Abstract 1216: Thiol-modification induced reactivation of mutant p53 protein in human cancers using a nontoxic stabilized homoglutathione disulfide

Loss of functional p53 gene is a frequent and most critical event in the genesis and progression of half of all human cancers. Conversion of the non-DNA binding and misfolded-p53 mutant proteins to their wild-type conformation using small molecules to induce apoptosis is currently an intense area of research. Although a few compounds with this property have been described, their abilities to alter the p53 structure in animal models have not been documented and they appear toxic as well. Human cancers harbor elevated levels of oxidative stress and are vulnerable to even slight redox oscillations. To exploit this susceptibility, we have designed a glutathione mimic, namely, homoglutathione disulfide that has been stabilized with the addition of small amounts of cisplatin (1000:10). This compound perturbs the cellular redox, alters kinase activities and induces protein-glutathionylation. Since the human p53 contains several redox-sensitive cysteines in the DNA-binding domain, we explored the possible reactivation of mutant p53 (R273H) present in HT29 and SW620 colon cancer cells by the stabilized hGSSG.Pt (hGTX). Electrophoretic mobility shift assays demonstrated a concentration and time-dependent restoration of DNA-binding for the mutant p53 in HT29 cells by hGTX. DNA affinity immunoblotting, immunoprecipitation and direct western blotting using specific antibodies against the mutant (Mab240) or wild type p53 (Mab1620) or pan p53 (MabDo1) confirmed a progressive generation of wild type p53 and a gradual decline of mutant p53 levels in HT29 cells. The half-life of the altered p53 remained similar to the endogenous mutant form. Treatment of HT29 nuclear extracts with hGTX also induced the binding of mutant p53 with DNA, indicating that thiolation of specific cysteines mediates the reactivation. RT-PCR and western analyses showed significant upregulation of many p53 target genes (2-5 fold). Furthermore, when nude mice bearing the HT29 xenografts were administered HGTX (100 mg/kg daily for 7 days), marked p53 reactivation with concomitant reduction of mutant p53 was observed in tumor tissues. Treatment of hGTX also modulated redox sensitive DNA repair systems like MGMT and Topoisomerase-I. A highly significant amplification of cytotoxicity was induced by anticancer drugs in association with hGTX. We conclude that the alteration of redox milieu following hGTX modifies the reactive thiols in the p53 protein to restore the native conformation. These findings acquired with a nontoxic endogenous metabolite suggest therapeutic applications in combination with chemotherapy agents. Molecular modeling and mass spectrometry studies are underway to determine the structural changes and residues altered by hGTX treatment (Supported by grants from CPRIT (RP130266), NIH (R03CA125872) to KSS. Citation Format: Debasish Basak, Kalkunte S. Srivenugopal. Thiol-modification induced reactivation of mutant p53 protein in human cancers using a nontoxic stabilized homoglutathione disulfide. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1216. doi:10.1158/1538-7445.AM2015-1216