Induction of Vascular Endothelial Growth Factor Expression and Hypoxia-inducible Factor 1α Protein by the Oxidative Stressor Arsenite

Abstract

Recent evidence suggests that vascular endothelial growth factor (VEGF) expression is up-regulated by oxidative stressors through activation of hypoxia-inducible Factor 1 (HIF-1). To investigate whether this is a general phenomenon, we studied the effects of the sulfhydryl reagent arsenite on VEGF expression in human ovarian cancer cells. Arsenite potently induces the production of reactive oxygen species (ROS) in several cell systems and directly interacts with sulfhydryl groups of cellular thiols. We report that arsenite induces VEGF mRNA and protein levels in normoxic H134 and OVCAR-3 cells. Arsenite also increases HIF-1␣ protein levels, suggesting a role for HIF-1 in the induction of VEGF expression. Pretreatment with the ROS inhibitors catalase and mannitol attenuated arsenite-induced ROS production, but did not affect induction of VEGF mRNA and HIF-1␣ protein. In contrast, pretreatment with the thiol antioxidants glutathione or N-acetylcysteine completely abrogated both effects, whereas a potentiation was observed by depletion of intracellular glutathione. These results demonstrate that arsenite-induced VEGF mRNA and HIF-1␣ protein expression is independent of increased ROS production but critically regulated by the cellular reduced glutathione content. In addition, these data suggest the involvement of a thiol-sensitive mechanism in the regulation of VEGF mRNA expression and HIF-1␣ protein in human ovarian cancer cells.

Evidence for a Role of p38 Kinase in Hypoxia-inducible Factor 1-independent Induction of Vascular Endothelial Growth Factor Expression by Sodium Arsenite

“…Hybridization of total RNA to a 136-nucleotide human ␥-actin antisense probe gives rise to a protected fragment of 130 nucleotide, whereas hybridization to the 185-nucleotide murine ␥-actin antisense probe is expected to result in the protection of a fragment of 151 nucleotides. It should be noted that hybridization of total RNA to the 301-nucleotide human VEGF 165 antisense probe and the 228-nucleotide murine VEGF 164 can give rise to fragments of different sizes because of protection by VEGF mRNAs of different isoforms (1,34,41,42). Protection by human VEGF 165 mRNA and murine VEGF 164 mRNA is most efficient, giving rise to protected fragments of 252 and 195 nucleotides, respectively.…”

“…In a previous study on the role of oxidative stress in the regulation of VEGF, we showed that sodium arsenite (NaAsO 2 ) induces HIF-1␣ protein and VEGF mRNA and protein levels in the human ovarian cancer cell lines OVCAR-3 and H134 (34). Arsenite induces oxidative stress by binding to thiol groups of cellular proteins and by increasing the production of ROS.…”

“…Arsenite induces oxidative stress by binding to thiol groups of cellular proteins and by increasing the production of ROS. Because the effects of sodium arsenite on HIF-1␣ protein and VEGF expression are independent of increased ROS production, we hypothesized that they may be mediated through binding of arsenite to thiol (SH) groups of the HIF-1␣ protein itself or of components of signal transduction pathways involved in HIF-1 or VEGF regulation (34).…”

“…The RNase protection assay was carried out as described (34). Hybridization of total RNA to a 136-nucleotide human ␥-actin antisense probe gives rise to a protected fragment of 130 nucleotide, whereas hybridization to the 185-nucleotide murine ␥-actin antisense probe is expected to result in the protection of a fragment of 151 nucleotides.…”

“…RNase Protection Assay-Generation of human ␥-actin and VEGF 165 antisense probes has been described elsewhere (34). pcDNA3 vectors (Invitrogen) containing a fragment of murine VEGF 164 or ␥-actin cDNA were used as templates for the synthesis of murine VEGF 164 and ␥-actin antisense probes.…”