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)

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

Human colon carcinoma HT29 cells sensitive (WT) and resistant (HT/M and HT/S) to ethacrynic acid (EA) were used to investigate the role of NAD(P)H:quinone oxidoreductase1 (NQO1) in drug resistance. Significant decreases in the levels of NQO1 activity were observed in resistant cells as compared with the sensitive cells. However, the decreased activities of NQO1 in resistant cells were found to be due to inhibition of the enzyme by EA. Human NQO1 cDNA-derived protein in monkey kidney COS1 cell extract was used to demonstrate that in vitro inhibition of NQO1 activity by EA was rapid, reversible and concentration dependent, with an IC50 value of 250 microM. These results suggest that NQO1 may not have a role in EA resistance of human colon carcinoma HT29 cells and that EA is an inhibitor of NQO1 activity.
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PMID:Inhibition of NAD(P)H:quinone oxidoreductase1 in ethacrynic acid-resistant human colon carcinoma cells. 801 Sep 86

Diaziridinylbenzoquinones such as 3,6-diaziridinyl-1,4-benzoquinone (DZQ) and its 2,5-methyl analog (MeDZQ) require bioreductive activation in order to elicit their cytotoxic activities. In this study, we have mapped the intracellular alkylation sites induced by DZQ and MeDZQ in a single copy gene at the nucleotide level using ligation-mediated polymerase chain reaction. We have performed this analysis in two human colon carcinoma cells, one proficient (HT-29) and one deficient (BE) in DT-diaphorase (DTD) activity. In the DTD-proficient HT-29 cell line, DZQ and MeDZQ were found to alkylate both 5'-(A/T)G(C)-3' and 5'-(A/T)A-3' sequences. This is consistent with the nucleotide preferences observed when DZQ and MeDZQ are activated by purified DTD to reactive metabolites capable of alkylating DNA in vitro (C-S. Lee, J. A. Hartley, M. D. Berardini, J. Butler, D. Siegel, D. Ross, and N. W. Gibson. Biochemistry, 31: 3019-3025, 1992). Surprisingly in the DTD-deficient BE cell line a pattern of alkylation induced by DZQ and MeDZQ similar to that observed in the DTD-proficient HT-29 cells was observed. This suggests that reductive enzymes other than DTD can be involved in activating DZQ and MeDZQ to DNA-reactive species in vivo.
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PMID:Mapping of DNA alkylation sites induced by aziridinylbenzoquinones in human cells by ligation-mediated polymerase chain reaction. 813 69

7-N-((2-([2-(gamma-L-Glutamylamino)ethyl]dithio)ethyl))mitomycin C (KW-2149) is an analogue of mitomycin C (MMC) and has prominent activities against various tumors. We studied the antitumor effects of KW-2149 in MMC-resistant variants of human colon carcinoma HT-29 (HT-29/MMC) and mouse hepatoma Hepa-I (C4, B13NBii1) cells, which are deficient in DT-diaphorase and cytochrome P450 reductase, respectively. These enzymes mediate the reductive activation of MMC in the cells. Although HT-29/MMC and C4, B13NBii1 cells showed significant resistance to MMC, they showed sensitivity tl KW-2149 comparable to their parental tumors, indicating that DT-diaphorase and cytochrome P450 reductase could not be involved in the activation of KW-2149. In studying the activation mechanism of KW-2149, we found that glutathione (GSH) and cysteine significantly enhanced the cytotoxicity of KW-2149 in HT-29 cells. The DNA adduct of KW-2149 was increased when HT-29 cells or the isolated nuclei of the cells were incubated with KW-2149 in the presence of physiological concentrations of GSH and cysteine. KW-2149 alkylated calf thymus DNA in the presence of GSH and cysteine in vitro. These results indicate that activation of KW-2149 by thiol molecules, unlike MMC, could be an important activation mechanism of KW-2149 to form DNA adduct and to exert its cytotoxicity. This is the reason why KW-2149 is effective against MMC-resistant tumors with deficiencies in the MMC activation enzymes.
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PMID:Nonenzymatic reductive activation of 7-N-((2-([2-(gamma-L-glutamylamino)ethyl]dithio)ethyl))mitomycin C by thiol molecules: a novel mitomycin C derivative effective on mitomycin C-resistant tumor cells. 816 87

