Glioblastoma is the deadliest and most prevalent brain tumor. It is considered to be a death sentence as the glioblastoma patients hardly survive more than 1 year following diagnosis. There is no effective therapeutic strategy for the treatment of glioblastoma. So, new and innovative therapeutic strategies need to be devised for the treatment of this deadly disease. In this investigation, we used rat C6 glioblastoma cells to examine the efficacy of a combination of a retinoid and a cytotoxic cytokine or cytostatic agent for controlling their growth. The malignant property of glioblastoma is often potentiated by the activation of telomerase activity. We hypothesized that retinoid induced differentiation of C6 glioblastoma cells and down regulation of telomerase activity would decrease the proliferative potential and increase drug sensitivity for apoptosis. Treatment of C6 cells with 1 μM all-trans retinoic acid (ATRA) or 1 μM 13-cis retinoic acid (13-CRA) for 14 days induced astrocytic differentiation morphologically and biochemically, and also down regulated telomerase activity. Differentiated C6 cells decreased proliferation and increased sensitivity to interferon-gamma (IFN-γ) and taxol (TXL) for apoptosis. Wright staining and ApopTag assay identified, respectively, morphological and biochemical features of apoptosis in C6 cells following exposure to 500 units/ml IFN-γ for 48 h or 0.1 μM TXL for 24 h. Fura-2 assay detected an increase in intracellular free Ca2+ in apoptotic C6 cells, indicating involvement of Ca2+-dependent events in apoptosis. Western blot analyses showed an increase in Bax:Bcl-2 ratio, cytochrome c release from mitochondria, conversion of pro-caspase-9 to active caspase-9, and overexpression and activation of calpain and caspase-3 to cleave 270 kD α-spectrin at specific sites for generation of 145 kD and 120 kD spectrin break down products (SBDPs), respectively, in course of apoptosis. Activation of caspase-3 was also confirmed in the increased generation of 20 kD and 12 kD fragments. Following exposure to IFN-γ or TXL, apoptosis related events were found to be highly prominent in the differentiated C6 cells. Moreover, treatment of undifferentiated and differentiated C6 cells with TXL caused the phosphorylation of Bcl-2 and thus inactivated its anti-apoptotic property, further promoting the commitment of cells to apoptosis. Taken together, all the results of this investigation suggested that first retinoid induced astrocytic differentiation with down regulation of telomerase activity and then induction of apoptosis with a cytotoxic or a cytostatic agent could be an effective combination therapeutic strategy for controlling the growth of glioblastoma cells. This investigation was supported in part by the R01 grants from the NCI and NINDS of the NIH, and also a grant from the State of SC.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]