• Journal of Life Science
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• v.26 no.2
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• pp.174-180
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• 2016

Dicumarol is a coumarin derivative isolated from sweet clover (Melilotus alba), and has anti-coagulant activity with the inhibitory activity of NAD(P)H quinone oxidoreductase1 (NQO1). NQO1 catalyzes the two-electron reduction of quinones to hydroquinones. Dicumarol competes with NAD(P)H for binding to NQO1, resulting in the inhibition of NQO1 enzymatic activity. The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. The expression of MMPs is regulated by cytokines and signal transduction pathways, including those activated by phorbol myristate acetate (PMA). However, the effects of dicumarol on metalloproteinase (MMP)-9 expression and activity are not investigated here. This study investigated whether dicumarol inhibits MMP-9 expression and activity in PMA-treated human renal carcinoma Caki cells. Dicumarol markedly inhibited the PMA-induced MMP-9 mRNA expression and MMP-9 activity. NF-κB and AP1 promoter activity, which is important in MMP-9 expression, also decreased in dicumarol-treated cells. Furthermore, dicumarol markedly suppressed the ability of PMA-mediated migration in Caki cells. When the relevance of NQO1 in the dicumarol-mediated inhibitory effect on PMA-induced MMP9 activity was elucidated, knock-down of NQO1 with siRNA was found to have no effect on PMA-induced MMP9 activity, suggesting that the stimulating effect of dicumarol on PMA-induced MMP9 activity is independent of NQO1 activity. Taken together, the present studies suggested that dicumarol may inhibit PMA-induced migration via down-regulation of MMP-9 expression and activity.

• Journal of Life Science
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• v.26 no.5
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• pp.531-536
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• 2016

We previously showed that NAD(P)H:quinone oxidoreductase 1 (NQO1) knockout (KO) mice exhibited spontaneous inflammation with markedly increased mucosal permeability in the gut, and that NQO1 is functionally associated with regulating tight junctions in the mucosal epithelial cells that govern the mucosal barrier. Here, we confirm the role of NQO1 in the formation of tight junctions by human colonic epithelial cells (HT29). We treated HT29 cells with a chemical inhibitor of NQO1 (dicumarol; 10 μM), and examined the effect on the transepithelial resistance of epithelial cells and the protein expression levels of ZO1 and occludin (two known regulators of tight junctions between gut epithelial cells). The dicumarol-induced inhibition of NQO1 markedly reduced transepithelial resistance (a measure of tight junctions) and decreased the levels of the tested tight junction proteins. In vivo, luminal injection of dicumarol significantly increased mucosal permeability and decreased ZO1 and occludin protein expression levels in mouse guts. However, in contrast to the previous report that the epithelial cells of NQO1 KO mice showed marked down-regulations of the transcripts encoding ZO1 and occludin, these transcript levels were not affected in dicumarol-treated HT29 cells. This result suggests that the NQO1-depedent regulation of tight junction molecules may involve multiple processes, including both transcriptional regulation and protein degradation processes such as those governed by the ubiquitination/proteasomal, and/or lysosomal systems.

• The Korean Journal of Zoology
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• v.20 no.2
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• pp.101-107
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• 1977

The effects of sodium azide, cAMP, G-strophanthin and dicumarol on the ATP-ase activity and Ca uptake of the fragmented sarcoplasmic reticulum of skeletal muscle were studied and the effects were compared with respect to the enzymatic activity and Ca transport. Sodium azide (0.05 mM) and G-strophanthin (0.25mM) caused no inhibition on either ATPase activity or Ca uptake. cAMP($1\\times10^{-6}\\sim5\\times10^{-4}$) had no effect on ATPase activity while inhibited Ca uptake. Dicumarol (0.05 mM) did not inhibit ATPase activity but caused a decreased Ca uptake of heavier fraction (8,000-12,000xG) of the reticulum fragments.

• Journal of Life Science
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• v.27 no.8
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• pp.873-878
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• 2017

We previously reported that NAD(P)H:quinone oxidoreductase 1 (NQO1)-knockout (KO) mice exhibited spontaneous inflammation in the gut. We also found that NQO1-KO mice showed highly increased inflammatory responses compared with NQO1-WT control mice when subjected to DSS-induced experimental colitis. In a Clostridium difficile toxin-induced mouse enteritis model, NQO1-KO mice were also sensitive compared with NQO1-WT mice. Moreover, numerous studies have shown that NQO1 is functionally associated with immune regulation. Here, we assessed whether NQO1 defects can alter macrophage activation. We found that peritoneal macrophages isolated from NQO1-KO mice produced more IL-6 and $TNF-{\alpha}$ than those isolated from NQO1-WT mice. Moreover, the dicumarol-induced inhibition of NQO1 significantly increased IL-6 and $TNF-{\alpha}$ production in peritoneal macrophages isolated from NQO1-WT mice, as well as in the cultured mouse macrophage cell line, RAW264.7. These results indicate that NQO1 may negatively regulate the activation of macrophages. Knockout or chemical inhibition of NQO1 markedly reduced the expression of $I{\kappa}B$ (inhibitor of $NF{\kappa}B$) in both mouse peritoneal macrophages and RAW264.7 cells. Finally, RAW264.7 cells treated with dicumarol exhibited morphological changes reflecting macrophage activation. Our results suggest that NQO1 may suppress the $NF{\kappa}B$ pathways in macrophages, thereby suppressing the activation of these cells. Thus, immunosuppressive activity may be among the many possible functions of NQO1.

