Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521-526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 +/- 4.6 nmol/min/mg; cytosolic activity, 94.3 +/- 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b5 reductase (15.6 +/- 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 +/- 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P < 0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol (IC50 approx. 50 microM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.
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PMID:The enzymology of doxorubicin quinone reduction in tumour tissue. 147 82

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

DT diaphorase (NAD(P)H dehydrogenase (quinone), EC 1.6.99.2) isolated from Walker 256 rat carcinoma cells can convert CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) to a cytotoxic DNA interstrand cross-linking agent. This is achieved by reduction of the 4-nitro group of CB 1954 to produce the hydroxylamino species, a bioactivation which accounts for the much greater sensitivity of Walker cells to CB 1954 when compared with other cells which are unable to carry out this reduction (Knox et al., Biochem Pharmacol 37: 4661-4669 and 4671-4677, 1988). As predicted from their measured DT diaphorase activities a number of rat hepatoma and hepatocyte cell lines were also shown to be sensitive to CB 1954. However, no CB 1954-sensitive cell lines of human origin were found, although levels of DT diaphorase similar to those in the sensitive rat cells were present in these cells. The human cells were as sensitive as rat cells to the active form of CB 1954 (5-(aziridin-1-yl)-4-hydroxyla mino-2-nitrobenzamide). DT diaphorase, purified to homogeneity from human Hep G2 cells, did metabolize CB 1954 to this 4-hydroxylamino product, but the rate of CB 1954 reduction and thus production of the cytotoxic product, was much lower than that of purified Walker enzyme (ratio of Kcat = 6.4). In addition, CB 1954 could be considered an inhibitor of, rather than a substrate for, the human form of DT diaphorase. The purified rat and human DT diaphorases possessed otherwise similar biochemical and molecular properties. These findings explain the decreased sensitivity towards CB 1954 of human cell lines when compared to rat cell lines.
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PMID:The differences in kinetics of rat and human DT diaphorase result in a differential sensitivity of derived cell lines to CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) 190 Dec 7

A form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) has been isolated from Walker 256 rat carcinoma cells. This enzyme can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Knox et al. Biochem Pharmacol, 37: 4661-4669 and 4671-4677, 1988). 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and AICA [5(4)-aminoimidazole-4(5)-carboxamide] have previously been reported to be antagonists of the anti-tumour effects of CB 1954. We have shown that both these compounds are inhibitors of the above enzyme and that AICA protects against both the cytotoxicity and the formation of DNA interstrand crosslinks, produced by CB 1954 in Walker cells. Similarly, known inhibitors of NAD(P)H dehydrogenase (quinone) such as dicoumarol, also reduced the cytotoxicity and DNA-interstrand crosslinking of CB 1954 in Walker cells. Caffeine was shown to be a novel inhibitor of NAD(P)H dehydrogenase (quinone) and also elicited the above protective effects. All of the above inhibitors were also shown to potentiate the toxic effects of menadione against the Walker cell. This quinone is known to be detoxified by NAD(P)H dehydrogenase (quinone) and thus emphasises the ability of these compounds to inhibit this enzyme within the cell.
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PMID:Caffeine, aminoimidazolecarboxamide and dicoumarol, inhibitors of NAD(P)H dehydrogenase (quinone) (DT diaphorase), prevent both the cytotoxicity and DNA interstrand crosslinking produced by 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) in Walker cells. 248 Jul 94

An NAD(P)H quinone reductase isolated from Walker rat 256 carcinoma cells has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell parameters a = 168.15 A, b = 105.09 A and c = 67.38 A and contain four monomeric or two dimeric enzyme molecules per asymmetric unit. Diffraction extends beyond 2.3 A resolution.
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PMID:Preliminary crystallographic data for NAD(P)H quinone reductase isolated from the Walker 256 rat carcinoma cell line. 249 46

A nitroreductase enzyme has been isolated from Walker 256 rat carcinoma cells which can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986; Knox RJ et al., Biochem Pharmacol 37: 4661-4669, 1988). The enzyme has now been identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) by comparison of partial protein sequences, coenzymes, substrate and inhibitor specificities, and spectroscopic data. 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and 5(4)-aminoimidazole-4(5)-carboxamide were shown to be inhibitors of the isolated Walker cell enzyme. This observation could explain the reported antagonistic action of the aminoimidazole carboxamides to the antitumour effects of CB 1954.
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PMID:The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2). 314 86

