Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.1 (NADPH-diaphorase)
 3,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Walker cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The basis of the sensitivity is that CB 1954 forms DNA interstrand crosslinks in Walker cells but not in insensitive cells. Crosslink formation is due to the aerobic reduction of CB 1954 to form 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide by the enzyme DT diaphorase. The 4-hydroxylamine can not crosslink DNA directly but requires further activation by a non-enzymatic reaction with a thioester (such as acetyl coenzyme A). As predicted from their measured DT diaphorase activities, a number of rat hepatoma and hepatocyte cell lines are also sensitive to CB 1954. However, no CB 1954-sensitive tumours or cell lines of human origin have been found. This is because the rate of reduction of CB 1954 by the human form of DT diaphorase is much lower than that of the Walker enzyme (ratio of kcat = 6.4). To overcome this intrinsic resistance of human cells towards CB 1954 a number of strategies have been developed. First, analogues have been developed that are more rapidly reduced by the human form of CB 1954. Second, the cytotoxicity of CB 1954 can be potentiated by reduced pyridinium compounds. Third, a CB 1954 activating enzyme can be targeted to human tumours by conjugating it to an antibody (ADEPT). A nitroreductase enzyme has been isolated from E. coli that can bioactivate CB 1954 much more rapidly than Walker DT diaphorase and is very suitable for ADEPT. Thus CB 1954 may have a role in the therapy of human tumours.
Cancer Metastasis Rev 1993 Jun
PMID:The bioactivation of CB 1954 and its use as a prodrug in antibody-directed enzyme prodrug therapy (ADEPT). 837 21

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is an anti-tumour agent that has a highly selective toxicity to hypoxic cells. In this study we delineate the role of several different bioreductive enzymes in the metabolism of SR 4233 by two tumour cell lines HT 1080 (human fibrosarcoma) and SCCVII (mouse carcinoma). Enzyme kinetics demonstrates similar KM of HT 1080 and SCCVII cell sonicates and differing Vmax. Among all cofactors tested, NADPH was the most important one in reducing SR 4233 by both tumour cell sonicates. NADH was the second most important cofactor while hypoxanthine and N-methylnicotinamide were less involved in the reduction of SR 4233. Carbon monoxide inhibited the reduction by about 60% suggesting that cytochrome P-450 may play a major role in the reduction of SR 4233 under hypoxia in both SCCVII and HT 1080 cells. DT diaphorase is also involved, particularly in HT 1080 cells, in this drug reduction. The level of functional cytochrome P-450, cytochrome P-450 reductase activity and DT diaphorase activity in both cell lines were assayed. These enzyme levels were all higher in SCCVII cells than in HT 1080 cells. This result correlated the higher Vmax of SR 4233 reduction in SCCVII cells than in HT 1080 cells.
Br J Cancer 1993 Feb
PMID:Metabolism of the bioreductive cytotoxin SR 4233 by tumour cells: enzymatic studies. 843 60

Structural modifications to the photoinactive benzophenoxazine Nile blue A have led to three novel derivatives which include 5-ethylamino-9-diethylaminobenzo[a]phenoxazinium (EtNBA), 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium (EtNBS), and 5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium (EtNBSe) chlorides. The incorporation of sulfur and selenium into the benzophenoxazine moiety results in lipophilic, red-absorbing (650-660 nm) chromophores which possess significantly increased singlet oxygen yields (0.025 and 0.65, respectively, compared to 0.005 for EtNBA). This study examines the photosensitizing efficacies and pharmacokinetics in vitro in the EMT-6 murine mammary sarcoma cell line as well as the physicochemical, photochemical, and redox properties of these new analogues. Comparisons with Photofrin II, the only photosensitizer available clinically, were made in an attempt to high-light their different pharmacological characteristics. The photodynamic activity of the benzophenoxazine dyes correlates with their ability to generate the phototoxin singlet oxygen and increases in the following order: EtNBA < EtNBS << EtNBSe. At an extracellular dye concentration of 0.5 microM, the light dose required to kill approximately 50% of the cells was 2.0 and < 0.5 J/cm2 for the sulfur and selenium dyes, respectively. The light dose required to kill approximately 50% of the cells for both EtNBA and Photofrin II could not be determined because of their weak phototoxic effect under these conditions. At a light dose of 3.3 J/cm2, EtNBSe is approximately 1000 times more phototoxic than Photofrin II. All three benzophenoxazine derivatives are characterized by a similar uptake/efflux pattern in vitro consisting of a rapid and extensive cellular accumulation followed by a slow efflux rate. Contrary to their rapid uptake, 50% of the accumulated EtNBS and EtNBSe is retained intracellularly after a 6-h period in dye-free medium. Video-enhanced fluorescence microscopy corroborates the rapid uptake measurements as well as indicating the intracellular localization of the dyes in both living and thermally inactivated cells. Low extracellular dye concentrations (0.05 microM) result in a punctate fluorescence pattern in the perinuclear region, while higher dye concentrations (> 0.1 microM) lead to additional fluorescence in the cytoplasm, cytomembranes, and other organelles but apparently not the nucleus. Absorption spectrometry revealed that living cells rapidly reduce the dyes to their colorless leuko form (photoinactive) if oxygen is not readily available in the environment. It is shown that the cellular reduction is an enzymatic process and that an oxygen-free and cell-free medium containing both the coenzyme NADH and the hydride transfer enzyme diaphorase is capable of reducing the dyes to the colorless leuko form.(ABSTRACT TRUNCATED AT 400 WORDS)
Cancer Res 1993 Jun 01
PMID:Phototoxicity, redox behavior, and pharmacokinetics of benzophenoxazine analogues in EMT-6 murine sarcoma cells. 849 21

