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.
Highlights
More than 40,000 women die from breast cancer each year in the United States alone despite significant advances towards targeted therapies and screening efforts [1]
Because reactive oxygen species (ROS) are implicated in apoptosis induction by a variety of natural anticancer agents [27], we questioned if proapoptotic response to Withaferin A (WA) was mediated by ROS generation
These results indicated that while WA treatment caused ROS production in breast cancer cells, human mammary epithelial cell line (HMEC) were resistant to pro-oxidant effect of this agent
Summary
More than 40,000 women die from breast cancer each year in the United States alone despite significant advances towards targeted therapies and screening efforts [1]. Previous research has identified some of the risk factors associated with breast cancer, including family history, Li-Fraumeni syndrome, atypical hyperplasia of the breast, late-age at first full-term pregnancy, early menarche, and late menopause [2,3]. Because some of these risk factors are not adjustable (e.g., genetic predisposition), novel strategies for reduction of breast cancer risk are clinically desirable. This objective is partially fulfilled with selective estrogen-receptor (ER) modulators (e.g., tamoxifen and raloxifene). Natural products have received increased attention in recent years for the discovery of novel cancer chemopreventive and therapeutic agents [6]
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