Glioblastoma is the most common and highly malignant brain tumor, which is characterized by the poorly differentiated astroglial cells and their uncontrolled proliferation in the brain. It is not yet significantly amenable to any conventional chemotherapeutic regimen and thus remains a big challenge to both the basic scientists and clinicians. In the current investigation, we used an innovative combination therapeutic approach for controlling the growth of human malignant glioblastoma U87MG cells. We tested the hypothesis that treatment of human malignant glioblastoma cells with a retinoid would induce astrocytic differentiation and down regulate telomerase activity and thereby enhance sensitivity to apoptosis inducing agents. Treatment of U87MG cells with 1 μM all-trans retinoic acid (ATRA) or 1 μM 13-cis-retinoic acid (13-CRA) for 7 days induced astrocytic differentiation morphologically as determined by methylene blue staining, and biochemically as evidenced by overexpression of glial fibrillary acidic protein (GFAP) at the mRNA and protein levels. The telomerase repeat amplification protocol (TRAP) applied to ATRA and 13-CRA treated glioblastoma cells detected a decrease in the activity of telomerase, an enzyme responsible for lending the unlimited capability of proliferation to many cancers including glioblastomas. Cells pretreated with ATRA or 13-CRA were then exposed to 500 units/ml interferon-gamma (IFN-γ) for 48 h or 0.5 μM taxol (TXL) for 24 h for determination of the extent of apoptotic death. Wright staining and light microscopy identified the morphological features of mostly apoptosis in human glioblastoma cells. Apoptosis was associated with an increase in intracellular free Ca2+, as determined by fura-2 assay. Western blot analyses showed alterations in levels of Bax and Bcl-2 resulting in an increase in Bax:Bcl-2 ratio, 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), repectively, during apoptosis. Phospholylation of Bcl-2 indicated inactivation of its anti-apoptotic property after treatment with TXL. Activation of caspase-3 was further confirmed by a colorimetric assay using a caspase-3-specific synthetic peptide substrate. Proteolytic activities of both calpain and caspase-3 appeared to contribute significantly to apoptotic death glioblastoma cells. The results from this investigation suggested that a treatment strategy with a combination of retinoic acid (RA) and IFN-γ, or RA and TXL could be highly effective for controlling the malignant growth of glioblastoma. This investigation was supported in part by the R01 grants from the NCI and NINDS of the NIH (Bethesda, MD), and also a grant from the State of SC.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]