Reduction of mitomycin C is catalysed by human recombinant NRH:quinone oxidoreductase 2 using reduced nicotinamide adenine dinucleotide as an electron donating co-factor

Jamieson, David; Tung, Alvin; Knox, Richard J.; Boddy, Alan V.

doi:10.1038/sj.bjc.6603414

Cited by 24 publications

(22 citation statements)

References 16 publications

(17 reference statements)

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“…This enzyme has been shown to be important for the prodrug activation of mitomycin C [38,39], an anticancer antibiotic used for treatment of bladder cancer and other tumours, and for product activation of the synthetic anticancer agent CB1954 that is currently in clinical trials [40]. QR2 is capable of metabolically activating various quinones and other compounds, via either 2- or 4-electron reductions, which can ultimately lead to cytotoxicity and cell death [38,41].…”

Section: Resultsmentioning

confidence: 99%

Pleiotropic mechanisms facilitated by resveratrol and its metabolites

Calamini

Ratia

Malkowski

et al. 2010

Biochemical Journal

155

126

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Resveratrol has demonstrated cancer chemopreventive activity in animal models and some clinical trials are underway. In addition, resveratrol was shown to promote cell survival, increase lifespan and mimic caloric restriction, thereby improving health and survival of mice on high-calorie diet. All of these effects are potentially mediated by the pleiotropic interactions of resveratrol with different enzyme targets including COX-1 (cyclo-oxygenase-1) and COX-2, NAD+-dependent histone deacetylase SIRT1 (sirtuin 1) and QR2 (quinone reductase 2). Nonetheless, the health benefits elicited by resveratrol as a direct result of these interactions with molecular targets have been questioned, since it is rapidly and extensively metabolized to sulfate and glucuronide conjugates, resulting in low plasma concentrations. To help resolve these issues, we tested the ability of resveratrol and its metabolites to modulate the function of some known targets in vitro. In the present study, we have shown that COX-1, COX-2 and QR2 are potently inhibited by resveratrol, and that COX-1 and COX-2 are also inhibited by the resveratrol 4′-O-sulfate metabolite. We determined the X-ray structure of resveratrol bound to COX-1 and demonstrate that it occupies the COX active site similar to other NSAIDs (non-steroidal anti-inflammatory drugs). Finally, we have observed that resveratrol 3- and 4′-O-sulfate metabolites activate SIRT1 equipotently to resveratrol, but that activation is probably a substrate-dependent phenomenon with little in vivo relevance. Overall, the results of this study suggest that in vivo an interplay between resveratrol and its metabolites with different molecular targets may be responsible for the overall beneficial health effects previously attributed only to resveratrol itself.

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Section: Resultsmentioning

confidence: 99%

Pleiotropic mechanisms facilitated by resveratrol and its metabolites

Calamini

Ratia

Malkowski

et al. 2010

Biochemical Journal

155

126

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“…Belcourt et al (13) and Seow et al (14) also used clonogenic assays to assess cytotoxicity and it may be that this assay selects for cells with altered sensitivity to the drug. Selected clones may express other enzymes reported to activate MMC such as DT-diaphorase (17, 35), NRH:quinone oxidoreductase 2 (37, 38), NADPH-ferrodoxin reductase (39) or cytochrome b5 reductase (9); they may also express antioxidants that detoxify and protect against ROS-induced damage. In this regard, antioxidant enzymes including glutathione peroxidase, superoxide dismutase and catalase have all been shown to reduce or abolish MMC cytotoxicity in tumor cells in vitro (29, 40).…”

Section: Discussionmentioning

confidence: 99%

“…This is supported by our findings that hypoxia has no effect on MMC-induced cytotoxicity. Other enzymes in CHO-OR cells that can mediate the two electron reduction of MMC include NRH:quinone oxidoreductase (37, 38). We also cannot exclude the possibility that other enzymes mediating the one electron reduction of MMC including NADH-cytochrome b5 reductase, xanthine oxidase and nitric oxide synthase also mediate cytotoxicity in these cells.…”

Section: Discussionmentioning

confidence: 99%

Distinct Roles of Cytochrome P450 Reductase in Mitomycin c Redox Cycling and Cytotoxicity