• BMB Reports
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• v.32 no.2
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• pp.127-132
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• 1999

The NAD(P)H-quinone oxidoreductase (EC 1. 6. 99. 2) was purified from S. cerevisiae. The native molecular weight of the enzyme is approximately 111 kDa and is composed of five identical subunits with molecular weights of 22 kDa each. The optimum pH of the enzyme is pH 6.0 with 1,4-benzoquinone as a substrate. The apparent $k_m$ for 1,4-benzoquinone and 1,4- naphthoquinone are 1.3 mM and $14.3\;{\mu}M$, respectively. Its activity is greatly inhibited by $Cu^{2+}$ and $Hg^{2+}$ ions, nitrofurantoin, dicumarol, and Cibacron blue 3GA. The purified NAD(P)H-quinone oxidoreductase was found capable of reducing aromatic nitroso compounds as well as a variety of quinones, and can utilize either NADH or NADPH as a source of reducing equivalents. The nitroso reductase activity of the purified NAD(P)H-quinone oxidoreductase is strongly inhibited by dicumarol.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.638-639
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• 2000

Quinone reductase was purified to electrophoretic homogeneity from bovine liver by using ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration chromatography. The enzyme utilized either NADH or NADPH as the electron donor. The optimum pH of the enzyme was pH 8.5, and the activity of the enzyme was greatly inhibited by $Cu^{2+}$ and $Hg^{2+}$ ions, dicumarol and cibacron blue 3GA. The enzyme catalyzed the reduction of several quinones and other artificial electron acceptors. Furthermore, the enzyme catalyzed NAD(P)H-dependent reduction of azobenzene or 4-nitroso-N,N-dimethylaniline. The apparent $K_m$ for 1,4-benzoquinone, azobenzene, and 4-nitroso-N,N-dimethylaniline was 1.64mM, 0.524mM and 0.225mM, respectively. The reduction of azobenzene or 4-nitroso-N,N-dimethylaniline by quinone reductase was strongly inhibited by dicumarol or cibacron blue 3GA, potent inhibitors of quinone reductase.

• Journal of Life Science
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• v.12 no.3
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• pp.281-287
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• 2002

Quinone reductase was purified to homogeneity from bovine liver and the purified enzyme catalyzed the reduction of phenanthrenequinone as well as benzo- and naphthoquinones. The enzyme catalyzed the biotransformation of arylnitroso nitroso compound and the reaction product was identified by TLC, GC, CC-MS and NMR. The reaction was almost entirely inhibitable by Cibacron blue 3GA or dicumarol, potent inhibitors of mammalian quinone reductase.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.4
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• pp.216-220
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• 2002

1,4-Benzoquinone reductase was purified to electrophoretic homogeneity from bovine liver, and the purified enzyme found to have a molecular mass of 29 kDa, as determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis The enzyme exhibited pH optimum between 8.0 and 8.5. The apparent fm for 1,4-benzoqulnone was 1.643 mM, and the apparent Km for NADH was 1.837 mM. Various divalent cations, such as Hg$\^$2+/, Cu$\^$2+/, and Zn$\^$2+/, exhibited strong inhibitory effects. The enzyme activity was also strongly inhibited by quercetin, dicumarol, and benzoic acid. Incubation of the enzyme with N-bromosuccinimide and pyridoxal 5’-phosphate led to inhibitions of 100% and 99%, respectively. Accordingly, these results suggest that trypto-phan and Iysine residues are Involved at or near the active sites of the enzyme.

• BMB Reports
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• v.34 no.3
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• pp.225-229
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• 2001

Besides a variety of quinones, purified bovine liver quinone reductase catalyzed the reduction of N,N-p-nitrosoaniline to N,N-dimethyl-p-phenylenediamine. The formation of N,N-dimethyl-p-phenylenediamine was identified by TLC, GC, GC-MS and NMR. Quinone reductase can utilize either NADH or NADPH as a source of reducing equivalents. The apparent Km for 1,4-benzoquinone and N,N-dimethyl-p-nitrosoaniline was 1.64 mM and 0.22 mM, respectively The reduction of N,N-dimethyl-p-nitrosoaniline was almost entirely hampered by dicumarol or Cibacron blue 3GA, potent inhibitors of mammalian quinone reductase. During the bovine liver quinone reductase-catalyzed reduction of N,N-dimethyl-p-nitrosoaniline, benzoquinonediiminium ion was produced.

• BMB Reports
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• v.33 no.4
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• pp.321-325
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• 2000

Quinone reductase was purified to homogeneity from bovine liver by using ammonium sulfate fractionation, ionexchange chromatography, and gel filtration chromatography. The enzyme utilized either NADH or NADPH as the electron donor. The enzyme catalyzed the reduction of several quinones and other artificial electron acceptors. Furthermore, the enzyme catalyzed NAD(P)H-dependent reduction of azobenzene. The apparent Km for 1,4-benzoquinone and azobenzene was 1.64 mM and 0.524 mM, respectively. The reduction of azobenzene by quinone reductase was almost entirely inhibited by dicumarol or Cibacron blue 3GA, potent inhibitors of the mammalian quinone reductase. In the presence of 1.0${\mu}M$ Cibacron blue 3GA, azoreductase activity was lowered by 45%, and almost complete inhibition was seen above 2.0 ${\mu}M$ Cibacron blue 3GA.