Studies on the mechanism of toxicity of the novel NQO1-directed antitumor agent, RH1.

摘要

NAD(P)H:Quinone Oxidoreductase 1 (NQO1), a flavoprotein, is classified as an obligate two-electron reductase. It is characterized by its use of either NADH or NADPH as reducing cofactors and its inhibition by dicoumarol. Much of the data generated on NQO1 focused on its detoxification of natural and exogenous quinones to hydroquinones to yield substrates for Phase II conjugation reactions. Two-electron reduction is not always a detoxification mechanism however, as certain quinones can result in the formation of toxic hydroquinones. Considerable attention has been focused on antitumor quinones containing aziridinyl functional groups as reduction of the quinone can result in the facile protonation of the aziridine ring leading to ring opening and alkylation of nucleophilic sites.; The overall purpose of these studies was to determine if RH1, a novel chemotherapeutic quinone compound was bioactivated to an alkylating species by NQO1 and to define the resulting in vitro and in vivo consequences. In addition, the impact of the NQO1*2/*2 polymorphism on the potential use of RH1 as an antitumor quinone was investigated in these studies.; Isogenic cell lines differing only in expression of wild-type NQO1 were developed to provide a model for evaluation of the role of NQO1 in the bioactivation of antitumor quinones. After preliminary screening, one isogenic cell line pair, MDA468 human breast adenocarcinoma cells and NQ16 cells that were transfected with wild-type NQO1 cDNA, were fully characterized and used in these studies. The use of a mechanism-based inhibitor of NQO1, ES936, was validated in cellular systems. ES936 provided a pharamacological tool to define the role of NQO1 bioactivation of antitumor quinones. The potential role of a polymorphic variant of NQO1, NQO1*2, in bioactivation of antitumor quinones in MDA468 cells was investigated. The effects of NQO1 on RH1 induced growth inhibition, DNA cross-linking, apoptosis and cell cycle alterations in the MDA isogenic cell line pair were defined. An isogenic human tumor xenograft model was developed to evaluate the ability of RH1 to induce NQO1-dependent growth inhibition or tumor regression. These data add to the evidence that NQO1 may be exploited as an activating enzyme for chemotherapeutic quinones.