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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. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: The Hsp70 family stress chaperone mortalin plays an important role in carcinogenesis and hence has emerged as a candidate target for cancer drug development. Mortaparibs (Mortaparib, MortaparibPlus, and MortaparibMild) are chemically synthesized small molecules isolated as inhibitors of mortalin–p53 interaction. They also downregulate mortalin and PARP1 expression and block cancer signaling in multiple ways. In this study, we demonstrate (i) the anticancer activity of MortaparibMild in p53-null cancer cells and (ii) combinations of three Mortaparibs for enhancing their potency for treating the hyper-proliferation, -migration, and -stress phenotypes of cancer cells. Mortalin, a member of the Hsp70 family of proteins, is commonly enriched in many types of cancers. It promotes carcinogenesis and metastasis in multiple ways of which the inactivation of the tumor suppressor activity of p53 has been firmly established. The downregulation of mortalin and/or disruption of mortalin–p53 interactions by small molecules has earlier been shown to activate p53 function yielding growth arrest/apoptosis in cancer cells. Mortaparibs (Mortaparib, MortaparibPlus, and MortaparibMild) are chemical inhibitors of mortalin isolated by cell-based two-way screening involving (i) a shift in the mortalin staining pattern from perinuclear (characteristics of cancer cells) to pancytoplasmic (characteristics of normal cells) and (ii) the nuclear enrichment of p53. They have similar structures and also cause the inhibition of PARP1 and hence were named Mortaparibs. In the present study, we report the anticancer and anti-metastasis activity of MortaparibMild (4-[(4-amino-5-thiophen-2-yl-1,2,4-triazol-3-yl)sulfanylmethyl]-N-(4-methoxyphenyl)-1,3-thiazol-2-amine) in p53-null cells. By extensive molecular analyses of cell proliferation, growth arrest, and apoptosis pathways, we demonstrate that although it causes relatively weaker cytotoxicity compared to Mortaparib and MortaparibPlus, its lower concentrations were equally potent to inhibit cell migration. We developed combinations (called MortaparibMix-AP, MortaparibMix-AM, and MortaparibMix-AS) consisting of different ratios of three Mortaparibs for specifically enhancing their anti-proliferation, anti-migration, and antistress activities, respectively. Based on the molecular analyses of control and treated cells, we suggest that the three Mortaparibs and their mixtures may be considered for further laboratory and clinical studies validating their use for the treatment of cancer as well as prevention of its relapse and metastasis. [ABSTRACT FROM AUTHOR]