Abstract 852: Apoptosis induction by ayurvedic medicine constituent withaferin A in human breast cancer cells is mediated by reactive oxygen species-dependent activation of Bak

Abstract

Abstract Withaferin A (WA) is a promising cancer chemopreventive constituent of Ayurvedic medicine plant Withania somnifera that has been used safely for thousands of years in India for treatment of different ailments. We have shown previously that WA treatment suppresses viability of cultured as well as xenografted human breast cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. Here, we demonstrate that the WA-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species (ROS)-dependent activation of Bak. WA treatment caused ROS production in MDA-MB-231 (an estrogen-independent cell line with mutant p53) and MCF-7 cells (an estrogen-responsive cell line with wild-type p53) in a concentration- and time-dependent manner as judged by flow cytometry and fluorescence microscopy using a chemical probe (MitoSOX Red) and EPR spectroscopy using a cell permeable spin probe (1-hydroxy-3-methoxy-carbonyl-2,2,5,5-tetramethylpyrrolidine). WA-mediated ROS production, as well as, apoptosis [characterized by release of histone-associated DNA fragments into the cytosol, and cleavage of poly-(ADP-ribose) polymerase or procaspase-3], was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase (SOD) or Mn-SOD. Real-time measurements of oxygen consumption rate and extracellular acidification rate using a Seahorse Bioscience XF24 Extracellular Flux Analyzer revealed that the ROS production resulting from WA exposure was accompanied by inhibition of basal and reserve oxidative phosphorylation. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were significantly more resistant to WA-induced ROS production and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak, but only Bak activation was attenuated by Cu,Zn-SOD overexpression. Furthermore, RNA interference or genetic suppression of Bax and Bak expression conferred significant protection against WA-induced apoptosis. In conclusion, the present study provides novel insights into the molecular circuitry of WA-induced apoptosis involving ROS-dependent activation of Bak. This investigation was supported by the USPHS grant CA142604-01, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 852. doi:10.1158/1538-7445.AM2011-852