Mitomycin C (MMC), an alkylating anti-tumor agent, was activated by non-enzymatic and enzymatic mechanisms leading to DNA binding and adduct formation. However, it was enzymatically, not non-enzymatically, activated MMC which induced inter-strand DNA cross-linking, a major determinant of cell death. The enzymatic activation of MMC was catalyzed by microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic enzyme activities. Human P450 reductase, transiently expressed from its cDNA in the COSI cells, metabolically activated MMC to generate 9 specific MMC-DNA adducts and induced inter-strand DNA cross-linking. Co-chromatography of the MMC-DNA adducts generated by P450 reductase and sodium borohydride in separate experiments indicated that MMC was metabolized by P450 reductase to produce 2,7-diaminomitosenes that exhibited binding to deoxyguanosine. Several experiments indicated that cytosolic enzymes which catalyzed reductive activation of MMC and DNA cross-linking included NAD(P)H:quinone oxidoreductaseI (NQOI or DT diaphorase) when present in extremely high concentrations and a unique cytosolic activity. The unique cytosolic activity was present in several mammalian cells and mouse colon and liver but absent in mouse kidney. The unique activity had properties of a diaphorase but was distinct from NQOI because of a lack of correlation between NQOI (2,6-dichlorophenolindophenol reduction) activity and the amount of MMC-reductive activation leading to DNA cross-linking. This activity was also distinct from xanthine oxidoreductase and NADH-cytochrome b5 reductase, 2 other enzymes that catalyze metabolic activation of MMC, because the unique activity was not inhibited by allopurinol (an inhibitor of xanthine oxidoreductase) and its activity was the same with NADH and NADPH (cytochrome b5 reductase is specific to NADH).
Int J Cancer 1996 Jan 17
PMID:Non-enzymatic and enzymatic activation of mitomycin C: identification of a unique cytosolic activity. 856 27

We have studied the resection specimens from 5 patients with idiopathic megarectum and megacolon and 10 control subjects with non-obstructing colonic cancer. Histological staining with haematoxylin and eosin, and immunocytochemical staining for protein gene product 9.5 (PGP9.5), S100 protein, vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP), and histochemical localization of NADPH diaphorase was performed. The amount of VIP and CGRP present in samples was measured using an enzyme-linked immunosorbent assay. Patients with idiopathic megarectum and megacolon showed hypertrophy of the muscularis mucosae and muscularis externa. The architecture of the innervation as assessed by immunoreactivity for PGP9.5 and S100 protein appeared normal. There was a decrease in the density of innervation of the longitudinal muscle in rectal tissue from patients with idiopathic megarectum, with fewer VIP- and NADPH-diaphorase-containing nerves. In the muscularis mucosae and lamina propria of the rectal samples of patients with idiopathic megarectum, VIP immunoreactivity was higher and more NADPH-diaphorase-containing nerves were seen. CGRP-immunoreactive nerve fibres were only seen in the myenteric plexus. No CGRP-immunoreactive cell bodies were seen. In summary, there is an increase in VIP and nitric oxide containing fibres in the muscularis mucosae and lamina propria and a decrease in the longitudinal muscle in rectal tissue of patients with idiopathic megarectum. Both are NANC (nonadrenergic noncholinergic) inhibitory transmitters in the gut and the possible relationship of the changes in their density with gut function is discussed.
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PMID:Enteric innervation in idiopathic megarectum and megacolon. 900 20

Synthesis of nitric oxide (NO) has been shown in the glandular epithelium of human prostate, with highest levels in the peripheral zone. This location is believed to be the main source of prostatic cancer. The ability of stromal cells to produce NO may contribute to the malignant process. Since solid tumours are prone to hypoxia and malignant progression, experiments were undertaken to test the effect of respiratory block on the induction of nitric oxide synthase (NOS) by a Dunning rat prostatic epithelial line. A metastatic phenotype (Mat-LyLu) was treated in vitro with brief exposure to cyanide in order to mimic transient hypoxic stress. NADPH-diaphorase activities in paraformaldehyde-fixed cells was used to follow the expression of NOS. NADPH-diaphorase activity was found to be inducible by a range of factors, including mechanical damage and infection of cultures. Cyanide induced a dose-dependent staining that was statistically greater than in untreated cells. Consistent with diaphorase staining being a marker for the inducible isoform of NOS (iNOS), induction and enhancement of staining, respectively, was observed in response to treatment with lipopolysaccharide or withdrawal of dexamethasone supplement. Results demonstrate that prostatic epithelia can be triggered in culture to express iNOS by transient oxidative stress in the form of respiratory poisoning by NaCN. Paradoxically, nitric oxide production by epithelia within hypoxic zones of solid tumours may contribute to the promotion and/or inhibition of tumorigenesis.
Cancer Lett 1997 Dec 16
PMID:Transient block of respiratory chain by cyanide triggers NADPH-diaphorase activity (a marker for nitric oxide synthase) in Dunning rat prostatic epithelium. 945 79

