Abstract 566: Withaferin A causes autophagy in normal as well as cancerous human breast cells

Abstract

Abstract Withaferin A (WA), a promising anticancer constituent of traditional Indian (Ayurvedic) medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 (estrogen-independent) and MCF-7 (estrogen-responsive) human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction. We have shown recently that WA-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species (ROS) resulting from inhibition of mitochondrial respiration. Because ROS are frequently implicated in induction of autophagy, which is an evolutionary conserved process for bulk-degradation of macromolecules including organelles, we raised the question of whether WA treatment causes autophagy. In the present study, we have addressed this question using MDA-MB-231 and MCF-7 human breast cancer cells, a spontaneously immortalized and non-tumorigenic normal human mammary epithelial cell line (MCF-10A), and MDA-MB-231 xenografts from control and WA-treated mice. WA treatment resulted in autophagy induction in cancerous (MCF-7 and MDA-MB-231) as well as normal mammary epithelial cells (MCF-10A) as judged by transmission electron microscopy, cleavage of microtubule-associated protein 1 light chain 3 (LC3), formation of acidic vesicular organelles, and punctate pattern of LC3 localization. Immunohistochemical analysis revealed markedly higher expression of LC3 in MDA-MB-231 xenografts from female athymic mice administered i.p. with 4 mg/kg WA compared with control. Autophagy is a protective mechanism against apoptosis by several agents. Interestingly, inhibition of WA-induced autophagy either by a pharmacological agent (3-methyladenine) or siRNA knockdown of critical autophagy regulating proteins ATG5 or Beclin1 did not have any meaningful impact on growth inhibition or apoptotic cell death resulting from WA exposure in MDA-MB-231 or MCF-7 cells. Collectively, these results indicate that WA induces autophagy in breast cancer cells regardless of estrogen receptor expression and this response is not unique to cancerous breast cells. In addition, unlike many other agents, autophagy induction by WA has no influence on its ability to inhibit cell growth or cause apoptosis at least in breast cancer cells. This investigation was supported by US PHS grant CA142604 awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 566. doi:1538-7445.AM2012-566