Role of vincristine in the inhibition of angiogenesis in glioblastoma

Abstract

Objective: Vincristine, a microtubule-destabilizing drug, was found to exhibit anti-angiogenic effects and antitumoral activity. However, the precise mechanism by which vincristine inhibits angiogenesis in glioblastomas is not well understood. Our aim was to investigate whether vincristine affects vascular endothelial growth factor (VEGF) expression in glioblastoma cells and determine whether it is mediated by the downregulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha). Methods: We investigated the expression of HIF-1 alpha in glioblastoma tissues resected from patients and in human glioblastoma cell lines using immunohistochemistry, Western blot analysis, and immunocytochemistry. In addition to an MTT assay assessing the effect of vincristine on cell proliferation and viability, the effects of vincristine on VEGF mRNA expression and HIF-1 alpha protein were examined using real-time RT-PCR and Western blot analysis under 1% O-2 (hypoxia). Results: HIF-1 alpha was expressed in the majority of glioblastoma tissues and was detected mainly in the nucleus. Strong immunoreactivity for HIF-1 alpha was found often in the hypercellular zones. Under hypoxic conditions, HIF-1a protein levels in the glioblastoma cell lines increased, primarily localizing into the nucleus similar to glioblastoma tissues. Exposure of glioblastoma cells to vincristine resulted in enrichment of the G(2)-M fraction of the cell cycle, which suggests that vincristine-mediated growth inhibition of glioblastoma is correlated with mitotic inhibition. Using doses lower than those found to reduce the viability and proliferation of cells by 50% (IC50), vincristine decreased both the expression of VEGF mRNA and the level of HIF-1 alpha protein in hypoxic glioblastoma cells. In addition, following exposure to vincristine, the expression of VEGF mRNA was correlated with HIF-1 alpha protein levels. Conclusions: Our results suggest that the mechanism by which vincristine elicits an anti-angiogenic effect in glioblastomas under hypoxic conditions might be mediated, in part, by HIF-1 alpha inhibition.