allopurinol and Glioma

During chemical hypoxia induced by cobalt chloride (CoCl2), hypoxia-inducible factor 1alpha (HIF1-alpha) mediates the induction of a variety of genes including erythropoietin and vascular endothelial growth factor. We used glioma cells with oxidative phosphorylation-dependent (D54-MG) and glycolytic-dependent (U251-MG) phenotypes to monitor HIF1-alpha regulation in association with redox responsiveness to CoCl2 treatment. We showed that CoCl2 increased xanthine oxidase (XO)-derived reactive oxygen species (ROS), which causes accumulation of HIF1-alpha protein in U251-MG cells. Under these conditions, blockade of XO activity by pharmacologic (N-acetyl-L-cysteine or allopurinol) or molecular (by small interfering RNA) approaches significantly attenuated HIF1-alpha expression. Exogenous H2O2 stabilizes HIF1-alpha protein. XO was present in these cells and was the primary source of free radicals. We also showed higher XO activity in cells exposed to CoCl2 compared with cells grown in normoxia. From the experiments shown here, we concluded that ROS were indeed generated in D54-MG cells exposed to CoCl2 but it was unlikely that ROS participated in the hypoxic signal transduction pathways in this cell type. Possibly, cell type-dependent and stimulus-dependent factors may control ROS dependency or redox sensitivity of HIF1-alpha and thus HIF1-alpha activation either directly or by induction of specific signaling cascades. Our findings reveal that XO-derived ROS is a novel and critical component of HIF1-alpha regulation in U251-MG cells, pointing toward a more general role of this transcription factor in tumor progression.

Topics: Acetylcysteine; Cell Line, Tumor; Cobalt; Free Radicals; Glioma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Reactive Oxygen Species; RNA, Small Interfering; Transfection; Xanthine Oxidase

1. Following ischaemic reperfusion, large amounts of superoxide anion (.O2-), hydroxyl radical (.OH) and H2O2 are produced, resulting in brain oedema and changes in cerebral vascular permeability. We have found that H2O2 (100 microM) induces a significant intracellular acidosis in both cultured rat cerebellar astrocytes (0.37 +/- 0.04 pH units) and C6 glioma cells (0.33 +/- 0.07 pH units). 2. Two membrane-crossing ferrous iron chelators, phenanthroline and deferoxamine, almost completely inhibited H2O2-induced intracellular acidosis, while the non-membrane-crossing iron chelator apo-transferrin had no effect. Furthermore, the acidosis was completely inhibited by two potent membrane-crossing .OH scavengers, N-(2-mercaptopropionyl)-glycine (N-MPG) and dimethyl thiourea (DMTU). Since .OH can be produced during iron-catalysed H2O2 breakdown (Fenton reaction), we have shown that a large reduction in pH1 in glial cells can result from the production of intracellular .OH via H2O2 oxidation. 3. We have ruled out the possible involvement of: (i) an increase in intracellular Ca2+ levels; and (ii) inhibition of oxidative phosphorylation. 4. Our results suggest that .OH inhibits glycolysis, leading to ATP hydrolysis and intracellular acidosis. This conclusion is based on the following observations: (i) in glucose-free medium, or in the presence of iodoacetate or 2-deoxy-D-glucose, H2O2-induced acidosis is completely suppressed; (ii) H2O2 and iodoacetate both produce an increase in levels of intracellular free Mg2+, an indicator of ATP breakdown; and (iii) direct measurement of intracellular ATP levels and lactate production show 50 and 55% reductions in ATP content and lactate production, respectively, following treatment with 100 microM H2O2. 5. Inhibition of the pH1 regulators (i.e. the Na(+)-H+ exchange and possibly the Na(+)-HCO3(-)-dependent pH1 transporters) resulting from H2O2-induced intracellular ATP reduction may also be involved in the H2O2-evoked intracellular acidosis in glial cells.

Topics: Acidosis; Adenosine Triphosphate; Animals; Astrocytes; Catalase; Cells, Cultured; Cerebellum; Citric Acid Cycle; Deferoxamine; Electron Transport; Female; Glioma; Glycolysis; Hydrogen Peroxide; Hypoxanthine; Lactates; Male; Oxidative Stress; Phenanthrolines; Rats; Rats, Wistar; Siderophores; Sodium-Hydrogen Exchangers; Superoxide Dismutase; Transferrin; Tumor Cells, Cultured; Xanthine Oxidase