Aluminum oxide nanoparticles alter cell cycle progression through CCND1 and EGR1 gene expression in human mesenchymal stem cells.

PubMed

Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

2016-05-01

Aluminum oxide nanoparticles (Al2 O3 -NPs) are important ceramic materials that have been used in a variety of commercial and industrial applications. However, the impact of acute and chronic exposure to Al2 O3 -NPs on the environment and on human health has not been well studied. In this investigation, we evaluated the cytotoxic effects of Al2 O3 -NPs on human mesenchymal stem cells (hMSCs) by using a cell viability assay and observing cellular morphological changes, analyzing cell cycle progression, and monitoring the expression of cell cycle response genes (PCNA, EGR1, E2F1, CCND1, CCNC, CCNG1, and CYCD3). The Al2 O3 -NPs reduced hMSC viability in a dose- and time-dependent manner. Nuclear condensation and fragmentation, chromosomal DNA fragmentation, and cytoplasmic vacuolization were observed in Al2 O3 -NP-exposed cells. The nuclear morphological changes indicated that Al2 O3 -NPs alter cell cycle progression and gene expression. The cell cycle distribution revealed that Al2 O3 -NPs cause cell cycle arrest in the sub-G0-G1 phase, and this is associated with a reduction in the cell population in the G2/M and G0/G1 phases. Moreover, Al2 O3 -NPs induced the upregulation of cell cycle response genes, including EGR1, E2F1, and CCND1. Our results suggested that exposure to Al2 O3 -NPs could cause acute cytotoxic effects in hMSCs through cell cycle regulatory genes. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Antitumorigenic effect of atmospheric-pressure dielectric barrier discharge on human colorectal cancer cells via regulation of Sp1 transcription factor

NASA Astrophysics Data System (ADS)

Han, Duksun; Cho, Jin Hyoung; Lee, Ra Ham; Bang, Woong; Park, Kyungho; Kim, Minseok S.; Shim, Jung-Hyun; Chae, Jung-Il; Moon, Se Youn

2017-02-01

Human colorectal cancer cell lines (HT29 and HCT116) were exposed to dielectric barrier discharge (DBD) plasma at atmospheric pressure to investigate the anticancer capacity of the plasma. The dose- and time-dependent effects of DBDP on cell viability, regulation of transcription factor Sp1, cell-cycle analysis, and colony formation were investigated by means of MTS assay, DAPI staining, propidium iodide staining, annexin V-FITC staining, Western blot analysis, RT-PCR analysis, fluorescence microscopy, and anchorage-independent cell transformation assay. By increasing the duration of plasma dose times, significant reductions in the levels of both Sp1 protein and Sp1 mRNA were observed in both cell lines. Also, expression of negative regulators related to the cell cycle (such as p53, p21, and p27) was increased and of the positive regulator cyclin D1 was decreased, indicating that the plasma treatment led to apoptosis and cell-cycle arrest. In addition, the sizes and quantities of colony formation were significantly suppressed even though two cancer promoters, such as TPA and epidermal growth factor, accompanied the plasma treatment. Thus, plasma treatment inhibited cell viability and colony formation by suppressing Sp1, which induced apoptosis and cell-cycle arrest in these two human colorectal cancer cell lines.

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.

Tectonic-1 contributes to the growth and migration of prostate cancer cells in vitro

PubMed Central

WANG, ZHIJUN; GAO, YI; LIU, YUSHAN; CHEN, JIE; WANG, JUNKAI; GAN, SISHUN; XU, DANFENG; CUI, XINGANG

2015-01-01

Tectonic-1 (TCTN1) is an upstream gene involved in embryonic development. The aim of the present study was to investigate the effect of the TCTN1 gene on the viability and migration of prostate cancer cells. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to silence the expression of TCTN1 in PC-3 and DU145 prostate cancer cells. Cell viability and proliferation were measured using MTT and colony formation assays, and the distribution of cells in phases of the cell cycle was determined using flow cytometry. Cell migration was detected using a Transwell assay. The results demonstrated that TCTN1 was widely expressed in several human prostate cancer cell lines. Knockdown of the TCTN1 gene by RNA interference markedly suppressed cell viability and colony formation in the PC-3 and DU145 cell lines. Cell cycle progression was also arrested by TCTN1 silencing. In addition, knockdown of the TCTN1 gene led to the inhibition of cell migration in the two cell lines. These findings confirmed the direct association between the TCTN1 gene and prostate cancer growth in vitro. With further understanding and clinical investigation, this indicates the potential for future development of a novel marker for early detection and gene therapy for prostate cancer. PMID:26310786

Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy

PubMed Central

Wu, Qing; Lin, Wei-Dong; Liao, Guan-Qun; Zhang, Li-Guo; Wen, Shun-Qian; Lin, Jia-Ying

2015-01-01

AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action. METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level. RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface. CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity. PMID:25624718

Glucose capped silver nanoparticles induce cell cycle arrest in HeLa cells.

PubMed

Panzarini, Elisa; Mariano, Stefania; Vergallo, Cristian; Carata, Elisabetta; Fimia, Gian Maria; Mura, Francesco; Rossi, Marco; Vergaro, Viviana; Ciccarella, Giuseppe; Corazzari, Marco; Dini, Luciana

2017-06-01

This study aims to determine the interaction (uptake and biological effects on cell viability and cell cycle progression) of glucose capped silver nanoparticles (AgNPs-G) on human epithelioid cervix carcinoma (HeLa) cells, in relation to amount, 2×10 3 or 2×10 4 NPs/cell, and exposure time, up to 48h. The spherical and well dispersed AgNPs (30±5nm) were obtained by using glucose as reducing agent in a green synthesis method that ensures to stabilize AgNPs avoiding cytotoxic soluble silver ions Ag + release. HeLa cells take up abundantly and rapidly AgNPs-G resulting toxic to cells in amount and incubation time dependent manner. HeLa cells were arrested at S and G2/M phases of the cell cycle and subG1 population increased when incubated with 2×10 4 AgNPs-G/cell. Mitotic index decreased accordingly. The dissolution experiments demonstrated that the observed effects were due only to AgNPs-G since glucose capping prevents Ag + release. The AgNPs-G influence on HeLa cells viability and cell cycle progression suggest that AgNPs-G, alone or in combination with chemotherapeutics, may be exploited for the development of novel antiproliferative treatment in cancer therapy. However, the possible influence of the cell cycle on cellular uptake of AgNPs-G and the mechanism of AgNPs entry in cells need further investigation. Copyright © 2017 Elsevier B.V. All rights reserved.

Astaxanthin Inhibits Proliferation of Human Gastric Cancer Cell Lines by Interrupting Cell Cycle Progression.

PubMed

Kim, Jung Ha; Park, Jong-Jae; Lee, Beom Jae; Joo, Moon Kyung; Chun, Hoon Jai; Lee, Sang Woo; Bak, Young-Tae

2016-05-23

Astaxanthin is a carotenoid pigment that has antioxidant, antitumoral, and anti-inflammatory properties. In this in vitro study, we investigated the mechanism of anticancer effects of astaxanthin in gastric carcinoma cell lines. The human gastric adenocarcinoma cell lines AGS, KATO-III, MKN-45, and SNU-1 were treated with various concentrations of astaxanthin. A cell viability test, cell cycle analysis, and immunoblotting were performed. The viability of each cancer cell line was suppressed by astaxanthin in a dose-dependent manner with significantly decreased proliferation in KATO-III and SNU-1 cells. Astaxanthin increased the number of cells in the G0/G1 phase but reduced the proportion of S phase KATO-III and SNU-1 cells. Phosphorylated extracellular signal-regulated kinase (ERK) was decreased in an inverse dose-dependent correlation with astaxanthin concentration, and the expression of p27(kip-1) increased the KATO-III and SNU-1 cell lines in an astaxanthin dose-dependent manner. Astaxanthin inhibits proliferation by interrupting cell cycle progression in KATO-III and SNU-1 gastric cancer cells. This may be caused by the inhibition of the phosphorylation of ERK and the enhanced expression of p27(kip-1).

Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

PubMed

de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

2013-12-31

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the importance of new approaches for IFN potency determination. Copyright © 2013 Elsevier B.V. All rights reserved.

The flavonoid quercetin transiently inhibits the activity of taxol and nocodazole through interference with the cell cycle

PubMed Central

Samuel, Temesgen; Fadlalla, Khalda; Turner, Timothy; Yehualaeshet, Teshome E.

2010-01-01

Quercetin is a flavonoid with anticancer properties. In this study, we examined the effects of quercetin on cell cycle, viability and proliferation of cancer cells, either singly or in combination with the microtubule-targeting drugs taxol and nocodazole. Although quercetin induced cell death in a dose dependent manner, 12.5-50μM quercetin inhibited the activity of both taxol and nocodazole to induce G2/M arrest in various cell lines. Quercetin also partially restored drug-induced loss in viability of treated cells for up to 72 hours. This antagonism of microtubule-targeting drugs was accompanied by a delay in cell cycle progression and inhibition of the buildup of cyclin-B1 at the microtubule organizing center of treated cells. However, quercetin did not inhibit the microtubule targeting of taxol or nocodazole. Despite the short-term protection of cells by quercetin, colony formation and clonogenicity of HCT116 cells were still suppressed by quercetin or quercetin-taxol combination. The status of cell adherence to growth matrix was critical in determining the sensitivity of HCT116 cells to quercetin. We conclude that while long-term exposure of cancer cells to quercetin may prevent cell proliferation and survival, the interference of quercetin with cell cycle progression diminishes the efficacy of microtubule-targeting drugs to arrest cells at G2/M. PMID:21058190

Magnolol pretreatment attenuates heat stress-induced IEC-6 cell injury.

PubMed

Mei, Chen; He, Sha-Sha; Yin, Peng; Xu, Lei; Shi, Ya-Ran; Yu, Xiao-Hong; Lyu, An; Liu, Feng-Hua; Jiang, Lin-Shu

2016-06-01

Heat stress (HS) is an important environmental stressor that adversely influences livestock during the summer. The aim of this study was to investigate whether magnolol protects against HS-induced intestinal epithelial cell injury. An intestinal epithelial cell line (IEC-6) was subjected to HS at 42 °C, with and without magnolol pretreatment. Cell injury was detected by monitoring lactate dehydrogenase (LDH) release. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was used to assess cell proliferation and viability, including identifying effective concentrations of magnolol. Flow cytometry confirmed G1-phase cell-cycle arrest and its alleviation by magnolol. Active DNA synthesis was measured by incorporation of nucleic acid 5-ethynyl-2'-deoxyuridine (EdU). G1-phase cell-cycle-related gene expression was assessed by real-time reverse transcription polymerase chain reaction (RT-PCR) and levels of G1-phase-related proteins by Western blotting. HS induced IEC-6 cell injury and decreased cell viability, as demonstrated by data from LDH and MTS assays, respectively. Based on a number of criteria, IEC-6 cells subjected to HS were arrested in the G1 phase of the cell cycle. Magnolol pretreatment decreased HS-induced cell injury through relief of this cell-cycle arrest. Magnolol pretreatment attenuates HS-induced injury in IEC-6 cells. Magnolol is potentially promising as a protective strategy for HS in livestock.

Gamma Irradiation Upregulates B-cell Translocation Gene 2 to Attenuate Cell Proliferation of Lung Cancer Cells Through the JNK and NF-κB Pathways.

PubMed

Wang, Peihe; Cai, Yuanyuan; Lin, Dongju; Jiang, Yingxiao

2017-08-07

Gamma ray can promote cancer cell apoptosis and cell cycle arrest. It is often used in the clinical treatment of tumors, including lung cancer. In this study, we aimed to explore the role of gamma ray treatment and its correlation with BTG2 in cell proliferation, apoptosis, and cell cycle arrest regulation in a lung cancer cell line. A549 cell viability, apoptosis rate, and cell cycle were investigated after gamma ray treatment. We then used siRNA for BTG2 to detect the effect of BTG2 knockdown on the progress of gamma ray-treated lung cancer cells. Finally, we investigated the signaling pathway by which gamma ray might regulate BTG2. We found that gamma ray inhibited A549 cell viability and promoted apoptosis and cell cycle arrest, while BTG2 knockdown could relieve the effect caused by gamma ray on A549 cells. Moreover, we confirmed that the effect of BTG2 partly depends on p53 expression and gamma ray-promoting BTG2 expression through the JNK/NF-κB signaling pathway. Our study assessed the possible mechanism of gamma ray in tumor treatment and also investigated the role of BTG2 in gamma ray therapy. All these findings might give a deep understanding of the effect of gamma ray on the progression of lung cancer involving BTG2.

Antitumor Effects of Flavopiridol on Human Uterine Leiomyoma In Vitro and in a Xenograft Model

PubMed Central

Lee, Hyun-Gyo; Baek, Jong-Woo; Shin, So-Jin; Kwon, Sang-Hoon; Cha, Soon-Do; Park, Won-Jin; Chung, Rosa; Choi, Eun-Som; Lee, Gun-Ho

2014-01-01

Dysregulated cyclin-dependent kinases (CDKs) are considered a potential target for cancer therapy. Flavopiridol is a potent CDK inhibitor. In this study, the antiproliferative effect of the flavonoid compound flavopiridol and its mechanism in human uterine leiomyoma cells were investigated. The present study focused on the effect of flavopiridol in cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Cell viability and cell proliferation assays were conducted. Flow cytometry was performed to determine the effect of flavopiridol on cell cycle. The expression of cell cycle regulatory-related proteins was evaluated by Western blotting. Cell viability and proliferation of uterine leiomyoma cells were significantly reduced by flavopiridol treatment in a dose-dependent manner. Flow cytometry results showed that flavopiridol induced G1 phase arrest. Flavopiridol-induced growth inhibition in uterine leiomyoma cells was associated with increased expression of p21cip/wafl and p27kip1 in a dose-dependent manner. Downregulation of CDK2/4 and Cyclin A with a concomitant increase in dephosphorylation of retinoblastoma was observed. This study demonstrates that flavopiridol inhibits cell proliferation by initiating G1 cell cycle arrest in human uterine leiomyoma. We also found that flavopiridol is effective in inhibiting xenografted human uterine leiomyoma growth. These results indicate that flavopiridol could prove to be a promising chemopreventive and therapeutic agent for human uterine leiomyoma. PMID:24572052

Effects of Different Zinc Species on Cellar Zinc Distribution, Cell Cycle, Apoptosis and Viability in MDAMB231 Cells.

PubMed

Wang, Yan-hong; Zhao, Wen-jie; Zheng, Wei-juan; Mao, Li; Lian, Hong-zhen; Hu, Xin; Hua, Zi-chun

2016-03-01

Intracellular metal elements exist in mammalian cells with the concentration range from picomoles per litre to micromoles per litre and play a considerable role in various biological procedures. Element provided by different species can influence the availability and distribution of the element in a cell and could lead to different biological effects on the cell's growth and function. Zinc as an abundant and widely distributed essential trace element, is involved in numerous and relevant physiological functions. Zinc homeostasis in cells, which is regulated by metallothioneins, zinc transporter/SLC30A, Zrt-/Irt-like proteins/SLC39A and metal-response element-binding transcription factor-1 (MTF-1), is crucial for normal cellular functioning. In this study, we investigated the influences of different zinc species, zinc sulphate, zinc gluconate and bacitracin zinc, which represented inorganic, organic and biological zinc species, respectively, on cell cycle, viability and apoptosis in MDAMB231 cells. It was found that the responses of cell cycle, apoptosis and death to different zinc species in MDAMB231 cells are different. Western blot analysis of the expression of several key proteins in regulating zinc-related transcription, cell cycle, apoptosis, including MTF-1, cyclin B1, cyclin D1, caspase-8 and caspase-9 in treated cells further confirmed the observed results on cell level.

Effects of γ-radiation on cell growth, cell cycle and promoter methylation of 22 cell cycle genes in the 1321NI astrocytoma cell line.

PubMed

Alghamian, Yaman; Abou Alchamat, Ghalia; Murad, Hossam; Madania, Ammar

2017-09-01

DNA damage caused by radiation initiates biological responses affecting cell fate. DNA methylation regulates gene expression and modulates DNA damage pathways. Alterations in the methylation profiles of cell cycle regulating genes may control cell response to radiation. In this study we investigated the effect of ionizing radiation on the methylation levels of 22 cell cycle regulating genes in correlation with gene expression in 1321NI astrocytoma cell line. 1321NI cells were irradiated with 2, 5 or 10Gy doses then analyzed after 24, 48 and 72h for cell viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu bromide) assay. Flow cytometry were used to study the effect of 10Gy irradiation on cell cycle. EpiTect Methyl II PCR Array was used to identify differentially methylated genes in irradiated cells. Changes in gene expression was determined by qPCR. Azacytidine treatment was used to determine whether DNA methylation affectes gene expression. Our results showed that irradiation decreased cell viability and caused cell cycle arrest at G2/M. Out of 22 genes tested, only CCNF and RAD9A showed some increase in DNA methylation (3.59% and 3.62%, respectively) after 10Gy irradiation, and this increase coincided with downregulation of both genes (by 4 and 2 fold, respectively). with azacytidine confirmed that expression of CCNF and RAD9A genes was regulated by methylation. 1321NI cell line is highly radioresistant and that irradiation of these cells with a 10Gy dose increases DNA methylation of CCNF and RAD9A genes. This dose down-regulates these genes, favoring G2/M arrest. Copyright © 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

PubMed Central

Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

2016-01-01

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells. PMID:27383047

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria.

PubMed

Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

2016-07-07

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.

E2F function in muscle growth is necessary and sufficient for viability in Drosophila

PubMed Central

Zappia, Maria Paula; Frolov, Maxim V.

2016-01-01

The E2F transcription factor is a key cell cycle regulator. However, the inactivation of the entire E2F family in Drosophila is permissive throughout most of animal development until pupation when lethality occurs. Here we show that E2F function in the adult skeletal muscle is essential for animal viability since providing E2F function in muscles rescues the lethality of the whole-body E2F-deficient animals. Muscle-specific loss of E2F results in a significant reduction in muscle mass and thinner myofibrils. We demonstrate that E2F is dispensable for proliferation of muscle progenitor cells, but is required during late myogenesis to directly control the expression of a set of muscle-specific genes. Interestingly, E2f1 provides a major contribution to the regulation of myogenic function, while E2f2 appears to be less important. These findings identify a key function of E2F in skeletal muscle required for animal viability, and illustrate how the cell cycle regulator is repurposed in post-mitotic cells. PMID:26823289

[1-9-NαC]-crourorb A1 isolated from Croton urucurana latex induces G2/M cell cycle arrest and apoptosis in human hepatocarcinoma cells.

PubMed

de Matos Cândido-Bacani, Priscila; Ezan, Frédéric; de Oliveira Figueiredo, Patrícia; Matos, Maria de Fátima Cepa; Rodrigues Garcez, Fernanda; Silva Garcez, Walmir; Baffet, Georges

2017-05-05

[1-9-NαC]-crourorb A1 is a cyclic peptide isolated from Croton urucurana Baillon latex, found in midwestern Brazil, that has been shown to exert cytotoxic effects against a panel of cancer cell lines. However, the underlying mechanisms responsible for the crourorb A1-induced cytotoxicity in cancer cells remain unknown. In this study, the effects of crourorb A1 on the viability, apoptosis, cell cycle and migration of Huh-7 (human hepatocarcinoma) cells were investigated. We evaluated the viability of Huh-7 cells treated with crourorb A1 in 2D and 3D collagen cultures and found that cells in 3D culture exhibited increased resistance to crourorb A1 compared to cells in 2D culture (IC 50 : 62μg/ml versus 35.75μg/ml). Crourorb A1 treatment decreases the viability of Huh-7 cells in a dose- and time-dependent manner and is associated with the induction of apoptosis, in the absence of necrotic cells, through the activation of caspase-3/7 and increased expression of the pro-apoptotic proteins Bak, Bid, Bax, Puma, Bim, and Bad. The effects of crourorb A1 are also associated with G2/M phase cell cycle arrest and increases in cyclin-dependent kinase (CDK1) and cyclin B1 expression. A significant reduction in Huh-7 cell migration induced by crourorb A1 was also observed in the presence of mitomycin C. Finally, we showed that the JNK/MAP pathway, but not ERK signaling, is involved in crourorb A1-induced hepatocarcinoma cell mortality. Copyright © 2017 Elsevier B.V. All rights reserved.

Telomerase reverse transcriptase (TERT) expression and role of vincristine sulfate in mouse model of malignancy related peritoneal ascites: an experimental metastatic condition.

PubMed

Chaklader, M; Das, P; Pereira, J A; Chatterjee, S; Basak, P; Law, A; Banerjee, T; Chauhan, S; Law, S

2011-06-01

To evaluate the efficacy of intraperitoneal vincristine administration into ascitic sarcoma-180 bearing mice as a model of human malignant ascites regarding various peritoneal/retroperitoneal sarcomatosis, and to evaluate the flowcytometric telomerase reverse transcriptase expression for the diagnostic and prognostic purposes. Present study included disease induction by intraperitoneal homologous ascitic sarcoma-180 transplantation followed by in vivo intraperitoneal drug administration to study mitotic index, flowcytometric cell cycle and telomerase reverse transcriptase expression pattern, erythrosin-B dye exclusion study for malignant cell viability assessment. Besides, in vitro malignant ascite culture in presence and absence of vincristine sulfate and survival study were also taken into consideration. Intraperitoneal vincristine administration (concentration 0.5 mg/kg body weight) significantly diminished the mitotic index in diseased subjects in comparison to untreated control subjects. Treated group of animals showed increased life span and median survival time. Cell viability assessment during the course of drug administration also revealed gradual depression on cell viability over time. Flowcytometric cell cycle analysis showed a good prognostic feature of chemotherapeutic administration schedule by representing high G2/M phase blocked cells along with reduced telomerase reverse transcriptase positive cells in treated animals. We conclude that long term administration of vincristine sulfate in small doses could be a good pharmacological intervention in case of malignant peritoneal ascites due to sarcomatosis as it indirectly reduced the level of telomerase reverse transcriptase expression in malignant cells by directly regulating cell cycle and simultaneously increased the life expectancy of the diseased subjects.

Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

NASA Astrophysics Data System (ADS)

Zhao, Guoping; Chen, Shaopeng; Zhao, Ye; Zhu, Lingyan; Huang, Pei; Bao, Lingzhi; Wang, Jun; Wang, Lei; Wu, Lijun; Wu, Yuejin; Xu, An

2010-02-01

Magnetic resonance image (MRI) systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields (SMF). With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid (AL) cells, Chinese hamster ovary (CHO) cells, DNA double-strand break repair deficient mutant (XRS-5) cells, and human primary skin fibroblasts (AG1522) cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster cells after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

Purine analogue ENERGI-F706 induces apoptosis of 786-O renal carcinoma cells via 5'-adenosine monophosphate-activated protein kinase activation.

PubMed

Hsu, Chao-Yu; Lin, Chun-Hsiang; Lin, Jiun-Tsai; Cheng, Yi-Fang; Chen, Han-Min; Kao, Shao-Hsuan

2015-09-01

Purine compounds are known to activate 5'-adenosine monophosphate-activated protein kinase (AMPK), which has important roles in treatments for renal cell carcinoma. The present study was aimed to investigate the effects of the purine analogue ENERGI‑F706 on the human renal carcinoma cell line 786‑O and the underlying mechanisms. The results revealed that ENERGI‑F706 (0.2‑0.6 mg/ml) significantly decreased the cell viability to up to 36.4±2.4% of that of the control. Compared to 786‑O cells, ENERGI‑F706 exerted less suppressive effects on the viability of the human non‑tumorigenic renal cell line HK‑2. Flow cytometric analysis showed that ENERGI‑F706 contributed to cell cycle arrest at S‑phase and triggered apoptosis of 786‑O cells. Immunoblot analysis revealed that anti‑apoptotic B‑cell lymphoma 2 (Bcl‑2) levels were reduced and pro‑apoptotic Bcl‑2‑associated X protein levels were diminished. In addition, activation of caspase‑9, caspase‑3 and poly(adenosine diphosphate ribose) polymerase (PARP) was promoted in 786‑O cells in response to ENERGI‑F706. Effects of ENERGI‑F706 on AMPK cascades were investigated and the results showed that ENERGI‑F706 enhanced phosphorylation of AMPKα (T172) and p53 (S15), a downstream target of AMPK. In addition, the AMPK activation, p53 (S15) phosphorylation, reduction of Bcl‑2, cleavage of caspase‑3 and PARP as well as suppressed cell viability induced by ENERGI‑F706 were reversed in the presence of AMPK inhibitor compound C (dorsomorphin). In conclusion, the findings of the present study revealed that ENERGI‑F706 significantly suppressed the viability of 786‑O cells via induction of cell cycle arrest and apoptosis, attributing to AMPK and p53 activation and subsequent cell cycle regulatory and apoptotic signaling. It was therefore indicated that ENERGI‑F706 may be suitable for the treatment of renal cell carcinoma.

Effects of PPARα inhibition in head and neck paraganglioma cells.

PubMed

Florio, Rosalba; De Lellis, Laura; di Giacomo, Viviana; Di Marcantonio, Maria Carmela; Cristiano, Loredana; Basile, Mariangela; Verginelli, Fabio; Verzilli, Delfina; Ammazzalorso, Alessandra; Prasad, Sampath Chandra; Cataldi, Amelia; Sanna, Mario; Cimini, Annamaria; Mariani-Costantini, Renato; Mincione, Gabriella; Cama, Alessandro

2017-01-01

Head and neck paragangliomas (HNPGLs) are rare tumors that may cause important morbidity, because of their tendency to infiltrate the skull base. At present, surgery is the only therapeutic option, but radical removal may be difficult or impossible. Thus, effective targets and molecules for HNPGL treatment need to be identified. However, the lack of cellular models for this rare tumor hampers this task. PPARα receptor activation was reported in several tumors and this receptor appears to be a promising therapeutic target in different malignancies. Considering that the role of PPARα in HNPGLs was never studied before, we analyzed the potential of modulating PPARα in a unique model of HNPGL cells. We observed an intense immunoreactivity for PPARα in HNPGL tumors, suggesting that this receptor has an important role in HNPGL. A pronounced nuclear expression of PPARα was also confirmed in HNPGL-derived cells. The specific PPARα agonist WY14643 had no effect on HNPGL cell viability, whereas the specific PPARα antagonist GW6471 reduced HNPGL cell viability and growth by inducing cell cycle arrest and caspase-dependent apoptosis. GW6471 treatment was associated with a marked decrease of CDK4, cyclin D3 and cyclin B1 protein expression, along with an increased expression of p21 in HNPGL cells. Moreover, GW6471 drastically impaired clonogenic activity of HNPGL cells, with a less marked effect on cell migration. Notably, the effects of GW6471 on HNPGL cells were associated with the inhibition of the PI3K/GSK3β/β-catenin signaling pathway. In conclusion, the PPARα antagonist GW6471 reduces HNPGL cell viability, interfering with cell cycle and inducing apoptosis. The mechanisms affecting HNPGL cell viability involve repression of the PI3K/GSK3β/β-catenin pathway. Therefore, PPARα could represent a novel therapeutic target for HNPGL.

The synthetic ligand of peroxisome proliferator-activated receptor-gamma ciglitazone affects human glioblastoma cell lines.

PubMed

Strakova, Nicol; Ehrmann, Jiri; Dzubak, Petr; Bouchal, Jan; Kolar, Zdenek

2004-06-01

Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that ligand activation of peroxisome proliferator-activated receptor (PPAR)-gamma can induce differentiation and inhibit proliferation of several cancer cells. In this study, we have investigated whether one PPARgamma ligand in particular, ciglitazone, inhibits cell viability and, additionally, whether it affects the cell cycle and apoptosis of human glioblastoma cell lines T98G, U-87 MG, A172, and U-118 MG. All glioblastoma cell lines were found to express PPARgamma protein, and following treatment with ciglitazone, localization was unchanged. Ciglitazone inhibited viability in a dose-dependent manner in all four tested glioblastoma cells after 24 h of treatment. Analysis of the cell cycle showed arrest in the G(1) phase and partial block in G(2)/M phase of the cell cycle. Cyclin D1 and cyclin B expression was decreased. Phosphorylation of Rb protein dropped as well. We found that ciglitazone was followed by increased expression of p27(Kip1) and p21(Waf1/Cip1). It also led to apoptosis induction: bax expression in T98G was elevated. Expression of the antiapoptotic protein bcl-2 was reduced in U-118 MG and U-87 MG and showed a slight decrease in A172 cells. Flow cytometry confirmed the induction of apoptosis. Moreover, PPARgamma ligand decreased telomerase activity in U-87 MG and U-118 MG cell lines. Our results demonstrate that ciglitazone inhibits the viability of human glioblastoma cell lines via induction of apoptosis; as a result, this ligand may offer potential new therapy for the treatment of central nervous system neoplasms.

Changes in Oscillatory Dynamics in the Cell Cycle of Early Xenopus laevis Embryos

PubMed Central

Tsai, Tony Y.-C.; Theriot, Julie A.; Ferrell, James E.

2014-01-01

During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development. PMID:24523664

Astaxanthin Inhibits Proliferation of Human Gastric Cancer Cell Lines by Interrupting Cell Cycle Progression

PubMed Central

Kim, Jung Ha; Park, Jong-Jae; Lee, Beom Jae; Joo, Moon Kyung; Chun, Hoon Jai; Lee, Sang Woo; Bak, Young-Tae

2016-01-01

Background/Aims Astaxanthin is a carotenoid pigment that has antioxidant, antitumoral, and anti-inflammatory properties. In this in vitro study, we investigated the mechanism of anticancer effects of astaxanthin in gastric carcinoma cell lines. Methods The human gastric adenocarcinoma cell lines AGS, KATO-III, MKN-45, and SNU-1 were treated with various concentrations of astaxanthin. A cell viability test, cell cycle analysis, and immunoblotting were performed. Results The viability of each cancer cell line was suppressed by astaxanthin in a dose-dependent manner with significantly decreased proliferation in KATO-III and SNU-1 cells. Astaxanthin increased the number of cells in the G0/G1 phase but reduced the proportion of S phase KATO-III and SNU-1 cells. Phosphorylated extracellular signal-regulated kinase (ERK) was decreased in an inverse dose-dependent correlation with astaxanthin concentration, and the expression of p27kip-1 increased the KATO-III and SNU-1 cell lines in an astaxanthin dose-dependent manner. Conclusions Astaxanthin inhibits proliferation by interrupting cell cycle progression in KATO-III and SNU-1 gastric cancer cells. This may be caused by the inhibition of the phosphorylation of ERK and the enhanced expression of p27kip-1. PMID:26470770

Effects of air transient spark discharge and helium plasma jet on water, bacteria, cells, and biomolecules.

PubMed

Hensel, Karol; Kučerová, Katarína; Tarabová, Barbora; Janda, Mário; Machala, Zdenko; Sano, Kaori; Mihai, Cosmin Teodor; Ciorpac, Mitică; Gorgan, Lucian Dragos; Jijie, Roxana; Pohoata, Valentin; Topala, Ionut

2015-06-06

Atmospheric pressure DC-driven self-pulsing transient spark (TS) discharge operated in air and pulse-driven dielectric barrier discharge plasma jet (PJ) operated in helium in contact with water solutions were used for inducing chemical effects in water solutions, and the treatment of bacteria (Escherichia coli), mammalian cells (Vero line normal cells, HeLa line cancerous cells), deoxyribonucleic acid (dsDNA), and protein (bovine serum albumin). Two different methods of water solution supply were used in the TS: water electrode system and water spray system. The effects of both TS systems and the PJ were compared, as well as a direct exposure of the solution to the discharge with an indirect exposure to the discharge activated gas flow. The chemical analysis of water solutions was performed by using colorimetric methods of UV-VIS absorption spectrophotometry. The bactericidal effects of the discharges on bacteria were evaluated by standard microbiological plate count method. Viability, apoptosis and cell cycle were assessed in normal and cancerous cells. Viability of cells was evaluated by trypan blue exclusion test, apoptosis by Annexin V-FITC/propidium iodide assay, and cell cycle progression by propidium iodide/RNase test. The effect of the discharges on deoxyribonucleic acid and protein were evaluated by fluorescence and UV absorption spectroscopy. The results of bacterial and mammalian cell viability, apoptosis, and cell cycle clearly show that cold plasma can inactivate bacteria and selectively target cancerous cells, which is very important for possible future development of new plasma therapeutic strategies in biomedicine. The authors found that all investigated bio-effects were stronger with the air TS discharge than with the He PJ, even in indirect exposure.

The viability of MCM-41 as separator in secondary alkaline cells

NASA Astrophysics Data System (ADS)

Meskon, S. R.; Othman, R.; Ani, M. H.

2018-01-01

The viability of MCM-41 membrane as a separator material in secondary alkaline cell is investigated. The inorganic membrane was employed in an alkaline nickel-zinc system. MCM-41 mesoporous material consists of arrays of hexagonal nano-pore channels. The membrane was synthesized using sol-gel route from parent solution comprising of quarternary ammonium surfactant, cethyltrimethylammonium bromide C16H33(CH3)3NBr (CTAB), hydrochloric acid (HCl), deionized water (H2O), ethanol (C2H5OH), and tetraethylortosilicate (TEOS). Both the anodic zinc/zinc oxide and cathodic nickel hydroxide electrodeposited film were coated with MCM-41 membrane. The Ni/MCM-41/Zn alkaline cell was then subjected to 100-cycle durability test and the structural stability of MCM-41 separator throughout the progression of the charge-discharge cycles is studied. X-ray diffraction (XRD) analysis on the dismantled cell shows that MCM-41 began to transform to lamellar MCM-50 on the 5th cycle and transformed almost completely on the 25th cycle. The phase transformation of MCM-41 hexagonal structure into gel-like MCM-50 prevents the mesoporous cell separator from diminished in the caustic alkaline surround. This work has hence demonstrated MCM-41 membrane is viable to be employed in secondary alkaline cells.

Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID

PubMed Central

Le, Quy; Maizels, Nancy

2015-01-01

AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome. PMID:26355458

Magnolol pretreatment attenuates heat stress-induced IEC-6 cell injury*

PubMed Central

Mei, Chen; He, Sha-sha; Yin, Peng; Xu, Lei; Shi, Ya-ran; Yu, Xiao-hong; Lyu, An; Liu, Feng-hua; Jiang, Lin-shu

2016-01-01

Objective: Heat stress (HS) is an important environmental stressor that adversely influences livestock during the summer. The aim of this study was to investigate whether magnolol protects against HS-induced intestinal epithelial cell injury. Materials and methods: An intestinal epithelial cell line (IEC-6) was subjected to HS at 42 °C, with and without magnolol pretreatment. Cell injury was detected by monitoring lactate dehydrogenase (LDH) release. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was used to assess cell proliferation and viability, including identifying effective concentrations of magnolol. Flow cytometry confirmed G1-phase cell-cycle arrest and its alleviation by magnolol. Active DNA synthesis was measured by incorporation of nucleic acid 5-ethynyl-2'-deoxyuridine (EdU). G1-phase cell-cycle-related gene expression was assessed by real-time reverse transcription polymerase chain reaction (RT-PCR) and levels of G1-phase-related proteins by Western blotting. Results: HS induced IEC-6 cell injury and decreased cell viability, as demonstrated by data from LDH and MTS assays, respectively. Based on a number of criteria, IEC-6 cells subjected to HS were arrested in the G1 phase of the cell cycle. Magnolol pretreatment decreased HS-induced cell injury through relief of this cell-cycle arrest. Conclusions: Magnolol pretreatment attenuates HS-induced injury in IEC-6 cells. Magnolol is potentially promising as a protective strategy for HS in livestock. PMID:27256675

DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by crocin from kashmiri saffron in a human pancreatic cancer cell line.

PubMed

Bakshi, Hamid; Sam, Smitha; Rozati, Roya; Sultan, Phalisteen; Islam, Tajamul; Rathore, Babita; Lone, Zahoor; Sharma, Manik; Triphati, Jagrati; Saxena, Ramesh Chand

2010-01-01

Apoptosis, a widely important mechanism that contributes to cell growth reduction, is reported to be induced by Crocus sativus in different cancer types. The present study was designed to elucidate apoptosis induction by crocin, a main component of Crocus sativus in a human pancreatic cancer cell line (BxPC-3). Cell viability was measured by MTT assay, Hoechest33258 staining was used to detect the chromatin condensation characteristic of apoptosis, and DNA fragmentation was assessed by gel electrophoresis and cell cycle analysis by flow cytometry. Crocin induced apoptosis and G1-phase cell cycle arrest of BxPC-3 cells, while decreasing cell viability in a dose dependent and time dependent manner. Cells treated with 10μg/L crocin exhibited apoptotic morphology (brightly blue-fluorescent condensed nuclei on Hoechst 33258 staining) and reduction of volume. DNA analysis revealed typical ladders as early as 12 hours after treatment indicative of apoptosis. Our preclinical study demonstrated a pancreatic cancer cell line to be highly sensitive to crocin-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of crocin action are not yet clearly understood, it appears to have potential as a therapeutic agent.

Induction of cell death in renal cell carcinoma with combination of D-fraction and vitamin C.

PubMed

Alexander, Bobby; Fishman, Andrew I; Eshghi, Majid; Choudhury, Muhammad; Konno, Sensuke

2013-09-01

Although several conventional therapeutic options for advanced renal cell carcinoma (RCC) are currently available, the unsatisfactory outcomes demand establishing more effective interventions. D-fraction (PDF), a bioactive proteoglucan of Maitake mushroom, demonstrates anticancer and immunomodulatory activities, which are also shown to be potentiated by vitamin C (VC). We thus hypothesized that a combination of PDF and VC (PDF + VC) could be an alternative approach to more effectively inhibit the growth of RCC. We examined the dose-dependent effects of PDF + VC on RCC cell viability and also performed biochemical assays to explore the growth regulatory mechanism. Human RCC, ACHN cell line, was employed and exposed to varying concentrations of PDF or VC and their combinations. Cell viability at specified times was determined by MTT assay. Lipid peroxidation assay, cell cycle analysis, and Western blot analysis were also performed. PDF or VC alone led to the significant reduction in cell viability at 72 hours with PDF >500 µg/mL and VC ≥300 µM. When various combinations of PDF and VC were tested, the combination of the ineffective concentrations of PDF (300 µg/mL) and VC (200 µM) resulted in ~90% cell death in 24 hours. Lipid peroxidation assay then indicated significantly (~2.5 fold) elevated oxidative stress with this PDF + VC. Cell cycle analysis also indicated a G1 cell cycle arrest following a 6-hour PDF + VC treatment. Western blots further revealed a downregulation of Bcl2, an upregulation of Bax, and proteolytic activation of PARP (poly[ADP-ribose] polymerase) in PDF + VC-treated cells, indicating induction of apoptosis. The present study demonstrates that the combination of PDF and VC can become highly cytotoxic, inducing severe cell death in ACHN cells. This cytotoxic mechanism appears to be primarily attributed to oxidative stress, accompanied by a G1 cell cycle arrest. Such cell death induced by PDF + VC could be more likely linked to apoptosis, as indicated by the modulation of apoptosis regulators (Bcl2, Bax, and PARP). Therefore, as PDF and VC may work synergistically to induce apoptotic cell death, they may have clinical implications in an alternative, improved therapeutic modality for advanced RCC.

Ovarian Expression, Localization, and Function of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period of the Human Menstrual Cycle1

PubMed Central

Rosewell, Katherine L.; Li, Feixue; Puttabyatappa, Muraly; Akin, James W.; Brännström, Mats; Curry, Thomas E.

2013-01-01

ABSTRACT Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability. PMID:24048576

Ovarian expression, localization, and function of tissue inhibitor of metalloproteinase 3 (TIMP3) during the periovulatory period of the human menstrual cycle.

PubMed

Rosewell, Katherine L; Li, Feixue; Puttabyatappa, Muraly; Akin, James W; Brännström, Mats; Curry, Thomas E

2013-11-01

Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability.

Switching of metabolic programs in response to light availability is an essential function of the cyanobacterial circadian output pathway

PubMed Central

Puszynska, Anna M; O'Shea, Erin K

2017-01-01

The transcription factor RpaA is the master regulator of circadian transcription in cyanobacteria, driving genome-wide oscillations in mRNA abundance. Deletion of rpaA has no effect on viability in constant light conditions, but renders cells inviable in cycling conditions when light and dark periods alternate. We investigated the mechanisms underlying this viability defect, and demonstrate that the rpaA- strain cannot maintain appropriate energy status at night, does not accumulate carbon reserves during the day, and is defective in transcription of genes crucial for utilization of carbohydrate stores at night. Reconstruction of carbon utilization pathways combined with provision of an external carbon source restores energy charge and viability of the rpaA- strain in light/dark cycling conditions. Our observations highlight how a circadian output pathway controls and temporally coordinates essential pathways in carbon metabolism to maximize fitness of cells facing periodic energy limitations. DOI: http://dx.doi.org/10.7554/eLife.23210.001 PMID:28430105

Galangin Induces Apoptosis in MCF-7 Human Breast Cancer Cells Through Mitochondrial Pathway and Phosphatidylinositol 3-Kinase/Akt Inhibition.

PubMed

Liu, Dan; You, Pengtao; Luo, Yan; Yang, Min; Liu, Yanwen

2018-06-07

The study aimed to investigate the molecular mechanism of inhibition of proliferation and apoptosis induction by galangin against MCF-7 human breast cancer cells. Cell Counting Kit-8 assay was used to assess cell viability and flow cytometry was used to detect cell apoptosis. The expression level of apoptosis-related proteins (cleaved-caspase-9, cleaved-caspase-8, cleaved-caspase-3, Bad, cleaved-Bid, Bcl-2, Bax, p-phosphatidylinositol 3-kinase [PI3K], and p-Akt) and cell cycle-related proteins (cyclin D3, cyclin B1, cyclin-dependent kinases CDK1, CDK2, CDK4, p21, p27, p53) were evaluated by Western blotting. Galangin increased the expression of Bax and decreased the expression of Bcl-2 in a concentration-dependent manner, inhibited cell viability, and induced apoptosis. Meanwhile, the expression of cleavage of caspase-9, caspase-8, caspase-3, Bid, and Bad increased significantly while the expression of p-PI3K and p-Akt proteins decreased. In addition, the protein levels of cyclin D3, cyclin B1, CDK1, CDK2, and CDK4 were downregulated while the expression levels of p21, p27, and p53 were upregulated significantly. Galangin could suppress the viability of MCF-7 cells and induce cell apoptosis via the mitochondrial pathway and PI3K/Akt inhibition as well as cell cycle arrest. © 2018 S. Karger AG, Basel.

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells.

PubMed

Guo, Jian-Ru; Chen, Qian-Qian; Lam, Christopher Wai-Kei; Zhang, Wei

2015-07-26

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells). MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells. Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.

Effects of Bauhinia championii (Benth.) Benth. polysaccharides on the proliferation and cell cycle of chondrocytes.

PubMed

Cai, Liangliang; Ye, Hongzhi; Yu, Fangrong; Li, Huiting; Chen, Jiashou; Liu, Xianxiang

2013-05-01

It has been recently shown that polysaccharides isolated from plants exhibit a number of beneficial therapeutic properties. Bauhinia championii (Benth.) Benth. has been widely used for the clinical treatment of knee osteoarthritis (OA) in China. However, the underlying molecular mechanisms of knee OA treatment have yet to be elucidated. In the present study, we investigated the effects of Bauhinia championii (Benth.) Benth. polysaccharides (BCBPs) on the proliferation and cell cycle of chondrocytes on 4-week-old male Sprague Dawley rats. Immunohistochemical staining was used to identify chondrocytes and an MTT assay was used to evaluate cell viability. Flow cytometry was used for cell cycle analysis. The mRNA and protein expression levels of cyclin D1, CDK4 and CDK6 in chondrocytes were detected using reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively. The data demonstrate that BCBP treatment increased the viability of chondrocytes. In addition, BCBP treatment reduced the cell population in the G0/G1 phase, whereas the cell population was increased in the S phase. Furthermore, BCBP treatment enhanced the expression of cyclin D1, CDK4 and CDK6. These results indicate that BCBP treatment promotes cell proliferation by accelerating the G1/S transition.

Iodine-131 treatment of thyroid cancer cells leads to suppression of cell proliferation followed by induction of cell apoptosis and cell cycle arrest by regulation of B-cell translocation gene 2-mediated JNK/NF-κB pathways.

PubMed

Zhao, L M; Pang, A X

2017-01-16

Iodine-131 (131I) is widely used for the treatment of thyroid-related diseases. This study aimed to investigate the expression of p53 and BTG2 genes following 131I therapy in thyroid cancer cell line SW579 and the possible underlying mechanism. SW579 human thyroid squamous carcinoma cells were cultured and treated with 131I. They were then assessed for 131I uptake, cell viability, apoptosis, cell cycle arrest, p53 expression, and BTG2 gene expression. SW579 cells were transfected with BTG2 siRNA, p53 siRNA and siNC and were then examined for the same aforementioned parameters. When treated with a JNK inhibitor of SP600125 and 131I or with a NF-κB inhibitor of BMS-345541 and 131I, non-transfected SW579 cells were assessed in JNK/NFκB pathways. It was observed that 131I significantly inhibited cell proliferation, promoted cell apoptosis and cell cycle arrest. Both BTG2 and p53 expression were enhanced in a dose-dependent manner. An increase in cell viability by up-regulation in Bcl2 gene, a decrease in apoptosis by enhanced CDK2 gene expression and a decrease in cell cycle arrest at G0/G1 phase were also observed in SW579 cell lines transfected with silenced BTG2 gene. When treated with SP600125 and 131I, the non-transfected SW579 cell lines significantly inhibited JNK pathway, NF-κB pathway and the expression of BTG2. However, when treated with BMS-345541 and 131I, only the NF-κB pathway was suppressed. 131I suppressed cell proliferation, induced cell apoptosis, and promoted cell cycle arrest of thyroid cancer cells by up-regulating B-cell translocation gene 2-mediated activation of JNK/NF-κB pathways.

Anticancer effects of phenoxazine derivatives revealed by inhibition of cell growth and viability, disregulation of cell cycle, and apoptosis induction in HTLV-1-positive leukemia cells.

PubMed

Miyano-Kurosaki, Naoko; Ikegami, Kou; Kurosaki, Kunihiko; Endo, Takahiko; Aoyagi, Hoshimi; Hanami, Mari; Yasumoto, Jun; Tomoda, Akio

2009-05-01

Adult T-cell leukemia (ATL) is a malignant tumor of human CD4(+) T cells infected with a human retrovirus, T lymphotropic virus type-1 (HTLV-1). The aim of the present study was to investigate the apoptotic effects of phenoxazines, 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha,8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-aminophenoxazine-3-one (Phx-3) on a T cell leukemia cell line from ATL patients, MT-1 cells; HTLV-1 transformed T-cell lines, HUT-102 cells and MT-2 cells; and an HTLV-1-negative rat sarcoma cell line, XC cells. Among these phenoxazines, Phx-3 at concentrations of less than 10 microg/ml extensively inhibited growth and cell viability; arrested cell cycles at sub G(0)/G(1) phase; and augmented apoptosis of MT-1, HUT-102, and MT-2 cells. However, these phenoxazines did not affect the cell viability of an HTLV-1-negative rat sarcoma cell line, XC cells, and phytohemaggutinin-activated human peripheral blood mononuclear cells, although they markedly inhibited the growth of these cells. The transmission of HTLV-1 from HTLV-1-positive cells (MT-2 cells) to HTLV-1-negative cells (XC cells) was considered to be prevented by Phx-1, Phx-2, or Phx-3 because the syncytium formation between these cells was inhibited markedly in the presence of these phenoxazines. The present results suggest that Phx-1, Phx-2, and, in particular, Phx-3 may be useful as therapeutic agents against ATL, which is extremely refractory to current therapies.

Effects of PPARα inhibition in head and neck paraganglioma cells

PubMed Central

Florio, Rosalba; di Giacomo, Viviana; Di Marcantonio, Maria Carmela; Cristiano, Loredana; Basile, Mariangela; Verginelli, Fabio; Verzilli, Delfina; Ammazzalorso, Alessandra; Prasad, Sampath Chandra; Cataldi, Amelia; Sanna, Mario; Cimini, Annamaria; Mariani-Costantini, Renato; Mincione, Gabriella; Cama, Alessandro

2017-01-01

Head and neck paragangliomas (HNPGLs) are rare tumors that may cause important morbidity, because of their tendency to infiltrate the skull base. At present, surgery is the only therapeutic option, but radical removal may be difficult or impossible. Thus, effective targets and molecules for HNPGL treatment need to be identified. However, the lack of cellular models for this rare tumor hampers this task. PPARα receptor activation was reported in several tumors and this receptor appears to be a promising therapeutic target in different malignancies. Considering that the role of PPARα in HNPGLs was never studied before, we analyzed the potential of modulating PPARα in a unique model of HNPGL cells. We observed an intense immunoreactivity for PPARα in HNPGL tumors, suggesting that this receptor has an important role in HNPGL. A pronounced nuclear expression of PPARα was also confirmed in HNPGL-derived cells. The specific PPARα agonist WY14643 had no effect on HNPGL cell viability, whereas the specific PPARα antagonist GW6471 reduced HNPGL cell viability and growth by inducing cell cycle arrest and caspase-dependent apoptosis. GW6471 treatment was associated with a marked decrease of CDK4, cyclin D3 and cyclin B1 protein expression, along with an increased expression of p21 in HNPGL cells. Moreover, GW6471 drastically impaired clonogenic activity of HNPGL cells, with a less marked effect on cell migration. Notably, the effects of GW6471 on HNPGL cells were associated with the inhibition of the PI3K/GSK3β/β-catenin signaling pathway. In conclusion, the PPARα antagonist GW6471 reduces HNPGL cell viability, interfering with cell cycle and inducing apoptosis. The mechanisms affecting HNPGL cell viability involve repression of the PI3K/GSK3β/β-catenin pathway. Therefore, PPARα could represent a novel therapeutic target for HNPGL. PMID:28594934

Exogenous hydrogen sulfide exerts proliferation, anti-apoptosis, migration effects and accelerates cell cycle progression in multiple myeloma cells via activating the Akt pathway.

PubMed

Zheng, Dong; Chen, Ziang; Chen, Jingfu; Zhuang, Xiaomin; Feng, Jianqiang; Li, Juan

2016-10-01

Hydrogen sulfide (H2S), regarded as the third gaseous transmitter, mediates and induces various biological effects. The present study investigated the effects of H2S on multiple myeloma cell progression via amplifying the activation of Akt pathway in multiple myeloma cells. The level of H2S produced in multiple myeloma (MM) patients and healthy subjects was measured using enzyme-linked immunosorbent assay (ELISA). MM cells were treated with 500 µmol/l NaHS (a donor of H2S) for 24 h. The expression levels of phosphorylated-Akt (p-Akt), Bcl-2 and caspase-3 were measured by western blot assay. Cell viability was detected by Cell Counting Kit 8 (CCK-8). The cell cycle was analyzed by flow cytometry. Our results show that the concentration of H2S was higher in MM patients and that it increased in parallel with disease progression. Treating MM cells with 500 µmol/l NaHS for 24 h markedly increased the expression level of Bcl-2 and the activation of p-Akt, however, the expression level of caspase-3 was decreased, cell viability was increased, and cell cycle progression was accelerated in MM cells. NaHS also induced migration in MM cells in transwell migration assay. Furthermore, co-treatment of MM cells with 500 µmol/l NaHS and 50 µmol/l LY294002 for 24 h significantly overset these effects. In conclusion, our findings demonstrate that the Akt pathway contributes to NaHS-induced cell proliferation, migration and acceleration of cell cycle progression in MM cells.

Cytotoxic effect of the serotonergic drug 1-(1-Naphthyl)piperazine against melanoma cells.

PubMed

Menezes, Ana Catarina; Carvalheiro, Manuela; Ferreira de Oliveira, José Miguel P; Ascenso, Andreia; Oliveira, Helena

2018-03-01

1-(1-Naphthyl)piperazine (1-NPZ) is a serotonergic derivative of quipazine acting both as antagonist and agonist of different serotonin receptors, with promising results for the management of skin cancer. In this work, we studied the effect of 1-NPZ on human MNT-1 melanoma cells by evaluating its effects on cell viability, ability to form colonies, cell cycle dynamics, reactive oxygen species (ROS) production and apoptosis. Treatment of MNT-1 cells with 1-NPZ for 24h decreased cell viability and induced apoptosis in a dose-dependent manner. Activity against melanoma was confirmed with a different melanoma cell line, SK-MEL-28. Simultaneously, 1-NPZ affected cell cycle progression by mediating a S-phase delay. Higher levels of ROS were also detected in MNT-1 cells after treatment with 1-NPZ. Furthermore, 1-NPZ significantly increased the expression of cyclooxygenase-2 in MNT-1 cells. These findings suggest that 1-NPZ pretreatment is able to induce oxidative stress, and consequently apoptotic cell death in melanoma cells. In conclusion, this study demonstrates the cytotoxic and genotoxic potential of 1-NPZ against melanoma cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts

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

Tsurumi, Chizuko; Firat, Elke; Gaedicke, Simone

2009-09-04

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPIImore » expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.« less

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts.

PubMed

Tsurumi, Chizuko; Firat, Elke; Gaedicke, Simone; Huai, Jisen; Mandal, Pankaj Kumar; Niedermann, Gabriele

2009-09-04

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.

The postmitotic Saccharomyces cerevisiae after spaceflight showed higher viability

NASA Astrophysics Data System (ADS)

Yi, Zong-Chun; Li, Xiao-Fei; Wang, Yan; Wang, Jie; Sun, Yan; Zhuang, Feng-Yuan

2011-06-01

The budding yeast Saccharomyces cerevisiae has been proposed as an ideal model organism for clarifying the biological effects caused by spaceflight conditions. The postmitotic S. cerevisiae cells onboard Practice eight recoverable satellite were subjected to spaceflight for 15 days. After recovery, the viability, the glycogen content, the activities of carbohydrate metabolism enzymes, the DNA content and the lipid peroxidation level in yeast cells were analyzed. The viability of the postmitotic yeast cells after spaceflight showed a three-fold increase as compared with that of the ground control cells. Compared to the ground control cells, the lipid peroxidation level in the spaceflight yeast cells markedly decreased. The spaceflight yeast cells also showed an increase in G2/M cell population and a decrease in Sub-G1 cell population. The glycogen content and the activities of hexokinase and succinate dehydrogenase significantly decreased in the yeast cells after spaceflight. In contrast, the activity of malate dehydrogenase showed an obvious increase after spaceflight. These results suggested that microgravity or spaceflight could promote the survival of postmitotic S. cerevisiae cells through regulating carbohydrate metabolism, ROS level and cell cycle progression.

Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress.

PubMed

Soares, Júlia Ribeiro; José Tenório de Melo, Edésio; da Cunha, Maura; Fernandes, Kátia Valevski Sales; Taveira, Gabriel Bonan; da Silva Pereira, Lidia; Pimenta, Samy; Trindade, Fernanda Gomes; Regente, Mariana; Pinedo, Marcela; de la Canal, Laura; Gomes, Valdirene Moreira; de Oliveira Carvalho, André

2017-01-01

Plant defensins were discovered at beginning of the 90s'; however, their precise mechanism of action is still unknown. Herein, we studied ApDef 1 -Saccharomyces cerevisiae interaction. ApDef 1 -S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assay was repeated with hydroxyurea synchronized-yeast and pretreated with CCCP. Plasma membrane permeabilization, ROS induction, chromatin condensation, and caspase activation analyses were assessed through Sytox green, DAB, DAPI and FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructural analysis was done by electron microscopy. ApDef 1 caused S. cerevisiae cell death and MIC was 7.8μM. Whole cell population died after 18h of ApDef 1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef 1 induced death. ApDef 1 -S. cerevisiae interaction resulted in membrane permeabilization, H 2 O 2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. ApDef 1 -S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via. We show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membrane potential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would be an indirect event of the ApDef 1 -S. cerevisiae interaction. Copyright © 2016 Elsevier B.V. All rights reserved.

Cibotium barometz polysaccharides stimulate chondrocyte proliferation in vitro by promoting G1/S cell cycle transition

PubMed Central

Fu, Changlong; Zheng, Chunsong; Lin, Jie; Ye, Jinxia; Mei, Yangyang; Pan, Caibin; Wu, Guangwen; Li, Xihai; Ye, Hongzhi; Liu, Xianxiang

2017-01-01

Cibotium barometz polysaccharides (CBPS) are one of the most important bioactive components extracted from the Cibotium barometz plant, which belongs to the Dicksoniaceae family. It has been widely used for the treatment of orthopedic diseases in traditional Chinese medicine. However, the molecular mechanisms behind the therapeutic effects of CBPS remain to be clarified. In the present study, the concentration of CBPS was detected by phenol-vitriol colorimetry. Furthermore, the effects stimulated by CBPS on the viability and G1/S cell cycle transition in primary chondrocytes from Sprague-Dawley rats were investigated. A cell viability assay demonstrated that chondrocyte proliferation may be enhanced by CBPS in a dose- and time-dependent manner. The mechanism underlying the promotion of chondrocyte cell cycle was suggested to involve the stimulation of G1 to S phase transition. To further confirm the results, reverse transcription-quantitative polymerase chain reaction and western blot analyses were used to detect the expression of mRNA and protein levels of cyclin D1, cyclin-dependent kinase 4 and retinoblastoma protein. The results suggested that CBPS may stimulate chondrocyte proliferation via promoting G1/S cell cycle transition. Since osteoarthritis is characterized by deficient proliferation in chondrocytes, the present study indicates that CBPS may potentially serve as a novel method for the treatment of osteoarthritis. PMID:28358416

ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox.

PubMed

Zhang, Di; Ren, Li; Chen, Guan-Qun; Zhang, Jie; Reed, Barbara M; Shen, Xiao-Hui

2015-09-01

Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 (·-) , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA-GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

Synergistic effects of the sesquiterpene lactone, EPD, with cisplatin and paclitaxel in ovarian cancer cells.

PubMed

van Haaften, Caroline; Boot, Arnoud; Corver, Willem E; van Eendenburg, Jaap D H; Trimbos, Baptist J M Z; van Wezel, Tom

2015-04-25

Ovarian cancer remains still the leading cause of death of gynecological malignancy, in spite of first-line chemotherapy with cisplatin and paclitaxel. Although initial response is favorably, relapses are common and prognosis for women with advanced disease stays poor. Therefore efficacious approaches are needed. Previously, an anti-cancer agent, EPD exhibited potent cytotoxic effects towards ovarian cancer and not towards normal cells. Cell viability and cell cycle analysis studies were performed with EPD, in combination with cisplatin and/or paclitaxel, using the ovarian carcinoma cell lines: SK-OV-3, OVCAR-3, JC, JC-pl and normal fibroblasts. Cell viability was measured using Presto Blue and cell cycle analysis using a flow cytometer. Apoptosis was measured in JC and JC-pl , using the caspase 3 assay kit. In JC-pl, SK-OV-3 and JC, synergistic interactions between either EPD and cisplatin or EPD and paclitaxel were observed. For the first time the effects of EPD on the cell cycle of ovarian cancer cells and normal cells was studied. EPD and combinations of EPD with cisplatin and/ or paclitaxel showed cell cycle arrest in the G2/M phase. The combination of EPD and cisplatin showed a significant synergistic effect in cell line JC-pl, while EPD with paclitaxel showed synergistic interaction in JC. Additionally, synergistic drug combinations showed increased apoptosis. Our results showed a synergistic effect of EPD and cisplatin in an ovarian drug resistant cell line as well as a synergistic effect of EPD and paclitaxel in two other ovarian cell lines. These results might enhance clinical efficacy, compared to the existing regimen of paclitaxel and cisplatin.

Effect of Procyanidin-rich Extract from Natural Cocoa Powder on Cellular Viability, Cell Cycle Progression, and Chemoresistance in Human Epithelial Ovarian Carcinoma Cell Lines

PubMed Central

Taparia, Shruti; Khanna, Aparna

2016-01-01

Background: Over the last 400 years, cocoa and chocolate have been described as having potential medicinal value, being consumed as a beverage or eaten as food. Concentration–dependant, antiproliferation, and cytotoxic effects of some of their polyphenolic constituents have been demonstrated against various cancers. Such an effect remains to be demonstrated in ovarian cancer Objective: To investigate the effect of cocoa procyanidins against ovarian cancer in vitro using OAW42 and OVCAR3 cell lines. Materials and Methods: Cocoa procyanidins were extracted and enriched from non alkalized cocoa powder. The polyphenolic content and antioxidant activity were determined. Effect on cell viability was determined after the treatment with ≤1000 μg/mL cocoa procyanidin-rich extract on OAW42 and OVCAR3 and normal human dermal fibroblasts. Similarly, chemosensitization effect was determined by pretreating cancer cell lines with extract followed by doxorubicin hydrochloride treatment. The effect of treatment on cell cycle and P-glycoprotein (P-gp) expression was determined using flow cytometry. Results: The cocoa extract showed high polyphenolic content and antioxidant activity. Treatment with extract caused cytotoxicity and chemosensitization in OAW42 and OVCAR3 cell lines. Normal dermal fibroblasts showed an increase in cell viability post treatment with extract. Treatment with extract affected the cell cycle and an increasing percentage of cells in hypodiploid sub-G1/G0 phase was observed. Treatment of OVCAR3 with the extract caused reduction of P-gp expression. Conclusion: Cocoa procyanidins were found to be selectively cytotoxic against epithelial ovarian cancer, interfered with the normal cell cycle and sensitized cells to subsequent chemotherapeutic treatment. Chemosensitization was found to be associated with P-gp reduction in OVCAR3 cells. SUMMARY Among the naturally occurring flavonoids, procyanidins have been shown to be effective against cancersNon alkalized cocoa powder is one of the richest sources of procyanidinsCocoa procyanidin-rich extract (CPRE) caused cytotoxicity and chemosensitization in ovarian carcinoma cell lines OAW42 and OVCAR3CPRE affected normal cell cycle progressionCPRE also downregulated P-glycoprotein, which mediates chemoresistance in multidrug-resistant OVCAR3 cell line. Abbreviations used: P-gp: P-glycoprotein, CPRE: Cocoa procyanidin rich extract, DMAC: 4-dimethylaminocinnamaldehyde, DPPH: Diphenylpicrylhydrazyl, ABTS: 2,2’;-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), PI: Propidium iodide, FITC: Fluorescein isothiocyanate, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, TLC: Thin layer chromatography, HPTLC: High-performance thin layer chromatography. PMID:27279694

Uncoupling reproduction from metabolism extends chronological lifespan in yeast

PubMed Central

Nagarajan, Saisubramanian; Kruckeberg, Arthur L.; Schmidt, Karen H.; Kroll, Evgueny; Hamilton, Morgan; McInnerney, Kate; Summers, Ryan; Taylor, Timothy; Rosenzweig, Frank

2014-01-01

Studies of replicative and chronological lifespan in Saccharomyces cerevisiae have advanced understanding of longevity in all eukaryotes. Chronological lifespan in this species is defined as the age-dependent viability of nondividing cells. To date this parameter has only been estimated under calorie restriction, mimicked by starvation. Because postmitotic cells in higher eukaryotes often do not starve, we developed a model yeast system to study cells as they age in the absence of calorie restriction. Yeast cells were encapsulated in a matrix consisting of calcium alginate to form ∼3 mm beads that were packed into bioreactors and fed ad libitum. Under these conditions cells ceased to divide, became heat shock and zymolyase resistant, yet retained high fermentative capacity. Over the course of 17 d, immobilized yeast cells maintained >95% viability, whereas the viability of starving, freely suspended (planktonic) cells decreased to <10%. Immobilized cells exhibited a stable pattern of gene expression that differed markedly from growing or starving planktonic cells, highly expressing genes in glycolysis, cell wall remodeling, and stress resistance, but decreasing transcription of genes in the tricarboxylic acid cycle, and genes that regulate the cell cycle, including master cyclins CDC28 and CLN1. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously up-regulated in the immobilized state, and an immobilized rim15 knockout strain fails to exhibit the long-lived, growth-arrested phenotype, suggesting that altered regulation of the Rim15-mediated nutrient-sensing pathway plays an important role in extending yeast chronological lifespan under calorie-unrestricted conditions. PMID:24706810

Anticancer activity of 7-epiclusianone, a benzophenone from Garcinia brasiliensis, in glioblastoma.

PubMed

Sales, Leilane; Pezuk, Julia Alejandra; Borges, Kleiton Silva; Brassesco, María Sol; Scrideli, Carlos Alberto; Tone, Luiz Gonzaga; dos Santos, Marcelo Henrique; Ionta, Marisa; de Oliveira, Jaqueline Carvalho

2015-10-30

Glioblastoma is the most common tumor of the central nervous system and one of the hardest tumors to treat. Consequently, the search for novel therapeutic options is imperative. 7-epiclusianone, a tetraprenylated benzophenone isolated from the epicarp of the native plant Garcinia brasiliensis, exhibits a range of biological activities but its prospect anticancer activity is underexplored. Thus, the aim of the present study was to evaluate the influence of 7-epiclusianone on proliferation, clonogenic capacity, cell cycle progression and induction of apoptosis in two glioblastoma cell lines (U251MG and U138MG). Cell viability was measured by the MTS assay; for the clonogenic assay, colonies were stained with Giemsa and counted by direct visual inspection; For cell cycle analysis, cells were stained with propidium iodide and analyzed by cytometry; Cyclin A expression was determined by immunoblotting; Apoptotic cell death was determined by annexin V fluorescein isothiocyanate labeling and Caspase-3 activity in living cells. Viability of both cell lines was drastically inhibited; moreover, the colony formation capacity was significantly reduced, demonstrating long-term effects even after removal of the drug. 7-epiclusianone treatment at low concentrations also altered cell cycle progression, decreased the S and G2/M populations and at higher concentrations increased the number of cells at sub-G1, in concordance with the increase of apoptotic cells. The present study demonstrates for the first time the anticancer potential of 7-epiclusianone against glioblastoma cells, thus meriting its further investigation as a potential therapeutic agent.

12-Chloracetyl-PPD, a novel dammarane derivative, shows anti-cancer activity via delay the progression of cell cycle G2/M phase and reactive oxygen species-mediate cell apoptosis.

PubMed

Wang, Xu De; Sun, Yuan Yuan; Zhao, Chen; Qu, Fan Zhi; Zhao, Yu Qing

2017-03-05

(20R)-Dammarane-3β, 12β, 20, 25-tetrol (25-OH-PPD) is a ginsenoside isolated from Panax ginseng (C. A. Meyer). This compound exhibits anti-cancer activities on many human cancer cell lines. In this study, we investigated anti-cancer mechanisms of 12β-O-( L -Chloracetyl)-dammar-20(22)-ene-3β,25-diol(12-Chloracetyl-PPD), a modified 25-OH-PPD. We found that compound 12-Chloracetyl-PPD resulted in a concentration-dependent inhibition of viability in prostate, breast, and gastric cancer cells, without affecting the viability of normal cell (human gastric epithelial cell line-GES-1, hair follicle dermal papilla cell line-HHDPC and rat myocardial cell line-H9C2). In MDA-MB-435 and C4-2B cancer cells, 12-Chloracetyl-PPD induced G2/M cell cycle arrest, down-regulated mouse double minute 2 (MDM2) expression, up-regulated p53 expression, triggered apoptosis, and stimulated reactive oxygen species production. Apoptosis can be attenuated by the reactive oxygen species scavenger N-acetylcysteine. Our results suggested that compound 12-Chloracetyl-PPD showed obvious anti-cancer activity based on delaying cell cycle arrest and inducing cell apoptosis by reactive oxygen species production, which supported development of 12-Chloracetyl-PPD as a potential agent for cancer chemotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.

shRNA-mediated EMMPRIN silencing inhibits human leukemic monocyte lymphoma U937 cell proliferation and increases chemosensitivity to adriamycin.

PubMed

Gao, Hui; Jiang, Qixiao; Han, Yantao; Peng, Jianjun; Wang, Chunbo

2015-03-01

EMMPRIN is a widely distributed cell surface glycoprotein, which plays an important role in tumor progression and confers resistance to some chemotherapeutic drugs. Recent studies have shown that EMMPRIN overexpression indicates poor prognosis in acute myeloid leukemia (AML). However, little was known on the role of EMMPRIN in leukemia. Human leukemia cell line U937 was stably transfected with a EMMPRIN-targeted shRNA-containing vector to investigate the effect of EMMPRIN on cellular functions. EMMPRIN expression was monitored by qRT-PCR and Western blotting. Cell viability and proliferation were determined by trypan blue exclusion and BrdU labeling, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. Cytotoxicity of chemotherapeutic agent adriamycin on cells was assessed by MTT assay. Knockdown of EMMPRIN gene significantly inhibited cell viability and decreased cell proliferation. Fluorescence-activated cell-sorting analysis revealed that the reduced EMMPRIN expression resulted in cell cycle arrest at G1 phase and induced apoptosis. Meanwhile, western blotting analysis showed that EMMPRIN knockdown was associated with downregulation of cell cycle- and apoptosis-related molecules including cyclin D1, cyclin E, as well as increase in cleavage of caspase-3 and PARP. This study also showed that silencing of EMMPRIN sensitized U937 cells to Adriamycin. EMMPRIN is involved in proliferation, growth, and chemosensitivity of human AML line U937, indicating that EMMPRIN may be a promising therapeutic target for AML.

Withagulatin A inhibits hepatic stellate cell viability and procollagen I production through Akt and Smad signaling pathways

PubMed Central

Liu, Qiong; Chen, Jing; Wang, Xu; Yu, Liang; Hu, Li-hong; Shen, Xu

2010-01-01

Aim: To investigate the effects of the natural product Withagulatin A on hepatic stellate cell (HSC) viability and type I procollagen production. The potential mechanism underlying the pharmacological actions was also explored. Methods: The effect of Withagulatin A on cell viability was evaluated in HSC and LX-2 cells using a sulforhodamine B (SRB) assay. Cell cycle distribution was analyzed using flow cytometry. Type I procollagen gene expression was determined using real-time PCR. Regulation of signaling molecules by Withagulatin A was detected using Western blotting. Results: Primary rat HSCs and the human hepatic stellate cell line LX-2 treated with Withagulatin A (0.625-20 μmol/L) underwent a dose-dependent decrease in cell viability, which was associated with S phase arrest and the induction of cell apoptosis. In addition, the natural product decreased phosphorylation of the Akt/mTOR/p70S6K pathway that controls cell proliferation and survival. Furthermore, Withagulatin A (1, 2 μmol/L) inhibited transforming growth factor-β (TGF-β) stimulated type I procollagen gene expression, which was attributable to the suppression of TGF-β stimulated Smad2 and Smad3 phosphorylation. Conclusion: Our results demonstrated that Withagulatin A potently inhibited HSC viability and type I procollagen production, thereby implying that this natural product has potential use in the development of anti-fibrogenic reagents for the treatment of hepatic fibrosis. PMID:20644552

Preservation media, durations and cell concentrations of short-term storage affect key features of human adipose-derived mesenchymal stem cells for therapeutic application.

PubMed

Zhang, Fengli; Ren, Huaijuan; Shao, Xiaohu; Zhuang, Chao; Chen, Yantian; Qi, Nianmin

2017-01-01

Adipose-derived mesenchymal stem cells (ADSCs) have shown great potential in the treatment of various diseases. However, the optimum short-term storage condition of ADSCs in 2∼8 °C is rarely reported. This study aimed at optimizing a short-term storage condition to ensure the viability and function of ADSCs before transplantation. Preservation media and durations of storage were evaluated by cell viability, apoptosis, adhesion ability and colony-forming unit (CFU) capacity of ADSCs. The abilities of cell proliferation and differentiation were used to optimize cell concentrations. Optimized preservation condition was evaluated by cell surface markers, cell cycle and immunosuppressive capacity. A total of 5% human serum albumin in multiple electrolytes (ME + HSA) was the optimized medium with high cell viability, low cluster rate, good adhesion ability and high CFU capacity of ADSCs. Duration of storage should be limited to 24 h to ensure the quality of ADSCs before transplantation. A concentration of 5 × 10 6 cells/ml was the most suitable cell concentration with low late stage apoptosis, rapid proliferation and good osteogenic and adipogenic differentiation ability. This selected condition did not change surface markers, cell cycle, indoleamine 2, 3-dioxygenase 1 (IDO1) gene expression and kynurenine (Kyn) concentration significantly. In this study, ME + HSA was found to be the best medium, most likely due to the supplement of HSA which could protect cells, the physiological pH (7.4) of ME and sodium gluconate ingredient in ME which could provide energy for cells. Duration should be limited to 24 h because of reduced nutrient supply and increased waste and lactic acid accumulation during prolonged storage. To keep cell proliferation and limit lactic acid accumulation, the proper cell concentration is 5× 10 6 cells/ml. Surface markers, cell cycle and immunosuppressive capacity did not change significantly after storage using the optimized condition, which confirmed our results that this optimized short-term storage condition of MSCs has a great potential for the application of cell therapy.

Exploring the effects of low-level laser therapy on fibroblasts and tumor cells following gamma radiation exposure.

PubMed

Ramos Silva, Camila; Cabral, Fernanda Viana; de Camargo, Claudinei Francisco Morais; Núñez, Silvia Cristina; Mateus Yoshimura, Tania; de Lima Luna, Arthur Cássio; Maria, Durvanei Augusto; Ribeiro, Martha Simões

2016-12-01

Ionizing radiation (IR) induces DNA damage and low-level laser therapy (LLLT) has been investigated to prevent or repair detrimental outcomes resulting from IR exposure. Few in vitro studies, however, explore the biological mechanisms underlying those LLLT benefits. Thus, in this work, fibroblasts and tumor cells are submitted to IR with doses of 2.5 Gy and 10 Gy. After twenty-four-h, the cells are exposed to LLLT with fluences of 30 J cm -2 , 90 J cm -2 , and 150 J cm -2 . Cellular viability, cell cycle phases, cell proliferation index and senescence are evaluated on days 1 and 4 after LLLT irradiation. For fibroblasts, LLLT promotes - in a fluence-dependent manner - increments in cell viability and proliferation, while a reduction in the senescence was observed. Regarding tumor cells, no influences of LLLT on cell viability are noticed. Whereas LLLT enhances cell populations in S and G 2 /M cell cycle phases for both cellular lines, a decrease in proliferation and increase in senescence was verified only for tumor cells. Putting together, the results suggest that fibroblasts and tumor cells present different responses to LLLT following exposure to gamma-radiation, and these promising results should stimulate further investigations. Senescence of tumor cells and fibroblasts on the 4 th day after ionizing radiation (IR) and low-level laser therapy (LLLT) exposures. The number of senescent cells increased significantly for tumor cells (a) while for fibroblasts no increment was observed (b). The blue collor indicates senescence activity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Berberine sensitizes nasopharyngeal carcinoma cells to radiation through inhibition of Sp1 and EMT.

PubMed

Wang, Jun; Kang, Min; Wen, Qin; Qin, Yu-Tao; Wei, Zhu-Xin; Xiao, Jing-Jian; Wang, Ren-Sheng

2017-04-01

Nasopharyngeal carcinoma (NPC) is a tumor of epithelial origin with radiotherapy as its standard treatment. However, radioresistance remains a critical issue in the treatment of NPC. This study aimed to investigate the effect of berberine on the proliferation, cell cycle regulation, apoptosis, radioresistance of NPC cells and whether specificity protein 1 (Sp1) is a functional target of berberine. Our results showed that treatment with berberine reduced the proliferation and viability of CNE-2 cells in a dose- and time‑dependent manner. Berberine induced cell cycle arrest in the G0/G1 phase and apoptosis. In CNE-2 cells exposed to gamma‑ray irradiation, berberine reduced cell viability at various concentrations (25, 50, 75 and 100 µmol/l). Berberine significantly decreased mRNA and protein expression of Sp1 in the CNE-2 cells. Mithramycin A, a selective Sp1 inhibitor, enhanced the radiosensitivity and the rate of apoptosis in the CNE-2 cells. Berberine inhibited transforming growth factor-β (TGF-β)-induced tumor invasion and suppressed epithelial-to-mesenchymal transition (EMT) process, as evidenced by increased E-cadherin and decreased vimentin proteins. Sp1 may be required for the TGF-β1-induced invasion and EMT by berberine. In conclusion, berberine demonstrated the ability to suppress proliferation, induce cell cycle arrest and apoptosis, and enhance radiosensitivity of the CNE-2 NPC cells. Sp1 may be a target of berberine which is decreased during the radiosensitization of berberine.

Roles of glucose in photoreceptor survival.

PubMed

Chertov, Andrei O; Holzhausen, Lars; Kuok, Iok Teng; Couron, Drew; Parker, Ed; Linton, Jonathan D; Sadilek, Martin; Sweet, Ian R; Hurley, James B

2011-10-07

Vertebrate photoreceptor neurons have a high demand for metabolic energy, and their viability is very sensitive to genetic and environmental perturbations. We investigated the relationship between energy metabolism and cell death by evaluating the metabolic effects of glucose deprivation on mouse photoreceptors. Oxygen consumption, lactate production, ATP, NADH/NAD(+), TCA cycle intermediates, morphological changes, autophagy, and viability were evaluated. We compared retinas incubated with glucose to retinas deprived of glucose or retinas treated with a mixture of mitochondrion-specific fuels. Rapid and slow phases of cell death were identified. The rapid phase is linked to reduced mitochondrial activity, and the slower phase reflects a need for substrates for cell maintenance and repair.

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.

Study on the Mechanism of Cell Cycle Checkpoint Kinase 2 (CHEK2) Gene Dysfunction in Chemotherapeutic Drug Resistance of Triple Negative Breast Cancer Cells.

PubMed

Luo, Li; Gao, Wei; Wang, Jinghui; Wang, Dingxue; Peng, Xiaobo; Jia, Zhaoyang; Jiang, Ye; Li, Gongzhuo; Tang, Dongxin; Wang, Yajie

2018-05-15

BACKGROUND This study aimed to investigate the mechanism of CHEK2 gene dysfunction in drug resistance of triple negative breast cancer (TNBC) cells. MATERIAL AND METHODS To perform our study, a stable CHEK2 wild type (CHEK2 WT) or CHEK2 Y390C mutation (CHEK2 Y390C) expressed MDA-MB-231 cell line was established. MTT assay, cell apoptosis assay and cell cycle assay were carried out to analyze the cell viability, apoptosis, and cell cycle respectively. Western blotting and qRT-PCR were applied for related protein and gene expression detection. RESULTS We found that the IC50 value of DDP (Cisplatin) to CHEK2 Y390C expressed MDA-MB-231 cells was significantly higher than that of the CHEK2 WT expressed cells and the control cells. After treatment with DDP for 48 h, cells expressing CHEK2 WT showed lower cell viability than that of the CHEK2 Y390C expressed cells and the control cells; compared with the CHEK2 Y390C expressed cells and the control cells, cells expressing CHEK2 WT showed significant G1/S arrest. Meanwhile, we found that compared with the CHEK2 Y390C expressed cells and the control cells, cell apoptosis was significantly increased in CHEK2 WT expressed cells. Moreover, our results suggested that cells expressing CHEK2 WT showed higher level of p-CDC25A, p-p53, p21, Bax, PUMA, and Noxa than that of the CHEK2 Y390C expressed cells and the control cells. CONCLUSIONS Our findings indicated that CHEK2 Y390C mutation induced the drug resistance of TNBC cells to chemotherapeutic drugs through administrating cell apoptosis and cell cycle arrest via regulating p53 activation and CHEK2-p53 apoptosis pathway.

Toxicological characterization of ZnO nanoparticles in malignant and non-malignant cells.

PubMed

Moratin, Helena; Scherzad, Agmal; Gehrke, Thomas; Ickrath, Pascal; Radeloff, Katrin; Kleinsasser, Norbert; Hackenberg, Stephan

2018-04-01

The increasing usage of zinc oxide nanoparticles (ZnO-NPs) in industrial applications as well as in consumer products raises concern regarding their potential adverse effects to a greater extend. Numerous studies have demonstrated toxic properties of NPs, however there is still a lack of knowledge concerning the underlying mechanisms. This study was designed to systematically investigate cytotoxicity, apoptosis, cell cycle alterations, and genotoxicity induced by ZnO-NP. Moreover, it was an aim of the investigations to specify the diverse effects of nanoparticle exposure in malignant in comparison with non-malignant cells. Therefore, human head and neck squamous cell carcinoma-derived FaDu cells were incubated with 4-20 µg/ml of ZnO-NPs for 1-48 hr and tested for cell viability, cell cycle alterations, apoptosis and caspase-3 gene expression as a sensitive marker of molecular apoptotic processes with regard to time- and dose-dependent effects. Human mesenchymal bone marrow stem cells were used as non-malignant representatives to examine oxidative stress-related genotoxicity. Results showed a significant reduction in cell viability as well as dose- and time-dependent increase of apoptotic cells following nanoparticle treatment. Likewise, caspase-3 gene expression enhanced already before first apoptotic cells were detectable. It could be observed that doses that were cytotoxic in tumor cells did not reduce viability in stem cells. However, the same concentrations already induced significant DNA damage. The findings of the study suggest to keep a more critical eye on the use of nanoparticles as anti-cancer agents. Yet, additional in vivo studies are needed to assess safety concerns for consumers and patients. Environ. Mol. Mutagen. 59:247-259, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Effects of short-chain chlorinated paraffins exposure on the viability and metabolism of human hepatoma HepG2 cells.

PubMed

Geng, Ningbo; Zhang, Haijun; Zhang, Baoqin; Wu, Ping; Wang, Feidi; Yu, Zhengkun; Chen, Jiping

2015-03-03

Short-chain chlorinated paraffins (SCCPs) have attracted considerable attention for their characteristic of persistent organic pollutants. However, very limited information is available for their toxic effects at environmentally relevant doses, limiting the evaluation of their health risks. In this study, cell viability assay and targeted metabolomic approach was used to evaluate the environmental dose (<100 μg/L) effect of SCCPs on HepG2 cells. Cell viability was found to be decreased with increases in exposure dose of SCCPs. Exposure for 48 h to C10-CPs resulted in a significant reduction in cell viability compared with 24 h, even at 1 μg/L. SCCPs exposure altered the intracellular redox status and caused significant metabolic disruptions. As a kind of peroxisome proliferator, SCCPs specifically stimulated the β-oxidation of unsaturated fatty acids and long-chain fatty acids. Meanwhile, SCCPs exposure disturbed glycolysis and amino acid metabolism, and led to the up-regulation of glutamate metabolism and urea cycle. The toxic effects of SCCPs might mainly involve the perturbation of energy production, protein biosynthesis, fatty acid metabolism, and ammonia recycling.

The MEK1/2 Inhibitor AZD6244 Sensitizes BRAF-Mutant Thyroid Cancer to Vemurafenib.

PubMed

Song, Hao; Zhang, Jinna; Ning, Liang; Zhang, Honglai; Chen, Dong; Jiao, Xuelong; Zhang, Kejun

2018-05-08

BACKGROUND [i]BRAF[/i]V600E mutation occurs in approximately 45% of papillary thyroid cancer (PTC) cases, and 25% of anaplastic thyroid cancer (ATC) cases. Vemurafenib/PLX4032, a selective BRAF inhibitor, suppresses extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) signaling and shows beneficial effects in patients with metastatic melanoma harboring the [i]BRAFV600E[/i] mutation. However, the response to vemurafenib is limited in BRAF-mutant thyroid cancer. The present study evaluated the effect of vemurafenib in combination with the selective MEK1/2 inhibitor AZD6244 on cell survival and explored the mechanism underlying the combined effect of vemurafenib and AZD6244 on thyroid cancer cells harboring BRAFV600E. MATERIAL AND METHODS Thyroid cancer 8505C and BCPAP cells harboring the [i]BRAFV600E[/i] mutation were exposed to vemurafenib (0.01, 0.1, and 1 µM) and AZD6244 (0.01, 0.1, and 1 µM) alone or in the indicated combinations for the indicated times. Cell viability was detected by the MTT assay. Cell cycle distribution and induction of apoptosis were detected by flow cytometry. The expression of cyclin D1, P27, (P)-ERK1/2 was evaluated by Western blotting. The effect of vemurafenib or AZD6244 or their combination on the growth of 8505C cells was examined in orthotopic xenograft mouse models [i]in vivo[/i]. RESULTS Vemurafenib alone did not increase cell apoptosis, whereas it decreased cell viability by promoting cell cycle arrest in BCPAP and 8505C cells. AZD6244 alone increased cell apoptosis by inducing cell cycle arrest in BCPAP and 8505C cells. Combination treatment with AZD6244 and vemurafenib significantly decreased cell viability and increased apoptosis in both BCPAP and 8505C cells compared with the effects of each drug alone. AZD6244 alone abolished phospho-ERK1/2 (pERK1/2) expression at 48 h, whereas vemurafenib alone downregulated pERK1/2 at 4-6 h, with rapid recovery of expression, reaching the highest level at 24-48 h. Combined treatment for 48 h completely inhibited pERK1/2 expression. Combination treatment with vemurafenib and AZD6244 inhibited cell growth and induced apoptosis by causing cell-cycle arrest, with the corresponding changes in the expression of the cell cycle regulators p27Kip1 and cyclin D1. Co-administration of vemurafenib and AZD6244 [i]in vivo[/i] had a significant synergistic antitumor effect in a nude mouse model. CONCLUSIONS Vemurafenib activated pERK1/2 and induced vemurafenib resistance in thyroid cancer cells. Combination treatment with vemurafenib and AZD6244 inhibited ERK signaling and caused cell cycle arrest, resulting in cell growth inhibition. Combination treatment in patients with thyroid cancer harboring the [i]BRAFV600E[/i] mutation may overcome vemurafenib resistance and enhance the therapeutic effect.

Ethanol addition enhances acid treatment to eliminate Lactobacillus fermentum from the fermentation process for fuel ethanol production.

PubMed

Costa, M A S; Cerri, B C; Ceccato-Antonini, S R

2018-01-01

Fermentation is one of the most critical steps of the fuel ethanol production and it is directly influenced by the fermentation system, selected yeast, and bacterial contamination, especially from the genus Lactobacillus. To control the contamination, the industry applies antibiotics and biocides; however, these substances can result in an increased cost and environmental problems. The use of the acid treatment of cells (water-diluted sulphuric acid, adjusted to pH 2·0-2·5) between the fermentation cycles is not always effective to combat the bacterial contamination. In this context, this study aimed to evaluate the effect of ethanol addition to the acid treatment to control the bacterial growth in a fed-batch system with cell recycling, using the industrial yeast strain Saccharomyces cerevisiae PE-2. When only the acid treatment was used, the population of Lactobacillus fermentum had a 3-log reduction at the end of the sixth fermentation cycle; however, when 5% of ethanol was added to the acid solution, the viability of the bacterium was completely lost even after the first round of cell treatment. The acid treatment +5% ethanol was able to kill L. fermentum cells without affecting the ethanol yield and with a low residual sugar concentration in the fermented must. In Brazilian ethanol-producing industry, water-diluted sulphuric acid is used to treat the cell mass at low pH (2·0) between the fermentative cycles. This procedure reduces the number of Lactobacillus fermentum from 10 7 to 10 4  CFU per ml. However, the addition of 5% ethanol to the acid treatment causes the complete loss of bacterial cell viability in fed-batch fermentation with six cell recycles. The ethanol yield and yeast cell viability are not affected. These data indicate the feasibility of adding ethanol to the acid solution replacing the antibiotic use, offering a low cost and a low amount of residue in the biomass. © 2017 The Society for Applied Microbiology.

Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

PubMed Central

2010-01-01

Background α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action. Methods MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells. Results α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G2/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G2/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells. Conclusions This study for the first time identifies effects of α-santalol in G2/M phase arrest and describes detailed mechanisms of G2/M phase arrest by this agent, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models. PMID:20682067

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

PubMed

Jayme, Cristiano Ceron; de Paula, Leonardo Barcelos; Rezende, Nayara; Calori, Italo Rodrigo; Franchi, Leonardo Pereira; Tedesco, Antonio Claudio

2017-11-15

DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Curcumin enhances the effects of irinotecan on colorectal cancer cells through the generation of reactive oxygen species and activation of the endoplasmic reticulum stress pathway.

PubMed

Huang, Yan-Feng; Zhu, Da-Jian; Chen, Xiao-Wu; Chen, Qi-Kang; Luo, Zhen-Tao; Liu, Chang-Chun; Wang, Guo-Xin; Zhang, Wei-Jie; Liao, Nv-Zhu

2017-06-20

Although initially effective against metastatic colorectal cancer (CRC), irinotecan-based chemotherapy leads to resistance and adverse toxicity. Curcumin is well known for its anti-cancer effects in many cancers, including CRC. Here, we describe reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress as important mechanisms by which curcumin enhances irinotecan's effects on CRC cells. CRC cell lines were treated with curcumin and/or irinotecan for 24 h, and then evaluated using cell proliferation assays, cell apoptosis assays, cell cycle analysis, intracellular Ca2+ measurements, ROS measurements and immunoblotting for key ER stress-related proteins. We found that cell viability was inhibited and apoptosis was increased, accompanied by ROS generation and ER stress activation in CRC cells treated with curcumin alone or in combination with irinotecan. Blocking ROS production attenuated the expression of two markers of ER stress: binding of immunoglobulin protein (BIP) and CCAAT/enhancer-binding protein homologous protein (CHOP). Blocking CHOP expression using RNA interference also inhibited ROS generation. These results demonstrated that curcumin could enhance the effects of irinotecan on CRC cells by inhibiting cell viability and inducing cell cycle arrest and apoptosis, and that these effects may be mediated, in part, by ROS generation and activation of the ER stress pathway.

Green synthesis of platinum nanoparticles that induce cell death and G2/M-phase cell cycle arrest in human cervical cancer cells.

PubMed

Alshatwi, Ali A; Athinarayanan, Jegan; Vaiyapuri Subbarayan, Periasamy

2015-01-01

Platinum-based chemotherapeutic drugs, including cisplatin, carboplatin, and oxaliplatin, have been used to manage cancer in spite of dose-dependent side effects, including nephrotoxicity, neurotoxicity and ototoxicity. These disadvantages have prompted the development of new strategies for cancer therapy that utilize functionalized nanoparticles as nanomedicines. In the present investigation, we have synthesized platinum nanoparticles using tea polyphenol (TPP) as both a reducing and surface modifying agent. The crystalline nature and morphology of the prepared TPP-functionalized platinum nanoparticles (TPP@Pt) were analyzed using X-ray diffraction (XRD) and transmission electron microscopy (TEM). The XRD results revealed that the TPP@Pt had a crystalline nature with a face-centered cubic structure. TEM imaging suggested that the TTP@Pt are flower shaped with a well-dispersed 30-60 nm-sized TPP@Pt formation. Cervical cancer cells (SiHa) were then treated with different concentrations of TPP@Pt. The effects of TPP@Pt on cell viability, nuclear morphology and cell cycle distribution were investigated. A cell viability assay revealed that the proliferation of SiHa cells was inhibited by TPP@Pt. Propidium iodide nuclear staining indicated that TPP@Pt induced nuclear fragmentation and chromatin condensation. Treatment with TPP@Pt significantly increased the percentage of cells in the G2/M phase, which indicates induced cell cycle arrest in the G2/M phase and an increased number of cells in the subG0 cell death phase. These findings highlight a potential use of TPP@Pt in cervical cancer treatment.

7 Methyl indole ethyl isothiocyanate causes ROS mediated apoptosis and cell cycle arrest in endometrial cancer cells.

PubMed

Kristjansdottir, Katrin; Kim, Kyukwang; Choi, Joong Sub; Horan, Timothy C; Brard, Laurent; Moore, Richard G; Singh, Rakesh K

2012-08-01

Chemotherapy options for advanced endometrial cancer are limited and newer therapeutic agents are urgently needed. This study describes the therapeutic potential of 7 Methyl-indole ethyl isothiocyanate (7Me-IEITC) in endometrial cancer cell lines. 7Me-IEITC was synthesized in our laboratory. The cell viability of 7Me-IEITC treated ECC-1 and KLE endometrial cancer cell was determined by MTS assay. Morphology and apoptosis were further confirmed by DAPI-staining and TUNEL assay. The measurement of reactive oxygen species (ROS), mitochondrial transmembrane depolarization potential (ΔΨm) and cell cycle phase was determined by FACS analysis. Expression of proteins involved in apoptosis, survival and cell-cycle progression was analyzed by Western blotting. 7Me-IEITC reduced the viability of the ECC-1 and KLE cancer cell-lines (IC(50)~2.5-10 μM) in a dose dependent fashion. 7Me-IEITC treatment caused mitochondrial transmembrane potential reduction, elevated the production of ROS, leading to activation of apoptosis in endometrial cancer KLE and ECC-1 cells. 7Me-IEITC treatment activated Bad, suppressed Bcl2 phosphorylation followed by PARP-1 deactivation and caspase 3 and 7 activation. 7Me-IEITC treatment arrested the progression of KLE cells in S-phase and caused CDC25 and cyclin-D1 downregulation. Pre-treatment with ascorbic acid abrogated 7Me-IEITC induced apoptosis in ECC-1 and KLE cells, suggesting that 7Me-IEITC mediated cytotoxicity is primarily through ROS production. 7Me-IEITC demonstrated promising cytotoxic effects in endometrial cancer cell line model. Copyright © 2012 Elsevier Inc. All rights reserved.

Inhibiting heat shock protein 90 and the ubiquitin-proteasome pathway impairs metabolic homeostasis and leads to cell death in human pancreatic cancer cells.

PubMed

Belalcazar, Astrid; Shaib, Walid L; Farren, Matthew R; Zhang, Chao; Chen, Zhengjia; Yang, Lily; Lesinski, Gregory B; El-Rayes, Bassel F; Nagaraju, Ganji Purnachandra

2017-12-15

Heat shock protein 90 (HSP90) and the ubiquitin-proteasome pathway play crucial roles in the homeostasis of pancreatic cancer cells. This study combined for the first time the HSP90 inhibitor ganetespib (Gan) and the proteasome inhibitor carfilzomib (Carf) to target key mechanisms of homeostasis in pancreatic cancer. It was hypothesized that Gan plus Carf would elicit potent antitumor activity by modulating complementary homeostatic processes. In vitro and in vivo effects of this combination on mechanisms of cell growth and viability were evaluated with human pancreatic cancer cell lines (MIA PaCa-2 and HPAC). Combined treatment with Gan and Carf significantly decreased cell viability. The mechanism varied by cell line and involved G 2 -M cell-cycle arrest accompanied by a consistent reduction in key cell-cycle regulatory proteins and concomitant upregulation of p27. Further studies revealed increased autophagy markers, including the upregulation of autophagy related 7 and light chain 3 cleavage, and evidence of apoptosis (increased Bax expression and processing of caspase 3). Immunoblot analyses confirmed the modulation of other pathways that influence cell viability, including phosphoinositide 3-kinase/Akt and nuclear factor κB. Finally, the treatment of athymic mice bearing HPAC tumors with Gan and Carf significantly reduced tumor growth in vivo. An immunoblot analysis of freshly isolated tumors from animals at the end of the study confirmed in vivo modulation of key signaling pathways. The results reveal Gan plus Carf to be a promising combination with synergistic antiproliferative, apoptotic, and pro-autophagy effects in preclinical studies of pancreatic cancer and will further the exploration of the utility of this treatment combination in clinical trials. Cancer 2017;123:4924-33. © 2017 American Cancer Society. © 2017 American Cancer Society.

Effects of Long-Term 50Hz Power-Line Frequency Electromagnetic Field on Cell Behavior in Balb/c 3T3 Cells

PubMed Central

An, Guang-Zhou; Xu, Hui; Zhou, Yan; Du, Le; Miao, Xia; Jiang, Da-Peng; Li, Kang-Chu; Guo, Guo-Zhen; Zhang, Chen; Ding, Gui-Rong

2015-01-01

Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn’t change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein. PMID:25695503

Effects of long-term 50Hz power-line frequency electromagnetic field on cell behavior in Balb/c 3T3 cells.

PubMed

An, Guang-Zhou; Xu, Hui; Zhou, Yan; Du, Le; Miao, Xia; Jiang, Da-Peng; Li, Kang-Chu; Guo, Guo-Zhen; Zhang, Chen; Ding, Gui-Rong

2015-01-01

Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn't change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein.

The green tea catechin epigallocatechin gallate induces cell cycle arrest and shows potential synergism with cisplatin in biliary tract cancer cells.

PubMed

Mayr, Christian; Wagner, Andrej; Neureiter, Daniel; Pichler, Martin; Jakab, Martin; Illig, Romana; Berr, Frieder; Kiesslich, Tobias

2015-06-23

The green tea catechin epigallocatechin gallate (EGCG) was shown to effectively inhibit tumor growth in various types of cancer including biliary tract cancer (BTC). For most BTC patients only palliative therapy is possible, leading to a median survival of about one year. Chemoresistance is a major problem that contributes to the high mortality rates of BTC. The aim of this study was to investigate the cytotoxic effect of EGCG alone or in combination with cisplatin on eight BTC cell lines and to investigate the cellular anti-cancer mechanisms of EGCG. The effect of EGCG treatment alone or in combination with the standard chemotherapeutic cisplatin on cell viability was analyzed in eight BTC cell lines. Additionally, we analyzed the effects of EGCG on caspase activity, cell cycle distribution and gene expression in the BTC cell line TFK-1. EGCG significantly reduced cell viability in all eight BTC cell lines (p < 0.05 or p < 0.01, respectively, for most cell lines and EGCG concentrations > 5 μM). Combined EGCG and cisplatin treatment showed a synergistic cytotoxic effect in five cell lines and an antagonistic effect in two cell lines. Furthermore, EGCG reduced the mRNA levels of various cell cycle-related genes, while increasing the expression of the cell cycle inhibitor p21 and the apoptosis-related death receptor 5 (p < 0.05). This observation was accompanied by an increase in caspase activity and cells in the sub-G1 phase of the cell cycle, indicating induction of apoptosis. EGCG also induced a down-regulation of expression of stem cell-related genes and genes that are associated with an aggressive clinical character of the tumor, such as cd133 and abcg2. EGCG shows various anti-cancer effects in BTC cell lines and might therefore be a potential anticancer drug for future studies in BTC. Additionally, EGCG displays a synergistic cytotoxic effect with cisplatin in most tested BTC cell lines. Graphical abstract Summary illustration.

Panaxadiol, a purified ginseng component, enhances the anti-cancer effects of 5-fluorouracil in human colorectal cancer cells.

PubMed

Li, Xiao-Li; Wang, Chong-Zhi; Mehendale, Sangeeta R; Sun, Shi; Wang, Qi; Yuan, Chun-Su

2009-11-01

Colorectal cancer is a major cause of morbidity and mortality for cancer worldwide. Although 5-fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents in first-line therapy for colorectal cancer, serious side effects limit its clinical usefulness. Panaxadiol (PD) is the purified sapogenin of ginseng saponins, which exhibit anti-tumor activity. In this study, we investigated the possible synergistic anti-cancer effects of PD and 5-FU on a human colorectal cancer cell line, HCT-116. Cell viability was evaluated by an MTS cell proliferation assay. Morphological observation was performed by crystal violet cell viability staining assay. Cell cycle distribution and apoptotic effects were analyzed by flow cytometry after staining with PI/RNase or Annexin V/PI. Cell growth was markedly suppressed in HCT-116 cells treated by 5-FU (20-100 microM) for 24 or 48 h with time-dependent effects. The significant suppression on HCT-116 cell proliferation was observed after treatment with PD (25 microM) for 24 and 48 h. Panaxadiol (25 microM) markedly (P < 0.05) enhanced the anti-proliferative effects of 5-FU (5, 10, 20 microM) on HCT-116 cells compared to single treatment of 5-FU for 24 and 48 h. Flow cytometric analysis on DNA indicated that PD and 5-FU selectively arrested cell cycle progression in the G1 phase and S phase (P < 0.01), respectively, compared to the control condition. Combination use of 5-FU with PD significantly (P < 0.001) increased cell cycle arrest in the S phase compared to that treated by 5-FU alone. The combination of 5-FU and PD significantly enhanced the percentage of apoptotic cells when compared with the corresponding cell groups treated by 5-FU alone (P < 0.001). Panaxadiol enhanced the anti-cancer effects of 5-FU on human colorectal cancer cells through the regulation of cell cycle transition and the induction of apoptotic cells.

Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells

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

Srivastava, Janmejai K.; Department of Urology, University Hospitals of Cleveland, Cleveland, OH 44106; Gupta, Sanjay

2006-07-28

One of the requisite of cancer chemopreventive agent is elimination of damaged or malignant cells through cell cycle inhibition or induction of apoptosis without affecting normal cells. In this study, employing normal human prostate epithelial cells (PrEC), virally transformed normal human prostate epithelial cells (PZ-HPV-7), and human prostate cancer cells (LNCaP, DU145, and PC-3), we evaluated the growth-inhibitory and apoptotic effects of tocotrienol-rich fraction (TRF) extracted from palm oil. TRF treatment to PrEC and PZ-HPV-7 resulted in almost identical growth-inhibitory responses of low magnitude. In sharp contrast, TRF treatment resulted in significant decreases in cell viability and colony formation inmore » all three prostate cancer cell lines. The IC{sub 5} values after 24 h TRF treatment in LNCaP, PC-3, and DU145 cells were in the order 16.5, 17.5, and 22.0 {mu}g/ml. TRF treatment resulted in significant apoptosis in all the cell lines as evident from (i) DNA fragmentation (ii) fluorescence microscopy, and (iii) cell death detection ELISA, whereas the PrEC and PZ-HPV-7 cells did not undergo apoptosis, but showed modestly decreased cell viability only at a high dose of 80 {mu}g/ml. In cell cycle analysis, TRF (10-40 {mu}g/ml) resulted in a dose-dependent G0/G1 phase arrest and sub G1 accumulation in all three cancer cell lines but not in PZ-HPV-7 cells. These results suggest that the palm oil derivative TRF is capable of selectively inhibiting cellular proliferation and accelerating apoptotic events in prostate cancer cells. TRF offers significant promise as a chemopreventive and/or therapeutic agent against prostate cancer.« less

Investigation of welded interconnection of large area wraparound contacted silicon solar cells

NASA Technical Reports Server (NTRS)

Lott, D. R.

1984-01-01

An investigation was conducted to evaluate the welding and temperature cycle testing of large area 5.9 x 5.9 wraparound silicon solar cells utilizing printed circuit substrates with SSC-155 interconnect copper metals and the LMSC Infrared Controlled weld station. An initial group of 5 welded modules containing Phase 2 developmental 5.9 x 5.9 cm cells were subjected to cyclical temperatures of + or 80 C at a rate of 120 cycles per day. Anomalies were noted in the adhesion of the cell contact metallization; therefore, 5 additional modules were fabricated and tested using available Phase I cells with demonstrated contact integrity. Cycling of the later module type through 12,000 cycles indicated the viability of this type of lightweight flexible array concept. This project demonstrated acceptable use of an alternate interconnect copper in combination with large area wraparound cells and emphasized the necessity to implement weld pull as opposed to solder pull procedures at the cell vendors for cells that will be interconnected by welding.

Magnolol Inhibits the Growth of Non-Small Cell Lung Cancer via Inhibiting Microtubule Polymerization.

PubMed

Shen, Jia; Ma, Hailin; Zhang, Tiancheng; Liu, Hui; Yu, Linghua; Li, Guosheng; Li, Huishuang; Hu, Meichun

2017-01-01

The tubulin/microtubule system, which is an integral component of the cytoskeleton, plays an essential role in mitosis. Targeting mitotic progression by disturbing microtubule dynamics is a rational strategy for cancer treatment. Microtubule polymerization assay was performed to examine the effect of Magnolol (a novel natural phenolic compound isolated from Magnolia obovata) on cellular microtubule polymerization in human non-small cell lung cancer (NSCLC) cells. Cell cycle analysis, mitotic index assay, cell proliferation assay, colony formation assay, western blotting analysis of cell cycle regulators, Annexin V-FITC/PI staining, and live/dead viability staining were carried out to investigate the Magnolol's inhibitory effect on proliferation and viability of NSCLS cells in vitro. Xenograft model of human A549 NSCLC tumor was used to determine the Magnolol's efficacy in vivo. Magnolol treatment effectively inhibited cell proliferation and colony formation of NSCLC cells. Further study proved that Magnolol induced the mitotic phase arrest and inhibited G2/M progression in a dose-dependent manner, which were mechanistically associated with expression alteration of a series of cell cycle regulators. Furthermore, Magnolol treatment disrupted the cellular microtubule organization via inhibiting the polymerization of microtubule. We also found treatment with NSCLC cells with Magnolol resulted in apoptosis activation through a p53-independent pathway, and autophgy induction via down-regulation of the Akt/mTOR pathway. Finally, Magnolol treatment significantly suppressed the NSCLC tumor growth in mouse xenograft model in vivo. These findings identify Magnolol as a promising candidate with anti-microtubule polymerization activity for NSCLC treatment. © 2017 The Author(s). Published by S. Karger AG, Basel.

The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle.

PubMed

Pramila, Tata; Wu, Wei; Miles, Shawna; Noble, William Stafford; Breeden, Linda L

2006-08-15

Transcription patterns shift dramatically as cells transit from one phase of the cell cycle to another. To better define this transcriptional circuitry, we collected new microarray data across the cell cycle of budding yeast. The combined analysis of these data with three other cell cycle data sets identifies hundreds of new highly periodic transcripts and provides a weighted average peak time for each transcript. Using these data and phylogenetic comparisons of promoter sequences, we have identified a late S-phase-specific promoter element. This element is the binding site for the forkhead protein Hcm1, which is required for its cell cycle-specific activity. Among the cell cycle-regulated genes that contain conserved Hcm1-binding sites, there is a significant enrichment of genes involved in chromosome segregation, spindle dynamics, and budding. This may explain why Hcm1 mutants show 10-fold elevated rates of chromosome loss and require the spindle checkpoint for viability. Hcm1 also induces the M-phase-specific transcription factors FKH1, FKH2, and NDD1, and two cell cycle-specific transcriptional repressors, WHI5 and YHP1. As such, Hcm1 fills a significant gap in our understanding of the transcriptional circuitry that underlies the cell cycle.

MiR-374b Promotes Proliferation and Inhibits Apoptosis of Human GIST Cells by Inhibiting PTEN through Activation of the PI3K/Akt Pathway.

PubMed

Long, Zi-Wen; Wu, Jiang-Hong; Hong, Cai-; Wang, Ya-Nong; Zhou, Ye

2018-06-14

Gastrointestinal stromal tumours (GIST) are the most common mesenchymal tumors of the gastrointestinal (GI) tract. In order to investigate a new treatment fot GIST, we hypothesized the effect of miR-374b targeting PTEN gene-mediated PI3K/Akt signal transduction pathway on proliferation and apoptosis of human gastrointestinal stromal tumor (GIST) cells. We obtained GIST tissues and adjacent normal tissues from 143 patients with GIST to measure the levels of miR- 374b, PTEN, PI3K, Akt, caspase9, Bax, MMP2, MMP9, ki67, PCNA, P53 and cyclinD1. Finally, cell viability, cell cycle and apoptosis were detected. According to the KFGG analysis of DEGs, PTEN was involved in a variety of signaling pathways and miRs were associated with cancer development. The results showed that MiR-374b was highly expressed, while PTEN was downregulated in the GIST tissues. The levels of miR-374b, PI3K, AKT and PTEN were related to tumor diameter and pathological stage. Additionally, miR-374b increased the mRNA and protein levels of PI3K, Akt, MMP2, MMP9, P53 and cyclinD1, suggesting that miR-374b activates PI3K/Akt signaling pathway in GIST-T1 cells. Moreover, MiR- 374b promoted cell viability, migration, invasion, and cell cycle entry, and inhibited apoptosis in GIST cells. Taken together, the results indicated that miR-374b promotes viability and inhibits apoptosis of human GIST cells by targeting PTEN gene through the PI3K/Akt signaling pathway. Thus, this study provides a new potential target for GIST treatment.

Diosmin reduces cell viability of A431 skin cancer cells through apoptotic induction.

PubMed

Buddhan, Rajamanickam; Manoharan, Shanmugam

2017-01-01

Aim of the present study was to evaluate the in vitro cytotoxic potential of the diosmin in A431 skin cancer cells. The cytotoxic (anti-cell proliferative) potential of diosmin in A431 cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (cell viability), dual staining (apoptotic induction), dichloro-dihydro-fluorescein diacetate assay (reactive oxygen species [ROS] generation), DNA fragmentation study, Western blotting analysis (apoptotic markers expression) and flow cytometry (cell cycle arrest). Diosmin reduced the cell viability of A431 cells in a dose-dependent fashion and the inhibitory concentration 50% value was attained at 45 μg/ml using MTT assay. Diosmin at a concentration of 45 μg/ml generated excessive ROS in A431 cells, as compared to untreated cells. Diosmin treated A431 cells also revealed multiple DNA fragments than the untreated cells. Diosmin upregulated the expression of p53, caspases 3 and 9 and downregulated the expression of Bcl-2, matrix metalloproteinases-2 and 9 in A431 cells. The cytotoxic or anti-cell proliferative potential of diosmin is due to its ROS-mediated apoptotic induction potential, as well as due to its role in the inhibition of invasion in the A431 cells.

The Biological Effects of Bilirubin Photoisomers

PubMed Central

Jasprova, Jana; Dal Ben, Matteo; Vianello, Eleonora; Goncharova, Iryna; Urbanova, Marie; Vyroubalova, Karolina; Gazzin, Silvia; Tiribelli, Claudio; Sticha, Martin; Cerna, Marcela; Vitek, Libor

2016-01-01

Although phototherapy was introduced as early as 1950’s, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells. PMID:26829016

Yongdamsagan-tang, a traditional herbal formula, inhibits cell growth through the suppression of proliferation and inflammation in benign prostatic hyperplasia epithelial-1 cells.

PubMed

Park, Eunsook; Lee, Mee-Young; Seo, Chang-Seob; Jeon, Woo-Young; Shin, Hyeun-Kyoo

2017-09-14

Benign prostatic hyperplasia (BPH), also called benign enlargement of the prostate, is a progressive disease that is observed in most elderly men. Yongdamsagan-tang, a traditional herbal formula, is used commonly for the treatment of inflammation-related diseases. Although the therapeutic efficacy of Yongdamsagan-tang against BPH in vivo was reported previously, its underlying mechanisms are not clearly understood. In this study, we investigated the effect of Yongdamsagan-tang water extract (YSTE) and its mechanism on the growth of human BPH epithelial BPH-1 cells. YSTE was extracted from 11 herbaceous plants and its chemical composition was analyzed by High-performance liquid chromatography (HPLC). YSTE was treated in the epithelial BPH-1 cell line and then cell lysates or supernant were used to evaluate cell viability, cell cycle, proliferation and cytokine production. HPLC revealed that Baicalin and gentiopicroside were involved as the major compounds of YSTE. YSTE treatment in BPH-1 cells repressed cell viability in a dose-dependent manner. Regarding the inhibitory mechanisms of YSTE on cell growth, YSTE inhibited cell proliferation via a decrease in endogenous cyclin D1 protein levels and arrest at the S phase during cell-cycle progression. Furthermore, YSTE treatment in BPH-1 cells suppressed prostaglandin E 2 production and cyclooxygenase-2 (COX-2) protein levels. The secretion of the proinflammatory cytokines, interleukin-8 and interleukin-6, was also reduced by YSTE treatment. YSTE in BPH-1 cells showed antiproliferative and anti-inflammatory activities via cell-cycle arrest and downregulation of COX-2 expression, respectively. Taken together, the results of the present study will enhance our understanding of the mechanisms underlying the effect of YSTE in BPH. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Antiproliferative Activity of Egg Yolk Peptides in Human Colon Cancer Cells.

PubMed

Yousr, Marwa N; Aloqbi, Akram A; Omar, Ulfat M; Howell, Nazlin K

2017-01-01

Egg yolk peptides were successfully prepared from egg yolk protein by-products after lecithin extraction. Defatted egg yolk protein was hydrolyzed with pepsin and pancreatin and purified by gel filtration to produce egg yolk gel filtration fraction (EYGF-33) with antiproliferative activity. The highlight of this study was that the peptide EYGF-33 (1.0 mg/ml) significantly inhibits cell viability of colon cancer cells (Caco-2) with no inhibitory effects on the viability of human colon epithelial normal cells (HCEC) after 48 h. Reduced cell viability can be explained by cell cycle arrest in the S-phase in which DNA replication normally takes place. EYGF-33 significantly enhanced the production of superoxide anions in the mitochondria of Caco-2 cells; this could activate a mitochondrial apoptotic pathway leading to typical Poly Adenosine diphosphate-ribose polymerase (PARP) cleavage as observed in the Western blot result. The induction of apoptotic cell death by EYGF-33 was supported by the externalization of phosphatidylserine (PS). However, further elucidation of the mechanism of EYGF-33-mediated apoptosis would provide further support for its use as a potential therapeutic and chemopreventive agent.

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

PubMed Central

Beaumont, Kimberley A.; Anfosso, Andrea; Ahmed, Farzana

2015-01-01

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions. PMID:26779761

Notch3-specific inhibition using siRNA knockdown or GSI sensitizes paclitaxel-resistant ovarian cancer cells.

PubMed

Kang, Haeyoun; Jeong, Ju-Yeon; Song, Ji-Ye; Kim, Tae Heon; Kim, Gwangil; Huh, Jin Hyung; Kwon, Ah-Young; Jung, Sang Geun; An, Hee Jung

2016-07-01

Notch signaling plays an important role in ovarian cancer chemoresistance, which is responsible for recurrence. Gamma-secretase inhibitor (GSI) is a broad-spectrum Notch inhibitor, but it has serious side effects. The efficacy of Notch3-specific inhibition in paclitaxel-resistant ovarian cancers was assessed in this study, which has not yet been evaluated relative to GSI. To analyze the effect of Notch3-specific inhibition on paclitaxel-resistant ovarian cancers, we compared cell viability, apoptosis, cell migration, angiogenesis, cell cycle, and spheroid formation after treatment with either Notch3 siRNA or GSI in paclitaxel-resistant SKpac cells and parental SKOV3 cells. Expression levels of survival, cell cycle, and apoptosis-related proteins were measured and compared between groups. Notch3 was significantly overexpressed in chemoresistant cancer tissues and cell lines relative to chemosensitive group. In paclitaxel-resistant cancer cells, Notch inhibition significantly reduced viability, migration, and angiogenesis and increased apoptosis, thereby boosting sensitivity to paclitaxel. Spheroid formation was also significantly reduced. Both Notch3 siRNA-treated cells and GSI-treated cells arrested in the G2/M phase of the cell cycle. Proteins of cell survival, cyclin D1 and cyclin D3 were reduced, whereas p21 and p27 were elevated. Both GSI and Notch3 siRNA treatment reduced expression of anti-apoptotic proteins (BCL-W, BCL2, and BCL-XL) and increased expression of pro-apoptotic proteins (Bad, Bak, Bim, Bid, and Bax). These results indicate that Notch3-specific inhibition sensitizes paclitaxel-resistant cancer cells to paclitaxel treatment, with an efficacy comparable to that of GSI. This approach would be likely to avoid the side effects of broad-spectrum GSI treatment. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-replication

DTIC Science & Technology

2006-04-01

replication in yeast cells. In the prior reporting period we demonstrated that re-replication induces a rapid and significant decrease in cell viability...repair, DNA replication, checkpoint, cell cycle, yeast , RAD9 16. SECURITY CLASSIFICATION OF: 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON...initiation, our laboratory has been able to conditionally induce varying amounts of re- replication in yeast cells. Effectively, cells enter, but do not

The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for neuroendocrine tumors: Results from multiple cell line models

PubMed Central

Rentsch, Jakob; Freitag, Helma; Detjen, Katharina; Briest, Franziska; Möbs, Markus; Weissmann, Victoria; Siegmund, Britta; Auernhammer, Christoph J.; Aristizabal Prada, Elke Tatjana; Lauseker, Michael; Grossman, Ashley; Exner, Samantha; Fischer, Christian; Grötzinger, Carsten

2017-01-01

Background/Aims The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110α inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines. Methods Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA. Results BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately. Conclusion Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus. PMID:28800359

The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for neuroendocrine tumors: Results from multiple cell line models.

PubMed

Nölting, Svenja; Rentsch, Jakob; Freitag, Helma; Detjen, Katharina; Briest, Franziska; Möbs, Markus; Weissmann, Victoria; Siegmund, Britta; Auernhammer, Christoph J; Aristizabal Prada, Elke Tatjana; Lauseker, Michael; Grossman, Ashley; Exner, Samantha; Fischer, Christian; Grötzinger, Carsten; Schrader, Jörg; Grabowski, Patricia

2017-01-01

The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110α inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines. Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA. BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately. Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus.

Molecular effects of the phosphatidylinositol-3-kinase inhibitor NVP-BKM120 on T and B-cell acute lymphoblastic leukaemia.

PubMed

Pereira, João Kleber Novais; Machado-Neto, João Agostinho; Lopes, Matheus Rodrigues; Morini, Beatriz Corey; Traina, Fabiola; Costa, Fernando Ferreira; Saad, Sara Teresinha Olalla; Favaro, Patricia

2015-09-01

Constitutive activation of the PI3K pathway in T cell acute lymphoblastic leukaemia (T-ALL) has been reported and in a mouse model, PI3K activation, together with MYC, cooperates in Burkitt lymphoma (BL) pathogenesis. We investigated the effects of NVP-BKM120, a potent pan-class I PI3K inhibitor, in lymphoblastic leukaemia cell lines. Effects of NVP-BKM120 on cell viability, clonogenicity, apoptosis, cell cycle, cell signalling and autophagy were assessed in vitro on T-ALL (Jurkat and MOLT-4) and BL (Daudi and NAMALWA) cell lines. NVP-BKM120 treatment decreased cell viability and clonogenic growth in all tested cells. Moreover, the drug arrested cell cycling in association with a decrease in Cyclin B1 protein levels, and increased apoptosis. Immunoblotting analysis of cells treated with the drug revealed decreased phosphorylation, in a dose-dependent manner, of AKT, mTOR, P70S6K and 4EBP1, with stable total protein levels. Additionally, we observed a dose-dependent decrease in BAD phosphorylation, in association with augmented BAX:BCL2 ratio. Quantification of autophagy showed a dose-dependent increase in acidic vesicular organelles in all cells tested. In summary, our present study establishes that NVP-BKM120 presents an effective antitumour activity against T-ALL and BL cell lines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Knockdown of OY-TES-1 by RNAi causes cell cycle arrest and migration decrease in bone marrow-derived mesenchymal stem cells.

PubMed

Cen, Yan-Hui; Guo, Wen-Wen; Luo, Bin; Lin, Yong-Da; Zhang, Qing-Mei; Zhou, Su-Fang; Luo, Guo-Rong; Xiao, Shao-Wen; Xie, Xiao-Xun

2012-10-01

OY-TES-1 is a member of the CTA (cancer-testis antigen) group expressed in a variety of cancer and restrictedly expressed in adult normal tissues, except for testis. To determine whether MSCs (mesenchymal stem cells) express OY-TES-1 and its possible roles on MSCs, OY-TES-1 expression in MSCs isolated from human bone marrow was tested with RT (reverse transcription)-PCR, immunocytochemistry and Western blot. Using RNAi (RNA interference) technology, OY-TES-1 expression was knocked down followed by analysing cell viability, cell cycle, apoptosis and migration ability. MSCs expressed OY-TES-1 at both mRNA and protein levels. The down-regulation of OY-TES-1 expression in these MSCs caused cell growth inhibition, cell cycle arrest, apoptosis induction and migration ability attenuation. Through these primary results it was suggested that OY-TES-1 may influence the biological behaviour of MSCs.

The BMI1 inhibitor PTC-209 is a potential compound to halt cellular growth in biliary tract cancer cells

PubMed Central

Mayr, Christian; Wagner, Andrej; Loeffelberger, Magdalena; Bruckner, Daniela; Jakab, Martin; Berr, Frieder; Di Fazio, Pietro; Ocker, Matthias; Neureiter, Daniel; Pichler, Martin; Kiesslich, Tobias

2016-01-01

BMI1 is a core component of the polycomb repressive complex 1 (PRC1) and is up-regulated in biliary tract cancer (BTC), contributing to aggressive clinical features. In this study we investigated the cytotoxic effects of PTC-209, a recently developed inhibitor of BMI1, in BTC cells. PTC-209 reduced overall viability in BTC cell lines in a dose-dependent fashion (0.04 - 20 μM). Treatment with PTC-209 led to slightly enhanced caspase activity and stop of cell proliferation. Cell cycle analysis revealed that PTC-209 caused cell cycle arrest at the G1/S checkpoint. A comprehensive investigation of expression changes of cell cycle-related genes showed that PTC-209 caused significant down-regulation of cell cycle-promoting genes as well as of genes that contribute to DNA synthesis initiation and DNA repair, respectively. This was accompanied by significantly elevated mRNA levels of cell cycle inhibitors. In addition, PTC-209 reduced sphere formation and, in a cell line-dependent manner, aldehyde dehydrogease-1 positive cells. We conclude that PTC-209 might be a promising drug for future in vitro and in vivo studies in BTC. PMID:26623561

Glucose-dependent growth arrest of leukemia cells by MCT1 inhibition: Feeding Warburg's sweet tooth and blocking acid export as an anticancer strategy.

PubMed

Pivovarova, Aleksandra I; MacGregor, Gordon G

2018-02-01

This study aims to investigate the utilization of The Warburg Effect, cancer's "sweet tooth" and natural greed for glucose to enhance the effect of monocarboxylate transporter inhibition on cellular acidification. By simulating hyperglycemia with high glucose we may increase the effectiveness of inhibition of lactate and proton export on the dysregulation of cell pH homeostasis causing cell death or disruption of growth in cancer cells. MCT1 and MCT4 expression was determined in MCF7 and K562 cell lines using RT-PCR. Cell viability, growth, intracellular pH and cell cycle analysis was measured in the cell lines grown in 5 mM and 25 mM glucose containing media in the presence and absence of the MCT1 inhibitor AR-C155858 (1 μM) and the NHE1 inhibitor cariporide (10 μM). The MCT1 inhibitor, AR-C155858 had minimal effect on the viability, growth and intracellular pH of MCT4 expressing MCF7 cells. AR-C155858 had no effect on the viability of the MCT1 expressing K562 cells, but decreased intracellular pH and cell proliferation, by a glucose-dependent mechanism. Inhibition of NHE1 on its own had a no effect on cell growth, but together with AR-C155858 showed an additive effect on inhibition of cell growth. In cancer cells that only express MCT1, increased glucose concentrations in the presence of an MCT1 inhibitor decreased intracellular pH and reduced cell growth by G1 phase cell-cycle arrest. Thus we propose a transient hyperglycemic-clamp in combination with proton export inhibitors be evaluated as an adjunct to cancer treatment in clinical studies. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The triterpenoids of Hibiscus syriacus induce apoptosis and inhibit cell migration in breast cancer cells.

PubMed

Hsu, Ren-Jun; Hsu, Yao-Chin; Chen, Shu-Pin; Fu, Chia-Lynn; Yu, Jyh-Cherng; Chang, Fung-Wei; Chen, Ying-Hsin; Liu, Jui-Ming; Ho, Jar-Yi; Yu, Cheng-Ping

2015-03-14

Breast cancer-related mortality increases annually. The efficacy of current breast cancer treatments is limited, and they have numerous side effects and permit high recurrence. Patients with estrogen receptor (ER)-negative or triple-negative breast cancer are particularly difficult to treat. Treatment for this type of cancer is lacking, and its prognosis is poor, necessitating the search for alternative treatments. This study screened Chinese herb Hibiscus syriacus extracts and identified a novel anti-cancer drug for patients with ER-negative breast cancer. The inhibitory effects on cell viability and migration were evaluated for each compound, and the molecular regulatory effects were evaluated on both mRNA and protein levels. We found several triterpenoids including betulin (K02) and its derivatives (K03, K04, and K06) from H. syriacus inhibited human triple-negative breast cancer cell viability and migration but revealed smaller cytotoxic effects on normal mammalian epithelial cells. Betulin and its derivatives induced apoptosis by activating apoptosis-related genes. In addition, they activated p21 expression, which induced cell cycle arrest in breast cancer cells. Betulin (K02) and betulinic acid (K06) had stronger inhibitory effects on cell viability and migration than K03 and K04. H. syriacus extracts might inhibit breast cancer cell viability and induce apoptosis by activating p53 family regulated pathways and inhibiting AKT activation. H. syriacus extracts may provide important insight into the development of novel alternative therapies for breast cancer.

VEGF improves survival of mesenchymal stem cells in infarcted hearts

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

Pons, Jennifer; Huang Yu; Arakawa-Hoyt, Janice

2008-11-14

Bone marrow-derived mesenchymal stem cells (MSC) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether vascular endothelial growth factor (VEGF) improve MSC viability in infracted hearts. We found long-term culture increased MSC-cellular stress: expressing more cell cycle inhibitors, p16{sup INK}, p21 and p19{sup ARF}. VEGF treatment reduced cellular stress, increased pro-survival factors, phosphorylated-Akt and Bcl-xL expression and cell proliferation. Co-injection of MSCs with VEGF to MI hearts increased cell engraftment and resulted in better improvement of cardiac function than that injected with MSCs ormore » VEGF alone. In conclusion, VEGF protects MSCs from culture-induce cellular stress and improves their viability in ischemic myocardium, which results in improvements of their therapeutic effect for the treatment of MI.« less

In vitro evidence of glucose-induced toxicity in GnRH secreting neurons: high glucose concentrations influence GnRH secretion, impair cell viability, and induce apoptosis in the GT1-1 neuronal cell line.

PubMed

Pal, Lubna; Chu, Hsiao-Pai; Shu, Jun; Topalli, Ilir; Santoro, Nanette; Karkanias, George

2007-10-01

To evaluate for direct toxic effects of high glucose concentrations on cellular physiology in GnRH secreting immortalized GT1-1 neurons. Prospective experimental design. In vitro experimental model using a cell culture system. GT1-1 cells were cultured in replicates in media with two different glucose concentrations (450 mg/dL and 100 mg/dL, respectively) for varying time intervals (24, 48, and 72 hours). Effects of glucose concentrations on GnRH secretion by the GT1-1 neurons were evaluated using a static culture model. Cell viability, cellular apoptosis, and cell cycle events in GT1-1 neurons maintained in two different glucose concentrations were assessed by flow cytometry (fluorescence-activated cell sorter) using Annexin V-PI staining. Adverse influences of high glucose concentrations on GnRH secretion and cell viability were noted in cultures maintained in high glucose concentration (450 mg/dL) culture medium for varying time intervals. A significantly higher percentage of cells maintained in high glucose concentration medium demonstrated evidence of apoptosis by a fluorescence-activated cell sorter. We provide in vitro evidence of glucose-induced cellular toxicity in GnRH secreting GT1-1 neurons. Significant alterations in GnRH secretion, reduced cell viability, and a higher percentage of apoptotic cells were observed in GT1-1 cells maintained in high (450 mg/dL) compared with low (100 mg/dL) glucose concentration culture medium.

Aloe vera inhibits proliferation of human breast and cervical cancer cells and acts synergistically with cisplatin.

PubMed

Hussain, Arif; Sharma, Chhavi; Khan, Saniyah; Shah, Kruti; Haque, Shafiul

2015-01-01

Many of the anti-cancer agents currently used have an origin in natural sources including plants. Aloe vera is one such plant being studied extensively for its diverse health benefits, including cancer prevention. In this study, the cytotoxic potential of Aloe vera crude extract (ACE) alone or in combination with cisplatin in human breast (MCF-7) and cervical (HeLa) cancer cells was studied by cell viability assay, nuclear morphological examination and cell cycle analysis. Effects were correlated with modulation of expression of genes involved in cell cycle regulation, apoptosis and drug metabolism by RT-PCR. Exposure of cells to ACE resulted in considerable loss of cell viability in a dose- and time-dependent fashion, which was found to be mediated by through the apoptotic pathway as evidenced by changes in the nuclear morphology and the distribution of cells in the different phases of the cell cycle. Interestingly, ACE did not have any significant cytotoxicity towards normal cells, thus placing it in the category of safe chemopreventive agent. Further, the effects were correlated with the downregulation of cyclin D1, CYP 1A1, CYP 1A2 and increased expression of bax and p21 in MCF-7 and HeLa cells. In addition, low dose combination of ACE and cisplatin showed a combination index less than 1, indicating synergistic growth inhibition compared to the agents applied individually. In conclusion, these results signify that Aloe vera may be an effective anti-neoplastic agent to inhibit cancer cell growth and increase the therapeutic efficacy of conventional drugs like cispolatin. Thus promoting the development of plant-derived therapeutic agents appears warranted for novel cancer treatment strategies.

The antiproliferative effect of indomethacin-loaded lipid-core nanocapsules in glioma cells is mediated by cell cycle regulation, differentiation, and the inhibition of survival pathways

PubMed Central

Bernardi, Andressa; Frozza, Rudimar L; Hoppe, Juliana B; Salbego, Christianne; Pohlmann, Adriana R; Battastini, Ana Maria O; Guterres, Sílvia S

2013-01-01

Despite recent advances in radiotherapy, chemotherapy, and surgical techniques, glioblastoma multiforme (GBM) prognosis remains dismal. There is an urgent need for new therapeutic strategies. Nanoparticles of biodegradable polymers for anticancer drug delivery have attracted intense interest in recent years because they can provide sustained, controlled, and targeted delivery. Here, we investigate the mechanisms involved in the antiproliferative effect of indomethacin-loaded lipid-core nanocapsules (IndOH-LNC) in glioma cells. IndOH-LNC were able to reduce cell viability by inducing apoptotic cell death in C6 and U138-MG glioma cell lines. Interestingly, IndOH-LNC did not affect the viability of primary astrocytes, suggesting that this formulation selectively targeted transformed cells. Mechanistically, IndOH-LNC induced inhibition of cell growth and cell-cycle arrest to be correlated with the inactivation of AKT and β-catenin and the activation of GSK-3β. IndOH-LNC also induced G0/G1 and/or G2/M phase arrest, which was accompanied by a decrease in the levels of cyclin D1, cyclin B1, pRb, and pcdc2 and an increase in the levels of Wee1 CDK inhibitor p21WAF1. Additionally, IndOH-LNC promoted GBM cell differentiation, observed as upregulation of glial fibrillary acidic protein (GFAP) protein and downregulation of nestin and CD133. Taken together, the crosstalk among antiproliferative effects, cell-cycle arrest, apoptosis, and cell differentiation should be considered when tailoring pharmacological interventions aimed at reducing glioma growth by using formulations with multiples targets, such as IndOH-LNC. PMID:23440594

HBX Protein-Induced Downregulation of microRNA-18a is Responsible for Upregulation of Connective Tissue Growth Factor in HBV Infection-Associated Hepatocarcinoma.

PubMed

Liu, Xiaomin; Zhang, Yingjian; Wang, Ping; Wang, Hongyun; Su, Huanhuan; Zhou, Xin; Zhang, Lamei

2016-07-16

BACKGROUND This study was designed to improve our understanding of the role of miR-18a and its target (connective tissue growth factor (CTGF), which are mediators in HBX-induced hepatocellular carcinoma (HCC). MATERIAL AND METHODS We first investigated the expression of several candidate microRNAs (miRNAs) reported to have been aberrantly expressed between HepG2 and HepG2.2.15, which is characterized by stable HBV infection, while the CTGF is identified as a target of miR-18a. Furthermore, the expression of CTGF evaluated in HepG2 was transfected with HBX, while the HepG2.2.15 was transfected with miR-18a and CTGF siRNA. We examined the cell cycle at the same time. RESULTS We found that the expression of miR-18a was abnormally reduced in the HBV-positive HCC tissue samples compared with HBV-negative HCC samples. Through the use of a luciferase reporter system, we also identified CTGF 3'UTR (1046-1052 bp) as the exact binding site for miR-18a. We also observed a clear increase in CTGF mRNA and protein expression levels in HBV-positive HCC human tissue samples in comparison with the HBV-negative controls, indicating a possible negatively associated relationship between miR-18a and CTGF. Furthermore, we investigated the effect of HBX overexpression on miR-18a and CTGF, as well as the viability and cell cycle status of HepG2 cells. In addition, we found that HBX introduction downregulated miR-18a, upregulated CTGF, elevated the viability, and promoted cell cycle progression. We transfected HepG2.2.15 with miR-18a mimics and CTGF siRNA, finding that upregulated miR-18a and downregulated CTGF suppress the viability and cause cell cycle arrest. CONCLUSIONS Our study shows the role of the CTGF gene as a target of miR-18a, and identifies the function of HBV/HBX/miR-18a/CTGF as a key signaling pathway mediating HBV infection-induced HCC.

Tri-ortho-cresyl phosphate induces autophagy of rat spermatogonial stem cells.

PubMed

Liu, Meng-Ling; Wang, Jing-Lei; Wei, Jie; Xu, Lin-Lin; Yu, Mei; Liu, Xiao-Mei; Ruan, Wen-Li; Chen, Jia-Xiang

2015-02-01

Tri-ortho-cresyl phosphate (TOCP) has been widely used as plasticizers, plastic softeners, and flame retardants in industry and reported to have a deleterious effect on the male reproductive system in animals besides delayed neurotoxicity. Our preliminary results found that TOCP could disrupt the seminiferous epithelium in the testis and inhibit spermatogenesis, but the precise mechanism is yet to be elucidated. This study shows that TOCP inhibited viability of rat spermatogonial stem cells in a dose-dependent manner. TOCP could not lead to cell cycle arrest in the cells; the mRNA levels of p21, p27, p53, and cyclin D1 in the cells were also not affected by TOCP. Meanwhile, TOCP did not induce apoptosis of rat spermatogonial stem cells. After treatment with TOCP, however, both LC3-II and the ratio of LC3-II/LC3-I were markedly increased; autophagy proteins ATG5 and beclin 1 were also increased after treatment with TOCP, indicating that TOCP could induce autophagy in the cells. Ultrastructural observation under the transmission electron microscopy indicated that autophagic vesicles in the cytoplasm containing extensively degraded organelles such as mitochondria and endoplasmic reticulum increased significantly after the cells were treated with TOCP. In summary, we have shown that TOCP can inhibit viability of rat spermatogonial stem cells and induce autophagy of the cells, without affecting cell cycle and apoptosis. © 2015 Society for Reproduction and Fertility.

Knockdown of EphB1 receptor decreases medulloblastoma cell growth and migration and increases cellular radiosensitization

PubMed Central

Timofeeva, Olga; Pasquale, Elena B.; Hirsch, Kellen; MacDonald, Tobey J.; Dritschilo, Anatoly; Lee, Yi Chien; Henkemeyer, Mark; Rood, Brian; Jung, Mira; Wang, Xiao-Jing; Kool, Marcel

2015-01-01

The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target. PMID:25879388

Knockdown of EphB1 receptor decreases medulloblastoma cell growth and migration and increases cellular radiosensitization.

PubMed

Bhatia, Shilpa; Baig, Nimrah A; Timofeeva, Olga; Pasquale, Elena B; Hirsch, Kellen; MacDonald, Tobey J; Dritschilo, Anatoly; Lee, Yi Chien; Henkemeyer, Mark; Rood, Brian; Jung, Mira; Wang, Xiao-Jing; Kool, Marcel; Rodriguez, Olga; Albanese, Chris; Karam, Sana D

2015-04-20

The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target.

Microrna-199a-5p Functions as a Tumor Suppressor via Suppressing Connective Tissue Growth Factor (CTGF) in Follicular Thyroid Carcinoma.

PubMed

Sun, Dawei; Han, Shen; Liu, Chao; Zhou, Rui; Sun, Weihai; Zhang, Zhijun; Qu, Jianjun

2016-04-11

BACKGROUND The objective of this study was to explore the role of miR-199a-5p in the development of thyroid cancer, including its anti-proliferation effect and downstream signaling pathway. MATERIAL AND METHODS We conducted qRT-PCR analysis to detect the expressions of several microRNAs in 42 follicular thyroid carcinoma patients and 42 controls. We identified CTGF as target of miR-491, and viability and cell cycle status were determined in FTC-133 cells transfected with CTGF siRNA, miR-199a mimics, or inhibitors. RESULTS We identified an underexpression of miR-199a-5p in follicular thyroid carcinoma tissue samples compared with controls. Then we confirmed CTGF as a target of miR-199a-5p thyroid cells by using informatics analysis and luciferase reporter assay. Additionally, we found that mRNA and protein expression levels of CTGF were both clearly higher in malignant tissues than in benign tissues. miR-199a-5p mimics and CTGF siRNA similarly downregulated the expression of CTGF, and reduced the viability of FTC-133 cells by arresting the cell cycle in G0 phase. Transfection of miR-199a-5p inhibitors increased the expression of CTGF and promoted the viability of the cells by increasing the fraction of cells in G2/M and S phases. CONCLUSIONS Our study proves that the CTGF gene is a target of miR-199a-5p, demonstrating the negatively related association between CTGF and miR-199a. These findings suggest that miR-199a-5p might be a novel therapeutic target in the treatment of follicular thyroid carcinoma.

Curcumin inhibits growth potential by G1 cell cycle arrest and induces apoptosis in p53-mutated COLO 320DM human colon adenocarcinoma cells.

PubMed

Dasiram, Jade Dhananjay; Ganesan, Ramamoorthi; Kannan, Janani; Kotteeswaran, Venkatesan; Sivalingam, Nageswaran

2017-02-01

Curcumin, a natural polyphenolic compound and it is isolated from the rhizome of Curcuma longa, have been reported to possess anticancer effect against stage I and II colon cancer. However, the effect of curcumin on colon cancer at Dukes' type C metastatic stage III remains still unclear. In the present study, we have investigated the anticancer effects of curcumin on p53 mutated COLO 320DM human colon adenocarcinoma cells derived from Dukes' type C metastatic stage. The cellular viability and proliferation were assessed by trypan blue exclusion assay and MTT assay, respectively. The cytotoxicity effect was examined by lactate dehydrogenase (LDH) cytotoxicity assay. Apoptosis was analyzed by DNA fragmentation analysis, Hoechst and propidium iodide double fluorescent staining and confocal microscopy analysis. Cell cycle distribution was performed by flow cytometry analysis. Here we have observed that curcumin treatment significantly inhibited the cellular viability and proliferation potential of p53 mutated COLO 320DM cells in a dose- and time-dependent manner. In addition, curcumin treatment showed no cytotoxic effects to the COLO 320DM cells. DNA fragmentation analysis, Hoechst and propidium iodide double fluorescent staining and confocal microscopy analysis revealed that curcumin treatment induced apoptosis in COLO 320DM cells. Furthermore, curcumin caused cell cycle arrest at the G1 phase, decreased the cell population in the S phase and induced apoptosis in COLO 320DM colon adenocarcinoma cells. Together, these data suggest that curcumin exerts anticancer effects and induces apoptosis in p53 mutated COLO 320DM human colon adenocarcinoma cells derived from Dukes' type C metastatic stage. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Uncovering the Role of BMP Signaling in Melanocyte Development and Melanoma Tumorigenesis

DTIC Science & Technology

2015-06-01

Cell Cycle, one lecture (0.5hr), one discussion group (2hr) 2011, 2015 Stem Cell and Regenerative Biology. Co-coordinator, two lectures and 2011-2012...of GDF6 levels has profound effects on cell viability and tumorigenic potential. Task 2: Use established screening procedures in zebrafish to...Molecular Medicine and Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School Albert Sherman Center, AS6.1041

Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines.

PubMed

Wierzbicki, Piotr M; Kogut-Wierzbicka, Marzena; Ruczynski, Jaroslaw; Siedlecka-Kroplewska, Kamila; Kaszubowska, Lucyna; Rybarczyk, Agnieszka; Alenowicz, Magdalena; Rekowski, Piotr; Kmiec, Zbigniew

2014-01-01

Cell penetrating peptides (CPPs) have the ability to translocate through cell membranes with high efficiency and therefore can introduce biological agents with pharmaceutical properties into the cell. Transportan (TP) and its shorter analog transportan 10 (TP10) are among the best studied CPPs, however, their effects on viability of and cargo introduction into colorectal cancer (CRC) cells have yet not been investigated. The aim of our study was to evaluate the cytotoxic effects of TP and TP10 on representative CRC lines and the efficiency of protein (streptavidin) and siRNA cargo delivery by TP-biotinylated derivatives (TP-biot). HT29 (early stage CRC model) and HCT116 (metastatic CRC model) cell lines were incubated with TP, TP10, TP-biot1, TP-biot13 and TP10-biot1. The effects of studied CPPs on cell viability and cell cycle were assessed by MTT and annexin V assays. The uptake of streptavidin-FITC complex into cells was determined by flow cytometry and fluorescence microscopy, with the inhibition of cellular vesicle trafficking by brefeldin A. The efficiency of siRNA for SASH1 gene delivery was measured by quantitative PCR (qPCR). Since up to 10 µM concentrations of each CPP showed no significant cytotoxic effect, the concentrations of 0.5-5 µM were used for further analyses. Within this concentration range none of the studied CPPs affected cell viability and cell cycle. The efficient and endocytosis-independent introduction of streptavidin-FITC complex into cells was observed for TP10-biot1 and TP-biot1 with the cytoplasmic location of the fluorescent cargo; decreased SASH1 mRNA level was noticed with the use of siRNA and analyzed CPPs. We conclude that TP, TP10 and their biotinylated derivatives can be used as efficient delivery vehicles of small and large cargoes into CRC cells.

Interference of peritoneal dialysis fluids with cell cycle mechanisms.

PubMed

Büchel, Janine; Bartosova, Maria; Eich, Gwendolyn; Wittenberger, Timo; Klein-Hitpass, Ludger; Steppan, Sonja; Hackert, Thilo; Schaefer, Franz; Passlick-Deetjen, Jutta; Schmitt, Claus P

2015-01-01

Peritoneal dialysis fluids (PDF) differ with respect to osmotic and buffer compound, and pH and glucose degradation products (GDP) content. The impact on peritoneal membrane integrity is still insufficiently described. We assessed global genomic effects of PDF in primary human peritoneal mesothelial cells (PMC) by whole genome analyses, quantitative real-time polymerase chain reaction (RT-PCR) and functional measurements. PMC isolated from omentum of non-uremic patients were incubated with conventional single chamber PDF (CPDF), lactate- (LPDF), bicarbonate- (BPDF) and bicarbonate/lactate-buffered double-chamber PDF (BLPDF), icodextrin (IPDF) and amino acid PDF (APDF), diluted 1:1 with medium. Affymetrix GeneChip U133Plus2.0 (Affymetrix, CA, USA) and quantitative RT-PCR were applied; cell viability was assessed by proliferation assays. The number of differentially expressed genes compared to medium was 464 with APDF, 208 with CPDF, 169 with IPDF, 71 with LPDF, 45 with BPDF and 42 with BLPDF. Out of these genes 74%, 73%, 79%, 72%, 47% and 57% were downregulated. Gene Ontology (GO) term annotations mainly revealed associations with cell cycle (p = 10(-35)), cell division, mitosis, and DNA replication. One hundred and eighteen out of 249 probe sets detecting genes involved in cell cycle/division were suppressed, with APDF-treated PMC being affected the most regarding absolute number and degree, followed by CPDF and IPDF. Bicarbonate-containing PDF and BLPDF-treated PMC were affected the least. Quantitative RT-PCR measurements confirmed microarray findings for key cell cycle genes (CDK1/CCNB1/CCNE2/AURKA/KIF11/KIF14). Suppression was lowest for BPDF and BLPDF, they upregulated CCNE2 and SMC4. All PDF upregulated 3 out of 4 assessed cell cycle repressors (p53/BAX/p21). Cell viability scores confirmed gene expression results, being 79% of medium for LPDF, 101% for BLPDF, 51% for CPDF and 23% for IPDF. Amino acid-containing PDF (84%) incubated cells were as viable as BPDF (86%). In conclusion, PD solutions substantially differ with regard to their gene regulating profile and impact on vital functions of PMC, i.e. on cells known to be essential for peritoneal membrane homeostasis. Copyright © 2015 International Society for Peritoneal Dialysis.

Interference of Peritoneal Dialysis Fluids with Cell Cycle Mechanisms

PubMed Central

Büchel, Janine; Bartosova, Maria; Eich, Gwendolyn; Wittenberger, Timo; Klein-Hitpass, Ludger; Steppan, Sonja; Hackert, Thilo; Schaefer, Franz; Passlick-Deetjen, Jutta; Schmitt, Claus P.

2015-01-01

♦ Introduction: Peritoneal dialysis fluids (PDF) differ with respect to osmotic and buffer compound, and pH and glucose degradation products (GDP) content. The impact on peritoneal membrane integrity is still insufficiently described. We assessed global genomic effects of PDF in primary human peritoneal mesothelial cells (PMC) by whole genome analyses, quantitative real-time polymerase chain reaction (RT-PCR) and functional measurements. ♦ Methods: PMC isolated from omentum of non-uremic patients were incubated with conventional single chamber PDF (CPDF), lactate- (LPDF), bicarbonate- (BPDF) and bicarbonate/lactate-buffered double-chamber PDF (BLPDF), icodextrin (IPDF) and amino acid PDF (APDF), diluted 1:1 with medium. Affymetrix GeneChip U133Plus2.0 (Affymetrix, CA, USA) and quantitative RT-PCR were applied; cell viability was assessed by proliferation assays. ♦ Results: The number of differentially expressed genes compared to medium was 464 with APDF, 208 with CPDF, 169 with IPDF, 71 with LPDF, 45 with BPDF and 42 with BLPDF. Out of these genes 74%, 73%, 79%, 72%, 47% and 57% were downregulated. Gene Ontology (GO) term annotations mainly revealed associations with cell cycle (p = 10-35), cell division, mitosis, and DNA replication. One hundred and eighteen out of 249 probe sets detecting genes involved in cell cycle/division were suppressed, with APDF-treated PMC being affected the most regarding absolute number and degree, followed by CPDF and IPDF. Bicarbonate-containing PDF and BLPDF-treated PMC were affected the least. Quantitative RT-PCR measurements confirmed microarray findings for key cell cycle genes (CDK1/CCNB1/CCNE2/AURKA/KIF11/KIF14). Suppression was lowest for BPDF and BLPDF, they upregulated CCNE2 and SMC4. All PDF upregulated 3 out of 4 assessed cell cycle repressors (p53/BAX/p21). Cell viability scores confirmed gene expression results, being 79% of medium for LPDF, 101% for BLPDF, 51% for CPDF and 23% for IPDF. Amino acid-containing PDF (84%) incubated cells were as viable as BPDF (86%). ♦ Conclusion: In conclusion, PD solutions substantially differ with regard to their gene regulating profile and impact on vital functions of PMC, i.e. on cells known to be essential for peritoneal membrane homeostasis. PMID:25082841

An integrated system for synchronous culture of animal cells under controlled conditions.

PubMed

Mendoza-Pérez, Elena; Hernández, Vanessa; Palomares, Laura A; Serrato, José A

2016-01-01

The cell cycle has fundamental effects on cell cultures and their products. Tools to synchronize cultured cells allow the study of cellular physiology and metabolism at particular cell cycle phases. However, cells are most often arrested by methods that alter their homeostasis and are then cultivated in poorly controlled environments. Cell behavior could then be affected by the synchronization method and culture conditions used, and not just by the particular cell cycle phase under study. Moreover, only a few viable cells are recovered. Here, we designed an integrated system where a large number of cells from a controlled bioreactor culture is separated by centrifugal elutriation at high viabilities. In contrast to current elutriation methods, cells are injected directly from a bioreactor into an injection loop, allowing the introduction of a large number of cells into the separation chamber without stressful centrifugation. A low pulsation peristaltic pump increases the stability of the elutriation chamber. Using this approach, a large number of healthy cells at each cell cycle phase were obtained, allowing their direct inoculation into fully instrumented bioreactors. Hybridoma cells synchronized and cultured in this system behaved as expected for a synchronous culture.

Arctigenin anti-tumor activity in bladder cancer T24 cell line through induction of cell-cycle arrest and apoptosis.

PubMed

Yang, Shucai; Ma, Jing; Xiao, Jianbing; Lv, Xiaohong; Li, Xinlei; Yang, Huike; Liu, Ying; Feng, Sijia; Zhang, Yafang

2012-08-01

Bladder cancer is the most common neoplasm in the urinary system. This study assesses arctigenin anti-tumor activity in human bladder cancer T24 cells in vitro and the underlying molecular events. The flow cytometry analysis was used to detect cell-cycle distribution and apoptosis. Western blotting was used to detect changes in protein expression. The data showed that arctigenin treatment reduced viability of bladder cancer T24 cells in a dose- and time-dependent manner after treatment with arctigenin (10, 20, 40, 80, and 100 μmol/L) for 24 hr and 48 hr. Arctigenin treatment clearly arrested tumor cells in the G1 phase of the cell cycle. Apoptosis was detected by hoechst stain and flow cytometry after Annexin-V-FITC/PI double staining. Early and late apoptotic cells were accounted for 2.32-7.01% and 3.07-7.35%, respectively. At the molecular level, arctigenin treatment decreased cyclin D1 expression, whereas CDK4 and CDK6 expression levels were unaffected. Moreover, arctigenin selectively altered the phosphorylation of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 and activated phosphorylation of p38 significantly in a dose-dependent manner. These results suggest that arctigenin may inhibit cell viability and induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the anti-tumor effect of arctigenin. The data from the current study demonstrate the usefulness of arctigenin in bladder cancer T24 cells, which should further be evaluated in vivo before translation into clinical trials for the chemoprevention of bladder cancer. Copyright © 2012 Wiley Periodicals, Inc.

Anticancer activity of taraxerol acetate in human glioblastoma cells and a mouse xenograft model via induction of autophagy and apoptotic cell death, cell cycle arrest and inhibition of cell migration.

PubMed

Hong, Jing-Fang; Song, Ying-Fang; Liu, Zheng; Zheng, Zhao-Cong; Chen, Hong-Jie; Wang, Shou-Sen

2016-06-01

The aim of the present study was to investigate the in vitro and in vivo anticancer and apoptotic effects of taraxerol acetate in U87 human glioblastoma cells. The effects on cell cycle phase distribution, cell cycle-associated proteins, autophagy, DNA fragmentation and cell migration were assessed. Cell viability was determined using the MTT assay, and phase contrast and fluorescence microscopy was utilized to determine the viability and apoptotic morphological features of the U87 cells. Flow cytometry using propidium iodide and Annexin V-fluorescein isothiocyanate demonstrated the effect of taraxerol acetate on the cell cycle phase distribution and apoptosis induction. Western blot analysis was performed to investigate the effect of the taraxerol acetate on cell cycle‑associated proteins and autophagy‑linked LC3B‑II proteins. The results demonstrated that taraxerol acetate induced dose‑ and time‑dependent cytotoxic effects in the U87 cells. Apoptotic induction following taraxerol acetate treatment was observed and the percentage of apoptotic cells increased from 7.3% in the control cells, to 16.1, 44.1 and 76.7% in the 10, 50 and 150 µM taraxerol acetate‑treated cells, respectively. Furthermore, taraxerol acetate treatment led to sub‑G1 cell cycle arrest with a corresponding decrease in the number of S‑phase cells. DNA fragments were observed as a result of the gel electrophoresis experiment following taraxerol acetate treatment. To investigate the inhibitory effects of taraxerol acetate on the migration of U87 cell, a wound healing assay was conducted. The number of cells that migrated to the scratched area decreased significantly following treatment with taraxerol acetate. In addition, taraxerol acetate inhibited tumor growth in a mouse xenograft model. Administration of 0.25 and 0.75 µg/g taraxerol acetate reduced the tumor weight from 1.2 g in the phosphate‑buffered saline (PBS)‑treated group (control) to 0.81 and 0.42 g, respectively. Similarly, 0.25 and 0.75 µg/g taraxerol acetate injection reduced the tumor volume from 1.3 cm3 in the PBS-treated group (control) to 0.67 and 0.25 cm3, respectively.

A Model of Yeast Cell-Cycle Regulation Based on a Standard Component Modeling Strategy for Protein Regulatory Networks.

PubMed

Laomettachit, Teeraphan; Chen, Katherine C; Baumann, William T; Tyson, John J

2016-01-01

To understand the molecular mechanisms that regulate cell cycle progression in eukaryotes, a variety of mathematical modeling approaches have been employed, ranging from Boolean networks and differential equations to stochastic simulations. Each approach has its own characteristic strengths and weaknesses. In this paper, we propose a "standard component" modeling strategy that combines advantageous features of Boolean networks, differential equations and stochastic simulations in a framework that acknowledges the typical sorts of reactions found in protein regulatory networks. Applying this strategy to a comprehensive mechanism of the budding yeast cell cycle, we illustrate the potential value of standard component modeling. The deterministic version of our model reproduces the phenotypic properties of wild-type cells and of 125 mutant strains. The stochastic version of our model reproduces the cell-to-cell variability of wild-type cells and the partial viability of the CLB2-dbΔ clb5Δ mutant strain. Our simulations show that mathematical modeling with "standard components" can capture in quantitative detail many essential properties of cell cycle control in budding yeast.

A Model of Yeast Cell-Cycle Regulation Based on a Standard Component Modeling Strategy for Protein Regulatory Networks

PubMed Central

Laomettachit, Teeraphan; Chen, Katherine C.; Baumann, William T.

2016-01-01

To understand the molecular mechanisms that regulate cell cycle progression in eukaryotes, a variety of mathematical modeling approaches have been employed, ranging from Boolean networks and differential equations to stochastic simulations. Each approach has its own characteristic strengths and weaknesses. In this paper, we propose a “standard component” modeling strategy that combines advantageous features of Boolean networks, differential equations and stochastic simulations in a framework that acknowledges the typical sorts of reactions found in protein regulatory networks. Applying this strategy to a comprehensive mechanism of the budding yeast cell cycle, we illustrate the potential value of standard component modeling. The deterministic version of our model reproduces the phenotypic properties of wild-type cells and of 125 mutant strains. The stochastic version of our model reproduces the cell-to-cell variability of wild-type cells and the partial viability of the CLB2-dbΔ clb5Δ mutant strain. Our simulations show that mathematical modeling with “standard components” can capture in quantitative detail many essential properties of cell cycle control in budding yeast. PMID:27187804

Relevance of HCN2-expressing human mesenchymal stem cells for the generation of biological pacemakers.

PubMed

Bruzauskaite, Ieva; Bironaite, Daiva; Bagdonas, Edvardas; Skeberdis, Vytenis Arvydas; Denkovskij, Jaroslav; Tamulevicius, Tomas; Uvarovas, Valentinas; Bernotiene, Eiva

2016-04-30

The transfection of human mesenchymal stem cells (hMSCs) with the hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2) gene has been demonstrated to provide biological pacing in dogs with complete heart block. The mechanism appears to be the generation of the ion current (If) by the HCN2-expressing hMSCs. However, it is not clear how the transfection process and/or the HCN2 gene affect the growth functions of the hMSCs. Therefore, we investigated survival, proliferation, cell cycle, and growth on a Kapton® scaffold of HCN2-expressing hMSCs. hMSCs were isolated from the bone marrow of healthy volunteers applying a selective cell adhesion procedure and were identified by their expression of specific surface markers. Cells from passages 2-3 were transfected by electroporation using commercial transfection kits and a pIRES2-EGFP vector carrying the pacemaker gene, mouse HCN2 (mHCN2). Transfection efficiency was confirmed by enhanced green fluorescent protein (EGFP) fluorescence, quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). After hMSCs were transfected, their viability, proliferation, If generation, apoptosis, cell cycle, and expression of transcription factors were measured and compared with non-transfected cells and cells transfected with pIRES2-EGFP vector alone. Intracellular mHCN2 expression after transfection increased from 22.14 to 62.66 ng/mg protein (p < 0.05). Transfection efficiency was 45 ± 5 %. The viability of mHCN2-transfected cells was 82 ± 5 %; they grew stably for more than 3 weeks and induced If current. mHCN2-transfected cells had low mitotic activity (10.4 ± 1.24 % in G2/M and 83.6 ± 2.5 % in G1 phases) as compared with non-transfected cells (52-53 % in G2/M and 31-35 % in G1 phases). Transfected cells showed increased activation of nine cell cycle-regulating transcription factors: the most prominent upregulation was of AMP-dependent transcription factor ATF3 (7.11-fold, p = 0.00056) which regulates the G1 phase. mHCN2-expressing hMSCs were attached and made anchorage-dependent connection with other cells without transmigration through a 12.7-μm thick Kapton® HN film with micromachined 1-3 μm diameter pores. mHCN2-expressing hMSCs preserved the major cell functions required for the generation of biological pacemakers: high viability, functional activity, but low proliferation rate through the arrest of cell cycle in the G1 phase. mHCN2-expressing hMSCs attached and grew on a Kapton® scaffold without transmigration, confirming the relevance of these cells for the generation of biological pacemakers.

Mechanisms involved in the cytotoxic action of Brazilian propolis and caffeic acid against HEp-2 cells and modulation of P-glycoprotein activity.

PubMed

da Silva, Lívia M; Frión-Herrera, Yahima; Bartolomeu, Ariane R; Gorgulho, Carolina Mendonça; Sforcin, José M

2017-11-01

The effects of propolis and phenolic compounds (caffeic acid - Caf; dihydrocinnamic acid - Cin; p-coumaric acid - Cou) in the same quantity found in our propolis sample were investigated on human laryngeal epidermoid carcinoma (HEp-2) cells. Cell viability, apoptosis/necrosis and cell cycle arrest, P53 and CASPASE-3 gene expression, generation of reactive oxygen species (ROS) and the ability of propolis to induce doxorubicin (DOX) efflux using a P-glycoprotein (P-gp) inhibitor (verapamil) were assayed. Propolis exerted a cytotoxic effect on HEp-2 cells, whereas isolated compounds had no effect on cell viability. Higher concentrations were tested and Caf induced late apoptosis or necrosis in HEp-2 cells, while propolis induced apoptosis, both probably due to ROS generation. P53 expression was downregulated by propolis but not by Caf. CASPASE-3 expression was correlated with induction of both early and late apoptosis, with both propolis and Caf alone upregulating its expression. Propolis induced cell cycle arrest at G2/M phase and Caf at S phase. Propolis but not Caf may act as a P-gp inhibitor by modulating P-gp activity and inhibiting DOX efflux. Propolis exerted cytotoxic effects on HEp-2 cells, and the mechanisms are discussed, showing its potential as an antitumour drug. © 2017 Royal Pharmaceutical Society.

Effect of a cryopreservation protocol on the proliferation of stem cells from human exfoliated deciduous teeth.

PubMed

Ginani, Fernanda; Soares, Diego Moura; Rabêlo, Luciana Maria; Rocha, Hugo Alexandre Oliveira; de Souza, Lélia Batista; Barboza, Carlos Augusto Galvão

2016-11-01

The aim of the present study was to evaluate the influence of a cryopreservation protocol on the proliferation and viability of stem cells from human exfoliated deciduous teeth (SHEDs). Cells from the pulp of three deciduous teeth were isolated and characterized to confirm their stem cell nature. In second passage, part of the cells were submitted to normal conditions of cell culture (Control group), while part of the cells were maintained in 10% DMSO diluted in foetal bovine serum and submitted to the following cryopreservation protocol: 2 h at 4 °C, 18 h at -20 °C and then at -80 °C for two intervals (30 days - Cryopreservation I; and 180 days Cryopreservation II). Cell proliferation and cell cycle were evaluated at intervals of 24, 48 and 72 h after plating, and apoptosis-related events were analyzed at 72 h. All groups exhibited an increase in the number of cells, and no significant differences between the cryopreserved and control groups were observed (p > .05). The distribution of cells in the cell cycle phases was consistent with cell proliferation, and the percentage of viable cells was higher than 99% in all groups, indicating that cell viability was not affected by the cryopreservation protocol throughout the experiment. The proposed cryopreservation protocol is adequate for the storage of SHED, permitting their use in future experimental studies.

DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by Ruta graveolens in human colon cancer cells.

PubMed

Arora, Shagun; Tandon, Simran

2015-01-01

In the present study, we investigated the anti-cancer effect of various potencies of Ruta graveolens (Ruta) on COLO-205 cell line, as evidenced by cytotoxicity, migration, clonogenecity, morphological and biochemical changes and modification in the levels of genes associated with apoptosis and cell cycle. On treatment of COLO-205 cells maximal effects were seen with mother tincture (MT) and 30C potencies, wherein decrease in cell viability along with reduced clonogenecity and migration capabilities were noted. In addition morphological and biochemical alterations such as nuclear changes (fragmented nuclei with condensed chromatin) and DNA ladder-like pattern (increased amount of fragmented DNA) in COLO-205 cells indicating apoptotic related cell death were seen. The expression of apoptosis and cell-cycle related regulatory genes assessed by reverse transcriptase-PCR revealed an up-regulation of caspase 9, caspase-3, Bax, p21 and p27 expression and down-regulation of Bcl-2 expression in treated cells. The mode of cell death was suggestive of intrinsic apoptotic pathway along with cell cycle arrest at the G2/M of the cell cycle. Our findings indicate that phytochemicals present in Ruta showed potential for natural therapeutic product development for colon carcinoma. Copyright © 2014 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.

Low-level laser irradiation induces in vitro proliferation of stem cells from human exfoliated deciduous teeth.

PubMed

Ginani, Fernanda; Soares, Diego Moura; de Oliveira Rocha, Hugo Alexandre; de Souza, Lélia Batista; Barboza, Carlos Augusto Galvão

2018-01-01

The aim of this study was to evaluate the effect of low-level laser irradiation (LLLI) on the proliferation and viability of stem cells from human exfoliated deciduous teeth (SHED). Cells were irradiated or not (control) with an InGaAlP laser diode (660 nm, 30 mW, continuous action mode) using two different energy densities (0.5 J/cm 2 -16 s; 1.0 J/cm 2 -33 s). Irradiation was performed at 0 and 48 h, with the laser probe fixed at a distance of 0.5 cm from the cells. Cell proliferation was analyzed at 0, 24, 48, and 72 h by the Trypan blue exclusion method and MTT assay. Cell cycle and Ki67 expression were analyzed by flow cytometry. Apoptosis-related events were evaluated by expression of annexin V/PI and nuclear morphological changes by staining with DAPI. Differences between groups at each time were analyzed by the Kruskal-Wallis and Mann-Whitney tests, adopting a level of significance of 5% (p < 0.05). The results showed that an energy density of 1.0 J/cm 2 promoted an increase in cell proliferation at 48 and 72 h compared to the control and 0.5 J/cm 2 groups. Cell cycle analysis revealed a predominance of cells in the S and G2/M phases in the irradiated groups. This finding was confirmed by the increased expression of Ki67. Low positive staining for annexin V and PI was observed in all groups, and no nuclear changes were detected, indicating that cell viability was not affected by the energy densities tested. It can be concluded that the LLLI parameters used (660 nm, 30 mW, 1.0 J/cm 2 ) promote the proliferation of SHEDs and the maintenance of cell viability.

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

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

Ding, Li; College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158; Huang, Yong

2014-03-07

Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressedmore » cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.« less

Licoricidin inhibits the growth of SW480 human colorectal adenocarcinoma cells in vitro and in vivo by inducing cycle arrest, apoptosis and autophagy

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

Ji, Shuai

Licorice (Glycyrrhiza uralensis Fisch.) possesses significant anti-cancer activities, but the active ingredients and underlying mechanisms have not been revealed. By screening the cytotoxic activities of 122 licorice compounds against SW480 human colorectal adenocarcinoma cells, we found that licoricidin (LCD) inhibited SW480 cell viability with an IC{sub 50} value of 7.2 μM. Further studies indicated that LCD significantly induced G1/S cell cycle arrest and apoptosis in SW480 cells, accompanied by inhibition of cyclins/CDK1 expression and activation of caspase-dependent pro-apoptotic signaling. Meanwhile, LCD promoted autophagy in SW480 cells, and activated AMPK signaling and inhibited Akt/mTOR pathway. Overexpression of a dominant-negative AMPKα2 abolishedmore » LCD-induced inhibition of Akt/mTOR, autophagic and pro-apoptotic signaling pathways, and significantly reversed loss of cell viability, suggesting activation of AMPK is essential for the anti-cancer activity of LCD. In vivo anti-tumor experiments indicated that LCD (20 mg/kg, i.p.) significantly inhibited the growth of SW480 xenografts in nude mice with an inhibitory rate of 43.5%. In addition, we obtained the glycosylated product LCDG by microbial transformation, and found that glycosylation slightly enhanced the in vivo anti-cancer activities of LCD. This study indicates that LCD could inhibit SW480 cells by inducing cycle arrest, apoptosis and autophagy, and is a potential chemopreventive or chemotherapeutic agent against colorectal cancer. - Highlights: • Molecular mechanisms for cytotoxic activity of licoricidin (LCD) were investigated. • LCD promoted autophagy of SW480 cells through AMPK and Akt/mTOR signaling pathways. • Both LCD and its glucoside showed in vivo anti-colorectal cancer activities.« less

Live cell imaging reveals marked variability in myoblast proliferation and fate

PubMed Central

2013-01-01

Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

The seleno-organic compound ebselen impairs mitochondrial physiology and induces cell death in AR42J cells.

PubMed

Santofimia-Castaño, Patricia; Garcia-Sanchez, Lourdes; Ruy, Deborah Clea; Fernandez-Bermejo, Miguel; Salido, Gines M; Gonzalez, Antonio

2014-09-17

Ebselen is a seleno-organic compound that causes cell death in several cancer cell types. The mechanisms underlying its deleterious effects have not been fully elucidated. In this study, the effects of ebselen (1 μM-40 μM) on AR42J tumor cells have been examined. Cell viability was studied using AlamarBlue(®) test. Cell cycle phase determination was carried out by flow cytometry. Changes in intracellular free Ca(2+) concentration were followed by fluorimetry analysis of fura-2-loaded cells. Distribution of mitochondria, mitochondrial Ca(2+) concentration and mitochondrial membrane potential were monitored by confocal microscopy of cells loaded with Mitotracker Green™ FM, rhod-2 or TMRM respectively. Caspase-3 activity was calculated following the luorogenic substrate ACDEVD-AMC signal with a spectrofluorimeter. Results show that cell viability decreased in the presence of ebselen. An increase in the number of cells in the S-phase of the cell cycle was observed. Ebselen induced a concentration-dependent mobilization of Ca(2+) from agonist- and thapsigargin-sensitive Ca(2+) pools. Ebselen induced also a transient increase in mitochondrial Ca(2+) concentration, a progressive decrease of the mitochondrial membrane potential and a disruption of the mitochondrial network. Finally, a concentration-dependent increase in caspase-3 activity was detected. We conclude that ebselen exerts deleterious actions on the cells that involve the impairment of mitochondrial physiology and the activation of caspase-3-mediated apoptotic pathway. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

6-Gingerol inhibits osteosarcoma cell proliferation through apoptosis and AMPK activation.

PubMed

Fan, Jingzhang; Yang, Xin; Bi, Zhenggang

2015-02-01

6-Gingerol, a major component of ginger, is demonstrated to possess a variety of pharmacological activities. Despite demonstration of its anti-cancer activity, the exact mechanism underlying the effects of 6-gingerol against sarcoma remains sketchy. In the present study, we investigated the anti-cancer effects of 6-gingerol on osteosarcoma cells. MTT assay was performed to determine cell viability. Phosphorylation and protein levels were determined by immunoblotting. Cell cycle was determined using flow cytometry. Quantitative polymerase chain reaction was employed to determine the changes in the messenger RNA (mRNA) expression of genes. Treatment with 6-gingerol resulted in a significant decrease in the viability of osteosarcoma cells in a dose-dependent fashion. In parallel, the number of cells arrested at the sub-G1 cell cycle phase was significantly increased. The results showed that 6-gingerol induced activation of caspase cascades and regulated cellular levels of Bcl2 and Bax. Moreover, 6-gingerol activated AMP-activated protein kinase (AMPK) signaling associated with the apoptotic pathways. Our findings suggest that 6-gingerol suppresses the growth of osteosarcoma cells. The anti-cancer activity is attributed to the activation of apoptotic signaling and the inhibition of anti-apoptotic signaling incorporating with 6-gingerol-induced AMPK activation. The study identifies a new molecular mechanism by which AMPK is involved in anti-cancer effects of 6-gingerol.

An essential cell cycle regulation gene causes hybrid inviability in Drosophila

PubMed Central

Phadnis, Nitin; Baker, EmilyClare P.; Cooper, Jacob C.; Frizzell, Kimberly A.; Hsieh, Emily; de la Cruz, Aida Flor A.; Shendure, Jay; Kitzman, Jacob O.; Malik, Harmit S.

2015-01-01

Speciation, the process by which new biological species arise, involves the evolution of reproductive barriers such as hybrid sterility or inviability between populations. However, identifying hybrid incompatibility genes remains a key obstacle in understanding the molecular basis of reproductive isolation. We devised a genomic screen, which identified a cell cycle regulation gene as the cause of male inviability in hybrids between Drosophila melanogaster and D. simulans. Ablation of the D. simulans allele of this gene is sufficient to rescue the adult viability of hybrid males. This dominantly acting cell cycle regulator causes mitotic arrest and, thereby, inviability of male hybrid larvae. Our genomic method provides a facile means to accelerate the identification of hybrid incompatibility genes in other model and non-model systems. PMID:26680200

Comparative proteomics of two life cycle stages of stable isotope-labeled Trypanosoma brucei reveals novel components of the parasite's host adaptation machinery.

PubMed

Butter, Falk; Bucerius, Ferdinand; Michel, Margaux; Cicova, Zdenka; Mann, Matthias; Janzen, Christian J

2013-01-01

Trypanosoma brucei developed a sophisticated life cycle to adapt to different host environments. Although developmental differentiation of T. brucei has been the topic of intensive research for decades, the mechanisms responsible for adaptation to different host environments are not well understood. We developed stable isotope labeling by amino acids in cell culture in trypanosomes to compare the proteomes of two different life cycle stages. Quantitative comparison of 4364 protein groups identified many proteins previously not known to be stage-specifically expressed. The identification of stage-specific proteins helps to understand how parasites adapt to different hosts and provides new insights into differences in metabolism, gene regulation, and cell architecture. A DEAD-box RNA helicase, which is highly up-regulated in the bloodstream form of this parasite and which is essential for viability and proper cell cycle progression in this stage is described as an example.

Long non‑coding RNA AK001796 contributes to cisplatin resistance of non‑small cell lung cancer.

PubMed

Liu, Bin; Pan, Chun-Feng; Ma, Teng; Wang, Jun; Yao, Guo-Liang; Wei, Ke; Chen, Yi-Jiang

2017-10-01

Cisplatin (DDP)‑based chemotherapy is the most widely used therapy for non‑small cell lung cancer (NSCLC). However, the existence of chemoresistance has become a major limitation in its efficacy. Long non‑coding RNAs (lncRNAs) have been shown to be involved in chemotherapy drug resistance. The aim of the present study was to investigate the biological role of lncRNA AK001796 in cisplatin‑resistant NSCLC A549/DDP cells. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis was performed to monitor the differences in the expression of AK001796 in cisplatin-resistant (A549/DDP) cells and parental A549 cells. Cellular sensitivity to cisplatin and cell viability were examined using an MTT assay. Cell apoptosis and cell cycle distribution were measured using flow cytometry. The expression levels of cell cycle proteins cyclin C (CCNC), baculoviral IAP repeat containing 5 (BIRC5), cyclin‑dependent kinase 1 (CDK1) and G2 and S phase‑expressed 1 (GTSE1) were assessed using RT‑qPCR and western blot analyses. It was found that the expression of AK001796 was increased in A549/DDP cells, compared with that in A549 cells. The knockdown of AK001796 by small interfering RNA reduced cellular cisplatin resistance and cell viability, and resulted in cell‑cycle arrest, with a marked increase in the proportion of A549/DDP cells in the G0/G1 phase. By contrast, the knockdown of AK001796 increased the number of apoptotic cancer cells during cisplatin treatment. It was also shown that the knockdown of AK001796 positively induced the expression of cell apoptosis‑associated factors, CCNC and BIRC5, and suppressed the expression of cell cycle‑associated factors, CDK1 and GTSE5. Taken together, these findings indicated that lncRNA AK001796 increased the resistance of NSCLC cells to cisplatin through regulating cell apoptosis and cell proliferation, and thus provides an attractive therapeutic target for NSCLC.

Fungal-Induced Cell Cycle Impairment, Chromosome Instability and Apoptosis via Differential Activation of NF-κB

PubMed Central

Ben-Abdallah, Mariem; Sturny-Leclère, Aude; Avé, Patrick; Louise, Anne; Moyrand, Frédérique; Weih, Falk; Janbon, Guilhem; Mémet, Sylvie

2012-01-01

Microbial pathogens have developed efficient strategies to compromise host immune responses. Cryptococcus neoformans is a facultative intracellular pathogen, recognised as the most common cause of systemic fungal infections leading to severe meningoencephalitis, mainly in immunocompromised patients. This yeast is characterized by a polysaccharide capsule, which inhibits its phagocytosis. Whereas phagocytosis escape and macrophage intracellular survival have been intensively studied, extracellular survival of this yeast and restraint of host innate immune response are still poorly understood. In this study, we have investigated whether C. neoformans affected macrophage cell viability and whether NF-κB (nuclear factor-κB), a key regulator of cell growth, apoptosis and inflammation, was involved. Using wild-type (WT) as well as mutant strains of C. neoformans for the pathogen side, and WT and mutant cell lines with altered NF-κB activity or signalling as well as primary macrophages for the host side, we show that C. neoformans manipulated NF-κB-mediated signalling in a unique way to regulate macrophage cell fate and viability. On the one hand, serotype A strains reduced macrophage proliferation in a capsule-independent fashion. This growth decrease, which required a critical dosage of NF-κB activity, was caused by cell cycle disruption and aneuploidy, relying on fungal-induced modification of expression of several cell cycle checkpoint regulators in S and G2/M phases. On the other hand, C. neoformans infection induced macrophage apoptosis in a capsule-dependent manner with a differential requirement of the classical and alternative NF-κB signalling pathways, the latter one being essential. Together, these findings shed new light on fungal strategies to subvert host response through uncoupling of NF-κB activity in pathogen-controlled apoptosis and impairment of cell cycle progression. They also provide the first demonstration of induction of aneuploidy by a fungal pathogen, which may have wider implications for human health as aneuploidy is proposed to promote tumourigenesis. PMID:22396644

Triptolide abrogates growth of colon cancer and induces cell cycle arrest by inhibiting transcriptional activation of E2F.

PubMed

Oliveira, Amanda; Beyer, Georg; Chugh, Rohit; Skube, Steven J; Majumder, Kaustav; Banerjee, Sulagna; Sangwan, Veena; Li, Lihua; Dawra, Rajinder; Subramanian, Subbaya; Saluja, Ashok; Dudeja, Vikas

2015-06-01

Despite significant progress in diagnostics and therapeutics, over 50 thousand patients die from colorectal cancer annually. Hence, there is urgent need for new lines of treatment. Triptolide, a natural compound isolated from the Chinese herb Tripterygium wilfordii, is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer. Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of colorectal cancer. For mechanistic studies, colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate dehydrogenase release, and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F-dependent genes, E2F1- retinoblastoma protein (Rb) binding, and proteins levels of regulator of G1-S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay. Triptolide decreased colon cancer cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than doubles the median survival of animals with liver metastases from colon cancer. Mechanistically, we demonstrate that at low concentrations triptolide induces apoptotic cell death but at higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding. Therefore, we conclude that Triptolide and Minnelide are effective against colon cancer in multiple pre-clinical models.

Fungal-induced cell cycle impairment, chromosome instability and apoptosis via differential activation of NF-κB.

PubMed

Ben-Abdallah, Mariem; Sturny-Leclère, Aude; Avé, Patrick; Louise, Anne; Moyrand, Frédérique; Weih, Falk; Janbon, Guilhem; Mémet, Sylvie

2012-01-01

Microbial pathogens have developed efficient strategies to compromise host immune responses. Cryptococcus neoformans is a facultative intracellular pathogen, recognised as the most common cause of systemic fungal infections leading to severe meningoencephalitis, mainly in immunocompromised patients. This yeast is characterized by a polysaccharide capsule, which inhibits its phagocytosis. Whereas phagocytosis escape and macrophage intracellular survival have been intensively studied, extracellular survival of this yeast and restraint of host innate immune response are still poorly understood. In this study, we have investigated whether C. neoformans affected macrophage cell viability and whether NF-κB (nuclear factor-κB), a key regulator of cell growth, apoptosis and inflammation, was involved. Using wild-type (WT) as well as mutant strains of C. neoformans for the pathogen side, and WT and mutant cell lines with altered NF-κB activity or signalling as well as primary macrophages for the host side, we show that C. neoformans manipulated NF-κB-mediated signalling in a unique way to regulate macrophage cell fate and viability. On the one hand, serotype A strains reduced macrophage proliferation in a capsule-independent fashion. This growth decrease, which required a critical dosage of NF-κB activity, was caused by cell cycle disruption and aneuploidy, relying on fungal-induced modification of expression of several cell cycle checkpoint regulators in S and G2/M phases. On the other hand, C. neoformans infection induced macrophage apoptosis in a capsule-dependent manner with a differential requirement of the classical and alternative NF-κB signalling pathways, the latter one being essential. Together, these findings shed new light on fungal strategies to subvert host response through uncoupling of NF-κB activity in pathogen-controlled apoptosis and impairment of cell cycle progression. They also provide the first demonstration of induction of aneuploidy by a fungal pathogen, which may have wider implications for human health as aneuploidy is proposed to promote tumourigenesis.

Fasting inhibits hepatic stellate cells activation and potentiates anti-cancer activity of Sorafenib in hepatocellular cancer cells.

PubMed

Lo Re, Oriana; Panebianco, Concetta; Porto, Stefania; Cervi, Carlo; Rappa, Francesca; Di Biase, Stefano; Caraglia, Michele; Pazienza, Valerio; Vinciguerra, Manlio

2018-02-01

Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown. A 24 hr fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. A 24 hr fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation, and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib. Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells. Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use. © 2017 Wiley Periodicals, Inc.

Susceptibility of Hep3B cells in different phases of cell cycle to tBid.

PubMed

Ma, Shi-Hong; Chen, George G; Ye, Caiguo; Leung, Billy C S; Ho, Rocky L K; Lai, Paul B S

2011-01-01

tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC. Copyright © 2010 Elsevier B.V. All rights reserved.

Effects of the organophosphate insecticides phosmet and chlorpyrifos on trophoblast JEG-3 cell death, proliferation and inflammatory molecule production.

PubMed

Guiñazú, Natalia; Rena, Viviana; Genti-Raimondi, Susana; Rivero, Virginia; Magnarelli, Gladis

2012-04-01

Epidemiological data have associated environmental organophosphate insecticide (OP) exposure during pregnancy with fetal growth deficits. To better understand OP injury that may adversely affect pregnancy, we used the JEG-3 choriocarcinoma cell line, which provide a recognized in vitro model to study placental function. The effects of the OP phosmet (Pm) and chlorpyrifos (Cp) on JEG-3 cells viability, proliferation, cell cycle and inflammatory molecule production were evaluated. Both insecticides affected cellular viability in a concentration- and time-dependent manner, inducing apoptosis and decreasing [(3)H]-thymidine incorporation. However, only Pm reduced DNA synthesis independently of cellular death and decreased the cell percentage at the S-phase. Unlike apoptosis, TNFα production varied with the concentration tested, suggesting that other TNFα independent mechanisms might trigger cell death. No induction of the inflammatory molecule nitric oxide was detected. The mRNA levels of pro-inflammatory IL-6, IL-17 and the anti-inflammatory IL-13 cytokines were differentially modulated. These findings show that Pm and Cp generate a specific toxicity signature, altering cell viability and inducing an inflammatory cytokine profile, suggesting that trophoblasts may represent a possible target for OP adverse effects. Copyright © 2012 Elsevier Ltd. All rights reserved.

A novel live cell imaging system reveals a reversible hydrostatic pressure impact on cell cycle progression.

PubMed

Brooker, Holly R; Gyamfi, Irene A; Wieckowska, Agnieszka; Brooks, Nicholas J; Mulvihill, Daniel P; Geeves, Michael A

2018-06-21

Life is dependent upon the ability of a cell to rapidly respond to changes in environment. Small perturbations in local environments change the ability of molecules to interact and hence communicate. Hydrostatic pressure provides a rapid non-invasive, fully-reversible method for modulating affinities between molecules both in vivo and in vitro We have developed a simple fluorescence imaging chamber that allows intracellular protein dynamics and molecular events to be followed at pressures up to 200 bar in living cells. Using yeast we investigate the impact of hydrostatic pressure upon cell growth and cell cycle progression. While 100 bar has no affect upon viability, it induces a delay in chromosome segregation, resulting in the accumulation of long-undivided-bent cells, consistent with disruption of the cytoskeletons. This delay is independent of stress signalling and induces synchronisation of cell-cycle progression. Equivalent affects were observed in Candida albicans , with pressure inducing a reversible cell-cycle delay and hyphal growth. We present a simple novel non-invasive fluorescence microscopy based approach to transiently impact molecular dynamics to visualise, dissect and study signalling pathways and cellular processes in living cells. © 2018. Published by The Company of Biologists Ltd.

Effect of ellagic acid on proliferation, cell adhesion and apoptosis in SH-SY5Y human neuroblastoma cells.

PubMed

Fjaeraa, Christina; Nånberg, Eewa

2009-05-01

Ellagic acid, a polyphenolic compound found in berries, fruits and nuts, has been shown to possess growth-inhibiting and apoptosis promoting activities in cancer cell lines in vitro. The objective of this study was to investigate the effect of ellagic acid in human neuroblastoma SH-SY5Y cells. In cultures of SH-SY5Y cells incubated with ellagic acid, time- and concentration-dependent inhibitory effects on cell number were demonstrated. Ellagic acid induced cell detachment, decreased cell viability and induced apoptosis as measured by DNA strand breaks. Ellagic acid-induced alterations in cell cycle were also observed. Simultaneous treatment with all-trans retinoic acid did not rescue the cells from ellagic acid effects. Furthermore, the results suggested that pre-treatment with all-trans retinoic acid to induce differentiation and cell cycle arrest did not rescue the cells from ellagic acid-induced cell death.

Nelfinavir induces radiation sensitization in pituitary adenoma cells

PubMed Central

Zeng, Jing; See, Alfred P.; Aziz, Khaled; Thiyagarajan, Saravanan; Salih, Tarek; Gajula, Rajendra P.; Armour, Michael; Phallen, Jillian; Terezakis, Stephanie; Kleinberg, Lawrence; Redmond, Kristen; Hales, Russell K.; Salvatori, Roberto; Quinones-Hinojosa, Alfredo; Tran, Phuoc T.; Lim, Michael

2017-01-01

Pituitary adenomas with local invasion and high secretory activity remain a therapeutic challenge. The HIV protease inhibitor nelfinavir is a radiosensitizer in multiple tumor models. We tested nelfinavir as a radiosensitizer in pituitary adenoma cells in vitro and in vivo. We examined the effect of nelfinavir with radiation on in vitro cell viability, clonogenic survival, apoptosis, prolactin secretion, cell cycle distribution and the PI3K-AKT-mTOR pathway. We evaluated tumor growth delay and confirmed nelfinavir’s effect on the PI3K-AKT-mTOR pathway in a hind-flank model. Nelfinavir sensitized pituitary adenoma cells to ionizing radiation as shown by viability assays and clonogenic assay with an enhancement ratio of 1.2 (p < 0.05). There is increased apoptotic cell death, as determined by annexin-V expression and cleaved caspase-3 levels. Nelfinavir does not affect prolactin secretion or cell cycle distribution. In vivo, untreated tumors reached 4-fold volume in 12 d, 17 d with nelfinavir treatment, 27 d with radiation 6 Gy, and 41 d with nelfinavir plus radiation (one-way ANOVA p < 0.001). Decreased phospho-S6 on protein gel blotting in vitro and immunohistochemistry in vivo demonstrated nelfinavir inhibition of the PI3K-AKT-mTOR pathway. Our data suggests a promising combination therapy with nelfinavir plus radiation in pituitary adenomas, which should be investigated in clinical studies. PMID:21811091

Nelfinavir induces radiation sensitization in pituitary adenoma cells.

PubMed

Zeng, Jing; See, Alfred P; Aziz, Khaled; Thiyagarajan, Saravanan; Salih, Tarek; Gajula, Rajendra P; Armour, Michael; Phallen, Jillian; Terezakis, Stephanie; Kleinberg, Lawrence; Redmond, Kristen; Hales, Russell K; Salvatori, Roberto; Quinones-Hinojosa, Alfredo; Tran, Phuoc T; Lim, Michael

2011-10-01

Pituitary adenomas with local invasion and high secretory activity remain a therapeutic challenge. The HIV protease inhibitor nelfinavir is a radiosensitizer in multiple tumor models. We tested nelfinavir as a radiosensitizer in pituitary adenoma cells in vitro and in vivo. We examined the effect of nelfinavir with radiation on in vitro cell viability, clonogenic survival, apoptosis, prolactin secretion, cell cycle distribution, and the PI3K-AKT-mTOR pathway. We evaluated tumor growth delay and confirmed nelfinavir's effect on the PI3K-AKT-mTOR pathway in a hind-flank model. Nelfinavir sensitized pituitary adenoma cells to ionizing radiation as shown by viability assays and clonogenic assay with an enhancement ratio of 1.2 (p < 0.05). There is increased apoptotic cell death, as determined by annexin-V expression and cleaved caspase-3 levels. Nelfinavir does not affect prolactin secretion or cell cycle distribution. In vivo, untreated tumors reached 4-fold volume in 12 days, 17 days with nelfinavir treatment, 27 days with radiation 6 Gy, and 41 days with nelfinavir plus radiation (one-way ANOVA p < 0.001). Decreased phospho-S6 on Western blotting in vitro and immunohistochemistry in vivo demonstrated nelfinavir inhibition of the PI3K-AKT-mTOR pathway. Our data suggests a promising combination therapy with nelfinavir plus radiation in pituitary adenomas, which should be investigated in clinical studies.

Boron neutron capture therapy induces cell cycle arrest and cell apoptosis of glioma stem/progenitor cells in vitro.

PubMed

Sun, Ting; Zhang, Zizhu; Li, Bin; Chen, Guilin; Xie, Xueshun; Wei, Yongxin; Wu, Jie; Zhou, Youxin; Du, Ziwei

2013-08-06

Glioma stem cells in the quiescent state are resistant to clinical radiation therapy. An almost inevitable glioma recurrence is due to the persistence of these cells. The high linear energy transfer associated with boron neutron capture therapy (BNCT) could kill quiescent and proliferative cells. The present study aimed to evaluate the effects of BNCT on glioma stem/progenitor cells in vitro. The damage induced by BNCT was assessed using cell cycle progression, apoptotic cell ratio and apoptosis-associated proteins expression. The surviving fraction and cell viability of glioma stem/progenitor cells were decreased compared with differentiated glioma cells using the same boronophenylalanine pretreatment and the same dose of neutron flux. BNCT induced cell cycle arrest in the G2/M phase and cell apoptosis via the mitochondrial pathway, with changes in the expression of associated proteins. Glioma stem/progenitor cells, which are resistant to current clinical radiotherapy, could be effectively killed by BNCT in vitro via cell cycle arrest and apoptosis using a prolonged neutron irradiation, although radiosensitivity of glioma stem/progenitor cells was decreased compared with differentiated glioma cells when using the same dose of thermal neutron exposure and boronophenylalanine pretreatment. Thus, BNCT could offer an appreciable therapeutic advantage to prevent tumor recurrence, and may become a promising treatment in recurrent glioma.

Controlled initiation of chromosomal replication in Escherichia coli requires functional Hda protein.

PubMed

Camara, Johanna Eltz; Skarstad, Kirsten; Crooke, Elliott

2003-05-01

Regulatory inactivation of DnaA helps ensure that the Escherichia coli chromosome is replicated only once per cell cycle, through accelerated hydrolysis of active replication initiator ATP-DnaA to inactive ADP-DnaA. Analysis of deltahda strains revealed that the regulatory inactivation of DnaA component Hda is necessary for maintaining controlled initiation but not for cell growth or viability.

[Effect of shikonin, a phytocompound from Lithospermum erythrorhizon, on rat vascular smooth muscle cells proliferation and apoptosis in vitro].

PubMed

Zhang, Zhuo-qi; Cao, Xi-chuan; Zhang, Ling; Zhu, Wen-ling

2005-06-08

To study the anti-proliferation, pro-apoptosis and cell cycle blocking effects of shikonin on rat vascular smooth muscle cell (VSMC) in vitro. VSMCs were primarily cultured by explant method from the thoracic aorta of male SD rats. Shikonin of different concentration, 4, 2, 1, 0.5, 0.25, and 0 micromol/L was added. The cell viability was detected by MTT method. Cell growth curve was drawn by trypan blue exclusion method. (3)H-thymidine incorporation was used to calculate the inhibition rate of DNA synthesis. Flow cytometry was used to detect the cell cycle. Cell apoptosis was observed by fluorescence microscopy. Western blotting was performed to detect the expression of different cell apoptosis and cell cycle regulatory proteins, such as cyclin D(1) and E, proliferating cell nuclear antigen (PCNA), p21(waf1/cip1), p27(kip1), and p53. Compared with control group, shikonin had no obvious cytotoxic effect on cell viability at the concentration of 0.25-1 micromol/L (P > 0.05). While it could inhibit, both time- and dose-dependently, the growth of VSMC, which was predominant of 1 micromol/L at 72 h (1.9 x 10(5)/well vs 5.8 x 10(5)/well, P < 0.05), and DNA synthesis was also significantly inhibited in a time- and dose-dependent manner with inhibition rate varied from 33 to 98% (P < 0.05 or P < 0.01). 1 micromol/L shikonin significantly blocked the cell cycle progression in proliferative VSMC, decreased S, G(2)/M phase (P < 0.05) and increased G(0)/G(1) phase (P < 0.05) to quiescent level with sub-G(1) apoptotic distribution at 48 h (10.9% +/- 0.3%). Shikohin at the concentration of 1-2 micromol/L significantly increased the percentage of apoptotic cells in a time- and dose-dependent manner compared with control group (2.8%-23.7% vs 0.2%-0.4%, P < 0.05), and typical apoptotic nuclear morphological changes were observed. 1 micromol/L shikonin significantly down-regulated cyclin D(1), E and PCNA expression, up-regulated p21(wif1/cip1) expression, and did not obviously influence the p27(kip1) and p53 expression. Shikonin inhibits the proliferation, promotes the apoptosis and blocks cell cycle progression of VSMC. These effects are associated with the expression changes of cell cycle regulatory proteins.

DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-replication

DTIC Science & Technology

2007-04-01

Conway, A., Lockhart, D. J., Davis, R. W., Brewer , B. J., and Fangman, W. L. (2001). Replication dynamics of the yeast genome. Science 294, 115–121... Brewer , B. J. (2001). An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint. Mol. Cell 7, 705–713. Vas, A., Mok, W., and...replication in yeast cells. We have demonstrated that re-replication induces a rapid and significant decrease in cell viability and a cellular DNA damage

Induction of Apoptosis and Antiproliferative Activity of Naringenin in Human Epidermoid Carcinoma Cell through ROS Generation and Cell Cycle Arrest

PubMed Central

Jafri, Asif; Ahmad, Sheeba; Afzal, Mohammad; Arshad, Md

2014-01-01

A natural predominant flavanone naringenin, especially abundant in citrus fruits, has a wide range of pharmacological activities. The search for antiproliferative agents that reduce skin carcinoma is a task of great importance. The objective of this study was to analyze the anti-proliferative and apoptotic mechanism of naringenin using MTT assay, DNA fragmentation, nuclear condensation, change in mitochondrial membrane potential, cell cycle kinetics and caspase-3 as biomarkers and to investigate the ability to induce reactive oxygen species (ROS) initiating apoptotic cascade in human epidermoid carcinoma A431 cells. Results showed that naringenin exposure significantly reduced the cell viability of A431 cells (p<0.01) with a concomitant increase in nuclear condensation and DNA fragmentation in a dose dependent manner. The intracellular ROS generation assay showed statistically significant (p<0.001) dose-related increment in ROS production for naringenin. It also caused naringenin-mediated epidermoid carcinoma apoptosis by inducing mitochondrial depolarization. Cell cycle study showed that naringenin induced cell cycle arrest in G0/G1 phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis. This study confirms the efficacy of naringenin that lead to cell death in epidermoid carcinoma cells via inducing ROS generation, mitochondrial depolarization, nuclear condensation, DNA fragmentation, cell cycle arrest in G0/G1 phase and caspase-3 activation. PMID:25330158

MicroRNA-205 targets SMAD4 in non-small cell lung cancer and promotes lung cancer cell growth in vitro and in vivo.

PubMed

Zeng, Yuanyuan; Zhu, Jianjie; Shen, Dan; Qin, Hualong; Lei, Zhe; Li, Wei; Liu, Zeyi; Huang, Jian-An

2017-05-09

Despite advances in diagnosis and treatment, the survival of non-small cell lung cancer (NSCLC) patients remains poor; therefore, improved understanding of the disease mechanism and novel treatment strategies are needed. Downregulation of SMAD4 and dysregulated expression of miR-205 have been reported. However, the relationship between them remains unclear. We investigated the effect of microRNA (miR)-205 on the expression of SMAD4 in NSCLC. Knockdown and overexpression of SMAD4 promoted or suppressed cellular viability and proliferation, and accelerated or inhibited the cell cycle in NSCLC cells, respectively. The 3'-untranslated region (3'-UTR) of SMAD4 was predicted as a target of miR-205. Luciferase assays validated that miR-205 binds directly to the SMAD4 3'-UTR. Protein and mRNA expression analyses confirmed that miR-205 overexpression in NSCLC cells inhibited the expression of SMAD4 mRNA and protein. In human NSCLC tissues, increased miR-205 expression was observed frequently and was inversely correlated with decreased SMAD4 expression. Ectopic expression of miR-205 in NSCLC cells suppressed cellular viability and proliferation, accelerated the cell cycle, and promoted tumor growth of lung carcinoma xenografts in nude mice. Our study showed that miR-205 decreased SMAD4 expression, thus promoting NSCLC cell growth. Our findings highlighted the therapeutic potential of targeting miR-205 in NSCLC treatment.

MicroRNA-205 targets SMAD4 in non-small cell lung cancer and promotes lung cancer cell growth in vitro and in vivo

PubMed Central

Qin, Hualong; Lei, Zhe; Li, Wei; Liu, Zeyi; Huang, Jian-an

2017-01-01

Despite advances in diagnosis and treatment, the survival of non-small cell lung cancer (NSCLC) patients remains poor; therefore, improved understanding of the disease mechanism and novel treatment strategies are needed. Downregulation of SMAD4 and dysregulated expression of miR-205 have been reported. However, the relationship between them remains unclear. We investigated the effect of microRNA (miR)-205 on the expression of SMAD4 in NSCLC. Knockdown and overexpression of SMAD4 promoted or suppressed cellular viability and proliferation, and accelerated or inhibited the cell cycle in NSCLC cells, respectively. The 3′-untranslated region (3′-UTR) of SMAD4 was predicted as a target of miR-205. Luciferase assays validated that miR-205 binds directly to the SMAD4 3′-UTR. Protein and mRNA expression analyses confirmed that miR-205 overexpression in NSCLC cells inhibited the expression of SMAD4 mRNA and protein. In human NSCLC tissues, increased miR-205 expression was observed frequently and was inversely correlated with decreased SMAD4 expression. Ectopic expression of miR-205 in NSCLC cells suppressed cellular viability and proliferation, accelerated the cell cycle, and promoted tumor growth of lung carcinoma xenografts in nude mice. Our study showed that miR-205 decreased SMAD4 expression, thus promoting NSCLC cell growth. Our findings highlighted the therapeutic potential of targeting miR-205 in NSCLC treatment. PMID:28199217

Disinfection byproduct formation during biofiltration cycle: Implications for drinking water production.

PubMed

Delatolla, R; Séguin, C; Springthorpe, S; Gorman, E; Campbell, A; Douglas, I

2015-10-01

The goal of this study was to investigate the potential of biofiltration to reduce the formation potential of disinfection byproducts (DBPs). Particularly, the work investigates the effect of the duration of the filter cycle on the formation potential of total trihalomethanes (TTHM) and five species of haloacetic acids (HAA5), dissolved oxygen (DO), organic carbon, nitrogen and total phosphorous concentrations along with biofilm coverage of the filter media and biomass viability of the attached cells. The study was conducted on a full-scale biologically active filter, with anthracite and sand media, at the Britannia water treatment plant (WTP), located in Ottawa, Ontario, Canada. The formation potential of both TTHMs and HAA5s decreased due to biofiltration. However the lowest formation potentials for both groups of DBPs and or their precursors were observed immediately following a backwash event. Hence, the highest percent removal of DBPs was observed during the early stages of the biofiltration cycle, which suggests that a higher frequency of backwashing will reduce the formation of DBPs. Variable pressure scanning electron microscopy (VPSEM) analysis shows that biofilm coverage of anthracite and sand media increases as the filtration cycle progressed, while biomass viability analysis demonstrates that the percentage of cells attached to the anthracite and sand media also increases as the filtration cycle progresses. These results suggest that the development and growth of biofilm on the filters increases the DPB formation potential. Copyright © 2015 Elsevier Ltd. All rights reserved.

An essential cell cycle regulation gene causes hybrid inviability in Drosophila.

PubMed

Phadnis, Nitin; Baker, EmilyClare P; Cooper, Jacob C; Frizzell, Kimberly A; Hsieh, Emily; de la Cruz, Aida Flor A; Shendure, Jay; Kitzman, Jacob O; Malik, Harmit S

2015-12-18

Speciation, the process by which new biological species arise, involves the evolution of reproductive barriers, such as hybrid sterility or inviability between populations. However, identifying hybrid incompatibility genes remains a key obstacle in understanding the molecular basis of reproductive isolation. We devised a genomic screen, which identified a cell cycle-regulation gene as the cause of male inviability in hybrids resulting from a cross between Drosophila melanogaster and D. simulans. Ablation of the D. simulans allele of this gene is sufficient to rescue the adult viability of hybrid males. This dominantly acting cell cycle regulator causes mitotic arrest and, thereby, inviability of male hybrid larvae. Our genomic method provides a facile means to accelerate the identification of hybrid incompatibility genes in other model and nonmodel systems. Copyright © 2015, American Association for the Advancement of Science.

A Role for Cytosolic Fumarate Hydratase in Urea Cycle Metabolism and Renal Neoplasia

PubMed Central

Adam, Julie; Yang, Ming; Bauerschmidt, Christina; Kitagawa, Mitsuhiro; O’Flaherty, Linda; Maheswaran, Pratheesh; Özkan, Gizem; Sahgal, Natasha; Baban, Dilair; Kato, Keiko; Saito, Kaori; Iino, Keiko; Igarashi, Kaori; Stratford, Michael; Pugh, Christopher; Tennant, Daniel A.; Ludwig, Christian; Davies, Benjamin; Ratcliffe, Peter J.; El-Bahrawy, Mona; Ashrafian, Houman; Soga, Tomoyoshi; Pollard, Patrick J.

2013-01-01

Summary The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target. PMID:23643539

A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia.

PubMed

Adam, Julie; Yang, Ming; Bauerschmidt, Christina; Kitagawa, Mitsuhiro; O'Flaherty, Linda; Maheswaran, Pratheesh; Özkan, Gizem; Sahgal, Natasha; Baban, Dilair; Kato, Keiko; Saito, Kaori; Iino, Keiko; Igarashi, Kaori; Stratford, Michael; Pugh, Christopher; Tennant, Daniel A; Ludwig, Christian; Davies, Benjamin; Ratcliffe, Peter J; El-Bahrawy, Mona; Ashrafian, Houman; Soga, Tomoyoshi; Pollard, Patrick J

2013-05-30

The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Allium Roseum L. Extract Exerts Potent Suppressive Activities on Chronic Myeloid Leukemia K562 Cell Viability Through the Inhibition of BCR-ABL, PI3K/Akt, and ERK1/2 Pathways and the Abrogation of VEGF Secretion.

PubMed

Souid, Soumaya; Najjaa, Hanen; Riahi-Chebbi, Ichrak; Haoues, Meriam; Neffati, Mohamed; Arnault, Ingrid; Auger, Jacques; Karoui, Habib; Essafi, Makram; Essafi-Benkhadir, Khadija

2017-01-01

Use of plant extracts, alone or combined to the current chemotherapy as chemosensitizers, has emerged as a promising strategy to overcome tumor drug resistance. Here, we investigated the anticancer activity of Allium roseum L. extracts, a wild edible species in North Africa, on human Chronic Myeloid Leukemia (CML) K562 cells. The dehydrated aqueous extract (DAE) disturbed the cell cycle progression and induced the apoptosis of K562 cells. Chemical analysis of DAE showed a diversity of organosulfur compounds S-alk(en)yl-cysteine sulfoxides (RCSO) and high amount of allicin, suggesting that such molecule may be behind its antitumor effect. DAE was efficient in inhibiting K562 cell viability. DAE inhibitory effect was associated with the dephosphorylation of the BCR-ABL kinase and interfered with ERK 1/2 , Akt, and STAT5 pathways. Furthermore, we found that DAE-induced inactivation of Akt kinase led to the activation of its target FOXO3 transcription factor, enhancing the expression of FOXO3-regulated proapoptotic effectors, Bim and Bax, and cell cycle inhibitor p27. Finally, we found that DAE reduced the secretion of vascular endothelial growth factor. Overall, our data suggest that A. roseum extract has great potential as a nontoxic cheap and effective alternative to conventional chemotherapy.

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

Gabrielson, Marike; Reizer, Edwin; Stål, Olle

An increasing body of evidence is pointing towards mitochondrial regulation of the cell cycle. In a previous study of HER2-positive tumours we could demonstrate a common loss in the gene encoding for the mitochondrial transporter SLC25A43 and also a significant relation between SLC25A43 protein expression and S-phase fraction. Here, we investigated the consequence of suppressed SLC25A43 expression on cell cycle progression and proliferation in breast epithelial cells. In the present study, we suppressed SLC25A43 using siRNA in immortalised non-cancerous breast epithelial MCF10A cells and HER2-positive breast cancer cells BT-474. Viability, apoptosis, cell proliferation rate, cell cycle phase distribution, and nuclearmore » Ki-67 and p21, were assessed by flow cytometry. Cell cycle related gene expressions were analysed using real-time PCR. We found that SLC25A43 knockdown in MCF10A cells significantly inhibited cell cycle progression during G{sub 1}-to-S transition, thus significantly reducing the proliferation rate and fraction of Ki-67 positive MCF10A cells. In contrast, suppressed SLC25A43 expression in BT-474 cells resulted in a significantly increased proliferation rate together with an enhanced G{sub 1}-to-S transition. This was reflected by an increased fraction of Ki-67 positive cells and reduced level of nuclear p21. In line with our previous results, we show a role for SLC25A43 as a regulator of cell cycle progression and proliferation through a putative mitochondrial checkpoint. These novel data further strengthen the connection between mitochondrial function and the cell cycle, both in non-malignant and in cancer cells. - Highlights: • Proposed cell cycle regulation through the mitochondrial transporter SLC25A43. • SLC25A43 alters cell proliferation rate and cell cycle progression. • Suppressed SLC25A43 influences transcription of cell cycle regulatory genes.« less

Nur77 inhibits oxLDL induced apoptosis of macrophages via the p38 MAPK signaling pathway

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

Shao, Qin; Han, Fei; Peng, Shi

2016-03-18

The interaction between macrophages and oxLDL plays a crucial role in the initiation and progression of atherosclerosis. As a key initiator in a number of plaque promoting processes, oxLDL induces variable effects such as cell apoptosis or proliferation. Orphan nuclear receptor Nur77 is potently induced in macrophages by diverse stimuli, suggesting that it is of importance in vascular inflammation resulting in atherosclerosis, but whether Nur77 induction is detrimental or protective is unclear. In our study, we explore the role of Nur77 in the regulation of oxLDL-induced macrophage apoptosis and the signaling pathways that are involved. We found that oxLDL inducedmore » Nur77 expression in a dose and time dependent fashion, and cell viability was decreased in parallel. To determine whether Nur77 induction contributes to the loss of cell viability or is a protective mechanism, the effect of Nur77 overexpression was examined. Importantly, Nur77 overexpression inhibited the oxLDL-induced decrease of cell viability, inhibited the production of apoptotic bodies and restored DNA synthesis following oxLDL exposure. Furthermore, we found that Nur77 induction is mediated through the p38 MAPK signaling pathway. After pretreatment with SB203580, cell viability was decreased, the expression of CyclinA2 and PCNA was attenuated and the percentage of cell apoptosis was enhanced. Likewise, Nur77 overexpression increased the expression of the cell cycle genes PCNA and p21, and attenuated the increase in caspase-3. On the other hand, knockdown of Nur77 expression by specific siRNA resulted in the increased expression of caspase 3. The results demonstrate that Nur77 is induced by oxLDL via the p38 MAPK signaling pathway, which is involved in the regulation of cell survival. Nur77 enhanced cell survival via suppressing apoptosis, without affecting cell proliferation of activated macrophages, which may be beneficial in patients with atherosclerosis. - Highlights: • oxLDL could induce Nur77 expression. • Nur77 overexpression inhibited oxLDL-induced cell viability, production of apoptotic bodies and restored DNA synthesis. • Cell viability, CyclinA2 and PCNA expression and cell apoptosis were mediated through the p38 MAPK signaling pathway. • Nur77 overexpression mediated the expression of genes PCNA, p21, and caspase-3.« less

MicroRNA-130a-3p suppresses cell viability, proliferation and invasion in nasopharyngeal carcinoma by inhibiting CXCL12.

PubMed

Qu, Rongfeng; Sun, Yan; Li, Yarong; Hu, Chunmei; Shi, Guang; Tang, Yan; Guo, Dongrui

2017-01-01

Incidence of nasopharyngeal carcinoma (NPC) has remained high worldwide, posing a serious health problem. MicroRNAs (miRNAs) are a family of about 20-23 nucleotides small non-coding molecules, which play a significant role in NPC. In this study, we explored the molecular mechanisms of miR-130a-3p in inhibiting viability, proliferation, migration and invasion of NPC cells by suppressing CXCL12 . The relative expression of miR-130a-3p and CXCL12 mRNA expression in tissues and cells was measured by qRT-PCR. NPC cell line CNE-2Z was transfected with miR-130a-3p mimics, CXCL12 siRNA, cDNA- CXCL12 and negative control. Western Blot was performed to detect CXCL12 expression. The MTT assay was performed to study cell viability. The colony formation assay was done to test cell growth. Flow cytometry was conducted to analyze cell cycle and apoptosis. The Transwell assay was used to investigate cell migration and invasion. The results found that the up-regulation of miR-130a-3p or down-regulation of CXCL12 could inhibit viability, proliferation, migration and invasion of CNE-2Z cells. Luciferase-reporting system assay was performed to investigate miR-130a-3p could bind to the 3'UTR region of CXCL12 and the overexpression of miR-130a-3p could suppress CXCL12 expression. Collectively, our finding suggested demonstrated that miR-130a-3p could prohibit the progression of NPC by suppressing CXCL12 , which might serve as potential therapeutic targets for NPC.

MicroRNA-142-5p Overexpression Inhibits Cell Growth and Induces Apoptosis by Regulating FOXO in Hepatocellular Carcinoma Cells.

PubMed

Lou, Kexin; Chen, Ning; Li, Zhihong; Zhang, Bei; Wang, Xiuli; Chen, Ye; Xu, Haining; Wang, Dongwei; Wang, Hao

2017-01-02

Abnormal expression of microRNA (miR)-142-5p has been reported in hepatocellular carcinoma (HCC). However, little information is available regarding the functional role of miR-142-5p in HCC. We aimed to explore the effects of miR-142-5p aberrant expression on HCC cell growth and cell apoptosis, as well as the underlying mechanism. Human HCC cell lines HepG2 and SMMC-7721 cells were transfected with miR-142-5p mimic, inhibitor, or a corresponding negative control. Cell viability, cell cycle distribution, and cell apoptosis were then analyzed. In addition, protein expression of Forkhead box, class O (FOXO) 1 and 3, a Bcl-2-interacting mediator of cell death (Bim), procaspase 3, and activated caspase 3 was measured. After transfection with miR-142-5p inhibitor, FOXO1 and FOXO3 were overexpressed, and then the cell viability and cell apoptosis were determined again. The relative cell viability in both HepG2 and SMMC-7721 cells was significantly reduced by miR-142-5p overexpression (p < 0.05). miR-142-5p overexpression displayed a significant blockage at the G1/S transition and significantly increased the percentages of G0/G1 phase. Moreover, the results showed that miR-142-5p overexpression significantly induced cell apoptosis and statistically elevated the protein expression levels of FOXO1, FOXO3, Bim, procaspase 3, and activated caspase 3. However, the cells transfected with miR-142-5p inhibitor showed contrary results. Additionally, the effects of miR-142-5p inhibitor on cell viability and apoptosis were reversed by overexpression of FOXO. In conclusion, our results suggest that miR-142-5p overexpression shows an important protective role in HCC by inhibiting cell growth and inducing apoptosis. These effects might be by regulating FOXO expression in HCC cells.

Matrix-Dependent Regulation of AKT in Hepsin-Overexpressing PC3 Prostate Cancer Cells12

PubMed Central

Wittig-Blaich, Stephanie M; Kacprzyk, Lukasz A; Eismann, Thorsten; Bewerunge-Hudler, Melanie; Kruse, Petra; Winkler, Eva; Strauss, Wolfgang S L; Hibst, Raimund; Steiner, Rudolf; Schrader, Mark; Mertens, Daniel; Sültmann, Holger; Wittig, Rainer

2011-01-01

The serine-protease hepsin is one of the most prominently overexpressed genes in human prostate carcinoma. Forced expression of the enzyme in mice prostates is associated with matrix degradation, invasive growth, and prostate cancer progression. Conversely, hepsin overexpression in metastatic prostate cancer cell lines was reported to induce cell cycle arrest and reduction of invasive growth in vitro. We used a system for doxycycline (dox)-inducible target gene expression in metastasis-derived PC3 cells to analyze the effects of hepsin in a quantitative manner. Loss of viability and adhesion correlated with hepsin expression levels during anchorage-dependent but not anchorage-independent growth. Full expression of hepsin led to cell death and detachment and was specifically associated with reduced phosphorylation of AKT at Ser473, which was restored by growth on matrix derived from RWPE1 normal prostatic epithelial cells. In the chorioallantoic membrane xenograft model, hepsin overexpression in PC3 cells reduced the viability of tumors but did not suppress invasive growth. The data presented here provide evidence that elevated levels of hepsin interfere with cell adhesion and viability in the background of prostate cancer as well as other tissue types, the details of which depend on the microenvironment provided. Our findings suggest that overexpression of the enzyme in prostate carcinogenesis must be spatially and temporally restricted for the efficient development of tumors and metastases. PMID:21750652

Pirfenidone inhibits proliferation, arrests the cell cycle, and downregulates heat shock protein-47 and collagen type I in rat hepatic stellate cells in vitro.

PubMed

Xiang, Xian-Hong; Jiang, Tian-Peng; Zhang, Shuai; Song, Jie; Li, Xing; Yang, Jian-Yong; Zhou, Shi

2015-07-01

Pirfenidone (esbiret) is an established anti-fibrotic and anti-inflammatory drug used to treat idiopathic pulmonary fibrosis. In the present study, the dose-dependent effects of pirfenidone on the cell cycle, proliferation and expression of heat shock protein (HSP)-47 and collagen type I in a cultured rat hepatic stellate cell line (HSC-T6) were investigated. Following pirfenidone treatment, cell proliferation was determined using the cell counting kit-8 assay and the cell cycle was measured using flow cytometry. HSP-47 expression was estimated using western blot analysis and collagen type I mRNA was assessed using reverse transcription quantitative polymerase chain reaction. Pirfenidone induced significant dose-dependent inhibition of proliferation in HSC-T6 cells. Cell viability was unaffected by treatment with pirfenidone (0, 10 or 100 µM) for 24 and 72 h. However, after 24 h, HSC-T6 cells exhibited dose-dependent decreases in HSP-47 protein and collagen I mRNA levels. In conclusion, pirfenidone inhibited HSC-T6 cell proliferation, arrested the cell cycle and reduced the expression of HSP-47 and collagen type I, indicating that pirfenidone may be a promising drug in the treatment of liver fibrosis.

Cell cycle synchronization and analysis of apoptosis-related gene in skin fibroblasts from domestic cat (Felis silvestris catus) and kodkod (Leopardus guigna).

PubMed

Veraguas, D; Gallegos, P F; Castro, F O; Rodriguez-Alvarez, L

2017-10-01

The kodkod population is in constant decrease and the somatic cell nuclear transfer (SCNT) might help to preserve the genetic pool of this species. The cell cycle synchronization of donor cells plays a crucial role in SCNT. The objective of this research was to evaluate two different methods for quiescence induction, serum starvation (SS) and contact inhibition (CI), both for 1, 3 and 5 days, on skin fibroblast from domestic cat and kodkod. Flow cytometry analysis revealed that in domestic cat, SS and CI, both at 3 and 5 days, increased the percentage of fibroblasts in G0/G1 compared to growing cells (GC) (p < .05). In kodkod, only SS for 3 and 5 days and CI for 1 and 3 days increased the percentage of fibroblasts in G0/G1 compared to GC (p < .05). Viability analysis by differential staining revealed that SS for 5 days decreased the proportion of live fibroblasts in domestic cat and kodkod (p < .05). Regarding gene expression analysis, in domestic cat fibroblasts, no differences were found in the BAX/BCL2 ratio in SS and CI (both at 1, 3 and 5 days) compared to GC. In kodkod fibroblasts, BAX/BCL2 ratio was increased in CI at 3 and 5 days compared to SS at 3 and 5 days (p < .05). In conclusion, in kodkod fibroblasts SS for 5 days and CI after 3 days might have a negative impact on cellular viability. According to these results, we suggest SS for 3 days for cell cycle synchronization in kodkod fibroblasts. © 2017 Blackwell Verlag GmbH.

Bioethanol production from uncooked raw starch by immobilized surface-engineered yeast cells.

PubMed

Chen, Jyh-Ping; Wu, Kuo-Wei; Fukuda, Hideki

2008-03-01

Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.

Bioethanol Production from Uncooked Raw Starch by Immobilized Surface-engineered Yeast Cells

NASA Astrophysics Data System (ADS)

Chen, Jyh-Ping; Wu, Kuo-Wei; Fukuda, Hideki

Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.

Long noncoding RNA CCAT1 promotes cell proliferation and metastasis in human medulloblastoma via MAPK pathway.

PubMed

Gao, Ran; Zhang, Rui; Zhang, Cuicui; Zhao, Li; Zhang, Yue

2018-01-01

Medulloblastoma is the most common posterior fossa tumor in children and one that easily metastasizes. The mechanisms of how the medulloblastoma develops and progresses remain to be elucidated. The present study aimed to assess the role of long noncoding colon cancer-associated transcript-1 (lncRNA CCAT1) in cell proliferation and metastasis in human medulloblastoma. Levels of CCAT1 were measured in samples and cell lines of medulloblastoma. Cell cycle progression, cell viability assay, colony formation assay, wound-healing and Transwell assays Corning, Cambridge, MA, USA were used to investigate the viability and motility of cells. Western blot assay was used to investigate the levels of CCAT1 and other proteins. The initial findings indicated that CCAT1 was significantly up-regulated in clinical cancerous tissues and expressed differently in a series of medulloblastoma cell lines. CCAT1 knockdown significantly slowed cell proliferation rates and inhibited cell clonogenic potential in Daoy cells and D283 cells. Cell cycle progression was disrupted with cell proportions in the G0/G1 phase decreased and the proportion in the S phase and G2/M phases increased, in Daoy cells and D283 cells. Concordantly, medulloblastoma tumor cell growth rates were found to be impaired in xenotransplanted mice. After CCAT1 knockdown, cell wound recovery ability was significantly inhibited. Furthermore, the phosphorylated levels of MAPK, ERK and MEK, but not their total levels decreased after the down-regulation of CCAT1 in Daoy and D283 cells. Our results suggested that the lncRNA CCAT1 promotes cell proliferation and metastasis in human medulloblastoma by possibly regulating the MAPK pathway.

In vitro effects of histone deacetylase inhibitors and mitomycin C on tenon capsule fibroblasts and conjunctival melanoma cells.

PubMed

Cunneen, Thomas S; Conway, R Max; Madigan, Michele C

2009-04-01

To investigate the effects of mitomycin C and the histone deacetylase inhibitors sodium butyrate and trichostatin on the viability and growth of conjunctival melanoma cell lines and Tenon capsule fibroblasts. Cells were treated with a range of concentrations of sodium butyrate, trichostatin, and mitomycin C. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assays were performed 48 hours after treatment. Treated cells were stained with acridine orange/ethidium bromide to assess for cell death. Cell-cycle changes in histone deacetylase inhibitor-treated melanoma cells were quantified using flow cytometry. All agents induced dose-dependent cell death in the melanoma cell lines; however, sodium butyrate and trichostatin were relatively nontoxic to Tenon capsule fibroblasts. Acridine orange/ethidium bromide staining indicated that sodium butyrate and trichostatin induced apoptotic cell death. At low doses, sodium butyrate and trichostatin induced a G1 cell-cycle block in the melanoma cells. Sodium butyrate and trichostatin induced cell death in melanoma cells, comparable with mitomycin C, with minimal effect on Tenon capsule fibroblasts. In addition, they induced a G1 cell-cycle block. These findings support the need for further investigation into the in vivo efficacy of these agents.

Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting | Office of Cancer Genomics

Cancer.gov

The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest.

Pharmacologic inhibition of Pim kinases alters prostate cancer cell growth and resensitizes chemoresistant cells to taxanes.

PubMed

Mumenthaler, Shannon M; Ng, Patricia Y B; Hodge, Amanda; Bearss, David; Berk, Gregory; Kanekal, Sarath; Redkar, Sanjeev; Taverna, Pietro; Agus, David B; Jain, Anjali

2009-10-01

The serine/threonine family of Pim kinases function as oncogenes and have been implicated in prostate cancer progression, particularly in hormone-refractory prostate disease, as a result of their antiapoptotic function. In this study, we used a pharmacologic inhibitor targeting the Pim family members, SGI-1776, to determine whether modulation of Pim kinase activity could alter prostate cancer cell survival and modulate chemotherapy resistance. Extensive biochemical characterization of SGI-1776 confirmed its specificity for the three isoforms of the Pim family. Treatment of prostate cancer cells with SGI-1776 resulted in a dose-dependent reduction in phosphorylation of known Pim kinase substrates that are involved in cell cycle progression and apoptosis (p21(Cip1/WAF1) and Bad). Consequently, SGI-1776 compromised overall cell viability by inducing G(1) cell cycle arrest and triggering apoptosis. Overexpression of recombinant Pim-1 markedly increased sensitivity of SGI-1776-mediated prostate cancer cell apoptosis and p21(Cip1/WAF1) phosphorylation inhibition, reinforcing the specificity of SGI-1776. An additional cytotoxic effect was observed when SGI-1776 was combined with taxane-based chemotherapy agents. SGI-1776 was able to reduce cell viability in a multidrug resistance 1 protein-based taxane-refractory prostate cancer cell line. In addition, SGI-1776 treatment was able to resensitize chemoresistant cells to taxane-based therapies by inhibiting multidrug resistance 1 activity and inducing apoptosis. These findings support the idea that inhibiting Pim kinases, in combination with a chemotherapeutic agent, could play an important role in prostate cancer treatment by targeting the clinical problem of chemoresistance.

Cnidium officinale Makino extract induces apoptosis through activation of caspase-3 and p53 in human liver cancer HepG2 cells

PubMed Central

Hong, Heeok; An, Jeong Cheol; de La Cruz, Joseph F.; Hwang, Seong-Gu

2017-01-01

A number of diverse studies have reported the anticancer properties of Cnidium officinale Makino (CO). However, the apoptotic effect of this traditional medicinal herb in human hepatocellular carcinoma cells (HepG2) remains to be elucidated. Therefore, the present study investigated the ability of CO to reduce cell viability through apoptotic pathways. Cell viability was determined using the 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assay. CO extract-induced apoptosis in HepG2 cells was assessed by Hoechst 33258 staining. The cell cycle was monitored using fluorescence-activated cell sorting analysis with propidium iodide staining. Furthermore, the present study explored whether various signaling molecules associated with HepG2 cell death were affected by CO treatment, including caspase-3, B-cell lymphoma 2 (Bcl-2), tumor protein p53 (p53), cyclin-dependent kinase 4 (CDK4) and cyclin D. The expression levels of these genes were examined by reverse-transcription polymerase chain reaction and western blotting. The expression levels of caspase-3 and p53 were upregulated with CO extract treatment, whereas those of Bcl-2, CDK4 and cyclin D were significantly downregulated. Cleaved caspase-3 expression was upregulated following treatment with CO extract in a dose-dependent manner. Collectively, the data suggest that CO extract has the potential to induce apoptosis of HepG2 cells and may act by suppressing the cell cycle, which leads to caspase-3 cleavage and p53 signaling. PMID:28966688

Boron neutron capture therapy induces cell cycle arrest and cell apoptosis of glioma stem/progenitor cells in vitro

PubMed Central

2013-01-01

Background Glioma stem cells in the quiescent state are resistant to clinical radiation therapy. An almost inevitable glioma recurrence is due to the persistence of these cells. The high linear energy transfer associated with boron neutron capture therapy (BNCT) could kill quiescent and proliferative cells. Methods The present study aimed to evaluate the effects of BNCT on glioma stem/progenitor cells in vitro. The damage induced by BNCT was assessed using cell cycle progression, apoptotic cell ratio and apoptosis-associated proteins expression. Results The surviving fraction and cell viability of glioma stem/progenitor cells were decreased compared with differentiated glioma cells using the same boronophenylalanine pretreatment and the same dose of neutron flux. BNCT induced cell cycle arrest in the G2/M phase and cell apoptosis via the mitochondrial pathway, with changes in the expression of associated proteins. Conclusions Glioma stem/progenitor cells, which are resistant to current clinical radiotherapy, could be effectively killed by BNCT in vitro via cell cycle arrest and apoptosis using a prolonged neutron irradiation, although radiosensitivity of glioma stem/progenitor cells was decreased compared with differentiated glioma cells when using the same dose of thermal neutron exposure and boronophenylalanine pretreatment. Thus, BNCT could offer an appreciable therapeutic advantage to prevent tumor recurrence, and may become a promising treatment in recurrent glioma. PMID:23915425

BMI-1 suppression increases the radiosensitivity of oesophageal carcinoma via the PI3K/Akt signaling pathway.

PubMed

Yang, Xing-Xiao; Ma, Ming; Sang, Mei-Xiang; Zhang, Xue-Yuan; Liu, Zhi-Kun; Song, Heng; Zhu, Shu-Chai

2018-02-01

B-cell‑specific Moloney murine leukaemia virus integration site-1 (BMI-1) contributes to the growth of tumour cells post-irradiation (IR). The aim of the present study was to characterize the effects of BMI-1 on cell viability, radiosensitivity and its mechanisms of action in oesophageal squamous cell cancer (ESCC). Western blotting and immunohistochemistry were employed to evaluate the protein expression of BMI-1 in ESCC cells and specimens, respectively. Additionally, the protein expression levels of BMI-1, H2AK119ub and γH2AX in ESCC cells were detected following different doses of IR and at different times after IR. The protein expression levels of MDC1 and 53BP1 were also measured. Flow cytometry and MTT assays were used to determine cell cycle progression, apoptosis and cell viability. The phosphatidylinositol 3-kinase inhibitor LY294002 and the agonist IGF-1 were employed to suppress or induce the phosphorylation of Akt to determine whether BMI-1 induces radioresistance in ESCC cells via activation of the PI3K/Akt pathway. The expression of BMI-1 was higher in ESCC tissues and cells compared with that in normal oesophageal tissues and cells. In addition, BMI-1 was positively related to tumour size and lymph node metastases and negatively to the overall survival of ESCC patients. IR induced the expression of BMI-1, H2AK119ub and γH2AX in a dose- and time-dependent manner. BMI-1 knockdown lowered the expression of γH2AX, MDC1 and 53BP1, suppressed cell viability and increased radiosensitivity. G2/M phase arrest was eliminated; this was followed by an increased proportion of cells entering the G0/G1 phase after IR and BMI-1 knockdown via the upregulation of P16 and downregulation of cyclin D2 and cyclin-dependent kinase-4. Moreover, BMI-1 knockdown increased cell apoptosis, downregulated MCL-1 and p-Akt and upregulated Bax. Additionally, the inhibitory effect of the downregulation of p-Akt by LY294002 on tumour cell viability was identical to that of BMI-1 knockdown, while the kinase agonist IGF-1 reversed the effects of BMI-1 knockdown on cell viability and radiosensitivity. Taken together, BMI-1 knockdown induces radiosensitivity in ESCC and significantly inhibits cell viability, which may contribute to an increased proportion of cells in the G0/G1 phase and cell apoptosis via suppression of the PI3K/Akt signalling pathway.

Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationally in cell cycle arrest after DNA damage.

PubMed

Weinert, T A; Hartwell, L H

1990-12-01

In eucaryotic cells, incompletely replicated or damaged chromosomes induce cell cycle arrest in G2 before mitosis, and in the yeast Saccharomyces cerevisiae the RAD9 gene is essential for the cell cycle arrest (T.A. Weinert and L. H. Hartwell, Science 241:317-322, 1988). In this report, we extend the analysis of RAD9-dependent cell cycle control. We found that both induction of RAD9-dependent arrest in G2 and recovery from arrest could occur in the presence of the protein synthesis inhibitor cycloheximide, showing that the mechanism of RAD9-dependent control involves a posttranslational mechanism(s). We have isolated and determined the DNA sequence of the RAD9 gene, confirming the DNA sequence reported previously (R. H. Schiestl, P. Reynolds, S. Prakash, and L. Prakash, Mol. Cell. Biol. 9:1882-1886, 1989). The predicted protein sequence for the Rad9 protein bears no similarity to sequences of known proteins. We also found that synthesis of the RAD9 transcript in the cell cycle was constitutive and not induced by X-irradiation. We constructed yeast cells containing a complete deletion of the RAD9 gene; the rad9 null mutants were viable, sensitive to X- and UV irradiation, and defective for cell cycle arrest after DNA damage. Although Rad+ and rad9 delta cells had similar growth rates and cell cycle kinetics in unirradiated cells, the spontaneous rate of chromosome loss (in unirradiated cells) was elevated 7- to 21-fold in rad9 delta cells. These studies show that in the presence of induced or endogenous DNA damage, RAD9 is a negative regulator that inhibits progression from G2 in order to preserve cell viability and to maintain the fidelity of chromosome transmission.

An amino acidic adjuvant to augment cryoinjury of MCF-7 breast cancer cells.

PubMed

Wang, Chuo-Li; Teo, Ka Yaw; Han, Bumsoo

2008-08-01

One of the major challenges in cryosurgery is to minimize incomplete cryodestruction near the edge of the iceball. In the present study, the feasibility and effectiveness of an amino acidic adjuvant, glycine was investigated to enhance the cryodestruction of MCF-7 human breast cancer cell at mild freezing/thawing conditions via eutectic solidification. The effects of glycine addition on the phase change characteristics of NaCl-water binary mixture were investigated with a differential scanning calorimeter and cryo-macro/microscope. The results confirmed that a NaCl-glycine-water mixture has two distinct eutectic phase change events - binary eutectic solidification of water-glycine, and ternary eutectic solidification of NaCl-glycine-water. In addition, its effects on the cryoinjury of MCF-7 cells were investigated by assessing the post-thaw cellular viability after a single freezing/thawing cycle with various eutectic solidification conditions due to different glycine concentrations, end temperatures and hold times. The viability of MCF-7 cells in isotonic saline supplemented with 10% or 20% glycine without freezing/thawing remained higher than 90% (n=9), indicating no apparent toxicity was induced by the addition of glycine. With 10% glycine supplement, the viability of the cells frozen to -8.5 degrees C decreased from 85.9+/-1.8% to 38.5+/-1.0% on the occurrence of binary eutectic solidification of glycine-water (n=3 for each group). With 20% glycine supplement, the viability of the cells frozen to -8.5 degrees C showed similar trends to those with 10% supplement. However, as the end temperature was lowered to -15 degrees C, the viability drastically decreased from 62.5+/-2.0% to 3.6+/-0.7% (n=3 for each group). The influences of eutectic kinetics such as nucleation temperature, hold time and method were less significant. These results imply that the binary eutectic solidification of water-glycine can augment the cryoinjury of MCF-7 cells, and the extent of the eutectic solidification is significant.

Effects of Camphorquinone on Cytotoxicity, Cell Cycle Regulation and Prostaglandin E2 Production of Dental Pulp Cells: Role of ROS, ATM/Chk2, MEK/ERK and Hemeoxygenase-1

PubMed Central

Chang, Mei-Chi; Lin, Li-Deh; Wu, Min-Tsz; Chan, Chiu-Po; Chang, Hsiao-Hua; Lee, Ming-Shu; Sun, Tzu-Ying; Jeng, Po-Yuan; Yeung, Sin-Yuet; Lin, Hsueh-Jen; Jeng, Jiiang-Huei

2015-01-01

Camphorquinone (CQ) is a popularly-used photosensitizer in composite resin restoration. In this study, the effects of CQ on cytotoxicity and inflammation-related genes and proteins expression of pulp cells were investigated. The role of reactive oxygen species (ROS), ATM/Chk2/p53 and hemeoxygenase-1 (HO-1) and MEK/ERK signaling was also evaluated. We found that ROS and free radicals may play important role in CQ toxicity. CQ (1 and 2 mM) decreased the viability of pulp cells to about 70% and 50% of control, respectively. CQ also induced G2/M cell cycle arrest and apoptosis of pulp cells. The expression of type I collagen, cdc2, cyclin B, and cdc25C was inhibited, while p21, HO-1 and cyclooxygenase-2 (COX-2) were stimulated by CQ. CQ also activated ATM, Chk2, and p53 phosphorylation and GADD45α expression. Besides, exposure to CQ increased cellular ROS level and 8-isoprostane production. CQ also stimulated COX-2 expression and PGE2 production of pulp cells. The reduction of cell viability caused by CQ can be attenuated by N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD), but can be promoted by Zinc protoporphyin (ZnPP). CQ stimulated ERK1/2 phosphorylation, and U0126 prevented the CQ-induced COX-2 expression and prostaglandin E2 (PGE2) production. These results indicate that CQ may cause cytotoxicity, cell cycle arrest, apoptosis, and PGE2 production of pulp cells. These events could be due to stimulation of ROS and 8-isoprostane production, ATM/Chk2/p53 signaling, HO-1, COX-2 and p21 expression, as well as the inhibition of cdc2, cdc25C and cyclin B1. These results are important for understanding the role of ROS in pathogenesis of pulp necrosis and pulpal inflammation after clinical composite resin filling. PMID:26658076

The antiproliferative and apoptotic effects of apigenin on glioblastoma cells.

PubMed

Stump, Trevor A; Santee, Brittany N; Williams, Lauren P; Kunze, Rachel A; Heinze, Chelsae E; Huseman, Eric D; Gryka, Rebecca J; Simpson, Denise S; Amos, Samson

2017-07-01

Glioblastoma (GBM) is highly proliferative, infiltrative, malignant and the most deadly form of brain tumour. The epidermal growth factor receptor (EGFR) is overexpressed, amplified and mutated in GBM and has been shown to play key and important roles in the proliferation, growth and survival of this tumour. The goal of our study was to investigate the antiproliferative, apoptotic and molecular effects of apigenin in GBM. Proliferation and viability tests were carried out using the trypan blue exclusion, MTT and lactate dehydrogenase (LDH) assays. Flow cytometry was used to examine the effects of apigenin on the cell cycle check-points. In addition, we determined the effects of apigenin on EGFR-mediated signalling pathways by Western blot analyses. Our results showed that apigenin reduced cell viability and proliferation in a dose- and time-dependent manner while increasing cytotoxicity in GBM cells. Treatment with apigenin-induced is poly ADP-ribose polymerase (PARP) cleavage and caused cell cycle arrest at the G2M checkpoint. Furthermore, our data revealed that apigenin inhibited EGFR-mediated phosphorylation of mitogen-activated protein kinase (MAPK), AKT and mammalian target of rapamycin (mTOR) signalling pathways and attenuated the expression of Bcl-xL. Our results demonstrated that apigenin has potent inhibitory effects on pathways involved in GBM proliferation and survival and could potentially be used as a therapeutic agent for GBM. © 2017 Royal Pharmaceutical Society.

An APC/C-Cdh1 Biosensor Reveals the Dynamics of Cdh1 Inactivation at the G1/S Transition.

PubMed

Ondracka, Andrej; Robbins, Jonathan A; Cross, Frederick R

2016-01-01

B-type cyclin-dependent kinase activity must be turned off for mitotic exit and G1 stabilization. B-type cyclin degradation is mediated by the anaphase-promoting complex/cyclosome (APC/C); during and after mitotic exit, APC/C is dependent on Cdh1. Cdh1 is in turn phosphorylated and inactivated by cyclin-CDK at the Start transition of the new cell cycle. We developed a biosensor to assess the cell cycle dynamics of APC/C-Cdh1. Nuclear exit of the G1 transcriptional repressor Whi5 is a known marker of Start; APC/C-Cdh1 is inactivated 12 min after Whi5 nuclear exit with little measurable cell-to-cell timing variability. Multiple phosphorylation sites on Cdh1 act in a redundant manner to repress its activity. Reducing the number of phosphorylation sites on Cdh1 can to some extent be tolerated for cell viability, but it increases variability in timing of APC/C-Cdh1 inactivation. Mutants with minimal subsets of phosphorylation sites required for viability exhibit striking stochasticity in multiple responses including budding, nuclear division, and APC/C-Cdh1 activity itself. Multiple cyclin-CDK complexes, as well as the stoichiometric inhibitor Acm1, contribute to APC/C-Cdh1 inactivation; this redundant control is likely to promote rapid and reliable APC/C-Cdh1 inactivation immediately following the Start transition.

Replicative stress and alterations in cell cycle checkpoint controls following acetaminophen hepatotoxicity restrict liver regeneration.

PubMed

Viswanathan, Preeti; Sharma, Yogeshwar; Gupta, Priya; Gupta, Sanjeev

2018-03-05

Acetaminophen hepatotoxicity is a leading cause of hepatic failure with impairments in liver regeneration producing significant mortality. Multiple intracellular events, including oxidative stress, mitochondrial damage, inflammation, etc., signify acetaminophen toxicity, although how these may alter cell cycle controls has been unknown and was studied for its significance in liver regeneration. Assays were performed in HuH-7 human hepatocellular carcinoma cells, primary human hepatocytes and tissue samples from people with acetaminophen-induced acute liver failure. Cellular oxidative stress, DNA damage and cell proliferation events were investigated by mitochondrial membrane potential assays, flow cytometry, fluorescence staining, comet assays and spotted arrays for protein expression after acetaminophen exposures. In experimental groups with acetaminophen toxicity, impaired mitochondrial viability and substantial DNA damage were observed with rapid loss of cells in S and G2/M and cell cycle restrictions or even exit in the remainder. This resulted from altered expression of the DNA damage regulator, ATM and downstream transducers, which imposed G1/S checkpoint arrest, delayed entry into S and restricted G2 transit. Tissues from people with acute liver failure confirmed hepatic DNA damage and cell cycle-related lesions, including restrictions of hepatocytes in aneuploid states. Remarkably, treatment of cells with a cytoprotective cytokine reversed acetaminophen-induced restrictions to restore cycling. Cell cycle lesions following mitochondrial and DNA damage led to failure of hepatic regeneration in acetaminophen toxicity but their reversibility offers molecular targets for treating acute liver failure. © 2018 John Wiley & Sons Ltd.

Taurine Protects Mouse Spermatocytes from Ionizing Radiation-Induced Damage Through Activation of Nrf2/HO-1 Signaling.

PubMed

Yang, Wenjun; Huang, Jinfeng; Xiao, Bang; Liu, Yan; Zhu, Yiqing; Wang, Fang; Sun, Shuhan

2017-01-01

The increasing prevalence of ionizing radiation exposure has inevitably raised public concern over the potential detrimental effects of ionizing radiation on male reproductive system function. The detection of drug candidates to prevent reproductive system from damage caused by ionizing radiation is urgent. We aimed to investigate the protective role of taurine on the injury of mouse spermatocyte-derived cells (GC-2) subjected to ionizing radiation. mouse spermatocytes (GC-2 cells) were exposed to ionizing radiation with or without treatment of Taurine. The effect of ionizing radiation and Taurine treatment on GC-2 cells were evaluated by cell viability assay (CCK8), cell cycle and apoptosis. The relative protein abundance change was determined by Western blotting. The siRNA was used to explore whether Nrf2 signaling was involved in the cytoprotection of Taurine. Taurine significantly inhibited the decrease of cell viability, percentage of apoptotic cells and cell cycle arrest induced by ionizing radiation. Western blot analysis showed that taurine significantly limited the ionizing radiation-induced down-regulation of CyclinB1 and CDK1, and suppressed activation of Fas/FasL system pathway. In addition, taurine treatment significantly increased the expression of Nrf2 and HO-1 in GC-2 cells exposed to ionizing radiation, two components in antioxidant pathway. The above cytoprotection of Taurine was blocked by siNrf2. Our results demonstrate that taurine has the potential to effectively protect GC-2 cells from ionizing radiation- triggered damage via upregulation of Nrf2/HO-1 signaling. © 2017 The Author(s). Published by S. Karger AG, Basel.

Dihydroartemisinin and Curcumin Synergistically Induce Apoptosis in SKOV3 Cells Via Upregulation of MiR-124 Targeting Midkine.

PubMed

Zhao, Jiaojiao; Pan, Yuchen; Li, Xiujun; Zhang, Xuefang; Xue, Yaxian; Wang, Tingting; Zhao, Shuli; Hou, Yayi

2017-01-01

Women with advanced ovarian carcinoma are less likely to receive platinum-based chemotherapy and surgery due to a greater risk of cytotoxicity and poorer outcomes. We attempted to improve a promising therapy against ovarian cancer by using a combination of dihydroartemisinin (DHA) and curcumin (Cur). Human ovarian cancer SKOV3 cells were treated with DHA, Cur alone, or a combination of both. The viability of SKOV3 cells was measured by Cell Counting Kit-8 (CCK-8) and a colony formation assay. The cell cycle and apoptosis of SKOV3 cells were monitored by flow cytometry. The mRNA and protein expression levels of target genes were respectively examined by qRT-PCR and western blot. The biological effects of miR-124 on midkine (MK) were verified by a luciferase activity analysis. Combined treatment of DHA and Cur synergistically decreased cell viability, arrested cell cycle, and promoted apoptosis in SKOV3 cells. Moreover, it significantly attenuated the expression of oncogene MK and synergistically upregulated the expression of miR-124. Furthermore, miR-124 was verified to bind directly to the 3'-untranslated region of MK mRNA, resulting in mRNA degradation and reduced MK protein levels. The combination of DHA with Cur significantly inhibited tumor growth in xenograft nude mice without obvious toxicity. Co-treatment with DHA and Cur exhibited a synergistic anti-tumor effect on SKOV3 cells both in vitro and in vivo. © 2017 The Author(s). Published by S. Karger AG, Basel.

Turnover of Glycerolipid Metabolite Pool and Seed Viability

PubMed Central

Hu, Xiao-Long; Yu, Xiao-Mei; Chen, Hong-Ying

2018-01-01

Hydration–dehydration cycles can frequently cause stress to seeds, but can also be used to improve germination. However, the molecular basis of the stress caused is poorly understood. Herein, we examine the effects of hydration–dehydration cycles on seed viability and profile the membrane glycerolipid molecular species. We find that seed viability was not affected during the first two cycles, but significantly decreased as further cycles were applied, until all viability was lost. The abundances of seven glycerolipid classes increased and decreased through hydration and dehydration, respectively, but the phosphatidic acid and diacylglycerol abundances changed in the opposite sense, while total glycerolipid contents remained constant. This suggests that during hydration–dehydration cycles, turnover of glycerolipid metabolite pools take place, while no significant lipid synthesis or degradation is involved. As further hydration–dehydration cycles occurred, lipid unsaturation increased, plastidic lipids decreased, and phosphatidylserine acyl chains lengthened. The latter two could be lethal for seeds. Our findings reveal a novel model of membrane lipid changes, and provide new insights into the responses of seeds to hydration–dehydration cycles. PMID:29747431

The AhR is involved in the regulation of LoVo cell proliferation through cell cycle-associated proteins.

PubMed

Yin, Jiuheng; Sheng, Baifa; Han, Bin; Pu, Aimin; Yang, Kunqiu; Li, Ping; Wang, Qimeng; Xiao, Weidong; Yang, Hua

2016-05-01

Some ingredients in foods can activate the aryl hydrocarbon receptor (AhR) and arrest cell proliferation. In this study, we hypothesized that 6-formylindolo [3, 2-b] carbazole (FICZ) arrests the cell cycle in LoVo cells (a colon cancer line) through the AhR. The AhR agonist FICZ and the AhR antagonist CH223191 were used to treat LoVo cells. Real-time PCR and Western blot analyses were performed to detect the expression of the AhR, CYP1A1, CDK4, cyclinD1, cyclin E, CDK2, P27, and pRb. The distribution and activation of the AhR were detected with immunofluorescence. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometric analysis were performed to measure cell viability, cell cycle stage, and apoptosis. Our results show that FICZ inhibited LoVo cell proliferation by inducing G1 cell cycle arrest but had no effect on epithelial apoptosis. Further analysis found that FICZ downregulated cyclinD1 and upregulated p27 expression to arrest Rb phosphorylation. The downregulation of cyclinD1 and upregulation of p27 were abolished by co-treatment with CH223191. We conclude that the AhR, when activated by FICZ (an endogenous AhR ligand), can arrest the cell cycle and block LoVo cell proliferation. © 2016 International Federation for Cell Biology.

Discerning Viable from Nonviable Yersinia pestis pgm- and Bacillus anthracis Sterne using Propidium Monoazide in the Presence of White Powders

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

Hess, Becky M.; Kaiser, Brooke LD; Sydor, Michael A.

ABSTRACT Aims To develop and optimize an assay to determine viability status of Bacillus anthracis Sterne and Yersinia pestis pgm- strains in the presence of white powders by coupling propidium monoazide (PMA) treatment with real-time PCR (qPCR) analysis. Methods and Results PMA selectively enters nonviable cells and binds DNA, thereby increasing qPCR assay cycle threshold (CT) values compared to untreated samples. Dye concentration, cell number and fitness, incubation time, inactivation methods, and assay buffer were optimized for B. anthracis Sterne and Y. pestis pgm-. Differences in CT values in nonviable cells compared to untreated samples were consistently > 9 formore » both B. anthracis Sterne vegetative cells and Y. pestis pgm- in the presence and absence of three different white powders. Our method eliminates the need for a DNA extraction step prior to detection by qPCR. Conclusions The developed assay enables simultaneous identification and viability assessment for B. anthracis Sterne and Y. pestis pgm- under laboratory conditions, even in the presence of white powders. Eliminating the DNA extraction step that is typically used reduces total assay time and labor requirements for sample analysis. Significance and Impact of the Study The method developed for simultaneous detection and viability assessment for B. anthracis and Y. pestis can be employed in forming decisions about the severity of a biothreat event or the safety of food. Keywords Bacillus anthracis, Yersinia pestis, Propidium Monoazide, qPCR, White Powders, Rapid Viability Detection« less

Anti-tumor effects of osthole on ovarian cancer cells in vitro.

PubMed

Jiang, Guoqiang; Liu, Jia; Ren, Baoyin; Tang, Yawei; Owusu, Lawrence; Li, Man; Zhang, Jing; Liu, Likun; Li, Weiling

2016-12-04

Cnidium monnieri (L.) Cusson is a commonly used traditional Chinese medicine to treat gynecological disease in some countries. Osthole, an active O-methylated coumadin isolated from Cnidium monnieri (L.) Cusson, has been shown to induce various beneficial biochemical effects such as anti-seizure and anti-inflammatory effects. However, the anti-tumor mechanism of osthole is not well known. Here, we show that osthole inhibited the proliferation and migration of two widely used ovarian cancer cell lines, A2780 and OV2008 cells, in a dose-dependent manner. The study investigated the molecular mechanisms underlying ovarian cancer cells proliferation, apoptosis, cell cycle arrest and migration triggered by osthole. Ovarian cancer cell lines A2780, OV2008 and normal ovarian cell line IOSE80 were used as experimental model. MTT assay was employed to evaluate cell viability. Flow cytometry assays were performed to confirm apoptosis and cell cycle. We employed wound healing and transwell assays to delineate invasive and migratory potential triggered by osthole. MTT assays indicated that cell viability significantly decreased in ovarian cancer cells treated with osthole without effect on normal ovarian cells. Flow cytometric analysis revealed that osthole suppressed cells proliferation by promoting G2/M arrest and inducing apoptosis. The underlying mechanisms involved were regulation of the relative apoptotic protein Bcl-2, Bax and Caspase 3/9. In addition, wound healing and transwell assays revealed that the migratory potential and activity of matrix metalloproteinase MMP-2 and MMP-9 were markedly inhibited when cells were exposed to osthole. Our findings suggested that osthole has the potential to be used in novel anti-cancer therapeutic formulations for ovarian cancer treatment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells.

PubMed

Tang, Nou-Ying; Chueh, Fu-Shin; Yu, Chien-Chih; Liao, Ching-Lung; Lin, Jen-Jyh; Hsia, Te-Chun; Wu, King-Chuen; Liu, Hsin-Chung; Lu, Kung-Wen; Chung, Jing-Gung

2016-04-01

Glioblastoma multiforme (GBM) is a highly malignant devastating brain tumor in adults. Benzyl isothiocyanate (BITC) is one of the isothiocyanates that have been shown to induce human cancer cell apoptosis and cell cycle arrest. Herein, the effect of BITC on cell viability and apoptotic cell death and the genetic levels of human brain glioblastoma GBM 8401 cells in vitro were investigated. We found that BITC induced cell morphological changes, decreased cell viability and the induction of cell apoptosis in GBM 8401 cells was time-dependent. cDNA microarray was used to examine the effects of BITC on GBM 8401 cells and we found that numerous genes associated with cell death and cell cycle regulation in GBM 8401 cells were altered after BITC treatment. The results show that expression of 317 genes was upregulated, and two genes were associated with DNA damage, the DNA-damage-inducible transcript 3 (DDIT3) was increased 3.66-fold and the growth arrest and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was downregulated and two genes were associated with receptor for cell responses to stimuli, the EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) was inhibited 2.01-fold and the TNF receptor-associated protein 1 (TRAP1) was inhibited 2.08-fold. BITC inhibited seven mitochondria ribosomal genes, the mitochondrial ribosomal protein; tumor protein D52 (MRPS28) was inhibited 2.06-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein L23 (MRPL23) decreased 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein S12 (MRPS12) decreased 2.08-fold, the mitochondria ribosomal protein L12 (MRPL12) decreased 2.25-fold and the mitochondria ribosomal protein S34 (MRPS34) was decreased 2.30-fold in GBM 8401 cells. These changes of gene expression can provide the effects of BITC on the genetic level and are potential biomarkers for glioblastoma therapy.

Huaier aqueous extract induces apoptosis of human fibrosarcoma HT1080 cells through the mitochondrial pathway

PubMed Central

CUI, YANG; MENG, HONGMEI; LIU, WEIDONG; WANG, HUAN; LIU, QINGPENG

2015-01-01

In recent years, aqueous extract of Trametes robiniophila Murr. (Huaier), a traditional Chinese medicine, has been frequently used in China for complementary cancer therapy. However, the mechanisms underlying its anticancer effects have yet to be elucidated. The present study aimed to evaluate the ability of Huaier extract to inhibit proliferation, promote apoptosis and suppress mobility in the fibrosarcoma HT1080 cell line in vitro. The cells were treated with gradient doses of Huaier extract at concentrations of 0, 4, 8 or 16 mg/ml for 24, 48 or 72 h. The cell viability and motility were measured in vitro using MTT, invasive, migration and scratch assays. The distribution of the cell cycle and the extent of cellular apoptosis were analyzed by flow cytometry. The apoptotic pathways were detected using a mitochondrial membrane potential transition assay and western blotting. The results revealed that the cellular viability decreased significantly with increasing concentrations of Huaier extract. In addition, cell invasiveness and migration were also suppressed significantly. It was demonstrated that Huaier extract induced G2 cell-cycle arrest and cellular apoptosis in a time- and dose-dependent manner. The decreased mitochondrial membrane potential, the downregulation of B-cell lymphoma 2 and pro-caspase-3, and upregulation of Bcl-2-associated X protein, cleaved caspase-9 and caspase-3 suggested that Huaier extract induced the apoptosis of HT1080 cells through the mitochondrial pathway. The results of the present study indicate that Huaier extract is a potential complementary agent for the treatment of fibrosarcoma. PMID:25789006

Efficacy of the MEK Inhibitor Cobimetinib and its Potential Application to Colorectal Cancer Cells.

PubMed

Gong, Shu; Xu, Dongsheng; Zhu, Jialin; Zou, Fangdong; Peng, Rui

2018-05-22

Mutations in the Ras/Raf/MEK/ERK pathway are detected in 50% of colorectal cancer cases and play a crucial role in cancer development and progression. Cobimetinib is a MEK inhibitor approved for the treatment of advanced melanoma and inhibits the cell viability of other types of cancer cells. HCT116 colorectal cancer cells were treated with cobimetinib, and MTT assay, colony formation assay, and flow cytometry were used to evaluate cell viability, cell cycle, and apoptosis, respectively. The expression of genes associated with the cell cycle and apoptosis were evaluated by quantitative real-time PCR and western blotting. To explore use of cobimetinib in colorectal cancer treatment and further understand its mechanisms, RNA-seq technology was used to identify differentially expressed genes (DEGs) between cobimetinib-treated and untreated HCT116 cells. Furthermore, we compared these DEGs with Gene Expression Omnibus data from colorectal cancer tissues and normal colonic epithelial tissues. We found that cobimetinib not only inhibited cell proliferation but also induced G1 phase arrest and apoptosis in HCT116 colorectal cancer cells, suggesting that cobimetinib may useful in colorectal cancer therapy. After cobimetinib treatment, 3,495 DEGs were obtained, including 2,089 upregulated genes and 1,406 downregulated genes, and most of these DEGs were enriched in the cell cycle, DNA replication, and DNA damage repair pathways. Our results revealed that some genes with high expression in colorectal cancer tissues were downregulated by cobimetinib in HCT116 cells, including CCND1, E2F1, CDC25C, CCNE2, MYC, and PCNA. These genes have vital roles in DNA replication and the cell cycle. Furthermore, genes with low expression in colorectal cancer tissues were upregulated by cobimetinib, including PRKCA, PI3K, RTK, and PKC. Based on our results, the PKC and PI3K pathways were activated after cobimetinib treatment, and inhibition of these two pathways can increase the cytotoxicity of cobimetinib in HCT116 cells. Notably, cobimetinib appeared to enhance the efficacy of 5-fluorouracil (5-FU) by decreasing TYMS expression, high expression of which is responsible for 5-FU resistance in colorectal cancer. Our results suggest the potential use of cobimetinib in colorectal cancer therapy. © 2018 The Author(s). Published by S. Karger AG, Basel.

Cytotoxic effect of artocarpin on T47D cells.

PubMed

Arung, Enos Tangke; Wicaksono, Britanto Dani; Handoko, Yohana Ayupriyanti; Kusuma, Irawan Wijaya; Shimizu, Kuniyoshi; Yulia, Dina; Sandra, Ferry

2010-10-01

In our screening projects for anticancer agents from natural resources, artocarpin [6-(3-methyl-1-butenyl)-5,2',4'-trihydroxy-3-isoprenyl-7-methoxyflavone] isolated from wood of jack fruit (Artocarpus heterophyllus) showed potent cytotoxic activity on human T47D breast cancer cells. The mode of action of artocarpin was evaluated by its effect on cell viability, nuclear morphology, cell cycle progression, expression of protein markers for apoptosis, and mitochondrial membrane potential (Delta psi m). These results showed that artocarpin caused a reduction of cell viability in a concentration-dependent manner and an alteration of cell and nuclear morphology. Moreover, the percentage of the sub-G1 phase formation was elevated dose-dependently. Artocarpin induced activation of caspase 8 and 10 as indicated by stronger signal intensity of cleaved-caspase 8 and weaker signal intensity of caspase 10 markers detected after artocarpin treatment. In addition, we also noticed the activation of caspase 3 by artocarpin. There were negligible changes in mitochondrial membrane potential (Delta psi m) due to artocarpin treatment. All together, these data indicated that artocarpin induced apoptosis in T47D cells possibly via an extrinsic pathway.

Comparision of Piceid and Resveratrol in Antioxidation and Antiproliferation Activities In Vitro

PubMed Central

Liu, Daozhou; Cui, Han; Zhang, Bangle; Zhou, Siyuan; Yang, Tiehong; Mei, Qibing

2013-01-01

Background The clinic therapeutic effect of resveratrol is limited due to its low oral bioavailability. Piceid, a precursor of resveratrol, is the most abundant form of resveratrol in nature. A number of studies have hypothesized that piceid may have the same bioactivities like those of resveratrol. The aim of this work is to compare piceid with resveratrol in antioxidation and antiproliferation activities in vitro. Methods The antioxidative effects of resveratrol and piceid were evaluated by phenanthroline-Fe2+ method and H2O2-induced oxidative injury cell model. The antiproliferation effects were determined by MTT method in human liver tumor HepG2 cells, human breast cancer MDA-MB-231 cells and MCF-7 cells. The effects of resveratrol and piceid on the cell cycle and the apoptosis were evaluated by flow cytometry. Additionally, the uptake profiles of resveratrol and piceid in cancer cells were observed using fluorescence microscopy and clarified by LC-MS/MS. Conclusion Piceid exhibited higher scavenging activity against hydroxyl radicals than resveratrol in vitro. Resveratrol showed a significant protective effect against H2O2-induced cell damage. What is more, resveratrol had biphasic effects on tumor cells. Resveratrol and piceid only showed significant cytotoxicity on tumor cells at high concentration (≥50 µmol/L), while low concentration of resveratrol (<30 µmol/L) increased the cell viability. The principal effect of resveratrol and piceid on the viability of tumor cells was caused by the cell cycle arrest, while the effect on apoptosis was relatively minor. The reason that piceid showed lower biological activity than resveratrol at the same concentration was probably because piceid was more difficult in being uptaken by cells. PMID:23342161

Impaired tRNA nuclear export links DNA damage and cell-cycle checkpoint.

PubMed

Ghavidel, Ata; Kislinger, Thomas; Pogoutse, Oxana; Sopko, Richelle; Jurisica, Igor; Emili, Andrew

2007-11-30

In response to genotoxic stress, cells evoke a plethora of physiological responses collectively aimed at enhancing viability and maintaining the integrity of the genome. Here, we report that unspliced tRNA rapidly accumulates in the nuclei of yeast Saccharomyces cerevisiae after DNA damage. This response requires an intact MEC1- and RAD53-dependent signaling pathway that impedes the nuclear export of intron-containing tRNA via differential relocalization of the karyopherin Los1 to the cytoplasm. The accumulation of unspliced tRNA in the nucleus signals the activation of Gcn4 transcription factor, which, in turn, contributes to cell-cycle arrest in G1 in part by delaying accumulation of the cyclin Cln2. The regulated nucleocytoplasmic tRNA trafficking thus constitutes an integral physiological adaptation to DNA damage. These data further illustrate how signal-mediated crosstalk between distinct functional modules, namely, tRNA nucleocytoplasmic trafficking, protein synthesis, and checkpoint execution, allows for functional coupling of tRNA biogenesis and cell-cycle progression.

The effects of baicalein on canine osteosarcoma cell proliferation and death.

PubMed

Helmerick, E C; Loftus, J P; Wakshlag, J J

2014-12-01

Flavonoids are a group of modified triphenolic compounds from plants with medicinal properties. Baicalein, a specific flavone primarily isolated from plant roots (Scutellaria baicalensis), is commonly used in Eastern medicine for its anti-inflammatory and antineoplastic properties. Previous research shows greater efficacy for baicalein than most flavonoids; however, there has been little work examining their effects on sarcoma cells, let alone canine cells. Three canine osteosarcoma cell lines (HMPOS, D17 and OS 2.4) were treated with baicalein to examine cell viability, cell cycle kinetics, anchorage-independent growth and apoptosis. Results showed that osteosarcoma cells were sensitive to baicalein at concentrations from approximately 1 to 25 μM. Modest cell cycle changes were observed in one cell line. Baicalein was effective in inducing apoptosis and did not prevent doxorubicin cell proliferation inhibition in all the cell lines. The mechanism for induction of apoptosis has not been fully elucidated; however, changes in mitochondrial permeability supersede the apoptotic response. © 2012 Blackwell Publishing Ltd.

Natriuretic peptide receptor A inhibition suppresses gastric cancer development through reactive oxygen species-mediated G2/M cell cycle arrest and cell death.

PubMed

Li, Zheng; Wang, Ji-Wei; Wang, Wei-Zhi; Zhi, Xiao-Fei; Zhang, Qun; Li, Bo-Wen; Wang, Lin-Jun; Xie, Kun-Ling; Tao, Jin-Qiu; Tang, Jie; Wei, Song; Zhu, Yi; Xu, Hao; Zhang, Dian-Cai; Yang, Li; Xu, Ze-Kuan

2016-10-01

Natriuretic peptide receptor A (NPRA), the major receptor for atrial natriuretic peptide (ANP), has been implicated in tumorigenesis; however, the role of ANP-NPRA signaling in the development of gastric cancer remains unclear. Immunohistochemical analyses indicated that NPRA expression was positively associated with gastric tumor size and cancer stage. NPRA inhibition by shRNA induced G2/M cell cycle arrest, cell death, and autophagy in gastric cancer cells, due to accumulation of reactive oxygen species (ROS). Either genetic or pharmacologic inhibition of autophagy led to caspase-dependent cell death. Therefore, autophagy induced by NPRA silencing may represent a cytoprotective mechanism. ROS accumulation activated c-Jun N-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). ROS-mediated activation of JNK inhibited cell proliferation by disturbing cell cycle and decreased cell viability. In addition, AMPK activation promoted autophagy in NPRA-downregulated cancer cells. Overall, our results indicate that the inhibition of NPRA suppresses gastric cancer development and targeting NPRA may represent a promising strategy for the treatment of gastric cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Photoactive platinum diimine complexes showing induced cancer cell death by apoptosis.

PubMed

Zhang, Zhigang; Dai, Ruihui

2017-02-01

Photoinduced cytotoxicity mediated by a triphenylenamine-modified platinum diimine complex in human breast adenocarcinoma cells has been studied by cell viability assay. The triphenylenamine-modified platinum diimine complex showed more potent cytotoxicity in light than its carboxylate-modified analogue. To gain insights into the mechanism of photodynamic activity of this class of platinum diimine complexes, flow cytometric analyses were performed. The results suggest that upon irradiation the two platinum diimine complexes studied could induce cell cycle arrest in G 2 /M or S phase, and both of them could induce cancer cell death by apoptosis.

