Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.5.2 (NQO1)
 6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of the two-electron reducing enzyme DT-diaphorase in the activation of mitomycin C under hypoxic conditions was investigated. Mitomycin C activity was compared in L5178Y murine lymphoblasts, which have low levels of DT-diaphorase activity, and L5178Y/HBM10 cells, which have elevated levels of enzyme activity. The cytotoxic and DNA cross-linking activities of mitomycin C were greater in L5178Y/HBM10 cells than in L5178Y cells. In L5178Y/HBM10 cells, dicoumarol, an inhibitor of DT-diaphorase, decreased cell kill and DNA cross-linking by mitomycin C in air but had no significant effect on these activities under hypoxia. By comparison, in L5178Y cells, dicoumarol had no effect on drug activity under either aerobic or hypoxic conditions. A model for the activation of mitomycin C by both one-electron and two-electron reduction is proposed. Our findings suggest that two-electron reduction by DT-diaphorase has only a limited role in the activation of mitomycin C under hypoxic conditions, although this enzyme appears to be an important contributor to drug activation under aerobic conditions.
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PMID:Role of NAD(P)H:(quinone acceptor) oxidoreductase (DT-diaphorase) in activation of mitomycin C under hypoxia. 137 99

15 human tumour cell lines (lung, breast and colon) have been evaluated for their sensitivity to the quinone based anti-cancer drugs Mitomycin C, Porfiromycin, and EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2-(IH-indole-4,7-dione)prop-beta- en-alpha-ol). Sensitivity has been compared with the intra-cellular levels of DT-diaphorase, an enzyme thought to be important in the reductive activation of these quinones. No correlation exists between levels of DT-diaphorase and sensitivity to Mitomycin C or Porfiromycin. However, for EO9 those cell lines showing highest levels of DT-diaphorase activity tend to be the most sensitive.
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PMID:The sensitivity of human tumour cells to quinone bioreductive drugs: what role for DT-diaphorase? 151 Jun 92

The role of DT-diaphorase (DTD, EC 1.6.99.2) in the bioreductive activation of mitomycin C was examined using purified rat hepatic DTD. The formation of adducts with reduced glutathione (GSH), binding of [3H]mitomycin C to DNA, and mitomycin C-induced DNA interstrand cross-linking were used as indicators of bioactivation. Mitomycin C was metabolized by DTD in a pH-dependent manner with increasing amounts of metabolism observed as the pH was decreased from 7.8 to 5.8. The major metabolite observed during DTD-mediated reduction of mitomycin C was 2,7-diaminomitosene. GSH adduct formation, binding of [3H]mitomycin C and mitomycin C-induced DNA interstrand cross-linking were observed during DTD-mediated metabolism. In agreement with the pH dependence of metabolism, increased bioactivation was observed at lower pH values. Temporal studies and experiments using authentic material showed that 2,7-diaminomitosene could be further metabolized by DTD resulting in the formation of mitosene adducts with GSH. DNA cross-linking during either chemical (sodium borohydride) or enzymatic (DTD) mediated reduction of mitomycin C could be observed at pH 7.4, but it increased as the pH was decreased to 5.8, showing the critical role of pH in the cross-linking process. These data provide unequivocal evidence that the obligate two-electron reductase DTD can bioactivate mitomycin C to reactive species which can form adducts with GSH and DNA and induce DNA cross-linking. The use of mitomycin C may be a viable approach to the therapy of tumors high in DTD activity, particularly when combined with strategies to lower tumor pH.
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PMID:Bioreductive activation of mitomycin C by DT-diaphorase. 151 Sep 75

The role of DT-diaphorase in bioreductive activation of mitomycin C was examined using HT-29 and BE human carcinoma cells which have high and low levels of DT-diaphorase activity, respectively. HT-29 cells were more sensitive to mitomycin C-induced cytotoxicity than the DT-diaphorase-deficient BE cell line. Mitomycin C induced DNA interstrand cross-linking in HT-29 cells but not in BE cells. Both mitomycin C-induced cytotoxicity and induction of DNA interstrand cross-links could be inhibited by pretreatment of HT-29 cells with dicoumarol. Metabolism of mitomycin C by HT-29 cell cytosol was pH dependent and increased as the pH was lowered to 5.8, the lowest pH tested. Metabolism of mitomycin C by HT-29 cytosol was inhibited by prior boiling of cytosol or by the inclusion of dicoumarol. Little metabolism was detected in BE cytosols. When purified rat hepatic DT-diaphorase was used, metabolism of mitomycin C increased as the pH was decreased and could be detected at pH 5.8, 6.4, 7.0, 7.4, but not at 7.8. Metabolism of mitomycin C was NADH dependent and inhibited by dicoumarol or by prior boiling of enzyme. An approximate 1:1 stoichiometry between NADH and mitomycin C removal was demonstrated and no oxygen consumption could be detected. Metabolism of mitomycin C by purified HT-29 DT-diaphorase was also dicoumarol inhibitable and pH dependent. The major metabolite formed during metabolism of mitomycin C by HT-29 cytosol, purified HT-29, and rat hepatic DT-diaphorase was characterized as 2,7-diaminomitosene. These data suggest that two-electron reduction of mitomycin C by DT-diaphorase may be an important determinant of mitomycin C-induced genotoxicity and cytotoxicity.
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PMID:Metabolism of mitomycin C by DT-diaphorase: role in mitomycin C-induced DNA damage and cytotoxicity in human colon carcinoma cells. 170 46

