Glioblastoma is the most malignant and prevalent brain tumor in humans. Various treatment strategies such as surgery, radiation therapy, chemotherapy, and/or an experimental therapy are being employed, still glioblastoma is not a curable cancer. Therefore, the search continues for an effective therapeutic strategy. Some recent studies suggest growth inhibitory effect of broccoli derived compound L-sulforaphane (L-SFN) on several cancers. In our investigation, we examined the capability of L-SFN for induction of apoptosis in human malignant glioblastoma cell lines T98G and U87MG, and also explored the activation of proteolytic mechanisms for mediation of apoptosis. Trypan blue dye exclusion test showed decreased viability of glioblastoma cells with increasing dose of L-SFN. Examination of Wright stained cells under the light microscopy identified apoptotic morphology following exposure to 20 μM and 40 μM L-SFN for 24 h. ApopTag assay detected DNA fragmentation in apoptotic cells. Fura-2 assay demonstrated an increase in intracellular free Ca2+, suggesting activation of Ca2+-dependent proteolytic pathways in apoptotic process. Western blot analyses indeed detected overexpression of 80 kD calpain, a Ca2+-dependent cysteine protease, and generation of active 76 kD calpain in apoptotic cells. Western blot analyses also suggested that activation of calpain could cleave 55 kD caspase-12 to generate active 40 kD caspase-12, which in turn could promote activation of caspase-3. Changes in expression of Bax and Bcl-2 proteins resulted in an increase in Bax:Bcl-2 ratio, showing a commitment of cells to apoptosis after exposure to L-SFN. As a consequence of increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol to contribute to the activation of caspase-9 that in turn activated caspase-3. In course of apoptosis, increased expression of Smac/Diablo suggested suppression of inhibitor-of-apoptosis proteins (IAPs), which otherwise could prevent activation of caspases. Morevover, decreased expression of p65 NFκB and increased expression of IκBα favored the process of apoptosis. Activation of calpain and caspase-3 cleaved 270 kD α-spectrin at specific sites generating, respectively, 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP. Further, activation of caspase-3 was confirmed in the fragmentation of inhibitor-of-caspase-3-activated-DNase (ICAD). Besides, over accumulation of apoptosis-inducing-factor (AIF) suggested an involvement of caspase-independent pathway of apoptosis as well. Taken together, all these results indicated activation of multiple molecular mechanisms for mediation of apoptosis in glioblastoma cells following exposure to L-SFN. 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]