Direct effects of phenformin on metabolism/bioenergetics and viability of SH-SY5Y neuroblastoma cells.

PubMed

Geoghegan, Fintan; Chadderton, Naomi; Farrar, G Jane; Zisterer, Daniela M; Porter, Richard K

2017-11-01

Phenformin, a member of the biguanides class of drugs, has been reported to be efficacious in cancer treatment. The focus of the current study was to establish whether there were direct effects of phenformin on the metabolism and bioenergetics of neuroblastoma SH-SY5Y cancer cells. Cell viability was assessed using the alamar blue assay, flow cytometry analysis using propidium iodide and annexin V stain and poly (ADP-ribose) polymerase analysis. Cellular and mitochondrial oxygen consumption was determined using a Seahorse Bioscience Flux analyser and an Oroboros Oxygraph respirometer. Cells were transfected using electroporation and permeabilized for in situ mitochondrial functional analysis using digitonin. Standard protocols were used for immunoblotting and proteins were separated on denaturing gels. Phenformin was effective in reducing the viability of SH-SY5Y cells, causing G 1 cell cycle arrest and inducing apoptosis. Bioenergetic analysis demonstrated that phenformin significantly decreased oxygen consumption in a dose- and time-dependent manner. The sensitivity of oxygen consumption in SH-SY5Y cells to phenformin was circumvented by the expression of NADH-quinone oxidoreductase 1, a ubiquinone oxidoreductase, suggesting that complex I may be a target of phenformin. As a result of this inhibition, adenosine monophosphate protein kinase is activated and acetyl-coenzyme A carboxylase is inhibited. To the best of our knowledge, the current study is the first to demonstrate the efficacy and underlying mechanism by which phenformin directly effects the survival of neuroblastoma cancer cells.

Direct effects of phenformin on metabolism/bioenergetics and viability of SH-SY5Y neuroblastoma cells

PubMed Central

Geoghegan, Fintan; Chadderton, Naomi; Farrar, G. Jane; Zisterer, Daniela M.; Porter, Richard K.

2017-01-01

Phenformin, a member of the biguanides class of drugs, has been reported to be efficacious in cancer treatment. The focus of the current study was to establish whether there were direct effects of phenformin on the metabolism and bioenergetics of neuroblastoma SH-SY5Y cancer cells. Cell viability was assessed using the alamar blue assay, flow cytometry analysis using propidium iodide and annexin V stain and poly (ADP-ribose) polymerase analysis. Cellular and mitochondrial oxygen consumption was determined using a Seahorse Bioscience Flux analyser and an Oroboros Oxygraph respirometer. Cells were transfected using electroporation and permeabilized for in situ mitochondrial functional analysis using digitonin. Standard protocols were used for immunoblotting and proteins were separated on denaturing gels. Phenformin was effective in reducing the viability of SH-SY5Y cells, causing G1 cell cycle arrest and inducing apoptosis. Bioenergetic analysis demonstrated that phenformin significantly decreased oxygen consumption in a dose- and time-dependent manner. The sensitivity of oxygen consumption in SH-SY5Y cells to phenformin was circumvented by the expression of NADH-quinone oxidoreductase 1, a ubiquinone oxidoreductase, suggesting that complex I may be a target of phenformin. As a result of this inhibition, adenosine monophosphate protein kinase is activated and acetyl-coenzyme A carboxylase is inhibited. To the best of our knowledge, the current study is the first to demonstrate the efficacy and underlying mechanism by which phenformin directly effects the survival of neuroblastoma cancer cells. PMID:29113281

Impact of thermal effects induced by ultrasound on viability of rat C6 glioma cells.

PubMed

Kujawska, T; Secomski, W; Bilmin, K; Nowicki, A; Grieb, P

2014-07-01

In order to have consistent and repeatable effects of sonodynamic therapy (SDT) on various cancer cells or tissue lesions we should be able to control a delivered ultrasound energy and thermal effects induced. The objective of this study was to investigate viability of rat C6 glioma cells in vitro depending on the intensity of ultrasound in the region of cells and to determine the exposure time inducing temperature rise above 43 °C, which is known to be toxic for cells. For measurements a planar piezoelectric transducer with a diameter of 20 mm and a resonance frequency of 1.06 MHz was used. The transducer generated tone bursts with 94 μs duration, 0.4 duty-cycle and initial intensity ISATA (spatial averaged, temporal averaged) varied from 0.33 W/cm(2) to 8 W/cm(2) (average acoustic power varied from 1 W to 24 W). The rat C6 glioma cells were cultured on a bottom of wells in 12-well plates, incubated for 24h and then exposed to ultrasound with measured acoustic properties, inducing or causing no thermal effects leading to cell death. Cell viability rate was determined by MTT assay (a standard colorimetric assay for assessing cell viability) as the ratio of the optical densities of the group treated by ultrasound to the control group. Structural cellular changes and apoptosis estimation were observed under a microscope. Quantitative analysis of the obtained results allowed to determine the maximal exposure time that does not lead to the thermal effects above 43 °C in the region of cells for each initial intensity of the tone bursts used as well as the threshold intensity causing cell death after 3 min exposure to ultrasound due to thermal effects. The averaged threshold intensity was found to be about 5.7 W/cm(2). Copyright © 2014 Elsevier B.V. All rights reserved.

Synergic carcinostatic effects of ascorbic acid and hyperthermia on Ehrlich ascites tumor cell.

PubMed

Saitoh, Y; Yoshimoto, T; Kato, S; Miwa, N

2015-06-01

In this study, we evaluated the carcinostatic effects of combined ascorbic acid (AsA) and a capacitive-resistive electric transfer (CRet) hyperthermic apparatus-induced hyperthermic treatment on Ehrlich ascites tumor (EAT) cells. EAT cells were exposed to various AsA (0-10 mM) concentrations for 1 h; they subsequently underwent CRet treatment for 15 min at 42 °C. Cell viability was assessed by the WST-8 assay 24 h after the combined treatment. Reactive oxygen species involvement was evaluated using catalase and tempol; caspase-3/7 activation was determined by their fluorescent substrates; cell proliferation were estimated by time-lapse observation. The effect on the cell cycle was analyzed by flow cytometry. Combined AsA and CRet treatment synergistically suppressed cell viability compared with either treatment alone, and these synergistically carcinostatic effects were evident even at noncytotoxic concentrations of AsA alone (≤ 2 mM). The carcinostatic effects of combined AsA and CRet treatment were attenuated in a dose-dependent manner by catalase addition, but not by the superoxide anion radical scavenger tempol. Time-lapse observation revealed that combined AsA and CRet treatment activated caspase-3/7 at 10-24 h after treatment, accompanied by significant cell growth suppression. Cell cycle analysis revealed that the rate of sub-G1-phase (apoptotic) cells was drastically increased at 12 h and 24 h, and that the G2/M-phase cells gradually increased at 6-24 h after treatment. These results indicate that combined AsA and CRet treatment synergistically inhibits EAT cell growth through G2/M arrest and apoptosis induction via H2O2 generation at lower AsA concentrations; this carcinostatic effect cannot be exerted by AsA alone.

Suppression of RRM2 inhibits cell proliferation, causes cell cycle arrest and promotes the apoptosis of human neuroblastoma cells and in human neuroblastoma RRM2 is suppressed following chemotherapy.

PubMed

Li, Junfeng; Pang, Jinglin; Liu, Yongdong; Zhang, Jing; Zhang, Chuanguang; Shen, Gang; Song, Lili

2018-07-01

Ribonucleotide reductase regulatory subunit M2 (RRM2) is a rate‑limiting enzyme for DNA synthesis and repair. RRM2 has vital roles in controlling the progression of cancer. In the present study, we investigated the RRM2 level in neuroblastoma tissues, analyzed its relationship with clinicopathological characteristics of neuroblastoma patients, and explored the effect of RRM2 on the biological functions of neuroblastoma cells. RRM2 levels in 67 pairs of neuroblastoma and matched adjacent non‑cancerous tissues were detected by qRT‑PCR, and its association with patient clinicopathological features was assessed. Using RRM2 siRNA, the role of RRM2 in cell viability was detected by CCK‑8 assay, and the effects on cell cycle distribution and cell apoptosis were detected by flow cytometry. Hoechst 33342 staining was also performed. For RRM2 protein detection in cells and tissues, western blot analyses were employed. Our results revealed that RRM2 expression was significant higher in neuroblastoma tissues than that noted in adjacent non‑cancerous tissues at both the mRNA and protein levels. The increased RRM2 level was significantly associated with clinical stage. RRM2 levels were suppressed in stage III and IV tumors in the chemotherapy subgroup, compared with levels noted in tumors in the preoperative non‑chemotherapy subgroup. RRM2 siRNA significantly inhibited cell viability in the SH‑5Y5Y cells, induced cell arrest in the G0/G1 phase, and enhanced cell apoptosis. Taken together, overexpression of RRM2 is associated with the genesis and progression of neuroblastoma, and may be a potential chemotherapeutic target.

HDAC inhibitors, MS-275 and salermide, potentiates the anticancer effect of EF24 in human pancreatic cancer cells.

PubMed

Yar Saglam, Atiye Seda; Yilmaz, Akin; Onen, Hacer Ilke; Alp, Ebru; Kayhan, Handan; Ekmekci, Abdullah

2016-01-01

Histone deacetylases (HDACs) play a major role in the regulation of chromatin structure and gene expression by changing acetylation status of histone and non-histone proteins. MS-275 (entinostat, MS) is a well-known benzamide-based HDACI and Salermide (SAL), a reverse amide compound HDACI, have antiproliferative effects on several human cancer cells. In this study, we aimed to investigate the effects of HDACIs (MS and SAL) alone and/or combined use with EF24 (EF), a novel synthetic curcumin analog, on human pancreatic cancer cell line (BxPC-3). In vitro, BxPC-3 cells were exposed to varying concentrations of MS, SAL with or without EF, and their effects on cell viability, acetylated Histone H3 and H4 levels, cytotoxicity, and cleaved caspase 3 levels, and cell cycle distribution were measured. The viability of BxPC-3 cells decreased significantly after treatment with EF, MS and SAL treatments. MS and SAL treatment increased the acetylation of histone H3 and H4 in a dose dependent manner. MS and SAL alone or combined with EF were increased the number of cells in G1 phase. In addition, treatment with agents significantly decreased the ratio of cell in G2/M phase. There were significant dose-dependent increases at cleaved Caspase 3 levels after MS treatment but not after SAL treatment. Our results showed that HDAC inhibitors (MS and SAL), when combined with EF, may effectively reduce pancreatic cancer cell (BxPC-3) progression and stop the cell cycle at G1 phase. Further molecular analyses are needed to understand the fundamental molecular consequences of HDAC inhibition in pancreas cancer cells.

The genetic covariance between life cycle stages separated by metamorphosis

PubMed Central

Aguirre, J. David; Blows, Mark W.; Marshall, Dustin J.

2014-01-01

Metamorphosis is common in animals, yet the genetic associations between life cycle stages are poorly understood. Given the radical changes that occur at metamorphosis, selection may differ before and after metamorphosis, and the extent that genetic associations between pre- and post-metamorphic traits constrain evolutionary change is a subject of considerable interest. In some instances, metamorphosis may allow the genetic decoupling of life cycle stages, whereas in others, metamorphosis could allow complementary responses to selection across the life cycle. Using a diallel breeding design, we measured viability at four ontogenetic stages (embryo, larval, juvenile and adult viability), in the ascidian Ciona intestinalis and examined the orientation of additive genetic variation with respect to the metamorphic boundary. We found support for one eigenvector of G (gobsmax), which contrasted larval viability against embryo viability and juvenile viability. Target matrix rotation confirmed that while gobsmax shows genetic associations can extend beyond metamorphosis, there is still considerable scope for decoupled phenotypic evolution. Therefore, although genetic associations across metamorphosis could limit that range of phenotypes that are attainable, traits on either side of the metamorphic boundary are capable of some independent evolutionary change in response to the divergent conditions encountered during each life cycle stage. PMID:24966319

Antitumor potential of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazoles in human bladder cancer cells.

PubMed

Tessmann, Josiane Weber; Buss, Julieti; Begnini, Karine Rech; Berneira, Lucas Moraes; Paula, Favero Reisdorfer; de Pereira, Claudio Martin Pereira; Collares, Tiago; Seixas, Fabiana Kömmling

2017-10-01

Bladder cancer is a genitourinary malignant disease common worldwide. Current chemotherapy is often limited mainly due to toxicity and drug resistance. Thus, there is a continued need to discover new therapies. Recently evidences shows that pyrazoline derivatives are promising antitumor agents in many types of cancers, but there are no studies with bladder cancer. In order to find potent and novel chemotherapy drugs for bladder cancer, a series of pyrazoline derivatives 2a-2d were tested for their antitumor activity in two human bladder cancer cell lines 5647 and T24. The MTT assay showed that the compounds 1-thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazole (2a) and 1-thiocarbamoyl-5-(4-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (2c) decrease the cell viability of 5637 cells. Molecular modeling indicated that these compounds had a good oral bioavailability and low toxicities. Clonogenic assay and flow cytometric analysis were used to assess colony formation, apoptosis induction and cell cycle distribution. Overall, our results suggest that pyrazoline 2a and 2c, with the substituents hydrogen and chlorine respectively, may decrease cell viability and colony formation of bladder cancer 5637 cell line by inhibition of cell cycle progression, and for pyrazoline 2a, by induction of apoptosis. As indicated by the physicochemical properties of these compounds, the steric factor influences the activity. Therefore, these pyrazoline derivatives can be considered promising anticancer agents for the treatment of bladder cancer. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

A methoxyflavanone derivative from the Asian medicinal herb (Perilla frutescens) induces p53-mediated G2/M cell cycle arrest and apoptosis in A549 human lung adenocarcinoma.

PubMed

Abd El-Hafeez, Amer Ali; Fujimura, Takashi; Kamei, Rikiya; Hirakawa, Noriko; Baba, Kenji; Ono, Kazuhisa; Kawamoto, Seiji

2017-07-14

Perilla frutescens is an Asian dietary herb consumed as an essential seasoning in Japanese cuisine as well as used for a Chinese medicine. Here, we report that a newly found methoxyflavanone derivative from P. frutescens (Perilla-derived methoxyflavanone, PDMF; 8-hydroxy-5,7-dimethoxyflavanone) shows carcinostatic activity on human lung adenocarcinoma, A549. We found that treatment with PDMF significantly inhibited cell proliferation and decreased viability through induction of G 2 /M cell cycle arrest and apoptosis. The PDMF stimulation induces phosphorylation of tumor suppressor p53 on Ser15, and increases its protein amount in conjunction with up-regulation of downstream cyclin-dependent kinase inhibitor p21 Cip1/Waf1 and proapoptotic caspases, caspase-9 and caspase-3. We also found that small interfering RNA knockdown of p53 completely abolished the PDMF-induced G 2 /M cell cycle arrest, and substantially abrogated its proapoptotic potency. These results suggest that PDMF represents a useful tumor-preventive phytochemical that triggers p53-driven G 2 /M cell cycle arrest and apoptosis.

Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm(®) gradient between freeze-thaw cycles.

PubMed

Alvarez-Rodríguez, Manuel; Alvarez, Mercedes; López-Urueña, Elena; Martínez-Rodriguez, Carmen; Borragan, Santiago; Anel-López, Luis; de Paz, Paulino; Anel, Luis

2013-12-01

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze-thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing-thawing cycles on sperm quality and to analyze three different elapsed times between freezing-thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing-thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen-thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality. Copyright © 2013 Elsevier Inc. All rights reserved.

Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis.

PubMed

Wang, Tao; Xie, Zi-ping; Huang, Zhan-sen; Li, Hao; Wei, An-yang; Di, Jin-ming; Xiao, Heng-jun; Zhang, Zhi-gang; Cai, Liu-hong; Tao, Xin; Qi, Tao; Chen, Di-ling; Chen, Jun

2015-10-01

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Downregulation of gasdermin D promotes gastric cancer proliferation by regulating cell cycle-related proteins.

PubMed

Wang, Wei Jie; Chen, Di; Jiang, Ming Zuo; Xu, Bing; Li, Xiao Wei; Chu, Yi; Zhang, Yu Jie; Mao, Ren; Liang, Jie; Fan, Dai Ming

2018-02-01

To explore the relationship between gasdermin D (GSDMD) and gastric cancer (GC) cell proliferation, and to determine whether the downregulated expression of GSDMD contributed to the tumorigenesis and proliferation of GC cells. GSDMD expressions in GC tissues and matched adjacent non-cancerous tissues were assessed by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry. The effect of GSDMD on cell proliferation in vitro was assessed by the colony formation assay and cell viability assays. In vivo, xenografted tumors in nude mice were evaluated. The cell cycle was analyzed by flow cytometry. In addition, the alterations of several cell cycle-related and cell signaling pathway proteins were analyzed by Western blot. GSDMD expression was decreased in GC, and the decreased expression of GSDMD could markedly promote the proliferation of tumors in vivo and in vitro. The downregulation of GSDMD accelerated S/G 2 cell transition by activating extracellular signal regulated kinase, signal transducer and activator of transcription 3 and phosphatidylinositol 3 kinase/protein kinase B signaling pathways and regulating cell cycle-related proteins in GC. GSDMD may protect against cell proliferation of GC, and it may be used as a diagnostic and treatment strategy for GC. © 2018 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

Long-term in vivo polychlorinated biphenyl 126 exposure induces oxidative stress and alters proteomic profile on islets of Langerhans

NASA Astrophysics Data System (ADS)

Loiola, Rodrigo Azevedo; Dos Anjos, Fabyana Maria; Shimada, Ana Lúcia; Cruz, Wesley Soares; Drewes, Carine Cristiane; Rodrigues, Stephen Fernandes; Cardozo, Karina Helena Morais; Carvalho, Valdemir Melechco; Pinto, Ernani; Farsky, Sandra Helena

2016-06-01

It has been recently proposed that exposure to polychlorinated biphenyls (PCBs) is a risk factor to type 2 diabetes mellitus (DM2). We investigated this hypothesis using long-term in vivo PCB126 exposure to rats addressing metabolic, cellular and proteomic parameters. Male Wistar rats were exposed to PCB126 (0.1, 1 or 10 μg/kg of body weight/day; for 15 days) or vehicle by intranasal instillation. Systemic alterations were quantified by body weight, insulin and glucose tolerance, and blood biochemical profile. Pancreatic toxicity was measured by inflammatory parameters, cell viability and cycle, free radical generation, and proteomic profile on islets of Langerhans. In vivo PCB126 exposure enhanced the body weight gain, impaired insulin sensitivity, reduced adipose tissue deposit, and elevated serum triglycerides, cholesterol, and insulin levels. Inflammatory parameters in the pancreas and cell morphology, viability and cycle were not altered in islets of Langerhans. Nevertheless, in vivo PCB126 exposure increased free radical generation and modified the expression of proteins related to oxidative stress on islets of Langerhans, which are indicative of early β-cell failure. Data herein obtained show that long-term in vivo PCB126 exposure through intranasal route induced alterations on islets of Langerhans related to early end points of DM2.

Regulation of Cell Cycle and Stress Responses to Hydrostatic Pressure in Fission Yeast

PubMed Central

George, Vinoj T.; Brooks, Gavin

2007-01-01

We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress. PMID:17699598

Efficacy of a Cell-Cycle Decoying Killer Adenovirus on 3-D Gelfoam®-Histoculture and Tumor-Sphere Models of Chemo-Resistant Stomach Carcinomatosis Visualized by FUCCI Imaging

PubMed Central

Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M.

2016-01-01

Stomach cancer carcinomatosis peritonitis (SCCP) is a recalcitrant disease. The goal of the present study was to establish an in vitro-in vivo-like imageable model of SCCP to develop cell-cycle-based therapeutics of SCCP. We established 3-D Gelfoam® histoculture and tumor-sphere models of SCCP. FUCCI-expressing MKN-45 stomach cancer cells were transferred to express the fluorescence ubiquinized cell-cycle indicator (FUCCI). FUCCI-expressing MKN-45 cells formed spheres on agarose or on Gelfoam® grew into tumor-like structures with G0/G1 cancer cells in the center and S/G2 cancer cells located in the surface as indicated by FUCCI imaging when the cells fluoresced red or green, respectively. We treated FUCCI-expressing cancer cells forming SCCP tumors in Gelfoam® histoculture with OBP-301, cisplatinum (CDDP), or paclitaxel. CDDP or paclitaxel killed only cycling cancer cells and were ineffective against G1/G2 MKN-45 cells in tumors growing on Gelfoam®. In contrast, the telomerase-dependent adenovirus OBP-301 decoyed the MKN-45 cells in tumors on Gelfoam® to cycle from G0/G1 phase to S/G2 phase and reduced their viability. CDDP- or paclitaxel-treated MKN-45 tumors remained quiescent and did not change in size. In contrast, OB-301 reduced the size of the MKN-45 tumors on Gelfoam®. We examined the cell cycle-related proteins using Western blotting. CDDP increased the expression of p53 and p21 indicating cell cycle arrest. In contrast, OBP-301 decreased the expression of p53 and p21 Furthermore, OBP-301 increased the expression of E2F and pAkt as further indication of cell cycle decoy. This 3-D Gelfoam® histoculture and FUCCI imaging are powerful tools to discover effective therapy of SCCP such as OBP-301. PMID:27673332

Inhibition of Aurora-A kinase induces cell cycle arrest in epithelial ovarian cancer stem cells by affecting NFκB pathway

PubMed Central

Alvero, Ayesha B; Visintin, Irene

2011-01-01

Recurrent ovarian cancer is resistant to conventional chemotherapy. A sub-population of ovarian cancer cells, the epithelial ovarian cancer stem cells (EOC stem cells) have stemness properties, constitutive NFκB activity, and represent the chemoresistant population. Currently, there is no effective treatment that targets these cells. Aurora-A kinase (Aurora-A) is associated with tumor initiation and progression and is overexpressed in numerous malignancies. The aim of this study is to determine the effect of Aurora-A inhibition in EOC stem cells. EOC stem cells were treated with the Aurora-A inhibitor, MK-5108. Cell growth was monitored by Incucyte real-time imaging system, cell viability was measured using the Celltiter 96 assay and cytokine levels were quantified using xMAP technology. The intracellular changes associated with MK-5108 treatment are: (1) polyploidy and cell cycle arrest; (2) inhibition of NFκB activity; (3) decreased cytokine production; and (4) nuclear accumulation of IκBα. Thus, inhibition of Aurora-A decreases cell proliferation in the EOC stem cells by inducing cell cycle arrest and affecting the NFκB pathway. As EOC stem cells represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition may effectively target the cancer stem cell population in ovarian cancer. PMID:21623171

Interaction of cholinesterase modulators with DNA and their cytotoxic activity.

PubMed

Janockova, Jana; Gulasova, Zuzana; Plsikova, Jana; Musilek, Kamil; Kuca, Kamil; Mikes, Jaromir; Culka, Lubomir; Fedorocko, Peter; Kozurkova, Maria

2014-03-01

This research was focused on a study of the binding properties of a series of cholinesterase reactivators compounds K075 (1), K027 (2) and inhibitors compounds K524, K009 and 7-MEOTA (3-5) with calf thymus DNA. The nature of the interactions between compounds 1-5 and DNA were studied using spectroscopic techniques (UV-vis, fluorescence spectroscopy and circular dichroism). The binding constants for complexes of cholinesterase modulators with DNA were determined from UV-vis spectroscopic titrations (K=0.5 × 10(4)-8.9 × 10(5)M(-1)). The ability of the prepared analogues to relax topoisomerase I was studied with electrophoretic techniques and it was proved that ligands 4 and 5 inhibited this enzyme at a concentration of 30 μM. The biological activity of the novel compounds was assessed through an examination of changes in cell cycle distribution, mitochondrial membrane potential and cellular viability. Inhibitors 3-5 exhibited a cytotoxic effect on HL-60 (human acute promyelocytic leukaemia) cell culture, demonstrated a tendency to affect mitochondrial physiology and viability, and also forced cells to accumulate in the G1/G0-phase of the cell cycle. The cholinesterase reactivators 1 and 2 were found relatively save from the point of view of DNA binding, whereas cholinesterase inhibitors 3-5 resulted as strong DNA binding agents that limit their plausible use. Copyright © 2013 Elsevier B.V. All rights reserved.

Cytotoxicity and genotoxicity of nanosilver in stable GADD45α promoter-driven luciferase reporter HepG2 and A549 cells.

PubMed

Che, Bizhong; Luo, Qiulin; Zhai, Bingzhong; Fan, Guoqiang; Liu, Zhiyong; Cheng, Kaiming; Xin, Lili

2017-09-01

The intense commercial application of silver nanoparticles (AgNPs) has been raising concerns about their potential adverse health effects to human. This study aimed to explore the potency of AgNPs to induce GADD45α gene, an important stress sensor, and its relationships with the cytotoxicity and genotoxicity elicited by AgNPs. Two established HepG2 and A549 cell lines containing the GADD45α promoter-driven luciferase reporter were treated with increasing concentrations of AgNPs for 48 hours. After the treatment, transcriptional activation of GADD45α indicated by luciferase activity, cell viability, cell cycle arrest, and levels of genotoxicity were determined. The uptake and intracellular localization of AgNPs, cellular Ag doses as well as Ag + release were also detected. AgNPs could activate GADD45α gene at the transcriptional level as demonstrated by the dose-dependent increases in luciferase activity in both the reporter cells. The relative luciferase activity was greater than 12× the control level in HepG2-luciferase cells at the highest concentration tested where the cell viability decreased to 17.0% of the control. These results was generally in accordance with the positive responses in cytotoxicity, cell cycle arrest of Sub G1 and G2/M phase, Olive tail moment, micronuclei frequency, and the cellular Ag content. The cytotoxicity and genotoxicity of AgNPs seems to occur mainly via particles uptake and the subsequent liberation of ions inside the cells. And furthermore, the GADD45α promoter-driven luciferase reporter cells, especially the HepG2-luciferase cells, could provide a new and valuable tool for predicting nanomaterials genotoxicity in humans. © 2017 Wiley Periodicals, Inc.

Chlorpyrifos promotes colorectal adenocarcinoma H508 cell growth through the activation of EGFR/ERK1/2 signaling pathway but not cholinergic pathway.

PubMed

Suriyo, Tawit; Tachachartvanich, Phum; Visitnonthachai, Daranee; Watcharasit, Piyajit; Satayavivad, Jutamaad

2015-12-02

Aside from the effects on neuronal cholinergic system, epidemiological studies suggest an association between chlorpyrifos (CPF) exposure and cancer risk. This in vitro study examined the effects of CPF and its toxic metabolite, chlorpyrifos oxon (CPF-O), on the growth of human colorectal adenocarcinoma H508, colorectal adenocarcinoma HT-29, normal colon epithelial CCD841, liver hepatocellular carcinoma HepG2, and normal liver hepatocyte THLE-3 cells. The results showed that CPF (5-100 μM) concentration-dependently increased viability of H508 and CCD841 cells in serum-free conditions. This increasing trend was not found in HT-29, HepG2 and THLE-3 cells. In contrast, CPF-O (50-100 μM) reduced the viability of all cell lines. Cell cycle analysis showed the induction of cells in the S phase, and EdU incorporation assay revealed the induction of DNA synthesis in CPF-treated H508 cells indicating that CPF promotes cell cycle progression. Despite the observation of acetylcholinesterase activity inhibition and reactive oxygen species (ROS) generation, atropine (a non-selective muscarinic acetylcholine receptor antagonist) and N-acetylcysteine (a potent antioxidant) failed to inhibit the growth-promoting effect of CPF. CPF increased the phosphorylation of epidermal growth factor receptor (EGFR) and its downstream effector, extracellular signal regulated kinase (ERK1/2), in H508 cells. AG-1478 (a specific EGFR tyrosine kinase inhibitor) and U0126 (a specific MEK inhibitor) completely mitigated the growth promoting effect of CPF. Altogether, these results suggest that EGFR/ERK1/2 signaling pathway but not cholinergic pathway involves in CPF-induced colorectal adenocarcinoma H508 cell growth. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

In vitro assessment of anti-proliferative effect induced by α-mangostin from Cratoxylum arborescens on HeLa cells

PubMed Central

El habbash, Aisha I.; Ibrahim, Mohamed Yousif; Yahayu, Maizatulakmal; Omer, Fatima Abd Elmutaal; Abd Rahman, Mashitoh; Nordin, Noraziah; Lian, Gwendoline Ee Cheng

2017-01-01

Natural medicinal products possess diverse chemical structures and have been an essential source for drug discovery. Therefore, in this study, α-mangostin (AM) is a plant-derived compound was investigated for the apoptotic effect on human cervical cancer cells (HeLa). The cytotoxic effects of AM on the viability of HeLa and human normal ovarian cell line (SV40) were evaluated by using MTT assay. Results showed that AM inhibited HeLa cells viability at concentration- and time-dependent manner with IC50 value of 24.53 ± 1.48 µM at 24 h. The apoptogenic effects of AM on HeLa were assessed using fluorescence microscopy analysis. The effect of AM on cell proliferation was also studied through clonogenic assay. ROS production evaluation, flow cytometry (cell cycle) analysis, caspases 3/7, 8, and 9 assessment and multiple cytotoxicity assays were conducted to determine the mechanism of cell apoptosis. This was associated with G2/M phase cell cycle arrest and elevation in ROS production. AM induced mitochondrial apoptosis which was confirmed based on the significant increase in the levels of caspases 3/7 and 9 in a dose-dependent manner. Furthermore, the MMP disruption and increased cell permeability, concurrent with cytochrome c release from the mitochondria to the cytosol provided evidence that AM can induce apoptosis via mitochondrial-dependent pathway. AM exerted a remarkable antitumor effect and induced characteristic apoptogenic morphological changes on HeLa cells, which indicates the occurrence of cell death. This study reveals that AM could be a potential antitumor compound on cervical cancer in vitro and can be considered for further cervical cancer preclinical and in vivo testing. PMID:28740747

Attenuation of Oxidative Stress-Induced Cell Apoptosis in Schwann RSC96 Cells by Ocimum Gratissimum Aqueous Extract

PubMed Central

Chao, Pei-Yu; Lin, James A.; Ye, Je-Chiuan; Hwang, Jin-Ming; Ting, Wei-Jen; Huang, Chih-Yang; Liu, Jer-Yuh

2017-01-01

Objectives:Cell transplantation therapy of Schwann cells (SCs) is a promising therapeutic strategy after spinal cord injury. However, challenges such as oxidative stress hinder satisfactory cell viability and intervention for enhancing SCs survival is critical throughout the transplantation procedures. Ocimum gratissimum, widely used as a folk medicine in many countries, has therapeutic and anti-oxidative properties and may protect SCs survival. Methods:We examined the protective effects of aqueous O. gratissimum extract (OGE) against cell damage caused by H2O2-induced oxidative stress in RSC96 Schwann cells. Results:Our results showed that the RSC96 cells, damaged by H2O2 oxidative stress, decreased their viability up to 32% after treatment with different concentrations of up to 300 μM H2O2, but OGE pretreatment (150 or 200 μg/mL) increased cell viability by approximately 62% or 66%, respectively. Cell cycle analysis indicated a high (43%) sub-G1 cell population in the H2O2-treated RSC96 cells compared with untreated cells (1%); whereas OGE pretreatment (150 and 200 μg/mL) of RSC96 cells significantly reduced the sub-G1 cells (7% and 8%, respectively). Furthermore, Western blot analysis revealed that OGE pretreatment inhibited H2O2-induced apoptotic protein caspase-3 activation and PARP cleavage, as well as it reversed Bax up-regulation and Bcl-2 down-regulation. The amelioration of OGE of cell stress and stress-induced apoptosis was proved by the HSP70 and HSP72 decrease. Conclusion: Our data suggest that OGE may minimize the cytotoxic effects of H2O2-induced SCs apoptosis by modulating the apoptotic pathway and could potentially supplement cell transplantation therapy. PMID:28824312

Evaluation of results of cell electrophoresis experiments on space shuttle STS-3 including pre-flight and post-flight laboratory experiments

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

The objectives of the red blood cell experiments were to provide a visual check on the electrophoretic process and especially electroosmotic flow in space as well as to provide test separations of non-degradable standard particles for comparison with the separations of the three viable cell types studied on the Apollo-Soyuz Test Project. Determination of the maximum concentrations of cells that can be separated in column electrophore was a significant goal. Two of the eight columns were available for red cell experiments, so two concentrations of human and rabbit RBC mixtures were used. The objectives of another experiment were to evaluate the reproducibility of microgravity electrophoretic separation of living kidney cells, to separate cells with highly viability despite two freeze-thaw cycles, and to optimize the physical conditions of cell separation. Owing to the uncertain heterogeneity of the starting material, the experimental design does not assess resolution in microgravity, but improved separability was sought in comparison to density-gradient electrophoresis or continuous-flow electrophoresis. Efforts were made to increase cell yield and cell viability and to assess reproducibility directly.

Formation of resting cells by non-spore-forming microorganisms as a strategy of long-term survival in the environment

NASA Astrophysics Data System (ADS)

Mulyukin, Andrei L.; Soina, Vera S.; Demkina, Elena V.; Kozlova, Alla N.; Suzina, Natalia E.; Dmitriev, Vladimir V.; Duda, Vitalii I.; El'-Registan, Galina I.

2003-01-01

Non-spore-forming bacteria of the genera Micrococcus and Arthrobacter, including the isolates from permafrost sediments, were found to be able to form cystlike cells under special conditions. Cystlike cells maintained the viability during long-term storage (for up to several years), had undetectable respiratory activity and the elevated resistance to heating and other unfavorable conditions, possessed the specific fine structure and morphology, and were formed in the life cycles of the microorganism. These properties allow cystlike cells to be attributed to a new type of resting microbial forms. Furthermore, the distinctive feature of resting cystlike cells was their low P/S ratios and high Ca/K ratios in comparison to vegetative cells as shown by X-ray microanalysis. The experimentally obtained bacterial cystlike cells with thickened and laminated cell walls and altered texture of the cytoplasm were similar to the cells abundant in native microbial populations isolated from permafrost sediments and ancient soils of the Kolyma lowland (Siberia, Russia). Due to the inherent elevated resistance to adverse conditions and maintenance of viability for prolonged periods, resting cystlike cells are likely to ensure long-term survival of non-spore-forming bacteria in cold environments.

Anti-apoptosis Effect of Decoy Receptor 3 in Cholangiocarcinoma Cell Line TFK-1

PubMed Central

Xu, Ying-Chen; Cui, Jing; Zhang, Li-Jun; Zhang, Dong-Xin; Xing, Bing-Chen; Huang, Xiong-Wei-Ye; Wu, Ji-Xiang; Liang, Chao-Jie; Li, Guang-Ming

2018-01-01

Background: Decoy receptor 3 (DcR3) is a protein with anti-apoptotic effect that belongs to the tumor necrosis factor receptor superfamily. DcR3 is highly expressed in a variety of malignant tumors including cholangiocarcinoma and its expression was found to be related to the clinical stage, the invasion, and the metastasis of the tumor. This in vitro study aimed to investigate the effect of downregulated expression of DcR3 on cell viability, cell apoptosis, and cell cycle in cholangiocarcinoma cell line TFK-1. Methods: Three different cell lines were cultured: human cholangiocarcinoma TFK-1, human biliary epithelial carcinoma HuCCT-1, and human cholangiocarcinoma RBE. The cholangiocarcinoma cell line with the highest expression of DcR3 was selected for further investigation. The expression of DcR3 was silenced/knocked down by transfection with DcR3-siRNA in the selected cell line. Various biological phenotype parameters such as cell viability, apoptosis, and cell cycle were observed. Results: The mRNA and protein levels of DcR3 were measured in the three cell lines, and TFK-1 was selected. After the treatment with DcR3-siRNA for 48 h, DcR3 mRNA and protein expression in the treatment group were 38.45% (P < 0.01) and 48.03% (P < 0.05) of that of the control, respectively. It was found that the cell viability decreased to 61.87% of the control group (P < 0.01) after the downregulation of DcR3 in cholangiocarcinoma cell line TFK-1 by transfection with DcR3-siRNA, while the percentage of apoptotic cells was 2.98 times as compared with the control group (P < 0.05). Compared with the control group the ratio of G0/G1 increased, and the ratio of G2/M decreased in the treatment group. However, the differences were not statistically significant. Conclusions: The effect of DcR3 on the growth and apoptosis of cholangiocarcinoma has been demonstrated. DcR3 is not only a predictive marker for malignant tumor but it is also likely to be a potential target for cancer gene therapy. Further studies should focus on exploring the binding ligand of DcR3, the signaling pathway involved, and the molecular mechanism for the regulation of DcR3 expression in cholangiocarcinoma. PMID:29271385

Anti-apoptosis Effect of Decoy Receptor 3 in Cholangiocarcinoma Cell Line TFK-1.

PubMed

Xu, Ying-Chen; Cui, Jing; Zhang, Li-Jun; Zhang, Dong-Xin; Xing, Bing-Chen; Huang, Xiong-Wei-Ye; Wu, Ji-Xiang; Liang, Chao-Jie; Li, Guang-Ming

2018-01-05

Decoy receptor 3 (DcR3) is a protein with anti-apoptotic effect that belongs to the tumor necrosis factor receptor superfamily. DcR3 is highly expressed in a variety of malignant tumors including cholangiocarcinoma and its expression was found to be related to the clinical stage, the invasion, and the metastasis of the tumor. This in vitro study aimed to investigate the effect of downregulated expression of DcR3 on cell viability, cell apoptosis, and cell cycle in cholangiocarcinoma cell line TFK-1. Three different cell lines were cultured: human cholangiocarcinoma TFK-1, human biliary epithelial carcinoma HuCCT-1, and human cholangiocarcinoma RBE. The cholangiocarcinoma cell line with the highest expression of DcR3 was selected for further investigation. The expression of DcR3 was silenced/knocked down by transfection with DcR3-siRNA in the selected cell line. Various biological phenotype parameters such as cell viability, apoptosis, and cell cycle were observed. The mRNA and protein levels of DcR3 were measured in the three cell lines, and TFK-1 was selected. After the treatment with DcR3-siRNA for 48 h, DcR3 mRNA and protein expression in the treatment group were 38.45% (P < 0.01) and 48.03% (P < 0.05) of that of the control, respectively. It was found that the cell viability decreased to 61.87% of the control group (P < 0.01) after the downregulation of DcR3 in cholangiocarcinoma cell line TFK-1 by transfection with DcR3-siRNA, while the percentage of apoptotic cells was 2.98 times as compared with the control group (P < 0.05). Compared with the control group the ratio of G0/G1increased, and the ratio of G2/M decreased in the treatment group. However, the differences were not statistically significant. The effect of DcR3 on the growth and apoptosis of cholangiocarcinoma has been demonstrated. DcR3 is not only a predictive marker for malignant tumor but it is also likely to be a potential target for cancer gene therapy. Further studies should focus on exploring the binding ligand of DcR3, the signaling pathway involved, and the molecular mechanism for the regulation of DcR3 expression in cholangiocarcinoma.

PHYSIOLOGICAL, CYTOLOGICAL AND BIOCHEMICAL STABILITY OF Medicago sativa L. CELL CULTURE AFTER 27 YEARS OF CRYOGENIC STORAGE.

PubMed

Volkova, L A; Urmantseva, V V; Popova, E V; Nosov, A M

2015-01-01

The efficiency of long-term cryogenic storage to prevent somaclonal variations in plant cell cultures and retain their major cytogenetic and biochemical traits remains under debate. In particular, it is not clear how stress conditions associated with cryopreservation, such as low temperature, dehydration and toxic action of some cryoprotectants (DMSO in particular), affect post-storage regrowth and genetic integrity of cell samples. We assessed growth, cytogenetic and biochemical characteristics of the peroxidase-producing strain of Medicago sativa L. cell culture recovered after 27 years of cryogenic storage as compared to the same culture before cryopreservation. In 1984, M. sativa L. cell culture was cryopreserved using programmed freezing and 7% DMSO as a cryoprotectant. In 2011, after rewarming in a water bath at 40 degree C for 90 s, cell culture was recovered and proliferated. Viability, growth profile, mitotic index, ploidy level, peroxidase activity and cell response to hypothermia and osmotic stress were compared between the recovered and the initial cell cultures using the records available from 1984. Viability of alfalfa cell culture after rewarming was below 20% but it increased to 80% by the 27th subculture cycle. Recovered culture showed higher mitotic activity and increased number of haploid and diploid cells compared to the initial cell line. Both peroxidase activity and response to abiotic stress in the recovered cell culture were similar to that of the initial culture. Cryopreservation by programmed freezing was effective at retaining the main characteristics of M. sativa undifferentiated cell culture after 27 years of storage. According to available data, this is longest period of successful cryopreservation of plant cell cultures reported so far. After storage, there was no evidence that DMSO had any detrimental effect on cell viability, growth or cytogenetics.

Determination of Optimum Operation Parameters for Low-Intensity Pulsed Ultrasound and Low-Level Laser Based Treatment to Induce Proliferation of Osteoblast and Fibroblast Cells.

PubMed

Coskun, Mehmet Emre; Coskun, Kubra Acikalin; Tutar, Yusuf

2018-05-01

The aim of this study was to determine the optimum operating parameters (pulse duration, energy levels, and application time) to promote induction of osteoblast and fibroblast cell proliferation and to maintain cell viability treated with low-intensity pulsed ultrasound (LIPUS) and low-level laser therapy (LLLT). The positive effects of LIPUS and LLLT on cellular activity have been reported in recent years. Comparisons between experimental parameters of previous studies are difficult because scientific studies reported frequencies and the duty cycles of LIPUS and wavelengths and doses of LLLT in a wide range of parameters. However, optimum amount of energy and optimum time exposure must be determined to induce bone and tissue cell proliferation for effective healing process and to avoid cell damage. Fibroblast and osteoblast cell cultures were irradiated with LIPUS (10-50% pulse and continuous mode at 1 and 3 MHz for 1, 3, and 5 min) and LLLT (4, 8, and 16 J at 50, 100, 200, 300, 400, and 500 mW). Cell cultures were analyzed using XTT assay. For both cell types, LIPUS treatment with 10% pulse (1:9 duty cycle), 3 MHz, and for 1 min and LLLT treatment over 100 mV for 4, 8, and 16 J modalities contributed to the growth, and may help bone repair and tissue healing process optimally. Bio-stimulating effects of LLLT irradiation promote proliferation and maintain cell viability better than LIPUS treatment without causing thermal response for both cell types, and the therapeutic modality above 200 mV has maximum effectiveness.

Curcumin induces apoptosis and cell cycle arrest via the activation of reactive oxygen species-independent mitochondrial apoptotic pathway in Smad4 and p53 mutated colon adenocarcinoma HT29 cells.

PubMed

Agarwal, Ayushi; Kasinathan, Akiladdevi; Ganesan, Ramamoorthi; Balasubramanian, Akhila; Bhaskaran, Jahnavi; Suresh, Samyuktha; Srinivasan, Revanth; Aravind, K B; Sivalingam, Nageswaran

2018-03-01

Curcumin is a natural dietary polyphenol compound that has various pharmacological activities such as antiproliferative and cancer-preventive activities on tumor cells. Indeed, the role reactive oxygen species (ROS) generated by curcumin on cell death and cell proliferation inhibition in colon cancer is poorly understood. In the present study, we hypothesized that curcumin-induced ROS may promote apoptosis and cell cycle arrest in colon cancer. To test this hypothesis, the apoptosis-inducing potential and cell cycle inhibition effect of ROS induced by curcumin was investigated in Smd4 and p53 mutated HT-29 colon adenocarcinoma cells. We found that curcumin treatment significantly increased the level of ROS in HT-29 cells in a dose- and time-dependent manner. Furthermore, curcumin treatment markedly decreased the cell viability and proliferation potential of HT-29 cells in a dose- and time-dependent manner. Conversely, generation of ROS and inhibitory effect of curcumin on HT-29 cells were abrogated by N-acetylcysteine treatment. In addition, curcumin treatment did not show any cytotoxic effects on HT-29 cells. Furthermore, curcumin-induced ROS generation caused the DNA fragmentation, chromatin condensation, and cell nuclear shrinkage and significantly increased apoptotic cells in a dose- and time-dependent manner in HT-29 cells. However, pretreatment of N-acetylcysteine inhibited the apoptosis-triggering effect of curcumin-induced ROS in HT-29 cells. In addition, curcumin-induced ROS effectively mediated cell cycle inhibition in HT-29 cells. In conclusion, our data provide the first evidence that curcumin induces ROS independent apoptosis and cell cycle arrest in colon cancer cells that carry mutation on Smad4 and p53. Copyright © 2018. Published by Elsevier Inc.

Molecular mechanisms of celery seed extract induced apoptosis via s phase cell cycle arrest in the BGC-823 human stomach cancer cell line.

PubMed

Gao, Lin-Lin; Feng, Lei; Yao, Shu-Tong; Jiao, Peng; Qin, Shu-Cun; Zhang, Wei; Zhang, Ya-Bin; Li, Fu-Rong

2011-01-01

Mechanisms of apoptosis in tumor cells is an important field of tumor therapy and cancer molecular biology. Loss of cell cycle control, leading to uncontrolled proliferation, is common in cancer. Therefore, the identification of potent and selective cyclin dependent kinase inhibitors is a priority for anti-cancer drug discovery. There are at least two major apoptotic pathways, initiated by caspase-8 and caspase-9, respectively, which can activate caspase cascades. Apoptosis triggered by activation of the mitochondrial-dependent caspase pathway represents the main programmed cell death mechanism. This is activated by various intracellular stresses that induce permeabilization of the mitochondrial membrane. Anti-tumor effects of celery seed extract (CSE) and related mechanisms regarding apoptosis were here investigated in human gastric cancer BGC-823 cells. CSE was produced by supercritical fluid extraction. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) assay and apoptosis by flow cytometry using Annexin/PI staining and DAPI staining and a laser scanning confocal microscope (LSCM). Cell cycling was evaluated using PI staining with flow cytometry and expression of cell cycle and apoptosis-related proteins cyclin A, CDK2, bcl-2 and bax was assessed by immunohistochemical staining. CSE had an anti-proliferation effect on human gastric cancer BGC-823 cells in a dose- and time-dependent manner. After treatment, the apoptotic rate significantly increased, with morphological changes typical of apoptosis observed with LSCM by DAPI staining. Cell cycle and apoptosis related proteins, such as cyclin A, CDK2 and bcl-2 were all down-regulated, whereas bax was up-regulated. The molecular determinants of inhibition of cell proliferation as well as apoptosis of CSE may be associated with cycle arrest in the S phase.

The effects of sulforaphane on canine osteosarcoma proliferation and invasion.

PubMed

Rizzo, V L; Levine, C B; Wakshlag, J J

2017-09-01

Recent evidence in in vitro and in vivo models suggests that sulforaphane (SFN), found in raw cruciferous vegetables, may have utility in chemoprevention, as an antineoplastic agent and as a free radical scavenger. The effects of SFN alone or with doxorubicin on cell viability were examined, as well as cell cycle kinetics, invasion capabilities and apoptosis in three canine osteosarcoma cell line (D17, OS 2.4 and HMPOS). Results showed that SFN could not induce cell death at potentially physiological concentrations (<50 μM), but significantly diminished cell invasion and downregulation of focal adhesion kinase (FAK) signaling. Modest cell cycle changes were observed in each cell line. When doxorubicin was used in conjunction with SFN, there was a protective effect to doxorubicin-induced cytotoxicity in D17 and OS 2.4 cells. Further studies examining SFN as a supplement are warranted, particularly in light of pro-proliferative and cytoprotective properties in canine osteosarcoma. © 2016 John Wiley & Sons Ltd.

Cell-based flavivirus infection (CFI) assay for the evaluation of dengue antiviral candidates using high-content imaging.

PubMed

Tan, Kah Hin; Ki, Kitti Chan Wing; Watanabe, Satoru; Vasudevan, Subhash G; Krishnan, Manoj

2014-01-01

Large-scale screening of antiviral compounds that target dengue virus life cycle requires a robust cell-based assay that is rapid, easy to conduct, and sensitive enough to be able to assess viral infectivity and cell viability so that antiviral efficacy can be measured. In this chapter we describe a method that uses high-content imaging to evaluate the in vitro antiviral efficacy in a modification to the cell-based flavivirus immunodetection (CFI) assay that was described previously in Wang et al. (Antimicrob Agents Chemother 53(5):1823-1831, 2009).

The soy-derived peptide Vglycin inhibits the growth of colon cancer cells in vitro and in vivo.

PubMed

Gao, Chang; Sun, Rui; Xie, Ya-Rong; Jiang, An-Li; Lin, Mei; Li, Min; Chen, Zheng-Wang; Zhang, Ping; Jin, Honglin; Feng, Jue-Ping

2017-05-01

Vglycin, a novel natural polypeptide isolated from pea seeds, possesses antidiabetic properties. Our previous studies have shown that Vglycin can induce the differentiation of human colon adenocarcinoma cells. We aimed to determine the anticancer activity of Vglycin against colon cancer cells and to elucidate related apoptosis-inducing mechanisms. Treatment with purified Vglycin significantly reduced growth, viability, and colony formation of CT-26, SW480, and NCL-H716 colon cancer cells in a dose-dependent manner while down-regulating the expression of proliferating cell nuclear antigen. Mouse xenograft studies showed a 38% inhibition of colon cancer growth in mice treated with Vglycin (20 mg/kg/day) at day 21. Furthermore, the potential mechanisms involved in Vglycin-induced cell apoptosis were examined using cell cycle studies, ultrastructural examination, as well as apoptosis-associated pathway analysis. The results showed that Vglycin significantly promoted apoptosis and G1/S phase cell cycle arrest. As revealed by Western blot, the expression of CDK2 and Cyclin D1 was down-regulated in all three Vglycin-treated colon cancer cells, indicating that the CDK2/Cyclin D1 cell cycle pathway involved in the initiation and progression of colon cancer. Moreover, the inhibition of Vglycin-induced cell proliferation in colon cancer cells was accompanied by alteration of the expression levels of the apoptosis-related proteins Bax, Bcl-2 and Mcl-1, and an increase of caspase-3 activity. Together, our results suggest that Vglycin may be another plant-derived peptide that suppresses colon cancer, supporting the continued investigation of Vglycin as therapeutic agent for colon cancer. Impact statement The antidiabetic properties and the capability of inducing differentiation of human colon adenocarcinoma cells of Vglycin have been reported in our previous studies. However, the anticancer potential of Vglycin on colon cancer cells and its possible related mechanisms were still unknown. In this study, we found that Vglycin could reduce growth, viability, and colony formation or colony size of CT-26, SW480, and NCL-H716 colon cancer cells. Moreover, Vglycin decreased tumor volume by 38% in xenograft mice transplanted with CT-26 cells. The mechanisms of these phenomena may be due to the down-regulated CDK2 and Cyclin D1, G1/S phase cell cycle arrest, and the dysregulated expression of Bax, Bcl-2, and Mcl-1. The findings highlight the anticancer potential of Vglycin against colon cancer cells, and suggest Vglycin may be another colon cancer potential suppressive component of plant-derived peptides.

Comparative Evaluation of Silibinin Effects on Cell Cycling and Apoptosis in Human Breast Cancer MCF-7 and T47D Cell Lines.

PubMed

Jahanafrooz, Zohreh; Motameh, Nasrin; Bakhshandeh, Behnaz

2016-01-01

Silibinin is a natural polyphenol with high antioxidant and anticancer properties. In this study, its influence on two of the most commonly employed human breast cancer cell lines, MCF-7 and T47D, and one non-malignant MCF-10A cell line, were investigated and compared. Cell viability, the cell cycle distribution and apoptosis induction were analyzed by MTT and flow cytometry, respectively. The effect of silibinin on PTEN, Bcl-2, P21, and P27 mRNAs expression was also investigated by real-time RT-PCR. It was found that silibinin caused G1 cell cycle arrest in MCF-7 and MCF-10A cells but had no effect on the T47D cell cycle. Silibinin induced cytotoxic and apoptotic effects in T47D cells more than the MCF-7 cells and had no cytotoxic effect in MCF-10A cells under the same conditions. Silibinin upregulated PTEN in MCF-7 and caused slightly increased P21 mRNA expression in T47D cells and slightly increased PTEN and P21 expression in MCF-10A cells. Bcl-2 expression decreased in all of the examined cells under silibinin treatment. P27 mRNA expression upregulated in T47D and MCF-10A cells under silibinin treatment. PTEN mRNA in T47D and P21 and P27 mRNAsin MCF-7 were not affected by silibinin. These results suggest that silibinin has mostly different inhibitory effects in breast cancer cells and might be an effective anticancer agent for some cells linked to influence on cell cycle progression.

Inhibition of highly productive HIV-1 infection in T cells, primary human macrophages, microglia, and astrocytes by Sargassum fusiforme

PubMed Central

Paskaleva, Elena E; Lin, Xudong; Li, Wen; Cotter, Robin; Klein, Michael T; Roberge, Emily; Yu, Er K; Clark, Bruce; Veille, Jean-Claude; Liu, Yanze; Lee, David Y-W; Canki, Mario

2006-01-01

Background The high rate of HIV-1 mutation and increasing resistance to currently available antiretroviral (ART) therapies highlight the need for new antiviral agents. Products derived from natural sources have been shown to inhibit HIV-1 replication during various stages of the virus life cycle, and therefore represent a potential source of novel therapeutic agents. To expand our arsenal of therapeutics against HIV-1 infection, we investigated aqueous extract from Sargassum fusiforme (S. fusiforme) for ability to inhibit HIV-1 infection in the periphery, in T cells and human macrophages, and for ability to inhibit in the central nervous system (CNS), in microglia and astrocytes. Results S. fusiforme extract blocked HIV-1 infection and replication by over 90% in T cells, human macrophages and microglia, and it also inhibited pseudotyped HIV-1 (VSV/NL4-3) infection in human astrocytes by over 70%. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5)-tropic HIV-1, was dose dependant and long lasting, did not inhibit cell growth or viability, was not toxic to cells, and was comparable to inhibition by the nucleoside analogue 2', 3'-didoxycytidine (ddC). S. fusiforme treatment blocked direct cell-to-cell infection spread. To investigate at which point of the virus life cycle this inhibition occurs, we infected T cells and CD4-negative primary human astrocytes with HIV-1 pseudotyped with envelope glycoprotein of vesicular stomatitis virus (VSV), which bypasses the HIV receptor requirements. Infection by pseudotyped HIV-1 (VSV/NL4-3) was also inhibited in a dose dependant manner, although up to 57% less, as compared to inhibition of native NL4-3, indicating post-entry interferences. Conclusion This is the first report demonstrating S. fusiforme to be a potent inhibitor of highly productive HIV-1 infection and replication in T cells, in primary human macrophages, microglia, and astrocytes. Results with VSV/NL4-3 infection, suggest inhibition of both entry and post-entry events of the virus life cycle. Absence of cytotoxicity and high viability of treated cells also suggest that S. fusiforme is a potential source of novel naturally occurring antiretroviral compounds that inhibit HIV-1 infection and replication at more than one site of the virus life cycle. PMID:16725040

Tea polyphenols induce S phase arrest and apoptosis in gallbladder cancer cells

PubMed Central

Wang, Jiaqi; Pan, Yixuan; Hu, Jiacheng; Ma, Qiang; Xu, Yi; Zhang, Yijian; Zhang, Fei; Liu, Yingbin

2018-01-01

Gallbladder cancer (GBC) is the most common malignancy in the biliary tract. Without effective treatment, its prognosis is notoriously poor. Tea polyphenols (TPs) have many pharmacological and health benefits, including antioxidant, anti-inflammatory, anti-tumor, anti-thrombotic, antibacterial, and vasodilatory properties. However, the anti-cancer effect of TPs in human gallbladder cancer has not yet been determined. Cell viability and colony formation assay were used to investigate the cell growth. Cell cycle and apoptosis were evaluated by flow cytometry analysis. Western blot assay was used to detect the expression of proteins related to cell cycle and apoptosis. Human tumor xenografts were used to examine the effect of TPs on gallbladder cancer cells in vivo. TPs significantly inhibited cell growth of gallbladder cancer cell lines in a dose- and time-dependent manner. Cell cycle progression in GBC cells was blocked at the S phase by TPs. TPs also induced mitochondrial-related apoptosis in GBC cells by upregulating Bax, cleaved caspase-3, and cleaved PARP expressions and downregulating Bcl-2, cyclin A, and Cdk2 expressions. The effects of TPs on GBC were further proven in vivo in a mouse xenograft model. Our study is the first to report that TPs inhibit GBC cell growth and these compounds may have potential as novel therapeutic agents for treating gallbladder cancer. PMID:29513793

Development of cell-based quantitative evaluation method for cell cycle-arrest type cancer drugs for apoptosis by high precision surface plasmon resonance sensor

NASA Astrophysics Data System (ADS)

Ona, Toshihiro; Nishijima, Hiroshi; Kosaihira, Atsushi; Shibata, Junko

2008-04-01

In vitro rapid and quantitative cell-based assay is demanded to verify the efficacy prediction of cancer drugs since a cancer patient may have unconventional aspects of tumor development. Here, we show the rapid and non-label quantitative verifying method and instrumentation of apoptosis for cell cycle-arrest type cancer drugs (Roscovitine and D-allose) by reaction analysis of living liver cancer cells cultured on a sensor chip with a newly developed high precision (50 ndeg s -1 average fluctuation) surface plasmon resonance (SPR) sensor. The time-course cell reaction as the SPR angle change rate for 10 min from 30 min cell culture with a drug was significantly related to cell viability. By the simultaneous detection of differential SPR angle change and fluorescence by specific probes using the new instrument, the SPR angle was related to the nano-order potential decrease in inner mitochondrial membrane potential. The results obtained are universally valid for the cell cycle-arrest type cancer drugs, which mediate apoptosis through different cell-signaling pathways, by a liver cancer cell line of Hep G2 (P<0.001). This system towards the application to evaluate personal therapeutic potentials of drugs using cancer cells from patients in clinical use.

Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM–Chk1/2–Cdc25C pathway

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

Ma, Yong-Cheng; Su, Nan; Shi, Xiao-Jing

2015-01-15

Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore,more » Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway. - Highlights: • Jaridonin induced G2/M phase arrest through induction of redox imbalance. • Jaridonin increased the level of ROS through depleting glutathione in cell. • ATM–Chk1/2–Cdc25C were involved in Jaridonin-induced cell cycle arrest. • Jaridonin selectively inhibited cancer cell viability and cell cycle progression.« less

A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro.

PubMed

Zhao, Li-Ping; Xu, Tian-Min; Kan, Mu-Jie; Xiao, Ye-Chen; Cui, Man-Hua

2016-05-01

Urokinase-type plasminogen activator (uPA) acts by breaking down the basement membrane and is involved in cell proliferation, migration and invasion. These actions are mediated by binding to the uPA receptor (uPAR) via its growth factor domain (GFD). The present study evaluated the effects of uPAg-KPI, a fusion protein of uPA-GFD and a kunitz protease inhibitor (KPI) domain that is present in the amyloid β-protein precursor. Using SKOV-3 cells, an ovarian cancer cell line, we examined cell viability, migration, invasion and also protein expression. Furthermore, we examined wound healing, and migration and invasion using a Transwell assay. Our data showed that uPAg-KPI treatment reduced the viability of ovarian cancer SKOV-3 cells in both a concentration and time-dependent manner by arresting tumor cells at G1/G0 phase of the cell cycle. The IC50 of uPAg-KPI was 0.5 µg/µl after 48 h treatment. At this concentration, uPAg-KPI also inhibited tumor cell colony formation, wound closure, as well as cell migration and invasion capacity. At the protein level, western blot analysis demonstrated that uPAg-KPI exerted no significant effect on the expression of total extracellular signal-regulated kinase (ERK)1/ERK2 and AKT, whereas it suppressed levels of phosphorylated ERK1/ERK2 and AKT. Thus, we suggest that this novel uPAg-KPI fusion protein reduced cell viability, colony formation, wound healing and the invasive ability of human ovarian cancer SKOV-3 cells in vitro by regulating ERK and AKT signaling. Further studies using other cell lines will confirm these findings.

A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro

PubMed Central

ZHAO, LI-PING; XU, TIAN-MIN; KAN, MU-JIE; XIAO, YE-CHEN; CUI, MAN-HUA

2016-01-01

Urokinase-type plasminogen activator (uPA) acts by breaking down the basement membrane and is involved in cell proliferation, migration and invasion. These actions are mediated by binding to the uPA receptor (uPAR) via its growth factor domain (GFD). The present study evaluated the effects of uPAg-KPI, a fusion protein of uPA-GFD and a kunitz protease inhibitor (KPI) domain that is present in the amyloid β-protein precursor. Using SKOV-3 cells, an ovarian cancer cell line, we examined cell viability, migration, invasion and also protein expression. Furthermore, we examined wound healing, and migration and invasion using a Transwell assay. Our data showed that uPAg-KPI treatment reduced the viability of ovarian cancer SKOV-3 cells in both a concentration and time-dependent manner by arresting tumor cells at G1/G0 phase of the cell cycle. The IC50 of uPAg-KPI was 0.5 µg/µl after 48 h treatment. At this concentration, uPAg-KPI also inhibited tumor cell colony formation, wound closure, as well as cell migration and invasion capacity. At the protein level, western blot analysis demonstrated that uPAg-KPI exerted no significant effect on the expression of total extracellular signal-regulated kinase (ERK)1/ERK2 and AKT, whereas it suppressed levels of phosphorylated ERK1/ERK2 and AKT. Thus, we suggest that this novel uPAg-KPI fusion protein reduced cell viability, colony formation, wound healing and the invasive ability of human ovarian cancer SKOV-3 cells in vitro by regulating ERK and AKT signaling. Further studies using other cell lines will confirm these findings. PMID:27035617

A novel resveratrol-salinomycin combination sensitizes ER-positive breast cancer cells to apoptosis.

PubMed

Venkatadri, Rajkumar; Iyer, Anand Krishnan V; Kaushik, Vivek; Azad, Neelam

2017-08-01

Resveratrol is a dietary compound that has been widely reported for its anticancer activities. However, successful extrapolation of its effects to pre-clinical studies is met with limited success due to inadequate bioavailability. We investigated the potential of combination therapy to improve the efficacy of resveratrol in a more physiologically relevant dose range. The effect of resveratrol on canonical Wnt signaling was evaluated by Western blotting. Wnt modulators HLY78 (activator) and salinomycin (inhibitor) were evaluated in combination with resveratrol for their effect on breast cancer cell viability (MTT assay), cell cycle progression and apoptosis (Western blotting). Bliss independency model was used to evaluate combinatorial effects of resveratrol-salinomycin combination. Resveratrol downregulated canonical Wnt signaling proteins in treated breast cancer cells (MCF-7, MDA-MB-231 and MDA-MB-468) in the dose range of 50-200μM, which also affected cellular viability. However, at very low doses (0-50μM), resveratrol exhibited no cellular toxicity. Co-treatment with salinomycin significantly potentiated the anti-cancer effects of resveratrol, whereas HLY78 co-treatment had minimal effect. Bliss independency model revealed that Wnt inhibition synergistically potentiates the effects of resveratrol in MCF-7 and BT474 cells. Significantly downregulated canonical Wnt signaling proteins and marker of epithelial-mesenchymal transition (EMT), vimentin were observed in cells treated with resveratrol-salinomycin combination. Cell cycle arrest, caspase activation and apoptosis induction in cells treated with resveratrol-salinomycin combination further confirmed the efficacy of the combination. We report a novel resveratrol-salinomycin combination for targeting ER-positive breast cancer cells and present evidence for successful pre-clinical implementation of resveratrol. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

A study of the effects of citrate-coated silver nanoparticles on RAW 264.7 cells using a toolbox of cytotoxic endpoints

NASA Astrophysics Data System (ADS)

Bastos, V.; Duarte, I. F.; Santos, C.; Oliveira, H.

2017-05-01

Citrate-coated silver nanoparticles (citrate-AgNPs) are among the most commonly used nanomaterials, widely present in industrial and biomedical products. In this study, the cytotoxicity of 30-nm citrate-AgNPs on the macrophage cell line RAW 264.7 was evaluated, using a battery of cytotoxicity endpoints (viability, oxidative stress, and cytostaticity/clastogenicity), at 24 and 48 h of exposure. Citrate-AgNPs decreased cell proliferation and viability only at 75 μg/mL, suggesting a low sensitivity of RAW cells to lower doses of these AgNPs. After 24 h of exposure, ROS content decreased in cells exposed to 60 μg/mL AgNPs (IC20 value), corroborating the high tolerance of these cells to citrate-AgNPs. However, these cells suffered an impairment of the cell cycle, shown by an increase at the sub-G1 phase. This increase of the sub-G1 population was correlated with an increase of DNA fragmentation, suggesting an increase of apoptosis. Thus, our data are important to understand the effects of low concentrations (IC20) of citrate-AgNPs on in vitro vital macrophage functions.

The genetic covariance between life cycle stages separated by metamorphosis.

PubMed

Aguirre, J David; Blows, Mark W; Marshall, Dustin J

2014-08-07

Metamorphosis is common in animals, yet the genetic associations between life cycle stages are poorly understood. Given the radical changes that occur at metamorphosis, selection may differ before and after metamorphosis, and the extent that genetic associations between pre- and post-metamorphic traits constrain evolutionary change is a subject of considerable interest. In some instances, metamorphosis may allow the genetic decoupling of life cycle stages, whereas in others, metamorphosis could allow complementary responses to selection across the life cycle. Using a diallel breeding design, we measured viability at four ontogenetic stages (embryo, larval, juvenile and adult viability), in the ascidian Ciona intestinalis and examined the orientation of additive genetic variation with respect to the metamorphic boundary. We found support for one eigenvector of G: (gobsmax ), which contrasted larval viability against embryo viability and juvenile viability. Target matrix rotation confirmed that while gobsmax shows genetic associations can extend beyond metamorphosis, there is still considerable scope for decoupled phenotypic evolution. Therefore, although genetic associations across metamorphosis could limit that range of phenotypes that are attainable, traits on either side of the metamorphic boundary are capable of some independent evolutionary change in response to the divergent conditions encountered during each life cycle stage. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

First siRNA library screening in hard-to-transfect HUVEC cells

PubMed Central

Zumbansen, Markus; Altrogge, Ludger M; Spottke, Nicole UE; Spicker, Sonja; Offizier, Sheila M; Domzalski, Sandra BS; St Amand, Allison L; Toell, Andrea; Leake, Devin; Mueller-Hartmann, Herbert A

2010-01-01

Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa® Nucleofector® 96-well Shuttle® System for siRNA screening in primary cells. Lonza's Clonetics® HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME® siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector® 96-well Shuttle® System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection. PMID:20628494

Curcumin-mediated regulation of Notch1/hairy and enhancer of split-1/survivin: molecular targeting in cholangiocarcinoma.

PubMed

Koprowski, Steven; Sokolowski, Kevin; Kunnimalaiyaan, Selvi; Gamblin, T Clark; Kunnimalaiyaan, Muthusamy

2015-10-01

Cholangiocarcinoma (CCA) is highly malignant and characterized by poor prognosis with chemotherapeutic resistance. Therefore, continued development of novel, effective approaches are needed. Notch expression is markedly upregulated in CCA, but the utility of Notch1 inhibition is not defined. Based on recent findings, we hypothesized that curcumin, a polyphenolic phytochemical, suppresses CCA growth in vitro via inhibition of Notch1 signaling. Established CCA cell lines CCLP-1 and SG-231 were treated with varying concentrations of curcumin (0-20 μM). Viability was assessed through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and clonogenic assays. Evaluation of apoptosis was determined via Western analysis for apoptotic markers and Caspase-Glo 3/7 assay. Cell lysates were further analyzed via Western blotting for Notch1/HES-1/survivin pathway expression, cell cycle progression, and survival. Curcumin-treated CCA cells exhibited reduced viability compared with control treatment. Statistically significant reductions in cell viability were observed with curcumin treatment at concentrations of 7.5, 10, and 15 μM by approximately 10%, 48%, and 56% for CCLP-1 and 13%, 25%, and 50% for SG-231, respectively. On Western analysis, concentrations of ≥10 μM showed reductions in Notch1, HES-1, and survivin. Apoptosis was evidenced by an increase in expression of cleaved poly [ADP] ribose polymerase and an increase in caspase activity. Cyclin D1 (cell cycle progression) expression levels were also reduced with treatment. Curcumin effectively induces CCA (CCLP-1 and SG-231) growth suppression and apoptosis at relatively low treatment concentrations when compared with the previous research. A concomitant reduction of Notch1, HES-1, and survivin expression in CCA cell lines provides novel evidence for a potential antitumorigenic mechanism-of-action. To our knowledge, this is the first report showing reduction in HES-1 expression via protein analysis after treatment with curcumin. Such findings merit further investigation of curcumin-mediated inhibition of Notch signaling in CCA either alone or in combination with chemotherapeutic agents. Copyright © 2015 Elsevier Inc. All rights reserved.

Tracking chemical changes in a live cell: Biomedical applications of SR-FTIR spectromicroscopy

DOE PAGES

Holman, Hoi-Ying N.; Martin, Michael C.; McKinney, Wayne R.

2003-01-01

Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy is a newly emerging bioanalytical and imaging tool. This unique technique provides mid-infrared (IR) spectra, hence chemical information, with high signal-to-noise at spatial resolutions as fine as 3 to 10 microns. Thus it enables researchers to locate, identify, and track specific chemical events within an individual living mammalian cell. Mid-IR photons are too low in energy (0.05-0.5 eV) to either break bonds or to cause ionization. In this review, we show that the synchrotron IR beam has no detectable effects on the short- and long-term viability, reproductive integrity, cell-cycle progression, and mitochondrial metabolismmore » in living human cells, and produces only minimal sample heating (<0.5°C). We will then present several examples demonstrating the application potentials of SR-FTIR spectromicroscopy in biomedical research. These will include monitoring living cells progressing through the cell cycle, including death, and cells reacting to dilute concentrations of toxins.« less

American cranberry (Vaccinium macrocarpon) extract affects human prostate cancer cell growth via cell cycle arrest by modulating expression of cell cycle regulators.

PubMed

Déziel, Bob; MacPhee, James; Patel, Kunal; Catalli, Adriana; Kulka, Marianna; Neto, Catherine; Gottschall-Pass, Katherine; Hurta, Robert

2012-05-01

Prostate cancer is one of the most common cancers in the world, and its prevalence is expected to increase appreciably in the coming decades. As such, more research is necessary to understand the etiology, progression and possible preventative measures to delay or to stop the development of this disease. Recently, there has been interest in examining the effects of whole extracts from commonly harvested crops on the behaviour and progression of cancer. Here, we describe the effects of whole cranberry extract (WCE) on the behaviour of DU145 human prostate cancer cells in vitro. Following treatment of DU145 human prostate cancer cells with 10, 25 and 50 μg ml⁻¹ of WCE, respectively for 6 h, WCE significantly decreased the cellular viability of DU145 cells. WCE also decreased the proportion of cells in the G2-M phase of the cell cycle and increased the proportion of cells in the G1 phase of the cell cycle following treatment of cells with 25 and 50 μg ml⁻¹ treatment of WCE for 6 h. These alterations in cell cycle were associated with changes in cell cycle regulatory proteins and other cell cycle associated proteins. WCE decreased the expression of CDK4, cyclin A, cyclin B1, cyclin D1 and cyclin E, and increased the expression of p27. Changes in p16(INK4a) and pRBp107 protein expression levels also were evident, however, the changes noted in p16(INK4a) and pRBp107 protein expression levels were not statistically significant. These findings demonstrate that phytochemical extracts from the American cranberry (Vaccinium macrocarpon) can affect the behaviour of human prostate cancer cells in vitro and further support the potential health benefits associated with cranberries.

Alpha-ketoglutarate enhances freeze-thaw tolerance and prevents carbohydrate-induced cell death of the yeast Saccharomyces cerevisiae.

PubMed

Bayliak, Maria M; Hrynkiv, Olha V; Knyhynytska, Roksolana V; Lushchak, Volodymyr I

2018-01-01

Stress resistance and fermentative capability are important quality characteristics of baker's yeast. In the present study, we examined protective effects of exogenous alpha-ketoglutarate (AKG), an intermediate of the tricarboxylic acid cycle and amino acid metabolism, against freeze-thaw and carbohydrate-induced stresses in the yeast Saccharomyces cerevisiae. Growth on AKG-supplemented medium prevented a loss of viability and improved fermentative capacity of yeast cells after freeze-thaw treatment. The cells grown in the presence of AKG had higher levels of amino acids (e.g., proline), higher metabolic activity and total antioxidant capacity, and higher activities of catalase, NADP-dependent glutamate dehydrogenase and glutamine synthase compared to control ones. Both synthesis of amino acids and enhancement of antioxidant system capacity could be involved in AKG-improved freeze-thaw tolerance in S. cerevisiae. Cell viability dramatically decreased under incubation of stationary-phase yeast cells in 2% glucose or fructose solutions (in the absence of the other nutrients) as compared with incubation in distilled water or in 10 mM AKG solution. The decrease in cell viability was accompanied by acidification of the medium, and decrease in cellular respiration, aconitase activity, and levels of total protein and free amino acids. The supplementation with 10 mM AKG effectively prevented carbohydrate-induced yeast death. Protective mechanisms of AKG could be associated with the intensification of respiration and prevention of decreasing protein level as well as with direct antioxidant AKG action.

Cannabidiol Reduces Leukemic Cell Size - But Is It Important?

PubMed

Kalenderoglou, Nikoletta; Macpherson, Tara; Wright, Karen L

2017-01-01

The anti-cancer effect of the plant-derived cannabinoid, cannabidiol, has been widely demonstrated both in vivo and in vitro . However, this body of preclinical work has not been translated into clinical use. Key issues around this failure can be related to narrow dose effects, the cell model used and incomplete efficacy. A model of acute lymphoblastic disease, the Jurkat T cell line, has been used extensively to study the cannabinoid system in the immune system and cannabinoid-induced apoptosis. Using these cells, this study sought to investigate the outcome of those remaining viable cells post-treatment with cannabidiol, both in terms of cell size and tracking any subsequent recovery. The phosphorylation status of the mammalian Target of Rapamycin (mTOR) signaling pathway and the downstream target ribosomal protein S6, were measured. The ability of cannabidiol to exert its effect on cell viability was also evaluated in physiological oxygen conditions. Cannabidiol reduced cell viability incompletely, and slowed the cell cycle with fewer cells in the G2/M phase of the cell cycle. Cannabidiol reduced phosphorylation of mTOR, PKB and S6 pathways related to survival and cell size. The remaining population of viable cells that were cultured in nutrient rich conditions post-treatment were able to proliferate, but did not recover to control cell numbers. However, the proportion of viable cells that were gated as small, increased in response to cannabidiol and normally sized cells decreased. This proportion of small cells persisted in the recovery period and did not return to basal levels. Finally, cells grown in 12% oxygen (physiological normoxia) were more resistant to cannabidiol. In conclusion, these results indicate that cannabidiol causes a reduction in cell size, which persists post-treatment. However, resistance to cannabidiol under physiological normoxia for these cells would imply that cannabidiol may not be useful in the clinic as an anti-leukemic agent.

Cannabidiol Reduces Leukemic Cell Size – But Is It Important?

PubMed Central

Kalenderoglou, Nikoletta; Macpherson, Tara; Wright, Karen L.

2017-01-01

The anti-cancer effect of the plant-derived cannabinoid, cannabidiol, has been widely demonstrated both in vivo and in vitro. However, this body of preclinical work has not been translated into clinical use. Key issues around this failure can be related to narrow dose effects, the cell model used and incomplete efficacy. A model of acute lymphoblastic disease, the Jurkat T cell line, has been used extensively to study the cannabinoid system in the immune system and cannabinoid-induced apoptosis. Using these cells, this study sought to investigate the outcome of those remaining viable cells post-treatment with cannabidiol, both in terms of cell size and tracking any subsequent recovery. The phosphorylation status of the mammalian Target of Rapamycin (mTOR) signaling pathway and the downstream target ribosomal protein S6, were measured. The ability of cannabidiol to exert its effect on cell viability was also evaluated in physiological oxygen conditions. Cannabidiol reduced cell viability incompletely, and slowed the cell cycle with fewer cells in the G2/M phase of the cell cycle. Cannabidiol reduced phosphorylation of mTOR, PKB and S6 pathways related to survival and cell size. The remaining population of viable cells that were cultured in nutrient rich conditions post-treatment were able to proliferate, but did not recover to control cell numbers. However, the proportion of viable cells that were gated as small, increased in response to cannabidiol and normally sized cells decreased. This proportion of small cells persisted in the recovery period and did not return to basal levels. Finally, cells grown in 12% oxygen (physiological normoxia) were more resistant to cannabidiol. In conclusion, these results indicate that cannabidiol causes a reduction in cell size, which persists post-treatment. However, resistance to cannabidiol under physiological normoxia for these cells would imply that cannabidiol may not be useful in the clinic as an anti-leukemic agent. PMID:28392768

Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function.

PubMed

Kwiatkowski, Nicholas; Jelluma, Nannette; Filippakopoulos, Panagis; Soundararajan, Meera; Manak, Michael S; Kwon, Mijung; Choi, Hwan Geun; Sim, Taebo; Deveraux, Quinn L; Rottmann, Sabine; Pellman, David; Shah, Jagesh V; Kops, Geert J P L; Knapp, Stefan; Gray, Nathanael S

2010-05-01

Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and for the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, the development of a potent, selective small-molecule inhibitor of Mps1 should facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 cocrystal structures of new, selective small-molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells having extra centrosomes (an abnormality found in some cancers), Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.

Antioxidant and Renoprotective Effects of Mushroom Extract: Implication in Prevention of Nephrolithiasis

PubMed Central

Schulman, Ariel; Chaimowitz, Matthew; Choudhury, Muhammad; Eshghi, Majid; Konno, Sensuke

2016-01-01

Background The pathogenesis of nephrolithiasis (kidney stone) remains elusive, while several therapeutic options are available but not effective as we expected. Accumulating data yet suggest that oxidative stress (generation of oxygen free radicals) may play a primary role in its occurrence. Particularly, calcium oxalate (CaOx) is a key element in the most common form (> 75%) of kidney stones, and its crystal form known as CaOx monohydrate (COM) has been shown to exert oxidative stress, facilitating CaOx stone formation. Hence, diminishing oxidative stress with certain antioxidants could be a potential strategic approach. We are interested in a bioactive extract of Poria mushroom, PE, which has been shown to have antioxidant and renoprotective activities. Accordingly, we investigated if PE might have antioxidant activity that would have implication in prevention of kidney stone formation. Methods Renal epithelial LLC-PK1 cells were employed and exposed to COM or hydrogen peroxide (H2O2) as a positive control capable of exerting oxidative stress. Possible antioxidant and protective effects of PE against oxidative stress (exerted by COM or H2O2) were assessed by cell viability test and lipid peroxidation (LPO) assay. To explore its protective mechanism, two glycolytic parameters, hexokinase (HK) activity and ATP synthesis, were examined and cell cycle analysis was also performed. Results Both H2O2 and COM led to a significant (P < 0.05) reduction in cell viability, accompanied by severe oxidative stress assessed by LPO assay. Such oxidative stress also caused the significant decline in HK activity and cellular ATP level, indicating the inhibition of glycolysis. Cell cycle analysis further indicated that oxidative stress interfered with cell cycle, inducing a G1 cell cycle arrest that presumably results in the cessation of cell proliferation. However, PE was capable of significantly preventing or diminishing all these cellular effects mediated through oxidative stress (exerted by H2O2 and COM). Conclusions The present study shows that the mushroom extract PE appears to have antioxidant and renoprotective effects against oxidative stress exerted by COM in renal cells. Therefore, PE with antioxidant activity is considered a promising natural agent that may have clinical implications in prevention of nephrolithiasis primarily induced by oxidative stress. PMID:27829958

Encapsulation in lipid-core nanocapsules overcomes lung cancer cell resistance to tretinoin.

PubMed

Schultze, Eduarda; Ourique, Aline; Yurgel, Virginia Campello; Begnini, Karine Rech; Thurow, Helena; de Leon, Priscila Marques Moura; Campos, Vinicius Farias; Dellagostin, Odir Antônio; Guterres, Silvia R; Pohlmann, Adriana R; Seixas, Fabiana Kömmling; Beck, Ruy Carlos Ruver; Collares, Tiago

2014-05-01

Tretinoin is a retinoid derivative that has an antiproliferative effect on several kinds of tumours. Human lung adenocarcinoma epithelial cell lines (A549) exhibit a profound resistance to the effects of tretinoin. Nanocarriers seem to be a good alternative to overcomecellular resistance to drugs. The aim of this study was to test whether tretinoin-loaded lipid-core nanocapsules exert anantitumor effect on A549 cells. A549 cells were incubated with free tretinoin (TTN), blank nanocapsules (LNC) and tretinoin-loaded lipid-core nanocapsules (TTN-LNC). Data from evaluation of DNA content and Annexin V binding assay by flow cytometry showed that TTN-LNC induced apoptosis and cell cycle arrest at the G1-phase while TTN did not. TTN-LNC showed higher cytotoxic effects than TTN on A549 cells evaluated by MTT and LIVE/DEAD cell viability assay. Gene expression profiling identified up-regulated expression of gene p21 by TTN-LNC, supporting the cell cycle arrest effect. These results showed for the first time that TTN-LNC are able to overcome the resistance of adenocarcinoma cell line A549 to treatment with TTN by inducing apoptosis and cell cycle arrest, providing support for their use in applications in lung cancer therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

A chemical screen in diverse breast cancer cell lines reveals genetic enhancers and suppressors of sensitivity to PI3K isotype-selective inhibition

PubMed Central

Torbett, Neil E; Luna, Antonio; Knight, Zachary A.; Houk, Andrew; Moasser, Mark; Weiss, William; Shokat, Kevan M.; Stokoe, David

2011-01-01

Synopsis The Phosphoinositide-3-kinase (PI3K) pathway regulates cell proliferation, survival and migration and is consequently of great interest for targeted cancer therapy. Using a panel of small molecule PI3K isoform-selective inhibitors in a diverse set of breast cancer cell lines, we demonstrate that the biochemical and biological responses were highly variable and dependent on the genetic alterations present. p110α inhibitors were generally effective in inhibiting the phosphorylation of Akt and S6, two downstream components of PI3K signaling, in most cell lines examined. In contrast, 110β selective inhibitors only reduced Akt phosphorylation in PTEN mutant cell lines, and was associated with a lesser decrease in S6 phosphorylation. PI3K inhibitors reduced cell viability by causing a cell cycle arrest in the G1 phase of the cell cycle, with multi-targeted inhibitors causing the most potent effects. Cells expressing mutant Ras were resistant to the cell cycle effects of PI3K inhibition, which could be reversed using inhibitors of Ras signaling pathways. Taken together our data indicates that these compounds, alone or in suitable combinations, may be useful as breast cancer therapeutics, when used in appropriate genetic contexts. PMID:18498248

Influence of carbon sources on the viability and resuscitation of Acetobacter senegalensis during high-temperature gluconic acid fermentation.

PubMed

Shafiei, Rasoul; Zarmehrkhorshid, Raziyeh; Mounir, Majid; Thonart, Philippe; Delvigne, Frank

2017-05-01

Much research has been conducted about different types of fermentation at high temperature, but only a few of them have studied cell viability changes during high-temperature fermentation. In this study, Acetobacter senegalensis, a thermo-tolerant strain, was used for gluconic acid production at 38 °C. The influences of different carbon sources and physicochemical conditions on cell viability and the resuscitation of viable but nonculturable (VBNC) cells formed during fermentation were studied. Based on the obtained results, A. senegalensis could oxidize 95 g l - 1 glucose to gluconate at 38 °C (pH 5.5, yield 83%). However, despite the availability of carbon and nitrogen sources, the specific rates of glucose consumption (q s ) and gluconate production (q p ) reduced progressively. Interestingly, gradual q s and q p reduction coincided with gradual decrease in cellular dehydrogenase activity, cell envelope integrity, and cell culturability as well as with the formation of VBNC cells. Entry of cells into VBNC state during stationary phase partly stemmed from high fermentation temperature and long-term oxidation of glucose, because just about 48% of VBNC cells formed during stationary phase were resuscitated by supplementing the culture medium with an alternative favorite carbon source (low concentration of ethanol) and/or reducing incubation temperature to 30 °C. This indicates that ethanol, as a favorable carbon source, supports the repair of stressed cells. Since formation of VBNC cells is often inevitable during high-temperature fermentation, using an alternative carbon source together with changing physicochemical conditions may enable the resuscitation of VBNC cells and their use for several production cycles.

Effect of sodium butyrate on cell proliferation and cell cycle in porcine intestinal epithelial (IPEC-J2) cells.

PubMed

Qiu, Yueqin; Ma, Xianyong; Yang, Xuefen; Wang, Li; Jiang, Zongyong

2017-04-01

Conflicting results have been reported that butyrate in normal piglets leads either to an increase or to a decrease of jejunal villus length, implying a possible effect on the proliferation of enterocytes. No definitive study was found for the biological effects of butyrate in porcine jejunal epithelial cells. The present study used IPEC-J2 cells, a non-transformed jejunal epithelial line to evaluate the direct effects of sodium butyrate on cell proliferation, cell cycle regulation, and apoptosis. Low concentrations (0.5 and 1 mM) of butyrate had no effect on cell proliferation. However, at 5 and 10 mM, sodium butyrate significantly decreased cell viability, accompanied by reduced levels of p-mTOR and PCNA protein. Sodium butyrate, in a dose-dependent manner, induced cell cycle arrest in G0/G1 phase and reduced the numbers of cells in S phase. In addition, relative expression of p21, p27, and pro-apoptosis bak genes, and protein levels of p21Waf1/Cip1, p27Kip1, cyclinD3, CDK4, and Cleave-caspase3 were increased by higher concentrations of sodium butyrate (1, 5, 10 mM), and the levels of cyclinD1 and CDK6 were reduced by 5 and 10 mM butyrate. Butyrate increased the phosphorylated form of the signaling molecule p38 and phosphorylated JNK. In conclusion, the present in vitro study indicated that sodium butyrate inhibited the proliferation of IPEC-J2 cells by inducing cell cycle arrest in the G0/G1 phase of cell cycles and by increasing apoptosis at high concentrations.

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

PubMed

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

2013-08-01

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

Effects of silk sericin on the proliferation and apoptosis of colon cancer cells.

