2-Oxoglutarate Down-Regulates Expression of Vascular Endothelial Growth Factor and Erythropoietin through Decreasing Hypoxia Inducible Factor-1 α and Inhibits Angiogenesis.

In oxygenated cells, hypoxia inducible factor-1 (HIF-1)α subunits are rapidly degraded by a mechanism that involves ubiquitination by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex using 2-oxoglutarate as a substrate. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. The interaction between human pVHL and a specific domain of the HIF-1α subunit is regulated through hydroxylation of proline residues 402 and 564 by HIF-1α proryl-hydroxylase (PHD). N-oxalyl glycine acts as a competitive inhibitor of HIF-PHDs and this inhibition is in competition with 2-oxoglutarate. We examined the effect of 2-oxoglutarate on the production of vascular endothelial growth factor (VEGF) and erythropoietin (Epo). The expression of VEGF and Epo protein were dose-dependently downregulated in Hep3B cells by the addition of 2-oxoglutarate. The enhancer activity of the HIF-1 binding site of Epo and the promoter activity of VEGF-luciferase were also dose-dependently downregulated by the addition of 2-oxoglutarate. Gel mobility shift assays revealed that the addition of 2-oxoglutarate dose-dependently inhibited HIF-1 binding activity, but did not affect GATA binding activity. Western blot analysis revealed that 2-oxoglutarate dose-dependently inhibited HIF-1α protein in Hep3B, Hela and SW480 cells in hypoxic conditions. However MG132 (the proteasome inhibitor) rescued the inhibition of HIF-1α protein expression by 2-oxoglutarate. Furthermore, under hypoxic conditions, 2-oxoglutarate dose-dependently inhibited tube formation in in vitro angiogenesis assays. These results indicate that 2-oxoglutarate treatment may be useful for the inhibition of tumor angiogenesis through decreasing HIF-1α protein, HIF-1 binding activity, the promoter activity of VEGF and enhancer activity of Epo, and the production of VEGF and Epo. More studies to determine if 2-oxoglutarate inhibits tumor angiogenesis in vivo mouse assay are in progress.