Non-enzymatic and enzymatic activation of mitomycin C: Identification of a unique cytosolic activity

Abstract

Mitomycin C (MMC), an alkylating anti-tumor agent, was activated by non-enzymatic and enzymatic mechanisms leading to DNA binding and adduct formation. However, it was enzymatically, not non-enzymatically, activated MMC which induced inter-strand DNA cross-linking, a major determinant of cell death. The enzymatic activation of MMC was catalyzed by microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic enzyme activities. Human P450 reductase, transiently expressed from its cDNA in the COS I cells, metabolically activated MMC to generate 9 specific MMC-DNA adducts and induced inter-strand DNA cross-linking. Co-chromatography of the MMC-DNA adducts generated by P450 reductase and sodium borohydride in separate experiments indicated that MMC was metabolized by P450 reductase to produce 2,7-diaminomitosenes that exhibited binding to deoxyguanosine. Several experiments indicated that cytosolic enzymes which catalyzed reductive activation of MMC and DNA cross-linking included NAD(P)H:quinone oxidoreductase, (NQO, or DT diaphorase) when present in extremely high concentrations and a unique cytosolic activity. The unique cytosolic activity was present in several mammalian cells and mouse colon and liver but absent in mouse kidney. The unique activity had properties of a diaphorase but was distinct from NQO, because of a lack of correlation between NQO, (2,6-dichlorophenolindophenol reduction) activity and the amount of MMC-reductive activation leading to DNA cross-linking. This activity was also distinct from xanthine oxidoreductase and NADHcytochrome b5 reductase, 2 other enzymes that catalyze metabolic activation of MMC, because the unique activity was not inhibited by allopurinol (an inhibitor of xanthine oxidoreduttase) and i t s activity was the same with NADH and NADPH (cytochrome b5 reductase is specific to NADH).o 1996 Wiley-Liss, Inc.Mitomycin C (MMC), a prototypical alkylating anti-tumor agent isolated from Streptomyces caespitotus, is therapeutically effective against tumors of human and experimental animals (Venveij and Pinedo, 1990). It was believed that the reductive metabolism resulting in the generation of electrophilic alkylating species capable of interacting with cellular macromolecules, especially DNA, was a pre-requisite for the cytotoxic as well as the therapeutic effect of MMC. At least 6 different enzymes have been shown to be capable of reductively activating MMC: NADH:cytochrome C reductase (EC 1.6.99.3), NADH:b5 reductase, NADPH:cytochrome P450 reductase (EC 1.6.2.4), xanthine dehydrogenase (EC 1.2.1.37), xanthine oxidase (EC 1.2.3.2) and NAD(P)H:quinone oxidoreductase 1 (NQOI; DT diaphorase; EC 1.6.99.2) (reviewed in Workman, 1994). While the majority of the studies concerning the enzymatic activation of MMC focused on the cytosolic NQO1, the actual involvement of this enzyme in the suggested reaction remains undetermined (Schlager and Powis, 1988;Pritsos et al., 1987; reviewed in Workman, 1994). Recent studies have addressed the involvement of NQOl in MMC activation by demo...

A New Mechanism of Mammalian Cell Resistance to the Toxic Effects of DNA-Binding Agents

“…MMC is a best-known member of the pyrroloquinone family of alkylating compounds, which are used in combination therapies against various malignancies. It is considered that, at first, MMC binds with DNA noncovalently, and that activation is required before it can alkylate DNA and exert its toxic effects (12,13). We suggest that MMC could be removed by a DNA-clearing system before it alkylates the DNA.…”

Curcumin reduced the side effects of mitomycin C by inhibiting GRP58‐mediated DNA cross‐linking in MCF‐7 breast cancer xenografts

“…MMC-induced DNA cross-linking assays were performed as described. (19,20) Briefly, two strands of 23 base pairs of oligonucleotides containing the MMC binding site were synthesized and used for the cross-linking experiments. The nucleotide sequence of the 23 base pair oligonucleotide was: sense strand 5¢-CTA CAT CGT GTC ATG CAC AGG AT-3¢ and anti-sense strand 5¢-A GAT CCT GTG CAT GAC ACG ATG T-3¢.…”