PubMed

Kaewkorn, Waraporn; Limpeanchob, Nanteetip; Tiyaboonchai, Waree; Pongcharoen, Sutatip; Sutheerawattananonda, Manote

2012-01-01

Sericin is a silk protein woven from silkworm cocoons (Bombyx mori). In animal model, sericin has been reported to have anti-tumoral action against colon cancer. The mechanisms underlying the activity of sericin against cancer cells are not fully understood. The present study investigated the effects of sericin on human colorectal cancer SW480 cells compared to normal colonic mucosal FHC cells. Since the size of the sericin protein may be important for its activity, two ranges of molecular weight were tested. Sericin was found to decrease SW480 and FHC cell viability. The small sericin had higher anti-proliferative effects than that of the large sericin in both cell types. Increased apoptosis of SW480 cells is associated with increased caspase-3 activity and decreased Bcl-2 expression. The anti-proliferative effect of sericin was accompanied by cell cycle arrest at the S phase. Thus, sericin reduced SW480 cell viability by inducing cell apoptosis via caspase-3 activation and down-regulation of Bcl-2 expression. The present study provides scientific data that support the protective effect of silk sericin against cancer cells of the colon and suggests that this protein may have significant health benefits and could potentially be developed as a dietary supplement for colon cancer prevention.

Estrogen and progesterone promote breast cancer cell proliferation by inducing cyclin G1 expression.

PubMed

Tian, J-M; Ran, B; Zhang, C-L; Yan, D-M; Li, X-H

2018-01-23

Breast cancer is the most common cause of cancer among women in most countries (WHO). Ovarian hormone disorder is thought to be associated with breast tumorigenesis. The present study investigated the effects of estrogen and progesterone administration on cell proliferation and underlying mechanisms in breast cancer MCF-7 cells. It was found that a single administration of estradiol (E2) or progesterone increased MCF-7 cell viability in a dose-dependent manner and promoted cell cycle progression by increasing the percentage of cells in the G2/M phase. A combination of E2 and progesterone led to a stronger effect than single treatment. Moreover, cyclin G1 was up-regulated by E2 and/or progesterone in MCF-7 cells. After knockdown of cyclin G1 in MCF-7 cells using a specific shRNA, estradiol- and progesterone-mediated cell viability and clonogenic ability were significantly limited. Additionally, estradiol- and progesterone-promoted cell accumulation in the G2/M phase was reversed after knockdown of cyclin G1. These data indicated that estrogen and progesterone promoted breast cancer cell proliferation by inducing the expression of cyclin G1. Our data indicated that novel therapeutics against cyclin G1 are promising for the treatment of estrogen- and progesterone-mediated breast cancer progression.

A systematic analysis of the PARP protein family identifies new functions critical for cell physiology

PubMed Central

Vyas, Sejal; Chesarone-Cataldo, Melissa; Todorova, Tanya; Huang, Yun-Han; Chang, Paul

2013-01-01

The poly(ADP-ribose) polymerase (PARP) family of proteins use NAD+ as their substrate to modify acceptor proteins with adenosine diphosphate-ribose (ADPr) modifications. The function of most PARPs under physiological conditions is unknown. Here, to better understand this protein family, we systematically analyze the cell cycle localization of each PARP and of poly(ADP-ribose), a product of PARP activity, then identify the knock-down phenotype of each protein and perform secondary assays to elucidate function. We show that most PARPs are cytoplasmic, identify cell cycle differences in the ratio of nuclear to cytoplasmic poly(ADP-ribose), and identify four phenotypic classes of PARP function. These include the regulation of membrane structures, cell viability, cell division, and the actin cytoskeleton. Further analysis of PARP14 shows that it is a component of focal adhesion complexes required for proper cell motility and focal adhesion function. In total, we show that PARP proteins are critical regulators of eukaryotic physiology. PMID:23917125

Anticancer effect of Polyphyllin Ι in colorectal cancer cells through ROS-dependent autophagy and G2/M arrest mechanisms.

PubMed

Yu, Si; Wang, Lijiao; Cao, Zhixing; Gong, Daoyin; Liang, Qianyi; Chen, Hanting; Fu, Huizhu; Wang, Wenwen; Tang, Xue; Xie, Zihao; He, Yang; Peng, Cheng; Li, Yuzhi

2018-06-01

Polyphyllin Ι is a steroidal saponin isolated from the rhizoma of Paris polyphylla. In the present study, we aimed to investigate the anticancer effects of polyphyllin Ι in colorectal cancer and to elucidate the potential underlying molecular mechanisms. Using, CCK8 assay, flow cytometry, laser confocal microscope analysis and western blot, the anticancer effects of the polyphyllin Ι were analysed in colorectal cells. Our results indicate that polyphyllin Ι significantly decreased cell viability of HCT 116 cells and induced autophagy. Furthermore, we found that polyphyllin Ι induced autophagy in an ROS-dependent cell death and not related with PI3 K/AKT/mTOR pathway. We also provide evidence that excessive ROS triggered by polyphyllin Ι could induce G2/M phase arrest via regulating cycle proteins expression of cell cycle regulators, such as p21 and cyclinB1. In conclusion, polyphyllin Ι exhibit anticancer effect through ROS-dependent autophagy and induces G2/M arrest in colorectal cancer.

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

Kuncharin, Yanin; Sangphech, Naunpun; Kueanjinda, Patipark

The Notch signaling pathway plays important roles in tumorigenesis in a context-dependent manner. In human cervical cancer, alterations in Notch signaling have been reported, and both tumor-suppressing and tumor-promoting roles of Notch signaling have been proposed; however, the precise molecular mechanisms governing these roles in cervical cancer remain controversial. MAML is a transcriptional co-activator originally identified by its role in Notch signaling. Recent evidence suggests that it also plays a role in other signaling pathways, such as the p53 and {beta}-catenin pathways. MAML is required for stable formation of Notch transcriptional complexes at the promoters of Notch target genes. Chromosomalmore » translocations affecting MAML have been shown to promote tumorigenesis. In this study, we used a truncated dominant-negative MAML1 (DN-MAML) to investigate the role of MAML in HPV-positive cervical cancer cell lines. Three human cervical cancer cell lines (HeLa, SiHa and CaSki) expressed all Notch receptors and the Notch target genes Hes1 and MAML1. Among these 3 cell lines, constitutive appearance of cleaved Notch1 was found only in CaSki cells, which suggests that Notch1 is constitutively activated in this cell line. Gamma secretase inhibitor (GSI) treatment, which suppresses Notch receptor activation, completely abrogated this form of Notch1 but had no effect on cell viability. Overexpression of DN-MAML by retroviral transduction in CaSki cells resulted in significant decreases in the mRNA levels of Hes1 and Notch1 but had no effects on the levels of MAML1, p53 or HPV E6/E7. DN-MAML expression induced increased viability of CaSki cells without any effect on cell cycle progression or cell proliferation. In addition, clonogenic assay experiments revealed that overexpression of DN-MAML resulted in increased colony formation compared to the overexpression of the control vector. When the status of the NF-{kappa}B pathway was investigated, CaSki cells overexpressing DN-MAML exhibited loss of phospho-I{kappa}B{alpha}, decreased total I{kappa}B{alpha} and nuclear localization of NF-{kappa}B p65, which suggests that the NF-{kappa}B pathway is hyperactivated. Furthermore, increased level of cleaved Notch1 was detected when DN-MAML was expressed. When DN-MAML-overexpressing cells were treated with GSI, significantly decreased cell viability was observed, indicating that inhibition of Notch signaling using GSI treatment and DN-MAML expression negatively affects cell viability. Taken together, targeting Notch signaling using DN-MAML and GSI treatment may present a novel method to control cell viability in cervical cancer cells.« less

Double strand breaks and cell-cycle arrest induced by the cyanobacterial toxin cylindrospermopsin in HepG2 cells.

PubMed

Alja, Štraser; Filipič, Metka; Novak, Matjaž; Žegura, Bojana

2013-08-21

The newly emerging cyanobacterial cytotoxin cylindrospermopsin (CYN) is increasingly found in surface freshwaters, worldwide. It poses a potential threat to humans after chronic exposure as it was shown to be genotoxic in a range of test systems and is potentially carcinogenic. However, the mechanisms of CYN toxicity and genotoxicity are not well understood. In the present study CYN induced formation of DNA double strand breaks (DSBs), after prolonged exposure (72 h), in human hepatoma cells, HepG2. CYN (0.1-0.5 µg/mL, 24-96 h) induced morphological changes and reduced cell viability in a dose and time dependent manner. No significant increase in lactate dehydrogenase (LDH) leakage could be observed after CYN exposure, indicating that the reduction in cell number was due to decreased cell proliferation and not due to cytotoxicity. This was confirmed by imunocytochemical analysis of the cell-proliferation marker Ki67. Analysis of the cell-cycle using flow-cytometry showed that CYN has an impact on the cell cycle, indicating G0/G1 arrest after 24 h and S-phase arrest after longer exposure (72 and 96 h). Our results provide new evidence that CYN is a direct acting genotoxin, causing DSBs, and these facts need to be considered in the human health risk assessment.

Survival, recovery and microcystin release of Microcystis aeruginosa in cold or dark condition

NASA Astrophysics Data System (ADS)

Ding, Yi; Gan, Nanqin; Liu, Jin; Zheng, Lingling; Li, Lin; Song, Lirong

2017-03-01

Microcystis often dominates phytoplankton in eutrophic lakes and must survive a long period of cold or dark conditions. However, the survival strategies of Microcystis to withstand cold or dark stress are less well known. In this study, we conducted experiments on the responses of two toxic Microcystis aeruginosa strains (FACHB-905 and FACHB-915) and their microcystin release in conditions of low temperature (15°C or 4°C, with illumination) or darkness, and subsequent recovery in standard conditions (25°C with illumination). On exposure to 15°C, a small decrease in cell viability was observed, but the cell number increased gradually, suggesting that M. aeruginosa FACHB-905 and FACHB-915 cells seem in general tolerant in 15°C. Interestingly, our results show that a higher carotenoid content and microcystin release potentially enhance the fitness of surviving cells at 15°C. M. aeruginosa cells exposed to lower temperature light stress (4°C) did not completely lose viability and retained the ability to reinitiate growth. In darkness, the maximum quantum yield ( F v/ F m) and the maximum electron transport rate (ETRmax) values and cell viability of M. aeruginosa cells gradually decreased with time. During the recovery period, the photosynthetic efficiency of M. aeruginosa reverted to the normal level. Additionally, M. aeruginosa FACHB-905 and FACHB-915 exposed to low temperature had increased caspase-3-like activity and DNA fragmentation, which suggests the occurrence of a type of cell death in M. aeruginosa cells under cold stress similar to programmed cell death. Overall, our findings could confer certain advantages on the Microcystis for surviving cold or dark conditions encountered in the annual cycle, and help explain its repeated occurrence in water blooms in large and shallow lakes.

The induction of apoptosis and autophagy in human hepatoma SMMC-7721 cells by combined treatment with vitamin C and polysaccharides extracted from Grifola frondosa.

PubMed

Zhao, Fei; Zhao, Jin; Song, Lei; Zhang, Ya-Qing; Guo, Zhong; Yang, Ke-Hu

2017-11-01

Polysaccharides extracted from the mushroom Grifola frondosa (GFP) are a potential anticancer agent. The objective of this study was to investigate the effect of GFP and vitamin C (VC) alone and in combination on the viability of human hepatocarcinoma SMMC-7721 and HepG2 cells. Studies designed to detect cell apoptosis and autophagy were also conducted to investigate the mechanism. Results from the cell viability assay indicated that a combination of GFP (0.2 or 0.25 mg/mL) and VC (0.3 mmol/L) (GFP/VC) led to 52.73 and 53.93% reduction in cell viability of SMMC-7721 and HepG2 cells separately after 24 h. Flow cytometric analysis indicated that GFP/VC treatment induced cell cycle arrest at the G2/M phase, and apoptosis occurred in approximately 43.62 and 42.46% of the SMMC-7721 and HepG2 cells separately. Moreover, results of Hoechst33258 and monodansylcadaverine staining, and transmission electron microscopy, showed that GFP/VC induced apoptosis and autophagy in SMMC-7721 and HepG2 cells. Western blot analysis showed changes in the expression of apoptosis-related proteins [upregulation of BAX and caspase-3, downregulation of Bcl-2, and activation of poly-(ADP-ribose)-polymerase] and autophagy protein markers (upregulation of beclin-1 and microtubule-associated protein 1A/1B light chain-3). We also demonstrated that the expression of both Akt and p-Akt was enhanced, suggesting the PI3K/Akt/mTOR pathway might not be involved in this process. Our study shows that the combined application of GFP and VC induced cell apoptosis and autophagy in vitro, and might have antitumor activity in vivo.

Histone Deacetylase Inhibitors Enhance Cytotoxicity Towards Breast Tumors While Preserving the Wound-Healing Function of Adipose-Derived Stem Cells.

PubMed

Koko, Kiavash R; Chang, Shaohua; Hagaman, Ashleigh L; Fromer, Marc W; Nolan, Ryan S; Gaughan, John P; Zhang, Ping; Carpenter, Jeffrey P; Brown, Spencer A; Matthews, Martha; Bird, Dorothy

2017-06-01

Paclitaxel improves the oncologic response of breast cancer resections; however, it may negatively affect the wound-healing potential of human adipose-derived stem cells (hASCs) for fat grafting and reconstructive surgery. Histone deacetylase inhibitors (HDACis) modify the epigenetic regulation of gene expression and stabilize microtubules similarly to paclitaxel, thus, creating a synergistic mechanism of cell cycle arrest. We aim to combine these drugs to enhance cytotoxicity towards breast cancer cells, while preserving the wound-healing function of hASCs for downstream reconstructive applications. Triple negative breast cancer cells (MBA-MB-231) and hASCs (institutional review board-approved clinical isolates) were treated with a standard therapeutic dose of paclitaxel (1.0 μM) or with low-dose paclitaxel (0.1 μM) combined with the HDACi suberoylanilide hydroxamic acid or trichostatin A. Cell viability, gene expression, apoptosis, and wound-healing/migration were measured via methylthiazol tetrazolium assay, quantitative real-time polymerase chain reaction, annexin V assay, and fibroblast scratch assay, respectively. Combined HDACi and low-dose paclitaxel therapy maintained cytotoxicity towards breast cancer cells and preserved adipose-derived stem cell viability. Histone deacetylase inhibitor demonstrated selective anti-inflammatory effects on adipose-derived stem cell gene expression and decreased expression of the proapoptotic gene FAS. Furthermore, HDACi therapy did not increase relative apoptosis within hASCs. A scratch assay demonstrated enhanced wound healing among injured fibroblasts indirectly co-cultured with HDACi-treated hASCs. Combining HDACi with low-dose paclitaxel improved cytotoxicity towards breast cancer cells and preserved hASC viability. Furthermore, enhanced wound healing was observed by improved migration in a fibroblast scratch assay. These results suggest that the addition of HDACi to taxane chemotherapy regimens may improve oncologic results and wound-healing outcomes after reconstructive surgery.

Pentoxifylline and the proteasome inhibitor MG132 induce apoptosis in human leukemia U937 cells through a decrease in the expression of Bcl-2 and Bcl-XL and phosphorylation of p65.

PubMed

Bravo-Cuellar, Alejandro; Hernández-Flores, Georgina; Lerma-Díaz, José Manuel; Domínguez-Rodríguez, Jorge Ramiro; Jave-Suárez, Luis F; De Célis-Carrillo, Ruth; Aguilar-Lemarroy, Adriana; Gómez-Lomeli, Paulina; Ortiz-Lazareno, Pablo Cesar

2013-02-28

In Oncology, the resistance of the cancerous cells to chemotherapy continues to be the principal limitation. The nuclear factor-kappa B (NF-κB) transcription factor plays an important role in tumor escape and resistance to chemotherapy and this factor regulates several pathways that promote tumor survival including some antiapoptotic proteins such as Bcl-2 and Bcl-XL. In this study, we investigated, in U937 human leukemia cells, the effects of PTX and the MG132 proteasome inhibitor, drugs that can disrupt the NF-κB pathway. For this, we evaluated viability, apoptosis, cell cycle, caspases-3, -8, -9, cytochrome c release, mitochondrial membrane potential loss, p65 phosphorylation, and the modification in the expression of pro- and antiapoptotic genes, and the Bcl-2 and Bcl-XL antiapoptotic proteins. The two drugs affect the viability of the leukemia cells in a time-dependent manner. The greatest percentage of apoptosis was obtained with a combination of the drugs; likewise, PTX and MG132 induce G1 phase cell cycle arrest and cleavage of caspases -3,-8, -9 and cytochrome c release and mitochondrial membrane potential loss in U937 human leukemia cells. In these cells, PTX and the MG132 proteasome inhibitor decrease p65 (NF-κB subunit) phosphorylation and the antiapoptotic proteins Bcl-2 and Bcl-XL. We also observed, with a combination of these drugs overexpression of a group of the proapoptotic genes BAX, DIABLO, and FAS while the genes BCL-XL, MCL-1, survivin, IκB, and P65 were downregulated. The two drugs used induce apoptosis per se, this cytotoxicity was greater with combination of both drugs. These observations are related with the caspases -9, -3 cleavage and G1 phase cell cycle arrest, and a decrease in p65 phosphorylation and Bcl-2 and Bcl-XL proteins. As well as this combination of drugs promotes the upregulation of the proapoptotic genes and downregulation of antiapoptotic genes. These observations strongly confirm antileukemic potential.

Effect of sulphur mustard on human skin cell lines with differential agent sensitivity.

PubMed

Simpson, Rachel; Lindsay, Christopher D

2005-01-01

The ability of sulphur mustard (HD) to induce DNA damage places limits on the efficacy of approaches aimed at protecting human cells from the cytotoxic effects of HD using a variety of protective agents such as thiol-containing esters and protease inhibitors. In the present study, potential alternative strategies were investigated by examining the differential effects of HD on G361, SVK14, HaCaT and NCTC 2544 human skin cells. The G361 cell line was more resistant to the cytotoxic effects of HD than the NCTC, HaCaT and SVK14 cell lines at HD doses of >3 and <100 microM HD as determined by the MTT assay. At 72 h after exposure to 60 microM HD there was up to an 8.8-fold difference (P < 0.0001) between G361 and SVK14 cell culture viability. Buthionine sulphoximine (BSO) pretreatment increased the sensitivity of all four cell lines to HD. A substantial proportion of the resistance of G361 cells to HD was attributable to BSO-mediated effects on antioxidant-mediated metabolism, although G361 cultures still retained a high degree of viability at 30 microM HD following BSO pretreatment. Cell cycle analysis confirmed that SVK14 cells were relatively more sensitive to HD, as shown by the 2.1-fold reduction (P < 0.0001) in the percentage of cells in G0/G1 phase 24 h after HD exposure compared with control cultures. This compared well with a 1.2-fold increase (P < 0.05) in the percentage of G361 cells in G0/G1 phase following HD exposure, suggesting the existence of a more efficient G0/G1 checkpoint control mechanism in this cell line. Manipulation of the cell cycle using various modulating agents did not increase the resistance of cell lines to the cytotoxic effects of HD. Crown copyright 2005

Inhibition of EGFR nuclear shuttling decreases irradiation resistance in HeLa cells.

PubMed

Wei, Hong; Zhu, Zijie; Lu, Longtao

2017-01-01

Cervical cancer is a leading cause of mortality in women worldwide. The resistance to irradiation at the advanced stage is the main reason for the poor prognosis and high mortality. This work aims to elucidate the molecular mechanism underlying the radio-resistance. In this study, we determined the pEGFR-T654 and pDNA-PK-T2609 expression level changes in irradiated HeLa cells treated with T654 peptide, a nuclear localization signal (NLS) inhibitor, to inhibit EGFR nuclear transport. Cell viability, cell cycle and migratory capacity were analyzed. Xenograft animal model was used to evaluate the effect of EGFR nuclear transport inhibition on the tumor growth in vivo. The enhanced translocation of nuclear EGFR in the irradiated HeLa cells correlated with the increasing level of pEGFR-T654 and pDNA-PK-T2609. Inhibition of EGFR nuclear translocation by NLS peptide inhibitor attenuated DNA damage repair in the irradiated HeLa cells, decreased cell viability and promoted cell death through arrest at G0 phase. NLS peptide inhibitor impaired the migratory capacity of irradiated HeLa cells, and negatively affected tumorigenesis in xenograft mice. This work puts forward a potential molecular mechanism of the irradiation resistance in cervical cancer cells, providing a promising direction towards an efficient therapy of cervical cancer.

Flavagline analog FL3 induces cell cycle arrest in urothelial carcinoma cell of the bladder by inhibiting the Akt/PHB interaction to activate the GADD45α pathway.

PubMed

Yuan, Gangjun; Chen, Xin; Liu, Zhuowei; Wei, Wensu; Shu, Qinghai; Abou-Hamdan, Hussein; Jiang, Lijuan; Li, Xiangdong; Chen, Rixin; Désaubry, Laurent; Zhou, Fangjian; Xie, Dan

2018-02-07

Prohibitin 1 (PHB) is a potential target for the treatment of urothelial carcinoma of the bladder (UCB). FL3 is a newly synthesized agent that inhibits cancer cell proliferation by targeting the PHB protein; however, the effect of FL3 in UCB cells remains unexplored. FL3 was identified to be a potent inhibitor of UCB cell viability using CCK-8 (cell counting kit-8) assay. Then a series of in vitro and in vivo experiments were conducted to further demonstrate the inhibitory effect of FL3 on UCB cell proliferation and to determine the underlying mechanisms. FL3 inhibited UCB cell proliferation and growth both in vitro and in vivo. By targeting the PHB protein, FL3 inhibited the interaction of Akt and PHB as well as Akt-mediated PHB phosphorylation, which consequently decreases the localization of PHB in the mitochondria. In addition, FL3 treatment resulted in cell cycle arrest in the G2/M phase, and this inhibitory effect of FL3 could be mimicked by knockdown of PHB. Through the microarray analysis of mRNA expression after FL3 treatment and knockdown of PHB, we found that the mRNA expression of the growth arrest and DNA damage-inducible alpha (GADD45α) gene were significantly upregulated. When knocked down the expression of GADD45α, the inhibitory effect of FL3 on cell cycle was rescued, suggesting that FL3-induced cell cycle inhibition is GADD45α dependent. Our data provide that FL3 inhibits the interaction of Akt and PHB, which in turn activates the GADD45α-dependent cell cycle inhibition in the G2/M phase.

[Combined effects of interferon γ and γ ray irradiation on A549 cells in vitro].

PubMed

Xia, Hui; Zhang, Yi-ming; Yu, Chang-hai; Zhang, Wen; Zhang, Bao-shi; Fang, Fang

2012-02-07

To define the role of interferon-γ on radiotherapy of lung cancer and explore a new way to clinical treatment. A549 cells were exposed to γ ray with or without IFN-γ co-treatment. MTT assay was performed to evaluate cell viability. Western blot was used to observe the expression of P53 protein. The results showed that co-treatment of IFN-γ decreased the cell viability significantly compared with the γ ray irradiation group (71.4% ± 2.1% vs 44.1% ± 3.1%, n = 7, P < 0.01). In addition, the expression of P53 protein also increased significantly after co-treatment (P < 0.01); Furthermore, the cell cycle was changed obviously in co-treatment group compared with γ ray irradiation group, S phase increased (12.9% vs 20.9%, n = 5, P < 0.05) and also blocked the G2/M phase (28.8% vs 38.9%, n = 5, P < 0.05). The results suggested that γ ray irradiation combined with IFN-γ can increase the efficiency of radiotherapy on A549 cells and there is much broad prospect in the clinical treatment of lung cancer.

Retinoids, retinoid analogs, and lactoferrin interact and differentially affect cell viability of 2 bovine mammary cell types in vitro.

PubMed

Wang, Y; Baumrucker, C R

2010-07-01

Two bovine mammary cell types (BME-UV1 and MeBo cells) were used to evaluate the effect of natural retinoids, retinoid analogs, and bovine lactoferrin (bLf) on cell viability in vitro. Experiments with Alamar Blue showed a linear relationship between fluorescence and cell viability index. The BME-UV1 cells exhibited twice the metabolic activity but required half the doubling time of the MeBo cells. The BME-UV1 cells were very sensitive to all-trans retinoic acid (atRA) inhibition of cell viability (P<0.05) and exhibited a dose-dependent inhibition with 9-cisRA (9cRA; P<0.05). The MeBo cells exhibited some inhibition with these natural ligands (P<0.05), but they were not as sensitive. The addition of bLf had similar inhibitory effects (P<0.05) on cell viability of the 2 mammary cell types. Applications of RA receptor (RAR) agonist indicated that the stimulation of the RAR in both mammary cell types was highly effective in inhibition of cell viability (P<0.05), whereas the application of an RAR antagonist stimulated MeBo cell viability (P<0.05) and inhibited BME-UV1 cell viability (P<0.05). Finally, the use of the RAR antagonist in conjunction with bLf indicated a rescue of the bLf effect in the MeBo cells, suggesting that bLf is acting through the RAR receptor. Conversely, bLf reverted inhibition of cell viability by 9cRA in the BME-UV1 cell type (P<0.05). We conclude that RAR interaction in bovine mammary cell types regulates cell viability in vitro; we hypothesize that the natural ligands mediate regulation of bovine mammary cell viability in vivo and that bLf can either enhance or reverse the retinoid-induced inhibition of cell viability, depending on the type of bovine mammary cell studied.

Involvement of two specific causes of cell mortality in freeze-thaw cycles with freezing to -196 degrees C.

PubMed

Dumont, Frédéric; Marechal, Pierre-André; Gervais, Patrick

2006-02-01

The purpose of this study was to examine cell viability after freezing. Two distinct ranges of temperature were identified as corresponding to stages at which yeast cell mortality occurred during freezing to -196 degrees C. The upper temperature range was related to the temperature of crystallization of the medium, which was dependent on the solute concentration; in this range mortality was prevented by high solute concentrations, and the proportion of the medium in the vitreous state was greater than the proportion in the crystallized state. The lower temperature range was related to recrystallization that occurred during thawing. Mortality in this temperature range was increased by a high cooling rate and/or high solute concentration in the freezing medium and a low temperature (less than -70 degrees C). However, a high rate of thawing prevented yeast mortality in this lower temperature range. Overall, it was found that cell viability could be conserved better under freezing conditions by increasing the osmotic pressure of the medium and by using an increased warming rate.

Involvement of Two Specific Causes of Cell Mortality in Freeze-Thaw Cycles with Freezing to −196°C

PubMed Central

Dumont, Frédéric; Marechal, Pierre-André; Gervais, Patrick

2006-01-01

The purpose of this study was to examine cell viability after freezing. Two distinct ranges of temperature were identified as corresponding to stages at which yeast cell mortality occurred during freezing to −196°C. The upper temperature range was related to the temperature of crystallization of the medium, which was dependent on the solute concentration; in this range mortality was prevented by high solute concentrations, and the proportion of the medium in the vitreous state was greater than the proportion in the crystallized state. The lower temperature range was related to recrystallization that occurred during thawing. Mortality in this temperature range was increased by a high cooling rate and/or high solute concentration in the freezing medium and a low temperature (less than −70°C). However, a high rate of thawing prevented yeast mortality in this lower temperature range. Overall, it was found that cell viability could be conserved better under freezing conditions by increasing the osmotic pressure of the medium and by using an increased warming rate. PMID:16461684

YC-1 induces G0/G1 phase arrest and mitochondria-dependent apoptosis in cisplatin-resistant human oral cancer CAR cells.

PubMed

Lee, Miau-Rong; Lin, Chingju; Lu, Chi-Cheng; Kuo, Sheng-Chu; Tsao, Je-Wei; Juan, Yu-Ning; Chiu, Hong-Yi; Lee, Fang-Yu; Yang, Jai-Sing; Tsai, Fuu-Jen

2017-06-01

Oral cancer is a serious and fatal disease. Cisplatin is the first line of chemotherapeutic agent for oral cancer therapy. However, the development of drug resistance and severe side effects cause tremendous problems clinically. In this study, we investigated the pharmacologic mechanisms of YC-1 on cisplatin-resistant human oral cancer cell line, CAR. Our results indicated that YC-1 induced a concentration-dependent and time-dependent decrease in viability of CAR cells analyzed by MTT assay. Real-time image analysis of CAR cells by IncuCyte™ Kinetic Live Cell Imaging System demonstrated that YC-1 inhibited cell proliferation and reduced cell confluence in a time-dependent manner. Results from flow cytometric analysis revealed that YC-1 promoted G 0 /G 1 phase arrest and provoked apoptosis in CAR cells. The effects of cell cycle arrest by YC-1 were further supported by up-regulation of p21 and down-regulation of cyclin A, D, E and CDK2 protein levels. TUNEL staining showed that YC-1 caused DNA fragmentation, a late stage feature of apoptosis. In addition, YC-1 increased the activities of caspase-9 and caspase-3, disrupted the mitochondrial membrane potential (AYm) and stimulated ROS production in CAR cells. The protein levels of cytochrome c, Bax and Bak were elevated while Bcl-2 protein expression was attenuated in YC-1-treated CAR cells. In summary, YC-1 suppressed the viability of cisplatin-resistant CAR cells through inhibiting cell proliferation, arresting cell cycle at G 0 /G 1 phase and triggering mitochondria-mediated apoptosis. Our results provide evidences to support the potentially therapeutic application of YC-1 on fighting against drug resistant oral cancer in the future. © Author(s) 2017. This article is published with open access by China Medical University.

Enhancing the activity of cannabidiol and other cannabinoids in vitro through modifications to drug combinations and treatment schedules.

PubMed

Scott, Katherine Ann; Shah, Sini; Dalgleish, Angus George; Liu, Wai Man

2013-10-01

Cannabinoids are the bioactive components of the Cannabis plant that display a diverse range of therapeutic qualities. We explored the activity of six cannabinoids, used both alone and in combination in leukaemic cells. Cannabinoids were cytostatic and caused a simultaneous arrest at all phases of the cell cycle. Re-culturing pre-treated cells in drug-free medium resulted in dramatic reductions in cell viability. Furthermore, combining cannabinoids was not antagonistic. We suggest that the activities of some cannabinoids are influenced by treatment schedules; therefore, it is important to carefully select the most appropriate strategy in order to maximise their efficacy.

Collagen Matrix Density Drives the Metabolic Shift in Breast Cancer Cells.

PubMed

Morris, Brett A; Burkel, Brian; Ponik, Suzanne M; Fan, Jing; Condeelis, John S; Aguirre-Ghiso, Julio A; Castracane, James; Denu, John M; Keely, Patricia J

2016-11-01

Increased breast density attributed to collagen I deposition is associated with a 4-6 fold increased risk of developing breast cancer. Here, we assessed cellular metabolic reprogramming of mammary carcinoma cells in response to increased collagen matrix density using an in vitro 3D model. Our initial observations demonstrated changes in functional metabolism in both normal mammary epithelial cells and mammary carcinoma cells in response to changes in matrix density. Further, mammary carcinoma cells grown in high density collagen matrices displayed decreased oxygen consumption and glucose metabolism via the tricarboxylic acid (TCA) cycle compared to cells cultured in low density matrices. Despite decreased glucose entry into the TCA cycle, levels of glucose uptake, cell viability, and ROS were not different between high and low density matrices. Interestingly, under high density conditions the contribution of glutamine as a fuel source to drive the TCA cycle was significantly enhanced. These alterations in functional metabolism mirrored significant changes in the expression of metabolic genes involved in glycolysis, oxidative phosphorylation, and the serine synthesis pathway. This study highlights the broad importance of the collagen microenvironment to cellular expression profiles, and shows that changes in density of the collagen microenvironment can modulate metabolic shifts of cancer cells. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

A Critical Review on the Effect of Docosahexaenoic Acid (DHA) on Cancer Cell Cycle Progression.

PubMed

Newell, Marnie; Baker, Kristi; Postovit, Lynne M; Field, Catherine J

2017-08-17

Globally, there were 14.1 million new cancer diagnoses and 8.2 million cancer deaths in 2012. For many cancers, conventional therapies are limited in their successes and an improved understanding of disease progression is needed in conjunction with exploration of alternative therapies. The long chain polyunsaturated fatty acid, docosahexaenoic acid (DHA), has been shown to enhance many cellular responses that reduce cancer cell viability and decrease proliferation both in vitro and in vivo. A small number of studies suggest that DHA improves chemotherapy outcomes in cancer patients. It is readily incorporated into cancer cell membranes and, as a result there has been considerable research regarding cell membrane initiated events. For example, DHA has been shown to mediate the induction of apoptosis/reduction of proliferation in vitro and in vivo. However, there is limited research into the effect of DHA on cell cycle regulation in cancer cells and the mechanism(s) by which DHA acts are not fully understood. The purpose of the current review is to provide a critical examination of the literature investigating the ability of DHA to stall progression during different cell cycle phases in cancer cells, as well as the consequences that these changes may have on tumour growth, independently and in conjunction with chemotherapy.

Effects of single and combined cell treatments based on low pH and high concentrations of ethanol on the growth and fermentation of Dekkera bruxellensis and Saccharomyces cerevisiae.

PubMed

Bassi, Ana Paula Guarnieri; da Silva, Jéssica Carolina Gomes; Reis, Vanda Renata; Ceccato-Antonini, Sandra Regina

2013-09-01

The alcoholic fermentation in Brazil displays some peculiarities because the yeast used is recycled in a non-aseptic process. After centrifugation, the cells are treated with acid to control the bacterial growth. However, it is difficult to manage the indigenous yeasts without affecting the main culture of Saccharomyces cerevisiae. This work evaluated how the cell treatment could be modified to combat contaminant yeasts based on the differential sensitivities to low pH and high concentrations of ethanol displayed by an industrial strain of S. cerevisiae and three strains of Dekkera bruxellensis, which are common contaminant yeasts in Brazilian fermentation processes. The tests were initially performed in rich medium with a low pH or a high concentration of ethanol to analyse the yeast growth profile. Then, the single and combined effects of low pH and ethanol concentration on the yeast cell viability were evaluated under non-proliferative conditions. The effects on the fermentation parameters were also verified. S. cerevisiae grew best when not subjected to the stresses, but this yeast and D. bruxellensis had similar growth kinetics when exposed to a low pH or increased ethanol concentrations. However, the combined treatments of low pH (2.0) and ethanol (11 or 13 %) resulted in a decrease of D. bruxellensis cell viability almost three times higher than of S. cerevisiae, which was only slightly affected by all cell treatments. The initial viability of the treated cells was restored within 8 h of growth in sugar cane juice, with the exception of the combined treatment for D. bruxellensis. The ethanol-based cell treatment, in despite of slowing the fermentation, could decrease and maintain D. bruxellensis population under control while S. cerevisiae was taking over the fermentation along six fermentative cycles. These results indicate that it may be possible to control the growth of D. bruxellensis without major effects on S. cerevisiae. The cells could be treated between the fermentation cycles by the parcelled addition of 13 % ethanol to the tanks in which the yeast cream is treated with sulphuric acid at pH 2.0.

Recombinant Escherichia coli Trx-JZTX-III represses the proliferation of mouse hepatocellular carcinoma cells through induction of cell cycle arrest.

PubMed

Sun, Mei-Na; Zhao, Xue-Jiao; Zhao, Han-Dong; Zhang, Wei-Guang; Li, Feng-Lan; Chen, Ming-Zi; Li, Hui; Li, Guangchao

2013-06-01

The aim of the present study was to investigate the effects of recombinant Escherichia coli (E. coli) Trx-jingzhaotoxin (JZTX)-III on cell growth in the mouse hepatocellular carcinoma (HCC) cell line Hepa1-6. The JZTX-III gene sequence was synthesized and cloned into the pET-32a(+) vector to construct the recombinant fusion protein Trx-JZTX-III, which was subsequently purified. Hepa1-6 cells were treated with 0 to 1,000-µg/ml concentrations of Trx-JZTX-III; this was demonstrated to affect cell viability, as determined by the 3-(4,5-dimethylthiazol‑2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. The expression of the proliferating cell nuclear antigen (PCNA) protein was investigated using western blot analysis. A colony formation assay was used to determine Hepa1-6 cell proliferation, and the migration ability of cells was determined using a wound‑healing assay. Additionally, flow cytometry was employed to observe changes in the cell cycle. The MTT assay and quantification of PCNA expression indicated that recombinant E. coli Trx-JZTX-III significantly repressed the proliferation of Hepa1-6 cells. Colony formation and the migration of malignant cells was inhibited following treatment with recombinant E. coli Trx-JZTX-III. Flow cytometry showed that recombinant E. coli Trx-JZTX-III induced G0/G1 cell cycle arrest. In conclusion, recombinant E. coli Trx-JZTX-III functions as a tumor suppressor drug in mouse HCC and its underlying mechanism may involve the induction of G0/G1 cell cycle arrest.

Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro by inhibiting the p53-dependent mitochondrial apoptotic pathway.

PubMed

Lee, Yoon-Jin; Kim, Soo A; Lee, Sang-Han

2016-05-01

Intra-articular injection of local anesthetics (LAs) is a common procedure for therapeutic purposes. However, LAs have been found toxic to articular cartilage, and hyaluronan may attenuate this toxicity. In this study we investigated whether hyaluronan attenuated lidocaine-induced chondrotoxicity, and if so, to elucidate the underlying mechanisms. Human chondrocyte cell line SW1353 and newly isolated murine chondrocytes were incubated in culture medium containing hyaluronan and/or lidocaine for 72 h. Cell viability was evaluated using MTT assay. Cell apoptosis was detected with DAPI staining, caspase 3/7 activity assay and flow cytometry. Cell cycle distributions, ROS levels and mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. The expression of p53 and p53-regulated gene products was measured with Western blotting. Lidocaine (0.005%-0.03%) dose-dependently decreased the viability of SW1353 cells. This local anesthetic (0.015%, 0.025%) induced apoptosis, G2/M phase arrest and loss of ΔΨm, and markedly increased ROS production in SW1353 cells. Hyaluronan (50-800 μg/mL) alone did not affect the cell viability, but co-treatment with hyaluronan (200 μg/mL) significantly attenuated lidocaine-induced apoptosis and other abnormalities in SW1353 cells. Furthermore, co-treatment with lidocaine and hyaluronan significantly decreased the levels of p53 and its transcription targets Bax and p21 in SW1353 cells, although treatment with lidocaine alone did not significantly change these proteins. Similar results were obtained in ex vivo cultured murine chondrocytes. Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro through inhibiting the p53-dependent mitochondrial apoptotic pathway.

Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro by inhibiting the p53-dependent mitochondrial apoptotic pathway

PubMed Central

Lee, Yoon-Jin; Kim, Soo A; Lee, Sang-Han

2016-01-01

Aim: Intra-articular injection of local anesthetics (LAs) is a common procedure for therapeutic purposes. However, LAs have been found toxic to articular cartilage, and hyaluronan may attenuate this toxicity. In this study we investigated whether hyaluronan attenuated lidocaine-induced chondrotoxicity, and if so, to elucidate the underlying mechanisms. Methods: Human chondrocyte cell line SW1353 and newly isolated murine chondrocytes were incubated in culture medium containing hyaluronan and/or lidocaine for 72 h. Cell viability was evaluated using MTT assay. Cell apoptosis was detected with DAPI staining, caspase 3/7 activity assay and flow cytometry. Cell cycle distributions, ROS levels and mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. The expression of p53 and p53-regulated gene products was measured with Western blotting. Results: Lidocaine (0.005%−0.03%) dose-dependently decreased the viability of SW1353 cells. This local anesthetic (0.015%, 0.025%) induced apoptosis, G2/M phase arrest and loss of ΔΨm, and markedly increased ROS production in SW1353 cells. Hyaluronan (50−800 μg/mL) alone did not affect the cell viability, but co-treatment with hyaluronan (200 μg/mL) significantly attenuated lidocaine-induced apoptosis and other abnormalities in SW1353 cells. Furthermore, co-treatment with lidocaine and hyaluronan significantly decreased the levels of p53 and its transcription targets Bax and p21 in SW1353 cells, although treatment with lidocaine alone did not significantly change these proteins. Similar results were obtained in ex vivo cultured murine chondrocytes. Conclusion: Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro through inhibiting the p53-dependent mitochondrial apoptotic pathway. PMID:27041463

Valproic acid treatment response in vitro is determined by TP53 status in medulloblastoma.

PubMed

Mascaro-Cordeiro, Bruna; Oliveira, Indhira Dias; Tesser-Gamba, Francine; Pavon, Lorena Favaro; Saba-Silva, Nasjla; Cavalheiro, Sergio; Dastoli, Patrícia; Toledo, Silvia Regina Caminada

2018-05-22

Histone deacetylate inhibitors (HDACi), as valproic acid (VA), have been reported to enhance efficacy and to prevent drug resistance in some tumors, including medulloblastoma (MB). In the present study, we investigated VA role, combined to cisplatin (CDDP) in cell viability and gene expression of MB cell lines. Dose-response curve determined IC 50 values for each treatment: (1) VA single, (2) CDDP single, and (3) VA and CDDP combined. Cytotoxicity and flow cytometry evaluated cell viability after exposure to treatments. Quantitative PCR evaluated gene expression levels of AKT, CTNNB1, GLI1, KDM6A, KDM6B, NOTCH2, PTCH1, and TERT, before and after treatment. Besides, we performed next-generation sequencing (NGS) for PTCH1, TERT, and TP53 genes. The most effective treatment to reduce viability was combined for D283MED and ONS-76; and CDDP single for DAOY cells (p < 0.0001). TERT, GLI1, and AKT genes were overexpressed after treatments with VA. D283MED and ONS-76 cells presented variants in TERT and PTCH1, respectively and DAOY cell line presented a TP53 mutation. MB tumors belonging to SHH molecular subgroup, with TP53 MUT , would be the ones that present high risk in relation to VA use during the treatment, while TP53 WT MBs can benefit from VA therapy, both SHH and groups 3 and 4. Our study shows a new perspective about VA action in medulloblastoma cells, raising the possibility that VA may act in different patterns. According to the genetic background of MB cell, VA can stimulate cell cycle arrest and apoptosis or induce resistance to treatment via signaling pathways activation.

GPER mediates enhanced cell viability and motility via non-genomic signaling induced by 17β-estradiol in triple-negative breast cancer cells.

PubMed

Yu, Tenghua; Liu, Manran; Luo, Haojun; Wu, Chengyi; Tang, Xi; Tang, Shifu; Hu, Ping; Yan, Yuzhao; Wang, Zhiliang; Tu, Gang

2014-09-01

Triple-negative breast cancer (TNBC) is an aggressive breast cancer with a generally poor prognosis. Due to lack of specific targets for its treatment, an efficient therapy is needed. G protein-coupled estrogen receptor (GPER), a novel estrogen receptor, has been reported to be expressed in TNBC tissues. In this study, we investigated the effects of blocking non-genomic signaling mediated by the estrogen/GPER pathway on cell viability and motility in the TNBC cells. GPER was strongly expressed in the TNBC cell lines MDA-MB-468 and MDA-MB-436, and the estrogen-mediated non-genomic ERK signaling activated by GPER was involved in cell viability and motility of TNBC cells. Treatment with 17β-estradiol (E2), the GPER-specific agonist G-1 and tamoxifen (TAM) led to rapid activation of p-ERK1/2, but not p-Akt. Moreover, estrogen/GPER/ERK signaling was involved in increasing cell growth, survival, and migration/invasion by upregulating expression of cyclinA, cyclinD1, Bcl-2, and c-fos associated with the cell cycle, proliferation, and apoptosis. Immunohistochemical analysis of TNBC specimens showed a significantly different staining of p-ERK1/2 between GPER-positive tissues (58/66, 87.9%) and GPER-negative tissues (13/30, 43.3%). The positivity of GPER and p-ERK1/2 displayed a strong association with large tumor size and poor clinical stage, indicating that GPER/ERK signaling might also contribute to tumor progression in TNBC patients which corresponded with in vitro experimental data. Our findings suggest that inhibition of estrogen/GPER/ERK signaling represents a novel targeted therapy in TNBC. Copyright © 2014 Elsevier Ltd. All rights reserved.

Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos.

PubMed

Sugimura, Satoshi; Akai, Tomonori; Somfai, Tamás; Hirayama, Muneyuki; Aikawa, Yoshio; Ohtake, Masaki; Hattori, Hideshi; Kobayashi, Shuji; Hashiyada, Yutaka; Konishi, Kazuyuki; Imai, Kei

2010-12-01

We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

Peo Life Cycle Cost Accountability: Viability Of Foreign Suppliers For Weapon System Development

DTIC Science & Technology

2016-02-16

i AIR WAR COLLEGE AIR UNIVERSITY PEO LIFE CYCLE COST ACCOUNTABILITY: VIABILITY OF FOREIGN SUPPLIERS FOR WEAPON SYSTEM DEVELOPMENT By...to decrease, then recycling may become more economically feasible. The need for the U.S. to develop affordable technologies for recycling has become

Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis

PubMed Central

Huang, Fang; Liu, Qiaoyun; Xie, Shujun; Xu, Jian; Huang, Bo; Wu, Yihua; Xia, Dajing

2016-01-01

Cypermethrin is one of the most highly effective synthetic pyrethroid insecticides. The toxicity of cypermethrin to the reproductive and nervous systems has been well studied. However, little is known about the toxic effect of cypermethrin on immune cells such as macrophages. Here, we investigated the cytotoxicity of cypermethrin on macrophages and the underlying molecular mechanisms. We found that cypermethrin reduced cell viability and induced apoptosis in RAW 264.7 cells. Cypermethrin also increased reactive oxygen species (ROS) production and DNA damage in a dose-dependent manner. Moreover, cypermethrin-induced G1 cell cycle arrest was associated with an enhanced expression of p21, wild-type p53, and down-regulation of cyclin D1, cyclin E and CDK4. In addition, cypermethrin treatment activated MAPK signal pathways by inducing c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases 1/2 ERK1/2 phosphorylation, and increased the cleaved poly ADP-ribose polymerase (PARP). Further, pretreatment with antioxidant N-acetylcysteine (NAC) effectively abrogated cypermethrin-induced cell cytotoxicity, G1 cell cycle arrest, DNA damage, PARP activity, and JNK and ERK1/2 activation. The specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059) effectively reversed the phosphorylation level of JNK and ERK1/2, and attenuated the apoptosis. Taken together, these data suggested that cypermethrin caused immune cell death via inducing cell cycle arrest and apoptosis regulated by ROS-mediated JNK/ERK pathway. PMID:27322250

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

PubMed

Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

2015-05-01

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.

Cytotoxic Activity and Antiproliferative Effects of Crude Skin Secretion from Physalaemus nattereri (Anura: Leptodactylidae) on in vitro Melanoma Cells.

PubMed

Cruz e Carvalho, Andréa; Márquez, César Augusto Prías; Azevedo, Ricardo Bentes; Joanitti, Graziella Anselmo; Pires Júnior, Osmindo Rodrigues; Fontes, Wagner; Castro, Mariana S

2015-10-08

Anuran secretions are rich sources of bioactive molecules, including antimicrobial and antitumoral compounds. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against skin cancer cells, and to assess its cytotoxic action mechanisms on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduced the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage), reduction in mitochondrial membrane potential, increase in phosphatidylserine exposure, and cell cycle arrest in S-phase. Together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also subjected to chromatographic fractioning using RP-HPLC, and eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular mass components in a range compatible with peptides. Although the specific mechanisms causing the reduced cell viability and cytotoxicity after the treatment with crude secretion are still unknown, it may be considered that molecules, such as the peptides found in the secretion, are effective against B16F10 tumor cells. Considering the growing need for new anticancer drugs, data presented in this study strongly reinforce the validity of P. nattereri crude secretion as a rich source of new anticancer molecules.

Changes of Constituents and Activity to Apoptosis and Cell Cycle During Fermentation of Tea

PubMed Central

Zhao, Hang; Zhang, Min; Zhao, Lu; Ge, Ya-kun; Sheng, Jun; Shi, Wei

2011-01-01

Tea is believed to be beneficial for health, and the effects of the fermentation process on its contributions to apoptosis and cell cycle arrest of gastric cancer cells have not been completely investigated. In this study, the chemical components in green tea, black tea and pu-erh tea aqueous extracts were analyzed and compared. The polysaccharide and caffeine levels were substantially higher in the fermented black tea and pu-erh tea, while the polyphenol level was higher in the unfermented green tea. Hence, a treatment of tea aqueous extract and the components, which are emerging as promising anticancer agents, were pursued to determine whether this treatment could lead to enhance apoptosis and cell cycle arrest. In the human gastric cancer cell line SGC-7901, the cell viability and flow cytometry analysis for apoptotic cells indicated effects in a dose-dependent inhibition manner for the three tea treatment groups. The apoptosis rates were found to be elevated after 48 h of treatment with 31.2, 125, and 500 μg/mL of green tea extract, the higher catechins content may be involved in the mechanism. Cell cycle was arrested in S phase in the fermented black tea and pu-erh tea, and the populations were significantly decreased in G2/M phases, possibly due to the oxidation of tea polyphenols, which causes an increase of theabrownins. CCC-HEL-1 normal cells were not sensitive to tea extract. These findings suggest that the fermentation process causes changes of the compounds which might be involved in the changes of cell proliferation inhibition, apoptosis induction and cell cycle arrest. PMID:21673927

Changes of constituents and activity to apoptosis and cell cycle during fermentation of tea.

PubMed

Zhao, Hang; Zhang, Min; Zhao, Lu; Ge, Ya-Kun; Sheng, Jun; Shi, Wei

2011-01-01

Tea is believed to be beneficial for health, and the effects of the fermentation process on its contributions to apoptosis and cell cycle arrest of gastric cancer cells have not been completely investigated. In this study, the chemical components in green tea, black tea and pu-erh tea aqueous extracts were analyzed and compared. The polysaccharide and caffeine levels were substantially higher in the fermented black tea and pu-erh tea, while the polyphenol level was higher in the unfermented green tea. Hence, a treatment of tea aqueous extract and the components, which are emerging as promising anticancer agents, were pursued to determine whether this treatment could lead to enhance apoptosis and cell cycle arrest. In the human gastric cancer cell line SGC-7901, the cell viability and flow cytometry analysis for apoptotic cells indicated effects in a dose-dependent inhibition manner for the three tea treatment groups. The apoptosis rates were found to be elevated after 48 h of treatment with 31.2, 125, and 500 μg/mL of green tea extract, the higher catechins content may be involved in the mechanism. Cell cycle was arrested in S phase in the fermented black tea and pu-erh tea, and the populations were significantly decreased in G2/M phases, possibly due to the oxidation of tea polyphenols, which causes an increase of theabrownins. CCC-HEL-1 normal cells were not sensitive to tea extract. These findings suggest that the fermentation process causes changes of the compounds which might be involved in the changes of cell proliferation inhibition, apoptosis induction and cell cycle arrest.

Lycopene and Beta-Carotene Induce Growth Inhibition and Proapoptotic Effects on ACTH-Secreting Pituitary Adenoma Cells

PubMed Central

Leite de Oliveira, Felipe; Soares, Nathália; de Mattos, Rômulo Medina; Hecht, Fábio; Dezonne, Rômulo Sperduto; Vairo, Leandro; Goldenberg, Regina Coeli dos Santos; Gomes, Flávia Carvalho Alcântara; de Carvalho, Denise Pires; Gadelha, Mônica R.; Nasciutti, Luiz Eurico; Miranda-Alves, Leandro

2013-01-01

Pituitary adenomas comprise approximately 10–15% of intracranial tumors and result in morbidity associated with altered hormonal patterns, therapy and compression of adjacent sella turcica structures. The use of functional foods containing carotenoids contributes to reduce the risk of chronic diseases such as cancer and vascular disorders. In this study, we evaluated the influence of different concentrations of beta-carotene and lycopene on cell viability, colony formation, cell cycle, apoptosis, hormone secretion, intercellular communication and expression of connexin 43, Skp2 and p27kip1 in ACTH-secreting pituitary adenoma cells, the AtT20 cells, incubated for 48 and 96 h with these carotenoids. We observed a decrease in cell viability caused by the lycopene and beta-carotene treatments; in these conditions, the clonogenic ability of the cells was also significantly decreased. Cell cycle analysis revealed that beta-carotene induced an increase of the cells in S and G2/M phases; furthermore, lycopene increased the proportion of these cells in G0/G1 while decreasing the S and G2/M phases. Also, carotenoids induced apoptosis after 96 h. Lycopene and beta-carotene decreased the secretion of ACTH in AtT20 cells in a dose-dependent manner. Carotenoids blocked the gap junction intercellular communication. In addition, the treatments increased the expression of phosphorylated connexin43. Finally, we also demonstrate decreased expression of S-phase kinase-associated protein 2 (Skp2) and increased expression of p27kip1 in carotenoid-treated cells. These results show that lycopene and beta-carotene were able to negatively modulate events related to the malignant phenotype of AtT-20 cells, through a mechanism that could involve changes in the expression of connexin 43, Skp2 and p27kip1; and suggest that these compounds might provide a novel pharmacological approach to the treatment of Cushing’s disease. PMID:23667519

CDK1 promotes nascent DNA synthesis and induces resistance of cancer cells to DNA-damaging therapeutic agents

PubMed Central

Liao, Hongwei; Ji, Fang; Geng, Xinwei; Xing, Meichun; Li, Wen; Chen, Zhihua; Shen, Huahao; Ying, Songmin

2017-01-01

Cyclin dependent kinase 1 (CDK1) is essential for cell viability and plays a vital role in many biological events including cell cycle control, DNA damage repair, and checkpoint activation. Here, we identify an unanticipated role for CDK1 in promoting nascent DNA synthesis during S-phase. We report that a short duration of CDK1 inhibition, which does not perturb cell cycle progression, triggers a replication-associated DNA damage response (DDR). This DDR is associated with a disruption of replication fork progression and leads to genome instability. Moreover, we show that compromised CDK1 activity dramatically increases the efficacy of chemotherapeutic agents that kill cancer cells through perturbing DNA replication, including Olaparib, an FDA approved PARP inhibitor. Our study has revealed an important role for CDK1 in the DNA replication program, and suggests that the therapeutic targeting CDK1 may be a novel approach for combination chemotherapy. PMID:29207595

Structural insights into cell cycle control by essential GTPase Era.

PubMed

Ji, Xinhua

Era (Escherichia coli Ras-like protein), essential for bacterial cell viability, is composed of an N-terminal GTPase domain and a C-terminal KH domain. In bacteria, it is required for the processing of 16S ribosomal RNA (rRNA) and maturation of 30S (small) ribosomal subunit. Era recognizes 10 nucleotides ( 1530 GAUCACCUCC 1539 ) near the 3' end of 16S rRNA and interacts with helix 45 (h45, nucleotides 1506-1529). GTP binding enables Era to bind RNA, RNA binding stimulates Era's GTP-hydrolyzing activity, and GTP hydrolysis releases Era from matured 30S ribosomal subunit. As such, Era controls cell growth rate via regulating the maturation of the 30S ribosomal subunit. Ribosomes manufacture proteins in all living organisms. The GAUCA sequence and h45 are highly conserved in all three kingdoms of life. Homologues of Era are present in eukaryotic cells. Hence, the mechanism of bacterial Era action also sheds light on the cell cycle control of eukaryotes.

Induction of apoptosis and cell cycle arrest in human colon carcinoma cells by Corema album leaves.

PubMed

León-González, Antonio J; Manson, Margaret M; López-Lizaro, Miguel; Navarro, Inmaculada; Martín-Cordero, Carmen

2014-01-01

The leaves of Corema album (Ericaceae), an endemic shrub which grows in Atlantic coastal areas of the Iberian Peninsula, are rich in flavonoids and other secondary metabolites. Silica gel column chromatography of the ethyl acetate extract from dried leaves was performed and a flavonic active fraction was obtained. The cytotoxic activity of this fraction was assessed using the colon cancer cell lines HCT116 and HT29. After 48 hours of treatment, cell viability was determined with luminescence-based ATPLite assay, showing IC50 values of 7.2 +/- 0.7 and 6.8 +/- 1.2 microg/mL, respectively. The study by flow cytometry revealed that the cytotoxicity of this fraction was mediated, at least in part, by induction of apoptosis and G2/M cell cycle arrest. The active fraction was then subjected to Sephadex LH-20 chromatography and two flavonoids were separated and identified as the flavanone pinocembrin and 2',4'-dihydroxychalcone after UV, MS and NMR analysis.

Measuring cell cycle progression kinetics with metabolic labeling and flow cytometry.

PubMed

Fleisig, Helen; Wong, Judy

2012-05-22

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments. Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tubeimoside-1 induces oxidative stress-mediated apoptosis and G0/G1 phase arrest in human prostate carcinoma cells in vitro

PubMed Central

Yang, Jing-bo; Khan, Muhammad; He, Yang-yang; Yao, Min; Li, Yong-ming; Gao, Hong-wen; Ma, Tong-hui

2016-01-01

Aim: Tubeimoside-1 (TBMS1), a triterpenoid saponin extracted from the Chinese herbal medicine Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), has shown anticancer activities in various cancer cell lines. The aim of this study was to investigate the anticancer activity and molecular targets of TBMS1 in human prostate cancer cells in vitro. Methods: DU145 and P3 human prostate cancer cells were treated with TBMS1. Cell viability and apoptosis were detected. ROS generation, mitochondrial membrane potential and cell cycle profile were examined. Western blotting was used to measure the expression of relevant proteins in the cells. Results: TBMS1 (5–100 μmol/L) significantly suppressed the viability of DU145 and P3 cells with IC50 values of approximately 10 and 20 μmol/L, respectively. Furthermore, TBMS1 dose-dependently induced apoptosis and cell cycle arrest at G0/G1 phase in DU145 and P3 cells. In DU145 cells, TBMS1 induced mitochondrial apoptosis, evidenced by ROS generation, mitochondrial dysfunction, endoplasmic reticulum stress, modulated Bcl-2 family protein and cleaved caspase-3, and activated ASK-1 and its downstream targets p38 and JNK. The G0/G1 phase arrest was linked to increased expression of p53 and p21 and decreased expression of cyclin E and cdk2. Co-treatment with Z-VAD-FMK (pan-caspase inhibitor) could attenuate TBMS1-induced apoptosis but did not prevent G0/G1 arrest. Moreover, co-treatment with NAC (ROS scavenger), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor) or salubrinal (ER stress inhibitor) significantly attenuated TBMS1-induced apoptosis. Conclusion: TBMS1 induces oxidative stress-mediated apoptosis in DU145 human prostate cancer cells in vitro via the mitochondrial pathway. PMID:27292614

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells.

PubMed

Takahashi, Kyohei; Shibata, Tomohito; Oba, Tatsuya; Ishikawa, Tetsuya; Yoshikawa, Masahito; Tatsunami, Ryosuke; Takahashi, Kazuhiko; Tampo, Yoshiko

2009-02-13

Menadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs). BAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody. Intracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells. We conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.

The neem limonoids azadirachtin and nimbolide induce cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells.

PubMed

Priyadarsini, R Vidya; Murugan, R Senthil; Sripriya, P; Karunagaran, D; Nagini, S

2010-06-01

Limonoids from the neem tree (Azadirachta indica) have attracted considerable research attention in recent years owing to their potent antioxidant and anti-proliferative effects. The present study was designed to investigate the cellular and molecular mechanisms by which azadirachtin and nimbolide exert cytotoxic effects in the human cervical cancer (HeLa) cell line. Both azadirachtin and nimbolide significantly suppressed the viability of HeLa cells in a dose-dependent manner by inducing cell cycle arrest at G0/G1 phase accompanied by p53-dependent p21 accumulation and down-regulation of the cell cycle regulatory proteins cyclin B, cyclin D1 and PCNA. Characteristic changes in nuclear morphology, presence of a subdiploid peak and annexin-V staining pointed to apoptosis as the mode of cell death. Increased generation of reactive oxygen species with decline in the mitochondrial transmembrane potential and release of cytochrome c confirmed that the neem limonoids transduced the apoptotic signal via the mitochondrial pathway. Altered expression of the Bcl-2 family of proteins, inhibition of NF-kappaB activation and over-expression of caspases and survivin provide compelling evidence that azadirachtin and nimbolide induce a shift of balance toward a pro-apoptotic phenotype. Antioxidants such as azadirachtin and nimbolide that can simultaneously arrest the cell cycle and target multiple molecules involved in mitochondrial apoptosis offer immense potential as anti-cancer therapeutic drugs.

Esculetin Inhibits the Survival of Human Prostate Cancer Cells by Inducing Apoptosis and Arresting the Cell Cycle.

PubMed

Turkekul, Kader; Colpan, R Dilsu; Baykul, Talha; Ozdemir, Mehmet D; Erdogan, Suat

2018-03-01

Prostate cancer (PCa) is one of the most important causes of death in men and thus new therapeutic approaches are needed. In this study, antiproliferative and anti-migration properties of a coumarin derivative esculetin were evaluated. Human PCa cell lines PC3, DU145, and LNCaP were treated with various concentrations of esculetin for 24 to 72 hours, and cell viability was determined by the MTT test. Cell cycle and apoptosis were analyzed by using cell-based cytometer. Gene expression levels were assessed by reverse transcription and quantitative real-time PCR, cell migration was determined by the wound healing assay. The protein expression was measured by Western blotting. Esculetin inhibited cell proliferation in a dose- and time-dependent manner. Cell migration was inhibited by esculetin treatment. Administration of esculetin significantly reduced the cells survival, induced apoptosis and caused the G1 phase cell cycle arrest shown by image-based cytometer. The induced expression of cytochrome c , p53, p21 and p27, and down-regulated CDK2 and CDK4 may be the underlying molecular mechanisms of esculetin effect. Esculetin suppressed phosphorylation of Akt and enhanced protein expression of tumor-suppressor phosphatase and tensin homologue. Our findings showed that the coumarin derivative esculetin could be used in the management of PCa. However, further in vivo research is needed.

Esculetin Inhibits the Survival of Human Prostate Cancer Cells by Inducing Apoptosis and Arresting the Cell Cycle

PubMed Central

Turkekul, Kader; Colpan, R. Dilsu; Baykul, Talha; Ozdemir, Mehmet D.

2018-01-01

Background Prostate cancer (PCa) is one of the most important causes of death in men and thus new therapeutic approaches are needed. In this study, antiproliferative and anti-migration properties of a coumarin derivative esculetin were evaluated. Methods Human PCa cell lines PC3, DU145, and LNCaP were treated with various concentrations of esculetin for 24 to 72 hours, and cell viability was determined by the MTT test. Cell cycle and apoptosis were analyzed by using cell-based cytometer. Gene expression levels were assessed by reverse transcription and quantitative real-time PCR, cell migration was determined by the wound healing assay. The protein expression was measured by Western blotting. Results Esculetin inhibited cell proliferation in a dose- and time-dependent manner. Cell migration was inhibited by esculetin treatment. Administration of esculetin significantly reduced the cells survival, induced apoptosis and caused the G1 phase cell cycle arrest shown by image-based cytometer. The induced expression of cytochrome c, p53, p21 and p27, and down-regulated CDK2 and CDK4 may be the underlying molecular mechanisms of esculetin effect. Esculetin suppressed phosphorylation of Akt and enhanced protein expression of tumor-suppressor phosphatase and tensin homologue. Conclusions Our findings showed that the coumarin derivative esculetin could be used in the management of PCa. However, further in vivo research is needed. PMID:29629344

Pulsed or continuous electromagnetic field induce p53/p21-mediated apoptotic signaling pathway in mouse spermatogenic cells in vitro and thus may affect male fertility.

PubMed

Solek, Przemyslaw; Majchrowicz, Lena; Bloniarz, Dominika; Krotoszynska, Ewelina; Koziorowski, Marek

2017-05-01

The impact of electromagnetic field (EMF) on the human health and surrounding environment is a common topic investigated over the years. A significant increase in the electromagnetic field concentration arouses public concern about the long-term effects of EMF on living organisms associated with many aspects. In the present study, we investigated the effects of pulsed and continuous electromagnetic field (PEMF/CEMF) on mouse spermatogenic cell lines (GC-1 spg and GC-2 spd) in terms of cellular and biochemical features in vitro. We evaluated the effect of EMF on mitochondrial metabolism, morphology, proliferation rate, viability, cell cycle progression, oxidative stress balance and regulatory proteins. Our results strongly suggest that EMF induces oxidative and nitrosative stress-mediated DNA damage, resulting in p53/p21-dependent cell cycle arrest and apoptosis. Therefore, spermatogenic cells due to the lack of antioxidant enzymes undergo oxidative and nitrosative stress-mediated cytotoxic and genotoxic events, which contribute to infertility by reduction in healthy sperm cells pool. In conclusion, electromagnetic field present in surrounding environment impairs male fertility by inducing p53/p21-mediated cell cycle arrest and apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Curcumin enhances the anticancer effects of trichostatin a in breast cancer cells.

PubMed

Yan, Guang; Graham, Kimmer; Lanza-Jacoby, Susan

2013-05-01

Breast cancer patients with HER-2 positive or estrogen receptor negative tumors have a poor prognosis because these tumors are aggressive and respond poorly to standard therapies. Histone deacetylase (HDAC) inhibitors have been shown to decreased cell survival, which suggests that HDAC inhibitors may be developed for preventing and treating breast cancer. Curcumin has anti-inflammatory and proapoptotic effects in cancer cells. We determined whether the HDAC inhibitor, Tricostatin A (TSA) in combination with curcumin would produce greater antiproliferative and apoptotic effects than either agent alone. Increasing the concentration of curcumin from 10 to 20 µM enhanced the growth inhibitory effects of the combination in SkBr3 and 435eB breast cancer cells, which was accompanied by decreased viability along with decreased phosphorylation of ERK and Akt. The decreased cell viability observed in SkBr3 cells when curcumin was combined with TSA led to a G0/G1 cell cycle arrest and increased p21 and p27, and decreased Cyclin D1 protein expression. The combination induced cleavage of caspase 3 and poly(ADP-ribose) polymerase-1, suggesting that cell death occurred by apoptosis. There were no changes in protein expression of Bcl2, Bax, or Bcl-xL and decreased expression of p53. The combination increased protein expression of phosphorylated JNK and phosphorylated p38. Pharmacological inhibition of JNK, but not p38, attenuated the decreased viability induced by the curcumin and TSA combination. We conclude that p53 independent apoptosis induced by combining curcumin and TSA involves JNK activation. These findings provide a rationale for exploring the potential benefits of the combination of curcumin with TSA for treatment of breast cancer. Copyright © 2012 Wiley Periodicals, Inc.

A sex difference in oxidative stress and behavioral suppression induced by ethanol withdrawal in rats

PubMed Central

Jung, Marianna E.; Metzger, Daniel B.

2016-01-01

Ethanol withdrawal (EW) is referred to the abrupt termination of long-term heavy drinking, and provokes oxidative brain damage. Here, we investigated whether the cerebellum and hippocampus of female rats are less affected by prooxidant EW than male rats due to the antioxidant effect of 17β-estradiol (E2). Female and male rats received a four-week ethanol diet and three-week withdrawal per cycle for two cycles. Some female rats were ovariectomized with E2 or antioxidant (Vitamin E+Co-Q10) treatment. Measurements were cerebellum (Rotarod) and hippocampus (water-maze)-related behaviors, oxidative markers (O2•−, malondialdehyde, protein carbonyls), mitochondrial membrane swelling, and a key mitochondrial enzyme, cytochrome c oxidase (CcO). Separately, HT22 (hippocampal) cells were subjected to ethanol-exposure and withdrawal for two cycles to assess the effect of a CcO inhibitor on E2’s protection for mitochondrial respiration and cell viability. Ethanol-withdrawn female rats showed a smaller increase in oxidative markers in cerebellum and hippocampus than male rats, and E2 treatment decreased the oxidative markers. Compared to male counterparts, ethanol-withdrawn female rats showed better Rotarod but poorer water-maze performance, accompanied by more severe mitochondrial membrane swelling and CcO suppression in hippocampus. E2 or antioxidant treatment improved Rotarod but not water-maze performance. In the presence of a CcO inhibitor, E2 treatment failed to protect mitochondrial respiration and cell viability from EW. These data suggest that antioxidant E2 contributes to smaller oxidative stress in ethanol-withdrawn female than male rats. They also suggest that EW-induced severe mitochondrial damage in hippocampus may blunt E2’s antioxidant protection for hippocampus-related behavior. PMID:27503149

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.

Long non-coding RNA PICART1 suppresses proliferation and promotes apoptosis in lung cancer cells by inhibiting JAK2/STAT3 signaling.

PubMed

Zhao, J M; Cheng, W; He, X G; Liu, Y L; Wang, F F; Gao, Y F

2018-06-26

Lung cancer remains the most common cause of tumor-related death worldwide. Recent studies have revealed that long non-coding RNAs (lncRNAs) are involved in the development of various cancers, including lung cancer. This study aimed to investigate the effect and the molecular basis of lncRNA PICART1 on lung cancer. We first assessed the PICART1 expression in lung cancer in vitro and vivo by qRT-PCR. Then the expression of PICART1 in SPC-A-1 and NCI-H1975 cell lines was inhibited and overexpressed by transient transfections. Thereafter, cell viability, cell cycle, migration and apoptosis were respectively measured by MTT, Transwell and flow cytometry assay. Furthermore, qRT-PCR and western blot analysis were mainly performed to assess the expression levels of apoptosis- and migration-related proteins and JAK2/STAT3 pathway proteins. Tumor formation was measured by xenograft tumor model assay in vivo. PICART1 expression was down-regulated in human lung cancer tissues and cell lines. Knockdown of PICART1 increased cell viability of lung cancer cell lines. However, PICART1 overexpression inhibited cell cycle progression and promoted apoptosis in SPC-A-1 and NCI-H1975 cell lines. PICART1 overexpression also inhibited migration, as evidenced by up-regulation of E-cadherin, and down-regulation of Twist1, MMP2 and MMP9. Furthermore, we found PICART1 inhibition may regulate cell apoptosis and migration through activating JAK2/STAT3 pathway. In vivo experiments revealed that PICART1 knockdown significantly promoted tumor formation.This study demonstrates that PICART1 overexpression represents an anti-growth and anti-metastasis role in lung cancer cells. Additionally, PICART1 acts as a tumor suppressor may be via regulation of JAK2/STAT3 pathway.

Bacterial Acclimation Inside an Aqueous Battery.

PubMed

Dong, Dexian; Chen, Baoling; Chen, P

2015-01-01

Specific environmental stresses may lead to induced genomic instability in bacteria, generating beneficial mutants and potentially accelerating the breeding of industrial microorganisms. The environmental stresses inside the aqueous battery may be derived from such conditions as ion shuttle, pH gradient, free radical reaction and electric field. In most industrial and medical applications, electric fields and direct currents are used to kill bacteria and yeast. However, the present study focused on increasing bacterial survival inside an operating battery. Using a bacterial acclimation strategy, both Escherichia coli and Bacillus subtilis were acclimated for 10 battery operation cycles and survived in the battery for over 3 days. The acclimated bacteria changed in cell shape, growth rate and colony color. Further analysis indicated that electrolyte concentration could be one of the major factors determining bacterial survival inside an aqueous battery. The acclimation process significantly improved the viability of both bacteria E. coli and B. subtilis. The viability of acclimated strains was not affected under battery cycle conditions of 0.18-0.80 mA cm(-2) and 1.4-2.1 V. Bacterial addition within 1.0×10(10) cells mL(-1) did not significantly affect battery performance. Because the environmental stress inside the aqueous battery is specific, the use of this battery acclimation strategy may be of great potential for the breeding of industrial microorganisms.

Next-generation sequencing reveals low-dose effects of cationic dendrimers in primary human bronchial epithelial cells.

PubMed

Feliu, Neus; Kohonen, Pekka; Ji, Jie; Zhang, Yuning; Karlsson, Hanna L; Palmberg, Lena; Nyström, Andreas; Fadeel, Bengt

2015-01-27

Gene expression profiling has developed rapidly in recent years with the advent of deep sequencing technologies such as RNA sequencing (RNA Seq) and could be harnessed to predict and define mechanisms of toxicity of chemicals and nanomaterials. However, the full potential of these technologies in (nano)toxicology is yet to be realized. Here, we show that systems biology approaches can uncover mechanisms underlying cellular responses to nanomaterials. Using RNA Seq and computational approaches, we found that cationic poly(amidoamine) dendrimers (PAMAM-NH2) are capable of triggering down-regulation of cell-cycle-related genes in primary human bronchial epithelial cells at doses that do not elicit acute cytotoxicity, as demonstrated using conventional cell viability assays, while gene transcription was not affected by neutral PAMAM-OH dendrimers. The PAMAMs were internalized in an active manner by lung cells and localized mainly in lysosomes; amine-terminated dendrimers were internalized more efficiently when compared to the hydroxyl-terminated dendrimers. Upstream regulator analysis implicated NF-κB as a putative transcriptional regulator, and subsequent cell-based assays confirmed that PAMAM-NH2 caused NF-κB-dependent cell cycle arrest. However, PAMAM-NH2 did not affect cell cycle progression in the human A549 adenocarcinoma cell line. These results demonstrate the feasibility of applying systems biology approaches to predict cellular responses to nanomaterials and highlight the importance of using relevant (primary) cell models.

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

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

Byrne, Ann; McLaren, Rajashree P.; Mason, Paul

2010-01-15

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21{sup /Cip} and p27{sup /Kip1}. Mostmore » notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.« less

Non-invasive metabolomic profiling of embryo culture media and morphology grading to predict implantation outcome in frozen-thawed embryo transfer cycles.

PubMed

Li, Xiong; Xu, Yan; Fu, Jing; Zhang, Wen-Bi; Liu, Su-Ying; Sun, Xiao-Xi

2015-11-01

Assessment of embryo viability is a crucial component of in vitro fertilization and currently relies largely on embryo morphology and cleavage rate. Because morphological assessment remains highly subjective, it can be unreliable in predicting embryo viability. This study investigated the metabolomic profiling of embryo culture media using near-infrared (NIR) spectroscopy for predicting the implantation potential of human embryos in frozen-thawed embryo transfer (FET) cycles. Spent embryo culture media was collected on day 4 after thawed embryo transfer (n = 621) and analysed using NIR spectroscopy. Viability scores were calculated using a predictive multivariate algorithm of fresh embryos with known pregnancy outcomes. The mean viability indices of embryos resulting in clinical pregnancy following FET were significantly higher than those of non-implanted embryos and differed between the 0, 50, and 100 % implantation groups. Notably, the 0 % group index was significantly lower than the 100 % implantation group index (-0.787 ± 0.382 vs. 1.064 ± 0.331, P < 0.01). To predict implantation outcomes, we examined the area under the ROC curve (AUCROC), which was significantly higher for the viability than for the morphology score (0.94 vs. 0.55; P < 0.01); however, the AUCROCs for the composite and viability scores did not differ significantly (0.92 vs. 0.94; P > 0.05). NIR metabolomic profiling of thawed embryo culture media is independent of morphology and correlates with embryo implantation potential in FET cycles. The viability score alone or in conjunction with morphologic grading is a more objective marker for implantation outcome in FET cycles than morphology alone.

Antitumor activity of Brazilian red propolis fractions against Hep-2 cancer cell line.

PubMed

Frozza, Caroline Olivieri da Silva; Santos, Denis Amilton; Rufatto, Luciane Corbellini; Minetto, Luciane; Scariot, Fernando Joel; Echeverrigaray, Sergio; Pich, Claus Tröger; Moura, Sidnei; Padilha, Francine Ferreira; Borsuk, Sibele; Savegnago, Lucielli; Collares, Tiago; Seixas, Fabiana Kömmling; Dellagostin, Odir; Roesch-Ely, Mariana; Henriques, João Antonio Pêgas

2017-07-01

Continuous increases in the rates of tumor diseases have highlighted the need for identification of novel and inexpensive antitumor agents from natural sources. In this study, we investigated the effects of enriched fraction from hydroalcoholic Brazilian red propolis extract against Hep-2 cancer cell line. Initially 201 fractions were arranged in 12 groups according to their chromatographic characteristics (A-L). After an in vitro cell viability screening, J and L were further selected as promising enriched fractions for this study. The chemical characterization was performed and Biochanin A, Formononetin, and Liquiritigenin compounds were quantified. Through MTT viability assay and morphological changes observed by Giemsa and DAPI staining, the results showed that red propolis inhibited cancer cells growth. Flow cytometry results indicated effects that were partly mediated through programmed cell death as confirmed by externalization of phosphatidylserine, DNA cleaved assay, increase at SUB G1-G0 phase in cell cycle analysis and loss of mitochondrial membrane potential. In conclusion, our results demonstrated that red propolis enriched fractions promoted apoptotic effects in human cancer cells through the mechanisms involving mitochondrial perturbation. Therefore, red propolis fractions contain candidate agents for adjuvant cancer treatment, which further studies should elucidate the comprehensive mechanistic pathways. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Reversible inhibition of lysine specific demethylase 1 is a novel anti-tumor strategy for poorly differentiated endometrial carcinoma.

PubMed

Theisen, Emily R; Gajiwala, Snehal; Bearss, Jared; Sorna, Venkataswamy; Sharma, Sunil; Janat-Amsbury, Margit

2014-10-09

Endometrial cancer is the most common gynecologic malignancy. Type II endometrial carcinoma is often poorly differentiated and patients diagnosed with Type II disease (~11%) are disproportionately represented in annual endometrial cancer deaths (48%). Recent genomic studies highlight mutations in chromatin regulators as drivers in Type II endometrial carcinoma tumorigenesis, suggesting the use of epigenetic targeted therapies could provide clinical benefit to these patients. We investigated the anti-tumor efficacy of the LSD1 inhibitor HCI2509 in two poorly differentiated Type II endometrial cancer cell lines AN3CA and KLE. The effects of HCI2509 on viability, proliferation, anchorage-independent growth, global histone methylation, LSD1 target gene induction, cell cycle, caspase activation and TUNEL were assayed. KLE cells were used in an orthotopic xenograft model to assess the anti-tumor activity of HCI2509. Both AN3CA and KLE cells were sensitive to HCI2509 treatment with IC50s near 500 nM for cell viability. Inhibition of LSD1 with HCI2509 caused decreased proliferation and anchorage independent growth in soft agar, elevated global histone methylation, and perturbed the cell cycle in both cell lines. These effects were largely dose-dependent. HCI2509 treatment also caused apoptotic cell death. Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen. Tumor burden was distributed log-normally. Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034). Our findings demonstrate the anti-cancer properties of the LSD1 inhibitor HCI2509 on poorly differentiated endometrial carcinoma cell lines, AN3CA and KLE. HCI2509 showed single-agent efficacy in orthotopic xenograft studies. Continued studies are needed to preclinically validate LSD1 inhibition as a therapeutic strategy for endometrial carcinoma.

Assessing the impact of As-Cd-Pb metal mixture on cell transformation by two-stage Balb/c 3T3 cell assay.

PubMed

Rodríguez-Sastre, M A; Rojas, E; Valverde, M

2014-07-01

Human beings are exposed to metals as a consequence of various industrial activities, including glass production, agrochemical production, metallurgy and battery manufacture. New data about the possible mechanisms involved in the carcinogenic activity of these metals are constantly being reported. Exposure to complex mixtures of metals is more likely to occur than exposure to a single metal alone. Among these elements, arsenic, cadmium and lead are ubiquitous air and water pollutants that continue to threaten the quality of public health around the world. The aim of the present study was to evaluate the capability of a mixture of 2 µM NaAsO2, 2 µM CdCl2 and 5 µM Pb(C2H3O2)2·3H2O at relevant epidemiological concentrations to induce cell transformation processes. Transforming potential was determined by a murine two-stage Balb/c 3T3 cell assay. Cell viability, reactive oxygen species (ROS), DNA damage, cell cycle analysis, senescence, generation time and metallothionein expression were also evaluated. The results showed that the metal mixture induced morphological cell transformation only when acting as initiator stimuli of the process. A decrease in cell viability was observed at the promotion stage, a time during which ROS increase, especially when a metal mixture was applied as a promoter stimulant. Changes in DNA damage were not observed throughout the assay; however, we observed G1 cell cycle arrest. The metal mixture, acting as a promoter, is capable of inducing senescence, but metals employed as initiators with 12-O-tetradecanoylphorbol-13-acetate as a promoter are capable of causing avoidance of senescence and triggering the transformation potential of the cells. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

β(1,3)-Glucanosyl-Transferase Activity Is Essential for Cell Wall Integrity and Viability of Schizosaccharomyces pombe

PubMed Central

de Medina-Redondo, María; Arnáiz-Pita, Yolanda; Clavaud, Cécile; Fontaine, Thierry; del Rey, Francisco; Latgé, Jean Paul; Vázquez de Aldana, Carlos R.

2010-01-01

Background The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of β-glucans. The β(1,3)-glucan synthase complex synthesizes linear β(1,3)-glucans, which remain unorganized until they are cross-linked to other β(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding β(1,3)-glucanosyl-transferases -gas1+, gas2+, gas4+ and gas5+- are present in S. pombe, although their function has not been analyzed. Methodology/Principal Findings Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1Δ mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display β(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast. Conclusions/Significance We conclude that β(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth. PMID:21124977

Indole-3-carbinol induces G1 cell cycle arrest and apoptosis through aryl hydrocarbon receptor in THP-1 monocytic cell line.

PubMed

Mohammadi, Saeed; Seyedhosseini, Fakhri Sadat; Behnampour, Nasser; Yazdani, Yaghoub

2017-10-01

The role of aryl hydrocarbon receptor (AhR) in carcinogenesis has been studied recently. Indole-3-carbinol (I3C) is an AhR agonist and a potential anticancer agent. Here, we investigated the effects of I3C on cell cycle progression and apoptosis through activation of AhR on THP-1 acute myeloid leukemia (AML) cell line. MTT viability assay was used to measure the cytotoxic effects of I3C on THP-1 cells. Apoptosis and cell cycle assays were investigated using flow cytometry. Real time RT-PCR was conducted to measure the alterations in the expression of AhR gene, key genes associated with AhR activation (IL1β and CYP1A1) and major genes involved in cell cycle regulation and apoptosis including P27, P21, CDK2, P53, BCL2 and FasR. Our findings revealed that I3C inhibits the proliferation of THP-1 cells in a dose- and time-dependent manner with minimal toxicity over normal monocytes. The AhR target genes (CYP1A1, IL1β) were overexpressed upon I3C treatment (p < .05 to p < .001). The antiproliferative effects of I3C were in association with programed cell death. I3C downregulated BCL2 and upregulated FasR in THP-1 cells (p < .05 to p < .001). G1 cell cycle arrest was also observed using flow cytometry. G1-acting cell cycle genes (P21, P27 and P53) were overexpressed (p < .05 to p < .001), while CDK2 was downregulated upon I3C treatment (p < .01 to p < .001). I3C could exert its antileukemic effects through AhR activation which is associated with programed cell death and G1 cell cycle arrest in a dose- and time-dependent manner. Therefore, AhR could be targeted as a novel treatment possibility in AML.

Role of Hydroalcoholic Extract of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), in Facilitating Cellular Acclimatization in a Low-Oxygen Microenvironment.

PubMed

Koganti, Praveen; Tulsawani, Rajkumar; Sharma, Purva; Sharma, Manish; Arora, Shivani; Misra, Kshipra

2018-01-01

Ganoderma lucidum is known to exert many health benefits including effects to improve oxygen utilization. Therefore, this study was designed to evaluate the role of a hydroalcoholic G. lucidum extract in providing tolerance to HT22 cells grown under hypoxic conditions. HT22 cells were exposed to 0.5% O2 in the presence or absence of the extract for 24 hours. At the end of the exposure period, we performed cell viability assays, cell cycle analysis, and biochemical and protein expression studies. The extract-treated cells revealed less cell death, minimized caspase 3 and reactive oxygen species levels, and relieved G0/G1 cell cycle arrest compared with hypoxic cells cultured without the extract. Further, extract-treated cells showed improved expression of Nrf2, heme oxygenase 1, and metallothionein and stabilized levels of hypoxia-inducible factor 1α. Moreover, lower levels of nuclear factor-κB and tumor necrosis factor a were evident in extract-treated cells. Overall, the G. lucidum extract reduced hypoxia-induced cell death and augmented transcription factors (HIF-1α and Nrf2), conferring tolerance to hypoxia.

5-demethyltangeretin inhibits human nonsmall cell lung cancer cell growth by inducing G2/M cell cycle arrest and apoptosis.

PubMed

Charoensinphon, Noppawat; Qiu, Peiju; Dong, Ping; Zheng, Jinkai; Ngauv, Pearline; Cao, Yong; Li, Shiming; Ho, Chi-Tang; Xiao, Hang

2013-12-01

Tangeretin (TAN) and 5-demethyltangeretin (5DT) are two closely related polymethoxyflavones found in citrus fruits. We investigated growth inhibitory effects on three human nonsmall cell lung cancer (NSCLC) cells. Cell viability assay demonstrated that 5DT inhibited NSCLC cell growth in a time- and dose-dependent manner, and IC50 s of 5DT were 79-fold, 57-fold, and 56-fold lower than those of TAN in A549, H460, and H1299 cells, respectively. Flow cytometry analysis showed that 5DT induced extensive G2/M cell cycle arrest and apoptosis in NSCLC cells, while TAN at tenfold higher concentrations did not. The apoptosis induced by 5DT was further confirmed by activation of caspase-3 and cleavage of PARP. Moreover, 5DT dose-dependently upregulated p53 and p21(Cip1/Waf1), and downregulated Cdc-2 (Cdk-1) and cyclin B1. HPLC analysis revealed that the intracellular levels of 5DT in NSCLC cells were 2.7-4.9 fold higher than those of TAN after the cells were treated with 5DT or TAN at the same concentration. Our results demonstrated that 5DT inhibited NSCLC cell growth by inducing G2/M cell cycle arrest and apoptosis. These effects were much stronger than those produced by TAN, which is partially due to the higher intracellular uptake of 5DT than TAN. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of a discoloration-resistant calcium aluminosilicate cement on the viability and proliferation of undifferentiated human dental pulp stem cells.

PubMed

Niu, Li-na; Watson, Devon; Thames, Kyle; Primus, Carolyn M; Bergeron, Brian E; Jiao, Kai; Bortoluzzi, Eduardo A; Cutler, Christopher W; Chen, Ji-hua; Pashley, David H; Tay, Franklin R

2015-11-30

Discoloration-resistant calcium aluminosilicate cement has been formulated to overcome the timely problem of tooth discoloration reported in the clinical application of bismuth oxide-containing hydraulic cements. The present study examined the effects of this experimental cement (Quick-Set2) on the viability and proliferation of human dental pulp stem cells (hDPSCs) by comparing the cellular responses with commercially available calcium silicate cement (white mineral trioxide aggregate; WMTA) after different aging periods. Cell viability and proliferation were examined using assays that examined plasma membrane integrity, leakage of cytosolic enzyme, caspase-3 activity for early apoptosis, oxidative stress, mitochondrial metabolic activity and intracellular DNA content. Results of the six assays indicated that both Quick-Set2 and WMTA were initially cytotoxic to hDPSCs after setting for 24 h, with Quick-Set2 being comparatively less cytotoxic than WMTA at this stage. After two aging cycles, the cytotoxicity profiles of the two hydraulic cements were not significantly different and were much less cytotoxic than the positive control (zinc oxide-eugenol cement). Based on these results, it is envisaged that any potential beneficial effect of the discoloration-resistant calcium aluminosilicate cement on osteogenesis by differentiated hDPSCs is more likely to be revealed after outward diffusion and removal of its cytotoxic components.

