Molecular Insights into the Anticancer Activity of Withaferin-A: The Inhibition of Survivin Signaling

Simple Summary The inactivation of apoptotic signaling by inhibitors of apoptosis proteins (IAPs) is one of the important hallmarks of cancer cells. Survivin, a 16.5 kDa member of the IAP family, is commonly enriched in many cancers and is a potential therapeutic target. We investigated the Survivin-targeting potential of Withaferin-A (Wi-A) and Withanone (Wi-N), two major withanolides from Withania somnifera (Ashwagandha), using computational assays. The results were validated in various in vitro experimental assays using Wi-A-rich extracts from Ashwagandha leaves, suggesting their use as an important bioresource for cancer drug development.Abstract Survivin, a member of the IAP family, functions as a homodimer and inhibits caspases, the key enzymes involved in apoptosis. Several Survivin inhibitors, including YM-155, Debio1143, EM1421, LQZ-7I, and TL32711, have emerged as potential anticancer drugs awaiting validation in clinical trials. Due to the high cost and adverse side effects of synthetic drugs, natural compounds with similar activity have also been in demand. In this study, we conducted molecular docking assays to evaluate the ability of Wi-A and Wi-N to block Survivin dimerization. We found that Wi-A, but not Wi-N, can bind to and prevent the homodimerization of Survivin, similar to YM-155. Therefore, we prepared a Wi-A-rich extract from Ashwagandha leaves (Wi-AREAL). Experimental analyses of human cervical carcinoma cells (HeLa and ME-180) treated with Wi-AREAL (0.05-0.1%) included assessments of viability, apoptosis, cell cycle, migration, invasion, and the expression levels (mRNA and protein) of molecular markers associated with these phenotypes. We found that Wi-AREAL led to growth arrest mediated by the upregulation of p21WAF1 and the downregulation of several proteins (CDK1, Cyclin B, pRb) involved in cell cycle progression. Furthermore, Wi-AREAL treatment activated apoptosis signaling, as evidenced by reduced PARP-1 and Bcl-2 levels, increased procaspase-3, and elevated Cytochrome C. Additionally, treating cells with a nontoxic low concentration (0.01%) of Wi-AREAL inhibited migration and invasion, as well as EMT (epithelial-mesenchymal transition) signaling. By combining computational and experimental approaches, we demonstrate the potential of Wi-A and Wi-AREAL as natural inhibitors of Survivin, which may be helpful in cancer treatment.