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)

Glutathione (GSH) has been shown to modulate the cytotoxicity of a variety of chemotherapeutic agents. The effect of mitomycin C (MMC) treatment duration and the effect of GSH depletion on in vitro cytotoxicity against the human colon cancer cell line HT-29 was studied under aerobic conditions. Continuous-exposure experiments revealed that the cytotoxicity of 0.1 microM MMC, as measured by clonogenic cell survival, exhibited a shoulder until exposure time was at least 12 h, after which time exponential cytotoxicity was observed. Lowering GSH levels to less than 3% of control using buthionine sulfoximine (BSO) did not enhance cytotoxicity of MMC given for 1 h or continuously for less than 12 h. However, GSH depletion did enhance cytotoxicity of MMC given continuously for at least 12 h, with a dose-modifying factor at 1% survival of 1.4 for a 24-h treatment. GSH depletion under these conditions enhanced cytotoxicity of even minimally cytotoxic MMC concentrations (0.02 microM). Absolute levels of GSH-related enzymes, including glutathione-S-transferase, and the MMC-metabolizing enzyme DT-diaphorase did not change appreciably. A tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay was used to verify the results further and to determine the optimal sequence of BSO administration with a 24-h MMC treatment. BSO added simultaneously with MMC did not increase cytotoxicity, compared to MMC alone. BSO added and then removed prior to MMC was effective (dose-modifying factor at 50% survival = 1.3), but the greatest cytotoxicity was noted when BSO was present before and during MMC treatment (dose-modifying factor = 1.5). GSH depletion in another cell line (SW480) showed similar enhancement of 24-h MMC cytotoxicity. These studies show that aerobic cytotoxicity of MMC is improved by administration of the drug in continuous fashion for at least 12 h, as opposed to continuous administration for shorter periods or 1-h bolus administration. Cytotoxicity of continuous (at least 12-h) MMC treatment can be modestly enhanced by GSH depletion, which must precede MMC exposure in order to be effective.
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PMID:Effect of treatment duration and glutathione depletion on mitomycin C cytotoxicity in vitro. 151 28

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

Endothelial nitric oxide (NO) synthase, a unique NO synthase (NOS) isoform that is expressed constitutively by the vascular endothelium both in vivo and in vitro, is believed to be essential to systemic and/or local vascular integrity. NOS expression by endothelial cells may indicate vascular activation. We successfully established a simple method for the culture of microvascular endothelial cells from a small amount of tissue and investigated ulcerative colitis (UC), in which condition vascular factors have not been studied extensively. We cultured endothelial cells from the mesenteries of surgical patients with UC and assayed NOS activity by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Strong NOS activity was demonstrated in the cells from all UC patients (5/5), whereas no activity was detected in the cells from human umbilical veins and the mesenteries of colon cancer patients (0/10 and 0/5, respectively). This strong NOS activity was not diminished by incubation with a high concentration of glucocorticoid, suggesting that it was constitutive. These results indicate a close relationship of vascular activation (high NOS activity) with the pathogenesis of UC.
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PMID:High nitric oxide synthase activity in endothelial cells in ulcerative colitis. 755 Aug 72

Resistance may limit the clinical usefulness of a variety of chemotherapeutic drugs including mitomycin C (MMC). The MMC-sensitive HT-29 colon cancer cell line and its MMC-resistant subline, HT-29R13, were studied in vitro under aerobic conditions to help characterize the mechanisms associated with MMC resistance. HT-29R13 cells exhibit approximately 2-fold resistance to MMC compared with HT-29 cells and lack the typical multidrug-resistance pattern; resistance is stable in the absence of drug exposure. Levels of glutathione (GSH) and total glutathione-S-transferase (GST) activity were not different between the two cell lines; however, levels of GSH reductase and GSH peroxidase were increased significantly in HT-29R13. Although total GST activity was unchanged, GST-pi and GST-alpha isoenzyme expression as measured using western blot were increased significantly in HT-29R13 compared with HT-29. DT-diaphorase levels and topoisomerase II activity were decreased significantly in HT-29R13. Both cell lines had equal P-glycoprotein expression. Multiple drug resistance mechanisms are present in HT-29R13 including decreased drug activation (decreased DT-diaphorase), increased drug detoxification (increased GST-pi and GST-alpha, GSH reductase, GSH peroxidase), and decreased accessibility of DNA targets (decreased topoisomerase II). Further work will be necessary to determine the degree to which each of these mechanisms contribute to MMC resistance in this model.
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PMID:Biochemical characterization of a mitomycin C resistant colon cancer cell line variant. 790 34

