -LA dramatically decreased NSCLC cell proliferation by downregulating Grb2; in contrast, Grb2 overexpression significantly prevented α-LA-induced decrease in cell growth in vitro. Western blot analysis indicated that α-LA decreased the levels of phospho-EGFR, CDK2/4/6, Cyclins D3 and E1, which are associated with the inhibition of G1/S-phase transition. Additional experiments indicated that Grb2 inhibition partially abolished EGF-induced phospho-EGFR and phospho-ERK1/2 activity. In addition, α-LA exerted greater inhibitory effects than gefitinib on NSCLC cells by preventing EGF-induced EGFR activation. For the first time, these findings provide the first evidence that α-LA inhibits cell proliferation through Grb2 by suppressing EGFR phosphorylation and that MAPK/ERK is involved in this pathway. Copyright © 2017. Published by Elsevier Inc.

Che-1 gene silencing induces osteosarcoma cell apoptosis by inhibiting mutant p53 expression

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

Liu, Ming; Wang, Dan, E-mail: danwangwdd@163.com; Li, Ning

2016-04-22

The transcriptional cofactor Che-1 is an RNA polymerase II (Pol II) which is involved in tumorigenesis, such as breast cancer and multiple myeloma. Che-1 can also regulate mutant p53 expression, which plays roles in many types of cancer. In this study, we aimed to investigate the effects and specific mechanism of Che-1 in the regulation of osteosarcoma (OS) cell growth. We found that Che-1 is highly expressed in several kinds of OS cells compared with osteoblast hFOB1.19 cells. MTT and flow cytometry assays showed that Che-1 depletion by siRNA markedly suppressed MG-63 and U2OS cell proliferation and promoted apoptosis. The chromatinmore » immunoprecipitation (ChIP) assay verified the presence of Che-1 on the p53 promoter in MG-63 and U2OS cells carrying mutant p53. Further studies showed that Che-1 depletion inhibited mutant p53 expression. Notably, our study showed that the loss of Che-1 inhibits proliferation and promotes apoptosis in MG-63 cells by decreasing the level of mutant p53. Therefore, these findings open the possibility that silencing of Che-1 will have therapeutic benefit in OS. - Highlights: • Che-1 is highly expressed in several kinds of OS cells. • Che-1 depletion suppressed MG-63 and U2OS cell growth. • Che-1 is existed in the p53 promoter in MG-63 and U2OS cells. • Che-1 depletion inhibited mutant p53 expression. • Che-1 depletion inhibits cell growth by decreasing the level of mutant p53.« less

Glycogen Synthase Kinase-3 (GSK3) Inhibition Induces Prosurvival Autophagic Signals in Human Pancreatic Cancer Cells*

PubMed Central

Marchand, Benoît; Arsenault, Dominique; Raymond-Fleury, Alexandre; Boisvert, François-Michel; Boucher, Marie-Josée

2015-01-01

Glycogen synthase kinase-3 (GSK3) are ubiquitously expressed serine-threonine kinases involved in a plethora of functions ranging from the control of glycogen metabolism to transcriptional regulation. We recently demonstrated that GSK3 inhibition triggers JNK-cJUN-dependent apoptosis in human pancreatic cancer cells. However, the comprehensive picture of downstream GSK3-regulated pathways/functions remains elusive. Herein, counterbalancing the death signals, we show that GSK3 inhibition induces prosurvival signals through increased activity of the autophagy/lysosomal network. Our data also reveal a contribution of GSK3 in the regulation of the master transcriptional regulator of autophagy and lysosomal biogenesis, transcription factor EB (TFEB) in pancreatic cancer cells. Similarly to mammalian target of rapamycin (mTOR) inhibition, GSK3 inhibitors promote TFEB nuclear localization and leads to TFEB dephosphorylation through endogenous serine/threonine phosphatase action. However, GSK3 and mTOR inhibition impinge differently and independently on TFEB phosphorylation suggesting that TFEB is regulated by a panel of kinases and/or phosphatases. Despite their differential impact on TFEB phosphorylation, both GSK3 and mTOR inhibitors promote 14-3-3 dissociation and TFEB nuclear localization. Quantitative mass spectrometry analyses further reveal an increased association of TFEB with nuclear proteins upon GSK3 and mTOR inhibition suggesting a positive impact on TFEB transcriptional function. Finally, a predominant nuclear localization of TFEB is unveiled in fully fed pancreatic cancer cells, whereas a reduction in TFEB expression significantly impairs their capacity for growth in an anchorage-independent manner. In addition, TFEB-restricted cells are more sensitive to apoptosis upon GSK3 inhibition. Altogether, our data uncover new functions under the control of GSK3 in pancreatic cancer cells in addition to providing key insight into TFEB regulation. PMID:25561726

