Induction of calpain and intrinsic caspase pathways for apoptosis in human malignant neuroblastoma cell line SH-SY5Y following exposure to diallyl sulfide (DAS) and diallyl disulfide (DADS)

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Neuroblastoma is an early childhood malignancy that mostly occurs in the sympathetic nervous system arising from the immature neuroblasts of the adrenal glands, neck, chest, or spinal cord. Neuroblastoma is an aggressive metastatic disease, especially, when it is diagnosed in children older than 1 year of age. It is the first cause of disease-associated death in chlidren at pre-school age. Malignant neuroblastoma (N-type) continues to defy all currently available chemotherapeutic regimens. So, there is an urgent need for the development of new therapeutic strategies for successful treatment of malignant neuroblastoma in young children. In our current investigation, we employed N-type neuroblastoma cell line SH-SY5Y to examine the efficacy of the garlic derived organosulfur compounds such as diallyl sulfide (DAS) and diallyl disulfide (DADS) for induction of apoptotic death and for exploration of the proteolytic mechanisms involved in this process. Trypan blue dye exclusion test showed a decrease in cell viability with an increasing dose of DAS or DADS. Wright staining and examination of the cell morphology under the light microscopy identified prominent apoptotic features in SH-SY5Y cells following exposure to 50 μM and 100 μM DAS or DADS for 24 h. Further, ApopTag assay detected the DNA fragmentation in apoptotic cells following exposure to DAS and DADS. It appeared from Wright staining and ApopTag assay that DADS could induce more apoptosis than DAS. Apoptosis in SH-SY5Y cells in response to these compounds could be associated with an increase in intracellular free Ca2+, which was determined by fura-2 assay. Western blot analyses showed alterations in levels of expression of the Bax and Bcl-2 proteins resulting in an increase in Bax:Bcl-2 ratio, release of cytochrome c from the mitochondria, increased amounts of Smac/Diablo in the cytosol, and overexpression and activation of caspase-9 and caspase-3 - all of which clearly indicated an involvement of intrinsic pathway of apoptosis in SH-SY5Y cells following exposure to DAS or DADS. Moreover, increased proteolytic activities of calpain and caspase-3 cleaved 270 kD α-spectrin at the specific sites generating, respectively, 145 kD spectrin break down product (SBDP) and 120 kD SBDP in course of apoptosis. Taken together, the results from our current investigation strongly suggest an involvement and co-operation between calpain and intrinsic caspase pathways for mediation of apoptosis in SH-SY5Y cells following exposure to these organosulfur compounds derived from garlic. This investigation implies that the efficacy of DAS and DADS for induction of apoptosis should be further explored in animal models of malignant neuroblastoma. 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.