Since the cure of solid tumors is limited by the presence of cells with low oxygen contents, we have approached the development of treatment regimens and of new drugs for these tumors by investigating agents which are preferentially bioactivated under hypoxia. Major emphasis has been directed at studying the mode of action of the mitomycin antibiotics, as bioreductive alkylating agents. Using primarily the EMT6 mouse mammary carcinoma as a solid tumor model, we have found that mitomycin C and porfiromycin are preferentially toxic to cells with low oxygen contents. The mitomycin analog BMY-25282 is more toxic to hypoxic cells than are mitomycin C and porfiromycin; however, unlike these antibiotics, BMY-25282 is preferentially toxic to well-oxygenated cells. With these three mitomycins, we have observed a correlation between cytotoxicity to hypoxic cells, the rate of generation of reactive products, and the redox potentials of the drugs. Investigations of the enzymes in EMT6 cells that could possibly activate mitomycin C have revealed that cytochrome P-450 and xanthine oxidase are not present in measurable quantities and therefore are not responsible for activation of mitomycin C. Activities representative of NADPH-cytochrome c reductase and DT-diaphorase are present in these neoplastic cells. Comparison of these enzymatic activities in EMT6, CHO, and V79 cells with the rate of generation of reactive products under hypoxia shows a direct correlation between these two parameters, but there is no quantitative correlation between these two parameters and the amount of cytotoxicity. Use of purified NADPH-cytochrome c reductase and inhibitors of this enzyme demonstrated that NADPH-cytochrome c reductase can activate mitomycin C, but that it is probably not the only enzyme participating in this bioactivation in EMT6 cells. The DT-diaphorase inhibitor dicoumarol was employed to show that this enzyme is not involved in the activation of mitomycin C to a cytotoxic agent. Instead, DT-diaphorase appears to metabolize mitomycin C to a nontoxic product. This property has been exploited to develop a new treatment regimen for solid tumors. Using X-rays to eliminate well oxygenated cells of a solid tumor implant of the EMT6 carcinoma, we have found that the combination of dicoumarol plus mitomycin C is more toxic to hypoxic tumor cells in vivo than mitomycin C alone. Furthermore, knowledge of the biochemical mechanism of mitomycin C activation permits a prediction of which tumors can best be treated with this combination of drugs by measuring enzymatic activities in biopsy specimens.
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PMID:Chemotherapeutic attack of hypoxic tumor cells by the bioreductive alkylating agent mitomycin C. 393 22

The expression of nitric oxide synthase (NOS) was studied by NAD(P)H diaphorase histochemical localization method in (i) individual cells of the normal colonic mucosa (n = 13) which served as control, (ii) colonic polyps (n = 14), (iii) colonic carcinoma (n = 20) and (iv) peritumoral mucosa (2 and 5 or 10 cm away from the tumor). Four of the tumor specimens had normal epithelium adjacent to the cancer, which thus served as an internal control. The expression of NOS activity in colon cancer was significantly reduced as compared to the control group of individuals (P < 0.004); undetectable in 25%, diminished in 45%, normal in 30%. On comparing the expression in normal mucosa and polyps there was a significant reduction of the expression in polyps (P < 0.027); undetectable in 14%, reduced in 35%, normal in 51%. When compared to the peritumoral mucosa at 2 and 10 cm the tumor showed a significant reduction in expression of NOS activity (P < 0.001 and P < 0.0001 respectively). There was no significant difference seen in the expression at 2 and 10 cm (P = 0.329). The peritumoral mucosa at a distance of 2 cm away from the tumor when compared to the control mucosa showed no significant difference (P = 1.000), although there is a tendency to a high normal expression of NOS activity in the mucosa at a distance of 2 cm. Similarly, there was no significant difference between the control mucosa and the peritumoral mucosa obtained at a distance of 10 cm (P = 0.383). The expression of NOS activity in all tissues examined was abolished by preincubation of tissue with the selective NOS inhibitor L-NMMA but not with D-NMMA. Our data showed extensive and significant reduction as identified by the NAD(P)H diaphorase method in the expression of NOS activity, thereby reflecting the activity of nitric oxide in colon cancer and colonic polyps. The generalized suppression of this activity, which precedes the onset of overt neoplasia, may be an important event in colon carcinogenesis. This aberrant expression could also be compatible with the selective advantage to either tumor promotion and metastatic progression or to tumoricidal activity.
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PMID:Aberrant expression of nitric oxide synthase in human polyps, neoplastic colonic mucosa and surrounding peritumoral normal mucosa. 752 94

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives. In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine. This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA. In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases. Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E. coli enzyme and cofactor (NADH). Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme. None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells. For those compounds which were substrates for the E. coli nitroreductase, there was a positive correlation between kcat and IC50 ratio. Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1. This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system.
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PMID:Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase. 766 63

High-level cytosolic class-3 aldehyde dehydrogenase (ALDH-3)-mediated oxazaphosphorine-specific resistance (> 35-fold as judged by the concentrations of mafosfamide required to effect a 90% cell-kill) was induced in cultured human breast adenocarcinoma MCF-7/0 cells by growing them in the presence of 30 microM catechol for 5 days. Resistance was transient in that cellular sensitivity to mafosfamide was fully restored after only a few days when the inducing agent was removed from the culture medium. The operative enzyme was identified as a type-1 ALDH-3. Cellular levels of glutathione S-transferase and DT-diaphorase activities, but not of cytochrome P450 IA1 activity, were also elevated. Other phenolic antioxidants, e.g. hydroquinone and 2,6-di-tert-butyl-4-hydroxytoluene, also induced ALDH-3 activity when MCF-7/0 cells were cultured in their presence. Thus, the increased expression of a type-1 ALDH-3 and the other enzymes induced by these agents was most probably the result of transcriptional activation of the relevant genes via antioxidant responsive elements present in their 5'-flanking regions. Cellular levels of ALDH-3 activity were also increased when a number of other human tumor cell lines, e.g. breast adenocarcinoma MDA-MB-231, breast carcinoma T-47D and colon carcinoma HCT 116b, were cultured in the presence of catechol. These findings should be viewed as greatly expanding the number of recognized environmental and dietary agents that can potentially negatively influence the sensitivity of tumor cells to cyclophosphamide and other oxazaphosphorines.
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PMID:Phenolic antioxidant-induced overexpression of class-3 aldehyde dehydrogenase and oxazaphosphorine-specific resistance. 788 82


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