Novel Pathways of Oxidative and Nitrosative Inactivation of the Human MGMT Protein in Colon Cancer and Glioblastoma Cells: Increased Efficacy of Alkylating Agents In Vitro and In Vivo

Background: O⁶-Methylguanine-DNA methyltransferase (MGMT) is a unique antimutagenic DNA repair protein that plays a crucial role in conferring resistance to various alkylating agents in brain tumor therapy. In this study, we exploited the susceptibility of the active site Cys145 of MGMT for thiolation and nitrosylation, both of which inactivate the enzyme. Methods: We designed a redox perturbing glutathione mimetic, a platinated homoglutathione disulfide (hGTX) by adding small amounts of cisplatin (1000:10) and used a nitric oxide-donor spermine NONOate. N6022, a potent inhibitor of S-nitrosoglutathione reductase was used to extend the retention of nitrosylated MGMT in tumor cell culture and subcutaneous xenografts. Results: Both hGTX and spermine NONOate inhibited MGMT activity in HT29, SF188, T98G, and other brain tumor cells. There was a robust increase in the alkylation-induced DNA interstrand cross-linking, G2/M cell cycle arrest, cytotoxicity, and the levels of apoptotic markers when either of the agents was used with alkylating agents. In the nude mice bearing T98G and HT29-luc2 xenografts, combinations of hGTX and spermine NONOate with alkylating agents produced a marked reduction in MGMT protein and tumor growth delay and regressions. N6022 treatment increased the presence of nitrosylated MGMT for a longer time, thereby extending the DNA-repair deficient state both in cell culture and preclinical settings. Conclusions: Our findings highlight the options for redox-driven therapeutic strategies for MGMT and suggest that oxidative and/or nitrosative inactivation of DNA repair in combination with alkylating agents could be exploited.