Combining cetuximab with killer lymphocytes synergistically inhibits human cholangiocarcinoma cells in vitro.

PubMed

Morisaki, Takashi; Umebayashi, Masayo; Kiyota, Akifumi; Koya, Norihiro; Tanaka, Hiroto; Onishi, Hideya; Katano, Mitsuo

2012-06-01

We explored the possibility of combining adoptive immunotherapy with cytokine-activated killer (CAK) cells and the epidermal growth factor receptor monoclonal antibody, cetuximab, as a treatment for cholangiocarcinoma. CAK cells were cultured with a high-dose of interleukin-2 and anti-CD3 monoclonal antibodies. This cell population contained both activated CD16+/CD56+ (NK) cells and CD3+/NKG2D(high+) T-cells. The effect of CAK cells and cetuximab, alone and in combination, on the viability of human cholangiocarcinoma cells was evaluated. Culture of CAK cells alone, but not cetuximab alone, exhibited modest cytotoxicity toward cholangiocarcinoma cells. However, combining CAK cells with cetuximab significantly enhanced cytotoxicity. This enhancement was inhibited by the addition of excess human immunoglobulins, suggesting that antibody-dependent cytotoxicity, mediated by activated NK cells in the CAK cell culture was involved in this mechanism. Cetuximab may be used to enhance CAK cell therapeutic activity in patients with cholangiocarcinoma, by potentiating antibody-dependent cellular cytotoxicity.

Apoptosis induction in prostate cancer cells by a novel gene product, pHyde, involves caspase-3.

PubMed

Zhang, X; Steiner, M S; Rinaldy, A; Lu, Y

2001-09-20

A novel gene, pHyde, was recently cloned from Dunning rat prostate cancer cells. A recombinant adenovirus containing pHyde cDNA gene (AdpHyde) was generated to investigate the biological function of pHyde protein. AdpHyde inhibited the growth of human prostate cancer cells. Apoptosis was induced in AdpHyde transduced cells as demonstrated by DAPI (4', 6-diamino-2-phenylindole), TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling) staining, and flow cytometry assays. Apoptosis was also induced in human xenograft prostate cancer tumors growing in nude mice following treatment with AdpHyde. AdpHyde transduction resulted in a dose-dependent stimulation of caspase-3 activity in DU145 cells which was blocked by DEVD (succinyl-Asp-Glu-Val-Asp-aldehyde) and VAD (benzyloxycarbonyl - Val - Ala - Asp -fluoromethylketone), inhibitors specifically against caspase-3. Moreover, cancer cells that lacked expression of endogenous caspase-3 were not or barely inhibited by pHyde. These results taken together suggest that pHyde inhibits cancer growth by inducing apoptosis through a caspase-3 dependent pathway.

Phosphoproteomics Reveals Resveratrol-Dependent Inhibition of Akt/mTORC1/S6K1 Signaling

PubMed Central

2015-01-01

Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells. PMID:25311616

Kynurenine 3-monooxygenase mediates inhibition of Th17 differentiation via catabolism of endogenous aryl hydrocarbon receptor ligands.