Endothelial nitric oxide (NO) synthase (NOS) that is expressed constitutively on microvascular endothelium is believed to be essential to systemic and/or local vascular integrity. Endothelial cells (ECs) were reported to express inducible NOS (iNOS) under some conditions iNOS expression indicates vascular activation. In this study we examined microvascular activation using ECs obtained from patients with ulcerative colitis (UC). We cultured ECs from the mesenteries of surgical UC patients and assayed NOS activity by NADPH-diaphorase cytochemistry and immunocytochemistry with an anti-iNOS antibody. Strong NOS activity was demonstrated on the cells from UC patients (5/5), whereas no activity was detected on the cells from cancer patients and human umbilical vein endothelial cells (HUVEC 0/5, 0/5). Strong iNOS activity was detected by immunoreaction, and large amounts of NO generated were detected by the conversion from [14C]arginine to [14C]citrulline (HUVEC 624+/-376 vs. EC-UC 1,492+/-233 dpm). These results suggest the possibility that ECs express spontaneous and continuous iNOS in active UC. They indicate a close relationship of vascular activation with the pathogenesis of UC.
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PMID:Activation of microvascular endothelial cells in active ulcerative colitis and detection of inducible nitric oxide synthase. 987 1

Coexpression of cytochrome P450 monooxygenases (CYPs) and reductase was found in human gastric mucosa with intestinal metaplasia. Immunohistochemistry showed reactivity to P450 reductase in metaplastic epithelial cells and in pyloric gland cells in glands showing intestinal metaplasia. These cells exhibit NADPH-diaphorase activity. Reverse transcription-PCR analysis and Western blotting showed that CYP1A1 and CYP1A2 were expressed in specimens with intestinal metaplasia. Tissue distribution of CYP1A1 coincided with that of P450 reductase. However, immunoreactivity to CYP1A2 protein was localized only in the pyloric gland cells near the intestinal metaplastic gland. Salmonella typhimurium mutagen assay definitively revealed that microsomes prepared from gastric mucosa with intestinal metaplasia, in particular in the pyloric gland, functionally activated benzo(a)pyrene and 2-amino-3-methylimidazo [4,5-f]quinoline. These results indicate that carcinogen activation by CYP enzymes expressed in the gastric mucosa may contribute to carcinogenesis of the stomach.
Cancer Res 1999 Aug 15
PMID:Mutagenic activation of environmental carcinogens by microsomes of gastric mucosa with intestinal metaplasia. 1046 77

The modulating effect of thymoquinone (TQ) on benzo(a)pyrene (BP)-induced forestomach tumours was investigated in female Swiss albino mice, receiving oral administration of BP at a dose of 1 mg twice weekly for 4 weeks. Administration of 0.01% of TQ in drinking water 1 week before, during and after BP treatment until the end of the experiment resulted in significant suppression of BP-induced tumourigenesis when compared with the group receiving BP alone. TQ inhibited both BP-induced forestomach tumour incidence and multiplicity by 70% and 67%, respectively. Lipid peroxide accumulation and decreased glutathione (GSH) content and glutathione-S-transferase (GST) and DT diaphorase activities were observed in the liver of BP-treated tumour-bearing mice. TQ alone showed a significant induction in the enzyme activities of hepatic GST and DT diaphorase. Mice treated with TQ along with BP showed almost normal hepatic lipid peroxides and GSH levels, and normal enzyme activities compared to the control group. The present data may indicate the potential of TQ, the main constituent of the volatile oil of Nigella sativa seed, as a powerful chemopreventive agent against BP-induced forestomach tumours in mice. The possible modes of action of TQ may be through its antioxidant and anti-inflammatory activities, coupled with enhancement of detoxification processes.
Eur J Cancer Prev 1999 Oct
PMID:Inhibition of benzo(a)pyrene-induced forestomach carcinogenesis in mice by thymoquinone. 1054 99

Vasculogenesis was simultaneously studied with embryogenesis in in ovo chick embryo culture, which was harvested at 40 hours. Endodermal cells and vascular endothelial cells were studied using a new combination of stains, immunohistochemistry (for nuclei and basement membrane) and NADPH-diaphorase activity in whole-mounts, paraffin sections and etched semithin sections. The model can be used for the study of developmental process of blood vessels as well as embryonic physiology of blood vessels vis-a-vis organogenesis in response to different angiogenic agents, drug trials, cancer therapy by angiostatic chemicals/radiations and toxins. Considering that vasculogenesis/angiogenesis as one of the fundamental phenomena in physiology, pathophysiology, toxicology and pharmacology of developmental sciences, the model in developing embryo is presented.
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PMID:A simultaneous ex vivo model of embryogenesis: II. Vasculogenesis. 1077 79


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