Astrocytomas express high levels of vascular endothelial growth factor (VEGF) which are reduced through RAS pathway inhibition

Introduction: Vascular Endothelial Growth Factor (VEGF) is the major angiogenic factor in normal development and in the progression of numerous tumors, including glioblastoma multiforme (GBM). Ras-transformed intestinal epithelial cells express high levels of VEGF, but the relationship between Ras pathway activity and VEGF expression in astrocytomas has not been previously elucidated. Methods: VEGF secretion by human astrocytoma cell lines into the conditioned medium was evaluated using western blotting and enzyme-linked immunosorbent assay (ELISA). Ras pathway activity was inhibited genetically with the dominant negative RasN17 mutant, and pharmacologically with the farnesyl transferase inhibitor. Results: Western blotting revealed that astrocytoma cell lines secrete 8-20 times more VEGF than the Ras-transformed human colon cancer cell line H460 and 109-262 times more VEGF than NIH 3T3 (mouse) fibroblasts. Expression of the constitutively-phosphorylated mutant epidermal growth factor receptor p140EGF-R resulted in a 55% increase in VEGF expression. Inhibition of the Ras pathway using RasN17 resulted in 30-75% reductions in VEGF secretion. In addition to the 48% reduction in U87 cell count seen with FTI treatment (at 10 μM), ELISA demonstrated an additional 49% reduction in cell count-normalized VEGF secretion. Conclusions: This study demonstrates that increased Ras pathway activity in astrocytoma cells results in increased VEGF secretion, which is known to be critical for tumor growth through the induction of angiogenesis. Ras pathway inhibition reduces cell proliferation in addition to reducing VEGF secretion, suggesting that such therapy holds much promise in the management of GBMs.