PubMed

Stephens, Geoffrey L; Wang, Qun; Swerdlow, Bonnie; Bhat, Geetha; Kolbeck, Roland; Fung, Michael

2013-07-01

The aryl hydrocarbon receptor (AhR) is a key transcriptional regulator of Th17-cell differentiation. Although endogenous ligands have yet to be identified, evidence suggests that tryptophan metabolites can act as agonists for the AhR. Tryptophan metabolites are abundant in circulation, so we hypothesized that cell intrinsic factors might exist to regulate the exposure of Th17 cells to AhR-dependent activities. Here, we find that Th17 cells preferentially express kynurenine 3-monooxygenase (KMO), which is an enzyme involved in catabolism of the tryptophan metabolite kynurenine. KMO inhibition, either with a specific inhibitor or via siRNA-mediated silencing, markedly increased IL-17 production in vitro, whereas IFN-γ production by Th1 cells was unaffected. Inhibition of KMO significantly exacerbated disease in a Th17-driven model of autoimmune gastritis, suggesting that expression of KMO by Th17 cells serves to limit their continuous exposure to physiological levels of endogenous AhR ligands in vivo. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Smad4 inhibits cell migration via suppression of JNK activity in human pancreatic carcinoma PANC-1 cells.

PubMed

Zhang, Xueying; Cao, Junxia; Pei, Yujun; Zhang, Jiyan; Wang, Qingyang

2016-05-01

Smad4 is a common Smad and is a key downstream regulator of the transforming growth factor-β signaling pathway, in which Smad4 often acts as a potent tumor suppressor and functions in a highly context-dependent manner, particularly in pancreatic cancer. However, little is known regarding whether Smad4 regulates other signaling pathways involved in pancreatic cancer. The present study demonstrated that Smad4 downregulates c-Jun N-terminal kinase (JNK) activity using a Smad4 loss-of-function or gain-of-function analysis. Additionally, stable overexpression of Smad4 clearly affected the migration of human pancreatic epithelioid carcinoma PANC-1 cells, but did not affect cell growth. In addition, the present study revealed that upregulation of mitogen-activated protein kinase phosphatase-1 is required for the reduction of JNK activity by Smad4, leading to a decrease in vascular endothelial growth factor expression and inhibiting cell migration. Overall, the present findings indicate that Smad4 may suppress JNK activation and inhibit the tumor characteristics of pancreatic cancer cells.

Effects of resveratrol-related hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action.

PubMed

Cho, Dong-Im; Koo, Na-Youn; Chung, Woon Jae; Kim, Tae-Sung; Ryu, Shi Yong; Im, Suhn Young; Kim, Kyeong-Man

2002-09-13

NF-kappaB that plays an important role in iNOS expression is one of the targets of various potential anti-inflammatory agents including resveratrol. Resveratrol contains a structural similarity with estrogen, and there has been speculation about resveratrol as estrogen agonist. In this study, the mechanism and structural requirements of resveratrol and related hydroxystilbenes for the inhibition of LPS-induced nitric oxide production were studied in macrophage cells (RAW 264.7 and J774) by comparing its effect on LPS-induced NF-kappaB translocation and nitric oxide production, and by considering the possibility of involvement of an estrogen receptor. LPS-induced nitric oxide production was inhibited only when cells were treated with resveratrol prior to stimulation with LPS, suggesting that resveratrol does not affect the enzyme itself. A higher concentration of resveratrol than needed for the inhibition of nitric oxide production was required for the inhibition of NF-kappaB mobilization or iNOS expression. Estrogen and diethylstilbesterol, an estrogen agonist, caused only weak inhibition of nitric oxide production, and the effects of resveratrol were not noticeably blocked by ICI-182780, an estrogen antagonist. Structure-activity analysis of resveratrol and nine hydroxystilbenes suggests that the structural balance between oxygen functional groups on the benzene rings is important for their activity. Our results suggest that resveratrol might act on other cellular targets as well as NF-kappaB at the initial stage of gene expression. Unique structural features of hydroxystilbenes are needed for suppression of nitric oxide production and it is unlikely that estrogen receptor is involved in it.

Identification of 2,4-dihydroxy-5-pyrimidinyl imidothiocarbomate as a novel inhibitor to Y box binding protein-1 (YB-1) and its therapeutic actions against breast cancer.