L5178Y cells resistant to the model quinone antitumor agent, hydrolyzed benzoquinone mustard, were four-fold more sensitive to mitomycin C compared to parental cells. Mitomycin C also produced increased DNA-DNA crosslinking in these cells compared to parental L5178Y cells, but did not induce DNA double strand breaks in either cell line. The resistant cells have a 24-fold increased level of DT-diaphorase activity, an enzyme that produces two electron reduction of quinone groups. Dicoumarol, an inhibitor of DT-diaphorase, significantly inhibited crosslinking and cytotoxicity by mitomycin C in the quinone resistant cells. These findings suggest that DNA-DNA cross-linking may be a major contributor to mitomycin C cytotoxic activity in L5178Y cells, and that the hydroquinone of mitomycin C may play a major role in the crosslinking activity of this agent.
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PMID:Increased sensitivity of quinone resistant cells to mitomycin C. 247 83

It has been suggested that quinone reductase [NAD(P)H: (quinone-acceptor)oxidoreductase], also known as DT-diaphorase, protects hypoxic cells against mitomycin C cytotoxicity by metabolizing mitomycin C to less toxic metabolites. This hypothesis is based on an increase in mitomycin C's cytotoxicity in the presence of the potent quinone reductase inhibitor dicumarol. It has been suggested that under aerobic conditions the metabolism of mitomycin C by quinone reductase leads to the formation of cytotoxic metabolites. In the present study, mitomycin C was found not to be a substrate for partially purified quinone reductase from human kidney. Mitomycin C did not cause the oxidation of NADPH by quinone reductase and there was no utilization of mitomycin C and no appearance of its metabolites. Quinone reductase did not catalyze the formation of alkylating metabolites from mitomycin C, determined by the lack of formation of 4-(p-nitrobenzyl)pyridine conjugates. However, mitomycin C was a weak competitive inhibitor of quinone reductase with dichloroindophenol as the substrate, with Ki = 0.32 mM. Therefore, the alteration of mitomycin C's cytotoxicity by dicumarol in tumor cell lines appears to involve a mechanism other than the direct inhibition of mitomycin C reduction by quinone reductase.
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PMID:Mitomycin C is not metabolized by but is an inhibitor of human kidney NAD(P)H: (quinone-acceptor)oxidoreductase. 313 41

Mitomycin C (MMC) is a bioreductive antitumor agent that is activated by NADPH:cytochrome P450 reductase (EC 1.6.2.4) and NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) (DT-diaphorase). DT-diaphorase is a two-electron reducing enzyme that is induced by a variety of chemicals, including quinones. Doxorubicin (DOX) is an anthraquinone antitumor agent that has been used clinically with MMC for combination chemotherapy in breast cancer. In this study, we investigated whether DOX could selectively induce DT-diaphorase in tumor cells and whether combining this agent with MMC in an appropriate schedule could produce synergistic antitumor activity. Treatment of EMT6 murine mammary tumor cells with DOX resulted in a 40% increase in DT-diaphorase activity in these cells, but had no effect on this enzyme in murine bone marrow cells. Combination therapy with DOX and MMC produced a 1.4-fold level of synergistic cell kill in the tumor cells, but a similar level of synergy was also observed in normal bone marrow cells. Thus, DOX can selectively induce elevated levels of DT-diaphorase in tumor cells; however, the synergy observed by combining this agent with MMC appears to be unrelated to the induction of DT-diaphorase.
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PMID:Induction of DT-diaphorase by doxorubicin and combination therapy with mitomycin C in vitro. 748 45

