Anticancer efficacy of natural withanolides in triple-negative breast cancer through down regulation of BRCA1, cell-cycle arrest, and induction of apoptosis.

Abstract

291 Background: Triple negative breast cancer is often chemoresistant creating the need for novel improved drugs for this disease. Naturally derived withanolides such as withaferin A (WA) have shown potent preclinical efficacy in breast cancers. The purpose of this study is to better define the anticancer activity of WA and novel analogs(X001 and X003) containing a 19-OH substitution, in triple negative MDA-MB-231cells where BRCA1 is important for cell survival. Methods: Cells were treated with increasing doses of WA, X001, or X003 for 24 h. MTS and trypan blue exclusion assays were used to examine cell proliferation and viability. Cell cycle was examined using propidium iodide (PI) staining on flow cytometry (FC). Annexin-V/PI co-staining on FC was used to detect apoptosis. Western analysis was used to evaluate BRCA1 protein levels as well as to confirm apoptosis by PARP cleavage and caspase-3 activation. Results: X003, WA, and X001 inhibited MDA-MB-231 cell viability (IC50=125, 500 and 1300nM respectively). IC50 level of WA induced G2/M phase cell cycle arrest while 2.5μM also induced apoptosis at 24 h as well as a significant decrease in both total and phospho-Akt protein levels. In addition, WA decreased BRCA1, heat shock factor 1, and mutant p53 protein levels in a dose-dependent manner. 2.5 μM of WA decreased BRCA1 protein levels by 75% compared to controls at 24 h (p<0.01) while its natural analogs X003 and X001 induced apoptosis associated with BRCA1 down-regulation at 200nM (1.5xIC50) and 6.5μM (5xIC50) and a G2/M cell cycle arrest at only 50nM (50%IC50) and 2μM (1.5xIC50) respectively after 24 hours. Conclusions: Natural withanolides such as WA and its potent analog X003 demonstrate anticancer effects on triple negative breast cancer cells through cell cycle arrest, apoptosis and down-regulation of BRCA1 protein expression. Targeting BRCA1 in triple negative breast cancer with natural withanolides represents a novel approach for future translational studies in this difficult disease.