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 flavoenzyme DT-diaphorase has the potential either to bioactivate or to detoxify different bioreductive cytotoxins. Elucidation of structural features governing the ability to act as a substrate for DT-diaphorase should facilitate rational optimization or elimination of this reductive pathway for a particular class of bioreductive drug. We have examined structure-activity relationships governing both the cytotoxicity and the DT-diaphorase mediated reduction of two groups of bioreductive alkylating agents: (1) Indoloquinones related to EO9 [3-hydroxy-methyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)prop-beta - en-alpha-ol]; and (2) derivatives of diaziridinyl benzoquinone or diaziquone [2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone]. The rat U.K. 256 Walker tumor cell line and the human HT29 colon carcinoma line were studied because of their high DT-diaphorase content. Enzyme activity was measured spectrophotometrically by dicoumarol inhibitable cytochrome c reduction in the presence of drug, and aerobic cytotoxicity was assessed by the MTT assay. EO9 acted as a good substrate for both enzyme preparations and was highly potent in each cell line, especially in Walker tumor cells (ID50 0.039 nM). AZQ was also reduced efficiently and gave an ID50 of 6 nM in the Walker tumor line. Slight modifications in structure resulted in large variations in both DT-diaphorase metabolism and toxicity for both types of agent. There was a clear tendency for the most efficiently reduced analogues to exhibit greater cytotoxic potency. Inclusion of an aziridine moiety in the structure appears to be desirable, but not essential, for both rapid reduction and cytotoxicity. There was no evidence of active site-directed enzyme inhibition.
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PMID:Structure-activity relationships for DT-diaphorase reduction of hypoxic cell directed agents: indoloquinones and diaziridinyl benzoquinones. 154 32

We describe a rapid and direct assay of NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) activity in cultured cells suitable for identifying and purifying inducers of this detoxication enzyme. Hepa 1c1c7 murine hepatoma cells are plated in 96-well microtiter plates, grown for 24 h, and exposed to inducing agents for another 24 h. The cells are then lysed and quinone reductase activity is assayed by the addition of a reaction mixture containing an NADPH-generating system, menadione (2-methyl-1,4-naphthoquinone), and MTT [3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide]. Quinone reductase catalyzes the reduction of menadione to menadiol by NADPH, and MTT is reduced nonenzymatically by menadiol resulting in the formation of a blue color which can be quantitated on a microtiter plate absorbance reader. The reaction is more than 90% dicoumarol inhibitable and menadione dependent. The results are comparable to those obtained by harvesting cells from larger plates, preparing cytosols, and carrying out spectrophotometric measurements.
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PMID:Direct measurement of NAD(P)H:quinone reductase from cells cultured in microtiter wells: a screening assay for anticarcinogenic enzyme inducers. 338 6

To elucidate the mechanisms of acquired resistance to mitomycin C (MMC) in non-small cell lung cancer (NSCLC), we established two MMC-resistant NSCLC sublines by continuous exposure to MMC, using PC-9 as a parent cell line. The sublines, PC-9/MC2 and PC-9/MC4, were 6.4- and 10-fold more resistant to MMC than their parent cell line, respectively, at the IC50 value as determined by MTT assay. They exhibited cross-resistance to EO9, but were not resistant to cisplatin, vindesine, etoposide, carboquone, or KW-2149, a novel MMC derivative. They were collaterally sensitive to adriamycin and menadione. Accumulation of the drug was decreased in the resistant sublines to about 60% of that in the parent cells. Cytosolic DT-diaphorase (DTD) activities were decreased to 13.5 +/- 3.2 in PC9/MC2 and 1.3 +/- 0.6 in PC-9/MC4 from 261.5 +/- 92.7 nmol/min/mg protein in the parent PC-9. NADH:cytochrome b5 reductase activities in both of the resistant cell lines were significantly decreased as compared to that in the parent cell line. Addition of dicumarol resulted in a two-fold increase in IC50 value in PC-9, whereas the IC50 value showed no change in PC-9/MC4. Moreover, dicumarol did not affect the sensitivities to KW-2149 but decreased the sensitivities to EO9 in both the parent and the resistant cell lines. Formation of an alkylating metabolite was significantly decreased in the resistant cells, in parallel to the degree of resistance. We concluded that deficient drug activation due to decreased DTD activity was important as a mechanism of resistance to MMC in PC-9, a relatively DTD-rich NSCLC cell line.
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PMID:Establishment and characterization of non-small cell lung cancer cell lines resistant to mitomycin C under aerobic conditions. 779 Mar 19