To investigate the resistant mechanisms against MMC in human tumor cells, we isolated an MMC-resistant variant (HT-29/MMC) of HT-29 human colon carcinoma cells. HT-29/MMC cells showed 5-fold resistance to MMC as compared with the parental cell line but did not show cross-resistance to Adriamycin, vincristine, ACNU, bleomycin, or cisplatin. Treatment of the cells with dicoumarol, an inhibitor of DT-diaphorase, reduced the cytotoxicity of MMC in DT-diaphorase proficient HT-29 cells but not in HT-29/MMC cells. HT-29/MMC cells were 5 times more sensitive than HT-29 cells to menadione, which is detoxified by DT-diaphorase, DT-diaphorase was deficient in HT-29/MMC cells as determined by the enzyme activity and immunoblot analysis of the cytoplasmic proteins. Levels of cytochrome P-450 reductase and glutathione S-transferase, however, were comparable in both cell lines. The amount of [3H]-MMC found covalently bound to chromosomal DNA in HT-29/MMC cells was one-fourth that detected in HT-29 cells. Treatment with dicoumarol reduced the DNA-bound MMC in HT-29 cells but not in HT-29/MMC cells. These results indicate that the deficiency in DT-diaphorase, an activating enzyme of MMC, is one of the mechanisms of resistance in HT-29/MMC cells.
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PMID:Isolation and characterization of a mitomycin C-resistant variant of human colon carcinoma HT-29 cells. 750 23

Because of the elevated DT-diaphorase (DTD) activity in certain tumors such as human nonsmall cell lung cancer (NCSLC), DTD is a potential target on which to base the development of new antitumor compounds. Mitomycin C is the most effective single agent used for the therapy of NSCLC and is metabolized and bioactivated by DTD. Mitomycin C is a poor substrate for DTD, however, and its metabolism is pH-dependent. We have therefore focused on identifying more efficient substrates for DTD. We have developed a metabolic and cytotoxicity screen that identifies compounds which are efficiently bioactivated by DTD. This screen utilizes both aerobic and hypoxic conditions and cell lines with both elevated and deficient DTD activity as an index of selectivity. Using the screen described above, we have identified [3-hydroxy-5-aziridinyl-1-methyl-2-(1H-indole-4,7-indione)-prop-be ta-en- alpha-ol] (E09), 2,5-diaziridinyl-1,4-benzoquinone (MeDZQ), and streptonigrin as compounds that are most efficiently bioactivated by DTD and exert selective cytotoxicity. Although certain tumors such as NSCLC have elevated DTD activity, we have characterized a point mutation at position 609 in the DTD cDNA, which codes for a proline to serine change in the protein and leads to a loss of enzyme activity. We have characterized this mutation in both BE human colon carcinoma cells and H596 human NSCLC cells. This mutation and resulting lack of DTD activity complicates the use of agents designed to target DTD in tumors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Bioactivation of quinones by DT-diaphorase, molecular, biochemical, and chemical studies. 762 Feb 17

Mitomycin C (MC), a clinically used natural antitumor agent, was shown to form three monoconjugates (11a-13a) and two bisconjugates (14a, 15a) with GSH upon reductive activation by rat liver microsomes, purified NADPH-cytochrome c reductase, or NADH-cytochrome c reductase or chemical reduction using H2/PtO2. Rat liver cytosol/NADH activated MC only at acidic pH (5.8), resulting in the formation of a single GSH-MC monoconjugate, 13a. The reductase responsible for cytosolic activation of MC to form this conjugate was DT-diaphorase. GSH itself did not reduce MC, and unreduced MC did not form conjugates with GSH. A moderate catalytic effect by glutathione S-transferase was demonstrated on the cytosol-activated reaction. Mercaptoethanol and N-acetylcysteine gave analogous sets of five MC-thiol conjugates under cytochrome c reductase or H2/PtO2 activation conditions. The structures of all 15 MC-thiol conjugates (five each with GSH, mercaptoethanol, and N-acetylcysteine, respectively) were determined, using 1H-NMR, UV, and mass spectroscopies, combined with analytical chemical and radiolabeling methods. The mechanism of formation of the conjugates features SN2 displacement of the carbamate of the reduced MC by GS-. The MC-GSH conjugates were noncytotoxic to the tumor cells tested. The conjugation of GSH with activated MC is likely to represent detoxication in mammalian cells. As another effect, GSH accelerates the rate of reduction of MC by "slow" reducing agents such as cytochrome c reductases and H2/PtO2. A mechanism is proposed to explain this effect, which involves further reduction of the initially formed MC semiquinone free radical by GSH.
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PMID:Conjugation of glutathione and other thiols with bioreductively activated mitomycin C. Effect of thiols on the reductive activation rate. 807 71


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