We have earlier reported anticancer activity in Withaferin A (Wi-A), a withanolide derived from Ashwagandha (Withania somnifera) and caffeic acid phenethyl ester (CAPE), an active compound from New Zealand honeybee propolis. Whereas Wi-A was cytotoxic to both cancer and normal cells, CAPE has been shown to cause selective death of cancer cells. In the present study, we investigated the efficacy of Wi-A, CAPE, and their combination to ovarian and cervical cancer cells. Both Wi-A and CAPE were seen to activate tumor suppressor protein p53 by downregulation of mortalin and abrogation of its interactions with p53. Downregulation of mortalin translated to compromised mitochondria integrity and function that affected poly ADP-ribose polymerase1 (PARP1); a key regulator of DNA repair and protein-target for Olaparib, drugs clinically used for treatment of breast, ovarian and cervical cancers)-mediated DNA repair yielding growth arrest or apoptosis. Furthermore, we also compared the docking capability of Wi-A and CAPE to PARP1 and found that both of these could bind to the catalytic domain of PARP1, similar to Olaparib. We provide experimental evidences that (i) Wi-A and CAPE cause inactivation of PARP1-mediated DNA repair leading to accumulation of DNA damage and activation of apoptosis signaling by multiple ways, and (ii) a combination of Wi-A and CAPE offers selective toxicity and better potency to cancer cells.
p53 has an essential role in suppressing the carcinogenesis process by inducing cell cycle arrest/apoptosis/senescence. Mortalin/GRP75 is a member of the Hsp70 protein family that binds to p53 causing its sequestration in the cell cytoplasm. Hence, p53 cannot translocate to the nucleus to execute its canonical tumour suppression function as a transcription factor. Abrogation of mortalin-p53 interaction and subsequent reactivation of p53’s tumour suppression function has been anticipated as a possible approach in developing a novel cancer therapeutic drug candidate. A chemical library was screened in a high-content screening system to identify potential mortalin-p53 interaction disruptors. By four rounds of visual assays for mortalin and p53, we identified a novel synthetic small-molecule triazole derivative (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]-1,2,4-triazole, henceforth named MortaparibPlus). Its activities were validated using multiple bioinformatics and experimental approaches in colorectal cancer cells possessing either wild-type (HCT116) or mutant (DLD-1) p53. Bioinformatics and computational analyses predicted the ability of MortaparibPlus to competitively prevent the interaction of mortalin with p53 as it interacted with the p53 binding site of mortalin. Immunoprecipitation analyses demonstrated the abrogation of mortalin-p53 complex formation in MortaparibPlus-treated cells that showed growth arrest and apoptosis mediated by activation of p21WAF1, or BAX and PUMA signalling, respectively. Furthermore, we demonstrate that MortaparibPlus-induced cytotoxicity to cancer cells is mediated by multiple mechanisms that included the inhibition of PARP1, up-regulation of p73, and also the down-regulation of mortalin and CARF proteins that play critical roles in carcinogenesis. MortaparibPlus is a novel multimodal candidate anticancer drug that warrants further experimental and clinical attention.
Background Tumor suppressor p53 protein is frequently mutated in a large majority of cancers. These mutations induce local or global changes in protein structure thereby affecting its binding to DNA. The structural differences between the wild type and mutant p53 thus provide an opportunity to selectively target mutated p53 harboring cancer cells. Restoration of wild type p53 activity in mutants using small molecules that can revert the structural changes have been considered for cancer therapeutics. Methods We used bioinformatics and molecular docking tools to investigate the structural changes between the wild type and mutant p53 proteins (p53 V143A , p53 R249S , p53 R273H and p53 Y220C ) and explored the therapeutic potential of Withaferin A and Withanone for restoration of wild type p53 function in cancer cells. Cancer cells harboring the specific mutant p53 proteins were used for molecular assays to determine the mutant or wild type p53 functions. Results We found that p53 V143A mutation does not show any significant structural changes and was also refractory to the binding of withanolides. p53 R249S mutation critically disturbed the H-bond network and destabilized the DNA binding site. However, withanolides did not show any selective binding to either this mutant or other similar variants. p53 Y220C mutation created a cavity near the site of mutation with local loss of hydrophobicity and water network, leading to functionally inactive conformation. Mutated structure could accommodate withanolides suggesting their conformational selectivity to target p53 Y220C mutant. Using human cell lines containing specific p53 mutant proteins, we demonstrated that Withaferin A, Withanone and the extract rich in these withanolides caused restoration of wild type p53 function in mutant p53 Y220C cells. This was associated with induction of p21 WAF-1 -mediated growth arrest/apoptosis. Conclusion The study suggested that withanolides may serve as highly potent anticancer compounds for treatment of cancers harboring a p53 Y220C mutation. Electronic supplementary material The online version of this article (10.1186/s13046-019-1099-x) contains supplementary material, which is available to authorized users.
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