ascorbic-acid and pyrazolanthrone

Impaired insulin sensitivity (insulin resistance) is a common denominator in many metabolic disorders, exerting pleiotropic effects on skeletal muscle, liver, and adipose tissue function. Heme oxygenase-1 (HOX-1), the rate-limiting enzyme in heme catabolism, has recently been shown to confer an antidiabetic effect while regulating cellular redox-buffering capacity. Therefore, in the present study, we probed into the mechanisms underlying the effect of insulin on HOX-1 in C2 skeletal myoblasts. Hence, insulin was found to suppress C2 myoblasts viability via stimulation of oxidative stress, with HOX-1 counteracting this action. Insulin induced HOX-1 expression in a time- and dose-dependent manner, an effect attenuated by selective inhibitors of ERK1/2 (PD98059), Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine), and c-Jun terminal kinases 1 and 2 (SP600125) pathways. Furthermore, nuclear factor-κB role in insulin-induced HOX-1 up-regulation was verified, with ERK1/2, Src, and c-Jun terminal kinases 1 and 2 mediating p65-nuclear factor-κB subunit phosphorylation. Overall, our novel findings highlight for the first time the transduction mechanisms mediating HOX-1 induction in insulin-treated C2 myoblasts. This effect was established to be cell type specific because insulin failed to promote HOX-1 expression in HepG2 hepatoma cells. Deciphering the signaling networks involved in insulin-stimulated HOX-1 up-regulation is of prominent significance because it may potentially contribute to elucidation of the mechanisms involved in associated metabolic pathologies.

Topics: Animals; Anthracenes; Antioxidants; Ascorbic Acid; Blotting, Western; Caspase 3; Catalase; Cell Line; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Heme Oxygenase-1; Insulin; JNK Mitogen-Activated Protein Kinases; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myoblasts, Skeletal; NF-kappa B; Oxidative Stress; Phosphorylation; Protoporphyrins; Reactive Oxygen Species; Signal Transduction

Oxygen enhancement of tumor radiosensitivity is attributed to DNA damage by reactive oxygen species. The mechanism remains unclear but may involve mitochondria as major sources of oxygen and nitrogen radicals as well as central effectors of energy homeostasis and apoptosis. Here we used dihydrorhodamine and 2',7'-dichlorodihydrofluorescein to compare mitochondrial and total cell generation, respectively, of reactive oxygen or nitrogen species in cells irradiated at 5 Gy. Irradiation in the presence of oxygen selectively stimulated mitochondrial radical production in HeLa and MeWo cells, but in MCF7 cells radical production was more generalized. In all three cell lines oxygen impaired cell proliferation as measured by resazurin reduction 7 days after irradiation. Antioxidants N-acetylcysteine, ascorbic acid, and melatonin largely prevented dye oxidation during normoxic irradiation yet had no effect on oxygen-dependent irradiation injury. However, NO synthase inhibitor N(G)-monomethyl-L-arginine protected HeLa and MCF7 though not MeWo cells, consistent with their different levels of constitutive NO generation. SB203580 inhibition of p38 MAPK appreciably protected HeLa and marginally protected MCF7 cells against oxygen-dependent irradiation injury, while the less specific JNK/SAPK inhibitor SP600125 and ERK inhibitor U0126 had no effect. None of the inhibitors affected MeWo radiosensitivity. Therefore oxygen-enhanced radiosensitivity in these tumor cell lines does not depend on extensive production of oxygen radicals and is cell-type dependent. NO mediates oxygen-dependent injury in HeLa and MCF7 cells, by p38-dependent and MAPK-independent mechanisms, respectively. In MeWo cells this oxygen-enhanced radiosensitivity is independent of both NO and MAPK signaling.

Topics: Acetylcysteine; Anthracenes; Antioxidants; Apoptosis; Ascorbic Acid; Butadienes; Cell Line, Tumor; Cell Proliferation; DNA Damage; Enzyme Inhibitors; Fluoresceins; HeLa Cells; Humans; Imidazoles; MAP Kinase Signaling System; Melatonin; Mitochondria; Nitriles; Nitrites; Oxazines; Oxygen; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Radiation Tolerance; Rhodamines; Spectrometry, Fluorescence; Xanthenes