title: Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species.
abstract: Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Eun-Ryeong
authors.initials: ER
authors.lastname: Hahm
authors.forename: Michelle B
authors.initials: MB
authors.lastname: Moura
authors.forename: Eric E
authors.initials: EE
authors.lastname: Kelley
authors.forename: Bennett
authors.initials: B
authors.lastname: Van Houten
authors.forename: Sruti
authors.initials: S
authors.lastname: Shiva
authors.forename: Shivendra V
authors.initials: SV
authors.lastname: Singh
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D014450:Electron Transport Complex III / Q000378:metabolism; D005260:Female; D006801:Humans; D051379:Mice; D010085:Oxidative Phosphorylation / Q000187:drug effects; D020011:Protective Agents / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D054358:Withanolides / Q000737:chemistry / Q000494:pharmacology*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D020011:Protective Agents; D017382:Reactive Oxygen Species; D054358:Withanolides; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D000255:Adenosine Triphosphate; D013482:Superoxide Dismutase; D020741:rho GTP-Binding Proteins; D014450:Electron Transport Complex III; C009684:withaferin A