PubMed

Gunasekaran, Vinoth Prasanna; Nishi, Kumari; Sivakumar, Dakshinamurthy; Sivaraman, Thirunavukkarasu; Mathan, Ganeshan

2018-04-30

In spite of advances in breast cancer treatment and early diagnosis, drug toxicity, cancer relapse, multidrug resistance and metastasis are the major impediment to the developments of efficient drugs. However, unique druggable targets of cancer cells distinct from the normal cells provide new rationale in cancer treatment. Previous reports clearly emphasize the differential expression and localization of Y box binding protein-1 (YB-1) between normal breast tissues and different stages of breast cancer. Y box binding protein-1 is DNA as well as RNA binding protein involved in transcription and translation regulation of various proteins involved in cancer progression, apoptosis, cell cycle, epithelial to mesenchymal transition (EMT) and drug resistance. Particularly, during doxorubicin (DOX) treatment and cancer relapse conditions, YB-1 expression was very high in breast cancer tissues and localized in to nucleus which further favours DOX efflux and metastasis. Moreover, siRNA mediated silencing of YB-1 reduces breast cancer progression and metastasis. In this rationale, using an array of computational methods, 2,4-dihydroxy-5-pyrimidinyl imidothiocarbomate (DPI) has been screened out as a drug-likeness antagonist to the YB-1for cancer treatment. In this study, we determined that DPI was toxic to breast cancer cell lines as individual drug as well as in combination with DOX. Moreover, immunofluorescence and confocal studies showed that DPI decreases DOX induced YB-1 nuclear translocation and increases DOX accumulation in breast cancer cell line. A G1/G0 phase cell cycle arrest and apoptosis was also induced by DPI. Moreover, DPI modulated YB-1 downstream targets such as p53, caspase-3, CDK-1 which are involved in cell cycle progression and apoptosis. Further, metastatic functional analysis revealed that DPI inhibits cell adhesion, migration, invasion in aggressive metastatic cell line and inhibits angiogenesis in chick embryonic chorioallantoic membrane (CAM) model. Meanwhile, DPI alters the expression of YB-1 downstream targets which are involved in metastasis such as VEGFR, caveolin, E-cadherin, cytokeratins, desmin and vimentin in MDA-MB-231 xenograft in chick embryonic CAM membrane. The results clearly demonstrated that DPI inhibited YB-1 nuclear translocation, thereby exhibited anti-apoptotic, anti-proliferative and anti-metastatic activities and increases the therapeutic potential of commercial breast cancer drug doxorubicin. Copyright © 2017 Elsevier B.V. All rights reserved.

Reactive oxygen species mediate Cr(VI)-induced carcinogenesis through PI3K/AKT-dependent activation of GSK-3β/β-catenin signaling

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

Son, Young-Ok; Pratheeshkumar, Poyil; Wang, Lei

Cr(VI) compounds are known human carcinogens that primarily target the lungs. Cr(VI) produces reactive oxygen species (ROS), but the exact effects of ROS on the signaling molecules involved in Cr(VI)-induced carcinogenesis have not been extensively studied. Chronic exposure of human bronchial epithelial cells to Cr(VI) at nanomolar concentrations (10–100 nM) for 3 months not only induced cell transformation, but also increased the potential of these cells to invade and migrate. Injection of Cr(VI)-stimulated cells into nude mice resulted in the formation of tumors. Chronic exposure to Cr(VI) increased levels of intracellular ROS and antiapoptotic proteins. Transfection with catalase or superoxidemore » dismutase (SOD) prevented Cr(VI)-mediated increases in colony formation, cell invasion, migration, and xenograft tumors. While chronic Cr(VI) exposure led to activation of signaling cascades involving PI3K/AKT/GSK-3β/β-catenin and PI3K/AKT/mTOR, transfection with catalase or SOD markedly inhibited Cr(VI)-mediated activation of these signaling proteins. Inhibitors specific for AKT or β-catenin almost completely suppressed the Cr(VI)-mediated increase in total and active β-catenin proteins and colony formation. In particular, Cr(VI) suppressed autophagy of epithelial cells under nutrition deprivation. Furthermore, there was a marked induction of AKT, GSK-3β, β-catenin, mTOR, and carcinogenic markers in tumor tissues formed in mice after injection with Cr(VI)-stimulated cells. Collectively, our findings suggest that ROS is a key mediator of Cr(VI)-induced carcinogenesis through the activation of PI3K/AKT-dependent GSK-3β/β-catenin signaling and the promotion of cell survival mechanisms via the inhibition of apoptosis and autophagy. - Highlights: • Chronic exposure to Cr(VI) induces carcinogenic properties in BEAS-2B cells. • ROS play an important role in Cr(VI)-induced tumorigenicity of BEAS-2B cells. • PI3K/AKT/GSK-3β/β-catenin signaling involved in Cr(VI) carcinogenesis. • The inhibition of apoptosis and autophagy contributes to Cr(VI) carcinogenesis.« less

