dicumarol and 2-3-dimethoxy-1-4-naphthoquinone

Topics: Animals; Cytochrome-B(5) Reductase; Dicumarol; Enzyme Inhibitors; gamma-Glutamyltransferase; Gene Expression Regulation, Enzymologic; Glutamate-Cysteine Ligase; Glutathione; Glutathione Peroxidase; Humans; Hydrogen Peroxide; Molecular Structure; NAD(P)H Dehydrogenase (Quinone); NADPH-Ferrihemoprotein Reductase; Naphthoquinones; Nitric Oxide Synthase; Oxidation-Reduction; Protein Isoforms; Quinone Reductases; Quinones; Response Elements; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured; Vitamin K 3; Xenobiotics

The stress-activated protein kinases SAPK/JNK and p38/mHOG are activated by diverse classes of stress stimuli, many of which induce redox perturbations. We studied the effects of reactive quinones on stress signaling pathways. Menadione (2-methyl-1,4-naphthoquinone), which undergoes both one- and two-electron reduction, completely inhibited SAPK activity at high concentrations while activating SAPK at lower concentrations. Menadione activated p38/mHOG dose responsively. 2,3-Dimethyl-1,4-naphthoquinone (DMNQ), which preferentially undergoes two-electron reduction, had similar effects. In contrast, 1,4-naphthoquinone, which preferentially undergoes one-electron reduction, inhibited SAPK at high concentrations, but failed to activate SAPK at any concentration tested. In addition, this quinone activated p38 only at lower concentrations; high concentrations inhibited p38 activity. These activity profiles correlated with the activation state of the upstream kinase, indicating that the effects were mediated by an upstream step in the kinase pathway. The quinone reductase inhibitor dicoumarol blocked activation of SAPK by menadione and DMNQ, suggesting that two-electron reduction is important. Finally, addition of increasing amounts of hydrogen peroxide mimicked the effects of menadione and DMNQ, suggesting that hydrogen peroxide may be the relevant mediator. Differential activation of stress kinases by reactive quinones demonstrates that the cellular redox environment independently modulates these pathways.

Topics: Animals; Antifibrinolytic Agents; Dicumarol; Dose-Response Relationship, Drug; Electrons; Enzyme Activation; Enzyme Inhibitors; Hydrogen Peroxide; Mice; Mitogen-Activated Protein Kinases; Naphthoquinones; NIH 3T3 Cells; Osmosis; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Protein Isoforms; Quinones; Uncoupling Agents; Vitamin K 3

The human colon carcinoma cell lines Caco-2 and HT-29 were exposed to three structurally related naphthoquinones. Menadione (MEN), 1,4-naphthoquinone (NQ), and 2,3-dimethoxy-1,4-naphthoquinone (DIM) redoxcycle at similar rates, NQ is a stronger arylator than MEN, and DIM does not arylate thiols. The Caco-2 cell line was particularly vulnerable to NQ and MEN and displayed moderate toxic effects of DIM. The HT-29 cell line was only vulnerable to NQ and MEN after inhibition of DT-diaphorase (DTD) with dicoumarol, whereas dicoumarol did not affect the toxicity of quinones to Caco-2 cells. DTD activity in the HT-29 and Caco-2 cell lines, as estimated by the dicoumarol-sensitive reduction of 2,6-dichlorophenolindophenol, was 393.7 +/- 46.9 and 6.4 +/- 2.2 nmol NADPH x min(-1) x mg protein(-1), respectively. MEN depleted glutathione to a small extent in the HT-29 cell line, but a rapid depletion similar to Caco-2 cells was achieved when dicoumarol was added. The data demonstrated that the DTD-deficient Caco-2 cell line was more vulnerable to arylating or redoxcycling quinones than DTD-expressing cell lines. Exposure of the Caco-2 cell line to quinones produced a rapid rise in protein disulphides and oxidised glutathione. In contrast to NQ and DIM, no intracellular GSSG was observed with MEN. The relatively higher levels of ATP in MEN-exposed cells may account for the efficient extrusion of intracellular GSSG. The reductive potential of the cell as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction was only increased by MEN and not with NQ and DIM. We conclude that arylation is a major contributing factor in the toxicity of quinones. For this reason, NQ was the most toxic quinone, followed by MEN, and the pure redoxcycler DIM elicited modest toxicity in Caco-2 cells.

Topics: Adenosine Triphosphate; Cell Survival; Colonic Neoplasms; Dicumarol; Glutathione; Glutathione Disulfide; Humans; Kinetics; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Neoplasm Proteins; Sulfhydryl Compounds; Tumor Cells, Cultured; Vitamin K