We have reported the establishment of a mitomycin-C (MMC)-resistant non-small-cell lung-cancer cell line, PC-9/MC4. As determined by an MTT assay, this resistant cell line was found to be 4 times more sensitive to adriamycin (ADM) than was the parental PC-9. There were no significant differences in sensitivity to etoposide, mitoxantrone, daunomycin, epirubicin, pirarubicin, 9-aminoanthracycline or 3'-deamino-3'-morpholino-13-deoxo-10-hydroxy carminomycin. These data suggest that neither qualitative or quantitative changes in DNA topoisomerase II nor the enhanced repair of DNA can explain the differing sensitivity to ADM observed. No significant differences were found in the accumulation of ADM and glutathione (GSH) in these cell lines. Although total glutathione-S-transferase (GST) activity in PC-9/MC4 cells was lower than that observed in PC-9 cells and treatment with ethacrynic acid (EA) reduced sensitivity to ADM in both cell lines, relative resistance was unaffected. NADH-cytochrome b5 reductase (B5R) activity in PC-9/MC4 cells showed a 3-fold greater decrease than that in PC-9 cells, and DT-diaphorase (DTD) activity in PC-9/MC4 cells showed an approximately 200-fold greater decrease than that in PC-9 cells. Addition of dicumarol, an inhibitor of DTD, decreased the sensitivity of ADM of PC-9 but not of PC-9/MC4. DTD activity in the PC-9 cell line was inhibited by treatment with dicumarol while in PC-9/MC4 it remained unchanged. These data suggest that DT-diaphorase is a determinant of sensitivity to ADM in the 2 cell lines.
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PMID:DT-diaphorase as a determinant of sensitivity to adriamycin in non-small-cell lung-cancer cell lines. 792 20

Microculture tetrazolium assays (MTAs) rely upon the bioreduction of tetrazolium salts to their intensely coloured formazans. Although these assays are being extensively used, the intracellular mechanisms responsible for the formazan production are not known. MTAs currently provide the basis for uniquely precise in vitro bioassays for human growth hormone (hGH) which use the Nb2 cells. We have compared two contrasting tetrazolium salts, namely 3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) and 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-++ +sulfophenyl) tetrazolium, inner salt (MTS), in this system. An intermediate electron acceptor (IEA) is obligatory for the MTS- but not the MTT-bioassay. We report that inhibitors of DT-diaphorase abolished MTS- but not MTT-formazan production. We conclude that substitution of MTT with MTS/menadione resulted in formazan production via a different electron transfer pathway which is exclusively mediated by DT-diaphorase.
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PMID:Growth hormone-responsive DT-diaphorase-mediated bioreduction of tetrazolium salts. 883 14

Humans ingest about 1 g of flavonoids daily in their diet, and they are increasingly being associated with cytoprotective antitumour properties. The mechanism(s) responsible for these effects have not yet been elucidated but may involve interaction with xenobiotic metabolising enzymes to alter the metabolic activation of potential carcinogens. We have investigated the effect of the flavonoids, quercetin (Q), myricetin (M) and epicatechin (E) on the growth, morphology and enzyme activities of MCF7 human breast cancer cells. Of the three flavonoids studied only Q caused a decrease in cell protein content and decreased the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium). It also inhibited protein, DNA and RNA synthesis to the greatest extent. Q and M increased intracellular reduced glutathione (GSH) content, and Q altered the morphology of the cells after 24 h exposure to 25 microM. E and Q inhibited the O-deethylation of ethoxyresorufin (EROD) catalysed by cytochrome P450 CYPIA. In contrast, M increased the EROD reaction 2-fold. Q increased the activity of DT-diaphorase, NADPH cytochrome c reductase and glutathione reductase, while E increased only NADPH cytochrome c reductase activity. The effects on enzyme activities in vitro suggest that there is not only the potential for flavonoids to alter metabolic activation of carcinogens but also of therapeutically administered drugs in vivo. We are at present investigating the synergy between anti-cancer drugs and flavonoids in terms of anti-tumour efficacy.
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PMID:The effect of the flavonoids, quercetin, myricetin and epicatechin on the growth and enzyme activities of MCF7 human breast cancer cells. 992 Apr 63

The mode of action of mitomycin C (MMC)-induced cell death in lens epithelia was investigated using cell culture system. The cytotoxicity of MMC on alpha-TN4 mouse lens epithelial cells (LECs) was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay. To determine the type of cell death induced by MMC, we stained LECs with acridine orange and ethidium bromide, and performed TUNEL assay and electron microscopic examination. The activation of MMC was also investigated with N-acetly-L-cystein (NAC), a free radical scavenger, and dicumarol, an inhibitor of DT-diaphorase, to analyze one- and two-electron reduction pathways involved in the activation of MMC. MTT assay showed that 400 microg/ml of MMC decreased the cell viability of the control cells to 45%, and the cytotoxicity of MMC on alpha-TN4 cells was increased in a dose-dependent manner. Based on the results obtained from acridine orange and ethidium bromide staining, the TUNEL assay, and transmission electron microscopic examination, we confirmed that MMC-induced cell death, at 400 microg/ml of MMC, occurs with necrosis as well as apoptotis. The treatment of cells with 2 mM NAC for 1 h or 20 microM dicumarol for 30 min, prior to 5 min of MMC (400 microg/ml) treatment, restored the growth suppression to 76 and 80% of the control level, respectively. In addition, the cotreatment of cells with NAC and dicumarol restored cell viability to 90% of the control level. The cellular death in MMC-treated LECs showed the involvement of oxidative stress in this apoptosis accounting for 22% of the observed cell death at 400 microg/ml of MMC.
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PMID:Mitomycin C-induced cell death in mouse lens epithelial cells. 1229 94

