Glioblastoma is the most malignant and prevalent human brain tumor of astroglial origin. It is considered as the death sentence in patients due to its poor response or resistance to existing therapeutic agents. Therefore, the search continues for an effective therapy for controlling the growth of this devastating cancer. This investigation used all-trans retinoic acid (ATRA) and interferon-gamma (IFN-γ) alone and in combination for controlling the growth of human glioblastoma T98G xenografted in nude mice. For development of xenografts, 6-week old nude mice (about 20 g each) were subcutaneously injected with a (1:1) mixture of exponentially growing T98G cells (6 million cells/mouse) and Matrigel. The mice were allowed to develop the tumors. Animals with the 3-week old xenografts were randomly assigned to four different groups: control, ATRA, IFN-γ, and ATRA plus IFN-γ. Control animals did not receive any therapy. Other animals with xenografts received a daily dose of ATRA (1.5 µg/kg), IFN-γ (5000 units/kg), or ATRA (1.5 µg/kg) plus 4 h later IFN-γ (5000 units/kg) for 7 days. Histopathological examination showed characteristic growth of glioblastoma in control group, inhibition of tumor cell proliferation and astrocytic differentiation in ATRA group, induction of modest amount of apoptosis in IFN-γ group, and occurrence of differentiation and enhancement of apoptosis in ATRA plus IFN-γ group. Western blot analyses showed that ATRA plus IFN-γ treatment activated extrinsic pathway of apoptosis by activation of caspase-8 and cleavage of Bid to tBid and also down regulated anti-apoptotic hTERT, c-IAP2 and survivin, favoring the process of apoptosis. Western blotting showed activation of cysteine protease calpain and involvement of both mitochondrial caspase-independent pathway by robust release of AIF and caspase-dependent pathway by the activation of caspase-9 and caspase-3 in T98G xenografts for apoptosis following combination therapy with ATRA plus IFN-γ. Increased activities of calpain and caspase-3 degraded 270 kD α-spectrin at the specific sites to generate 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, respectively. Further, increased activity of caspase-3 cleaved inhibitor-of-caspase-activated DNase (ICAD) and release CAD for DNA fragmentation for apoptosis in tumors treated with the combination of ATRA and IFN-γ. In situ TUNEL and double immunofluorescent labelings detected apoptosis with increased expression of calpain, caspase-12, and caspase-3 in tumors treated with IFN-γ, or ATRA plus IFN-γ. Thus, we developed a combination therapy for increasing apoptosis in human glioblastoma in vivo. This investigation was supported in part by the R01 grants (CA-91460 and NS-57811).
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA