Glioblastoma (GBM) is one of the most chemoresistant and angiogenic types of tumors known. Several treatments have failed in altering survivals beyond 12 months of diagnosis. The broad objective of this proposal will be to investigate new approaches to inhibit angiogenesis in GBM using different animal models. Hypoxia-inducible factor l alpha (HIF-1a) becomes up regulated in hypoxic conditions and leads to angiogenesis via vascular endothelial growth factor (VEGF) expression. Normally wildtype p53 promotes MDM2-mediated ubiquitination and proteosomal degradation of the HIF-la protein, thus limiting VEGF-induced angiogenesis. Loss of wildtype p53 function has been associated with neovascularization and growth of xenografts in nude mice. We hypothesize that overexpression of MDM2 (seen in more than 50 percent of GBM), a gene directly regulated by p53 and also linked with resistance to chemotherapy in human GBM cell lines, could lead to the sequestration of p53 in p53-MDM2 complexes preventing the normal p53 MDM2-mediated degradation of HIF-1a. Here we show flavopiridol, a novel protein kinase inhibitor with reported antiangiogenic activity, down regulates HIF-1a expression in glioma cell lines in vitro, thus providing one mechanism for its antiangiogenic activity. The proposed studies will evaluate MDM2 antisense treatment with and without flavopiridol to promote down regulation of MDM2 expression in glioma cell lines and xenografts, up regulation of p53-mediated responses and down regulation of HIF-1a/VEGF expression to decrease angiogenic-signaling. This will be accomplished by 1) Use of in vitro studies to optimize MDM2 antisense and drug treatment conditions that promote p53 function and/or decrease HIF-la/VEGF expression, respectively, under hypoxic growth conditions. 2) Use of the murine glioma GL261 intracranial model of angiogenesis to determine the capacity of flavopiridol to decrease HIF-1a/VEGF expression and antiangiogenic activity in hypoxic conditions. 3,4) Use of nude mice xenografts both subcutaneously and intracranially to determine the in vivo activity of MDM2 antisense treatment with and without flavopiridol to inhibit angiogenesis and tumor growth in hypoxic conditions.