Resistance of hypoxic tumor cells to ionizing radiation and cytotoxic drugs has been attributed to changes in the reactivity and/or the half-times of reactive species in the altered redox environment. Exposure of eukaryotic cells to such hypoxic conditions results in the induction of the synthesis of several unrelated proteins. To investigate further the phenomenon of hypoxic cell resistance to cytotoxic drugs, we examined the effects of hypoxia on the expression of a group of enzymes involved in drug metabolism. Exposure of HT29 colon carcinoma cells to hypoxia resulted in a marked increase in the activity of DT-diaphorase and in glutathione content. The activity of glutathione transferase was not increased by this treatment. The response was proportional to the duration of hypoxia. After the cells were exposed to hypoxic conditions for 8 h, followed by restoration of an oxic environment, the elevation in enzyme activity and glutathione content reached a peak at 48 h (40 h after the restoration of an oxic environment) and returned to baseline at 72 h. Elevation of steady-state levels of DT-diaphorase and gamma-glutamylcysteine synthetase mRNA followed a similar time course, with > 10-fold increases over oxic cells at 24 h. The elevation of DT-diaphorase mRNA content was found to result both from transcriptional induction and from increased message stability. The magnitude and persistence of elevated detoxicating enzyme activity following a relatively short hypoxic exposure followed by reoxygenation suggest a novel potential mechanism of resistance to cytotoxic drugs in hypoxic tumors.
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PMID:Effects of hypoxia on detoxicating enzyme activity and expression in HT29 colon adenocarcinoma cells. 820 21

The metabolism, cytotoxicity, and genotoxicity of streptonigrin (SN) w ere determined in two human colon carcinoma cell lines: HT-29 with high NAD(P)H:quinone oxidoreductase (EC 1.6.99.2, DTD) activity and BE with undetectable DTD activity. Dicumarol-sensitive oxidation of NADH was observed with HT-29 cytosol, but not with BE cytosol. Oxygen consumption was also observed using HT-29 cytosol, but was absent with BE cytosol. Dicumarol inhibited oxygen consumption with HT-29 cytosol, but deferoxamine had no effect, suggesting that divalent metal cations were not necessary for efficient auto-oxidation of SN hydroquinone. In cytotoxicity studies, SN was much more toxic to the DTD-rich HT-29 cells than to the DTD-deficient BE cells. Deferoxamine decreased toxicity in both cell lines, implicating hydroxyl radicals produced during Fenton-type reactions as the toxic species. In the genotoxicity assay, SN induced a much higher incidence of DNA strand breaks in HT-29 cells than in BE cells, and deferoxamine protected against DNA strand breaks in both cell lines. Some evidence of DNA repair was also observed in the two cell lines. These results support an important role for DTD in the cytotoxicity of SN in the high DTD HT-29 colon carcinoma cell line.
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PMID:Role of NAD(P)H:quinone oxidoreductase (DT-diaphorase) in cytotoxicity and induction of DNA damage by streptonigrin. 861 1

DT-diaphorase (DTD) activity has been related to bioactivation and cytotoxicity of antitumor quinones. A pair of human colon adenocarcinoma cell lines, HT29 and BE, were used in this study to examine the role of DTD in antitumor quinone induced apoptosis. HT29 cells have elevated levels of DTD whereas BE cells lack functional DTD due to a point mutation which results in a complete lack of DTD activity. MeDZQ, a quinone that is efficiently bioactivated by DTD, induced apoptosis both in HT29 and BE cells, but with a much higher incidence in HT29, as assessed by morphological criteria and the formation of oligonucleosomal fragments of DNA. Two other quinone compounds which are also substrates for DTD, i.e. streptonigrin and mitomycin C, also preferentially induced apoptosis in HT29 cells, which could be inhibited by dicoumarol. Our data suggest that bioreductive activation of antitumor quinones by DTD results in induction of apoptosis in human colon carcinoma cells.
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PMID:Quinone-induced apoptosis in human colon adenocarcinoma cells via DT-diaphorase mediated bioactivation. 865 8

Bioreducible anti-tumour agents are prodrugs which are intended to be inactive in normal cells, but are able to undergo metabolic reduction in cancer cells to produce toxic species that can damage biomolecules. A series of N-oxides of heterocyclic aliphatic amines were designed and prepared as mentioned below as bioreducible drugs based on the reported anti-cancer activity of 2,6-bis(halomethyl)piperidines. In order to study structure-activity relationships in these conformationally restricted nitrogen mustards, samples of cis- and trans-2,6-dihydroxymethyl-N-methylpiperidine were prepared and converted into a number of carbamate or halogen derivatives. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation. The corresponding N-oxides were also prepared for comparison in cytotoxicity tests. In total, 21 new compounds were synthesized plus cis-N-methyl-2,6-bis(chloromethyl)-piperidine (prepared previously but lacking spectroscopic data) and tested against two human colon carcinoma cell lines, HT 29 (high DT-diaphorase) and BE (no DT-diaphorase), under oxic and hypoxic conditions. The majority of the free bases were equally toxic against both cell lines. The most toxic compounds were cis- and trans-N-methyl-2,6-bis(bromomethyl)piperidine with oxic IC50 values between 6 and 11 microM against both cell lines. The N-oxides were relatively non-toxic under both oxic and hypoxic conditions apart from the N-oxide of trans-N-methyl-2,6-bis(bromomethyl)-piperidine. Their low toxicity suggested that the N-oxides are not reduced under hypoxic conditions. We conclude that: (i) 2,6-disubstituted N-methylpiperidine derivatives are chemically versatile cytotoxic entities that are suitable for prodrugging to enhance their therapeutic selectivity; and (ii) N-oxide prodrugs of these compounds are deactivated chemically and display reduced cytotoxicity compared to the parent amines but are apparently not reduced under hypoxic conditions. At least in the colorectal cell lines tested the latter issue would need to be addressed by modifying the redox properties in future work to progress this approach.
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PMID:Synthesis and anti-cancer activity of 2,6-disubstituted N-methylpiperidine derivatives and their N-oxides. 883 8

NAD(P)H:quinone oxidoreductase (NQO1, EC 1.6.99.2) is an obligate two-electron reductase that can either bioactivate or detoxify quinones and has been proposed to play an important role in chemoprevention. We have previously characterized a homozygous point mutation in the BE human colon carcinoma cell line that leads to a loss of NQO1 activity. Sequence analysis showed that this mutation was at position 609 of the NQO1 cDNA, conferring a proline to serine substitution at position 187 of the NQO1 enzyme. Using polymerase chain reaction (PCR) analysis, we have found that the H596 human non-small-cell lung cancer (NSCLC) cell line has elevated NQO1 mRNA, but no detectable enzyme activity. Sequencing of the coding region of NQO1 from the H596 cells showed the presence of the identical homozygous point mutation present in the BE cell line. Expression and purification of recombinant wild-type and mutant protein from E. coli showed that mutant protein could be detected using immunoblot analysis and had 2% of the enzymatic activity of the wild-type protein. PCR and Northern blot analysis showed moderate to low levels of expression of the correctly sized transcript in the mutant cells. Immunoblot analysis also revealed that recombinant mutant protein was immunoreactive; however, the mutant protein was not detected in the cytosol of either BE or H596 cells, suggesting that the mutant proteins were either not translated or were rapidly degraded. The absence of any detectable, active protein, therefore, appears to be responsible for the lack of NQO1 activity in cells homozygous for the mutation. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for the mutation at position 609 conducted on 90 human lung tissue samples (45 matched sets of tumour and uninvolved tissue) revealed a 7% incidence of individuals homozygous for the mutation, and 42% heterozygous for the mutation. These data suggest that the mutation at position 609 represents a polymorphism in an important xenobiotic metabolizing enzyme, which has implications for cancer therapy, chemoprevention and chemoprotection.
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PMID:Characterization of a polymorphism in NAD(P)H: quinone oxidoreductase (DT-diaphorase). 900 Jun

It has been demonstrated that synthetic quinones, such as menadione, cause DNA damage in different cell systems, possibly being mediated by free radicals generated during redox cycling. It has been suggested that the damage caused could be related to tumor induction in different sites. To our knowledge it has not yet been demonstrated that the natural quinones, vitamin K1 and K2, exert the same activity. Using a colon carcinoma cell line, HT-29, we examined the extent of DNA damage induced by menadione, vitamin K1 and K2. Menadione caused significant DNA damage at low concentrations (25-200 microM) with a linear correlation of r = 0.95. In the presence of dicoumarol, a DT-diaphorase inhibitor, the damage was detected at concentrations five times lower indicating that free radicals generated during the redox cycling play a key role. Neither vitamin K1, incorporated in micelles, nor K2 caused detectable single strand breaks with respect to the controls either in the presence or in absence of dicoumarol. Our results demonstrate that, despite their redox cycling properties, the natural forms of vitamin K do not cause DNA damage in HT-29 cells as menadione does in the experimental conditions used.
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PMID:Quinone-induced DNA single strand breaks in a human colon carcinoma cell line. 905 88


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