ABSTRACT
NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic flavoprotein that utilizes NRH as electron donor. The present studies investigate the role of NQO2 in metabolic detoxification/activation of quinones and quinone based anti-tumor drugs. Chinese hamster ovary (CHO) cells stably overexpressing cDNA derived mouse NQO2 and mouse keratinocytes from DMBA-induced skin tumors in wild-type and NQO2-null mice were generated. The CHO cells overexpressing NQO2 and mouse keratinocytes expressing or deficient in NQO2 were treated with varying concentrations of mitomycin C (MMC), CB1954, MMC analog BMY25067, EO9, menadione and BP-3,6-quinone, in the absence and presence of NRH. The cytotoxicity of the drugs was evaluated by colony formation. The CHO cells overexpressing higher levels of mouse NQO2 showed significantly increased cytotoxicity to menadione, BP-3,6-quinone and to the anti-tumor drugs MMC and CB1954 when compared to CHO cells expressing endogenous NQO2. The cytotoxicity increased in presence of NRH. Similar results were also observed with BMY25067 and EO9 treatments, but to a lesser extent. The results on keratinocytes deficient in NQO2 supported the data from CHO cells. The inclusion of NRH had no effect on cytotoxicity of quinones and drugs in keratinocytes deficient in NQO2. Mouse NQO2 protein was expressed in bacteria, purified and used to study the role of NQO2 in MMC-induced DNA cross-linking. Bacterially expressed and purified NQO2 efficiently catalyzed MMC activation that led to DNA cross-linking. These results concluded that NQO2 plays a significant role in the metabolic activation of both quinones and anti-tumor drugs leading to cytotoxicity and cell death.
Subject(s)
Antineoplastic Agents/pharmacology , Biotransformation/drug effects , Quinone Reductases/metabolism , Quinones/pharmacology , Animals , Antineoplastic Agents/metabolism , Aziridines/metabolism , Aziridines/pharmacology , Benzopyrenes/metabolism , Benzopyrenes/pharmacology , CHO Cells , Cell Survival/drug effects , Cells, Cultured , Cricetinae , Cricetulus , Cross-Linking Reagents/metabolism , Cross-Linking Reagents/pharmacology , DNA, Complementary/genetics , Dose-Response Relationship, Drug , Hydroquinones/metabolism , Hydroquinones/pharmacology , Indolequinones/metabolism , Indolequinones/pharmacology , Keratinocytes/cytology , Keratinocytes/drug effects , Keratinocytes/metabolism , Mice , Mice, Knockout , Mitomycin/metabolism , Mitomycin/pharmacology , Mitomycins/metabolism , Mitomycins/pharmacology , Quinone Reductases/genetics , Quinones/metabolism , Transfection , Vitamin K 3/metabolism , Vitamin K 3/pharmacologyABSTRACT
Mitomycin C (MMC) is an anticancer drug that requires reductive activation to exert its toxicity. MMC is known to cross-link DNA that contributes significantly to the cytotoxicity and consequent cell death. Cytosolic NADPH:quinone oxidoreductase 1 (NQO1) and microsomal enzymes have been shown to mediate MMC-induced DNA cross-linking. However, NQO1 plays only a minor role, indicating presence of other cytosolic enzymes/proteins that contribute to this process. In this study, we have characterized a unique cytosolic activity in NQO1-null mice that catalyzed MMC-induced DNA cross-linking. This activity was cofactor independent and dicoumarol insensitive. The unique cytosolic activity was purified to homogeneity. The peptide sequencing of the purified protein identified the unique cytosolic activity as GRP58 (M(r) 58,000 glucose-regulatory protein), also known as GRp57/ER60/ERp61/HIP-70/Q2 and CPT. Immunodepletion of NQO1-null mice liver cytosol and partially purified fractions with anti-GRP58 antibody led to a complete loss of GRP58 protein and consequent significant reduction of MMC-induced DNA cross-linking. Mouse cDNA encoding GRP58 was isolated and sequenced. Chinese hamster ovary cells permanently overexpressing GRP58 showed increased MMC-induced DNA cross-linking and increased cytotoxicity on exposure to MMC. Bacterially expressed and purified GRP58 increased the MMC-induced DNA cross-linking when added to mouse cytosolic samples. A tissue array analysis indicated that GRP58 is ubiquitously expressed among mouse tissues, although at different levels. Expression analysis using matched human tumor/normal array revealed an up-regulation of GRP58 in breast, uterus, lung, and stomach tumors compared with normal tissues of similar origin.