The effect of pH and oxygen on DNA alkylation by mitomycin C (MMC) was studied with cell fractions and intact cells. The cell lines used were the HCT 116 human colon cancer cell line and a MMC-resistant subline (HCT 116-R30A) that has 5% of the quinone reductase activity present in the parent cell line. Microsomal fractions of the two cell lines catalyzed MMC-DNA adduct formation only under anaerobic conditions with equal efficiency. However, the pH of the reaction controlled the production of four identified and two unidentified adducts. Soluble fractions from each cell source catalyzed MMC-DNA adduct formation under aerobic and anaerobic conditions similarly. At higher pH, limited DNA adducts were produced by MMC activated by soluble fractions from either cell source. At lower pH, more DNA adducts were obtained with MMC activated by the soluble fraction of HCT 116 cells than with that activated by the soluble fraction of HCT 116-R30A cells. Four of these adducts were identified as N2-(2" beta,7"-diaminomitosene-1" alpha-yl)-2'-deoxyguanylic acid, N2-(2" beta,7"-diaminomitosen-1" beta-yl)-2'-deoxyguanylic acid, N2-(10"-decarbamoyl-2",7"-diaminomitosen-1" alpha-yl)-2'-deoxyguanylic acid, and N2-(2" beta,7"-diamino-10"-deoxyguanyl-N2-yl-mitosen-1" alpha-yl)-2'- deoxyguanylic acid. Acidic intracellular pH enhanced the cytotoxicity of MMC for HCT 116 cells, decreasing the IC50 from 0.3 +/- 0.04 microM to 0.1 +/- 0.03 microM, but pH had limited effect on the cytotoxicity of MMC for HCT 116-R30A cells. When intracellular pH was decreased, interstrand DNA cross-linking by MMC increased to a greater extent in HCT 116 cells than in HCT 116-R30A cells. Only two DNA adducts, each at low intensity, were detected in HCT 116-R30A cells treated at pH 6.0 and 7.6 and in HCT 116 cells treated at pH 7.6. However, six radioactive spots were detected in HCT 116 cells treated at pH 6.0. Three of these adducts were identified. This is the first direct evidence that acidic intracellular pH enhances MMC-DNA adduct formation in tumor cells containing high quinone reductase activity. Results from this study further confirm that pH and not enzyme is the determining factor in the distribution of types of MMC-DNA adducts. This study also indicates that low intracellular pH enhances the activity of quinone reductase in reducing MMC, which is important for aerobic cytotoxicity of MMC against tumor cells with high concentration of quinone reductase.
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PMID:Enzymatic and pH modulation of mitomycin C-induced DNA damage in mitomycin C-resistant HCT 116 human colon cancer cells. 831 19

