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

Abstract

NRH:Quinone Oxidoreductase 2 (NQO2) has been described as having no enzymatic activity with nicotinamide adenine dinucleotide (NADH) or NADPH as electron donating cosubstrates. Mitomycin C (MMC) is both a substrate for and a mechanistic inhibitor of the NQO2 homologue NQO1. NRH:quinone oxidoreductase 2 catalysed the reduction of MMC at pH 5.8 with NADH as a co-factor. This reaction results in species that inhibit the NQO2-mediated metabolism of CB1954. In addition, MMC caused an increase in DNA cross-links in a cell line transfected to overexpress NQO2 to an extent comparable to that observed with an isogenic NQO1-expressing cell line. These data indicate that NQO2 may contribute to the metabolism of MMC to cytotoxic species.

Figure 1

Reduction of MMC catalysed by hrNQO1 and hrNQO2 compared with BSA : lactose : PBS and no protein controls. At pH 5.8 the mean MMC reduced in the hrNQO1 reaction (30±3.4 μM) and the hrNQO2 (35±4.9 μM) reaction were significantly less than the MMC reduced in the no protein control (P=0.002 and 0.002, respectively, t-test). Reactions were carried out at pH indicated and room temperature for 30 min using 4 mM NADH, 100 μM MMC. Data are mean and s.e. of 5 (pH 5.8) or three independent experiments.

Figure 2

Reduction of MMC catalysed by hrNQO2±quercetin or dicoumarol. Incubation with 100 μM quercetin and hrNQO2 resulted in inhibition of reduction of MMC (P=0.002 compared with no inhibitor). Incubation with 100 μM dicoumarol and hrNQO2 resulted in partial inhibition of reduction of MMC (P=0.017 compared with no inhibitor). Reactions were carried out at pH 5.8, room temperature for 30 min using 100 μM MMC and 4 mM NADH. Data are mean and s.e. of three independent experiments.

Figure 3

Reduction of CB1954 at 37°C, pH 6.3 using NRH as an electron donor and 200 μl aliquots of a 30 min preincubation of 16.2 nM hrNQO2 with or without MMC or NADH as described in the text. MMC added (rhombi) represents the addition of MMC at time 0 of the CB1954 reaction (difference between MMC+NADH+ and MMC+NADH−, P=0.0033, 2 Way ANOVA). Lines are second-order curves used to calculate initial velocity. Data are the mean and s.e. of three independent experiments.

Figure 4

Comet olive tail moment of plasmid (F179, white), NQO2 (TH7, grey) or NQO1 (hdt, black) transformed cells following incubation with or without 10 μM MMC. Data represent the mean and s.e. from one of three independent experiments. ^***=P-value of <0.0001, t-test of mean compared with untreated control.