Wang

Gray

Mishin

et al. 2010

Molecular Cancer Therapeutics

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Mitomycin c (MMC), a quinone-containing anticancer drug, is known to redox cycle and generate reactive oxygen species. A key enzyme mediating MMC redox cycling is cytochrome P450 reductase, a microsomal NADPH-dependent flavoenzyme. In the present studies, CHO cells overexpressing this enzyme (CHO-OR cells) and corresponding control cells (CHO-WT cells) were used to investigate the role of cytochrome P450 reductase in the actions of MMC. In lysates from both cell types, MMC was found to redox cycle and generate H2O2; this activity was greater in CHO-OR cells (Vmax = 1.2 ± 0.1 nmol H2O2/min/mg protein in CHO-WT cells vs. 32.4 ± 3.9 nmol H2O2/min/mg protein in CHO-OR cells). MMC was also more effective in generating superoxide anion and hydroxyl radicals in CHO-OR cells, relative to CHO-WT cells. Despite these differences in MMC redox cycling, MMC-induced cytotoxicity, as measured by growth inhibition, was similar in the two cell types (IC50 = 72 ± 20 nM for CHO-WT and 75 ± 23 nM for CHO-OR cells), as was its ability to induce G2/M and S phase arrest. Additionally, in 9 different tumor cell lines, although a strong correlation was observed between MMC-induced H2O2 generation and cytochrome P450 reductase activity, there was no relationship between redox cycling and cytotoxicity. Hypoxia, which stabilizes MMC radicals generated by redox cycling, also had no effect on the sensitivity of tumor cells to MMC-induced cytotoxicity. These data indicate that NADPH cytochrome P450 reductase-mediated MMC redox cycling is not involved in cytotoxicity of this chemotherapeutic agent.

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“…This suggest that either the MDA468 cells have sufficient NRH to catalyze turnover of NQO2 or alternatively, that other pyridine nucleotides in the cell can serve as a co-factor for NQO2. Interestingly, recent work showed that NQO2 can catalyze the reduction of mitomycin C using NADH as co-factor (Jamieson et al, 2006). The respective efficiencies of NQO1 and NQO2 at bioactivation of RH1 require further study.…”

Section: Discussionmentioning

confidence: 99%

Dissecting the Role of Multiple Reductases in Bioactivation and Cytotoxicity of the Antitumor Agent 2,5-Diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1)

Yan

Kepa²,

Siegel³

et al. 2008

Mol Pharmacol

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2,5-Diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1) is a novel antitumor diaziridinyl benzoquinone derivative designed to be bioactivated by the two-electron reductase NAD(P)H:quinone oxidoreductase (NQO1) and is currently in clinical trials. NQO1 is expressed at high levels in many solid tumors. RH1 cytotoxicity has been shown previously to be NQO1-dependent. The purpose of this study was to investigate whether other reducing enzymes such as cytochrome b 5 reductase (b5R), cytochrome P450 reductase (P450R), dihydronicotinamide riboside:quinone oxidoreductase 2 (NQO2), and xanthine oxidase/xanthine dehydrogenase (XO/XDH) also contribute to the bioactivation and cytotoxicity of RH1 in human tumor cells. For these studies, we established a series of stable MDA468 breast cancer cell lines overexpressing various levels of NQO1, b5R, P450R, and NQO2 and compared RH1-induced growth inhibition [3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium and sulforhodamine B analysis] and interstrand DNA cross-linking (comet analysis) in both parental MDA468 cells and transfected clones. RH1 toxicity correlated with NQO1 and NQO2 but not with either b5R or P450R activity levels in the respective series of transfected MDA468 cell clones. Enzymatic assays showed that RH1 was an in vitro substrate for xanthine oxidase. However, XO/XDH protein and activity could not be detected in a variety of human tumor cell lines. These studies suggest that NQO1 and NQO2 are the principal enzymatic determinants of RH1 bioactivation in MDA468 tumor cells and that b5R, P450R, and XDH/XO are unlikely to play major roles. Our studies also suggest that NQO2 may be particularly relevant as a bioactivation system for RH1 in NQO1-deficient tumors such as leukemias and lymphomas.

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Reduction of mitomycin C is catalysed by human recombinant NRH:quinone oxidoreductase 2 using reduced nicotinamide adenine dinucleotide as an electron donating co-factor

Cited by 24 publications

References 16 publications

Pleiotropic mechanisms facilitated by resveratrol and its metabolites

Pleiotropic mechanisms facilitated by resveratrol and its metabolites

Distinct Roles of Cytochrome P450 Reductase in Mitomycin c Redox Cycling and Cytotoxicity

Dissecting the Role of Multiple Reductases in Bioactivation and Cytotoxicity of the Antitumor Agent 2,5-Diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1)

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