Apoptosis Induction, Disruption of Cytoskeleton Remodeling and <scp>EMT</scp> Inhibition as Major Mechanism(s) Underlying the Pleiotropic Action of <i>Withania somnifera</i> in Breast Cancer

The role of actin and its binding proteins has been discovered in cytoskeleton remodeling as well as in Epithelial-Mesenchymal Transition (EMT) of metastatic cells and apoptosis. Even minor changes in the biomolecular structure of actin and its ABPs (by binding of ligands) can lead to drastic changes in the cytoskeleton with far reaching effects per se. Agents targeting actin can, thus, be viewed as potential anti-metastatic agents. The effect of Withania somnifera (L.) Dunal (WS) on the cytoskeleton has remained relatively unexplored. The present study highlights the interaction between 20 WS phytoconstituents and 10 selected cytoskeletal proteins in silico with a view to validate and analyze the perturbation in growth and differentiation of breast cancer cells in vitro. Pharmacokinetic analyses revealed that the majority of WS phytoconstituents exhibited no violations of Lipinski's rule-of-five parameters. Withanolides A, B, D, M and O displayed the greatest binding affinity particularly for coronin1A, vimentin, gelsolin, ezrin and F-actin. MD simulations of 100 ns revealed maximum stable interaction(s) between Coronin-Viscosalactone B (VISCB) and Vimentin-Withanolide E (WITHE). The prepared methanolic extract of WS stem (WSME), characterized using LC-MS, revealed the presence of Withaferin A (WFA). Both WSME and WFA exhibited potent cytotoxicity against breast cancer MDA-MB-231 cells. WSME increased ROS levels, arrested the cell cycle in S and G2-M phases, decreased the expression of mesenchymal markers, namely, vimentin, N-cadherin and increased levels of the epithelial marker E-cadherin in treated MDA-MB-231 cells. These findings suggest that VISCB, WITHE, and WFA have the potential to emerge as potential antimetastatic agents against breast cancer in the future.