High consumption of fruits and vegetables which are abundant in dietary antioxidants has been linked to a reduced incidence of colorectal cancer. A potential mechanism of dietary anticarcinogenesis involves the induction of detoxifying phase II enzymes, including NAD(P)H:quinone reductase (QR) and glutathione-S-transferase (GST). This study therefore examined the ability of the dietary antioxidant vitamins beta-carotene, alpha-tocopherol and ascorbic acid to induce cellular expression of QR and GST activities in human colon cancer cells. Colo205 cells were cultured in the presence or absence of various concentrations (10(-10) to 10(-5) M) of each antioxidative micronutrient, then assessed for cytosolic QR and GST activities and cell growth. beta-Carotene, alpha-tocopherol and ascorbic acid each resulted in dose-dependent increases in QR activity, without adverse effects upon cell proliferation. To investigate whether the ability of beta-carotene to induce QR may be attributable to its conversion to vitamin A and/or to its antioxidant capacity as a carotenoid, retinol, retinoic acid, and lycopene were similarly tested for their capacity for enzyme induction. Although retinol and retinoic acid were both noted to be antiproliferative at higher concentrations (10(-6) to 10(-5) M), both retinoids stimulated QR at physiological concentrations. Lycopene, a carotenoid which is not converted to vitamin A, was devoid of biologic activity. By contrast with the effects upon QR, GST activity was unaffected by treatment with any of the micronutrients tested in this in vitro model. The results support a hypothesis that a high dietary consumption of vitamins A, E and C may confer partial protection against colorectal cancer by the induction of specific detoxifying enzymes. The antioxidant capacity of beta-carotene appears to have less biologic impact vis-a-vis QR induction than its function as a non-toxic reservoir of vitamin A. Measurements of QR activity within the colorectal mucosa may provide an index of cancer susceptibility, and may be an appropriate surrogate endpoint biomarker for colorectal cancer prevention studies involving diet modification or specific relevant micronutrients.
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PMID:Induction of NAD(P)H:quinone reductase by vitamins A, E and C in Colo205 colon cancer cells. 852 7

Maintenance of cellular homeostasis is a critical survival trait in tumors when exposed to anticancer drugs. Because conjugation and elimination of drugs and their metabolites is dependent upon sequential and coordinated pathways, acquired drug resistance through a gradual adaptive response would rarely be expected to be the consequence of changes in the expression of one gene product. We have used a number of drug-resistant human cell lines to characterize those genes that are implicated in maintaining a resistant phenotype. Human HT29 colon cancer cells chronically exposed to ethacrynic acid (EA) [a glutathione (GSH) and glutathione S-transferase (GST) modulator] have acquired resistance to the drug. Commensurate with resistance, EA is more effectively conjugated to GSH and effluxed from the resistant cells. Using directed and random (differential display) approaches, a number of detoxification and/or protective gene products have been shown to be expressed at elevated levels. These include: gamma-glutamyl cysteine synthetase (gamma-GCS, the rate-limiting enzyme in GSH biosynthesis); GST pi (the enzyme catalyzing the conjugation reaction); multidrug resistance associated protein (MRP) (the membrane pump responsible for effluxing the conjugate from the cell interior). In addition, other gene products not directly linked with EA metabolism were induced, including dihydrodiol dehydrogenase (an alpha-ketoreductase) (30-fold), DT-diaphorase (threefold), and a transcriptional regulator SSP 3521 (threefold). HL60 cells resistant to a GSH paralog Ter199 also show increased expression of some of these gene products. Furthermore, an adriamycin-resistant human HL60 cell line also shows overexpression of GST pi, gamma-GCS, and MRP, but in addition has approximately 20-fold more DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This enzyme is an early stress response gene that can phosphorylate and activate downstream transcription factors. Such overexpression could impact on the transcriptional control of the other detoxification gene products. Both adriamycin and a typical drug-GSH conjugate (APA-SG) are inhibitors of DNA-PK. Because cellular levels of these conjugates would presumably be a good indicator of stress, it would seem reasonable to speculate that DNA-PK may act as a receiver and transmitter of signals that are crucial to the drug-resistant phenotype. Additionally, this enzyme may prove to be a potentially important target for drug design based upon the inhibitory activity of GSH conjugates.
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PMID:Importance of glutathione and associated enzymes in drug response. 940 35

