Flavopiridol Induces Mitochondrial-Mediated Apoptosis in Murine Glioma GL261 Cells via Release of Cytochrome c and Apoptosis Inducing Factor

Abstract

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. Over 80% of adult high-grade astrocytomas show inactivation of the Rb tumor suppressor pathway. Therefore, one possible therapeutic strategy would be to directly modulate cyclin dependent kinase (CDK) activity resulting in inhibition of Rb phosphorylation and cell cycle progression. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to: 1. inhibit cell growth; 2. inhibit cell migration;3. decrease expression of CDK inhibitor cyclin D1, CDK4 and p21; 4. induce apoptosis in cells with high levels of p27 expression; and5. decrease the expression of the anti-apoptotic protein Bcl-2. The mechanism by which flavopiridol induces apoptosis is mitochondrial-mediated. We demonstrate by electron microscopy and immunohistochemistry that drug treatment induces mitochondrial damage that was accompanied by the release of cytochrome c into the cytosol together with the translocation of apoptosis inducing factor (AIF) into the nucleus. This finding in murine glioma cells differs markedly from the mechanism of flavopiridol-induced apoptosis cell death reported by us for human glioma cells (Alonso et al., Mol Cancer Ther 2003; 2:139) where drug treatment induced a caspase- and cytochrome c-independent pathway in the absence of detectable damage to mitochondria. In apoptotic human glioma cells only translocation of AIF into the nucleus occurred. Thus, the same drug induces apoptosis inkills different types of glioma cells by different mitochondrial-dependent pathways.