(+)-Nootkatone inhibits tumor necrosis factor α/interferon γ-induced production of chemokines in HaCaT cells

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

Choi, Hyeon-Jae; Lee, Jin-Hwee; Jung, Yi-Sook, E-mail: yisjung@ajou.ac.kr

Highlights: • (+)-Nootkatone inhibits TNF-α/IFN-γ-induced TARC and MDC expression in HaCaT cells. • PKCζ, p38 MAPK, or NF-κB mediate TNF-α/IFN-γ-induced TARC and MDC expression. • (+)-Nootkatone inhibits TNF-α/IFN-γ-induced activation of PKCζ, p38 MAPK, or NF-κB. • (+)-Nootkatone suppresses chemokine expression by inhibiting of PKCζ and p38 pathways. - Abstract: Chemokines are important mediators of cell migration, and thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are well-known typical inflammatory chemokines involved in atopic dermatitis (AD). (+)-Nootkatone is the major component of Cyperus rotundus. (+)-Nootkatone has antiallergic, anti-inflammatory, and antiplatelet activities. The purpose of this study was to investigate themore » effect of (+)-nootkatone on tumor necrosis factor α (TNF-α)/interferon γ (IFN-γ)-induced expression of Th2 chemokines in HaCaT cells. We found that (+)-nootkatone inhibited the TNF-α/IFN-γ-induced expression of TARC/CCL17 and MDC/CCL22 mRNA in HaCaT cells. It also significantly inhibited TNF-α/IFN-γ-induced activation of nuclear factor kappa B (NF-κB), p38 mitogen-activated protein kinase (MAPK), and protein kinase Cζ (PKCζ). Furthermore, we showed that PKCζ and p38 MAPK contributed to the inhibition of TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression by blocking IκBα degradation in HaCaT cells. Taken together, these results suggest that (+)-nootkatone may suppress TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression in HaCaT cells by inhibiting of PKCζ and p38 MAPK signaling pathways that lead to activation of NF-κB. We propose that (+)-nootkatone may be a useful therapeutic candidate for inflammatory skin diseases such as AD.« less

Labdane diterpenes protect against anoxia/reperfusion injury in cardiomyocytes: involvement of AKT activation

PubMed Central

Cuadrado, I; Fernández-Velasco, M; Boscá, L; de las Heras, B

2011-01-01

Several labdane diterpenes exert anti-inflammatory and cytoprotective actions; therefore, we have investigated whether these molecules protect cardiomyocytes in an anoxia/reperfusion (A/R) model, establishing the molecular mechanisms involved in the process. The cardioprotective activity of three diterpenes (T1, T2 and T3) was studied in the H9c2 cell line and in isolated rat cardiomyocyte subjected to A/R injury. In both cases, treatment with diterpenes T1 and T2 protected from A/R-induced apoptosis, as deduced by a decrease in the percentage of apoptotic and caspase-3 active positive cells, a decrease in the Bcl-2/Bax ratio and an increase in the expression of antiapoptotic proteins. Analysis of cell survival signaling pathways showed that diterpenes T1 and T2 added after A/R increased phospho-AKT and phospho-ERK 1/2 levels. These cardioprotective effects were lost when AKT activity was pharmacologically inhibited. Moreover, the labdane-induced cardioprotection involves activation of AMPK, suggesting a role for energy homeostasis in their mechanism of action. Labdane diterpenes (T1 and T2) also exerted cardioprotective effects against A/R-induced injury in isolated cardiomyocytes and the mechanisms involved activation of specific survival signals (PI3K/AKT pathways, ERK1/2 and AMPK) and inhibition of apoptosis. PMID:22071634

Overcoming adaptive resistance in mucoepidermoid carcinoma through inhibition of the IKK-β/IκBα/NFκB axis

PubMed Central

Wagner, Vivian P.; Martins, Marco A.T.; Martins, Manoela D.; Warner, Kristy A.; Webber, Liana P.; Squarize, Cristiane H.; Nör, Jacques E.; Castilho, Rogerio M.

2016-01-01

Patients with mucoepidermoid carcinoma (MEC) experience low survival rates and high morbidity following treatment, yet the intrinsic resistance of MEC cells to ionizing radiation (IR) and the mechanisms underlying acquired resistance remain unexplored. Herein, we demonstrated that low doses of IR intrinsically activated NFκB in resistant MEC cell lines. Moreover, resistance was significantly enhanced in IR-sensitive cell lines when NFκB pathway was stimulated. Pharmacological inhibition of the IKK-β/IκBα/NFκB axis, using a single dose of FDA-approved Emetine, led to a striking sensitization of MEC cells to IR and a reduction in cancer stem cells. We achieved a major step towards better understanding the basic mechanisms involved in IR-adaptive resistance in MEC cell lines and how to efficiently overcome this critical problem. PMID:27682876

Citrus limonin and its glucoside inhibit colon adenocarcinoma cell proliferation through apoptosis.

PubMed

Chidambara Murthy, Kotamballi N; Jayaprakasha, G K; Kumar, Vinod; Rathore, Keerti S; Patil, Bhimanagouda S

2011-03-23

The current study was an attempt to elucidate the mechanism of human colon cancer cell proliferation inhibition by limonin and limonin glucoside (LG) isolated from seeds of Citrus reticulata. The structures of purified compounds were confirmed by NMR and quantified using HPLC. These compounds of more than 95% purity were subjected to proliferation inhibition assay using human colon adenocarcinoma (SW480) cells. The IC50 value of 54.74 and 37.39 μM was observed for limonin and LG, respectively at 72 h. Following confirmation of proliferation inhibition, pattern of DNA fragmentation and activation of caspase-3 of the cells treated with limonoids suggest involvement of apoptosis. Furthermore, reduction in the transcription ratio of bcl2/bax and induction of cytochrome c release from mitochondria to cytosol with treatment of limonoids confirm the activation of intrinsic apoptosis pathway. The activity of Bax and Bcl2 was confirmed through analysis of mitochondrial membrane potential and intracellular calcium in the cells treated with limonin and LG; the net content of caspase-8 was not affected by limonoids. Results of the current study provide compelling evidence on the induction of mitochondria mediated intrinsic apoptosis by both limonin and LG in cultured SW480 cells for the first time.

Antifungal activity of geraniol and citronellol, two monoterpenes alcohols, against Trichophyton rubrum involves inhibition of ergosterol biosynthesis.

PubMed

Pereira, Fillipe de Oliveira; Mendes, Juliana Moura; Lima, Igara Oliveira; Mota, Kelly Samara de Lira; Oliveira, Wylly Araújo de; Lima, Edeltrudes de Oliveira

2015-02-01

Trichophyton rubrum is the most common fungus causing chronic dermatophytosis in humans. Antifungal activity of promising agents is of great interest. Geraniol and citronellol are monoterpenes with antimicrobial properties. This study aimed to investigate the inhibitory effects and possible mechanism of antifungal activity of geraniol and citronellol against strains of T. rubrum. The minimum inhibitory concentration (MIC) of each drug against 14 strains was determined by broth microdilution. The effects of the drugs on dry mycelial weight, conidial germination, infectivity on human nail fragments, and morphogenesis of T. rubrum were analyzed. The effects on the cell wall (test with sorbitol) and cell membrane (release of intracellular material and ergosterol biosynthesis) were investigated. MIC values of geraniol ranged between 16 and 256 µg/mL while citronellol showed MIC values from 8 to 1024 µg/mL. The drugs (MIC and 2 × MIC) inhibited the mycelial growth, conidia germination, and fungal growth on nail fragments. The drugs (half of MIC) induced the formation of wide, short, and crooked hyphae in T. rubrum morphology. With sorbitol, geraniol MIC was increased by 64-fold and citronellol by 32-fold. The drugs caused leakage of intracellular material and inhibited ergosterol biosynthesis. The results suggest that the drugs damage cell wall and cell membrane of T. rubrum through a mechanism that seems to involve the inhibition of the ergosterol biosynthesis. This study confirms that geraniol and citronellol can be regarded as potential drugs for controlling T. rubrum growth, with great potential against agents of dermatophytosis.