Dihydroisotanshinone I is a phenanthrenequinone derivative isolated from the roots of Salvia trijuga Diels. The present study demonstrated the hepatoprotective effect of dihydroisotanshinone I against menadione-induced cytotoxicity in a primary culture of rat hepatocytes. Pretreating the cells with dihydroisotanshinone I at concentrations ranging from 2.5 microM to 20 microM for 24 hours caused dose-dependent protection against hepatotoxicity induced by menadione. Intracellular glutathione level and activity of DT-diaphorase have been suggested to play important roles in menadione-induced cytotoxicity. However, treating the hepatocytes with 20 microM dihydroisotanshinone I for 24 hours did not cause a significant change in glutathione level and DT-diaphorase activity. On the contrary, adding dihydroisotanshinone I to freshly isolated hepatocytes at concentrations between 50 nM to 200 nM inhibited NADH-induced superoxide production dose-dependently as indicated by the decrease of lucigenin-amplified chemiluminescence. In addition, dihydroisotanshinone I at concentrations ranging from 5 microM to 20 microM inhibited tert-butyl hydroperoxide-induced lipid peroxidation dose-dependently in isolated hepatocytes as indicated by the level of malondialdehyde. These results suggest that the protective action of dihydroisotanshinone I against menadione-induced hepatotoxicity is attributed to its antioxidant properties including the free radical scavenging activity and inhibition of lipid peroxidation. Abbreviations. DTD:DT-diaphorase GSH:glutathione LDH:lactate dehydrogenase MDA:malondialdehyde MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide TBHP: tert-butyl hydroperoxide
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PMID:Dihydroisotanshinone I protects against menadione-induced toxicity in a primary culture of rat hepatocytes. 1249 33

The synthesis and biological evaluation of a homologous series of conjugates (9-13) of 2,5-diaziridinylbenzoquinone (DZQ) and 9-carbonylacridine, a DNA intercalating moiety, via a polymethylene unit (n=2-6) are described. In addition, the non-acridine compound 14, analogous to compound 12, and the 5-methyl-DZQ derivatized conjugate 15, an analog of compound 10, were also prepared. Through a Comet assay, compounds 9-13 were shown to produce DNA interstrand cross-links at submicromolar concentrations, consistent with K562 leukemia cells accumulating in the G2/M stage in the cell cycle. The cytotoxicity of compounds 9-15 was examined using a MTT assay on several human cancer cell lines, including chronic myeloid leukemia K562, the non-small cell lung cancers H596 and H460, and colon carcinoma cells BE and HT29. H460 and HT29 are rich in DT-diaphorase (DTD), and H596 and BE cells have negligible amounts of functional DTD. Under continuous exposure of drugs, except to the non-aziridine compound 19b, the IC50 values of all other compounds were determined to be in the range of 0.3-11.3 nM. Compound 10, which has a propyl linker group, was subjected to in vivo studies. When BDF1 mice with established mouse mammary carcinoma were treated with compound 10 (2 mg/kg at day 1 and 5 mg/kg at day 7), a significant delay (9-10 days) in cancer growth was recorded when compared to untreated controls. Furthermore, administration of compound 10 to nu/nu BDF1 mice bearing human lung cancer H460 xenograft (1.5 mg/kg for 10 for five consecutive days from day 13 and 17) also showed a significant reduction in tumor growth compared to untreated controls. The half-life of compound 10 in the presence of five different peptidases (porcine esterase, carboxypeptidase A, B and Y, and pepsin) was determined to be between 30 and 60 h.
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PMID:Synthesis and biological evaluation of novel diaziridinylquinone-acridine conjugates. 1450 82

We developed concise, accurate prediction models of the in vitro activity for 8 anticancer drugs (5-FU, CDDP, MMC, DOX, CPT-11, SN-38, TXL and TXT), along with individual clinical responses to 5-FU using expression data of 12 genes. We first performed cDNA microarray analysis and MTT assay of 19 human cancer cell lines to sort out genes which were correlative in expression levels with cytotoxicities of the 8 drugs; we selected 13 genes with proven functional significance to drug sensitivity from a huge number of potent prediction marker genes. The correlation significance of each was confirmed using expression data quantified by real-time RT-PCR, and finally 12 genes (ABCB1, ABCG2, CYP2C8, CYP3A4, DPYD, GSTP1, MGMT, NQO1, POR, TOP2A, TUBB and TYMS) were selected as more reliable predictors of drug response. Using multiple regression analysis, we fixed 8 prediction formulae which embraced the variable expressions of the 12 genes and arranged them in order, to predict the efficacy of the drugs by referring to the value of Akaike's information criterion for each sample. These formulae appeared to accurately predict the in vitro efficacy of the drugs. For the first clinical application model, we fixed prediction formulae for individual clinical response to 5-FU in the same way using 41 clinical samples obtained from 30 gastric cancer patients and found to be of predictive value in terms of survival, time to treatment failure and tumor growth. None of the 12 selected genes alone could predict such clinical responses.
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PMID:Concise prediction models of anticancer efficacy of 8 drugs using expression data from 12 selected genes. 1523 42


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