EO9 is a novel bioreductive drug which has recently undergone extensive clinical evaluation. Its mechanism of action remains to be clearly defined. Antitumour activity of EO9 has been determined in 2 human colon cancer xenografts (HT-29 and BE) and 2 murine colon adenocarcinomas (MAC 16 and 26) after intratumoural injection of 250 microg of drug. Levels of the major bioreductive enzymes (DT-diaphorase, cytochrome P-450 reductase and cytochrome b5 reductase) were measured in tumours using cytochrome c reduction and menadione as the intermediate electron acceptor. There was no correlation between chemosensitivity (T/C: HT-29, 15%; BE, 27%; MAC 16, 33% and MAC 26, 60%) and enzyme activity (r2 = 0.47 for DT-diaphorase, r2 = 0.1 for cytochrome P-450 reductase and r2 = 0.52 for cytochrome b5 reductase). Drug metabolism was followed in vitro using tumour homogenates incubated under aerobic and anaerobic conditions. Four metabolites were identified by HPLC and characterised bv UV-visible spectroscopy. With the exception of the hydrolysis product EO5A, all other metabolites appeared to be drug adducts. No correlation was observed between the kinetics of metabolite formation and antitumour activity. A good correlation (r2 = 0.86) was found with the rate of disappearance of parent drug and antitumour activity. These data show that the overall capacity of a tumour to metabolise EO9 is the most important determinant of antitumour activity rather than the expression of the major bioreductive enzymes and that the parent drug rather than a metabolite leads to the active form of the drug.
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PMID:Pharmacological and biochemical determinants of the antitumour activity of the indoloquinone EO9. 948 90

Maintenance of cellular homeostasis is a critical survival trait when cells are exposed to electrophilic chemicals. Because conjugation and elimination of these toxins is dependent upon sequential and coordinated metabolic pathways, acquired resistance through a gradual adaptive response would rarely be expected to be the consequence of changes in one gene product. Human HT29 colon cancer cells chronically exposed to EA have acquired resistance to the drug. Commensurate with resistance, EA is more effectively conjugated to GSH and effluxed from the resistant cells. Using directed and random (differential display) approaches, a number of detoxification and/or protective gene products have been shown to be expressed at elevated levels. These include gamma-GCS (approximately 3-fold), GST-pi (approximately 3-fold), MRP (approximately 3-fold), NQO1 (approximately 3-fold), DDH (20-fold), and SSP 3521, a transcriptional regulator (approximately 3-fold). Multiple mechanisms contribute to these increases, including enhanced transcriptional rate and prolonged mRNA and protein half lives. Further indications for the involvement of transcriptional regulators is found in HL60 adriamycin-resistant cells which overexpress MRP, GST-pi and gamma-GCS and also have 15-20-fold more DNA-dependent protein kinase. It is possible that this enzyme serves as an early stress response gene which may activate downstream transcription factors. Intriguingly, the catalytic subunit of DNA-dependent protein kinase has a high avidity for [35S]azidophenacyl-GSH. High levels of GSH conjugates indicate cell stress and it would seem reasonable to speculate that DNA-dependent protein kinase may serve as a receiver and transmitter of signals which contribute to drug resistance and maintain cell viability.
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PMID:Coordinate changes in expression of protective genes in drug-resistant cells. 967 55

A network composed of activation and inactivation pathways to regulate mitomycin C (MMC) action is suggested to exist in human cancer cells. COLO201 colon cancer cells were stably transfected with human NQO1 cDNA that encodes NAD(P)H:quinone oxidoreductase (DT-diaphorase, DTD), and a clonal cell line with about 57-fold elevated DTD activity was obtained. Northern analysis revealed that expression of the NADPH:cytochrome P450 reductase (P450 reductase) gene was decreased in the transfectant, COLO201/NQO1, associated with the increase of NQO1 expression. Biochemical characterization of the cells showed a significant increase of the glutathione (GSH) content concomitantly with the decrease of the P450 reductase activity. As a result of these coordinated modulations, sensitivity of COLO201/NQO1 to MMC was not increased as compared to the parent cells. Analyses of inhibition by specific inhibitors of DTD, P450 reductase and glutathione S-transferase (GST) in 5 human colon cancer cell lines including the transfectant showed that DTD and P450 reductase play significant roles in MMC activation in cells with sufficiently high DTD activity and with marginal DTD activity, respectively. In contrast, GST appeared to participate in MMC inactivation in cells with a high level of GST activity. These results indicated that DTD, P450 reductase, GSH and GST may act together compensatively or competitively, depending on their levels in cells, to determine the cellular sensitivity to MMC.
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PMID:Regulatory network of mitomycin C action in human colon cancer cells. 1039 Oct 98


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