Disulfiram is a direct and potent inhibitor of human O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells and mouse brain and markedly increases the alkylating DNA damage

Abstract

The alcohol aversion drug disulfiram (DSF) reacts and conjugates with the protein-bound nucleophilic cysteines and is known to elicit anticancer effects alone or improve the efficacy of many cancer drugs. We investigated the effects of DSF on human O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein and chemotherapy target that removes the mutagenic O(6)-akyl groups from guanines, and thus confers resistance to alkylating agents in brain tumors. We used DSF, copper-chelated DSF or CuCl2-DSF combination and found that all treatments inhibited the MGMT activity in two brain tumor cell lines in a rapid and dose-dependent manner. The drug treatments resulted in the loss of MGMT protein from tumor cells through the ubiquitin-proteasome pathway. Evidence showed that Cys145, a reactive cysteine, critical for DNA repair was the sole site of DSF modification in the MGMT protein. DSF was a weaker inhibitor of MGMT, compared with the established O(6)-benzylguanine; nevertheless, the 24-36h suppression of MGMT activity in cell cultures vastly increased the alkylation-induced DNA interstrand cross-linking, G2/M cell cycle blockade, cytotoxicity and the levels of apoptotic markers. Normal mice treated with DSF showed significantly attenuated levels of MGMT activity and protein in the liver and brain tissues. In nude mice bearing T98 glioblastoma xenografts, there was a preferential inhibition of tumor MGMT. Our studies demonstrate a strong and direct inhibition of MGMT by DSF and support the repurposing of this brain penetrating drug for glioma therapy. The findings also imply an increased risk for alkylation damage in alcoholic patients taking DSF.

Disulfiram when Combined with Copper Enhances the Therapeutic Effects of Temozolomide for the Treatment of Glioblastoma

“…Thus, the independent validation of activity against the patient-derived glioma cells, DSF's low nanomolar efficacy on these cells, BBB penetration, and its extensive multi-decade clinical use with very little evidence of adverse drug reactions made it desirable to pursue in a rigorous preclinical assessment regime. DSF requires copper for effective killing and inhibition of self-renewal of genetically distinct patient-derived BTICs As described above, DSF was found to be highly effective at killing a wide range of patient-derived BTICs, an observation consistent with recent in vitro reports using human glioma cell lines (17,18,41,43); however, in vivo testing has not been performed and discrepancies with respect to the IC 50 and the requirement of copper for its therapeutic effects exist (17,18). We found that while some patient-derived BTICs were sensitive to the treatment with DSF alone (Fig.…”

“…Of note, a recent study showed that DSF directly inhibited the expression of MGMT, an enzyme that removes O6 akyl groups from guanine, a repair function in error-free DNA replication (43). However, the reduction of MGMT occurred at concentrations of DSF significantly higher than the IC 50 of the BTICs.…”

“…DSF inhibits both the proteasome and reduces DNA repair capabilities DSF has been reported to have several biologic activities including its well-characterized activity as an aldehyde dehydrogenase (ALDH) inhibitor for the treatment of alcoholism, its ability to be an effective proteasome inhibitor (26S), and recently, its ability to inhibit the activity of MGMT (43). Moreover, there is growing literature to suggest that the anticancer properties of DSF do not rely solely on its ability to inhibit ALDH (45), a mechanism that does not require Cu.…”

Influence of fatty acid synthase inhibitor orlistat on the DNA repair enzyme O6-methylguanine-DNA methyltransferase in human normal or malignant cells in vitro

“…The finding that the compound is unable to downregulate consistently MGMT function within 2 h of treatment (Table I), does not support the hypothesis that orlistat, similarly to LM, could behave as pseudo-substrate able to bind and inactivate MGMT, followed by a proteasome-dependent degradation process (17,30,31).…”

“…The MGMT protein behaves as an acceptor of the methyl adduct that is transferred in a stoichiometric reaction from DNA O 6 -MeG to a cysteine residue associated with the active site of MGMT. It follows that the biochemical lesion is repaired, whereas MGMT is inactivated according to a sort of suicide process (21) leading to ubiquitination followed by proteasome-mediated degradation of the repair protein (17,30,31). Affected cells restore their MGMT levels through an entirely de novo synthesis of the protein.…”