Glioblastoma is the most malignant brain cancer in humans. It is not yet amenable to any currently available therapeutic regimen. In the past, various conventional chemotherapeutic agents have been used to control the growth of glioblastoma. But none of them showed significant success. So, the search continues to find out an innovative and effective therapeutic strategy for treating this deadly disease. Garlic derived organosulfur compounds such as diallyl sulfide (DAS) and diallyl disulfide (DADS) offer significant protection against chemical carcinogen induced cancers in animal models. The objective of this study was to investigate the capability of DAS and DADS for induction of apoptosis in human malignant glioblastoma cell lines T98G and U87MG. Trypan blue dye exclusion test showed a decrease in number of viable human glioblastoma cells with an increasing dose of DAS and DADS. Wright staining and light microscopy for examination of cell morphology indicated predominantly apoptotic features in human glioblastoma cells following exposure to 100 μM DAS and 100 μM DADS for 24 h. Notably, DADS demonstrated more pro-apoptotic effect than DAS. Fura-2 assay showed that apoptosis in both human glioblastoma cell lines was associated with an increase in intracellular free Ca2+, indicating participation of Ca2+-dependent molecular events in mediation of the apoptotic process. Changes in transcriptional level expression (mRNA) of all eight members of the inhibitor-of-apoptosis proteins (IAPs), also currently designated as the baculovirus IAP repeat containing (BIRC) proteins, were examined by the reverse transcriptase-polymerase chain reaction (RT-PCR). In addition to the RT-PCR experiments, Western blot analyses were performed to determine the changes in protein level expression of the selective BIRC proteins. Also, Western blot analyses showed changes in the levels of expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins resulting in an increase in Bax:Bcl-2 ratio, and overexpression and activation of calpain and caspase-3 leading to the cleavage of 270 kD α-spectrin at specific sites for generation of 145 kD spectrin break down products (SBDP) and 120 kD SBDP, respectively, in course of apoptosis in glioblastoma cells. The activation of caspase-3 was further confirmed in the increased generation of 20 kD and 12 kD fragments from 32 kD pro-caspase-3 and by a colorimetric assay using a caspase-3-specific synthetic peptide substrate. Taken together, these results from our investigation strongly suggest that garlic compounds such as DAS and DADS can be used as potential therapeutic agents for induction of proteolytic activities for apoptotic death in human 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]