Anti Tumor Effect of 2-Oxoglutarate through Inhibition of Angiogenesis in a Murine Tumor Model.

Hypoxia-inducible factor 1 (HIF-1) plays an essential role in tumor angiogenesis and growth by regulating the transcription of several genes in response to hypoxic stress and changes in growth factors. HIF-1 is a heterodimeric transcriptional activator and consists of inducible α and constitutive β subunits. In oxygenated cells, intracellular oxygen concentrations are directly sensed by proteins containing the prolyl hydroxylase domain (PHD), which tag HIF-1α subunits for polyubiquitination and proteasomal degradation by prolyl hydroxylation using 2-oxoglutarate (2-OX) and dioxygen. Our recent studies have shown that 2-OX reduces HIF-1α, erythropoietin, and vascular endothelial growth factor (VEGF) expression by hypoxia in the hepatoma cell line Hep3B (Matsumoto K et al.; J. Cell. Physiol., 2006). Similar results were obtained in Lewis lung cancer (LLC) cells in in vitro studies. Here, to address the clinical usefulness of 2-OX, we investigated its antitumor effect using a mouse dorsal air sac (DAS) assay and a murine tumor xenograft model. For the DAS assay, LLC cells suspended in PBS with 0, 7.5, or 15 mM 2-OX per Millipore chamber were implanted subcutaneously into C57BL/6J mice. The values of blood vessel area by LLC cells were 100 ± 20.8, 64.0 ± 9.4, and 45.6 ± 4.4%, respectively, by quantitative analysis with angiogenesis-measuring software. This result indicated that 2-OX clearly inhibited the growth of subcutaneous tumors. To elucidate the effect of 2-OX on tumor growth, 2-OX was administrated to C57BL/6J mice inoculated with LLC cells. LLC cells (1 × 10⁶) in PBS were implanted into the right flank region of 7-week-old mice. Daily intraperitoneal (i.p.) injections of 2-OX were started on the next day after implantation. From 6 to 12 days after implantation, we measured tumors with calipers and calculated volumes as (length × width²) × 0.5. LLC tumors were removed from mice 12 days after implantation for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical studies. Tumor volumes and weights 12 days after implantation were as follows: PBS alone, 330.8 ± 108.1 mm³ and 192.6 ± 66.4 mg; 50 mg/kg 2-OX, 128.8 ± 16.4 mm³ and 111.0 ± 64.5 mg; and 100 mg/kg 2-OX, 78.7 ± 43.7 mm³ and 88.8 ± 57.5 mg. Quantitative RT-PCR revealed that 2-OX treatment (100 mg/kg) decreased the expression levels of the VEGF gene 0.67-fold in tumor tissues compared with control. We observed quantitative differences in microvessel density (PBS alone, 100 ± 16.5%; 100 mg/kg 2-OX, 46.0 ± 13.5%) using immunostaining for the endothelial cell marker CD31. Intraperitoneal injection of 2-OX significantly inhibited tumor growth and angiogenesis in tumor tissues. Combination therapy is necessary for anti-angiogenic therapy in the human. To examine the effect of 2-OX in combination with 5-fluorouracil (5-FU) chemotherapy, we injected 5-FU i.p. on day 6 and/or 2-OX i.p. on each of days 6–15 in this mouse model. Tumor volumes and weights 15 days after implantation were as follows: PBS alone, 531.2 ± 144.9 mm³ and 306.3 ± 186.6 mg; 2-OX alone, 324.0 ± 156.5 mm³ and 308.7 ± 299.0 mg; 5-FU alone, 287.1 ± 155.2 mm³ and 204.7 ± 108.9 mg; and 5-FU + 2-OX, 98.7 ± 64.9 mm³ and 130.3 ± 113.5 mg. 5-FU combined with 2-OX significantly inhibited tumor growth in this model, which was accompanied by 53% reduction of VEGF gene expression in tumor tissues removed from mice 15 days after implantation, using quantitative RT-PCR analysis. These results suggest that 2-OX is a promising anti-angiogenic therapeutic agent. To examine whether the inhibitory effect of 2-OX is specific for PHD-containing proteins, an RNAi study will be performed.