Effects of a discoloration-resistant calcium aluminosilicate cement on the viability and proliferation of undifferentiated human dental pulp stem cells

PubMed Central

Niu, Li-na; Watson, Devon; Thames, Kyle; Primus, Carolyn M.; Bergeron, Brian E.; Jiao, Kai; Bortoluzzi, Eduardo A.; Cutler, Christopher W.; Chen, Ji-hua; Pashley, David H.; Tay, Franklin R.

2015-01-01

Discoloration-resistant calcium aluminosilicate cement has been formulated to overcome the timely problem of tooth discoloration reported in the clinical application of bismuth oxide-containing hydraulic cements. The present study examined the effects of this experimental cement (Quick-Set2) on the viability and proliferation of human dental pulp stem cells (hDPSCs) by comparing the cellular responses with commercially available calcium silicate cement (white mineral trioxide aggregate; WMTA) after different aging periods. Cell viability and proliferation were examined using assays that examined plasma membrane integrity, leakage of cytosolic enzyme, caspase-3 activity for early apoptosis, oxidative stress, mitochondrial metabolic activity and intracellular DNA content. Results of the six assays indicated that both Quick-Set2 and WMTA were initially cytotoxic to hDPSCs after setting for 24 h, with Quick-Set2 being comparatively less cytotoxic than WMTA at this stage. After two aging cycles, the cytotoxicity profiles of the two hydraulic cements were not significantly different and were much less cytotoxic than the positive control (zinc oxide–eugenol cement). Based on these results, it is envisaged that any potential beneficial effect of the discoloration-resistant calcium aluminosilicate cement on osteogenesis by differentiated hDPSCs is more likely to be revealed after outward diffusion and removal of its cytotoxic components. PMID:26617338

Simultaneous inhibition of pan-phosphatidylinositol-3-kinases and MEK as a potential therapeutic strategy in peripheral T-cell lymphomas.

PubMed

Martín-Sánchez, Esperanza; Rodríguez-Pinilla, Socorro M; Sánchez-Beato, Margarita; Lombardía, Luis; Domínguez-González, Beatriz; Romero, Diana; Odqvist, Lina; García-Sanz, Pablo; Wozniak, Magdalena B; Kurz, Guido; Blanco-Aparicio, Carmen; Mollejo, Manuela; Alves, F Javier; Menárguez, Javier; González-Palacios, Fernando; Rodríguez-Peralto, José Luis; Ortiz-Romero, Pablo L; García, Juan F; Bischoff, James R; Piris, Miguel A

2013-01-01

Peripheral T-cell lymphomas are very aggressive hematologic malignancies for which there is no targeted therapy. New, rational approaches are necessary to improve the very poor outcome in these patients. Phosphatidylinositol-3-kinase is one of the most important pathways in cell survival and proliferation. We hypothesized that phosphatidylinositol-3-kinase inhibitors could be rationally selected drugs for treating peripheral T-cell lymphomas. Several phosphatidylinositol-3-kinase isoforms were inhibited genetically (using small interfering RNA) and pharmacologically (with CAL-101 and GDC-0941 compounds) in a panel of six peripheral and cutaneous T-cell lymphoma cell lines. Cell viability was measured by intracellular ATP content; apoptosis and cell cycle changes were checked by flow cytometry. Pharmacodynamic biomarkers were assessed by western blot. The PIK3CD gene, which encodes the δ isoform of phosphatidylinositol-3-kinase, was overexpressed in cell lines and primary samples, and correlated with survival pathways. However, neither genetic nor specific pharmacological inhibition of phosphatidylinositol-3-kinase δ affected cell survival. In contrast, the pan-phosphatidylinositol-3-kinase inhibitor GDC-0941 arrested all T-cell lymphoma cell lines in the G1 phase and induced apoptosis in a subset of them. We identified phospho-GSK3β and phospho-p70S6K as potential biomarkers of phosphatidylinositol-3-kinase inhibitors. Interestingly, an increase in ERK phosphorylation was observed in some GDC -0941-treated T-cell lymphoma cell lines, suggesting the presence of a combination of phosphatidylinositol-3-kinase and MEK inhibitors. A highly synergistic effect was found between the two inhibitors, with the combination enhancing cell cycle arrest at G0/G1 in all T-cell lymphoma cell lines, and reducing cell viability in primary tumor T cells ex vivo. These results suggest that the combined treatment of pan-phosphatidylinositol-3-kinase + MEK inhibitors could be more effective than single phosphatidylinositol-3-kinase inhibitor treatment, and therefore, that this combination could be of therapeutic value for treating peripheral and cutaneous T-cell lymphomas.

Inactivation of Mirk/Dyrk1b Kinase Targets Quiescent Pancreatic Cancer Cells *

PubMed Central

Ewton, Daina Z.; Hu, Jing; Vilenchik, Maria; Deng, Xiaobing; Luk, Kin-chun; Polonskaia, Ann; Hoffman, Ann F.; Zipf, Karen; Boylan, John F.; Friedman, Eileen A.

2011-01-01

A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they re-enter the cell cycle. Earlier studies demonstrated that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G0 tumor cells, that Mirk uses several mechanisms to block cell cycling, and that Mirk increases expression of antioxidant genes which lower ROS levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G0 state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1 and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In prior studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase. PMID:21878655

Noninvasive Real-Time Assessment of Cell Viability in a Three-Dimensional Tissue.

PubMed

Mahfouzi, Seyed Hossein; Amoabediny, Ghassem; Doryab, Ali; Safiabadi-Tali, Seyed Hamid; Ghanei, Mostafa

2018-04-01

Maintaining cell viability within 3D tissue engineering scaffolds is an essential step toward a functional tissue or organ. Assessment of cell viability in 3D scaffolds is necessary to control and optimize tissue culture process. Monitoring systems based on respiration activity of cells (e.g., oxygen consumption) have been used in various cell cultures. In this research, an online monitoring system based on respiration activity was developed to monitor cell viability within acellular lung scaffolds. First, acellular lung scaffolds were recellularized with human umbilical cord vein endothelial cells, and then, cell viability was monitored during a 5-day period. The real-time monitoring system generated a cell growth profile representing invaluable information on cell viability and proliferative states during the culture period. The cell growth profile obtained by the monitoring system was consistent with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and glucose consumption measurement. This system provided a means for noninvasive, real-time, and repetitive investigation of cell viability. Also, we showed the applicability of this monitoring system by introducing shaking as an operating parameter in a long-term culture.

An authentic imaging probe to track cell fate from beginning to end.

PubMed

Lee, Seung Koo; Mortensen, Luke J; Lin, Charles P; Tung, Ching-Hsuan

2014-10-17

Accurate tracing of cell viability is critical for optimizing delivery methods and evaluating the efficacy and safety of cell therapeutics. A nanoparticle-based cell tracker is developed to image cell fate from live to dead. The particle is fabricated from two types of optically quenched polyelectrolytes, a life indicator and a death indicator, through electrostatic interactions. On incubation with cells, the fabricated bifunctional nanoprobes are taken up efficiently and the first colour is produced by normal intracellular proteolysis, reflecting the healthy status of the cells. Depending on the number of coated layers, the signal can persist for several replication cycles. However, as the cells begin dying, the second colour appears quickly to reflect the new cell status. Using this chameleon-like cell tracker, live cells can be distinguished from apoptotic and necrotic cells instantly and definitively.

Unravelling the potential of a new uracil phosphoribosyltransferase (UPRT) from Arabidopsis thaliana in sensitizing HeLa cells towards 5-fluorouracil.

PubMed

Narayanan, Sharmila; Sanpui, Pallab; Sahoo, Lingaraj; Ghosh, Siddhartha Sankar

2016-10-01

In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) revealed its lower binding energies for uracil and 5-fluorouracil (5-FU) as compared to those of bacterial UPRT indicating the prospective of AtUPRT in gene therapy implications. Hence, AtUPRT was cloned and stably expressed in cervical cancer cells (HeLa) to investigate the effect of prodrug 5-FU on these transfected cancer cells. The treatment of AtUPRT-expressing HeLa (HeLa-UPP) cells with 5-FU for 72h resulted in significant decrease in cell viability. Moreover, 5-FU was observed to induce apoptosis and perturb mitochondrial membrane potential in HeLa-UPP cells. While cell cycle analysis revealed significant S-phase arrest as a result of 5-FU treatment in HeLa-UPP cells, quantitative gene expression analysis demonstrated simultaneous upregulation of important cell cycle related genes, cyclin D1 and p21. The survival fractions of non-transfected, vector-transfected and AtUPRT-transfected HeLa cells, following 5-FU treatment, were calculated to be 0.425, 0.366 and 0.227, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Viability and proliferation potential of adipose-derived stem cells following labeling with a positron-emitting radiotracer.

PubMed

Elhami, Esmat; Goertzen, Andrew L; Xiang, Bo; Deng, Jixian; Stillwell, Chris; Mzengeza, Shadreck; Arora, Rakesh C; Freed, Darren; Tian, Ganghong

2011-07-01

Adipose-derived stem cells (ASCs) have promising potential in regenerative medicine and cell therapy. Our objective is to examine the biological function of the labeled stem cells following labeling with a readily available positron emission tomography (PET) tracer, (18)F-fluoro-2-deoxy-D: -glucose (FDG). In this work we characterize labeling efficiency through assessment of FDG uptake and retention by the ASCs and the effect of FDG on cell viability, proliferation, transdifferentiation, and cell function in vitro using rat ASCs. Samples of 10(5) ASCs (from visceral fat tissue) were labeled with concentrations of FDG (1-55 Bq/cell) in 0.75 ml culture medium. Label uptake and retention, as a function of labeling time, FDG concentration, and efflux period were measured to determine optimum cell labeling conditions. Cell viability, proliferation, DNA structure damage, cell differentiation, and other cell functions were examined. Non-labeled ASC samples were used as a control for all experimental groups. Labeled ASCs were injected via tail vein in several healthy rats and initial cell biodistribution was assessed. Our results showed that FDG uptake and retention by the stem cells did not depend on FDG concentration but on labeling and efflux periods and glucose content of the labeling and efflux media. Cell viability, transdifferentiation, and cell function were not greatly affected. DNA damage due to FDG radioactivity was acute, but reversible; cells managed to repair the damage and continue with cell cycles. Over all, FDG (up to 25 Bq/cell) did not impose severe cytotoxicity in rat ASCs. Initial biodistribution of the FDG-labeled ASCs was 80% + retention in the lungs. In the delayed whole-body images (2-3 h postinjection) there was some activity distribution resembling typical FDG uptake patterns. For in vivo cell tracking studies with PET tracers, the parameter of interest is the amount of radiotracer that is present in the cells being labeled and consequent biological effects. From our study we developed a labeling protocol for labeling ASCs with a readily available PET tracer, FDG. Our results indicate that ASCs can be safely labeled with FDG concentration up to 25 Bq/cell, without compromising their biological function. A labeling period of 90 min in glucose-free medium and efflux of 60 min in complete media resulted in optimum label retention, i.e., 60% + by the stem cells. The initial biodistribution of the implanted FDG-labeled stem cells can be monitored using microPET imaging.

Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action.

PubMed

Chilampalli, Chandeshwari; Guillermo, Ruth; Zhang, Xiaoying; Kaushik, Radhey S; Young, Alan; Zeman, David; Hildreth, Michael B; Fahmy, Hesham; Dwivedi, Chandradhar

2011-10-20

Magnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development. UVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle. Magnolol pretreated groups (30, 60 μ g) before UVB treatments (30 mJ/cm2, 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice.Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells. Magnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various signaling pathways. Magnolol could be a potentially safe and potent anticarcinogenic agent against skin cancer.

Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action

PubMed Central

2011-01-01

Background Magnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development. Methods UVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle. Results Magnolol pretreated groups (30, 60 μ g) before UVB treatments (30 mJ/cm2, 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice. Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells. Conclusions Magnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various signaling pathways. Magnolol could be a potentially safe and potent anticarcinogenic agent against skin cancer. PMID:22014088

Caffeine induces sustained apoptosis of human gastric cancer cells by activating the caspase-9/caspase-3 signalling pathway

PubMed Central

Liu, Hanyang; Zhou, Yan; Tang, Liming

2017-01-01

Caffeine is one of the most widely consumed substances found in beverages, and has demonstrated anticancer effects in several types of cancer. The present study aimed to examine the anticancer effects of caffeine on gastric cancer (GC) cells (MGC-803 and SGC-7901) in vitro, and to determine whether the apoptosis-related caspase-9/−3 pathway is associated with these effects. The sustained antiproliferative effects of caffeine on gastric cancer were also investigated. GC cell viability and proliferation were evaluated using cell counting and colony forming assays, following treatment with various concentrations of caffeine. Flow cytometry was performed to assess cell cycle dynamics and apoptosis. Western blot analysis was conducted to detect the activity of the caspase-9/−3 pathway. The results indicated that caffeine treatment significantly suppressed GC cell growth and viability and induced apoptosis by activating the caspase-9/−3 pathway. Furthermore, the anticancer effects of caffeine appeared to be sustained, as the caspase-9/−3 pathway remained active following caffeine withdrawal. In conclusion, caffeine may function as a sustained anticancer agent by activating the caspase-9/−3 pathway, which indicates that it may be useful as a therapeutic candidate in gastric cancer. PMID:28677810

Simple Perfusion Apparatus (SPA) for Manipulation, Tracking and Study of Oocytes and Embryos

PubMed Central

Angione, Stephanie L.; Oulhen, Nathalie; Brayboy, Lynae M.; Tripathi, Anubhav; Wessel, Gary M.

2016-01-01

Objective To develop and implement a device and protocol for oocyte analysis at a single cell level. The device must be capable of high resolution imaging, temperature control, perfusion of media, drugs, sperm, and immunolabeling reagents all at defined flow-rates. Each oocyte and resultant embryo must remain spatially separated and defined. Design Experimental laboratory study Setting University and Academic Center for reproductive medicine. Patients/Animals Women with eggs retrieved for ICSI cycles, adult female FVBN and B6C3F1 mouse strains, sea stars. Intervention Real-time, longitudinal imaging of oocytes following fluorescent labeling, insemination, and viability tests. Main outcome measure(s) Cell and embryo viability, immunolabeling efficiency, live cell endocytosis quantitation, precise metrics of fertilization and embryonic development. Results Single oocytes were longitudinally imaged following significant changes in media, markers, endocytosis quantitation, and development, all with supreme control by microfluidics. Cells remained viable, enclosed, and separate for precision measurements, repeatability, and imaging. Conclusions We engineered a simple device to load, visualize, experiment, and effectively record individual oocytes and embryos, without loss of cells. Prolonged incubation capabilities provide longitudinal studies without need for transfer and potential loss of cells. This simple perfusion apparatus (SPA) provides for careful, precise, and flexible handling of precious samples facilitating clinical in vitro fertilization approaches. PMID:25450296

Hypericin-mediated photocytotoxic effect on HT-29 adenocarcinoma cells is reduced by light fractionation with longer dark pause between two unequal light doses.

PubMed

Sacková, Veronika; Kuliková, Lucia; Mikes, Jaromír; Kleban, Ján; Fedorocko, Peter

2005-01-01

The present study demonstrates the in vitro effect of hypericin-mediated PDT with fractionated light delivery. Cells were photosensitized with unequal light fractions separated by dark intervals (1 or 6 h). We compared the changes in viability, cell number, survival, apoptosis and cell cycle on HT-29 cells irradiated with a single light dose (12 J/cm(2)) to the fractionated light delivery (1 + 11 J/cm(2)) 24 and 48 h after photodynamic treatment. We found that a fractionated light regime with a longer dark period resulted in a decrease of hypericin cytotoxicity. Both cell number and survival were higher after light sensitization with a 6-h dark interval. DNA fragmentation occurred after a single light-dose application, but in contrast no apoptotic DNA formation was detected with a 6-h dark pause. After fractionation the percentage of cells in the G1 phase of the cell cycle was increased, while the proportion of cells in the G2 phase decreased as compared to a single light-dose application, i.e. both percentage of cells in the G1 and G2 phase of the cell cycle were near control levels. We presume that the longer dark interval after the irradiation of cells by first light dose makes them resistant to the effect of the second illumination. These findings confirm that the light application scheme together with other photodynamic protocol components is crucial for the photocytotoxicity of hypericin.

Cytotoxic Activity and Antiproliferative Effects of Crude Skin Secretion from Physalaemus nattereri (Anura: Leptodactylidae) on in vitro Melanoma Cells

PubMed Central

Cruz e Carvalho, Andréa; Prías Márquez, César Augusto; Azevedo, Ricardo Bentes; Joanitti, Graziella Anselmo; Pires Júnior, Osmindo Rodrigues; Fontes, Wagner; Castro, Mariana S.

2015-01-01

Anuran secretions are rich sources of bioactive molecules, including antimicrobial and antitumoral compounds. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against skin cancer cells, and to assess its cytotoxic action mechanisms on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduced the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage), reduction in mitochondrial membrane potential, increase in phosphatidylserine exposure, and cell cycle arrest in S-phase. Together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also subjected to chromatographic fractioning using RP-HPLC, and eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular mass components in a range compatible with peptides. Although the specific mechanisms causing the reduced cell viability and cytotoxicity after the treatment with crude secretion are still unknown, it may be considered that molecules, such as the peptides found in the secretion, are effective against B16F10 tumor cells. Considering the growing need for new anticancer drugs, data presented in this study strongly reinforce the validity of P. nattereri crude secretion as a rich source of new anticancer molecules. PMID:26457717

MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells

PubMed Central

Brito, Jose L.R.; Walker, Brian; Jenner, Matthew; Dickens, Nicholas J.; Brown, Nicola J.M.; Ross, Fiona M.; Avramidou, Athanasia; Irving, Julie A.E.; Gonzalez, David; Davies, Faith E.; Morgan, Gareth J.

2009-01-01

Background The recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear. Design and Methods The expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays. Results We found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples. Conclusions In conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival. PMID:19059936

Genistein induced anticancer effects on pancreatic cancer cell lines involves mitochondrial apoptosis, G0/G1cell cycle arrest and regulation of STAT3 signalling pathway.

PubMed

Bi, Yi-Liang; Min, Min; Shen, Wei; Liu, Yan

2018-01-15

Genistein is a natural flavonoid that has been reported to exhibit anticancer effects against different types of cancers which include, but are not limited to, breast and oral squamous cell carcinoma. The present study was designed to evaluate the anticancer effects of the natural flavonoid genistein against pancreatic cancer cell lines and to explore the underlying mechanism. Antiproliferative activity was investigated by MTT assay. Apoptosis was detected by DAPI and annexin V/PI staining. DNA damage was assessed by comet assay. Reactive oxygen species (ROS) and reduction of mitochondrial membrane potential (MMP) were determined by flow cytometry. Cell migration was examined by wound healing assay. Protien expressions were determined by western blotting. Antiproliferative assay revealed that genistein reduced the cell viability of pancreatic cancer cells in a dose dependent manner with an IC 50 of 20 and 25 µM against Mia-PaCa2 and PANC-1 cancer cell lines respectively. However, its antiproliferative effects were less pronounced against non-cancerous pancreatic ductal epithelial cell line (H6C7) as evident from the IC 50 of 120 µM. Genistein induced significant morphological changes in pancreatic cancer cells and triggered cell cycle arrest in G 0 /G 1 phase. DAPI staining and flow cytometric analysis revealed that genistein induced apoptosis in a dose dependent manner through generation of substantial amounts of ROS and reduction of MMP. However, treatment of the pancreatic cancer with genistein and ascorbic acid could abrogate the effects of genistein on cell viability. Protien expression analysis revealed that genistein upregulated cytosolic cytochrome c, Bax, cleaved Caspase-3 and cleaved caspase-9 expressions with concomitant downregulation of Bcl-2 expression. Moreover, genistein inhibited the phosphorylation of signal transducer and activator of transcription STAT3 proteins and downregulated the expression of survivin, cyclin D1 and ALDH1A1 in Mia-PaCa2 cells in a dose dependent manner. Interestingly, genistein could inhibit the cell migration potential of the Mia-PaCa2 cells which was further associated with the downregulation of metalloproteinases (MPP-2 and MPP-9). Taken together, we propose that genistein exerts anticancer activity in pancreatic cancer cells through induction of ROS mediated mitochondrial apoptosis, cell cycle arrest and regulation of STAT3 and may therefore prove beneficial in the management of pancreatic cancers cancer. Copyright © 2017 Elsevier GmbH. All rights reserved.

The effect of ultrasound-related stimuli on cell viability in microfluidic channels

PubMed Central

2013-01-01

Background In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli. Results Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 Vpp), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0–29 Vpp, at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 Vpp. There was a ~17% reduction in viability at 21 Vpp, corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 Vpp, corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 Vpp) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec. Conclusion The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that significantly reduce cell viability. Results from this study may be of potential interest to researchers in the field of US-induced intracellular drug delivery and ultrasonic manipulation of biological cells. PMID:23809777

The effect of ultrasound-related stimuli on cell viability in microfluidic channels.

PubMed

Ankrett, Dyan N; Carugo, Dario; Lei, Junjun; Glynne-Jones, Peter; Townsend, Paul A; Zhang, Xunli; Hill, Martyn

2013-06-28

In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli. Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 Vpp), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0-29 Vpp, at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 Vpp. There was a ~17% reduction in viability at 21 Vpp, corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 Vpp, corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 Vpp) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec. The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that significantly reduce cell viability. Results from this study may be of potential interest to researchers in the field of US-induced intracellular drug delivery and ultrasonic manipulation of biological cells.

Developmental kinetics of pig embryos by parthenogenetic activation or by handmade cloning.

PubMed

Li, J; Li, R; Liu, Y; Villemoes, K; Purup, S; Callesen, H

2013-10-01

The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time-lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time-lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non-viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro-handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre-implantation developmental kinetics should be observed. © 2013 Blackwell Verlag GmbH.

The dual targeting of insulin and insulin-like growth factor 1 receptor enhances the mTOR inhibitor-mediated antitumor efficacy in hepatocellular carcinoma

PubMed Central

Pivonello, Claudia; Negri, Mariarosaria; De Martino, Maria Cristina; Napolitano, Maria; de Angelis, Cristina; Provvisiero, Donatella Paola; Cuomo, Gaia; Auriemma, Renata Simona; Simeoli, Chiara; Izzo, Francesco; Colao, Annamaria; Hofland, Leo J.; Pivonello, Rosario

2016-01-01

Deregulation of mTOR and IGF pathways is frequent in hepatocellular carcinoma (HCC), thus mTOR and IGF1R represent suitable therapeutic targets in HCC. The aim of this study was to evaluate the effects of mTOR inhibitors (mTORi) and OSI-906, blocker of IGF1R/IR, on HCC cell proliferation, viability, migration and invasion, and alpha-fetoprotein (α-FP) secretion. In HepG2 and HuH-7 we evaluated, the expression of mTOR and IGF pathway components; the effects of Sirolimus, Everolimus, Temsirolimus and OSI-906 on cell proliferation; the effects of Sirolimus, OSI-906, and their combination, on cell secretion, proliferation, viability, cell cycle, apoptosis, invasion and migration. Moreover, intracellular mechanisms underlying these cell functions were evaluated in both cell lines. Our results show that HepG2 and HuH-7 present with the same mRNA expression profile with high levels of IGF2. OSI-906 inhibited cell proliferation at high concentration, while mTORi suppressed cell proliferation in a dose-time dependent manner in both cell lines. The co-treatment showed an additive inhibitory effect on cell proliferation and viability. This effect was not related to induction of apoptosis, but to G0/G1 phase block. Moreover, the co-treatment prevented the Sirolimus-induced AKT activation as escape mechanism. Both agents demonstrated to be differently effective in inhibiting α-FP secretion. Sirolimus, OSI-906, and their combination, blocked cell migration and invasion in HuH-7. These findings indicate that, co-targeting of IGF1R/IR and mTOR pathways could be a novel therapeutic approach in the management of HCC, in order to maximize antitumoral effect and to prevent the early development of resistance mechanisms. PMID:26756219

Activation of PPARγ mediates icaritin-induced cell cycle arrest and apoptosis in glioblastoma multiforme.

PubMed

Liu, Yongji; Shi, Ling; Liu, Yuan; Li, Peng; Jiang, Guoping; Gao, Xiaoning; Zhang, Yongbin; Jiang, Chuanwu; Zhu, Weiping; Han, Hongxing; Ju, Fang

2018-04-01

Glioblastoma multiforme (GBM) is the most prevalent primary malignancy of the brain. This study was designed to investigate whether icaritin exerts anti-neoplastic activity against GBM in vitro. Cell Counting Kit-8 (CCK-8) assay was utilized to examine the viability of GBM cells. The apoptotic cell population was measured by flow cytometry analysis. Cell cycle distribution was detected by flow cytometry as well. Western blot analysis was performed to examine the level of biomarker proteins in GBM cells. Levels of PPARγ mRNA and protein were detected by qPCR and western blot analysis, respectively. To examine the role of PPARγ in the anti-neoplastic activity of icaritin, PPARγ antagonist GW9662 or PPARγ siRNA was used. The activity of PPARγ was determined by DNA binding and luciferase assays. Our findings revealed that icaritin markedly suppresses cell growth in a dose-dependent and time-dependent fashion. The cell population at the G0/G1 phase of the cell cycle was significantly increased following icaritin treatment. Meanwhile, icaritin promoted apoptotic cell death in T98G and U87MG cells. Further investigation showed upregulation of PPARγ played a key role in the anti-neoplastic activities of icaritin. Moreover, our result demonstrated activation of AMPK signaling by icaritin mediated the modulatory effect of icaritin on PPARγ. Our results suggest the PPARγ may mediate anti-neoplastic activities against GBM. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Targeting the Wnt/β-catenin pathway in primary ovarian cancer with the porcupine inhibitor WNT974.

PubMed

Boone, Jonathan D; Arend, Rebecca C; Johnston, Bobbi E; Cooper, Sara J; Gilchrist, Scott A; Oelschlager, Denise K; Grizzle, William E; McGwin, Gerald; Gangrade, Abhishek; Straughn, J Michael; Buchsbaum, Donald J

2016-02-01

Preclinical studies in ovarian cancer have demonstrated upregulation of the Wnt/β-catenin pathway promoting tumor proliferation and chemoresistance. Our objective was to evaluate the effect of the Wnt/β-catenin pathway inhibitor, WNT974, in primary ovarian cancer ascites cells. Ascites cells from patients with papillary serous ovarian cancer were isolated and treated with 1 μM WNT974±100 μM carboplatin. Viability was evaluated with the ATPlite assay. The IC50 was calculated using a dose-response analysis. Immunohistochemistry (IHC) was performed on ascites cells and tumor. Expression of R-spondin 2 (RSPO2), RSPO3, PORCN, WLS, AXIN2, and three previously characterized RSPO fusion transcripts were assessed using Taqman assays. Sixty ascites samples were analyzed for response to WNT974. The ascites samples that showed a decrease in ATP concentration after treatment demonstrated no difference from the untreated cells in percent viability with trypan blue staining. Flow cytometry demonstrated fewer cells in the G2 phase and more in the G1 and S phases after treatment with WNT974. Combination therapy with WNT974 and carboplatin resulted in a higher percentage of samples that showed ≥30% reduction in ATP concentration than either single drug treatment. IHC analysis of Wnt pathway proteins suggests cell cycle arrest rather than cytotoxicity after WNT974 treatment. QPCR indicated that RSPO fusions are not prevalent in ovarian cancer tissues or ascites. However, higher PORCN expression correlated to sensitivity to WNT974 (P=0.0073). In conclusion, WNT974 produces cytostatic effects in patient ascites cells with primary ovarian cancer through inhibition of the Wnt/β-catenin pathway. The combination of WNT974 and carboplatin induces cytotoxicity plus cell cycle arrest in a higher percentage of ascites samples than with single drug treatment. RSPO fusions do not contribute to WNT974 sensitivity; however, higher PORCN expression indicates increased WNT974 sensitivity.

Expression of PFKFB3 and Ki67 in lung adenocarcinomas and targeting PFKFB3 as a therapeutic strategy.

PubMed

Li, Xiaoli; Liu, Jian; Qian, Li; Ke, Honggang; Yao, Chan; Tian, Wei; Liu, Yifei; Zhang, Jianguo

2018-01-11

Phosphofructokinase-2/fructose-2, 6-bisphosphatase 3 (PFKFB3) catalyzes the synthesis of F2,6BP, which is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1): the rate-limiting enzyme of glycolysis. During tumorigenesis, PFKFB3 increases glycolysis, angiogenesis, and tumor progression. In this study, our aim was to investigate the significance of PFKFB3 and Ki67 in human lung adenocarcinomas and to target PFKFB3 as a therapeutic strategy. In this study, we determined the expression levels of PFKFB3 mRNA and proteins in cancerous and normal lung adenocarcinomas by quantitative reverse transcription PCR (qRT-PCR), Western blot analysis, and tissue microarray immunohistochemistry analysis, respectively. In human adenocarcinoma tissues, PFKFB3 and Ki67 protein levels were related to the clinical characteristics and overall survival. Both PFKFB3 mRNA and protein were significantly higher in lung adenocarcinoma cells (all P < 0.05). A high expression of PFKFB3 and Ki67 were associated with the degree of differentiation, TNM staging, lymph node metastasis, and survival. A high expression of PFKFB3 protein was an independent prognostic marker in lung adenocarcinoma. Subsequently, 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) was used as a selective antagonist of PFKFB3. Glycolytic flux was determined by measuring glucose uptake, F2,6BP, and lactate production. Cell viability, cell cycle, cell apoptosis, cell migration, and invasion were analyzed by MTT, flow cytometry, Western blot analysis, wound healing assay, and transwell chamber assay. By targeting PFKFB3, it inhibited cell viability and glycolytic activity. It also caused apoptosis and induced cell cycle arrest. Furthermore, the migration and invasion of A549 cells was inhibited. We conclude that PFKFB3 bears an oncogene-like regulatory element in lung adenocarcinoma progression. In the treatment of lung adenocarcinoma, targeting PFKFB3 would be a promising therapeutic strategy.

Cellular Uptake and Tissue Biodistribution of Functionalized Gold Nanoparticles and Nanoclusters.

PubMed

Escudero-Francos, María A; Cepas, Vanesa; González-Menédez, Pedro; Badía-Laíño, Rosana; Díaz-García, Marta E; Sainz, Rosa M; Mayo, Juan C; Hevia, David

2017-02-01

In this study, the in vitro uptake by fibroblasts and in vivo biodistribution of 15 nm 11-mercaptoundecanoicacid-protected gold nanoparticles (AuNPs-MUA) and 3 nm glutathione- and 3 nm bovine serum albumin-protected gold nanoclusters (AuNCs@GSH and AuNCs@BSA, respectively) were evaluated. In vitro cell viability was examined after gold nanoparticle treatment for 48 h, based on MTT assays and analyses of morphological structure, the cycle cell, cellular doubling time, and the gold concentration in cells. No potential toxicity was observed at any studied concentration (up to 10 ppm) for AuNCs@GSH and AuNCs@BSA, whereas lower cell viability was observed for AuNPs-MUA at 10 ppm than for other treatments. Neither morphological damage nor modifications to the cell cycle and doubling time were detected after contact with nanoparticles. Associations between cells and AuNPs and AuNCs were demonstrated by inductively coupled plasma mass spectrometry (ICP-MS). AuNCs@GSH exhibited fluorescence emission at 611 nm, whereas AuNCs@BSA showed a band at 640 nm. These properties were employed to confirm their associations with cells by fluorescence confocal microscopy; both clusters were observed in cells and maintained their original fluorescence. In vivo assays were performed using 9 male mice treated with 1.70 μg Au/g body weight gold nanoparticles for 24 h. ICP-MS measurements showed a different biodistribution for each type of nanoparticle; AuNPs-MUA mainly accumulated in the brain, AuNCs@GSH in the kidney, and AuNCs@BSA in the liver and spleen. Spleen indexes were not affected by nanoparticle treatment; however, AuNCs@BSA increased the thymus index significantly from 1.28 to 1.79, indicating an immune response. These nanoparticles have great potential as organ-specific drug carriers and for diagnosis, photothermal therapy, and imaging.

MT-4 suppresses resistant ovarian cancer growth through targeting tubulin and HSP27.

PubMed

Pai, Hui Chen; Kumar, Sunil; Shen, Chien-Chang; Liou, Jing Ping; Pan, Shiow Lin; Teng, Che Ming

2015-01-01

In this study, the anticancer mechanisms of MT-4 were examined in A2780 and multidrug-resistant NCI-ADR/res human ovarian cancer cell lines. To evaluate the activity of MT-4, we performed in vitro cell viability and cell cycle assays and in vivo xenograft assays. Immunoblotting analysis was carried out to evaluate the effect of MT-4 on ovarian cancer. Tubulin polymerization was determined using a tubulin binding assay. MT-4 (2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-ethyl]-phenol), a derivative of moscatilin, can inhibit both sensitive A2780 and multidrug-resistant NCI-ADR/res cell growth and viability. MT-4 inhibited tubulin polymerization to induce G2/M arrest followed by caspase-mediated apoptosis. Further studies indicated that MT-4 is not a substrate of P-glycoprotein (p-gp). MT-4 also caused G2/M cell cycle arrest, accompanied by the upregulation of cyclin B, p-Thr161 Cdc2/p34, polo-like kinase 1 (PLK1), Aurora kinase B, and phospho-Ser10-histone H3 protein levels. In addition, we found that p38 MAPK pathway activation was involved in MT-4-induced apoptosis. Most importantly, MT-4 also decreased heat shock protein 27 expression and reduced its interaction with caspase-3, which inured cancer cells to chemotherapy resistance. Treatment of cells with SB203580 or overexpression of dominant negative (DN)-p38 or wild-type HSP27 reduced PARP cleavage caused by MT-4. MT-4 induced apoptosis through regulation of p38 and HSP27. Our xenograft models also show the in vivo efficacy of MT-4. MT-4 inhibited both A2780 and NCI-ADR/res cell growth in vitro and in vivo. These findings indicate that MT-4 could be a potential lead compound for the treatment of multidrug-resistant ovarian cancer.

Studies of yeast cell oxygenation and energetics by laser fluorometry of reduced nicotinamide adenine dinucleotide

NASA Astrophysics Data System (ADS)

Pan, Fu-shih; Chen, Stephen; Mintzer, Robert A.; Chen, Chin-Tu; Schumacker, Paul

1991-03-01

It is of fundamental importance for biological scientists to assess cellular energetics. Under aerobic conditions, the tricarboxylic acid cycle (TCA cycle) is coupled with the mitochondrial electron cascade pathway to provide the cell with energy. The nicotinamide adenine dinucleotide-conjugated pair (NAD and NADH) is the coenzyme in numerous important biomedical reactions which include several important dehydrogenase reactions in the TCA cycle. Based on Le Chatelier's principle, NADH will accumulate when this energy production mechanism is impaired. The relative amounts of NAD and NADH in a cell are defined as the redox state of the cell (Williamson et.al. 1967) which provides a valuable index of cellular energetics. The sum of the amounts of NAD and NADH in a cell may be assumed to be constant during a finite time; therefore, a reliable means of measuring the NADH concentration would provide us with a useful indicator of tissue viability. Traditionally, the quantities of NADH and NAD may be measured by chemical assay methods. We can avoid these tediois analyses by exploiting the significant difference between the ultraviolet absorption spectra of this redox pair. However, because of the opacity of biological samples and the interference of other biochemicals that also absorb ultraviolet radiation, measurement of NADH and NAD+ concentrations in vivo by absorption spectroscopy is not feasible.

Neuroblastoma cells depend on HDAC11 for mitotic cell cycle progression and survival

PubMed Central

Thole, Theresa M; Lodrini, Marco; Fabian, Johannes; Wuenschel, Jasmin; Pfeil, Sebastian; Hielscher, Thomas; Kopp-Schneider, Annette; Heinicke, Ulrike; Fulda, Simone; Witt, Olaf; Eggert, Angelika; Fischer, Matthias; Deubzer, Hedwig E

2017-01-01

The number of long-term survivors of high-risk neuroblastoma remains discouraging, with 10-year survival as low as 20%, despite decades of considerable international efforts to improve outcome. Major obstacles remain and include managing resistance to induction therapy, which causes tumor progression and early death in high-risk patients, and managing chemotherapy-resistant relapses, which can occur years after the initial diagnosis. Identifying and validating novel therapeutic targets is essential to improve treatment. Delineating and deciphering specific functions of single histone deacetylases in neuroblastoma may support development of targeted acetylome-modifying therapeutics for patients with molecularly defined high-risk neuroblastoma profiles. We show here that HDAC11 depletion in MYCN-driven neuroblastoma cell lines strongly induces cell death, mostly mediated by apoptotic programs. Genes necessary for mitotic cell cycle progression and cell division were most prominently enriched in at least two of three time points in whole-genome expression data combined from two cell systems, and all nine genes in these functional categories were strongly repressed, including CENPA, KIF14, KIF23 and RACGAP1. Enforced expression of one selected candidate, RACGAP1, partially rescued the induction of apoptosis caused by HDAC11 depletion. High-level expression of all nine genes in primary neuroblastomas significantly correlated with unfavorable overall and event-free survival in patients, suggesting a role in mediating the more aggressive biological and clinical phenotype of these tumors. Our study identified a group of cell cycle-promoting genes regulated by HDAC11, being both predictors of unfavorable patient outcome and essential for tumor cell viability. The data indicate a significant role of HDAC11 for mitotic cell cycle progression and survival of MYCN-amplified neuroblastoma cells, and suggests that HDAC11 could be a valuable drug target. PMID:28252645

Sparstolonin B, a Novel Plant Derived Compound, Arrests Cell Cycle and Induces Apoptosis in N-Myc Amplified and N-Myc Nonamplified Neuroblastoma Cells

PubMed Central

Kumar, Ambrish; Fan, Daping; DiPette, Donald J.; Singh, Ugra S.

2014-01-01

Neuroblastoma is one of the most common solid tumors and accounts for ∼15% of all the cancer related deaths in the children. Despite the standard therapy for advanced disease including chemotherapy, surgery, and radiation, the mortality rate remains high for these patients. Hence, novel therapeutic agents are desperately needed. Here we examined the anticancer activity of a novel plant-derived compound, sparstolonin B (SsnB; 8,5′-dihydroxy-4-phenyl-5,2′-oxidoisocoumarin) using neuroblastoma cell lines of different genetics. SsnB was recently isolated from an aquatic Chinese herb, Sparganium stoloniferum, and tubers of this herb have been used in traditional Chinese medicine for the treatment of several inflammatory diseases and cancers. Our cell viability and morphological analysis indicated that SsnB at 10 µM concentration significantly inhibited the growth of both N-myc amplified (SK-N-BE(2), NGP, and IMR-32 cells) and N-myc nonamplified (SH-SY5Y and SKNF-1 cells) neuroblastoma cells. The flow cytometric analyses suggested that SsnB arrests the cell cycle progression at G2-M phase in all neuroblastoma cell lines tested. Exposure of SsnB inhibited the compact spheroid formation and reduced the tumorigenicity of SH-SY5Y cells and SK-N-BE(2) cells in in vitro 3-D cell culture assays (anchorage-independent colony formation assay and hanging drop assay). SsnB lowers the cellular level of glutathione (GSH), increases generation of reactive oxygen species and activates the cleavage of caspase-3 whereas co-incubation of a GSH precursor, N-acetylcysteine, along with SsnB attenuates the inhibitory effects of SsnB and increases the neuroblastoma cell viability. Our results for the first time demonstrate that SsnB possesses anticancer activity indicating that SsnB-induced reactive oxygen species generation promotes apoptotic cell death in neuroblastoma cells of different genetic background. Thus these data suggest that SsnB can be a promising drug candidate in neuroblastoma therapy. PMID:24788776

Inhibition of Fatty Acid Metabolism Reduces Human Myeloma Cells Proliferation

PubMed Central

Tirado-Vélez, José Manuel; Joumady, Insaf; Sáez-Benito, Ana; Cózar-Castellano, Irene; Perdomo, Germán

2012-01-01

Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40–70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma. PMID:23029529

Comparative study on effects of two different types of titanium dioxide nanoparticles on human neuronal cells.

PubMed

Valdiglesias, Vanessa; Costa, Carla; Sharma, Vyom; Kiliç, Gözde; Pásaro, Eduardo; Teixeira, João Paulo; Dhawan, Alok; Laffon, Blanca

2013-07-01

Titanium dioxide (TiO2) are among most frequently used nanoparticles (NPs). They are present in a variety of consumer products, including food industry in which they are employed as an additive. The potential toxic effects of these NPs on mammal cells have been extensively studied. However, studies regarding neurotoxicity and specific effects on neuronal systems are very scarce and, to our knowledge, no studies on human neuronal cells have been reported so far. Therefore, the main objective of this work was to investigate the effects of two types of TiO₂ NPs, with different crystalline structure, on human SHSY5Y neuronal cells. After NPs characterization, a battery of assays was performed to evaluate the viability, cytotoxicity, genotoxicity and oxidative damage in TiO₂ NP-exposed SHSY5Y cells. Results obtained showed that the behaviour of both types of NPs resulted quite comparable. They did not reduce the viability of neuronal cells but were effectively internalized by the cells and induced dose-dependent cell cycle alterations, apoptosis by intrinsic pathway, and genotoxicity not related with double strand break production. Furthermore, all these effects were not associated with oxidative damage production and, consequently, further investigations on the specific mechanisms underlying the effects observed in this study are required. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biological Modulation of Mouse RPE Cells in Response to Subthreshold Diode Micropulse Laser Treatment.

PubMed

Li, Zhouyue; Song, Yanping; Chen, Xiao; Chen, Zhongshan; Ding, Qin

2015-11-01

Many clinical trials have demonstrated the effectiveness of subthreshold phototherapy with no visible damage in retinal vascular diseases, such as diabetic retinopathy. We aimed primarily to investigate the effect of subthreshold diode micropulse laser (SDM) treatment on mouse retinal pigmented epithelium (RPE) cells. The expression of angiogenesis-modulating cytokines in response to SDM was also explored. The least toxic laser dose was selected by measuring cell viability with MTT assay and 5 % duty cycle (DC) was chosen for use in further experiments. RPE cells were treated with laser-induced radiation ranging from 0 to 400 mW for 24 h. The apoptotic rate of RPE cells was assessed by flow cytometry. Expressions of vascular endothelial growth factor A (VEGF-A), transforming growth factor beta (TGF-β), basic fibroblast growth factor (bFGF), and pigment epithelium-derived factor (PEDF) were determined by Western Blotting and real-time PCR, respectively. After 24 h of laser irradiation, cell viability was reduced dose dependently and the effect was significant compared to the controls (P < 0.05). In addition, laser treatment with intensities of 100 and 200 mW with DC of 5 % produced no significant effect on cell viability and apoptosis as compared with the control group (P > 0.05). The protein and mRNA expressions of angiogenic stimulators (VEGF-A, TGF-β, and bFGF) were significantly down-regulated (P < 0.05), whereas those of the angiogenic inhibitor (PEDF) were up-regulated (P < 0.05). No significant difference was found between the cells treated with different intensities of laser radiation (P > 0.05). Our results showed that SDM treatment of the RPE cells suppressed the expression of choroid neovasculization-promoting cytokines and up-regulated the angiogenic inhibitor, PEDF without damaging the cells. Further investigation is needed to understand the mechanism and to optimize the use of SDM as a novel method of treatment for retinal vascular diseases.

Sensitization of breast cancer cells to doxorubicin via stable cell line generation and overexpression of DFF40.

PubMed

Bagheri, Fatemeh; Safarian, Shahrokh; Eslaminejad, Mohamadreza Baghaban; Sheibani, Nader

2015-12-01

There are a number of reports demonstrating a relationship between the alterations in DFF40 expression and development of some cancers. Here, increased DFF40 expression in T-47D cells in the presence of doxorubicin was envisaged for therapeutic usage. The T-47D cells were transfected with an eukaryotic expression vector encoding the DFF40 cDNA. Following incubation with doxorubicin, propidium iodide (PI) staining was used for cell cycle distribution analysis. The rates of apoptosis were determined by annexin V/PI staining. Apoptosis was also evaluated using the DNA laddering analysis. The viability of DFF40-transfected cells incubated with doxorubicin was significantly decreased compared with control cells. However, there were no substantial changes in the cell cycle distribution of pIRES2-DFF40 cells incubated with doxorubicin compared to control cells. The expression of DFF40, without doxorubicin incubation, had also no significant effect on the cell cycle distribution. There was no DNA laddering in cells transfected with the empty pIRES2 vector when incubated with doxorubicin. In contrast, DNA laddering was observed in DFF40 transfected cells in the presence of doxorubicin after 48 h. Also, the expression of DFF40 and DFF45 was increased in DFF40 transfected cells in the presence of doxorubicin enhancing cell death. Collectively our results indicated that co-treatment of DFF40-transfected cells with doxorubicin can enhance the killing of these tumor cells via apoptosis. Thus, modulation of DFF40 level may be a beneficial strategy for treatment of chemo-resistant cancers.

P53-dependent antiproliferative and pro-apoptotic effects of trichostatin A (TSA) in glioblastoma cells.

PubMed

Bajbouj, K; Mawrin, C; Hartig, R; Schulze-Luehrmann, J; Wilisch-Neumann, A; Roessner, A; Schneider-Stock, R

2012-05-01

Glioblastomas are known to be highly chemoresistant, but HDAC inhibitors (HDACi) have been shown to be of therapeutic relevance for this aggressive tumor type. We treated U87 glioblastoma cells with trichostatin A (TSA) to define potential epigenetic targets for HDACi-mediated antitumor effects. Using a cDNA array analysis covering 96 cell cycle genes, cyclin-dependent kinase inhibitor p21(WAF1) was identified as the major player in TSA-induced cell cycle arrest. TSA slightly inhibited proliferation and viability of U87 cells, cumulating in a G1/S cell cycle arrest. This effect was accompanied by a significant up-regulation of p53 and its transcriptional target p21(WAF1) and by down-regulation of key G1/S regulators, such as cdk4, cdk6, and cyclin D1. Nevertheless, TSA did not induce apoptosis in U87 cells. As expected, TSA promoted the accumulation of total acetylated histones H3 and H4 and a decrease in endogenous HDAC activity. Characterizing the chromatin modulation around the p21(WAF1) promoter after TSA treatment using chromatin immunoprecipitation, we found (1) a release of HDAC1, (2) an increase of acetylated H4 binding, and (3) enhanced recruitment of p53. p53-depleted U87 cells showed an abrogation of the G1/S arrest and re-entered the cell cycle. Immunofluorescence staining revealed that TSA induced the nuclear translocation of p21(WAF1) verifying a cell cycle arrest. On the other hand, a significant portion of p21(WAF1) was present in the cytoplasmic compartment causing apoptosis resistance. Furthermore, TSA-treated p53-mutant cell line U138 failed to show an induction in p21(WAF1), showed a deficient G2/M checkpoint, and underwent mitotic catastrophe. We suggest that HDAC inhibition in combination with other clinically used drugs may be considered an effective strategy to overcome chemoresistance in glioblastoma cells.

Down-Regulation of Glucose-Regulated Protein (GRP) 78 Potentiates Cytotoxic Effect of Celecoxib in Human Urothelial Carcinoma Cells

PubMed Central

Huang, Kuo-How; Kuo, Kuan-Lin; Chen, Shyh-Chyan; Weng, Te-I; Chuang, Yuan-Ting; Tsai, Yu-Chieh; Pu, Yeong-Shiau; Chiang, Chih-Kang; Liu, Shing-Hwa

2012-01-01

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has been reported to elicit anti-proliferative response in various tumors. In this study, we aim to investigate the antitumor effect of celecoxib on urothelial carcinoma (UC) cells and the role endoplasmic reticulum (ER) stress plays in celecoxib-induced cytotoxicity. The cytotoxic effects were measured by MTT assay and flow cytometry. The cell cycle progression and ER stress-associated molecules were examined by Western blot and flow cytometry. Moreover, the cytotoxic effects of celecoxib combined with glucose-regulated protein (GRP) 78 knockdown (siRNA), (−)-epigallocatechin gallate (EGCG) or MG132 were assessed. We demonstrated that celecoxib markedly reduces the cell viability and causes apoptosis in human UC cells through cell cycle G1 arrest. Celecoxib possessed the ability to activate ER stress-related chaperones (IRE-1α and GRP78), caspase-4, and CCAAT/enhancer binding protein homologous protein (CHOP), which were involved in UC cell apoptosis. Down-regulation of GRP78 by siRNA, co-treatment with EGCG (a GRP78 inhibitor) or with MG132 (a proteasome inhibitor) could enhance celecoxib-induced apoptosis. We concluded that celecoxib induces cell cycle G1 arrest, ER stress, and eventually apoptosis in human UC cells. The down-regulation of ER chaperone GRP78 by siRNA, EGCG, or proteosome inhibitor potentiated the cytotoxicity of celecoxib in UC cells. These findings provide a new treatment strategy against UC. PMID:22438966

Induction of apoptosis and cell cycle arrest in L-1210 murine lymphoblastic leukaemia cells by (2E)-3-(2-naphthyl)-1-(3'-methoxy-4'-hydroxy-phenyl)-2-propen-1-one.

PubMed

Pedrini, Fernanda Spezia; Chiaradia, Louise Domeneghini; Licínio, Marley Aparecida; de Moraes, Ana Carolina Rabello; Curta, Juliana Costa; Costa, Aline; Mascarello, Alessandra; Creczinsky-Pasa, Tânia Beatriz; Nunes, Ricardo José; Yunes, Rosendo Augusto; Santos-Silva, Maria Cláudia

2010-09-01

New compounds with biological targets and less cytotoxicity to normal cells are necessary for cancer therapy. In this work ten synthetic chalcones derived from 2-naphtaldehyde were evaluated for their cytotoxic effect in murine acute lymphoblastic leukemia cells L-1210. A series of ten chalcones derived from 2-naphtaldehyde and corresponding acetophenones were prepared by aldolic condensation, using methanol as solvent under basic conditions, at room temperature for 24 h. The cell viability was determined by MTT colorimeter method. The cell cycle phase analysis was carried out by flow cytometry after propidium iodide staining. The apoptosis induction was assessed by exposure to phosphatidylserine (ANNEXIN V-FITC). Cytometric analysis was performed to evaluate the expression of p53, Bcl-2 and Bax protein. The caspase-3 expression was studied by immunoblotting analysis. A preliminary screening of a series of ten chalcones derived from 2-naphtaldehyde showed that chalcone 8, (2E)-3-(2-naphtyl)-1-(3'-methoxy-4'-hydroxy-phenyl)-2-propen-1-one, had the highest cytotoxic effect (IC50 of 54 microM), but not in normal human lymphocytes. To better understand the cytotoxic mechanism of chalcone 8, its effect on cell cycle and apoptosis was assessed. Our results showed that chalcone 8 caused cell cycle arrest in the G2/M phase and a significant increase in the proportion of cells in the subG0/G1 phase. Our results also demonstrated that chalcone 8 promoted a modification in Bax:Bcl-2 ratio and increased p53 expression and caspase-3 activation. The studied chalcone 8 has cytotoxic effect against L-1210 lymphoblastic leukaemic cells, and this effect is associated with increase of p-53 and Bax expression.

Oridonin Up-regulates Expression of P21 and Induces Autophagy and Apoptosis in Human Prostate Cancer Cells

PubMed Central

Li, Xiang; Li, Xiang; Wang, Jiaxiong; Ye, Zaiyuan; Li, Ji-Cheng

2012-01-01

Background: Oridonin (ORI) could inhibit the proliferation and induce apoptosis in various cancer cell lines. However, the mechanism is not fully understood. Methods: Human prostate cancer (HPC) cells were cultured in vitro and cell viability was detected by the CCK-8 assay. The ultrastructure changes were observed under transmission electron microscope (TEM). Chemical staining with acridine orange (AO), MDC or DAPI was used to detect acidic vesicular organelles (AVOs) and alternation of DNA. Expression of LC3 and P21 was detected by Western Blot. Apoptotic rates and cell cycle arrest were detected by FACS. Results: Our study demonstrated that after ORI treatment, the proliferations of human prostate cancer (HPC) cell lines PC-3 and LNCaP were inhibited in a concentration and time-dependent manner. ORI induced cell cycle arrest at the G2/M phase. A large number of autophagosomes with double-membrane structure and acidic vesicular organelles (AVOs) were detected in the cytoplasm of HPC cells treated with ORI for 24 hours. ORI resulted in the conversion of LC3-I to LC3-II and recruitment of LC3-II to the autophagosomal membranes. Autophagy inhibitor 3-methyladenine (3-MA) reduced AVOs formation and inhibited LC3-I to LC3-II conversion. At 48 h, DNA fragmentation, chromatin condensation and disappearance of surface microvilli were detected in ORI-treated cells. ORI induced a significant increase in the number of apoptotic cells (PC-3: 5.4% to 27.0%, LNCaP: 5.3% to 31.0%). Promoting autophagy by nutrient starvation increased cell viability, while inhibition of autophagy by 3-MA promoted cell death. The expression of P21 was increased by ORI, which could be completely reversed by the inhibition of autophagy. Conclusions: Our findings indicated that autophagy occurred before the onset of apoptosis and protected cancer cells in ORI-treated HPC cells. P21 was involved in ORI-induced autophagy and apoptosis. Our results provide an experimental basis for understand the anti-tumor mechanism of ORI as treatment for prostate cancer. PMID:22745580

γ-Oryzanol reduces caveolin-1 and PCGEM1 expression, markers of aggressiveness in prostate cancer cell lines.

PubMed

Hirsch, Gabriela E; Parisi, Mariana M; Martins, Leo A M; Andrade, Claudia M B; Barbé-Tuana, Florencia M; Guma, Fátima T C R

2015-06-01

Prostate cancer is a leading cause of death among men due to the limited number of treatment strategies available for advanced disease. γ-oryzanol is a component of rice bran, rich in phytosterols, known for its antioxidant, anti-carcinogenic and endocrinological effects. It is known that γ-oryzanol may affect prostate cancer cells through the down regulation of the antioxidant genes and that phytosterols have anti-proliferative and apoptotic effects. There are evidences showing that some of the components of γ-oryzanol can modulate genes involved in the development and progression of prostate cancer, as caveolin-1 (Cav-1) and prostate specific androgen-regulated gene (PCGEM1). To determine the effects of γ-oryzanol on prostate cancer cell survival we evaluated the cell viability and biomass by MTT and sulforhodamine B assays, respectively. Cell death, cell cycle and pERK1/2 activity were assessed by flow cytometry. The changes in gene expression involved in the survival and progression of prostate cancer cav-1 and PCGEM1 genes were evaluated by quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR) and cav-1 protein by immunofluorescence followed by confocal microscopy analysis. We found that γ-oryzanol decreases cell viability and culture biomass by apoptosis and/or necrosis death in androgen unresponsive (PC3 and DU145) and responsive (LNCaP) cell lines, and signals through pERK1/2 in LNCaP and DU145 cells. γ-oryzanol also appears to block cell cycle progression at the G2/M in PC3 and LNCaP cells and at G0/G1 in DU145 cells. These effects were accompanied by a down regulation in the expression of the cav-1 in both androgen unresponsive cell lines and PCGEM1 gene in DU145 and LNCaP cells. In summary, we used biochemical and genetics approaches to demonstrate that γ-oryzanol show a promising adjuvant role in the treatment of prostate cancer. © 2015 Wiley Periodicals, Inc.

Anti-tumor effect of hot aqueous extracts from Sonchus oleraceus (L.) L. and Juniperus sabina L - Two traditional medicinal plants in China.

PubMed

Huyan, Ting; Li, Qi; Wang, Yi-Lin; Li, Jing; Zhang, Jian-Yang; Liu, Ya-Xiong; Shahid, Muhammad Riaz; Yang, Hui; Li, Huan-Qing

2016-06-05

Sonchus oleraceus (L.) L (SO) and Juniperus sabina L (JS) are traditional medicinal plants in China. And the aqueous extracts of them have been used to treat tumor, inflammatory diseases, infection and so on in Chinese folk culture. However, the underlying mechanisms of their anti-tumor activities have not been illustrated yet. This study aims to evaluate the inhibitory effects of aqueous extracts from SO and JS on tumor cells. The prepared aqueous extracts of SO and JS were used to treat HepG-2 and K562 tumor cells, while the human peripheral blood mononuclear cells (PBMCs) were set as normal control. The viabilities, cell cycle and apoptosis of tumor cells after extracts treatment were assessed, in addition the expression of apoptosis-related genes (FasL, caspase 3, 6, 7, 8, 9, and 10) were analyzed. Meanwhile, the adherence and migration of HepG-2 were tested, and the expression levels of MMPs and ICAM-1 were analyzed. On top of that, the pSTAT in the two cells were also analyzed and suggested the related signaling pathway that the extracts acted on with in these tumor cells. Results showed that aqueous extracts of SO and JS have inhibitory effects on HepG-2 and K562 cells by decreasing cell viability and inducing apoptosis via up-regulation of the expression of the apoptosis-related genes FasL, caspase 3 and caspase 9. The extracts had different IC50 on tumor cells and PBMCs, which could block the tumor cell cycle at the G(0)/G(1) stage and significantly inhibit the adherence of HepG-2 cells. The extracts inhibited migration of these cells by inhibiting the expression of ICAM-1, MMP-2 and MMP-9. Further study indicated that the inhibition of pSTAT1 and 3 might be responsible for the inhibitory effects of the extracts on tumor cells. The results of this study indicated that SO and JS extracts had the anti-tumor effects, which may be developed as novel anti-tumor drugs and used in cancer therapy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Glutamate oxaloacetate transaminase enables anaplerotic refilling of TCA cycle intermediates in stroke-affected brain

PubMed Central

Rink, Cameron; Gnyawali, Surya; Stewart, Richard; Teplitsky, Seth; Harris, Hallie; Roy, Sashwati; Sen, Chandan K.; Khanna, Savita

2017-01-01

Ischemic stroke results in excessive release of glutamate, which contributes to neuronal cell death. Here, we test the hypothesis that otherwise neurotoxic glutamate can be productively metabolized by glutamate oxaloacetate transaminase (GOT) to maintain cellular energetics and protect the brain from ischemic stroke injury. The GOT-dependent metabolism of glutamate was studied in primary neural cells and in stroke-affected C57-BL6 mice using magnetic resonance spectroscopy and GC-MS. Extracellular Glu sustained cell viability under hypoglycemic conditions and increased GOT-mediated metabolism in vitro. Correction of stroke-induced hypoxia using supplemental oxygen in vivo lowered Glu levels as measured by 1H magnetic resonance spectroscopy. GOT knockdown abrogated this effect and caused ATP loss in the stroke-affected brain. GOT overexpression increased anaplerotic refilling of tricarboxylic acid cycle intermediates in mouse brain during ischemic stroke. Furthermore, GOT overexpression not only reduced ischemic stroke lesion volume but also attenuated neurodegeneration and improved poststroke sensorimotor function. Taken together, our results show that GOT enables metabolism of otherwise neurotoxic extracellular Glu through a truncated tricarboxylic acid cycle under hypoglycemic conditions.—Rink, C., Gnyawali, S., Stewart, R., Teplitsky, S., Harris, H., Roy, S., Sen, C. K., Khanna, S. Glutamate oxaloacetate transaminase enables anaplerotic refilling of TCA cycle intermediates in stroke-affected brain. PMID:28096234

RNA interference of argininosuccinate synthetase restores sensitivity to recombinant arginine deiminase (rADI) in resistant cancer cells

PubMed Central

2011-01-01

Background Sensitivity of cancer cells to recombinant arginine deiminase (rADI) depends on expression of argininosuccinate synthetase (AS), a rate-limiting enzyme in synthesis of arginine from citrulline. To understand the efficiency of RNA interfering of AS in sensitizing the resistant cancer cells to rADI, the down regulation of AS transiently and permanently were performed in vitro, respectively. Methods We studied the use of down-regulation of this enzyme by RNA interference in three human cancer cell lines (A375, HeLa, and MCF-7) as a way to restore sensitivity to rADI in resistant cells. The expression of AS at levels of mRNA and protein was determined to understand the effect of RNA interference. Cell viability, cell cycle, and possible mechanism of the restore sensitivity of AS RNA interference in rADI treated cancer cells were evaluated. Results AS DNA was present in all cancer cell lines studied, however, the expression of this enzyme at the mRNA and protein level was different. In two rADI-resistant cell lines, one with endogenous AS expression (MCF-7 cells) and one with induced AS expression (HeLa cells), AS small interference RNA (siRNA) inhibited 37-46% of the expression of AS in MCF-7 cells. ASsiRNA did not affect cell viability in MCF-7 which may be due to the certain amount of residual AS protein. In contrast, ASsiRNA down-regulated almost all AS expression in HeLa cells and caused cell death after rADI treatment. Permanently down-regulated AS expression by short hairpin RNA (shRNA) made MCF-7 cells become sensitive to rADI via the inhibition of 4E-BP1-regulated mTOR signaling pathway. Conclusions Our results demonstrate that rADI-resistance can be altered via AS RNA interference. Although transient enzyme down-regulation (siRNA) did not affect cell viability in MCF-7 cells, permanent down-regulation (shRNA) overcame the problem of rADI-resistance due to the more efficiency in AS silencing. PMID:21453546

Comparisons of human amniotic mesenchymal stem cell viability in FDA-approved collagen-based scaffolds: Implications for engineered diaphragmatic replacement.

PubMed

Shieh, Hester F; Graham, Christopher D; Brazzo, Joseph A; Zurakowski, David; Fauza, Dario O

2017-06-01

We sought to examine amniotic fluid mesenchymal stem cell (afMSC) viability within two FDA-approved collagen-based scaffolds, as a prerequisite to clinical translation of afMSC-based engineered diaphragmatic repair. Human afMSCs were seeded in a human-derived collagen hydrogel and in a bovine-derived collagen sheet at 3 matching densities. Cell viability was analyzed at 1, 3, and 5days using an ATP-based 3D bioluminescence assay. Statistical comparisons were by ANOVA (P<0.05). There was a highly significant 3-way interaction between scaffold type, seeding density, and time in 3D culture as determinants of cell viability, clearly favoring the human hydrogel (P<0.001). In both scaffolds, cell viability was highest at the highest seeding density of 150,000 cells/mL. Time in 3D culture impacted cell viability at the optimal seeding density in the human hydrogel, with the highest levels on days 1 (P<0.001) and 5 (P=0.05) with no significant effect in the bovine sheet (P=0.39-0.96). Among clinically-approved cell delivery vehicles, mesenchymal stem cell viability is significantly enhanced in a collagen hydrogel when compared with a collagen sheet. Cell viability can be further optimized by seeding density and time in 3D culture. These data further support the regulatory viability of clinical trials of engineered diaphragmatic repair. N/A (animal and laboratory study). Copyright © 2017 Elsevier Inc. All rights reserved.

Biological effects of tritium on fish cells in the concentration range of international drinking water standards.

PubMed

Stuart, Marilyne; Festarini, Amy; Schleicher, Krista; Tan, Elizabeth; Kim, Sang Bog; Wen, Kendall; Gawlik, Jilian; Ulsh, Brant

2016-10-01

To evaluate whether the current Canadian tritium drinking water limit is protective of aquatic biota, an in vitro study was designed to assess the biological effects of low concentrations of tritium, similar to what would typically be found near a Canadian nuclear power station, and higher concentrations spanning the range of international tritium drinking water standards. Channel catfish peripheral blood B-lymphoblast and fathead minnow testis cells were exposed to 10-100,000 Bq l(-1) of tritium, after which eight molecular and cellular endpoints were assessed. Increased numbers of DNA strand breaks were observed and ATP levels were increased. There were no increases in γH2AX-mediated DNA repair. No differences in cell growth were noted. Exposure to the lowest concentrations of tritium were associated with a modest increase in the viability of fathead minnow testicular cells. Using the micronucleus assay, an adaptive response was observed in catfish B-lymphoblasts. Using molecular endpoints, biological responses to tritium in the range of Canadian and international drinking water standards were observed. At the cellular level, no detrimental effects were noted on growth or cycling, and protective effects were observed as an increase in cell viability and an induced resistance to a large challenge dose.

Direct concentration and viability measurement of yeast in corn mash using a novel imaging cytometry method.

PubMed

Chan, Leo L; Lyettefi, Emily J; Pirani, Alnoor; Smith, Tim; Qiu, Jean; Lin, Bo

2011-08-01

Worldwide awareness of fossil-fuel depletion and global warming has been increasing over the last 30 years. Numerous countries, including the USA and Brazil, have introduced large-scale industrial fermentation facilities for bioethanol, biobutanol, or biodiesel production. Most of these biofuel facilities perform fermentation using standard baker's yeasts that ferment sugar present in corn mash, sugar cane, or other glucose media. In research and development in the biofuel industry, selection of yeast strains (for higher ethanol tolerance) and fermentation conditions (yeast concentration, temperature, pH, nutrients, etc.) can be studied to optimize fermentation performance. Yeast viability measurement is needed to identify higher ethanol-tolerant yeast strains, which may prolong the fermentation cycle and increase biofuel output. In addition, yeast concentration may be optimized to improve fermentation performance. Therefore, it is important to develop a simple method for concentration and viability measurement of fermenting yeast. In this work, we demonstrate an imaging cytometry method for concentration and viability measurements of yeast in corn mash directly from operating fermenters. It employs an automated cell counter, a dilution buffer, and staining solution from Nexcelom Bioscience to perform enumeration. The proposed method enables specific fluorescence detection of viable and nonviable yeasts, which can generate precise results for concentration and viability of yeast in corn mash. This method can provide an essential tool for research and development in the biofuel industry and may be incorporated into manufacturing to monitor yeast concentration and viability efficiently during the fermentation process.

Pressure signatures can influence tissue response for individuals supported on an alternating pressure mattress.

PubMed

Chai, C Y; Sadou, O; Worsley, P R; Bader, D L

2017-08-01

Prolonged mechanical loading can lead to the breakdown of skin and underlying tissues which can, in turn, develop into a pressure ulcer. The benefits of pressure relief and/or redistribution to minimise risk have been well documented. Manufacturers have developed alternating air pressure mattresses (APAMs) to provide periodic relief for individuals on prolonged bed-rest. The present study describes the development of a control system, termed Pneumatic Manager which can vary the signature of an APAM, namely its pressure amplitude, cell profile and cycle period. An experimental array was designed to investigate the effects of varying these parameters, particularly with respect to its ability to maintain skin viability in a group of five healthy volunteers lying in a supine position. Transcutaneous gas (T c PO 2 /T c PCO 2 ) tensions at the sacrum were monitored. In addition, pressures and microclimate parameters at the loaded support interface were also measured. In the majority of test conditions the alternating support produced sacral T c PO 2 values, which either remained relatively high or fluctuated in concert with cycle period providing adequate viability. However, in 46% of cases at the extreme pressure amplitude of 100/0 mmHg, there was compromise to the skin viability at the sacrum, as reflected in depressed T c PO 2 levels associated with an elevation of T c PCO 2 levels above the normal range. In all cases, both the humidity and temperature levels increased during the test period. It is interesting to note that interface pressures at the sacrum rarely exceeded 60 mmHg. Although such studies need to be extended to involve bed-bound individuals, the results provide a design template for the optimum pressure signatures of APAM systems to ensure maintenance of skin viability during pronged loading. Copyright © 2017 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.

Ursodeoxycholic acid induces apoptosis of hepatocellular carcinoma cells in vitro.

PubMed

Zhu, Lei; Shan, Lu Juan; Liu, Yue Jian; Chen, Dan; Xiao, Xiao Guang; Li, Yan

2014-12-01

Ursodeoxycholic acid (UDCA) is widely used to treat chronic liver diseases, and its cytoprotective effect on normal hepatocytes has been shown. This study aimed to investigate the apoptotic effects of UDCA on hepatocellular carcinoma (HCC) cells and the underlying molecular events in vitro. HCC cells were treated by UDCA at different doses and periods of time to assess cell morphology, viability, apoptosis and gene expression using methyl thiazolyl tetrazolium (MTT), Annexin V/propidium iodide (PI) stain, transferase dUTP nick end labeling (TUNEL), enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and quantitative reverse transcription polymerase chain reaction, respectively. UDCA treatment reduced cell viability but induced HCC cell apoptosis in dose-dependent and time-dependent manners. UDCA arrested HepG2 cells at phase S of the cell cycle. At the gene levels, UDCA downregulated Bcl-2 and second mitochondria-derived activator of caspase (Smac) protein expressions, but upregulated Bax and Livin proteins in HCC cells. At the highest concentration, UDCA inhibited Livin mRNA expression but increased Smac and caspase-3 mRNA expressions as well as the activity of caspase-3 in HCC cells. The induction of HCC cell apoptosis by UDCA was dose-dependent and time-dependent and was mediated by the regulation of Bax to Bcl-2 ratio, the expressions of Smac and Livin, and caspase-3 expression and activity. © 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

Cytotoxic effects of pyrrolidine dithiocarbamate in small-cell lung cancer cells, alone and in combination with cisplatin.

PubMed

Tahata, Shinichi; Yuan, Bo; Kikuchi, Hidetomo; Takagi, Norio; Hirano, Toshihiko; Toyoda, Hiroo

2014-10-01

The cytocidal effect of pyrrolidine dithiocarbamate (PDTC) was investigated by focusing on cell viability, cell cycle arrest and apoptosis induction in small-cell lung cancer (SCLC) cell lines (NCI-H196 and NCI-H889). PDTC exhibited a much stronger dose-dependent cytotoxic activity against NCI-H196 compared to NCI-H889, while no such activity was observed in normal human embryonal lung fibroblast MRC-5 cells. Cell cycle arrest in S phase paralleled with suppression of c-myc expression without accompanying DNA fragmentation was observed in NCI-H196 cells. A transient increase in the intracellular ROS accompanied with an alteration of expression of oxidative stress-related genes was also confirmed in NCI-H196 cells. Furthermore, the addition of N-acetyl-l-cysteine (NAC), a free radical scavenger, not only abolished PDTC-trigger alterations of expression of these oxidative-related genes, but also almost completely abrogated PDTC-induced reduction in cell viability and morphological changes associated with cell damage. These results thus suggest that PDTC-induced cytotoxicity is attributed to its pro-oxidant activity. PDTC-induced cytotoxicity was further enhanced by CuCl2, however, abolished by bathocuproine disulfonate (BCPS), a non-permeable copper-specific chelator, supporting the plausibility that accumulation of intracellular Cu plays an important role in the cytotoxicity. Importantly, we demonstrated for the first time that PDTC downregulated the expression of ATP7A, known to be responsible for Cu efflux, but did not affect the expression of CTR1, known as a copper uptake transporter. Intriguingly, combination of much lower dose of cisplatin (5 µM) and non-toxic dose of PDTC (0.1 µM) synergistically induced a significant cytotoxicity in NCI-H196 cells. Given that ATP7A plays a critical role in the resistance of platinum-drug (such as cisplatin) representing a first-line treatment for SCLC, PDTC could be a promising candidate of adjunct therapeutic reagent for the patients requiring treatment with platinum-based regimens.

Bacterial Acclimation Inside an Aqueous Battery

PubMed Central

Dong, Dexian; Chen, Baoling; Chen, P.

2015-01-01

Specific environmental stresses may lead to induced genomic instability in bacteria, generating beneficial mutants and potentially accelerating the breeding of industrial microorganisms. The environmental stresses inside the aqueous battery may be derived from such conditions as ion shuttle, pH gradient, free radical reaction and electric field. In most industrial and medical applications, electric fields and direct currents are used to kill bacteria and yeast. However, the present study focused on increasing bacterial survival inside an operating battery. Using a bacterial acclimation strategy, both Escherichia coli and Bacillus subtilis were acclimated for 10 battery operation cycles and survived in the battery for over 3 days. The acclimated bacteria changed in cell shape, growth rate and colony color. Further analysis indicated that electrolyte concentration could be one of the major factors determining bacterial survival inside an aqueous battery. The acclimation process significantly improved the viability of both bacteria E. coli and B. subtilis. The viability of acclimated strains was not affected under battery cycle conditions of 0.18-0.80 mA cm-2 and 1.4-2.1 V. Bacterial addition within 1.0×1010 cells mL-1 did not significantly affect battery performance. Because the environmental stress inside the aqueous battery is specific, the use of this battery acclimation strategy may be of great potential for the breeding of industrial microorganisms. PMID:26070088

Amarogentin secoiridoid inhibits in vivo cancer cell growth in xenograft mice model and induces apoptosis in human gastric cancer cells (SNU-16) through G2/M cell cycle arrest and PI3K/Akt signalling pathway.

PubMed

Zhao, Jian-Guo; Zhang, Ling; Xiang, Xiao-Jun; Yu, Feng; Ye, Wan-Li; Wu, Dong-Ping; Wang, Jian-Fang; Xiong, Jian-Ping

2016-01-01

To investigate the in vitro and in vivo antitumor effects of amarogentin in SNU-16 human gastric cancer cells as well as in nude mice xenograft model. The effects of this compound on cell apoptosis, cell cycle phase distribution and PI3K/Akt and m-TOR signalling pathways were also studied in detail. MTT assay was used to study the effect of amarogentin on SNU-16 cell viability while clonogenic assay indicated the effect of the compound on colony formation tendency of these cells. Phase contrast microscopy revealed the effect on cellular morphology while flow cytometry was engaged to study the effects on cell apoptosis and cell cycle arrest. SNU-16 cancer cells were subcutaneously inoculated into nude mice to investigate the in vivo antitumor effects of amarogentin. Amarogentin induced potent, dose-dependent as well as time-dependent cytotoxic effects on the growth of SNU-16 human gastric cancer cells. Amarogentin also inhibited the colony forming capability of these tumor cells and its treatment led to morphological alterations in these cells in which the cells became withered and rounded, detached from one another and adopted irregular shapes while floating freely in the culture medium. In comparison to untreated control cells, the amarogentin treated cells with 10, 50 and 75 μM exhibited 32.5, 45.2 and 57.1 % apoptotic cells, respectively. Amarogentin induced potent and dose-dependent G2/M cell cycle arrest in these cells and led to downregulation of m-TOR, p-PI3K, PI3K, p-Akt and Akt and upregulation of cyclin D1 and cyclin E protein expressions. The tumor tissues obtained from the amarogentin-treated mice were much smaller than the tumor tissues derived from the control group. Amarogentin exerts potent in vitro and in vivo antitumor effects in SNU-16 cell model as well as in nude mice xenograft model. These antitumor effects were found to be mediated through apoptosis induction, G2/M cell cycle arrest and downregulation of PI3K/Akt/m-TOR signalling pathways.

Properties of Retinal Precursor Cells Grown on Vertically Aligned Multiwalled Carbon Nanotubes Generated for the Modification of Retinal Implant-Embedded Microelectrode Arrays

PubMed Central

Johnen, Sandra; Meißner, Frank; Krug, Mario; Baltz, Thomas; Endler, Ingolf; Mokwa, Wilfried; Walter, Peter

2016-01-01

Background. To analyze the biocompatibility of vertically aligned multiwalled carbon nanotubes (MWCNT), used as nanomodification to optimize the properties of prostheses-embedded microelectrodes that induce electrical stimulation of surviving retinal cells. Methods. MWCNT were synthesized on silicon wafers. Their growth was achieved by iron particles (Fe) or mixtures of iron-platinum (Fe-Pt) and iron-titanium (Fe-Ti) acting as catalysts. Viability, growth, adhesion, and gene expression of L-929 and retinal precursor (R28) cells were analyzed after nondirect and direct contact. Results. Nondirect contact had almost no influence on cell growth, as measured in comparison to reference materials with defined levels of cytotoxicity. Both cell types exhibited good proliferation properties on each MWCNT-coated wafer. Viability ranged from 95.9 to 99.8%, in which better survival was observed for nonfunctionalized MWCNT generated with the Fe-Pt and Fe-Ti catalyst mixtures. R28 cells grown on the MWCNT-coated wafers showed a decreased gene expression associated with neural and glial properties. Expression of the cell cycle-related genes CCNC, MYC, and TP53 was slightly downregulated. Cultivation on plasma-treated MWCNT did not lead to additional changes. Conclusions. All tested MWCNT-covered slices showed good biocompatibility profiles, confirming that this nanotechnology is a promising tool to improve prostheses bearing electrodes which connect with retinal tissue. PMID:27200182

Properties of Retinal Precursor Cells Grown on Vertically Aligned Multiwalled Carbon Nanotubes Generated for the Modification of Retinal Implant-Embedded Microelectrode Arrays.

PubMed

Johnen, Sandra; Meißner, Frank; Krug, Mario; Baltz, Thomas; Endler, Ingolf; Mokwa, Wilfried; Walter, Peter

2016-01-01

Background. To analyze the biocompatibility of vertically aligned multiwalled carbon nanotubes (MWCNT), used as nanomodification to optimize the properties of prostheses-embedded microelectrodes that induce electrical stimulation of surviving retinal cells. Methods. MWCNT were synthesized on silicon wafers. Their growth was achieved by iron particles (Fe) or mixtures of iron-platinum (Fe-Pt) and iron-titanium (Fe-Ti) acting as catalysts. Viability, growth, adhesion, and gene expression of L-929 and retinal precursor (R28) cells were analyzed after nondirect and direct contact. Results. Nondirect contact had almost no influence on cell growth, as measured in comparison to reference materials with defined levels of cytotoxicity. Both cell types exhibited good proliferation properties on each MWCNT-coated wafer. Viability ranged from 95.9 to 99.8%, in which better survival was observed for nonfunctionalized MWCNT generated with the Fe-Pt and Fe-Ti catalyst mixtures. R28 cells grown on the MWCNT-coated wafers showed a decreased gene expression associated with neural and glial properties. Expression of the cell cycle-related genes CCNC, MYC, and TP53 was slightly downregulated. Cultivation on plasma-treated MWCNT did not lead to additional changes. Conclusions. All tested MWCNT-covered slices showed good biocompatibility profiles, confirming that this nanotechnology is a promising tool to improve prostheses bearing electrodes which connect with retinal tissue.

Targeting loss of the Hippo signaling pathway in NF2-deficient papillary kidney cancers

PubMed Central

Ricketts, Christopher J.; Wei, Darmood; Yang, Youfeng; Baranes, Sarah M.; Gibbs, Benjamin K.; Ohanjanian, Lernik; Spencer Krane, L.; Scroggins, Bradley T.; Keith Killian, J.; Wei, Ming-Hui; Kijima, Toshiki; Meltzer, Paul S.; Citrin, Deborah E.; Neckers, Len; Vocke, Cathy D.; Marston Linehan, W.

2018-01-01

Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15–20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. This study investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines. NF2-deficency correlated with increased expression of YAP1 transcriptional targets and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC. PMID:29535838

Differential Control of Growth, Apoptotic Activity, and Gene Expression in Human Breast Cancer Cells by Extracts Derived from Medicinal Herbs Zingiber officinale

PubMed Central

Elkady, Ayman I.; Abuzinadah, Osama A.; Baeshen, Nabih A.; Rahmy, Tarek R.

2012-01-01

The present study aimed to examine the antiproliferative potentiality of an extract derived from the medicinal plant ginger (Zingiber officinale) on growth of breast cancer cells. Ginger treatment suppressed the proliferation and colony formation in breast cancer cell lines, MCF-7 and MDA-MB-231. Meanwhile, it did not significantly affect viability of nontumorigenic normal mammary epithelial cell line (MCF-10A). Treatment of MCF-7 and MDA-MB-231 with ginger resulted in sequences of events marked by apoptosis, accompanied by loss of cell viability, chromatin condensation, DNA fragmentation, activation of caspase 3, and cleavage of poly(ADP-ribose) polymerase. At the molecular level, the apoptotic cell death mediated by ginger could be attributed in part to upregulation of Bax and downregulation of Bcl-2 proteins. Ginger treatment downregulated expression of prosurvival genes, such as NF-κB, Bcl-X, Mcl-1, and Survivin, and cell cycle-regulating proteins, including cyclin D1 and cyclin-dependent kinase-4 (CDK-4). On the other hand, it increased expression of CDK inhibitor, p21. It also inhibited the expression of the two prominent molecular targets of cancer, c-Myc and the human telomerase reverse transcriptase (hTERT). These findings suggested that the ginger may be a promising candidate for the treatment of breast carcinomas. PMID:22969274

Growth inhibitory and proapoptotic effects of l-asparaginase from Fusarium culmorum ASP-87 on human leukemia cells (Jurkat).

PubMed

Meghavarnam, Anil K; Salah, Maryam; Sreepriya, Meenakshisundaram; Janakiraman, Savitha

2017-06-01

The objective of this study was to evaluate the anticancer properties of l-asparaginase purified from fungal isolate Fusarium culmorum ASP-87 against human T-cell leukemia cell line (Jurkat). The growth inhibitory and proapoptotic effects of purified l-asparaginase on Jurkat cell lines were investigated by determining its influence on cell viability, colony formation, DNA fragmentation, and cell cycle progression. The results revealed that purified l-asparaginase showed significant decrease in cell survival with IC 50 value of 90 μg/mL (9 IU/mL). The enzyme inhibited colony formation and showed characteristic laddering pattern on agarose gel thereby confirming the induction of apoptosis. Further, cell cycle analysis revealed that the enzyme induced apoptotic cell death by arresting the growth of cells at G 2 -M phase. However, the enzyme did not elicit any toxic effects on human erythrocytes. l-asparaginase purified from F. culmorum ASP-87 showed significant and selective cytotoxic and apoptotic effects on human T-cell leukemic cells in dose-dependent manner. Results of the study give leads for the anticancer effects of fungal l-asparaginase and its potential usefulness in the chemotherapy of leukemia. © 2016 Société Française de Pharmacologie et de Thérapeutique.

Quercetin suppresses HeLa cells by blocking PI3K/Akt pathway.

PubMed

Xiang, Tao; Fang, Yong; Wang, Shi-Xuan

2014-10-01

To explore the effect of quercetin on the proliferation and apoptosis of HeLa cells, HeLa cells were incubated with quercetin at different concentrations. Cell viability was evaluated by MTT assay, cell apoptosis was detected by Annexin-V/PI double labeled cytometry and DNA ladder assay. Cell cycle was flow cytometrically determined and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33258 staining and the apoptosis-related proteins in the HeLa cells were assessed by Western blotting. The results showed that quercetin significantly inhibited the growth of HeLa cells and induced obvious apoptosis in vitro in a time- and dose-dependent manner. Moreover, quercetin induced apoptosis of HeLa cells in cell cycle-dependent manner because quercetin could induce arrest of HeLa cells at G0/G1 phase. Quercetin treatment down-regulated the expression of the PI3K and p-Akt. In addition, quercetin could down-regulate expression of bcl-2, up-regulate Bax, but exerted no effect on the overall expression of Akt. We are led to conclude that quercetin induces apoptosis via PI3k/Akt pathways, and quercetin has potential to be used as an anti-tumor agent against human cervix cancer.

Dihydroartemisinin is an inhibitor of ovarian cancer cell growth.

PubMed

Jiao, Yang; Ge, Chun-min; Meng, Qing-hui; Cao, Jian-ping; Tong, Jian; Fan, Sai-jun

2007-07-01

To investigate the anticancer activity of dihydroartemisinin (DHA), a derivative of antimalaria drug artemisinin in a panel of human ovarian cancer cell lines. Cell growth was determined by the MTT viability assay. Apoptosis and cell cycle progression were evaluated by a DNA fragmentation gel electro-phoresis, flow cytometry assay, and TUNEL assay; protein and mRNA expression were analyzed by Western blotting and RT-PCR assay. Artemisinin and its derivatives, including artesunate, arteether, artemether, arteannuin, and DHA, exhibit anticancer growth activities in human ovarian cancer cells. Among them, DHA is the most effective in inhibiting cell growth. Ovarian cancer cell lines are more sensitive (5-10-fold) to DHA treatment compared to normal ovarian cell lines. DHA at micromolar dose levels exhibits a dose- and time-dependent cytotoxicity in ovarian cancer cell lines. Furthermore, DHA induced apoptosis and G2 cell cycle arrest, accompanied by a decrease of Bcl-xL and Bcl-2 and an increase of Bax and Bad. The promising results show for the first time that DHA inhibits the growth of human ovarian cancer cells. The selective inhibition of ovarian cancer cell growth, apoptosis induction, and G2 arrest provide in vitro evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of ovarian cancer.

Cell division is dispensable but not irrelevant in Streptomyces.

PubMed

McCormick, Joseph R

2009-12-01

In part, members of the genus Streptomyces have been studied because they produce many important secondary metabolites with antibiotic activity and for the interest in their relatively elaborate life cycle. These sporulating filamentous bacteria are remarkably synchronous for division and genome segregation in specialized aerial hyphae. Streptomycetes share some, but not all, of the division genes identified in the historic model rod-shaped organisms. Curiously, normally essential cell division genes are dispensable for growth and viability of Streptomyces coelicolor. Mainly, cell division plays a more important role in the developmental phase of life than during vegetative growth. Dispensability provides an advantageous genetic system to probe the mechanisms of division proteins, especially those with functions that are poorly understood.

Discovery of Potent Antiproliferative Agents Targeting EGFR Tyrosine Kinase Based on the Pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine Scaffold.

PubMed

Aziz, Yasmine Mohamed Abdel; Said, Mohamed Mokhtar; El Shihawy, Hosam Ahmed; Tolba, Mai Fathy; Abouzid, Khaled Abouzid Mohamed

2015-01-01

A series of pyridothieno[3,2-d]pyrimidin-4-amines was designed and synthesized as congeners to the classical 4-anilinoquinazolines as ATP-competitive epidermal growth factor receptor (EGFR) inhibitors. Compound 5a exhibited the most potent and selective inhibitory activity against EGFR with an IC50 value of 36.7 nM. Moreover, compounds 4b and 5a showed remarkable cell growth inhibition against leukemia, central nervous system cancer, and non-small cell lung cancer cell lines that overexpress EGFR, with growth inhibition of 50% (GI50) values of around 10 nM in the full U.S. National Cancer Institute 60 cell panel assay. Cell cycle studies indicated that compounds 4b and 5a induced significant cell cycle arrest in the S-phase and G0/G1, respectively, in addition to boosting P27(kip) expression. Compound 5a did not alter the viability of placental trophoblasts, which reflects its safety for normal cells. The standard COMPARE analyses demonstrated considerable correlation levels between compounds 4b and 5a and erlotinib, with pyridinium chlorochromate (PCC) values of 0.707 and 0.727, respectively.

