Abstract
We have shown previously that withaferin A (WA), a promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits growth of MCF‐7 and MDA‐MB‐231 human breast cancer cells in culture and MDA‐MB‐231 xenografts in vivo by causing apoptosis. However, the mechanism of WA‐induced apoptosis is not fully understood. The present study was designed to systematically determine the role of tumor suppressor p53 and estrogen receptor‐α (ER‐α) in proapoptotic response to WA using MCF‐7, T47D, and ER‐α overexpressing MDA‐MB‐231 cells as a model. WA treatment resulted in induction as well as increased S15 phosphorylation of p53 in MCF‐7 cells, but RNA interference of this tumor suppressor conferred modest protection at best against WA‐induced apoptosis. WA‐mediated growth inhibition and apoptosis induction in MCF‐7 cells were significantly attenuated in the presence of 17β‐estradiol (E2). Exposure of MCF‐7 cells to WA resulted in a marked decrease in protein levels of ER‐α (but not ER‐β) and ER‐α regulated gene product pS2, and this effect was markedly attenuated in the presence of E2. WA‐mediated down‐regulation of ER‐α protein expression correlated with a decrease in its nuclear level, suppression of its mRNA level, and inhibition of E2‐dependent activation of ERE2e1b‐luciferase reporter gene. Ectopic expression of ER‐α in the MDA‐MB‐231 cell line conferred partial but statistically significant protection against WA‐mediated apoptosis, but not G2/M phase cell cycle arrest. Collectively, these results indicate that WA functions as an anti‐estrogen, and the proapoptotic effect of this promising natural product is partially attenuated by p53 knockdown and E2‐ER‐α. © 2011 Wiley‐Liss, Inc.