Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species

• Published in: PloS one Vol. 6; no. 8; p. e23354
• Main Authors: Hahm, Eun-Ryeong, Moura, Michelle B, Kelley, Eric E, Van Houten, Bennett, Shiva, Sruti, Singh, Shivendra V
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10.08.2011; Public Library of Science (PLoS)
• Subjects: Activation; Adenosine Triphosphate - metabolism; Animals; Apoptosis; Apoptosis - drug effects; bcl-2 Homologous Antagonist-Killer Protein - metabolism; bcl-2-Associated X Protein - metabolism; Bioenergetics; Biology; Breast cancer; Breast Neoplasms - enzymology; Breast Neoplasms - pathology; Cancer; Cancer cells; Cancer treatment; Cell culture; Cell death; Cell Line, Tumor; Circuits; Cytosol; Deoxyribonucleic acid; DNA; Drug Resistance, Neoplasm - drug effects; Drug Screening Assays, Antitumor; Ectopic expression; Electron transport; Electron Transport Complex III - metabolism; Embryo fibroblasts; Epithelial cells; Female; Fibroblasts; Gene expression; Herbal medicine; Humans; Inhibition; Ischemia; Mammary gland; Medicinal plants; Medicine; Membrane potential; Metabolism; Mice; Microscopy; Mitochondrial DNA; Oxidative phosphorylation; Oxidative Phosphorylation - drug effects; Oxygen; Pharmacology; Phosphorylation; Physiology; Protective Agents - pharmacology; Proteins; Reactive oxygen species; Reactive Oxygen Species - metabolism; rho GTP-Binding Proteins - metabolism; Rodents; Superoxide dismutase; Superoxide Dismutase - metabolism; Superoxides; Withania somnifera; Withanolides - chemistry; Withanolides - pharmacology; Xenografts; Xenotransplantation; Zinc; Zinc compounds; United States > US; Pittsburgh Pennsylvania; Pennsylvania
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0023354

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.