Apigenin inhibits proliferation and invasion, and induces apoptosis and cell cycle arrest in human melanoma cells.

PubMed

Zhao, Guangming; Han, Xiaodong; Cheng, Wei; Ni, Jing; Zhang, Yunfei; Lin, Jingrong; Song, Zhiqi

2017-04-01

Malignant melanoma is the most invasive and fatal form of cutaneous cancer. Moreover it is extremely resistant to conventional chemotherapy and radiotherapy. Apigenin, a non-mutagenic flavonoid, has been found to exhibit chemopreventive and/or anticancerogenic properties in many different types of human cancer cells. Therefore, apigenin may have particular relevance for development as a chemotherapeutic agent for cancer treatment. In the present study, we investigated the effects of apigenin on the viability, migration and invasion potential, dendrite morphology, cell cycle distribution, apoptosis, phosphorylation of the extracellular signal-regulated protein kinase (ERK) and the AKT/mTOR signaling pathway in human melanoma A375 and C8161 cell lines in vitro. Apigenin effectively suppressed the proliferation of melanoma cells in vitro. Moreover, it inhibited cell migration and invasion, lengthened the dendrites, and induced G2/M phase arrest and apoptosis. Furthermore, apigenin promoted the activation of cleaved caspase-3 and cleaved PARP proteins and decreased the expression of phosphorylated (p)‑ERK1/2 proteins, p-AKT and p-mTOR. Consequently, apigenin is a novel therapeutic candidate for melanoma.

Differential effects of selective frankincense (Ru Xiang) essential oil versus non-selective sandalwood (Tan Xiang) essential oil on cultured bladder cancer cells: a microarray and bioinformatics study.

PubMed

Dozmorov, Mikhail G; Yang, Qing; Wu, Weijuan; Wren, Jonathan; Suhail, Mahmoud M; Woolley, Cole L; Young, D Gary; Fung, Kar-Ming; Lin, Hsueh-Kung

2014-01-01

Frankincense (Boswellia carterii, known as Ru Xiang in Chinese) and sandalwood (Santalum album, known as Tan Xiang in Chinese) are cancer preventive and therapeutic agents in Chinese medicine. Their biologically active ingredients are usually extracted from frankincense by hydrodistillation and sandalwood by distillation. This study aims to investigate the anti-proliferative and pro-apoptotic activities of frankincense and sandalwood essential oils in cultured human bladder cancer cells. The effects of frankincense (1,400-600 dilutions) (v/v) and sandalwood (16,000-7,000 dilutions) (v/v) essential oils on cell viability were studied in established human bladder cancer J82 cells and immortalized normal human bladder urothelial UROtsa cells using a colorimetric XTT cell viability assay. Genes that responded to essential oil treatments in human bladder cancer J82 cells were identified using the Illumina Expression BeadChip platform and analyzed for enriched functions and pathways. The chemical compositions of the essential oils were determined by gas chromatography-mass spectrometry. Human bladder cancer J82 cells were more sensitive to the pro-apoptotic effects of frankincense essential oil than the immortalized normal bladder UROtsa cells. In contrast, sandalwood essential oil exhibited a similar potency in suppressing the viability of both J82 and UROtsa cells. Although frankincense and sandalwood essential oils activated common pathways such as inflammatory interleukins (IL-6 signaling), each essential oil had a unique molecular action on the bladder cancer cells. Heat shock proteins and histone core proteins were activated by frankincense essential oil, whereas negative regulation of protein kinase activity and G protein-coupled receptors were activated by sandalwood essential oil treatment. The effects of frankincense and sandalwood essential oils on J82 cells and UROtsa cells involved different mechanisms leading to cancer cell death. While frankincense essential oil elicited selective cancer cell death via NRF-2-mediated oxidative stress, sandalwood essential oil induced non-selective cell death via DNA damage and cell cycle arrest.

Differential effects of selective frankincense (Ru Xiang) essential oil versus non-selective sandalwood (Tan Xiang) essential oil on cultured bladder cancer cells: a microarray and bioinformatics study

PubMed Central

2014-01-01

Background Frankincense (Boswellia carterii, known as Ru Xiang in Chinese) and sandalwood (Santalum album, known as Tan Xiang in Chinese) are cancer preventive and therapeutic agents in Chinese medicine. Their biologically active ingredients are usually extracted from frankincense by hydrodistillation and sandalwood by distillation. This study aims to investigate the anti-proliferative and pro-apoptotic activities of frankincense and sandalwood essential oils in cultured human bladder cancer cells. Methods The effects of frankincense (1,400–600 dilutions) (v/v) and sandalwood (16,000–7,000 dilutions) (v/v) essential oils on cell viability were studied in established human bladder cancer J82 cells and immortalized normal human bladder urothelial UROtsa cells using a colorimetric XTT cell viability assay. Genes that responded to essential oil treatments in human bladder cancer J82 cells were identified using the Illumina Expression BeadChip platform and analyzed for enriched functions and pathways. The chemical compositions of the essential oils were determined by gas chromatography–mass spectrometry. Results Human bladder cancer J82 cells were more sensitive to the pro-apoptotic effects of frankincense essential oil than the immortalized normal bladder UROtsa cells. In contrast, sandalwood essential oil exhibited a similar potency in suppressing the viability of both J82 and UROtsa cells. Although frankincense and sandalwood essential oils activated common pathways such as inflammatory interleukins (IL-6 signaling), each essential oil had a unique molecular action on the bladder cancer cells. Heat shock proteins and histone core proteins were activated by frankincense essential oil, whereas negative regulation of protein kinase activity and G protein-coupled receptors were activated by sandalwood essential oil treatment. Conclusion The effects of frankincense and sandalwood essential oils on J82 cells and UROtsa cells involved different mechanisms leading to cancer cell death. While frankincense essential oil elicited selective cancer cell death via NRF-2-mediated oxidative stress, sandalwood essential oil induced non-selective cell death via DNA damage and cell cycle arrest. PMID:25006348

Bilirubin Inhibits Neointima Formation and Vascular Smooth Muscle Cell Proliferation and Migration

PubMed Central

Peyton, Kelly J.; Shebib, Ahmad R.; Azam, Mohammad A.; Liu, Xiao-ming; Tulis, David A.; Durante, William

2012-01-01

Bilirubin is a heme metabolite generated by the concerted action of the enzymes heme oxygenase and biliverdin reductase. Although long considered a toxic byproduct of heme catabolism, recent preclinical, and clinical studies indicate the bilirubin exerts beneficial effects in the circulation. In the present study, we determined whether local administration of bilirubin attenuates neointima formation following injury of rat carotid arteries. In addition, the ability of bilirubin to regulate the proliferation and migration of human arterial smooth muscle cells (SMCs) was investigated. Local perivascular administration of bilirubin immediately following balloon injury of rat carotid arteries significantly attenuated neointima formation. Bilirubin-mediated inhibition of neointimal thickening was associated with a significant decrease in ERK activity and cyclin D1 and A protein expression, and an increase in p21 and p53 protein expression in injured blood vessels. Treatment of human aortic SMCs with bilirubin inhibited proliferation and migration in a concentration-dependent manner without affecting cell viability. In addition, bilirubin resulted in a concentration-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and this was paralleled by a decrease in the fraction of cells in the S and G2M phases of the cell cycle. Finally, bilirubin had no effect on mitochondrial function and ATP content of vascular SMCs. In conclusion, these studies demonstrate that bilirubin inhibits neointima formation after arterial injury and this is associated with alterations in the expression of cell cycle regulatory proteins. Furthermore, bilirubin blocks proliferation and migration of human arterial SMCs and arrests SMCs in the G0/G1 phase of the cell cycle. Bilirubin represents an attractive therapeutic agent in treating occlusive vascular disease. PMID:22470341

Antineoplastic Effects of α-Santalol on Estrogen Receptor-Positive and Estrogen Receptor-Negative Breast Cancer Cells through Cell Cycle Arrest at G2/M Phase and Induction of Apoptosis

PubMed Central

Santha, Sreevidya; Bommareddy, Ajay; Rule, Brittny; Guillermo, Ruth; Kaushik, Radhey S.; Young, Alan; Dwivedi, Chandradhar

2013-01-01

Anticancer efficacy and the mechanism of action of α-santalol, a terpenoid isolated from sandalwood oil, were investigated in human breast cancer cells by using p53 wild-type MCF-7 cells as a model for estrogen receptor(ER)-positive and p53 mutated MDA-MB-231 cells as a model for ER-negative breast cancer. α-Santalol inhibited cell viability and proliferation in a concentration and time-dependent manner in both cells regardless of their ER and/or p53 status. However, α-santalol produced relatively less toxic effect on normal breast epithelial cell line, MCF-10A. It induced G2/M cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cells. Cell cycle arrest induced by α-santalol was associated with changes in the protein levels of BRCA1, Chk1, G2/M regulatory cyclins, Cyclin dependent kinases (CDKs), Cell division cycle 25B (Cdc25B), Cdc25C and Ser-216 phosphorylation of Cdc25C. An up-regulated expression of CDK inhibitor p21 along with suppressed expression of mutated p53 was observed in MDA-MB-231 cells treated with α-santalol. On the contrary, α-santalol did not increase the expression of wild-type p53 and p21 in MCF-7 cells. In addition, α-santalol induced extrinsic and intrinsic pathways of apoptosis in both cells with activation of caspase-8 and caspase-9. It led to the activation of the executioner caspase-6 and caspase-7 in α-santalol-treated MCF-7 cells and caspase-3 and caspase-6 in MDA-MB-231 cells along with strong cleavage of poly(ADP-ribose) polymerase (PARP) in both cells. Taken together, this study for the first time identified strong anti-neoplastic effects of α-santalol against both ER-positive and ER-negative breast cancer cells. PMID:23451128

An experimental study on providing a scientific evidence for seven-time alcohol-steaming of Rhei Rhizoma when clinically used.

PubMed

Sim, Yeomoon; Oh, Hyein; Oh, Dal-Seok; Kim, Namkwon; Gu, Pil Sung; Choi, Jin Gyu; Kim, Hyo Geun; Kang, Tong Ho; Oh, Myung Sook

2015-10-27

Rhei Rhizoma (RR) has been widely used as laxative and processed to alter its therapeutic actions or reduce its side effects. In this study, we evaluated experimentally the clinical application guideline that RR should be alcohol-steamed seven times before being used in elderly patients, as described in Dongeuibogam, the most famous book on Korean traditional medicine. Unprocessed RR (RR-U) was soaked in rice wine, steamed and then fully dried (RR-P1). The process was repeated four (RR-P4) or seven times (RR-P7). Reversed-phase high-performance liquid chromatography was used to determine the RR-U, RR-P1, RR-P4 and RR-P7 (RRs) constituents. To evaluate the effect of RRs on liver toxicity, human hepatoma cells (HepG2) were treated with RRs at 100 μg/mL for 4 h and then cell viabilities were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. To confirm the effects in vivo, 5-week-old male Sprague-Dawley rats were treated with RRs at 3 g/kg/day for 21 days. Body weight and serum biochemical parameters were measured and liver histology was assessed. The levels of sennosides decreased in processed RRs in an iteration-dependent manner, while the emodin level was unaffected. In HepG2 cells, cell viability was reduced with RR-U, while the toxicity decreased according to the number of processing cycles. The changes in body weight, relative liver weight and liver enzymes of RR-U-treated rats were reduced in processed RRs-treated rats. Histopathological analysis indicated swelling and cholestasis improved following seven times alcohol-steaming cycles. These results provide experimental evidence that RR-P7 almost completely reduces RR hepatotoxicity.

Immunomodulatory role of Emblica officinalis in arsenic induced oxidative damage and apoptosis in thymocytes of mice

PubMed Central

2013-01-01

Background Arsenic is widely distributed in the environment and has been found to be associated with the various health related problems including skin lesions, cancer, cardiovascular and immunological disorders. The fruit extract of Emblica officinalis (amla) has been shown to have anti-oxidative and immunomodulatory properties. In view of increasing health risk of arsenic, the present study has been carried out to investigate the protective effect of amla against arsenic induced oxidative stress and apoptosis in thymocytes of mice. Methods Mice were exposed to arsenic (sodium arsenite 3 mg/kg body weight p.o.) or amla (500 mg/kg body weight p.o.) or simultaneously with arsenic and amla for 28 days. The antioxidant enzyme assays were carried out using spectrophotometer and generation of ROS, apoptotic parameters, change in cell cycle were carried out using flow cytometer following the standard protocols. Results Arsenic exposure to mice caused a significant increase in the lipid peroxidation, ROS production and decreased cell viability, levels of reduced glutathione, the activity of superoxide dismutase, catalase, cytochrome c oxidase and mitochondrial membrane potential in the thymus as compared to controls. Increased activity of caspase-3 linked with apoptosis assessed by the cell cycle analysis and annexin V/PI binding was also observed in mice exposed to arsenic as compared to controls. Co-treatment with arsenic and amla decreased the levels of lipid peroxidation, ROS production, activity of caspase-3, apoptosis and increased cell viability, levels of antioxidant enzymes, cytochrome c oxidase and mitochondrial membrane potential as compared to mice treated with arsenic alone. Conclusions The results of the present study exhibits that arsenic induced oxidative stress and apoptosis significantly protected by co-treatment with amla that could be due to its strong antioxidant potential. PMID:23889914

Apoptosis induction by 7-chloroquinoline-1,2,3-triazoyl carboxamides in triple negative breast cancer cells.

PubMed

Begnini, Karine Rech; Duarte, Wladimir R; da Silva, Liziane Pereira; Buss, Julieti H; Goldani, Bruna S; Fronza, Mariana; Segatto, Natália Vieira; Alves, Diego; Savegnago, Lucielli; Seixas, Fabiana Kömmling; Collares, Tiago

2017-07-01

Breast cancer is a major public health burden in both developed and developing countries and there is still a need to screen new molecules with different modes of actions. The aims of this study were to evaluate the selectivity profile, apoptotic cell death and cell cycle arrest induced by 7-chloroquinoline-1,2,3-triazoyl carboxamides derivatives in hormonal-dependent and hormonal-independent breast cancer cells. Results showed significantly decreased MCF-7 and MDA-MB-231 cells viability in vitro in a dose dependent manner after treatment with 7-chloroquinoline derivatives QTCA-1, QTCA-2 and QTCA-3. QTCA-1 displayed the highest cytotoxic activity from all the tested compounds in MDA-MB-231 with IC50 values of 20.60, 20.42 and 19.91μM in 24, 48 and 72h of treatment respectively. Apoptosis induction was also significantly higher in the hormonal-independent breast cancer cells, with 80.4% of dead cells in MDA-MB-231 and only 16.8% of dead in MCF-7 cells. As a result, G0/G1 cycle arrest was observed in MCF-7 cells and no cell cycle arrest at all was observed in MDA-MB-231 cells. Molecular docking showed a high affinity of QTCA-1 to PARP-1, Scr and PI3K/mTOR targets. These results suggest a strong activity of the 7-chloroquinoline derivative QTCA-1 in independent-hormonal cells and suggest selectivity for triple negative cells. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Piperlongumine potentiates the effects of gemcitabine in in vitro and in vivo human pancreatic cancer models

PubMed Central

Mohammad, Jiyan; Dhillon, Harsharan; Chikara, Shireen; Mamidi, Sujan; Sreedasyam, Avinash; Chittem, Kishore; Orr, Megan; Wilkinson, John C.; Reindl, Katie M.

2018-01-01

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers due to a late diagnosis and poor response to available treatments. There is a need to identify complementary treatment strategies that will enhance the efficacy and reduce the toxicity of currently used therapeutic approaches. We investigated the ability of a known ROS inducer, piperlongumine (PL), to complement the modest anti-cancer effects of the approved chemotherapeutic agent gemcitabine (GEM) in PDAC cells in vitro and in vivo. PDAC cells treated with PL + GEM showed reduced cell viability, clonogenic survival, and growth on Matrigel compared to control and individually-treated cells. Nude mice bearing orthotopically implanted MIA PaCa-2 cells treated with both PL (5 mg/kg) and GEM (25 mg/kg) had significantly lower tumor weight and volume compared to control and single agent-treated mice. RNA sequencing (RNA-Seq) revealed that PL + GEM resulted in significant changes in p53-responsive genes that play a role in cell death, cell cycle, oxidative stress, and DNA repair pathways. Cell culture assays confirmed PL + GEM results in elevated ROS levels, arrests the cell cycle in the G0/G1 phase, and induces PDAC cell death. We propose a mechanism for the complementary anti-tumor effects of PL and GEM in PDAC cells through elevation of ROS and transcription of cell cycle arrest and cell death-associated genes. Collectively, our results suggest that PL has potential to be combined with GEM to more effectively treat PDAC. PMID:29535819

Centchroman induces redox-dependent apoptosis and cell-cycle arrest in human endometrial cancer cells.

PubMed

Shyam, Hari; Singh, Neetu; Kaushik, Shweta; Sharma, Ramesh; Balapure, Anil K

2017-04-01

Centchroman (CC) or Ormeloxifene has been shown to induce apoptosis and cell cycle arrest in various types of cancer cells. This has, however, not been addressed for endometrial cancer cells where its (CC) mechanism of action remains unclear. This study focuses on the basis of antineoplasticity of CC by blocking the targets involved in the cell cycle, survival and apoptosis in endometrial cancer cells. Ishikawa Human Endometrial Cancer Cells were cultured under estrogen deprived medium, exposed to CC and analyzed for proliferation and apoptosis. Additionally, we also analyzed oxidative stress induced by CC. Cell viability studies confirmed the IC 50 of CC in Ishikawa cells to be 20 µM after 48 h treatment. CC arrests the cells in G0/G1 phase through cyclin D1 and cyclin E mediated pathways. Phosphatidylserine externalization, nuclear morphology changes, DNA fragmentation, PARP cleavage, and alteration of Bcl-2 family protein expression clearly suggest ongoing apoptosis in the CC treated cells. Activation of caspase 3 & 9, up-regulation of AIF and inhibition of apoptosis by z-VAD-fmk clearly explains the participation of the intrinsic pathway of programmed cell death. Further, the increase of ROS, loss of MMP, inhibition of antioxidant (MnSOD, Cu/Zn-SOD and GST) and inhibition of apoptosis with L-NAC suggests CC induced oxidative stress leading to apoptosis via mitochondria mediated pathway. Therefore, CC could be a potential therapeutic agent for the treatment of Endometrial Cancer adjunct to its utility as a contraceptive and an anti-breast cancer agent.

[Resting forms of gram negative chemolithoautotrophic bacteria Thioalkalivibrio versutus and Thioalkalimicrobium aerophilum].

PubMed

Loĭko, N G; Soina, V S; Sorokin, D Iu; Mitiushina, L L; El'-Registan, G I

2003-01-01

The haloalkaliphilic chemoautotrophic gram-negative bacteria Thioalkalivibrio versutus, strain AL2, and Thioalkalimicrobium aerophilum, strain AL3, were shown to possess the capacity to produce resting forms, namely cyst-like refractile cells (CRC), whose production was controlled by the level of the d1 extracellular factors, exhibiting the function of anabiosis autoinducers. The conditions were elucidated that promoted the formation of CRC in the developmental cycles of the cultures studied, in condensed cell suspensions undergoing autolysis, and under the action of exogenously introduced chemical analogues of anabiosis autoinducers (alkylhydroxybenzenes). The peculiarities of the fine structure of the resting cells obtained were studied. Distinctions were revealed (with respect to viability and thermotolerance) between the CRC formed under different conditions. The relationship between the growth strategy and survival strategy of extremophilic bacteria is discussed with taking into account the effect of the d1 autoregulatory factors. A new model of CRC formation is proposed: CRC production in the life cycle of bacteria developing under conditions of increased concentration of anabiosis autoinducers.

Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function.

PubMed

Lerit, Dorothy A; Jordan, Holly A; Poulton, John S; Fagerstrom, Carey J; Galletta, Brian J; Peifer, Mark; Rusan, Nasser M

2015-07-06

Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle-dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability.

Effect of Wasabi Component 6-(Methylsulfinyl)hexyl Isothiocyanate and Derivatives on Human Pancreatic Cancer Cells

PubMed Central

Chen, Yu-Jen; Huang, Yu-Chuen; Tsai, Tung-Hu

2014-01-01

The naturally occurring compound 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) was isolated from Wasabia japonica (Wasabi), a pungent spice used in Japanese food worldwide. The synthetic derivatives 6-(methylsulfenyl)hexyl isothiocyanate (I7447) and 6-(methylsulfonyl)hexyl isothiocyanate (I7557) are small molecule compounds derived from 6-MITC. This study aimed to evaluate the effect of these compounds on human pancreatic cancer cells. Human pancreatic cancer cell lines PANC-1 and BxPC-3 were used to perform an MTT assay for cell viability and Liu's stain for morphological observation. The cell cycle was analyzed by DNA histogram. Aldehyde dehydrogenase (ALDH) activity was used as a marker for cancer stem cells (CSC). Western blotting was performed for the expression of proteins related to CSC signaling. The results showed that compounds 6-MITC and I7557, but not I7447, inhibited viability of both PANC-1 and BxPC-3 cells. Morphological observation showed mitotic arrest and apoptosis in 6-MITC- and I7557-treated cells. These two compounds induced G2/M phase arrest and hypoploid population. Percentages of ALDH-positive PANC-1 cells were markedly reduced by 6-MITC and I7557 treatment. The expression of CSC signaling molecule SOX2, but not NOTCH1, ABCG2, Sonic hedgehog, or OCT4, was inhibited by 6-MITC and I7557. In conclusion, wasabi compounds 6-MITC and I7557 may possess activity against the growth and CSC phenotypes of human pancreatic cancer cells. PMID:24575144

Effect of Wasabi Component 6-(Methylsulfinyl)hexyl Isothiocyanate and Derivatives on Human Pancreatic Cancer Cells.

PubMed

Chen, Yu-Jen; Huang, Yu-Chuen; Tsai, Tung-Hu; Liao, Hui-Fen

2014-01-01

The naturally occurring compound 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) was isolated from Wasabia japonica (Wasabi), a pungent spice used in Japanese food worldwide. The synthetic derivatives 6-(methylsulfenyl)hexyl isothiocyanate (I7447) and 6-(methylsulfonyl)hexyl isothiocyanate (I7557) are small molecule compounds derived from 6-MITC. This study aimed to evaluate the effect of these compounds on human pancreatic cancer cells. Human pancreatic cancer cell lines PANC-1 and BxPC-3 were used to perform an MTT assay for cell viability and Liu's stain for morphological observation. The cell cycle was analyzed by DNA histogram. Aldehyde dehydrogenase (ALDH) activity was used as a marker for cancer stem cells (CSC). Western blotting was performed for the expression of proteins related to CSC signaling. The results showed that compounds 6-MITC and I7557, but not I7447, inhibited viability of both PANC-1 and BxPC-3 cells. Morphological observation showed mitotic arrest and apoptosis in 6-MITC- and I7557-treated cells. These two compounds induced G2/M phase arrest and hypoploid population. Percentages of ALDH-positive PANC-1 cells were markedly reduced by 6-MITC and I7557 treatment. The expression of CSC signaling molecule SOX2, but not NOTCH1, ABCG2, Sonic hedgehog, or OCT4, was inhibited by 6-MITC and I7557. In conclusion, wasabi compounds 6-MITC and I7557 may possess activity against the growth and CSC phenotypes of human pancreatic cancer cells.

In-vitro assessment of oxidative stress generated by orthodontic archwires.

PubMed

Spalj, Stjepan; Mlacovic Zrinski, Magda; Tudor Spalj, Vedrana; Ivankovic Buljan, Zorana

2012-05-01

Several metals undergo redox cycling, producing free radicals and generating oxidative stress. The purpose of this study was to investigate in-vitro oxidative stress of orthodontic archwires made of various alloys. Mouse fibroblast cells L929 were exposed to 6 types of archwires, and the concentration of the oxidative stress marker 8-hydroxy-2'-deoxyguanosine in DNA was evaluated. Trypan blue dye was used in the determination of cell viability and numbers. Standard nickel-titanium archwires generated the highest oxidative stress, significantly higher than all other wires and the controls (P <0.05), and coated nickel-titanium, copper-nickel-titanium, and cobalt-chromium were lower than nickel-titanium (P <0.05), but higher than titanium-molybdenum and the negative and absolute controls (P <0.05). Titanium-molybdenum and stainless steel generated the lowest stress. Nickel-titanium induced the lowest viability, lower than the negative and absolute controls and all other wires (P <0.05) except titanium-molybdenum. Stainless steel showed the highest viability. Nickel-titanium produced the highest inhibition of cell growth, higher than all samples (P <0.05) except the positive control and cobalt-chromium. The lowest inhibition was observed in stainless steel and titanium-molybdenum, lower than nickel-titanium, cobalt-chromium, and the positive control (P <0.05). All orthodontic archwires generate oxidative stress in vitro. Stainless steel archwires have the highest and nickel-titanium the lowest biocompatibility. Copyright © 2012 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

Inhibitory activity of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes) on transformed cells by human papillomavirus.

PubMed

Hernández-Márquez, Eva; Lagunas-Martínez, Alfredo; Bermudez-Morales, Victor H; Burgete-García, Ana I; León-Rivera, Ismael; Montiel-Arcos, Elizur; García-Villa, Enrique; Gariglio, Patricio; Madrid-Marina V, Vicente; Ondarza-Vidaurreta, Raul N

2014-01-01

In this study, we investigated the effects of the aqueous extracts of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum, obtained from three localities (China; and Morelos and Michoacan, Mexico) on cervical cells transformed by human papillomavirus (HeLa and SiHa) and C-33A cancer cells. The cells were plated in DMEM medium supplemented, and were incubated in the presence of different concentrations of G. lucidum for 24 h. Cell proliferation was determined by MTT colorimetric assay and viability by trypan blue assay. Inhibitory dose was determined (IC50) of the three different extracts of G. lucidum in the culture cell lines mentioned above. The apoptosis process was confirmed by nuclear DNA fragmentation and the cell cycle was determined by flow cytometry. The results showed that aqueous extracts G. lucidum obtained from three localities produced inhibition in the proliferation of VPH transformed cells; they also induced apoptosis and cell cycle arrest in HeLa, SiHa, and C-33A cancer cells. Therefore, it was found that aqueous extracts G. lucidum obtained from three different locations produced inhibitory effect on cancer cells and may have a potential therapeutic use for the prevention and treatment of this disease.

Ethanolic Neem (Azadirachta indica) Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and Potentiates the Therapeutic Index of Cisplatin

PubMed Central

Sharma, Chhavi; Vas, Andrea J.; Goala, Payal; Gheewala, Taher M.; Rizvi, Tahir A.

2014-01-01

The present study was designed to gain insight into the antiproliferative activity of ethanolic neem leaves extract (ENLE) alone or in combination with cisplatin by cell viability assay on human breast (MCF-7) and cervical (HeLa) cancer cells. Nuclear morphological examination and cell cycle analysis were performed to determine the mode of cell death. Further, to identify its molecular targets, the expression of genes involved in apoptosis, cell cycle progression, and drug metabolism was analyzed by RT-PCR. Treatment of MCF-7, HeLa, and normal cells with ENLE differentially suppressed the growth of cancer cells in a dose- and time-dependent manner through apoptosis. Additionally, lower dose combinations of ENLE with cisplatin resulted in synergistic growth inhibition of these cells compared to the individual drugs (combination index <1). ENLE significantly modulated the expression of bax, cyclin D1, and cytochrome P450 monooxygenases (CYP 1A1 and CYP 1A2) in a time-dependent manner in these cells. Conclusively, these results emphasize the chemopreventive ability of neem alone or in combination with chemotherapeutic treatment to reduce the cytotoxic effects on normal cells, while potentiating their efficacy at lower doses. Thus, neem may be a prospective therapeutic agent to combat gynecological cancers. PMID:24624140

Induction of cell death by pyropheophorbide-α methyl ester-mediated photodynamic therapy in lung cancer A549 cells.

PubMed

Tu, Ping-Hua; Huang, Wen-Jun; Wu, Zhan-Ling; Peng, Qing-Zhen; Xie, Zhi-Bin; Bao, Ji; Zhong, Ming-Hua

2017-03-01

Pyropheophorbide-α methyl ester (MPPa) was a promising photosensitizer with stable chemical structure, strong absorption, higher tissue selectivity and longer activation wavelengths. The present study investigated the effect of MPPa-mediated photodynamic treatment on lung cancer A549 cells as well as the underlying mechanisms. Cell Counting Kit-8 was employed for cell viability assessment. Reactive oxygen species levels were determined by fluorescence microscopy and flow cytometry. Cell morphology was evaluated by Hoechst staining and transmission electron microscopy. Mitochondrial membrane potential, cellular apoptosis and cell cycle distribution were evaluated flow-cytometrically. The protein levels of apoptotic effectors were examined by Western blot. We found that the photocytotoxicity of MPPa showed both drug- and light- dose dependent characteristics in A549 cells. Additionally, MPPa-PDT caused cell apoptosis by reducing mitochondrial membrane potential, increasing reactive oxygen species (ROS) production, inducing caspase-9/caspase-3 signaling activation as well as cell cycle arrest at G 0 /G 1 phase. These results suggested that MPPa-PDT mainly kills cells by apoptotic mechanisms, with overt curative effects, indicating that MPPa should be considered a potent photosensitizer for lung carcinoma treatment. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Apatinib has anti-tumor effects and induces autophagy in colon cancer cells.

PubMed

Lu, Wu; Ke, He; Qianshan, Ding; Zhen, Wang; Guoan, Xiang; Honggang, Yu

2017-09-01

Apatinib recently has been used to treat patients with gastric cancer, but the function of apatinib in colon cancer remains unclear. This study was conducted to investigate the impacts of apatinib on the biological function and its potential mechanism of colon cancer cells in vitro . The effect of apatinib in colon cancer cells were detected by assessing cell viability, migration and invasion capabilities. Apoptosis cells and the cell cycle distribution of colon cancer cells were analyzed by flow cytometry. The potential mechanism was investigated via autophagy related proteins and pathways in vitro . The proliferation, migration and invasion of colon cancer cells were inhibited when they were treated with different concentration of apatinib (20, 40 μM). When HCT116 and SW480 cells were treated with apatinib at the concentration of 20 μM, the apoptosis percentage were 3.7% and 5.8% respectively. As the drug concentration increased to 40μΜ, the the apoptosis percentage increased to 11.9% and 13.5%. Meanwhile, cell cycle was also altered. Furthermore, apatinib inhibited the expression of AKT-mTOR signaling pathway and increased the expression of LC3-II. Apatinib can significantly inhibit the malignant phenotype of colon cancer cells, and it was involved in regulation of autophagy.

Enhanced viability of corneal epithelial cells for efficient transport/storage using a structurally modified calcium alginate hydrogel.

PubMed

Wright, Bernice; Cave, Richard A; Cook, Joseph P; Khutoryanskiy, Vitaliy V; Mi, Shengli; Chen, Bo; Leyland, Martin; Connon, Che J

2012-05-01

Therapeutic limbal epithelial stem cells could be managed more efficiently if clinically validated batches were transported for 'on-demand' use. In this study, corneal epithelial cell viability in calcium alginate hydrogels was examined under cell culture, ambient and chilled conditions for up to 7 days. Cell viability improved as gel internal pore size increased, and was further enhanced with modification of the gel from a mass to a thin disc. Ambient storage conditions were optimal for supporting cell viability in gel discs. Cell viability in gel discs was significantly enhanced with increases in pore size mediated by hydroxyethyl cellulose. Our novel methodology of controlling alginate gel shape and pore size together provides a more practical and economical alternative to established corneal tissue/cell storage methods.

Efficient Intracellular Delivery of Molecules with High Cell Viability Using Nanosecond-Pulsed Laser-Activated Carbon Nanoparticles

PubMed Central

2015-01-01

Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5–9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability. PMID:24547946

Combination of hyperthermia and photodynamic therapy on mesenchymal stem cell line treated with chloroaluminum phthalocyanine magnetic-nanoemulsion

NASA Astrophysics Data System (ADS)

de Paula, Leonardo B.; Primo, Fernando L.; Pinto, Marcelo R.; Morais, Paulo C.; Tedesco, Antonio C.

2015-04-01

The present study reports on the preparation and the cell viability assay of two nanoemulsions loaded with magnetic nanoparticle and chloroaluminum phthalocyanine. The preparations contain equal amount of chloroaluminum phthalocyanine (0.05 mg/mL) but different contents of magnetic nanoparticle (0.15×1013 or 1.50×1013 particle/mL). The human bone marrow mesenchymal stem cell line was used as the model to assess the cell viability and this type of cell can be used as a model to mimic cancer stem cells. The cell viability assays were performed in isolated as well as under combined magnetic hyperthermia and photodynamic therapy treatments. We found from the cell viability assay that under the hyperthermia treatment (1 MHz and 40 Oe magnetic field amplitude) the cell viability reduction was about 10%, regardless the magnetic nanoparticle content within the magnetic nanoparticle/chloroaluminum phthalocyanine formulation. However, cell viability reduction of about 50% and 60% were found while applying the photodynamic therapy treatment using the magnetic nanoparticle/chloroaluminum phthalocyanine formulation containing 0.15×1013 or 1.50×1013 magnetic particle/mL, respectively. Finally, an average reduction in cell viability of about 66% was found while combining the hyperthermia and photodynamic therapy treatments.

Boswellia sacra essential oil induces tumor cell-specific apoptosis and suppresses tumor aggressiveness in cultured human breast cancer cells

PubMed Central

2011-01-01

Background Gum resins obtained from trees of the Burseraceae family (Boswellia sp.) are important ingredients in incense and perfumes. Extracts prepared from Boswellia sp. gum resins have been shown to possess anti-inflammatory and anti-neoplastic effects. Essential oil prepared by distillation of the gum resin traditionally used for aromatic therapy has also been shown to have tumor cell-specific anti-proliferative and pro-apoptotic activities. The objective of this study was to optimize conditions for preparing Boswellea sacra essential oil with the highest biological activity in inducing tumor cell-specific cytotoxicity and suppressing aggressive tumor phenotypes in human breast cancer cells. Methods Boswellia sacra essential oil was prepared from Omani Hougari grade resins through hydrodistillation at 78 or 100 oC for 12 hours. Chemical compositions were identified by gas chromatography-mass spectrometry; and total boswellic acids contents were quantified by high-performance liquid chromatography. Boswellia sacra essential oil-mediated cell viability and death were studied in established human breast cancer cell lines (T47D, MCF7, MDA-MB-231) and an immortalized normal human breast cell line (MCF10-2A). Apoptosis was assayed by genomic DNA fragmentation. Anti-invasive and anti-multicellular tumor properties were evaluated by cellular network and spheroid formation models, respectively. Western blot analysis was performed to study Boswellia sacra essential oil-regulated proteins involved in apoptosis, signaling pathways, and cell cycle regulation. Results More abundant high molecular weight compounds, including boswellic acids, were present in Boswellia sacra essential oil prepared at 100 oC hydrodistillation. All three human breast cancer cell lines were sensitive to essential oil treatment with reduced cell viability and elevated cell death, whereas the immortalized normal human breast cell line was more resistant to essential oil treatment. Boswellia sacra essential oil hydrodistilled at 100 oC was more potent than the essential oil prepared at 78 oC in inducing cancer cell death, preventing the cellular network formation (MDA-MB-231) cells on Matrigel, causing the breakdown of multicellular tumor spheroids (T47D cells), and regulating molecules involved in apoptosis, signal transduction, and cell cycle progression. Conclusions Similar to our previous observations in human bladder cancer cells, Boswellia sacra essential oil induces breast cancer cell-specific cytotoxicity. Suppression of cellular network formation and disruption of spheroid development of breast cancer cells by Boswellia sacra essential oil suggest that the essential oil may be effective for advanced breast cancer. Consistently, the essential oil represses signaling pathways and cell cycle regulators that have been proposed as therapeutic targets for breast cancer. Future pre-clinical and clinical studies are urgently needed to evaluate the safety and efficacy of Boswellia sacra essential oil as a therapeutic agent for treating breast cancer. PMID:22171782

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

PubMed Central

Johnson, M. Brittany; Criss, Alison K.

2013-01-01

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells. PMID:24056524

Role of PP2Cα in cell growth, in radio- and chemosensitivity, and in tumorigenicity

PubMed Central

Lammers, Twan; Peschke, Peter; Ehemann, Volker; Debus, Jürgen; Slobodin, Boris; Lavi, Sara; Huber, Peter

2007-01-01

Background PP2Cα is the representative member of the type 2C family of protein phosphatases, and it has recently been implicated in the regulation of p53-, TGFβ-, cyclin-dependent kinase- and apoptosis-signaling. To investigate the role of PP2Cα in cell growth and in radio- and chemosensitivity, wild type and PP2Cα siRNA-expressing MCF7 cells were subjected to several different viability and cell cycle analyses, both under basal conditions and upon treatment with radio- and chemotherapy. By comparing the growth of tumors established from both types of cells, we also evaluated the involvement of PP2Cα in tumorigenesis. Results It was found that knockdown of PP2Cα did not affect the proliferation, the clonogenic survival and the membrane integrity of MCF7 cells. In addition, it did not alter their radio- and chemosensitivity. For PP2Cα siRNA-expressing MCF7 cells, the number of cells in the G0/G1 phase of the cell cycle was reduced, the induction of the G1 block was attenuated, the number of cells in G2/M was increased, and the induction of the G2 block was enhanced. The tumorigenic potential of PP2Cα siRNA-expressing MCF7 cells was found to be higher than that of wild type MCF7 cells, and the in vivo proliferation of these cells was found to be increased. Conclusion Based on these findings, we conclude that PP2Cα is not involved in controlling cell growth and radio- and chemosensitivity in vitro. It does, however, play a role in the regulation of the cell cycle, in the induction of cell cycle checkpoints and in tumorigenesis. The latter notion implies that PP2Cα may possess tumor-suppressing properties, and it thereby sets the stage for more elaborate analyses on its involvement in the development and progression of cancer. PMID:17941990

Panobinostat induces apoptosis via production of reactive oxygen species and synergizes with topoisomerase inhibitors in cervical cancer cells.

PubMed

Wasim, Lubna; Chopra, Madhu

2016-12-01

Cervical cancer is the fourth major cause of cancer-related deaths in women worldwide and is the most common cancer in developing countries. Therefore, a search for novel treatment modalities is warranted. The present study is designed to investigate the effect of pan histone deacetylase inhibitor, 'panobinostat', on cervical cancer cells alone and in combination with topoisomerase inhibitors. We assessed the effect of panobinostat on two cervical cancer cell lines, HeLa and SiHa, for cell viability, apoptosis, oxidative stress and mitochondrial function using various assays. The results indicate that panobinostat reduces the viability of cervical cancer cells in a dose- and time-dependent manner; it arrests HeLa cells in G0/G1 and SiHa cells in G2/M phase of the cell cycle. Panobinostat induced apoptosis through an increase in the ROS production and the disruption of mitochondrial membrane potential. Concomitantly the expression of anti-apoptotic gene Bcl-xL was reduced, while levels of CDK inhibitor p21 and caspase-9 were increased. Panobinostat increased the acetylation of histone H3 indicating HDAC inhibition. In addition, panobinostat also showed synergistic effect with topoisomerase inhibitors mediated by increased activation of caspase-3/7 activity compared to that in cells treated with panobinostat alone. These results suggest a combination therapy using inhibitors of histone deacetylase and topoisomerase together could hold the promise for an effective targeted therapeutic strategy. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells.

PubMed

Xiao, Dong; Johnson, Candace S; Trump, Donald L; Singh, Shivendra V

2004-05-01

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, offers significant protection against cancer in animals induced by a variety of carcinogens. The present study demonstrates that PEITC suppresses proliferation of PC-3 cells in a dose-dependent manner by causing G(2)-M-phase cell cycle arrest and apoptosis. Interestingly, phenyl isothiocyanate (PITC), which is a structural analogue of PEITC but lacks the -CH(2) spacers that link the aromatic ring to the -N=C=S group, neither inhibited PC-3 cell viability nor caused cell cycle arrest or apoptosis. These results indicated that even a subtle change in isothiocyanate (ITC) structure could have a significant impact on its biological activity. The PEITC-induced cell cycle arrest was associated with a >80% reduction in the protein levels of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C (Cdc25C; 24 h after treatment with 10 micro M PEITC), which led to an accumulation of Tyr(15) phosphorylated (inactive) Cdk1. On the other hand, PITC treatment neither reduced protein levels of Cdk1 or Cdc25C nor affected Cdk1 phosphorylation. The PEITC-induced decline in Cdk1 and Cdc25C protein levels and cell cycle arrest were significantly blocked on pretreatment of PC-3 cells with proteasome inhibitor lactacystin. A 24 h exposure of PC-3 cells to 10 micro M PEITC, but not PITC, resulted in about 56% and 44% decrease in the levels of antiapoptotic proteins Bcl-2 and Bcl-X(L), respectively. However, ectopic expression of Bcl-2 failed to alter sensitivity of PC-3 cells to growth inhibition or apoptosis induction by PEITC. Treatment of cells with PEITC, but not PITC, also resulted in cleavage of procaspase-3, procaspase-9, and procaspase-8. Moreover, the PEITC-induced apoptosis was significantly attenuated in the presence of general caspase inhibitor and specific inhibitors of caspase-8 and caspase-9. In conclusion, our data indicate that PEITC-induced cell cycle arrest in PC-3 cells is likely due to proteasome-mediated degradation of Cdc25C and Cdk1, and ectopic expression of Bcl-2 fails to confer resistance to PEITC-induced apoptosis. Furthermore, the results of the present study point toward involvement of both caspase-8- and caspase-9-mediated pathways in apoptosis induction by PEITC.

The potential role of polyphenols in the modulation of skin cell viability by Aspalathus linearis and Cyclopia spp. herbal tea extracts in vitro.

PubMed

Magcwebeba, Tandeka Unathi; Riedel, Sylvia; Swanevelder, Sonja; Swart, Pieter; De Beer, Dalene; Joubert, Elizabeth; Andreas Gelderblom, Wentzel Christoffel

2016-11-01

The relationship between polyphenol constituents, antioxidant properties of aqueous and methanol extracts of green tea (Camellia sinensis), the herbal teas, rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.), against skin cell viability was investigated in vitro. The effect of extracts, characterised in terms of polyphenol content and antioxidant properties, on cell viability of premalignant, normal and malignant skin cells was determined. Phenolic composition, particularly high levels of potent antioxidants, of rooibos and green tea methanol extracts was associated with a strong reduction in cell viability specifically targeting premalignant cells. In contrast, the aqueous extracts of Cyclopia spp. were more effective in reducing cell viability. This correlated with a relatively high flavanol/proanthocyanidin content and ABTS radical cation scavenging capacity. The major green tea flavanol (epigallocatechin gallate) and rooibos dihydrochalcone (aspalathin) exhibited differential effects against cell viability, while the major honeybush xanthone (mangiferin) and flavanone (hesperidin) lacked any effect presumably due to a cytoprotective effect. The underlying mechanisms against skin cell viability are likely to involve mitochondrial dysfunction resulting from polyphenol-iron interactions. The polyphenol constituents and antioxidant parameters of herbal tea extracts are useful tools to predict their activity against skin cell survival in vitro and potential chemopreventive effects in vivo. © 2016 Royal Pharmaceutical Society.

The silence of p66(Shc) in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway.

PubMed

Zhang, Ling; Zhu, Shengtao; Shi, Xuesen; Sha, Weihong

2015-01-01

Colon cancer is the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. The previous studies demonstrated that p66(Shc) protein, a member of Shc family, is highly expressed in colon cancer cells, but the role of p66(Shc) in the progress of colon cancer still unknown. In this study, we found that p66(Shc) highly expressed in colon cancer tissue and colon cancer cell line SW620 cells, HCT8 cells, HCT116 cells and CaCO2 cells. The silence of p66(Shc) in HCT8 cells reduced the proliferation and accelerated the apoptosis, in addition, the expression of pro-apoptotic proteins caspase-3, caspase-9, Bax was enhanced and the expression of anti-apoptotic protein Bcl-2 was declined. Moreover, the cell cycle arrest in G0/G1 phase after HCT8 cells treated with p66(Shc) siRNA. Furthermore, after HCT8 cells treated with p66(Shc) siRNA, the phosphorylation of PI3K and AKT was significantly suppressed, and the expression of Mdm-2, a downstream of AKT, was obviously prohibited, while the expression of p53 was enhanced. These results indicate that the silence of p66(Shc) in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway, it may provide a promising approach to prevent the progress of colon cancer cell.

The silence of p66Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway

PubMed Central

Zhang, Ling; Zhu, Shengtao; Shi, Xuesen; Sha, Weihong

2015-01-01

Colon cancer is the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. The previous studies demonstrated that p66Shc protein, a member of Shc family, is highly expressed in colon cancer cells, but the role of p66Shc in the progress of colon cancer still unknown. In this study, we found that p66Shc highly expressed in colon cancer tissue and colon cancer cell line SW620 cells, HCT8 cells, HCT116 cells and CaCO2 cells. The silence of p66Shc in HCT8 cells reduced the proliferation and accelerated the apoptosis, in addition, the expression of pro-apoptotic proteins caspase-3, caspase-9, Bax was enhanced and the expression of anti-apoptotic protein Bcl-2 was declined. Moreover, the cell cycle arrest in G0/G1 phase after HCT8 cells treated with p66Shc siRNA. Furthermore, after HCT8 cells treated with p66Shc siRNA, the phosphorylation of PI3K and AKT was significantly suppressed, and the expression of Mdm-2, a downstream of AKT, was obviously prohibited, while the expression of p53 was enhanced. These results indicate that the silence of p66Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway, it may provide a promising approach to prevent the progress of colon cancer cell. PMID:26464652

Disrupted cell cycle control in cultured endometrial cells from patients with endometriosis harboring the progesterone receptor polymorphism PROGINS.

PubMed

D'Amora, Paulo; Maciel, Thiago Trovati; Tambellini, Rodrigo; Mori, Marcelo A; Pesquero, João Bosco; Sato, Helio; Girão, Manoel João Batista Castello; Guerreiro da Silva, Ismael Dale Cotrim; Schor, Eduardo

2009-07-01

Presently, little is understood about how endometriosis is established or maintained, or how genetic factors can predispose women to the disease. Because of the crucial role that the progesterone receptor polymorphism PROGINS plays in predisposing women to the development of endometriosis, we hypothesized that this variant may influence critical steps during endometrial cell metabolism that are involved in the pathogenesis of endometriosis. Eutopic endometria were collected from three sources: women with endometriosis who had a single PROGINS allele (from the progesterone receptor gene); women with endometriosis who had the wild-type progesterone receptor allele; and women without endometriosis who had the wild-type allele. Cells prepared from the eutopic endometria of these women were stimulated with both estradiol and progesterone, and then examined for cell proliferation, viability, and apoptosis. The cells from women with endometriosis that carried the PROGINS allele demonstrated increased proliferation, greater viability, and decreased apoptosis following progesterone treatment. In general, these parameters were very different as compared with those of women with endometriosis but without the PROGINS allele and women in the control group. This result indicates there is a reduced level of progesterone responsiveness in women who carry the PROGINS polymorphism. Because progesterone responsiveness is known to be an important characteristic of women with endometriosis, these data support the contention that the PROGINS polymorphism enhances the endometriosis phenotype.

Anticarcinogenic properties of medium chain fatty acids on human colorectal, skin and breast cancer cells in vitro.

PubMed

Narayanan, Amoolya; Baskaran, Sangeetha Ananda; Amalaradjou, Mary Anne Roshni; Venkitanarayanan, Kumar

2015-03-05

Colorectal cancer, breast cancer and skin cancer are commonly-reported cancer types in the U.S. Although radiation and chemotherapy are routinely used to treat cancer, they produce side effects in patients. Additionally, resistance to chemotherapeutic drugs has been noticed in cancers. Thus, there is a need for effective and safe bioprophylactics and biotherapeutics in cancer therapy. The medicinal value of goat milk has been recognized for centuries and is primarily attributed to three fatty acids, namely capric, caprylic and caproic acids. This research investigates the anticancer property of these fatty acids on human colorectal, skin and mammary gland cancer cells. The cancer cells were treated with various concentrations of fatty acids for 48 h, and cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Additionally, real-time quantitative PCR (RT-qPCR) was performed to elucidate the potential anti-cancer mechanisms of the three fatty acids under investigation. Capric, caprylic and caproic acids reduced cancer cell viability by 70% to 90% (p < 0.05) compared to controls. RT-qPCR data indicated that these natural molecules produced anticancer effects by down-regulating cell cycle regulatory genes and up-regulating genes involved in apoptosis. Future research will validate the anticancer effect of these fatty acids in an appropriate in vivo model.

Disrupted Cell Cycle Control in Cultured Endometrial Cells from Patients with Endometriosis Harboring the Progesterone Receptor Polymorphism PROGINS

PubMed Central

D'Amora, Paulo; Maciel, Thiago Trovati; Tambellini, Rodrigo; Mori, Marcelo A.; Pesquero, João Bosco; Sato, Helio; Girão, Manoel João Batista Castello; Guerreiro da Silva, Ismael Dale Cotrim; Schor, Eduardo

2009-01-01

Presently, little is understood about how endometriosis is established or maintained, or how genetic factors can predispose women to the disease. Because of the crucial role that the progesterone receptor polymorphism PROGINS plays in predisposing women to the development of endometriosis, we hypothesized that this variant may influence critical steps during endometrial cell metabolism that are involved in the pathogenesis of endometriosis. Eutopic endometria were collected from three sources: women with endometriosis who had a single PROGINS allele (from the progesterone receptor gene); women with endometriosis who had the wild-type progesterone receptor allele; and women without endometriosis who had the wild-type allele. Cells prepared from the eutopic endometria of these women were stimulated with both estradiol and progesterone, and then examined for cell proliferation, viability, and apoptosis. The cells from women with endometriosis that carried the PROGINS allele demonstrated increased proliferation, greater viability, and decreased apoptosis following progesterone treatment. In general, these parameters were very different as compared with those of women with endometriosis but without the PROGINS allele and women in the control group. This result indicates there is a reduced level of progesterone responsiveness in women who carry the PROGINS polymorphism. Because progesterone responsiveness is known to be an important characteristic of women with endometriosis, these data support the contention that the PROGINS polymorphism enhances the endometriosis phenotype. PMID:19497994

Silymarin inhibits melanoma cell growth both in vitro and in vivo by targeting cell cycle regulators, angiogenic biomarkers and induction of apoptosis.

PubMed

Vaid, Mudit; Singh, Tripti; Prasad, Ram; Katiyar, Santosh K

2015-11-01

Cutaneous malignant melanoma is the leading cause of death from skin diseases and is often associated with activating mutations of the proto-oncogene BRAF. To develop more effective strategies for the prevention or treatment of melanoma, we have examined the inhibitory effects of silymarin, a flavanoid from Silybum marianum, on melanoma cells. Using A375 (BRAF-mutated) and Hs294t (non BRAF-mutated but highly metastatic) human melanoma cell lines, we found that in vitro treatment with silymarin resulted in a dose-dependent: (i) reduction in cell viability; (ii) enhancement of either Go/G1 (A375) or G2-M (Hs294t) phase cell cycle arrest with corresponding alterations in cyclins and cyclin-dependent kinases; and (iii) induction of apoptosis. The silymarin-induced apoptosis of human melanoma cells was associated with a reduction in the levels of anti-apoptotic proteins (Bcl-2 and Bcl-xl), an increase in the levels of pro-apoptotic protein (Bax), and activation of caspases. Further, oral administration of silymarin (500 mg/kg body weight/2× a week) significantly inhibited (60%, P < 0.01) the growth of BRAF-mutated A375 melanoma tumor xenografts, and this was associated with: (i) inhibition of cell proliferation; (ii) induction of apoptosis of tumor cells; (iii) alterations in cell cycle regulatory proteins; and (iv) reduced expression of tumor angiogenic biomarkers in tumor xenograft tissues. These results indicate that silymarin may have a chemotherapeutic effect on human melanoma cell growth and warrant its further evaluation. © 2014 Wiley Periodicals, Inc.

Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

PubMed

Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

2013-01-01

Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC.

Inkjet printing Schwann cells and neuronal analogue NG108-15 cells.

PubMed

Tse, Christopher; Whiteley, Robert; Yu, Tong; Stringer, Jonathan; MacNeil, Sheila; Haycock, John W; Smith, Patrick J

2016-03-01

Porcine Schwann cells and neuronal analogue NG108-15 cells were printed using a piezoelectric-inkjet-printer with a nozzle diameter of 60 μm, within the range of 70-230 V, with analysis of viability and quality after printing. Neuronal and glial cell viabilities of >86% and >90% were detected immediately after printing and no correlation between voltage applied and cell viability could be seen. Printed neuronal cells were shown to produce neurites earlier compared to controls, and over several days, produced longer neurites which become most evident by day 7. The number of neurites becomes similar by day 7 also, and cells proliferate with a similar viability to that of non-printed cells (controls). This method of inkjet printing cells provides a technical platform for investigating neuron-glial cell interactions with no significant difference to cell viability than standard cell seeding. Such techniques can be utilized for lab-on-a-chip technologies and to create printed neural networks for neuroscience applications.

The effect of desferrioxamine on transferrin receptors, the cell cycle and growth rates of human leukaemic cells.

PubMed Central

Bomford, A; Isaac, J; Roberts, S; Edwards, A; Young, S; Williams, R

1986-01-01

The effect of the iron chelator, desferrioxamine, on transferrin binding, growth rates and the cell cycle was investigated in the human leukaemic cell line, K562. At all concentrations of the chelator (2-50 microM) binding of 125I-transferrin was increased by 24 h and reached a maximum at 72-96 h. Maximum binding (6-8-fold increased) occurred in cells treated with 20 microM-desferrioxamine, in contrast with control cells which, at 96 h, showed a 50% decrease over initial binding. Scatchard analysis at 4 degrees C showed that this increased binding was due to an increase in the number of receptors, as the Kd was similar in induced (1.8 nM) and control (1.5 nM) cells. After 96 h cells, cultured with 20 and 50 microM-desferrioxamine accumulated 59Fe from bovine transferrin at over twice the rate found with control cells, reflecting the increase in transferrin receptors. Although iron uptake was unimpaired by the chelator there was a dose-dependent inhibition of cell growth, with control cells completing three divisions in 96 h and those in 10 microM-desferrioxamine only two divisions. At the highest concentration (50 microM), cell division was abrogated although cell viability was maintained (85%). In contrast, DNA synthesis was not markedly affected, except at 50 microM-desferrioxamine when incorporation of [3H]thymidine was 52% of that in control cells. Flow cytometry revealed that there was a progressive accumulation of the cells in the active phases of their cycle (S, G2 + M). Desferrioxamine may increase transferrin receptors in two ways: by chelating a regulatory pool of iron within the cell, and by arresting cells in S phase when receptors are maximally expressed. PMID:3790074

Long term cryopreservation in 5% DMSO maintains unchanged CD34(+) cells viability and allows satisfactory hematological engraftment after peripheral blood stem cell transplantation.

PubMed

Abbruzzese, L; Agostini, F; Durante, C; Toffola, R T; Rupolo, M; Rossi, F M; Lleshi, A; Zanolin, S; Michieli, M; Mazzucato, M

2013-07-01

Peripheral blood stem cell cryopreservation is associated with cell damage and decreased viability. We evaluated the impact of up to 10 years of cryopreservation (5% DMSO) on viability of CD34(+) cells utilizing graft samples of consecutive patients (2002-2012) with different malignancies who underwent stem cell collection and transplantation. Viability of CD34(+) cells from oncohaematological patients measured after 5 weeks (97·2 ± 0·6%) or after 9-10 years of cryopreservation (95·9 ± 0·5%) was unaffected. Haemoglobin, granulocyte and platelet recovery after transplantation of long-term cryopreserved grafts occurred within 8-13 days. CD34(+) stem cells can be safely stored up to 9-10 years, without affecting cell viability and clinical effectiveness. © 2013 International Society of Blood Transfusion.

mTOR Activation by PI3K/Akt and ERK Signaling in Short ELF-EMF Exposed Human Keratinocytes

PubMed Central

Patruno, Antonia; Pesce, Mirko; Grilli, Alfredo; Speranza, Lorenza; Franceschelli, Sara; De Lutiis, Maria Anna; Vianale, Giovina; Costantini, Erica; Amerio, Paolo; Muraro, Raffaella; Felaco, Mario; Reale, Marcella

2015-01-01

Several reports suggest that ELF-EMF exposures interact with biological processes including promotion of cell proliferation. However, the molecular mechanisms by which ELF-EMF controls cell growth are not completely understood. The present study aimed to investigate the effect of ELF-EMF on keratinocytes proliferation and molecular mechanisms involved. Effect of ELF-EMF (50 Hz, 1 mT) on HaCaT cell cycle and cells growth and viability was monitored by FACS analysis and BrdU assay. Gene expression profile by microarray and qRT-PCR validation was performed in HaCaT cells exposed or not to ELF-EMF. mTOR, Akt and MAPKs expressions were evaluated by Western blot analysis. In HaCaT cells, short ELF-EMF exposure modulates distinct patterns of gene expression involved in cell proliferation and in the cell cycle. mTOR activation resulted the main molecular target of ELF-EMF on HaCaT cells. Our data showed the increase of the canonical pathway of mTOR regulation (PI3K/Akt) and activation of ERK signaling pathways. Our results indicate that ELF-EMF selectively modulated the expression of multiple genes related to pivotal biological processes and functions that play a key role in physio-pathological mechanisms such as wound healing. PMID:26431550

Phaleria macrocarpa (Boerl.) fruit induce G0/G1 and G2/M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 human breast cancer cell.

PubMed

Kavitha, Nowroji; Ein Oon, Chern; Chen, Yeng; Kanwar, Jagat R; Sasidharan, Sreenivasan

2017-04-06

Phaleria macrocarpa (Scheff) Boerl, is a well-known folk medicinal plant in Indonesia. Traditionally, P. macrocarpa has been used to control cancer, impotency, hemorrhoids, diabetes mellitus, allergies, liver and hearth disease, kidney disorders, blood diseases, acne, stroke, migraine, and various skin diseases. The purpose of this study was to determine the in situ cytotoxicity effect P. macrocarpa fruit ethyl acetate fraction (PMEAF) and the underlying molecular mechanism of cell death. MDA-MB-231 cells were incubated with PMEAF for 24h. Cell cycle and viability were examined using flow cytometry analysis. Apoptosis was determined using the Annexin V assay and also by fluorescence microscopy. Apoptosis protein profiling was detected by RayBio® Human Apoptosis Array. The AO/PI staining and flow cytometric analysis of MDA-MB-231 cells treated with PMEAF were showed apoptotic cell death. The cell cycle analysis by flow cytometry analysis revealed that the accumulation of PMEAF treated MDA-MB-231 cells in G 0 /G 1 and G 2 /M-phase of the cell cycle. Moreover, the PMEAF exert cytotoxicity by increased the ROS production in MDA-MB-231 cells consistently stimulated the loss of mitochondrial membrane potential (∆ Ψm ) and induced apoptosis cell death by activation of numerous signalling proteins. The results from apoptosis protein profiling array evidenced that PMEAF stimulated the expression of 9 pro-apoptotic proteins (Bax, Bid, caspase 3, caspase 8, cytochrome c, p21, p27, p53 and SMAC) and suppressed the 4 anti-apoptotic proteins (Bcl-2, Bcl-w, XIAP and survivin) in MDA-MB-231 cells. The results indicated that PMEAF treatment induced apoptosis in MDA-MB-231 cells through intrinsic mitochondrial related pathway with the participation of pro and anti-apoptotic proteins, caspases, G 0 /G 1 and G 2 /M-phases cell cycle arrest by p53-mediated mechanism. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Pre-exposure to 50 Hz-electromagnetic fields enhanced the antiproliferative efficacy of 5-fluorouracil in breast cancer MCF-7 cells

PubMed Central

Chen, Sha; Sun, Xiongshan; Guan, Xiao; Yang, Yao; Peng, Bingjie; Pan, Xiaodong; Li, Jinfang; Yi, Weijing; Li, Peng; Zhang, Hongwei; Feng, Dongfang; Chen, An; Li, Xiaohui; Yin, Zuoming

2018-01-01

Resistance to 5-fluorouracil (5-FU) and its induced immune suppression have prevented its extensive application in the clinical treatment of breast cancer. In this study, the combined effect of 50 Hz-EMFs and 5-FU in the treatment of breast cancer was explored. MCF-7 and MCF10A cells were pre-exposed to 50 Hz-EMFs for 0, 2, 4, 8 and 12 h and then treated with different concentrations of 5-FU for 24 h; cell viability was analyzed by MTT assay and flow cytometry. After pre-exposure to 50 Hz-EMFs for 12 h, apoptosis and cell cycle distribution in MCF-7 and MCF10A cells were detected via flow cytometry and DNA synthesis was measured by EdU incorporation assay. Apoptosis-related and cell cycle-related gene and protein expression levels were monitored by qPCR and western blotting. Pre-exposure to 50 Hz-EMFs for 12 h enhanced the antiproliferative effect of 5-FU in breast cancer cell line MCF-7 in a dose-dependent manner but not in normal human breast epithelial cell line MCF10A. Exposure to 50 Hz-EMFs had no effect on apoptosis and P53 expression of MCF-7 and MCF10A cells, whereas it promoted DNA synthesis, induced entry of MCF-7 cells into the S phase of cell cycle, and upregulated the expression levels of cell cycle-related proteins Cyclin D1 and Cyclin E. Considering the pharmacological mechanisms of 5-FU in specifically disrupting DNA synthesis, this enhanced inhibitory effect might have resulted from the specific sensitivity of MCF7 cells in active S phase to 5-FU. Our findings demonstrate the enhanced cytotoxic activity of 5-FU on MCF7 cells through promoting entry into the S phase of the cell cycle via exposure to 50 Hz-EMFs, which provides a novel method of cancer treatment based on the combinatorial use of 50 Hz-EMFs and chemotherapy. PMID:29617363

An approach for cell viability online detection based on the characteristics of lensfree cell diffraction fingerprint.

PubMed

Li, Guoxiao; Zhang, Rongbiao; Yang, Ning; Yin, Changsheng; Wei, Mingji; Zhang, Yecheng; Sun, Jian

2018-06-01

To overcome the drawbacks such as low automation and high cost, an approach for cell viability online detection is proposed, based on the extracted lensfree cell diffraction fingerprint characteristics. The cell fingerprints are acquired by a constructed large field-of-view (FOV) diffraction imaging platform without any lenses. The approach realizes distinguishing live and dead cells online and calculating cell viability index based on the number of live cells. With theoretical analysis and simulation, diffraction fingerprints of cells with different morphology are simulated and two characteristics are discovered to be able to reflect cell viability status effectively. Two parameters, fringe intensity contrast (FIC) and fringe dispersion (FD), are defined to quantify these two characteristics. They are verified to be reliable to identify live cells. In a cytotoxicity assay of different methyl mercury concentration on BRL cells, the proposed approach is used to detect cell viability. MTT method is also employed and the results of correlational analysis and Bland-Altman analysis prove the validity of the proposed approach. By comparison, it can be revealed that the proposed approach has some advantages over other present techniques. Therefore it may be widely used as a cell viability measurement method in drug screening, nutritional investigation and cell toxicology studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of Wannachawee Recipe with Antipsoriatic Activity on Suppressing Inflammatory Cytokine Production in HaCaT Human Keratinocytes

PubMed Central

Na Takuathung, Mingkwan; Wongnoppavich, Ariyaphong; Pitchakarn, Pornsiri; Panthong, Ampai; Khonsung, Parirat; Soonthornchareonnon, Noppamas

2017-01-01

Psoriasis is a chronic inflammatory and immune-mediated skin disease. The pathogenesis involves T cells activation via the IL-23/Th17 axis. Conventional treatments of psoriasis have adverse events influencing patients' adherence. Wannachawee Recipe (WCR) has been effectively used as Thai folk remedy for psoriasis patients; however, preclinical evidence defining how WCR works is still lacking. This study defined mechanisms for its antiproliferation and anti-inflammatory effects in HaCaT cells. The cytotoxicity and antiproliferation results from SRB and CCK-8 assays showed that WCR inhibited the growth and viability of HaCaT cells in a concentration-dependent manner. The distribution of cell cycle phases determined by flow cytometry showed that WCR did not interrupt cell cycle progression. Interestingly, RT-qPCR revealed that WCR significantly decreased the mRNA expression of IL-1β, IL-6, IL-8, IL-17A, IL-22, IL-23, and TNF-α but induced IL-10 expression in TNF-α- and IFN-γ-induced HaCaT cells. At the protein level determined by ELISA, WCR significantly reduced the secretion of IL-17A, IL-22, and IL-23. The WCR at low concentrations was proved to possess anti-inflammatory effect without cytotoxicity and it did not interfere with cell cycle of keratinocytes. This is the first study to provide convincing evidence that WCR is a potential candidate for development of effective psoriasis therapies. PMID:28900461

Effects of Wannachawee Recipe with Antipsoriatic Activity on Suppressing Inflammatory Cytokine Production in HaCaT Human Keratinocytes.

PubMed

Na Takuathung, Mingkwan; Wongnoppavich, Ariyaphong; Pitchakarn, Pornsiri; Panthong, Ampai; Khonsung, Parirat; Chiranthanut, Natthakarn; Soonthornchareonnon, Noppamas; Sireeratawong, Seewaboon

2017-01-01

Psoriasis is a chronic inflammatory and immune-mediated skin disease. The pathogenesis involves T cells activation via the IL-23/Th17 axis. Conventional treatments of psoriasis have adverse events influencing patients' adherence. Wannachawee Recipe (WCR) has been effectively used as Thai folk remedy for psoriasis patients; however, preclinical evidence defining how WCR works is still lacking. This study defined mechanisms for its antiproliferation and anti-inflammatory effects in HaCaT cells. The cytotoxicity and antiproliferation results from SRB and CCK-8 assays showed that WCR inhibited the growth and viability of HaCaT cells in a concentration-dependent manner. The distribution of cell cycle phases determined by flow cytometry showed that WCR did not interrupt cell cycle progression. Interestingly, RT-qPCR revealed that WCR significantly decreased the mRNA expression of IL-1 β , IL-6, IL-8, IL-17A, IL-22, IL-23, and TNF- α but induced IL-10 expression in TNF- α - and IFN- γ -induced HaCaT cells. At the protein level determined by ELISA, WCR significantly reduced the secretion of IL-17A, IL-22, and IL-23. The WCR at low concentrations was proved to possess anti-inflammatory effect without cytotoxicity and it did not interfere with cell cycle of keratinocytes. This is the first study to provide convincing evidence that WCR is a potential candidate for development of effective psoriasis therapies.

Free flow cell electrophoresis using zwitterionic buffer

NASA Technical Reports Server (NTRS)

Rodkey, R. Scott

1990-01-01

Studies of a zwitterionic buffer formulated for cell electrophoresis were done using the McDonnell-Douglas Continuous Flow Electrophoresis System. Standard buffers were analyzed for their stability in the electrical field and the results showed that both buffers tested were inherently unstable. Further, titration studies showed that the standards buffers buffered poorly at the pH employed for electrophoresis. The zwitterionic buffer buffered well at its nominal pH and was shown to be stable in the electrical field. Comparative studies of the buffer with standard cell separation buffers using formalin fixed rabbit and goose red blood cells showed that the zwitterionic buffer gave better resolution of the fixed cells. Studies with viable hybridoma cells showed that buffer Q supported cell viability equal to Hank's Balanced Salt Solution and that hybridoma cells in different stages of the growth cycle demonstrated reproducible differences in electrophoretic mobility.

14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression.

PubMed

Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S; Balyan, Renu; Borde, Lalit; Kundu, Tapas K; Dalal, Sorab N

2016-06-02

More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering.

Exosomal MicroRNA MiR-1246 Promotes Cell Proliferation, Invasion and Drug Resistance by Targeting CCNG2 in Breast Cancer.

PubMed

Li, Xiu Juan; Ren, Zhao Jun; Tang, Jin Hai; Yu, Qiao

2017-01-01

Treatment of breast cancer remains a clinical challenge. This study aims to validate exosomal microRNA-1246 (miR-1246) as a serum biomarker for breast cancer and understand the underlying mechanism in breast cancer progression. The expression levels of endogenous and exosomal miRNAs were examined by real time PCR, and the expression level of the target protein was detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study their uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-1246 was estimated by invasion assay and cell viability assay. In this study, we demonstrate that exosomes carrying microRNA can be transferred among different cell lines through direct uptake. miR-1246 is highly expressed in metastatic breast cancer MDA-MB-231 cells compared to non-metastatic breast cancer cells or non-malignant breast cells. Moreover, miR-1246 can suppress the expression level of its target gene, Cyclin-G2 (CCNG2), indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could enhance the viability, migration and chemotherapy resistance of non-malignant HMLE cells. Together, our results support an important role of exosomes and exosomal miRNAs in regulating breast tumor progression, which highlights their potential for applications in miRNA-based therapeutics. © 2017 The Author(s). Published by S. Karger AG, Basel.

Cell viability monitoring using Fano resonance in gold nanoslit array

NASA Astrophysics Data System (ADS)

Wu, Shu-Han; Hsieh, Shu-Yi; Lee, Kuang-Li; Weng, Ruei-Hung; Chiou, Arthur; Wei, Pei-Kuen

2013-09-01

Cell viability is a crucial issue in biological research. We present label-free monitoring of adhesion cells viability by gold nanoslits-based Fano resonance biosensors. Plastic multiple wells with gold nanoslits substrate were made using a thermal nanoimprint method. Adhesion cells in the wells were treated with doxorubicin for inducing cell death and compared with conventional colorimetric assay. The nanoslits method shows better respones of viability tests under low concentration and short interaction time due to its high surface sensitivies. The vinculin labelling indicates that the measured signals are in good agreement with the adhesion abilities of cells.

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.

A Vitex agnus-castus extract inhibits cell growth and induces apoptosis in prostate epithelial cell lines.

PubMed

Weisskopf, M; Schaffner, W; Jundt, G; Sulser, T; Wyler, S; Tullberg-Reinert, H

2005-10-01

Extracts of Vitex agnus-castus fruits (VACF) are described to have beneficial effects on disorders related to hyperprolactinemia (cycle disorders, premenstrual syndrome). A VACF extract has recently been shown to exhibit antitumor activities in different human cancer cell lines. In the present study, we explored the antiproliferative effects of a VACF extract with a particular focus on apoptosis-inducing and potential cytotoxic effects. Three different human prostate epithelial cell lines (BPH-1, LNCaP, PC-3) representing different disease stages and androgen responsiveness were chosen. The action of VACF on cell viability was assessed using the WST-8-tetrazolium assay. Cell proliferation in cells receiving VACF alone or in combination with a pan-caspase inhibitor (Z-VAD-fmk) was quantified using a Crystal Violet assay. Flow cytometric cell cycle analysis and measurement of DNA fragmentation using an ELISA method were used for studying the induction of apoptosis. Lactate dehydrogenase (LDH) activity was determined as a marker of cytotoxicity. The extract inhibited proliferation of all three cell lines in a concentration-dependent manner with IC (50) values below 10 microg/mL after treatment for 48 h. Cell cycle analysis and DNA fragmentation assays suggest that part of the cells were undergoing apoptosis. The VACF-induced decrease in cell number was partially inhibited by Z-VAD-fmk, indicating a caspase-dependent apoptotic cell death. However, the concentration-dependent LDH activity of VACF treated cells indicated cytotoxic effects as well. These data suggest that VACF contains components that inhibit proliferation and induce apoptosis in human prostate epithelial cell lines. The extract may be useful for the prevention and/or treatment not only of benign prostatic hyperplasia but also of human prostate cancer.

Eupatilin, a dietary flavonoid, induces G2/M cell cycle arrest in human endometrial cancer cells.

PubMed

Cho, Jung-Hoon; Lee, Jong-Gyu; Yang, Yeong-In; Kim, Ji-Hyun; Ahn, Ji-Hye; Baek, Nam-In; Lee, Kyung-Tae; Choi, Jung-Hye

2011-08-01

This study is the first to investigate the antiproliferative effect of eupatilin in human endometrial cancer cells. Eupatilin, a naturally occurring flavonoid isolated from Artemisia princeps, has anti-inflammatory, anti-oxidative, and anti-tumor activities. In the present study, we investigated the potential effect of eupatilin on cell growth and its molecular mechanism of action in human endometrial cancer cells. Eupatilin was more potent than cisplatin in inhibiting cell viability in the human endometrial cancer cell lines Hec1A and KLE. Eupatilin showed relatively low cytotoxicity in normal human endometrial cells HES and HESC cells when compared to cisplatin. Eupatilin induced G2/M phase cell cycle arrest in a time- and dose-dependent manner, as indicated by flow cytometry analysis. In addition, treatment of Hec1A cells with eupatilin resulted in a significant increase in the expression of p21(WAF1/CIP1) and in the phosphorylation of Cdc25C and Cdc2. Knockdown of p21 using specific siRNAs significantly compromised eupatilin-induced cell growth inhibition. Interestingly, levels of mutant p53 in Hec1A cells decreased markedly upon treatment with eupatilin, and p53 siRNA significantly increased p21 expression. Moreover, eupatilin modulated the phosphorylation of protein kinases ERK1/2, Akt, ATM, and Chk2. These results suggest that eupatilin inhibits the growth of human endometrial cancer cells via G2/M phase cell cycle arrest through the up-regulation of p21 by the inhibition of mutant p53 and the activation of the ATM/Chk2/Cdc25C/Cdc2 checkpoint pathway. Copyright © 2011 Elsevier Ltd. All rights reserved.

Shock Wave-Stimulated Periosteum for Cartilage Repair

DTIC Science & Technology

2013-12-01

were added to the Gtn-HPA prior to the gelation 6 process, at a cell density of 1×105 cells/ml. In the control groups, cells received no treatment...Mesenchymal Stem Cell Viability Viability test was performed 24 hours post- gelation using the Live/Dead assay. Viability/cytotoxicity kit was used (Molecular

Quercus Suber L. Cork Extracts Induce Apoptosis in Human Myeloid Leukaemia HL-60 Cells.

PubMed

Bejarano, Ignacio; Godoy-Cancho, Belén; Franco, Lourdes; Martínez-Cañas, Manuel A; Tormo, María A

2015-08-01

Quercus suber L. cork contains a diversity of phenolic compounds, mostly low molecular weight phenols. A rising number of reports support with convergent findings that polyphenols evoke pro-apoptotic events in cancerous cells. However, the literature related to the anti-cancer bioactivity of Q. suber L. cork extractives (QSE) is still limited. Herein, we aim to describe the antitumor potential displayed by cork extractives obtained by different extraction methods in the human promyelocytic leukaemia cells. In order to quantify the effects of QSE on cancer cells viability, phosphatidylserine exposure, caspase-3 activity, mitochondrial membrane potential and cell cycle were evaluated. The results indicated that the QSE present a time-dependent and dose-dependent cytotoxicity in the human promyelocytic leukaemia cells. Such a noxious effect leads these leukaemia cells to their death through apoptotic processes by altering the mitochondrial outer membrane potential, activating caspase-3 and externalizing phosphatidylserine. However, cells cycle progression was not affected by the treatments. This study contributes to open a new way to use this natural resource by exploiting its anti-cancer properties. Moreover, it opens new possibilities of application of cork by-products, being more efficient in the sector of cork-based agriculture. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Synergistic effects of combined treatment with histone deacetylase inhibitor suberoylanilide hydroxamic acid and TRAIL on human breast cancer cells

PubMed Central

Zhou, Weiqiang; Feng, Xiuyan; Han Han; Guo, Shanchun; Wang, Guangdi

2016-01-01

Previous studies showed that either histone deacetylase (HDAC) inhibitors or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in tumor cells including breast cancer. However, the underling mechanisms of combining HDAC inhibitors with TRAIL in the treatment of breast cancer are poorly understood. In this study, we determined the ability of SAHA and TRAIL as single agents or in combination to inhibit the growth and survival of MCF-7 and MDA-MB-231 breast cancer cells. Our results demonstrate that the distinct effects of SAHA or TRAIL individually and in combination on the proliferation, cell viability, apoptosis, cell cycle distribution, and morphological changes of MDA-MB-231 and MCF-7 cells. We further determined the different effects of SAHA or TRAIL alone and combining SAHA with TRAIL on the expression of a number of apoptosis-related molecules, cell cycle, growth factors and their receptors in cancer cells. Our results demonstrated that the combinatorial treatment of SAHA and TRAIL may target multiple pathways and serve as an effective therapeutic strategy against breast cancer. An improved understanding of the molecular mechanisms may facilitate either SAHA or TRAIL targeted use and the selection of suitable combinations. PMID:27292433

The role of adrenergic activation on murine luteal cell viability and progesterone production.

PubMed

Wang, Jing; Tang, Min; Jiang, Huaide; Wu, Bing; Cai, Wei; Hu, Chuan; Bao, Riqiang; Dong, Qiming; Xiao, Li; Li, Gang; Zhang, Chunping

2016-09-15

Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating β-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective β-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on β-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating β-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3β-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO. Copyright © 2016 Elsevier Inc. All rights reserved.

Optimization of low energy sonication treatment for granular activated carbon colonizing biomass assessment.

PubMed

Saccani, G; Bernasconi, M; Antonelli, M

2014-01-01

This study is aimed at optimizing a low energy sonication (LES) treatment for granular activated carbon (GAC)-colonizing biomass detachment and determination, evaluating detachment efficiency and the effects of ultrasound exposure on bacterial cell viability. GAC samples were collected from two filters fed with groundwater. Conventional heterotrophic plate count (HPC) and fluorescence microscopy with a double staining method were used to evaluate cell viability, comparing two LES procedures, without and with periodical bulk substitution. A 20 min LES treatment, with bulk substitution after cycles of 5 min as maximum treatment time, allowed to recover 87%/100% of attached biomass, protecting detached bacteria from ultrasound damaging effects. Observed viable cell inactivation rate was 6.5/7.9% cell/min, with membrane-compromised cell damage appearing to be even higher (11.5%/13.1% cell/min). Assessing bacterial detachment and damaging ultrasound effects, fluorescence microscopy turned out to be more sensitive compared to conventional HPC. The optimized method revealed a GAC-colonizing biomass of 9.9 x 10(7) cell/gGAC for plant 1 and 8.8 x 10(7) cell/gGAC for plant 2, 2 log lower than reported in literature. The difference between the two GAC-colonizing biomasses is higher in terms of viable cells (46.3% of total cells in plant 1 GAC-colonizing biomass compared to the 33.3% in plant 2). Studying influent water contamination through multivariate statistical analyses, apossible combined toxic and genotoxic effect of chromium VI and trichloroethylene was suggested as a reason for the lower viable cell fraction observed in plant 2 GAC-colonizing population.

Safety Profile of TiO2-Based Photocatalytic Nanofabrics for Indoor Formaldehyde Degradation

PubMed Central

Cui, Guixin; Xin, Yan; Jiang, Xin; Dong, Mengqi; Li, Junling; Wang, Peng; Zhai, Shumei; Dong, Yongchun; Jia, Jianbo; Yan, Bing

2015-01-01

Anatase TiO2 nanoparticles (TNPs) are synthesized using the sol-gel method and loaded onto the surface of polyester-cotton (65/35) fabrics. The nanofabrics degrade formaldehyde at an efficiency of 77% in eight hours with visible light irradiation or 97% with UV light. The loaded TNPs display very little release from nanofabrics (~0.0%) during a standard fastness to rubbing test. Assuming TNPs may fall off nanofabrics during their life cycles, we also examine the possible toxicity of TNPs to human cells. We found that up to a concentration of 220 μg/mL, they do not affect viability of human acute monocytic leukemia cell line THP-1 macrophages and human liver and kidney cells. PMID:26610470

6-Shogaol induces cell cycle arrest and apoptosis in human hepatoma cells through pleiotropic mechanisms.

PubMed

Wu, Jung-Ju; Omar, Hany A; Lee, Ying-Ray; Teng, Yen-Ni; Chen, Pin-Shern; Chen, Yu-Chung; Huang, Hsiao-Shan; Lee, Kuan-Han; Hung, Jui-Hsiang

2015-09-05

Shogaols are a group of the active constituents of ginger that have been identified to have various biological activities. The aim of the current study was to investigate the antitumor activity of 6-shogaol in hepatocellular carcinoma (HCC) and the possible involvement of reactive oxygen species as a putative mechanism of action. HCC cell lines, HepG2 and Huh-7, were used to study the in vitro anti-cancer activity of 6-shogaol via the application of various molecular biology techniques. Results showed that 6-shogaol effectively inhibited the cell viability, caused cell cycle arrest at G2/M phase and induced apoptosis in HCC cells as indicated by MTT assay, DAPI nuclear staining, annexin V assay, cell cycle analysis, and activation of caspase-3. Western blot analysis revealed the ability of 6-shogaol to target cancer survival signaling pathways mediated by mitogen-activated protein kinase (MAPK), 5' AMP-activated protein kinase (AMPK) and Akt. In addition, 6-Shogaol induced alteration of cyclin proteins expression and caused cleavage of protein kinase C delta. Furthermore, 6-Shogaol was able to induce the production of reactive oxygen species and endoplasmic reticulum (ER) stress-associated proteins and the consequent activation of autophagy in HepG2 cells. Taken together, the current study highlights evidences that 6-shogaol induces apoptosis, modulates cyclins expression and targets cancer survival signaling pathways in HCC cell lines, at least in part, via the production of reactive oxygen species. These findings support 6-shogaol's clinical promise as a potential candidate for HCC therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma.

PubMed

Dai, Bingling; Wang, Wenjie; Liu, Rui; Wang, Hongying; Zhang, Yanmin

2015-03-01

Taspine is an active compound in anticancer agent development. 12k was synthesized with taspine as lead compound bearing biphenyl scaffold and showed potent anticancer activity. Here, we investigated the effect of taspine derivative 12k on A549 lung cells. We showed that 12k not only decreased significantly A549 cell viability, A549 cell colony formation but also impaired A549 cell migration. Moreover, 12k treatment blocked cell cycle progression by increasing cell number in S phase to 42.80% for 6 μmol/L vs. 28.86% for control while decreasing cell number in G1 phase. Accordingly, this was associated with an increase protein expression of cyclin E and a decrease protein expression of cyclin D1, cyclin B1 and its associated CDK1 (cdc2). Meanwhile, we found that 12k induced A549 cell apoptosis, which was closely associated with the effect of the Bcl-2 family. Increase of Bad, Bak and Bax expression levels, decrease of Bcl-2 and Mcl-1 expression levels were observed. SiRNA knockdown of c-myc in A549 cells significantly attenuated tumor inhibition effects of 12k. In conclusion, our results demonstrate that 12k has an inhibitory effect on growth of A549 cell by inducing cell cycle arrest and apoptosis. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

High-efficient and high-content cytotoxic recording via dynamic and continuous cell-based impedance biosensor technology.

PubMed

Hu, Ning; Fang, Jiaru; Zou, Ling; Wan, Hao; Pan, Yuxiang; Su, Kaiqi; Zhang, Xi; Wang, Ping

2016-10-01

Cell-based bioassays were effective method to assess the compound toxicity by cell viability, and the traditional label-based methods missed much information of cell growth due to endpoint detection, while the higher throughputs were demanded to obtain dynamic information. Cell-based biosensor methods can dynamically and continuously monitor with cell viability, however, the dynamic information was often ignored or seldom utilized in the toxin and drug assessment. Here, we reported a high-efficient and high-content cytotoxic recording method via dynamic and continuous cell-based impedance biosensor technology. The dynamic cell viability, inhibition ratio and growth rate were derived from the dynamic response curves from the cell-based impedance biosensor. The results showed that the biosensors has the dose-dependent manners to diarrhetic shellfish toxin, okadiac acid based on the analysis of the dynamic cell viability and cell growth status. Moreover, the throughputs of dynamic cytotoxicity were compared between cell-based biosensor methods and label-based endpoint methods. This cell-based impedance biosensor can provide a flexible, cost and label-efficient platform of cell viability assessment in the shellfish toxin screening fields.

Metformin Induces Apoptosis and Cell Cycle Arrest Mediated by Oxidative Stress, AMPK and FOXO3a in MCF-7 Breast Cancer Cells

PubMed Central

Queiroz, Eveline A. I. F.; Puukila, Stephanie; Eichler, Rosangela; Sampaio, Sandra C.; Forsyth, Heidi L.; Lees, Simon J.; Barbosa, Aneli M.; Dekker, Robert F. H.; Fortes, Zuleica B.; Khaper, Neelam

2014-01-01

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration- dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G0-G1 phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRβ, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H2O2 increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role. PMID:24858012

Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

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

Nguyen Ngoc, Tam Dan; Son, Young-Ok; Lim, Shin-Saeng

2012-03-15

Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaFmore » induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.« less

Effect of sodium hypochlorite on human pulp cells: an in vitro study

PubMed Central

Essner, Mark D.; Javed, Amjad; Eleazer, Paul D.

2014-01-01

Background The purpose of this study was to determine the effect of sodium hypochlorite (NaOCl) on human pulp cells to provide an aid in determining its optimum concentration in maintaining the viability of remaining pulp cells in the revascularization of immature permanent teeth with apical periodontitis. Study design Human pulp tissue cells taken from extracted third molars were plated, incubated, and subjected to various concentrations of NaOCl (0.33%, 0.16%, 0.08%, and 0.04%) for 5-, 10-, and 15-minute time intervals to simulate possible contact times in vivo. The Cell Titer–Glo Luminescent Cell Viability Assay was used to determine the number of viable cells present in culture following treatment. Results The results showed an increase in cell viability with the lowering of NaOCl concentration. The use of 0.04% NaOCl was similar to the control, indicating nearly complete preservation of cell viability at all time intervals tested. As sodium hypochlorite concentration increased from 0.04% to 0.33%, cell viability decreased correspondingly. Conclusions The results indicate that the lowest concentration of NaOCl tested did not affect the viability of cells. This may prove beneficial in developing a new treatment protocol to help preserve existing vital pulp cells in revascularization cases. PMID:21821446

Anti-tumor effect of emodin on gynecological cancer cells.

