All-trans retinoic acid (ATRA) has been widely used for the treatment of malignant cancers because it is a potent inhibitor of cell proliferation and inducer of differentiation. Interferon-gamma (IFN-g) is a well-known cytotoxic cytokine that induces apoptosis in many malignant cancers including glioblastoma, which is the most common primary brain tumor in humans. Phosphatase and tensin homolog located on chromosome ten (PTEN) is a tumor suppression as it negatively regulates activation of protein kinase B (also known as Akt), which plays a prominent role in tumorigenesis. Mutation or deletion of PTEN has been found in as high as 80% malignant glioblastomas, which have aberrant signaling pathways passing through phoshphatidylinositol-3-kinase (PI3K) and Akt. Glioblastoma cells without functional PTEN are not easily amenable to apoptosis. We investigated the possibility of modulation of signal transduction pathways for induction of apoptosis in human glioblastoma T98G (PTEN-harboring) and U87MG (PTEN-deficient) cell lines after treatment with the combination of ATRA and IFN-g. Wright staining and light microscopy showed significant amounts of morphological features of apoptosis in both T98G and U87MG cell lines following treatment with the combination of ATRA and IFN-g for 18 h. Western blotting with an antibody capable of specifically detecting phosphorylation of Akt at Ser-473 showed no phosphorylation of Akt in T98G cells but time-dependent phosphorylation of Akt in U87MG cells. The phosphorylation of Akt in U87MG could be reversed by adding the PI3K inhibitor LY294002, indicating the involvement of PI3K activity in this process. Upregulation of the tumor suppressor PTEN, down regulation of anti-apoptotic Bcl-2, and mitochondrial release of cytochrome c favored apoptosis in T98G cells. On the other hand, luciferase reporter gene assay showed significant down regulation of the survival factor nuclear factor kappa B (NFkB) and Western blotting showed huge upregulation of pro-apoptotic Bax in apoptosis of U87MG cells. Increased calpain and caspaser-3 activities cleaved 270 kD alpha-spectrin at specific sites to generate 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, respectively, in course of apoptosis. Collectively, our results demonstrated that the combination of ATRA and IFN-g could modulate the signal transduction pathways so as to induce apoptosis in both T98G and U87MG cell lines. Therefore, the combination of ATRA and IFN-g can be considered as an effective therapeutic strategy for controlling the growth of both PTEN-harboring and PTEN-deficient glioblastoma cells. This investigation was supported in part by the R01 grants (CA-91460 and NS-57811) from the National Institutes of Health (Bethesda, MD, USA) and also another grant (SCIRF-0803) from the State of South Carolina.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA