DT-diaphorase: questionable role in mitomycin C resistance, but a target for novel bioreductive drugs?

Workman, Paul; Walton, Michael I.; Powis, Garth; Schlager, John J.

doi:10.1038/bjc.1989.364

Cited by 49 publications

(10 citation statements)

References 17 publications

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“…Although mitomycin C has been reported not to act as a substrate for DT-diaphorase (Workman et al, 1989), reduction is detectable at neutral pH (Pritsos et aL, 1987) and is much more rapid at an acid pH (Siegel et al, 199%). The greater aerobic sensitivity observed here of DT-diaphorase-rich HT29 compared with BE is consistent with that observed by Siegel et al (1990b).…”

Section: Discussionmentioning

confidence: 97%

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Unusually marked hypoxic sensitization to indoloquinone E09 and mitomycin C in a human colon‐tumour cell line that lacks DT‐diaphorase activity

Plumb

Workman

1994

Intl Journal of Cancer

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Studies with purified DT-diaphorase have shown that the enzyme is capable of catalyzing a two-electron reduction of the novel indoloquinone E 0 9 to a DNA-damaging alkylating species. The aim of this study was to determine to what extent DT-diaphorase may be involved in the metabolic activation of E09 and mitomycin C in both aerobic and hypoxic conditions. Two human colon-carcinoma cell lines were used; H l 2 9 has high levels of DT-diaphorase whilst BE lacks this activity because of a point mutation in the NQOl gene. In aerobic conditions the 2 cell lines show similar sensitivities to a number of qtotoxic drugs including cisplatin, doxorubicin and etoposide. They are equally sensitive to the benzotriazine di-N-oxide SR 4233 but H R 9 is more sensitive than BE to mitomycin C and E09. Sensitivity to SR 4233 is increased by about 100-fold for both cell lines in hypoxic conditions. DT-diaphorase-deficient BE cells show markedly increased sensitivity to mitomycin C and particularly E 0 9 in hypoxic conditions, whereas DTdiaphorase-rich H R 9 cells show little hypoxic sensitization to these agents unless exposed in the presence of dicoumarol.These results suggest that DT-diaphorase can reduce E09 and mitomycin C to potent cytotoxic species in aerobic conditions, and this activii predominates over the one-electron-reducing enzymes even in hypoxic conditions. In the absence of DTdiaphorase activity, E 0 9 and mitomycin C are reduced in hypoxic conditions, presumably by one-electron-reducing enzymes, to a similar or greater extent than is achieved with DT-diaphorase.8 1994 Wfq-Liss, Znc.The presence of hypoxic areas in solid tumours suggests a target for the development of selective anti-cancer agents. The main classes of bioreductive agents which show specificity for hypoxic cells are quinone, nitro and N-oxide compounds (Workman, 1992). Hypoxic cell specificity is dependent on preferential metabolic activation by reductase enzymes in hypoxic conditions (Workman, 1992). Mitomycin C is the prototype bioreductive agent which shows clinical activity against some solid tumours but exhibits little selectivity towards hypoxic tumour cells (Stratford and Stephens, 1989; Workman, 1992). It (Oostveen and Speckamp, 1987) is structurally related to mitomycin C (Fig. l), is less myelosuppressive (Hendriks et al., 1993) and is in clinical trial in Europe under the auspices of the EORTC. E09 also requires metabolic activation and has been shown to be a much better substrate for both human and rat DT-diaphorase than mitomycin C (Walton et al., 1991).In hypoxic conditions one-electron reduction of quinone bioreductive agents by enzymes such as cytochrome P450 reductase and xanthine oxidase results in the production of semiquinone free radicals. However, in aerobic conditions toxic oxygen radicals are produced due to back oxidation of these unstable metabolites. In contrast, obligate two-electron reduction by DT-diaphorase (Iyanagi and Yamazaki, 1970) results in production of more stable hydroquinone metabolites under both aerobic an...

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Section: Discussionmentioning

confidence: 97%

“…The problems with this approach have been discussed elsewhere (Workman et al, 1989;Preusch et al, 1991) and relate to the lack of specificity of dicoumarol as an inhibitor. The pair of cell lines BE and HT29 are an alternative model for such studies (Siegel et al, 1990U, b).…”

Section: Discussionmentioning

confidence: 97%

Unusually marked hypoxic sensitization to indoloquinone E09 and mitomycin C in a human colon‐tumour cell line that lacks DT‐diaphorase activity

Plumb

Workman

1994

Intl Journal of Cancer

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“…The role of NQOl in the reductive metabolism of MMC and other bioreductive agents has attracted much attention because of its ability to catalyze 2-electron reduction of quinones in the presence or absence of oxygen and because NQOl was found to be expressed 20-to 50-fold higher in certain tumor tissues as compared to normal tissues of the same origin (reviewed in Workman, 1994). However, the role of NQOl in the metabolism of MMC is a subject of great controversy (Schlager and Powis, 1988;Workman et al, 1989;Marshall et al, 1989a, b;Pritsos et al, 1987;Siege1 et al, 1992;Ross et al, 1994;Workman, 1994). Most of the available data supporting a role for NQOl in the reductive metabolism of MMC were obtained using cell lines expressing differential levels of dicoumarol-sensitive NQOl activity (Marshall et al, 1989a, b;Bizanek et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

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

Joseph

Jaiswal

1996

Int. J. Cancer

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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...

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“…In the late 1980s for example, there was considerable doubt as to whether or not MMC was actually a substrate for human NQO1. 27,28 It is now widely acknowledged that MMC is a substrate for NQO1 but only under mild acidic conditions. 29 Under physiological pH conditions, MMC serves as a suicide inhibitor of NQO1.…”

Section: Discussionmentioning

confidence: 99%

Immunohistochemical analysis of NAD(P)H:quinone oxidoreductase and NADPH cytochrome P450 reductase in human superficial bladder tumours: Relationship between tumour enzymology and clinical outcome following intravesical mitomycin C therapy

Basu

Brown

Flannigan

et al. 2004

Intl Journal of Cancer

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A central theme within the concept of enzyme-directed bioreductive drug development is the potential to predict tumour response based on the profiling of enzymes involved in the bioreductive activation process. Mitomycin C (MMC) is the prototypical bioreductive drug that is reduced to active intermediates by several reductases including NAD(P)H:quinone oxidoreductase (NQO1) and NADPH cytochrome P450 reductase (P450R). The purpose of our study was to determine whether NQO1 and P450R protein expression in a panel of low-grade, human superficial bladder tumours correlates with clinical response to MMC. A retrospective clinical study was conducted in which the response to MMC of 92 bladder cancer patients was compared to the immunohistochemical expression of NQO1 and P450R protein in archived paraffin-embedded bladder tumour specimens. A broad spectrum of NQO1 protein levels exists in bladder tumours between individual patients, ranging from intense to no immunohistochemical staining. In contrast, levels of P450R were similar with most tumours having moderate to high levels. All patients were chemotherapy naïve prior to receiving MMC and clinical response was defined as the time to first recurrence. Key words: NQO1; cytochrome P450 reductase; mitomycin C; bladder cancerThe ability to predict tumour response to chemotherapy has been and continues to be a major objective in cancer research with the emphasis currently being placed on the identification of molecular markers of tumour response. 1,2 Within the field of bioreductive drug development, the ability to individualise chemotherapy based on the activity of specific reductases and hypoxia in tumours forms one of the cornerstones of a concept known as enzyme-directed bioreductive drug development. 3 The key requirements for the successful clinical application of this concept include the development of drugs that are bioreductively activated by specific reductases and good correlations between tumour response and enzymology in experimental tumour models. Mitomycin C (MMC) is a clinically active agent used to treat a number of tumours and is regarded as the prototypical bioreductive drug. 4 Its mechanism of action has been extensively reviewed elsewhere [5][6][7][8] and involves a complex interplay between tumour physiology (oxygen tension and extracellular pH) and several reductase enzymes (both 1 and 2 electron reductases). The ability to predict cellular and tumour response to MMC based on tumour enzymology has been addressed by several research groups but the results are controversial, and there are conflicting reports on the relationship between tumour enzymology and chemosensitivity in the literature. This is particularly true in the case of the 2 electron reductase NAD(P)H:quinone oxidoreductase-1 (NQO1), which has been implicated in the bioreductive activation of MMC, especially under aerobic conditions. 5,6,9 Reports of good correlations between NQO1 activity (and mRNA expression) and response to MMC contrast sharply with reports of poor correlations in bot...

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DT-diaphorase: questionable role in mitomycin C resistance, but a target for novel bioreductive drugs?

Cited by 49 publications

References 17 publications

Unusually marked hypoxic sensitization to indoloquinone E09 and mitomycin C in a human colon‐tumour cell line that lacks DT‐diaphorase activity

Unusually marked hypoxic sensitization to indoloquinone E09 and mitomycin C in a human colon‐tumour cell line that lacks DT‐diaphorase activity

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

Immunohistochemical analysis of NAD(P)H:quinone oxidoreductase and NADPH cytochrome P450 reductase in human superficial bladder tumours: Relationship between tumour enzymology and clinical outcome following intravesical mitomycin C therapy

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