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Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells.

ABSTRACT: Withaferin A (WA), a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 ?M WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD). WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs) in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L). Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS) in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L) suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.

SUBMITTER: Nishikawa Y

PROVIDER: S-EPMC4521694 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells.

Nishikawa Yukihiro Y Okuzaki Daisuke D Fukushima Kohshiro K Mukai Satomi S Ohno Shouichi S Ozaki Yuki Y Yabuta Norikazu N Nojima Hiroshi H

PloS one 20150731 7

Withaferin A (WA), a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and K ...[more]

PMID: 26230090

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Project description:Objective: The purpose of this study was to evaluate the anticancer effects of Ophiopogonin D' (OPD', a natural product extracted from a traditional Chinese medicine (Radix Ophiopogonis) against androgen-independent prostate cancer cells and to explore the underlying molecular mechanism(s) of action. Methods: The CCK-8 assay was used to assess the viability of prostate cancer cells. The cell morphology was examined by an ultrastructural analysis via transmission electron microscopy. Cells in apoptosis (early and late stages) were detected using an Annexin V-FITC/propidium iodide kit with a FACSCaliber flow cytometer. JC-1, a cationic lipophilic probe, was employed to measure the mitochondrial membrane potential (MMP) of PC3 cells. Changes in the protein expression of RIPK1, C-RIPK1, caspase 8, cleaved-caspase 8, Bim, Bid, caspase 10, and cleaved-caspase 10 were evaluated by Western blotting. The mRNA expression of Bim was examined by quantitative real-time reverse transcription polymerase chain reaction. Z-VAD-FMK (a caspase inhibitor) and necrostatin-1 (a specific inhibitor of RIPK1) were utilized to determine whether the cell death was mediated by RIPK1 or caspases. PC3 and DU145 xenograft models in BALB/c nude mice were used to evaluate the anticancer activity of OPD' in vivo. Results: OPD' was shown to exert potent anti-tumor activity against PC3 cells. It induced apoptosis via a RIPK1-related pathway, increased the protein expression levels of RIPK1 and Bim, and decreased the levels of cleaved-RIPK1, caspase 8, cleaved-caspase 8, Bid, caspase 10, and cleaved-caspase 10. OPD' also increased the mRNA expression of Bim. The protein expression of Bim was decreased when cells were pre-treated with necrostatin-1. Treatment with OPD' inhibited the growth of PC3 and DU145 xenograft tumors in BALB/c nude mice. Conclusion: OPD' significantly inhibited the in vitro and in vivo growth of prostate cells via RIPK1, suggesting that OPD' may be developed as a potential anti-prostate cancer agent.

Project description:BackgroundCastration resistant prostate cancer (CRPC) develops as a consequence of hormone therapies used to deplete androgens in advanced prostate cancer patients. CRPC cells are able to grow in a low androgen environment and this is associated with anomalous activity of their endogenous androgen receptor (AR) despite the low systemic androgen levels in the patients. Therefore, the reactivated tumor cell androgen signaling pathway is thought to provide a target for control of CRPC. Previously, we reported that Hedgehog (Hh) signaling was conditionally activated by androgen deprivation in androgen sensitive prostate cancer cells and here we studied the potential for cross-talk between Hh and androgen signaling activities in androgen deprived and androgen independent (AI) prostate cancer cells.ResultsTreatment of a variety of androgen-deprived or AI prostate cancer cells with the Hh inhibitor, cyclopamine, resulted in dose-dependent modulation of the expression of genes that are regulated by androgen. The effect of cyclopamine on endogenous androgen-regulated gene expression in androgen deprived and AI prostate cancer cells was consistent with the suppressive effects of cyclopamine on the expression of a reporter gene (luciferase) from two different androgen-dependent promoters. Similarly, reduction of smoothened (Smo) expression with siRNA co-suppressed expression of androgen-inducible KLK2 and KLK3 in androgen deprived cells without affecting the expression of androgen receptor (AR) mRNA or protein. Cyclopamine also prevented the outgrowth of AI cells from androgen growth-dependent parental LNCaP cells and suppressed the growth of an overt AI-LNCaP variant whereas supplemental androgen (R1881) restored growth to the AI cells in the presence of cyclopamine. Conversely, overexpression of Gli1 or Gli2 in LNCaP cells enhanced AR-specific gene expression in the absence of androgen. Overexpressed Gli1/Gli2 also enabled parental LNCaP cells to grow in androgen depleted medium. AR protein co-immunoprecipitates with Gli2 protein from transfected 293T cell lysates.ConclusionsCollectively, our results indicate that Hh/Gli signaling supports androgen signaling and AI growth in prostate cancer cells in a low androgen environment. The finding that Gli2 co-immunoprecipitates with AR protein suggests that an interaction between these proteins might be the basis for Hedgehog/Gli support of androgen signaling under this condition.

Dataset Information

Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells.

Publications

Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells.

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