PubMed

Wang, Yaoxian; Yu, Hui; Zhang, Jin; Ge, Xin; Gao, Jing; Zhang, Yunyan; Lou, Ge

2015-10-01

Although an anti-tumor effect of emodin has been reported before, its effect on human gynecological cancer cells has so far not been studied. Here, we assessed the effect of emodin on cervical cancer-derived (Hela), choriocarcinoma-derived (JAR) and ovarian cancer-derived (HO-8910) cells, and investigated the possible underlying molecular and cellular mechanisms. The respective cells were treated with 0, 5, 10 or 15 μM emodin for 72 h. Subsequently, MTT and Transwell in vitro migration assays revealed that emodin significantly decreased the viability and invasive capacity of the gynecological cancer-derived cells tested. We found that emodin induced apoptosis and significantly decreased mitochondrial membrane potential and ATP release in these cells. We also found that emodin may exert its apoptotic effects via regulating the activity of caspase-9 and the expression of cleaved-caspase-3. Moreover, we found that emodin induced a cell cycle arrest at the G0/G1 phase, possibly through down-regulating the key cell cycle regulators Cyclin D and Cyclin E. Interestingly, emodin also led to autophagic cell death, as revealed by increased MAP LC3 expression, a marker of the autophagosome, and decreased expression of the autophagy regulators Beclin-1 and Atg12-Atg5. Finally, we found that the protein levels of both VEGF and VEGFR-2 were significantly decreased in emodin-treated cells, suggesting an anti-angiogenic effect of emodin on gynecological cancer-derived cells. Our results suggest that emodin exhibits an anti-tumor effect on gynecological cancer-derived cells, possibly through multiple mechanisms including the induction of apoptosis and autophagy, the arrest of the cell cycle, and the inhibition of angiogenesis. Our findings may provide a basis for the design of potential emodin-based strategies for the treatment of gynecological tumors.

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.

Are endothelial cell bioeffects from acoustic droplet vaporization proximity dependent?

NASA Astrophysics Data System (ADS)

Seda, Robinson; Li, David; Fowlkes, J. Brian; Bull, Joseph

2013-11-01

Acoustic droplet vaporization (ADV) produces gas microbubbles that provide a means of selective occlusion in gas embolotherapy. Vaporization and subsequent occlusion occur inside blood vessels supplying the targeted tissue, such as tumors. Theoretical and computational studies showed that ADV within a vessel can impart high fluid mechanical stresses on the vessel wall. Previous in vitro studies have demonstrated that vaporization at an endothelial layer may affect cell attachment and viability. The current study is aimed at investigating the role of vaporization distance away from the endothelial layer. HUVECs were cultured in OptiCell™ chambers until reaching confluence. Dodecafluoropentane microdroplets were added, attaining a 10:1 droplet to cell ratio. A single ultrasound pulse (7.5 MHz) consisting of 16 cycles (~ 2 μs) and a 5 MPa peak rarefactional pressure was used to produce ADV while varying the vaporization distance from the endothelial layer (0 μm, 500 μm, 1000 μm). Results indicated that cell attachment and viability was significantly different if the distance was 0 μm (at the endothelial layer). Other distances were not significantly different from the control. ADV will significantly affect the endothelium if droplets are in direct contact with the cells. Droplet concentration and flow conditions inside blood vessels may play an important role. This work was supported by NIH grant R01EB006476.

In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma.

PubMed

Fisher, T; Golan, H; Schiby, G; PriChen, S; Smoum, R; Moshe, I; Peshes-Yaloz, N; Castiel, A; Waldman, D; Gallily, R; Mechoulam, R; Toren, A

2016-03-01

Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ(9)-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl.

In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma

PubMed Central

Fisher, T.; Golan, H.; Schiby, G.; PriChen, S.; Smoum, R.; Moshe, I.; Peshes-Yaloz, N.; Castiel, A.; Waldman, D.; Gallily, R.; Mechoulam, R.; Toren, A.

2016-01-01

Background Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. Methods We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ9-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. Results Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. Conclusions Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl. PMID:27022310

Beta sitosterol and Daucosterol (phytosterols identified in Grewia tiliaefolia) perturbs cell cycle and induces apoptotic cell death in A549 cells.

PubMed

Rajavel, Tamilselvam; Mohankumar, Ramar; Archunan, Govindaraju; Ruckmani, Kandasamy; Devi, Kasi Pandima

2017-06-13

Lung cancer is the leading cause of cancer related deaths both in developed and developing countries. Since majority of the existing therapeutic methods harms both normal and malignant cells, a viable alternative is to switch into safe and beneficial traditional medicinal plants. Hence the present study was framed to identify selective anti-lung cancer agents from the medicinal plant Grewia tiliaefolia (GT). Cell viability experiments showed that benzene extract of GT (BGT) leaf effectively inhibited the growth of A549 cells, while being non-toxic to normal human lung L132 and PBMC cells. Ames and comet assays demonstrated that BGT is of non-mutagenic and non-genotoxic nature in untransformed cells. The hematological and histopathological profiles of the in vivo acute and sub-acute toxicity studies demonstrated that BGT is safe and tolerable. Importantly, western blot analysis and Annexin V-FITC staining confirmed that BGT promotes mitochondrial dependent apoptotic cell death in A549 cells by arresting cell cycle at G2/M phase. Bio-assay guided fractionation revealed the presence of phytosteols (β-sitosterol and daucosterol) which significantly inhibited the growth of A549 cells both alone and in combination. This study warrants that these phytosterols in alone or in combination can be considered as safe and potential drug candidates for lung cancer treatment.

Curcumin enhances the radiosensitivity of renal cancer cells by suppressing NF-κB signaling pathway.

PubMed

Li, Gang; Wang, Ziming; Chong, Tie; Yang, Jie; Li, Hongliang; Chen, Haiwen

2017-10-01

The radiation resistance of renal cell carcinoma (RCC) remains the primary obstacle to improve patient survival. This study aimed to investigate the effects of curcumin on the radiosensitivity of RCC cells. Human RCC cell (ACHN) was exposed to irradiation (IR) and/or curcumin treatment. Cell viability, DNA repair, cell cycle, and apoptosis, were evaluated by MTT, immunofluoresence staining and flow cytometry. Moreover, ACHN cells were xenografted into nude mice and subjected to IR and/or curcumin treatment. The expression of NF-κB signaling related proteins in ACHN cells and xenografts was detected by western blot analysis. The results showed that curcumin significantly increased radiosensitivity of ACHN cells by inhibiting the cell proliferation and DNA damage repair, causing cell cycle arrest at G2/M phase, inducing apoptosis in vitro, and suppressing the growth of xenografts in vivo. In addition, curcumin enhanced radiosensitivity was through markedly inhibiting IR-induced NF-κB signaling by modulating the related protein expressions including NF-κBP65, I-κB, VEGF, COX2, and Bcl-2 in ACHN cells, which was further strengthened by NF-κB inhibitor PDTC treatment. Thus, curcumin may confer radiosensitivity on RCC via inhibition of NF-κB activation and its downstream regulars, suggesting the potential application of curcumin as an adjuvant in radiotherapy of RCC. Copyright © 2017. Published by Elsevier Masson SAS.

The metastatic microenvironment: lung-derived factors control the viability of neuroblastoma lung metastasis.

PubMed

Maman, Shelly; Edry-Botzer, Liat; Sagi-Assif, Orit; Meshel, Tsipi; Yuan, Weirong; Lu, Wuyuan; Witz, Isaac P

2013-11-15

Recent data suggest that the mechanisms determining whether a tumor cell reaching a secondary organ will enter a dormant state, progress toward metastasis, or go through apoptosis are regulated by the microenvironment of the distant organ. In neuroblastoma, 60-70% of children with high-risk disease will ultimately experience relapse due to the presence of micrometastases. The main goal of this study is to evaluate the role of the lung microenvironment in determining the fate of neuroblastoma lung metastases and micrometastases. Utilizing an orthotopic mouse model for human neuroblastoma metastasis, we were able to generate two neuroblastoma cell populations-lung micrometastatic (MicroNB) cells and lung macrometastatic (MacroNB) cells. These two types of cells share the same genetic background, invade the same distant organ, but differ in their ability to create metastasis in the lungs. We hypothesize that factors present in the lung microenvironment inhibit the propagation of MicroNB cells preventing them from forming overt lung metastasis. This study indeed shows that lung-derived factors significantly reduce the viability of MicroNB cells by up regulating the expression of pro-apoptotic genes, inducing cell cycle arrest and decreasing ERK and FAK phosphorylation. Lung-derived factors affected various additional progression-linked cellular characteristics of neuroblastoma cells, such as the expression of stem-cell markers, morphology, and migratory capacity. An insight into the microenvironmental effects governing neuroblastoma recurrence and progression would be of pivotal importance as they could have a therapeutic potential for the treatment of neuroblastoma residual disease. Copyright © 2013 UICC.

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.

Lebein, a snake venom disintegrin, suppresses human colon cancer cells proliferation and tumor-induced angiogenesis through cell cycle arrest, apoptosis induction and inhibition of VEGF expression.

PubMed

Zakraoui, Ons; Marcinkiewicz, Cezary; Aloui, Zohra; Othman, Houcemeddine; Grépin, Renaud; Haoues, Meriam; Essafi, Makram; Srairi-Abid, Najet; Gasmi, Ammar; Karoui, Habib; Pagès, Gilles; Essafi-Benkhadir, Khadija

2017-01-01

Lebein, is an heterodimeric disintegrin isolated from Macrovipera lebetina snake venom that was previously characterized as an inhibitor of ADP-induced platelet aggregation. In this study, we investigated the effect of Lebein on the p53-dependent growth of human colon adenocarcinoma cell lines. We found that Lebein significantly inhibited LS174 (p53wt), HCT116 (p53wt), and HT29 (p53mut) colon cancer cell viability by inducing cell cycle arrest through the modulation of expression levels of the tumor suppression factor p53, cell cycle regulating proteins cyclin D1, CDK2, CDK4, retinoblastoma (Rb), CDK1, and cyclin-dependent kinase inhibitors p21 and p27. Interestingly, Lebein-induced apoptosis of colon cancer cells was dependent on their p53 status. Thus, in LS174 cells, cell death was associated with PARP cleavage and the activation of caspases 3 and 8 while in HCT116 cells, Lebein induced caspase-independent apoptosis through increased expression of apoptosis inducing factor (AIF). In LS174 cells, Lebein triggers the activation of the MAPK ERK1/2 pathway through induction of reactive oxygen species (ROS). It also decreased cell adhesion and migration to fibronectin through down regulation of α5β1 integrin. Moreover, Lebein significantly reduced the expression of two angiogenesis stimulators, Vascular Endothelial Growth Factor (VEGF) and Neuropilin 1 (NRP1). It inhibited the VEGF-induced neovascularization process in the quail embryonic CAM system and blocked the development of human colon adenocarcinoma in nude mice. Overall, our work indicates that Lebein may be useful to design a new therapy against colon cancer. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Curcumin analog EF24 induces apoptosis via ROS-dependent mitochondrial dysfunction in human colorectal cancer cells.

PubMed

He, Guodong; Feng, Chen; Vinothkumar, Rajamanickam; Chen, Weiqian; Dai, Xuanxuan; Chen, Xi; Ye, Qingqing; Qiu, Chenyu; Zhou, Huiping; Wang, Yi; Liang, Guang; Xie, Yubo; Wu, Wei

2016-12-01

Colorectal cancer is the most commonly diagnosed malignancy with high mortality rates worldwide. Improved therapeutic strategies with minimal adverse side effects are urgently needed. In this study, the anti-tumor effects of EF24, a novel analog of the natural compound curcumin, were evaluated in colorectal cancer cells. The anti-tumor activity of EF24 on human colon cancer lines (HCT-116, SW-620, and HT-29) was determined by measures of cell cycle arrest, apoptosis, and mitochondrial function. The contribution of ROS in the EF24-induced anti-tumor activity was evaluated by measures of H 2 O 2 and pretreatment with an ROS scavenger, NAC. The findings indicated that EF24 treatment dose-dependently inhibited cell viability and caused cell cycle arrest at G2/M phase in all the tested colon cancer cell lines. Furthermore, we demonstrated that EF24 treatment induced apoptosis effectively via enhancing intracellular accumulation of ROS in both HCT-116 and SW-620 cells, but with moderate effects in HT-29 cells. We found that EF24 treatment decreased the mitochondrial membrane potential in the colon cancer cells, leading to the release of mitochondrial cytochrome c. Also, EF24 induced activation of caspases 9 and 3, causing decreased Bcl-2 protein expression and Bcl-2/Bax ratio. Pretreatment with NAC, a ROS scavenger, abrogated the EF24-induced cell death, apoptosis, cell cycle arrest, and mitochondrial dysfunction, suggesting an upstream ROS generation which was responsible for the anticancer effects of EF24. Our findings support an anticancer mechanism by which EF24 enhanced ROS accumulation in colon cancer cells, thereby resulting in mitochondrial membrane collapse and activated intrinsic apoptotic signaling. Thus, EF24 could be a potential candidate for therapeutic application of colon cancer.

Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

PubMed

Park, Jina; Jin, Sung Il; Kim, Hyung Min; Ahn, Junhyoung; Kim, Yeon-Gu; Lee, Eun Gyo; Kim, Min-Gon; Shin, Yong-Beom

2015-02-15

We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers. Copyright © 2014 Elsevier B.V. All rights reserved.

Recombinant HE4 protein promotes proliferation of pancreatic and endometrial cancer cell lines.

PubMed

Lu, Qinsheng; Chen, Haibin; Senkowski, Christopher; Wang, Jianhao; Wang, Xue; Brower, Steven; Glasgow, Wayne; Byck, David; Jiang, Shi-Wen; Li, Jinping

2016-01-01

Pancreatic adenocarcinoma is one of the most deadly malignancies, and endometrial cancer represents the most common gynecologic cancer in the USA. Better understanding on the pathologic mechanisms and pathways is required for effective treatment of these malignancies. Recently, human epididymis protein 4 (HE4 or WFDC2), a secretory glycoprotein, was found to be overexpressed in pancreatic and endometrial cancers. In addition, studies have shown that HE4 overexpression in endometrial cancer cell lines led to faster cancer progression in a mouse subcutaneous model. These findings raise a question on the role(s) of secretory, extracellular HE4 in cancer development. In the present study, we found that treatment of pancreatic and endometrial cancer cell lines with purified, extracellular HE4 protein led to a significant increase in cell viability and proliferation. Moreover, extracellular HE4 protein was able to increase DNA synthesis, and modulate the mRNA and protein levels of cell cycle marker PCNA and cell cycle inhibitor p21. These effects appeared to be robust and sustainable and required a relatively low concentration of HE4 protein. The findings indicated the secreted, extracellular HE4 may carry some physiopathological functions. Via paracrine/endocrine actions, circulatory HE4 produced by malignant cells may contribute to pancreatic and endometrial cancer progression and/or metastasis.

Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing

PubMed Central

Krzyzanek, Vladislav; Mravec, Filip; Hrubanova, Kamila; Samek, Ota; Kucera, Dan; Benesova, Pavla; Marova, Ivana

2016-01-01

Accumulation of polyhydroxybutyrate (PHB) seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase), yeast (Saccharomyces cerevisiae) and bacterial cells (Cupriavidus necator) against the adverse effects of freezing-thawing cycles. Further, the viability of the frozen–thawed PHB accumulating strain of C. necator was compared to that of the PHB non-accumulating mutant. The presence of PHB granules in cells was revealed to be a significant advantage during freezing. This might be attributed to the higher intracellular level of 3-hydroxybutyrate in PHB accumulating cells (due to the action of parallel PHB synthesis and degradation, the so-called PHB cycle), but the cryoprotective effect of PHB granules seems to be more complex. Since intracellular PHB granules retain highly flexible properties even at extremely low temperatures (observed by cryo-SEM), it can be expected that PHB granules protect cells against injury from extracellular ice. Finally, thermal analysis indicates that PHB-containing cells exhibit a higher rate of transmembrane water transport, which protects cells against the formation of intracellular ice which usually has fatal consequences. PMID:27315285

Targeting survivin as a potential new treatment for chondrosarcoma of bone

PubMed Central

de Jong, Y; van Oosterwijk, J G; Kruisselbrink, A B; Briaire-de Bruijn, I H; Agrogiannis, G; Baranski, Z; Cleven, A H G; Cleton-Jansen, A-M; van de Water, B; Danen, E H J; Bovée, J V M G

2016-01-01

Chondrosarcomas are malignant cartilage-forming bone tumors, which are intrinsically resistant to chemo- and radiotherapy, leaving surgical removal as the only curative treatment option. Therefore, our aim was to identify genes involved in chondrosarcoma cell survival that could serve as a target for therapy. siRNA screening for 51 apoptosis-related genes in JJ012 chondrosarcoma cells identified BIRC5, encoding survivin, as essential for chondrosarcoma survival. Using immunohistochemistry, nuclear as well as cytoplasmic survivin expression was analyzed in 207 chondrosarcomas of different subtypes. Nuclear survivin has been implicated in cell-cycle regulation while cytoplasmic localization is important for its anti-apoptotic function. RT–PCR was performed to determine expression of the most common survivin isoforms. Sensitivity to YM155, a survivin inhibitor currently in phase I/II clinical trial for other tumors, was examined in 10 chondrosarcoma cell lines using viability assay, apoptosis assay and cell-cycle analysis. Survivin expression was found in all chondrosarcoma patient samples. Higher expression of nuclear and cytoplasmic survivin was observed with increasing histological grade in central chondrosarcomas. Inhibition of survivin using YM155 showed that especially TP53 mutant cell lines were sensitive, but no caspase 3/7 or PARP cleavage was observed. Rather, YM155 treatment resulted in a block in S phase in two out of three chondrosarcoma cell lines, indicating that survivin is more involved in cell-cycle regulation than in apoptosis. Thus, survivin is important for chondrosarcoma survival and chondrosarcoma patients might benefit from survivin inhibition using YM155, for which TP53 mutational status can serve as a predictive biomarker. PMID:27159675

Lunasin, with an arginine-glycine-aspartic acid motif, causes apoptosis to L1210 leukemia cells by activation of caspase-3.

PubMed

de Mejia, Elvira Gonzalez; Wang, Wenyi; Dia, Vermont P

2010-03-01

Lunasin is a novel chemopreventive peptide featuring a cell adhesion motif composed of arginine-glycine-aspartate (RGD) which has been associated to cytotoxicity to established cell lines. The objectives of this study were to determine the effect of lunasin on the viability of L1210 leukemia cells and to understand the underlying mechanisms involved. Pure lunasin and lunasin enriched soy flour (LES) caused cytotoxicity to L1210 leukemia cells with IC(50) of 14 and 16 microM (lunasin equivalent), respectively. Simulated gastrointestinal digestion showed that 25% of the original amount of lunasin survived 3 h of pepsin digestion and 3% of lunasin remained after sequential pepsin-pancreatin digestion for a total of 6 h. Cell cycle analysis showed that lunasin caused a dose-dependent G2 cell cycle arrest and apoptosis. Treatment of L1210 leukemia cells with 1 mg/mL of LES for 18 h led to an increase in the amount of apoptotic cells from 2 to 40%. Compared to untreated cells, treatment with 1 mg/mL LES showed a 6-fold increase on the expressions of caspases-8 and -9, and and a 12-fold increase on the expression of caspase-3. These results showed for the first time that lunasin, a naturally occurring peptide containing an RGD motif, caused apoptosis to L1210 leukemia cells through caspase-3 activation.

Decursin inhibits growth of human bladder and colon cancer cells via apoptosis, G1-phase cell cycle arrest and extracellular signal-regulated kinase activation.

PubMed

Kim, Wun-Jae; Lee, Se-Jung; Choi, Young Deuk; Moon, Sung-Kwon

2010-04-01

Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, has demonstrated anti-cancer properties. In the present study, we found that decursin inhibited cell viability in cultured human urinary bladder cancer 235J cells and colon cancer HCT116 cells. The inhibited proliferation was due to apoptotic induction, because both cells treated with decursin dose-dependently showed a sub-G1 phase accumulation and an increased cytoplasmic DNA-histone complex. Cell death caused by decursin was also associated with the down-regulation of anti-apoptotic factor Bcl-2 and the up-regulation of pro-apoptotic molecules cytochrome c, caspase 3 and Bax. Treatment of both types of cancer cells with decursin resulted in G1-phase cell cycle arrest, as revealed by FACS analyses. In addition, decursin increased protein levels of p21WAF1 with a decrease in cyclins and cyclin dependent kinases (CDKs). Furthermore, decursin induced the activation of extracellular signal-regulated kinases (ERK) in both cancer cell lines, with the notable exceptions of c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase. Finally, pretreatment with ERK-specific inhibitor PD98059 reversed decursin-induced p21WAF1 expression and decursin-inhibited cell growth. Thus, these findings suggest that decursin has potential therapeutic efficacy for the treatment of bladder and colon cancer.

C1 Domain-Targeted Isophthalate Derivatives Induce Cell Elongation and Cell Cycle Arrest in HeLa Cells

PubMed Central

Talman, Virpi; Tuominen, Raimo K.; Gennäs, Gustav Boije af; Yli-Kauhaluoma, Jari; Ekokoski, Elina

2011-01-01

Diacylglycerol (DAG)-mediated signaling pathways, such as those mediated by protein kinase C (PKC), are central in regulating cell proliferation and apoptosis. DAG-responsive C1 domains are therefore considered attractive drug targets. Our group has designed a novel class of compounds targeted to the DAG binding site within the C1 domain of PKC. We have previously shown that these 5-(hydroxymethyl)isophthalates modulate PKC activation in living cells. In this study we investigated their effects on HeLa human cervical cancer cell viability and proliferation by using standard cytotoxicity tests and an automated imaging platform with machine vision technology. Cellular effects and their mechanisms were further characterized with the most potent compound, HMI-1a3. Isophthalate derivatives with high affinity to the PKC C1 domain exhibited antiproliferative and non-necrotic cytotoxic effects on HeLa cells. The anti-proliferative effect was irreversible and accompanied by cell elongation. HMI-1a3 induced down-regulation of retinoblastoma protein and cyclins A, B1, D1, and E. Effects of isophthalates on cell morphology, cell proliferation and expression of cell cycle-related proteins were different from those induced by phorbol 12-myristate-13-acetate (PMA) or bryostatin 1, but correlated closely to binding affinities. Therefore, the results strongly indicate that the effect is C1 domain-mediated. PMID:21629792

Can human mesenchymal stem cells survive on a NiTi implant material subjected to cyclic loading?

PubMed

Habijan, T; Glogowski, T; Kühn, S; Pohl, M; Wittsiepe, J; Greulich, C; Eggeler, G; Schildhauer, T A; Köller, M

2011-06-01

Nickel-titanium shape memory alloys (NiTi-SMAs) exhibit mechanical and chemical properties which make them attractive candidate materials for various types of biomedical applications. However, the high nickel content of NiTi-SMAs may result in adverse tissue reactions, especially when they are considered for load-bearing implants. It is generally assumed that a protective titanium oxide layer separates the metallic alloy from its environment and that this explains the good biocompatibility of NiTi. Cyclic loading may result in failure of the protective oxide layer. The scientific objective of this work was to find out whether cyclic dynamic strain, in a range relevant for orthopedic implants, diminishes the biocompatibility of NiTi-SMAs. In order to analyze the biocompatibility of NiTi-SMA surfaces subjected to cyclic loading, NiTi-SMA tensile specimens were preloaded with mesenchymal stem cells, transferred to a sterile cell culture system and fixed to the pull rods of a tensile testing machine. Eighty-six thousand and four hundred strain cycles at 2% pseudoelastic strain were performed for a period of 24 h or 7 days. Cytokines (IL-6, IL-8 and VEGF) and nickel ion release were determined within the cell culture medium. Adherent cells on the tensile specimens were stained with calcein-AM and propidium iodide to determine cell viability. Dynamic loading of the tensile specimens did not influence the viability of adherent human mesenchymal stem cells (hMSCs) after 24 h or 7 days compared with the non-strained control. Dynamic cycles of loading and unloading did not affect nickel ion release from the tensile specimens. The release of IL-6 from hMSCs cultured under dynamic conditions was significantly higher after mechanical load (873 pg ml(-1)) compared with static conditions (323 pg ml(-1)). The present work demonstrates that a new type of mechanical in vitro cell culture experiment can provide information which previously could only be obtained in large animal experiments. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

PubMed

Sediq, A S; Klem, R; Nejadnik, M R; Meij, P; Jiskoot, Wim

2018-05-30

To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells.

EBV latent membrane protein 1 activates Akt, NFkappaB, and Stat3 in B cell lymphomas.

PubMed

Shair, Kathy H Y; Bendt, Katherine M; Edwards, Rachel H; Bedford, Elisabeth C; Nielsen, Judith N; Raab-Traub, Nancy

2007-11-01

Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFkappaB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFkappaB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.

The effect of simultaneous exposure of HEMn-DP and HEMn-LP melanocytes to nicotine and UV-radiation on the cell viability and melanogenesis

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

Delijewski, Marcin; Wrześniok, Dorota; Beberok, Ar

Nicotine is a main compound of tobacco plants and may affect more than a billion people all over the world that are permanently exposed to nicotine from cigarettes, various forms of smoking cessation therapies, electronic cigarettes or second-hand smoke. It is known that nicotine forms complexes with melanin what may lead to accumulation of this alkaloid in tissues of living organisms containing the pigment. This may affect the viability of cells and process of melanin biosynthesis that takes place in melanocytes. Although UV radiation is known to be a particular inductor of melanin biosynthesis, its simultaneous effect with nicotine onmore » this process as well as the viability of human cells containing melanin have not been assessed so far. The aim of this study was to examine the simultaneous impact of nicotine and UV radiation on viability and melanogenesis in cultured normal human melanocytes dark (HEMn-DP) and light (HEMn-LP) pigmented. Nicotine together with UV radiation induced concentration-dependent loss in melanocytes viability. The higher cell loss was observed in dark pigmented melanocytes in comparison to light pigmented cells. Simultaneous exposure of cells to nicotine and UV radiation also caused changes in melanization process in both tested cell lines. The data suggest that simultaneous exposure of melanocytes to nicotine and UV radiation up-regulates melanogenesis and affects cell viability. Observed processes are more pronounced in dark pigmented cells. - Highlights: • Nicotine and UVA induced concentration-dependent loss in melanocytes viability. • Nicotine and UVA modulated melanization process in melanocytes. • Changes in viability and melanization were more pronounced in dark pigmented cells.« less

Anethum graveolens (dill) - A medicinal herb induces apoptosis and cell cycle arrest in HepG2 cell line.

PubMed

Mohammed, Furkhan Ahmed; Elkady, Ayman I; Syed, Fareeduddin Quadri; Mirza, Muqtadir Baig; Hakeem, Khalid Rehman; Alkarim, Saleh

2018-06-12

The medicinal herb, Anethum graveolens L. (dill) is one of the potent culinary herbs used as an alternative form of medicine worldwide. The unguent topical Oil from the aerial parts of A. graveolens was found to be effective in the management of uterus cancer in ethnomedicine has been reported. The incidence and mortality rates of Hepatocellular carcinoma (HCC) are steadily rising worldwide, especially, in underdeveloped and developing countries. Moreover, HCC develops rapidly in patients with chronic cirrhosis or hepatitis, where the solid tumours/malignancies coexist with the inflammation. Recent studies have shown that the medicinal herb, Anethum graveolens, holds anticancer potential, which could be a promising approach for the treatment of various tumours. In the current study, we have analysed the antiproliferative effect of ethyl acetate fraction of Dill Seeds (EAFD) on HepG2 cell line. Cell viability and proliferation were observed by MTT assay; Morphological changes were studied using fluorescent stains like Hoechst 33342, acridine orange/ethidium bromide and JC-1 dye. Further, the pro-apoptotic activity was demonstrated through Annexin-V-FITC/ PI assay and cell cycle analysis. Different concentrations (0.1, 0.2, 0.4, 0.6, 0.8 mg/ml) of EAFD were studied. EAFD markedly suppressed the proliferation of HepG2 cells in a dose and time-dependent manner. The phase contrast and fluorescence microscopy revealed the morphological alterations like disruption, shrinkage, detachment and blebbing of cell membrane accompanied by nuclear condensation after exposure to EAFD. Radical scavenging activity was evidenced by measurement of ROS levels post-treatment. Modulation of mitochondrial membrane potential was exhibited leading to the activation of caspases 3/7 and 9 which is a committed step towards apoptosis. Annexin V-FITC/ PI assay and cell cycle, later confirmed the apoptosis and cell cycle arrest in 'G2/M' phase through flow cytometric analysis. In conclusion, a significant apoptogenic effect was exhibited by EAFD against HepG2 cells in inducing apoptosis and cell cycle arrest. Our findings indicate that the medicinal herb- Anethum graveolens, holds potential in treating hepatocellular carcinoma effectively. Copyright © 2018 Elsevier B.V. All rights reserved.

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

PubMed

Wigg, Alan J; Phillips, John W; Wheatland, Loretta; Berry, Michael N

2003-06-01

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

Artemisia leaf extract induces apoptosis in human endometriotic cells through regulation of the p38 and NFκB pathways.

PubMed

Kim, Ji-Hyun; Jung, Seung-Hyun; Yang, Yeong-In; Ahn, Ji-Hye; Cho, Jin-Gyeong; Lee, Kyung-Tae; Baek, Nam-In; Choi, Jung-Hye

2013-02-13

Artemisia leaves have long been used for the treatment of gynecological disorders, including infertility and dysmenorrhea, which can be commonly caused by endometriosis. In the present study, we investigated the effect of Artemisia princeps extract (APE) on the cell growth and apoptosis of human endometriotic cells. MTT assays and FACS analysis using PI and Annexin staining were performed to study cell viability, cell cycle progression, and apoptosis. We also explored the mechanism of APE-induced effects by evaluating the activation of caspases, Akt, p38, and NFκB. The expressions of XIAP, Bcl-2, and Bcl-xL were measured by real-time RT-PCR and Western blot analyses. APE significantly inhibited the cell viability of 11Z and 12Z human endometriotic epithelial cells. Interestingly, endometriotic cells were more sensitive to APE treatment than immortalized endometrial cells (HES). Treatment with APE induced apoptosis of 11Z cells in a time-dependent manner, as shown by accumulation of sub G1 and apoptotic cell populations. In addition, treatment with APE stimulated the activation of caspase -3, -8, and -9 in a dose- and time-dependent manner. Furthermore, p38 was activated by APE treatment, and the p38 inhibitor SB203580 markedly inhibited APE-induced cell death in 11Z cells. Moreover, treatment with APE suppressed the activation of NFκB and the expressions of anti-apoptotic factors such as XIAP, Bcl-2, and Bcl-xL. These results indicate that APE is a potential anti-endometriotic agent, acting to induce apoptosis of endometrial cells through the modulation of the p38 and NFκB pathways. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Gallium, a promising candidate to disrupt the vicious cycle driving osteolytic metastases.

PubMed

Strazic-Geljic, Ivana; Guberovic, Iva; Didak, Blanka; Schmid-Antomarchi, Heidy; Schmid-Alliana, Annie; Boukhechba, Florian; Bouler, Jean-Michel; Scimeca, Jean-Claude; Verron, Elise

2016-09-15

Bone metastases of breast cancer typically lead to a severe osteolysis due to an excessive osteoclastic activity. On the other hand, the semi-metallic element gallium (Ga) displays an inhibitory action on osteoclasts, and therefore on bone resorption, as well as antitumour properties. Thus, we explored in vitro Ga effects on osteoclastogenesis in an aggressive bone metastatic environment based on the culture of pre-osteoclast RAW 264.7 cells with conditioned medium from metastatic breast tumour cells, i.e. the breast tumour cell line model MDA-MB-231 and its bone-seeking clone MDA-231BO. We first observed that Ga dose-dependently inhibited the tumour cells-induced osteoclastic differentiation of RAW 264.7 cells. To mimic a more aggressive environment where pro-tumourigenic factors are released from bone matrix due to osteoclastic resorption, metastatic breast tumour cells were stimulated with TGF-β, a mayor cytokine in bone metastasis vicious cycle. In these conditions, we observed that Ga still inhibited cancer cells-driven osteoclastogenesis. Lastly, we evidenced that Ga affected directly and strongly the proliferation/viability of both cancer cell lines, as well as the expression of major osteolytic factors in MDA-231BO cells. With the exception of two small scale clinical studies from 1980s, this is the first time that antitumour properties of Ga have been specifically studied in the context of bone metastases. Our data strongly suggest that, through its action against the vicious cycle involving bone cells and tumour cells, Ga represents a relevant and promising candidate for the local treatment of bone metastases in patients with breast cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Coconut milk and probiotic milk as storage media to maintain periodontal ligament cell viability: an in vitro study.

PubMed

Saini, Divya; Gadicherla, Prahlad; Chandra, Prakash; Anandakrishna, Latha

2017-06-01

The viability of periodontal ligament (PDL) cells is a significant determinant of the long-term prognosis of replanted avulsed teeth. A storage medium is often required to maintain the viability of these cells during the extra-alveolar period. Many studies have been carried out to search for the most suitable storage medium for avulsed teeth, but an ideal solution has not yet been found. The purpose of the study was to compare and analyze the ability of coconut milk and probiotic milk to maintain PDL cell viability. In an in vitro setting, 69 caries free human premolars with normal periodontium that had been extracted for orthodontic purposes were randomly divided into two experimental groups on the basis of storage media used (i.e., coconut milk or probiotic milk) and a Hanks' balanced salt solution (HBSS) control group (23 samples per group). Immediately after extraction, the teeth were stored dry for 20 min and then immersed for 30 min in one of the storage media. The teeth were then subjected to collagenase-dispase assay and labeled with 0.5% trypan blue staining solution for determination of cell viability. The number of viable cells was counted under a light microscope and statistically analyzed using anova and post hoc Tukey test (P ≤ 0.05). Statistical analysis demonstrated there was a significant difference (P < 0.001) between coconut milk and probiotic milk as well as HBSS in maintaining cell viability. However, there was no significant difference between probiotic milk and HBSS in ability to maintain PDL cell viability (P > 0.05). Coconut milk may not be suitable as an interim transport media due to poor maintenance of cell viability. However, probiotic milk was able to maintain PDL cell viability as well as HBSS. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions.

PubMed

Branska, Barbora; Pechacova, Zora; Kolek, Jan; Vasylkivska, Maryna; Patakova, Petra

2018-01-01

Biobutanol production by clostridia via the acetone-butanol-ethanol (ABE) pathway is a promising future technology in bioenergetics , but identifying key regulatory mechanisms for this pathway is essential in order to construct industrially relevant strains with high tolerance and productivity. We have applied flow cytometric analysis to C. beijerinckii NRRL B-598 and carried out comparative screening of physiological changes in terms of viability under different cultivation conditions to determine its dependence on particular stages of the life cycle and the concentration of butanol. Dual staining by propidium iodide (PI) and carboxyfluorescein diacetate (CFDA) provided separation of cells into four subpopulations with different abilities to take up PI and cleave CFDA, reflecting different physiological states. The development of a staining pattern during ABE fermentation showed an apparent decline in viability, starting at the pH shift and onset of solventogenesis, although an appreciable proportion of cells continued to proliferate. This was observed for sporulating as well as non-sporulating phenotypes at low solvent concentrations, suggesting that the increase in percentage of inactive cells was not a result of solvent toxicity or a transition from vegetative to sporulating stages. Additionally, the sporulating phenotype was challenged with butanol and cultivation with a lower starting pH was performed; in both these experiments similar trends were obtained-viability declined after the pH breakpoint, independent of the actual butanol concentration in the medium. Production characteristics of both sporulating and non-sporulating phenotypes were comparable, showing that in C. beijerinckii NRRL B-598, solventogenesis was not conditional on sporulation. We have shown that the decline in C. beijerinckii NRRL B-598 culture viability during ABE fermentation was not only the result of accumulated toxic metabolites, but might also be associated with a special survival strategy triggered by pH change.

Viability of human fibroblasts in coconut water as a storage medium.

PubMed

Moreira-Neto, J J S; Gondim, J O; Raddi, M S G; Pansani, C A

2009-09-01

To evaluate the effectiveness of a new storage medium for avulsed teeth, coconut water, in maintaining the viability of human fibroblasts. Cell viability after different time periods was evaluated in the following storage media: coconut water, coconut water with sodium bicarbonate, milk, saline and still mineral water. Human fibroblasts were seeded in Eagle's minimal essential medium (EMEM) supplemented with 7.5% foetal calf serum. After trypsinisation, 100 microL of culture medium containing approximately 10(4) cells mL(-1) were collected and pipetted into the wells of 96-well plates, which were incubated overnight in 5% CO(2) and 95% air mixture at 37 degrees C. EMEM was then replaced by the storage media and the plates were incubated at 37 degrees C for 1, 2 and 4 h. Cell viability was determined using the neutral red assay. The proportions of viable cells after exposure to the storage media were analysed statistically by anova and the least significant difference (LSD) test (alpha = 5%). Milk had the greatest capacity to maintain cell viability (P < 0.05), followed by coconut water with sodium bicarbonate and saline. Coconut water was significantly worse at maintaining cell viability compared to milk, coconut water with sodium bicarbonate and saline. The smallest number of viable cells was observed for mineral water (P < 0.05). Coconut water was worse than milk in maintaining human fibroblast cell viability.

Impaired Angiogenic Potential of Human Placental Mesenchymal Stromal Cells in Intrauterine Growth Restriction.

PubMed

Mandò, Chiara; Razini, Paola; Novielli, Chiara; Anelli, Gaia Maria; Belicchi, Marzia; Erratico, Silvia; Banfi, Stefania; Meregalli, Mirella; Tavelli, Alessandro; Baccarin, Marco; Rolfo, Alessandro; Motta, Silvia; Torrente, Yvan; Cetin, Irene

2016-04-01

Human placental mesenchymal stromal cells (pMSCs) have never been investigated in intrauterine growth restriction (IUGR). We characterized cells isolated from placental membranes and the basal disc of six IUGR and five physiological placentas. Cell viability and proliferation were assessed every 7 days during a 6-week culture. Expression of hematopoietic, stem, endothelial, and mesenchymal markers was evaluated by flow cytometry. We characterized the multipotency of pMSCs and the expression of genes involved in mitochondrial content and function. Cell viability was high in all samples, and proliferation rate was lower in IUGR compared with control cells. All samples presented a starting heterogeneous population, shifting during culture toward homogeneity for mesenchymal markers and occurring earlier in IUGR than in controls. In vitro multipotency of IUGR-derived pMSCs was restricted because their capacity for adipocyte differentiation was increased, whereas their ability to differentiate toward endothelial cell lineage was decreased. Mitochondrial content and function were higher in IUGR pMSCs than controls, possibly indicating a shift from anaerobic to aerobic metabolism, with the loss of the metabolic characteristics that are typical of undifferentiated multipotent cells. This study demonstrates that the loss of endothelial differentiation potential and the increase of adipogenic ability are likely to play a significant role in the vicious cycle of abnormal placental development in intrauterine growth restriction (IUGR). This is the first observation of a potential role for placental mesenchymal stromal cells in intrauterine growth restriction, thus leading to new perspectives for the treatment of IUGR. ©AlphaMed Press.

Impaired Angiogenic Potential of Human Placental Mesenchymal Stromal Cells in Intrauterine Growth Restriction

PubMed Central

Mandò, Chiara; Razini, Paola; Novielli, Chiara; Anelli, Gaia Maria; Belicchi, Marzia; Erratico, Silvia; Banfi, Stefania; Meregalli, Mirella; Tavelli, Alessandro; Baccarin, Marco; Rolfo, Alessandro; Motta, Silvia

2016-01-01

Human placental mesenchymal stromal cells (pMSCs) have never been investigated in intrauterine growth restriction (IUGR). We characterized cells isolated from placental membranes and the basal disc of six IUGR and five physiological placentas. Cell viability and proliferation were assessed every 7 days during a 6-week culture. Expression of hematopoietic, stem, endothelial, and mesenchymal markers was evaluated by flow cytometry. We characterized the multipotency of pMSCs and the expression of genes involved in mitochondrial content and function. Cell viability was high in all samples, and proliferation rate was lower in IUGR compared with control cells. All samples presented a starting heterogeneous population, shifting during culture toward homogeneity for mesenchymal markers and occurring earlier in IUGR than in controls. In vitro multipotency of IUGR-derived pMSCs was restricted because their capacity for adipocyte differentiation was increased, whereas their ability to differentiate toward endothelial cell lineage was decreased. Mitochondrial content and function were higher in IUGR pMSCs than controls, possibly indicating a shift from anaerobic to aerobic metabolism, with the loss of the metabolic characteristics that are typical of undifferentiated multipotent cells. Significance This study demonstrates that the loss of endothelial differentiation potential and the increase of adipogenic ability are likely to play a significant role in the vicious cycle of abnormal placental development in intrauterine growth restriction (IUGR). This is the first observation of a potential role for placental mesenchymal stromal cells in intrauterine growth restriction, thus leading to new perspectives for the treatment of IUGR. PMID:26956210

Hyperosmolarity potentiates toxic effects of benzalkonium chloride on conjunctival epithelial cells in vitro

PubMed Central

Godefroy, David; Riancho, Luisa; Rostène, William; Baudouin, Christophe; Brignole-Baudouin, Françoise

2012-01-01

Purpose Benzalkonium chloride (BAK), the most commonly used preservative in eye drops, is known to induce ocular irritation symptoms and dry eye in long-term treated patients and animal models. As tear film hyperosmolarity is diagnostic of some types of dry eye disease, we determined in vitro on conjunctival epithelial cells the cytoxicity of BAK in hyperosmolar conditions through cell viability, apoptosis, and oxidative stress assays. Methods The Wong Kilbourne derivative of Chang conjunctival epithelial cells were cultured for 24 h or 48 h either in NaCl-induced hyperosmolar conditions (400–425–500 mOsM), in low concentrations of BAK (10−4%, 3.10−4%, and 5.10−4%), or in combination of both. We investigated cell viability through lysosomal integrity evaluation, cell death (cell membrane permeability and chromatin condensation), and oxidative stress (reactive oxygen species, superoxide anion) using spectrofluorimetry. Immunohistochemistry was performed for cytoskeleton shrinkage (phalloidin staining), mitochondrial permeability transition pore (cytochrome c release), the apoptosis effector active caspase-3, and the caspase-independent apoptosis factor AIF. We also observed early effects induced by the experimental conditions on the conjunctival cell layers using phase contrast imaging of live cells. Results As compared to standard culture solutions, hyperosmolar stress potentiated BAK cytotoxicity on conjunctival cells through the induction of oxidative stress; reduction of cell viability; cell membrane permeability increase; cell shrinkage with cell blebbing, as shown in phase contrast imaging of live cells; and chromatin condensation. Like BAK, but to a much lesser extent, hyperosmolarity increased cell death in a concentration-dependent manner through a caspase-dependent apoptosis characterized by a release of cytochrome c in the cytoplasm from mitochondria and the activation of caspase-3. Moreover, the caspase-independent apoptosis factor AIF was found translocated from mitochondria to the nucleus in both conditions. Conclusions This study showed increased cytotoxic effects of BAK in hyperosmotic conditions, with characteristic cell death processes, namely caspase-dependent and independent apoptosis and oxidative stress. As BAK is known to disrupt tear film, which could promote evaporative dry eye and tear hyperosmolarity, BAK could promote the conditions enhancing its own cytotoxicity. This in vitro hyperosmolarity model thus highlights the risk of inducing a vicious cycle and the importance of avoiding BAK in patients with dry eye conditions. PMID:22529703

Hyperosmolarity potentiates toxic effects of benzalkonium chloride on conjunctival epithelial cells in vitro.

PubMed

Clouzeau, Chloé; Godefroy, David; Riancho, Luisa; Rostène, William; Baudouin, Christophe; Brignole-Baudouin, Françoise

2012-01-01

Benzalkonium chloride (BAK), the most commonly used preservative in eye drops, is known to induce ocular irritation symptoms and dry eye in long-term treated patients and animal models. As tear film hyperosmolarity is diagnostic of some types of dry eye disease, we determined in vitro on conjunctival epithelial cells the cytoxicity of BAK in hyperosmolar conditions through cell viability, apoptosis, and oxidative stress assays. The Wong Kilbourne derivative of Chang conjunctival epithelial cells were cultured for 24 h or 48 h either in NaCl-induced hyperosmolar conditions (400-425-500 mOsM), in low concentrations of BAK (10(-4)%, 3.10(-4)%, and 5.10(-4)%), or in combination of both. We investigated cell viability through lysosomal integrity evaluation, cell death (cell membrane permeability and chromatin condensation), and oxidative stress (reactive oxygen species, superoxide anion) using spectrofluorimetry. Immunohistochemistry was performed for cytoskeleton shrinkage (phalloidin staining), mitochondrial permeability transition pore (cytochrome c release), the apoptosis effector active caspase-3, and the caspase-independent apoptosis factor AIF. We also observed early effects induced by the experimental conditions on the conjunctival cell layers using phase contrast imaging of live cells. As compared to standard culture solutions, hyperosmolar stress potentiated BAK cytotoxicity on conjunctival cells through the induction of oxidative stress; reduction of cell viability; cell membrane permeability increase; cell shrinkage with cell blebbing, as shown in phase contrast imaging of live cells; and chromatin condensation. Like BAK, but to a much lesser extent, hyperosmolarity increased cell death in a concentration-dependent manner through a caspase-dependent apoptosis characterized by a release of cytochrome c in the cytoplasm from mitochondria and the activation of caspase-3. Moreover, the caspase-independent apoptosis factor AIF was found translocated from mitochondria to the nucleus in both conditions. This study showed increased cytotoxic effects of BAK in hyperosmotic conditions, with characteristic cell death processes, namely caspase-dependent and independent apoptosis and oxidative stress. As BAK is known to disrupt tear film, which could promote evaporative dry eye and tear hyperosmolarity, BAK could promote the conditions enhancing its own cytotoxicity. This in vitro hyperosmolarity model thus highlights the risk of inducing a vicious cycle and the importance of avoiding BAK in patients with dry eye conditions.

Survival of microbial isolates from clouds toward simulated atmospheric stress factors

NASA Astrophysics Data System (ADS)

Joly, Muriel; Amato, Pierre; Sancelme, Martine; Vinatier, Virginie; Abrantes, Magali; Deguillaume, Laurent; Delort, Anne-Marie

2015-09-01

In the atmosphere, airborne microbial cells are exposed to conditions that are thought to affect their survival. Here, we investigated the survival of 5 microorganisms among the most represented in the cultivable community of clouds (4 bacteria affiliated to Pseudomonas, Sphingomonas and Arthrobacter and 1 yeast of Dioszegia) after exposition to different atmospheric factors generally considered stressful for cells: artificial solar light (10 h), oxidant (hydrogen peroxide: 0-1 mM for 90 min), osmotic shocks (0.1-2.5 M NaCl) and freeze-thaw cycles (6 cycles of 5 °C/-40 °C). Each condition was applied separately to cell suspensions, and survival rates were examined by culture. Survival was highly strain and stress dependent, with no relationship with pigmentation or ice nucleation activity. In all strains, solar light had no or mitigated influence, and exposition to H2O2 at the concentration measured in cloud water only slightly impacted viability (>70% of the cells survived). The strain Sphingomonas sp. was particularly impacted by osmotic shocks while repeated freeze-thaw was particularly damaging for Arthrobacter and Pseudomonas species. Overall, our results tend to indicate that in the atmosphere, the most stringent selection factors on living organisms are probably freeze-thaw and condensation/evaporation (osmotic shocks) cycles, whereas the impacts of oxidants and of solar light are limited.

Cytotoxic effect of achatinin(H) (lectin) from Achatina fulica against a human mammary carcinoma cell line (MCF7).

PubMed

Dharmu, Indra; Ramamurty, N; Kannan, Ramalingam; Babu, Mary

2007-01-01

The hemolymph-derived achatinin(H) (lectin) from Achatina fulica showed a marked cytotoxic effect on MCF7, a human mammary carcinoma cell line. IC(50) values as measured by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay for achatinin(H) ranged from 6 to 10 microg/ml in the MCF7 cells. MCF7 cells showed significant morphological changes leading to cell death. The above cell death was observed after 48 h of treatment with 8 microg/ml when compared to untreated cells. Alterations in the tumor marker enzymes, as well as in antioxidant enzymes, were observed after achatinin(H) treatment. The specificity and purity of the achatinin(H) was confirmed by the Western blot assay. Achatinin(H) binding to MCF7 cells was detected by anti-achatinin(H), and visualization of the achatinin(H) binding sites on confluent MCF7 cells was confirmed by flourescein isothiocyanate conjugated secondary antibody. MCF7-treated cells fluoresced, indicating the presence of achatinin(H) binding sites. Fluorescence-activated cell sorting analysis of the cell cycle showed a significant increase in S-phase in MCF7 cells after 48 h of achatinin(H) treatment. The cells were arrested in G(2)/M phase of the cell cycle after 48 h with significant changes in cell viability. Cellular damage was confirmed by agarose gel electrophoresis with the characteristic appearance of a DNA streak in treated MCF7 cells indicating the ongoing apoptosis.

Anti-mullerian hormone is expressed by endometriosis tissues and induces cell cycle arrest and apoptosis in endometriosis cells

PubMed Central

2014-01-01

Background The anti-mullerian hormone (AMH) is a member of the transforming growth factor β (TGF-β) superfamily, which is responsible of the regression of the mullerian duct. AMH is expressed in the normal endometrium, where, acting in a paracrine fashion, negatively regulates cellular viability. Our objective was to evaluate the in vitro effects of the treatment with AMH of endometriosic cells. Methods AMH expression in human endometriosis glands was evaluated by immunohistochemistry. RT-PCR has been used to quantify the expression levels of AMH and AMH RII isoforms, as well as of cytochrome P450 in both endometriosis epithelial and stromal cells Effects of AMH and AMH-cleaved treatment in endometriosis cells were evaluated by flow-cytometry analysis. Finally, it has been evaluated the effect of plasmin-digested AMH on cytochrome P450 activity. Results AMH and AMH RII isoforms, as well as cytochrome P450, were expressed in both endometriosis epithelial and stromal cells. Treatment of endometriosis stromal and epithelial cell growth with AMH was able to induce a decrease in the percentage of cells in S phase and increase percentage of cells in G1 and G2 phase; coherently, decreased cell viability and increased percentage of cells death fraction was observed. The plasmin-digested AMH was able to suppress most of the cytochrome P450 activity, causing an increase of pre-G1 phase and of apoptosis induction treating with plasmin-digested AMH in both cell lines, most marked in the epithelial cells. Conclusions The data produced suggest a possible use of AMH as therapeutic agents in endometriosis. PMID:24886254

Ultrasound-microbubble mediated cavitation of plant cells: effects on morphology and viability.

PubMed

Qin, Peng; Xu, Lin; Zhong, Wenjing; Yu, Alfred C H

2012-06-01

The interaction between ultrasound pulses and microbubbles is known to generate acoustic cavitation that may puncture biological cells. This work presents new experimental findings on the bioeffects of ultrasound-microbubble mediated cavitation in plant cells with emphasis on direct observations of morphological impact and analysis of viability trends in tobacco BY-2 cells that are widely studied in higher plant physiology. The tobacco cell suspensions were exposed to 1 MHz ultrasound pulses in the presence of 1% v/v microbubbles (10% duty cycle; 1 kHz pulse repetition frequency; 70 mm between probe and cells; 1-min exposure time). Few bioeffects were observed at low peak negative pressures (<0.4 MPa) where stable cavitation presumably occurred. In contrast, at 0.9 MPa peak negative pressure (with more inertial cavitation activities according to our passive cavitation detection results), random pores were found on tobacco cell wall (observed via scanning electron microscopy) and enhanced exogenous uptake into the cytoplasm was evident (noted in our fluorescein isothiocyanate dextran uptake analysis). Also, instant lysis was observed in 23.4% of cells (found using trypan blue staining) and programmed cell death was seen in 23.3% of population after 12 h (determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling [TUNEL]). These bioeffects generally correspond in trend with those for mammalian cells. This raises the possibility of developing ultrasound-microbubble mediated cavitation into a targeted gene transfection paradigm for plant cells and, conversely, adopting plant cells as experimental test-beds for sonoporation-based gene therapy in mammalian cells. Copyright © 2012 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

BID is a critical factor controlling cell viability regulated by IFN-α.

PubMed

Tsuno, Takaya; Mejido, Josef; Zhao, Tongmao; Phillips, Terry; Myers, Timothy G; Bekisz, Joseph; Zoon, Kathryn C

2012-01-01

Clinical applications of human interferon (IFN)-α have met with varying degrees of success. Nevertheless, key molecules in cell viability regulated by IFN-α have not been clearly identified. Our previous study indicated that IFN (α, β, and ω) receptor (IFNAR) 1/2- and IFN regulatory factor 9-RNA interference (RNAi) completely restored cell viability after IFN-α treatment in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-α. In this study, IFNAR1/2- and IFN regulatory factor 9-RNAi inhibited the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not of Fas ligand, after IFN-α treatment. In fact, TRAIL but not Fas ligand inhibited the viability of OVCAR3 cells. IFN-α notably upregulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. After TRAIL signaling, caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly restored cell viability in response to IFN-α and TRAIL in OVCAR3 cells. Furthermore, BID-RNAi prevented both IFN-α and TRAIL from collapsing the mitochondrial membrane potential (ΔΨm). Finally, we provided important evidence that BID overexpression led to significant inhibition of cell viability after IFN-α or TRAIL treatments in human lung carcinoma A549 cells resistant to IFN-α. Thus, this study suggests that BID is crucial for cell viability regulated by IFN-α which can induce mitochondria-mediated apoptosis, indicating a notable potential to be a targeted therapy for IFN-α resistant tumors.

Inhibition of Krebs cycle and activation of glyoxylate cycle in the course of chronological aging of Saccharomyces cerevisiae. Compensatory role of succinate oxidation.

PubMed

Samokhvalov, V; Ignatov, V; Kondrashova, M

2004-01-01

We investigated oxidative processes in mitochondria of Saccharomyces cerevisiae grown on ethanol in the course of chronological aging. We elaborated a model of chronological aging that avoids the influence of exhaustion of medium, as well as the accumulation of toxic metabolites during aging. A decrease in total respiration of cells and, even more, of the contribution of respiration coupled with ATP-synthesis was observed during aging. Aging is also related with the decrease of the contribution of malonate-insensitive respiration. Activities of citrate-synthase (CS), alpha-ketoglutarate dehydrogenase (KGDH) and malate dehydrogenase (MDH) were threefold decreased. The activity of NADP-dependent isocitrate dehydrogenase (NADP-ICDH) decreased more significantly, while the activity of NAD-dependent isocitrate dehydrogenase (NAD-ICDH) fell even greater, being completely inactivated on the third week of aging. In contrast, succinate dehydrogenase (SDH), enzymes of glyoxylate cycle (GCL) (isocitrate lyase (ICL) and malate synthase (MLS)), and enzymes of ethanol oxidation (alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ACDH)), were activated by 50% or more. The behavior of oxidative enzymes and metabolic pathways are apparently inherent to a more viable, long-lived cells in population, selected in the course of chronological aging. This selection allows cells to reveal the mechanism of their higher viability as caused by shunting of complete Krebs cycle by glyoxylate cycle, with a concomitant increased rate of the most efficient energy source, namely succinate formation and oxidation. Thiobarbituric-reactive species (TAR species) increased during aging. We supposed that to be the immediate cause of damage of a part of yeast population. These data show that a greater succinate contribution to respiration in more active cells is a general property of yeast and animal tissues.

Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.

PubMed

Ouyang, Liliang; Yao, Rui; Zhao, Yu; Sun, Wei

2016-09-16

3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and acceptable viability ranges, a combined parameters region was obtained. This study provides guidance for parameter optimization and the fine-tuning of 3D cell printing processes regarding both bioink printability and cell viability after bioplotting, especially for easily damaged cells, like ESCs.

Study of probiotic potential of four wild Lactobacillus rhamnosus strains.

PubMed

Tuo, Yanfeng; Zhang, Weiqin; Zhang, Lanwei; Ai, Lianzhong; Zhang, Yingchun; Han, Xue; Yi, Huaxi

2013-06-01

The four wild Lactobacillus rhamnosus strains were examined in vitro for resistance to simulated gastro and intestinal juices, adhesion to HT-29 cells, antagonistic activity against enteric pathogens and immunomodulating activity. The strains L. rhamnosus SB5L, J5L and IN1L were able to survive in simulated gastro juice while the strain L. rhamnosus SB31L lost viability exposed to simulated gastro juice for 3 h. The four strains had high viability in simulated small intestinal juice with little loss (<1.0 cycle reduction). The strains SB5L, J5L and IN1L antagonized against Escherichia coli ATCC 25922, Salmonella enterica serovar Typhimurium ATCC 14028, Shigella sonnei ATCC 25931. The strain L. rhamnosus IN1L had the highest adhesive capability to HT-29 cells in vitro (251 bacteria cells per 100 HT-29 cells) compared to the other three L. rhamnosus strains. The live bacteria, cell wall and DNA of the four L. rhamnosus induced the secretion of pro-inflammatory cytokines IL-12 (p70), IFN-γ and TNF-α by human peripheral blood mononuclear cells (PBMCs). The levels of IL-12 (p70), IFN-γ and TNF-α produced by stimulated PBMCs were significantly higher (P < 0.05) than those of the control. Those data indicated that the four L. rhamnosus strains have the potential as the probiotic for human being use, although further studies are still needed. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Mangiferin enhances the sensitivity of human multiple myeloma cells to anticancer drugs through suppression of the nuclear factor κB pathway.

PubMed

Takeda, Tomoya; Tsubaki, Masanobu; Kino, Toshiki; Kawamura, Ayako; Isoyama, Shota; Itoh, Tatsuki; Imano, Motohiro; Tanabe, Genzoh; Muraoka, Osamu; Matsuda, Hideaki; Satou, Takao; Nishida, Shozo

2016-06-01

Multiple myeloma (MM) is still an incurable hematological malignancy with a 5-year survival rate of ~35%, despite the use of various treatment options. The nuclear factor κB (NF-κB) pathway plays a crucial role in the pathogenesis of MM. Thus, inhibition of the NF-κB pathway is a potential target for the treatment of MM. In a previous study, we showed that mangiferin suppressed the nuclear translocation of NF-κB. However, the treatment of MM involves a combination of two or three drugs. In this study, we examined the effect of the combination of mangiferin and conventional anticancer drugs in an MM cell line. We showed that the combination of mangiferin and an anticancer drug decreased the viability of MM cell lines in comparison with each drug used separately. The decrease in the combination of mangiferin and an anticancer drug induced cell viability was attributed to increase the expression of p53 and Noxa and decreases the expression of XIAP, survivin, and Bcl-xL proteins via inhibition of NF-κB pathway. In addition, the combination treatment caused the induction of apoptosis, activation of caspase-3 and the accumulation of the cells in the sub-G1 phase of the cell cycle. Our findings suggest that the combination of mangiferin and an anticancer drug could be used as a new regime for the treatment of MM.

Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis

PubMed Central

Parajuli, Keshab R; Zhang, Qiuyang; Liu, Sen; Patel, Neil K; Lu, Hua; Zeng, Shelya X; Wang, Guangdi; Zhang, Changde; You, Zongbing

2014-01-01

Methoxyacetic acid (MAA) is a primary metabolite of ester phthalates that are used in production of consumer products and pharmaceutical products. MAA causes embryo malformation and spermatocyte death through inhibition of histone deacetylases (HDACs). Little is known about MAA’s effects on cancer cells. In this study, two immortalized human normal prostatic epithelial cell lines (RWPE-1 and pRNS-1-1) and four human prostate cancer cell lines (LNCaP, C4-2B, PC-3, and DU-145) were treated with MAA at different doses and for different time periods. Cell viability, apoptosis, and cell cycle analysis were performed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR, Western blot, and chromatin immunoprecipitation analyses. We found that MAA dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. MAA-induced apoptosis was due to down-regulation of the anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, also named cIAP1), leading to activation of caspases 7 and 3 and turning on the downstream apoptotic events. MAA-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and CDK2 expression at the late time. MAA up-regulated p21 expression through inhibition of HDAC activities, independently of p53/p63/p73. These findings demonstrate that MAA suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which suggests that MAA could be used as a potential therapeutic drug for prostate cancer. PMID:25606576

Long non-coding RNA CRNDE promotes tumor growth in medulloblastoma.

PubMed

Song, H; Han, L-M; Gao, Q; Sun, Y

2016-06-01

Medulloblastoma is the most common malignant brain tumor in children. Despite remarkable advances over the past decades, a novel therapeutic strategy is urgently required to increase long-term survival. This study aimed to understand the role of a long non-coding RNA (lncRNA), colorectal neoplasia differentially expressed (CRNDE), in medulloblastoma tumor growth. The transcript level of CRNDE was initially examined in dissected clinical tissues and cultured cancerous cells. Effects of CRNDE knockdown on cell viability and colony formation in vitro were assessed using the CCK-8 and colony formation assays, respectively. Cell cycle progression and survival were also determined after CRNDE knockdown. A xenograft mouse model of human medulloblastoma was established by injecting nude mice with medulloblastoma cells stably depleted of CRNDE expression. Our data suggest that transcript levels of CRNDE are elevated in clinical medulloblastoma tissues instead of in adjacent non-cancerous tissues. Knockdown of CRNDE significantly slowed cell proliferation rates and inhibited colony formation in Daoy and D341 cells. Tumor growth in vivo was also inhibited after CRNDE knockdown. Moreover, after knockdown of CRNDE, cell cycle progression was arrested in S phase and apoptosis was promoted by 15-20% in Daoy and D341 cells. In vivo data further showed that proliferating cell nuclei antigen (PCNA) was decreased, whereas the apoptosis initiator cleaved-caspase-3 was increased upon CRNDE knockdown in cancerous tissues from the mouse model. All these data suggest that CRNDE promotes tumor growth both in vitro and in vivo. This growth-promotion effect might be achieved via arresting cell cycle progression and inhibiting apoptosis. Therapeutics against CRNDE may be a novel strategy for the treatment of medulloblastoma.

Up-regulation of tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 in human colon cancer Caco-2 cells following repetitive exposure to dietary levels of a polyphenol-rich chokeberry juice.

PubMed

Bermúdez-Soto, María J; Larrosa, Mar; Garcia-Cantalejo, Jesús M; Espín, Juan C; Tomás-Barberan, Francisco A; García-Conesa, María T

2007-04-01

Consumption of berries and red fruits rich in polyphenols may contribute to the reduction of colon cancer through mechanisms not yet understood. In this study, we investigated the response of subconfluent Caco-2 cells (a human colon carcinoma model) to repetitive exposure (2 h a day for a 4-day period) of a subtoxic dose of a chokeberry (Aronia melanocarpa) juice containing mixed polyphenols. To mimic physiological conditions, we subjected the chokeberry juice to in vitro gastric and pancreatic digestion. The effects on viability, proliferation and cell cycle were determined, and changes in the expression of genes in response to the chokeberry treatment were screened using Affymetrix oligonucleotide microarrays. Exposure to the chokeberry juice inhibited Caco-2 cell proliferation by causing G(2)/M cell cycle arrest. We detected changes in the expression of a group of genes involved in cell growth and proliferation and cell cycle regulation, as well as those associated to colorectal cancer. A selection of these genes was further confirmed by quantitative RT-PCR. Among these, the tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), whose expression is known to be reduced in the majority of early adenomas and carcinomas, was up-regulated by the treatment both at the mRNA and protein levels (as shown by flow cytometry analysis). CEACAM1, with a significant regulatory role on cell proliferation of particular interest at early stages of cancer development, may be a potential target for chemoprevention by food components such as those present in polyphenol-rich fruits.

Adaptive Roles of SSY1 and SIR3 During Cycles of Growth and Starvation in Saccharomyces cerevisiae Populations Enriched for Quiescent or Nonquiescent Cells.

PubMed

Wloch-Salamon, Dominika M; Tomala, Katarzyna; Aggeli, Dimitra; Dunn, Barbara

2017-06-07

Over its evolutionary history, Saccharomyces cerevisiae has evolved to be well-adapted to fluctuating nutrient availability. In the presence of sufficient nutrients, yeast cells continue to proliferate, but upon starvation haploid yeast cells enter stationary phase and differentiate into nonquiescent (NQ) and quiescent (Q) cells. Q cells survive stress better than NQ cells and show greater viability when nutrient-rich conditions are restored. To investigate the genes that may be involved in the differentiation of Q and NQ cells, we serially propagated yeast populations that were enriched for either only Q or only NQ cell types over many repeated growth-starvation cycles. After 30 cycles (equivalent to 300 generations), each enriched population produced a higher proportion of the enriched cell type compared to the starting population, suggestive of adaptive change. We also observed differences in each population's fitness suggesting possible tradeoffs: clones from NQ lines were better adapted to logarithmic growth, while clones from Q lines were better adapted to starvation. Whole-genome sequencing of clones from Q- and NQ-enriched lines revealed mutations in genes involved in the stress response and survival in limiting nutrients ( ECM21 , RSP5 , MSN1 , SIR4 , and IRA2 ) in both Q and NQ lines, but also differences between the two lines: NQ line clones had recurrent independent mutations affecting the Ssy1p-Ptr3p-Ssy5p (SPS) amino acid sensing pathway, while Q line clones had recurrent, independent mutations in SIR3 and FAS1 Our results suggest that both sets of enriched-cell type lines responded to common, as well as distinct, selective pressures. Copyright © 2017 Wloch-Salamon et al.

Distinct Signaling Pathways Mediate Stimulation of Cell Cycle Progression and Prevention of Apoptotic Cell Death by Estrogen in Rat Pituitary Tumor PR1 Cells

PubMed Central

Caporali, Simona; Imai, Manami; Altucci, Lucia; Cancemi, Massimo; Caristi, Silvana; Cicatiello, Luigi; Matarese, Filomena; Penta, Roberta; Sarkar, Dipak K.; Bresciani, Francesco; Weisz, Alessandro

2003-01-01

Estrogens control cell growth and viability in target cells via an interplay of genomic and extragenomic pathways not yet elucidated. Here, we show evidence that cell proliferation and survival are differentially regulated by estrogen in rat pituitary tumor PR1 cells. Pico- to femtomolar concentrations of 17β-estradiol (E2) are sufficient to foster PR1 cell proliferation, whereas nanomolar concentrations of the same are needed to prevent cell death that occurs at a high rate in these cells in the absence of hormone. Activation of endogenous (PRL) or transfected estrogen-responsive genes occurs at the same, higher concentrations of E2 required to promote cell survival, whereas stimulation of cyclin D3 expression and DNA synthesis occur at lower E2 concentrations. Similarly, the pure antiestrogen ICI 182,780 inhibits estrogen response element-dependent trans-activation and cell death more effectively than cyclin-cdk activity, G1-S transition, or DNA synthesis rate. In antiestrogen-treated and/or estrogen-deprived cells, death is due predominantly to apoptosis. Estrogen-induced cell survival, but not E2-dependent cell cycle progression, can be prevented by an inhibitor of c-Src kinase or by blockade of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway. These data indicate the coexistence of two distinguishable estrogen signaling pathways in PR1 cells, characterized by different functions and sensitivity to hormones and antihormones. PMID:12960425

Effect of the oncolytic ECHO-7 virus Rigvir® on the viability of cell lines of human origin in vitro.

PubMed

Tilgase, Andra; Patetko, Liene; Blāķe, Ilze; Ramata-Stunda, Anna; Borodušķis, Mārtiņš; Alberts, Pēteris

2018-01-01

Background: The role of oncolytic viruses in cancer treatment is increasingly studied. The first oncolytic virus (Rigvir®, ECHO-7) was registered in Latvia over a decade ago. In a recent retrospective study Rigvir® decreased mortality 4.39-6.57-fold in stage IB-IIC melanoma patients. The aims of the present study are to test the effect of Rigvir® on cell line viability in vitro and to visualize the cellular presence of Rigvir® by immunocytochemistry. Methods: The cytolytic effect of Rigvir® on the viability of FM-9, RD, AGS, A549, HDFa, HPAF‑II, MSC, MCF7, HaCaT, and Sk-Mel-28 cell lines was measured using live cell imaging. PBMC viability was measured using flow cytometry. The presence of ECHO-7 virus was visualized using immunocytochemistry. Statistical difference between treatment groups was calculated using two-way ANOVA. Results: Rigvir® (10%, volume/volume) reduced cell viability in FM-9, RD, AGS, A549, HDFa, HPAF‑II and MSC cell lines by 67-100%. HaCaT cell viability was partly affected while Rigvir® had no effect on MCF7, Sk-Mel-28 and PBMC viability. Detection of ECHO-7 by immunocytochemistry in FM-9, RD, AGS, A549, HDFa, HPAF-II and Sk-Mel-28 cell lines suggests that the presence of Rigvir® in the cells preceded or coincided with the time of reduction of cell viability. Rigvir® (10%) had no effect on live PBMC count. Conclusions: The results suggest that Rigvir® in vitro reduces the viability of cells of human melanoma, rhabdomyosarcoma, gastric adenocarcinoma, lung carcinoma, pancreas adenocarcinoma but not in PBMC. The presence of Rigvir® in the sensitive cells was confirmed using anti-ECHO-7 antibodies. The present results suggest that a mechanism of action for the clinical benefit of Rigvir® is its cytolytic properties. The present results suggest that the effect of Rigvir® could be tested in other cancers besides melanoma. Further studies of possible Rigvir® entry receptors are needed.

Innovative Microcapsules for Pancreatic β-Cells Harvested from Mature Double-Transgenic Mice: Cell Imaging, Viability, Induced Glucose-Stimulated Insulin Measurements and Proinflammatory Cytokines Analysis.

PubMed

Mooranian, Armin; Tackechi, Ryu; Jamieson, Emma; Morahan, Grant; Al-Salami, Hani

2017-06-01

Recently we demonstrated that microencapsulation of a murine pancreatic β-cell line using an alginate-ursodeoxycholic acid (UDCA) matrix produced microcapsules with good stability and cell viability. In this study, we investigated if translation of this formulation to microencapsulation of primary β-cells harvested from mature double-transgenic healthy mice would also generate stable microcapsules with good cell viability. Islets of Langerhans were isolated from Ngn3-GFP/RIP-DsRED mice by intraductal collagenase P digestion and density gradient centrifugation, dissociated into single cells and the β-cell population purified by Fluorescence Activated Cell Sorting. β-cells were microencapsulated using either alginate-poly-l-ornithine (F1; control) or alginate-poly-l-ornithine-UDCA (F2; test) formulations. Microcapsules were microscopically examined and microencapsulated cells were analyzed for viability, insulin and cytokine release, 2 days post-microencapsulation. Microcapsules showed good uniformity and morphological characteristics and even cell distribution within microcapsules with or without UDCA. Two days post microencapsulation cell viability, mitochondrial ATP and insulin production were shown to be optimized in the presence of UDCA whilst production of the proinflammatory cytokine IL-1β was reduced. Contradictory to our previous studies, UDCA did not reduce production of any other pro-inflammatory biomarkers. These results suggest that UDCA incorporation improves microcapsules' physical and morphological characteristics and improves the viability and function of encapsulated mature primary pancreatic β-cells.

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

Cheng, Jing; Du, Yi-Fang; Xiao, Zhi-Yi

KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the inhibitory activity test on Hep G{sub 2} growth. We found that KYKZL-1 inhibited the growth of Hep G{sub 2} cells via inducing apoptosis. Further studies showed that KYKZL-1 activated caspase-3 through cytochrome c release from mitochondria and down regulation of Bcl-2/Bax ratio and reduced the high level of COX-2 and 5-LOX. As shown in its anti-inflammatory effect, KYKZL-1 also exhibited inhibitory effect on the PGE{sub 2} and LTB{sub 4} production in Hep G{sub 2} cells. Accordingly, exogenous addition of PGE{sub 2} or LTB{sub 4} reversed the decreasesmore » in cell viability. In addition, KYKZL-1 caused cell cycle arrest at the S–G{sub 2} checkpoint via the activation of p21{sup CIP1} protein and down-regulation of cyclin A expression. These data indicate that the growth inhibitory effect of KYKZL-1 is associated with inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest. Combined with our previous findings, KYKZL-1 exhibiting COX/5-LOX inhibition may be a promising potential agent not only for inflammation control but also for cancer prevention/therapy with an enhanced gastric safety profile. - Highlights: • KYKZL-1 is designed to exhibit COX/5-LOX dual inhibition. • KYKZL-1 resulted in apoptosis of Hep G{sub 2} cells. • KYKZL-1 activated caspase-3 through cytochrome c and bcl-2/bax ratio. • KYKZL-1 caused cell cycle arrest via modulation of p21{sup CIP1} and cyclin A level.« less

[Phloretin induces apoptosis of BEL-7402 cells in vitro].

PubMed

Luo, Hui; Wang, Ya-jun; Chen, Jie; Liu, Jiang-qin; Zhang, Hai-tao

2008-07-01

To examine the effect of phloretin on apoptosis of BEL-7402 cells. The viability changes of BEL- 7402 cells as a result of phloretin-induced toxicity were analyzed using MTT assay, and the cell morphology changes were observed with fluorescence microscope. Flow cytometry was used to analyze the cell cycle and mitochondrial membrane potential changes, and chromogenic substrate assay performed to detect caspase activity. Phloretin induced obvious cytotoxicity against BEL-7402 cells with IC50 of 89.23 microg/mL. The growth curve demonstrated decreased growth of the cells as phloretin concentration increased. Cell apoptosis occurred 24 h after treatment with 40-160 microg/mL phloretin. Morphological, the cells exposed to phloretin exhibited nuclear chromatin condensation and increased fluorescence intensity. The activity of caspase-9 reached the peak level 12 h after phloretin exposure, and leak levels of caspase-6 and caspase-3 activities occurred 18 and 24 h after the exposure, respectively. Phloretin can induce BEL-7402 cell apoptosis though the mitochondrial pathway.

Superior anti-tumor activity of the MDM2 antagonist idasanutlin and the Bcl-2 inhibitor venetoclax in p53 wild-type acute myeloid leukemia models.

PubMed

Lehmann, Christian; Friess, Thomas; Birzele, Fabian; Kiialainen, Anna; Dangl, Markus

2016-06-28

Venetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML). In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML. The effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments. Combination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway) in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that inhibition of the anti-apoptotic protein Mcl-1 contributed to the activity of venetoclax and idasanutlin, with earlier inhibition of Mcl-1 in response to combination treatment contributing to the superior combined activity. The role of Mcl-1 was confirmed by small hairpin RNA gene knockdown studies. Our findings provide functional and molecular insight on the superior anti-tumor activity of combined idasanutlin and venetoclax treatment in AML and support its further exploration in clinical studies.

CD9 monoclonal antibody-conjugated PEGylated liposomes for targeted delivery of rapamycin in the treatment of cellular senescence

NASA Astrophysics Data System (ADS)

Thuy Nguyen, Hanh; Thapa, Raj Kumar; Shin, Beom Soo; Jeong, Jee-Heon; Kim, Jae-Ryong; Yong, Chul Soon; Kim, Jong Oh

2017-03-01

Premature cellular senescence refers to the state of irreversible cell cycle arrest due to DNA damage or other stresses. In this study, CD9 monoclonal antibody (CD9mAb) was successfully conjugated to the surface of PEGylated liposomes for targeted delivery of rapamycin (LR-CD9mAb) to overcome senescence of CD9 receptor-overexpressing cells. LR-CD9mAb has a small particle size (143.3 ± 2.4 nm), narrow size distribution (polydispersity index: 0.220 ± 0.036), and negative zeta potential (-14.6 ± 1.2 mV). The uptake of CD9-targeted liposomes by premature senescent human dermal fibroblasts (HDFs) was higher than that by young HDFs, as displayed by confocal microscopic images. The senescence might not be reversed by treatment with rapamycin; however, the drug promoted cell proliferation and reduced the number of cells that expressed the senescence-associated-β-galactosidase (SA-β-gal). These effects were further confirmed by cell viability, cell cycle, and Western blotting analyses. Moreover, CD9-targeted liposomes showed better anti-senescence activity, in comparison with free rapamycin or the conventional liposomal formulation, suggesting the potential application of this system in further in vivo studies.

Chloroquine inhibits hepatocellular carcinoma cell growth in vitro and in vivo

PubMed Central

HU, TAO; LI, PEI; LUO, ZHONGGUANG; CHEN, XIAOYU; ZHANG, JINGYANG; WANG, CHUNYAO; CHEN, PING; DONG, ZIMING

2016-01-01

Recently, chloroquine (CQ) has been widely used to improve the efficacy of different chemotherapy drugs to treat tumors. However, the effects of single treatment of CQ on liver cancer have not been investigated. In the present study, we examined the effects of CQ on the growth and viability of liver cancer cells in vitro and in vivo, and revealed that CQ treatment triggered G0/G1 cell cycle arrest, induced DNA damage and apoptosis in a dose- and time-dependent manner in liver cancer cells. Moreover, administration of CQ to tumor-bearing mice suppressed the tumor growth in an orthotopic xenograft model of liver cancer. These findings extend our understanding and suggest that CQ could be repositioned as a treatment option for liver cancer as a single treatment or in combination. PMID:26530158

Laser and Non-Coherent Light Effect on Peripheral Blood Normal and Acute Lymphoblastic Leukemic Cells by Using Different Types of Photosensitizers

NASA Astrophysics Data System (ADS)

El Batanouny, Mohamed H.; Khorshid, Amira M.; Arsanyos, Sonya F.; Shaheen, Hesham M.; Abdel Wahab, Nahed; Amin, Sherif N.; El Rouby, Mahmoud N.; Morsy, Mona I.

2010-04-01

Photodynamic therapy (PDT) is a novel treatment modality of cancer and non-cancerous conditions that are generally characterized by an overgrowth of unwanted or abnormal cells. Irradiation of photosensitizer loaded cells or tissues leads via the photochemical reactions of excited photosensitizer molecules to the production of singlet oxygen and free radicals, which initiate cell death. Many types of compounds have been tested as photosensitizers, such as methylene blue (MB) and photopherin seemed to be very promising. This study involved 26 cases of acute lymphoblastic leukemia and 15 normal volunteers as a control group. The cell viability was measured by Light microscope and flowcytometer. Mode of cell death was detected by flowcytometer and electron microscope in selected cases. The viability percentage of normal peripheral blood mononuclear cells (PBMC) incubated with methylene blue (MB) alone or combined with photo irradiation with diode laser (as measured by light microscope) was significantly lower than that of untreated cases either measured after 1 hour (p<0.001) or 24 hours (p<0.001) post incubation. There was a significantly lower viability percentage of normal cells incubated with MB and photoirradiated with diode laser compared to normal cells treated with MB alone for either measured after 1 hour (p<0.001) or 24 hours (p<0.001) post incubation. The decrease in viability was more enhanced with increasing the incubation time. For normal cells incubated with photopherin either for 1/2 an hour or 1 hour, there was a weak cytotoxic effect compared to the effect on untreated cells. There was a significant decrease in viability percentage of cells incubated with photopherin either for 1/2 an hour or 1 hour and photoirradiated with He:Ne laser compared to normal untreated cells. The decrease in the cell viability percentage was significantly lower with the use of PDT (photopherin and He:Ne laser ) compared to either photopherin alone or He:Ne laser alone. The decrease in viability was more enhanced with increasing the incubation time. The same effects reported on normal cells were detected on leukemic cells on comparing different methods used. However a more pronounced decrease in cell viability was detected. The most efficient ways of decreasing viability of leukemic cells with much less effect on normal cells was the use of PDT of cell incubation with MB for 1 hour then photoirradiation with diode laser and PDT of cell incubation with photopherin for 1 hour then photoirradiation with He:Ne laser. Flowcytometer (FCM) was more sensitivite than the light microscope in detecting the decrease in cell viability, it also helped in determining the mode of cell death weather apoptosis, necrosis or combined apoptosis and necrosis. Apoptotic cell percentage was higher in PDT of MB and Diode laser or photopherin and He:Ne laser, treated ALL cells compared to untreated ALL cells after 1 hour but was significantly lower after 24 hours post irradiation. A significant increase in necrotic, combined necrotic and apoptotic cell percentages either measured 1 hour or 24 hours post PDT, compared to untreated ALL cells and PDT treated normal cells. Electron microscope helped in detecting early cellular apoptotic changes occurring in response to different therapeutic modalities used in this study. In conclusion, PDT proved to be an effective clinical modality in decreasing the number of leukemic cells when irradiated in vitro with appropriate laser and photosensitizer system. Both PDT systems used in this study were efficient in inducing cell death of leukemic cells compared to untreated leukemic cells. However, photopherin PDT system was more efficient in decreasing the cell viability. A significant decrease in viability percentage was detected when studying the effect of PDT on leukemic cells compared to that on normal cells. This suggests that PDT when applied clinically will selectively differentiate between leukemic cells and normal cells, offering a successful component in ALL therapy.

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

Matthews, Q; Lum, JJ; Isabelle, M

Purpose: To use label-free Raman spectroscopy (RS) for early treatment monitoring of tumour cell radioresistance. Methods: Three human tumour cell lines, two radioresistant (H460, SF{sub 2} = 0.57 and MCF7, SF{sub 2} = 0.70) and one radiosensitive (LNCaP, SF{sub 2} = 0.36), were irradiated with single fractions of 2, 4, 6, 8 or 10 Gy. In additional experiments, H460 and MCF7 cells were irradiated under co-treatment with the anti-diabetic drug metformin, a known radiosensitizing agent. Treated and control cultures were analyzed with RS daily for 3 days post-treatment. Single-cell Raman spectra were acquired from 20 live cells per sample, andmore » experiments were repeated in triplicate. The combined data sets were analyzed with principal component analysis using standard algorithms. Cells from each culture were also subjected to standard assays for viability, proliferation, cell cycle, and radiation clonogenic survival. Results: The radioresistant cells (H460, MCF7) exhibited a RS molecular radiation response signature, detectable as early as 1 day post-treatment, of which radiation-induced glycogen synthesis is a significant contributor. The radiosensitive cells (LNCaP) exhibited negligible glycogen synthesis. Co-treatment with metformin in MCF7 cells blocked glycogen synthesis, reduced viability and proliferation, and increased radiosensitivity. Conversely, metformin co-treatment in H460 cells did not produce these same effects; importantly, both radiation-induced synthesis of glycogen and radiosensitivity were unaffected. Conclusions: Label-free RS can detect early glycogen synthesis post-irradiation, a previously undocumented metabolic mechanism associated with tumour cell radioresistance that can be targeted to increase radiosensitivity. RS monitoring of intratumoral glycogen may provide new opportunities for personalized combined modality radiotherapy treatments.« less

The effects of the fungicides fenhexamid and myclobutanil on SH-SY5Y and U-251 MG human cell lines.

PubMed

Nagel, David A; Hill, Eric J; O'Neil, John; Mireur, Alexandra; Coleman, Michael D

2014-11-01

Mixtures of pesticides in foodstuffs and the environment are ubiquitous in the developed world and although agents are usually exhaustively tested individually, the toxicological implications of pesticide mixtures are underreported. In this study, the effects of two fungicides, fenhexamid and myclobutanil were investigated individually and in combination on two human cell lines, SH-SY5Y neuronal cells and U-251 MG glial cells. After 48h of incubation with increasing concentrations of pesticides ranging from 1 to 1000μM, gene expression profiles were studied in addition to toxicity end points, including cell viability, mitochondrial depolarisation as well as cellular glutathione maintenance. There were no significant differences between the susceptibility of the two cell lines in terms of cell viability assessment or mitochondrial membrane potential, when agents were administered either individually or in combination. By contrast, in the presence of the fungicides, the SH-SY5Y cells showed significantly greater susceptibility to oxidative stress in terms of total thiol depletion in comparison with the astrocytic cells. Treatment with the two pesticides led to significant changes in the cell lines' expression of several genes which regulate cell cycle control and growth (RB1, TIMP1) as well as responses to DNA attrition (ATM and CDA25A) and control of apoptosis (FAS). There was no evidence in this study that the combination of fenhexamid and myclobutanil was significantly more toxic than individual exposure, although gene expression changes suggested there may be differences in the sub-lethal response of both cell lines to both individual and combined exposure. Copyright © 2014 Elsevier B.V. All rights reserved.

Porous α-Fe2O3 nanostructures and their lithium storage properties as full cell configuration against LiFePO4

NASA Astrophysics Data System (ADS)

Veluri, P. S.; Shaligram, A.; Mitra, S.

2015-10-01

A two step approach for synthesis of porous α-Fe2O3 nanostructures has been realized via polyol method by complexing iron oxalate with ethylene glycol. Crystalline Fe2O3 samples with different porosities are obtained by calcination of Fe-Ethylene glycol complex at various temperatures. The as-prepared porous Fe2O3 structures exhibit promising lithium storage performance at high current rates. It is observed that the calcination temperature and the resultant porosity have a significant effect on capacity and cycling stability. Samples calcined at high temperature (600 °C) demonstrates stable cycle life with capacity retention of 1077 mAh g-1 at 500 mA g-1 current rate after 50 charge-discharge cycles. Samples calcined at temperatures of 500 and 600 °C display stable cycle life and high rate capability with reversible capacity of 930 mAh g-1 and 688 mAh g-1 at 5 A g-1, respectively. Impregnation of electrodes with electrolyte before cell fabrication shows enhanced electrochemical performance. The viability of Fe2O3 porous nanostructures as prospective anode material examined against commercial LiFePO4 cathode shows promising electrochemical performance.

Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei *

PubMed Central

McDermott, Suzanne M.; Guo, Xuemin; Carnes, Jason; Stuart, Kenneth

2015-01-01

Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. The mRNAs are differentially edited in bloodstream form (BF) and procyclic form (PF) life cycle stages, and this correlates with the differential utilization of glycolysis and oxidative phosphorylation between the stages. The mechanism that controls this differential editing is unknown. Editing is catalyzed by multiprotein ∼20S editosomes that contain endonuclease, 3′-terminal uridylyltransferase, exonuclease, and ligase activities. These editosomes also contain KREPB5 and KREPA3 proteins, which have no functional catalytic motifs, but they are essential for parasite viability, editing, and editosome integrity in BF cells. We show here that repression of KREPB5 or KREPA3 is also lethal in PF, but the effects on editosome structure differ from those in BF. In addition, we found that point mutations in KREPB5 or KREPA3 differentially affect cell growth, editosome integrity, and RNA editing between BF and PF stages. These results indicate that the functions of KREPB5 and KREPA3 editosome proteins are adjusted between the life cycle stages. This implies that these proteins are involved in the processes that control differential editing and that the 20S editosomes differ between the life cycle stages. PMID:26304125