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Diallyl trisulfide (DATS), a cancer chemopreventive constituent of processed garlic, inhibits growth of cancer cells in culture and in vivo by causing apoptosis but the sequence of events leading to cell death is poorly defined. Here, we demonstrate that DATS-induced apoptosis in LNCaP human prostate cancer cells is initiated by reactive oxygen species (ROS) and regulated by Bax/Bak and X-linked inhibitor of apoptosis (XIAP) proteins. The DATS-mediated apoptosis in LNCaP cells was accompanied by mitochondrial swelling (revealed by transmission electron microscopy), disruption of mitochondrial membrane potential (revealed by flow cytometry after staining the cells with membrane potential sensitive dye JC-1), and cytosolic release of apoptogenic molecules (revealed by immunofluorescence microscopy for cytochrome c and Smac/DIABLO). The DATS-induced cell death in LNCaP cells correlated with induction of Bax and Bak, mitochondrial translocation (activation) of Bax, and a decrease in protein levels of Bcl-2 and Bcl-xL. The SV40 immortalized mouse embryonic fibroblasts (MEFs) derived from Bax and/or Bak knockout mice, but not Bid knockout mice, were significantly more resistant to DATS-induced apoptosis compared with the MEFs derived from wild-type mice. Moreover, combined knockdown of Bax and Bak proteins using siRNA conferred statistically significant protection against DATS-induced apoptosis in LNCaP cells. On the other hand, ectopic expression of either Bcl-2 or Bcl-xL in LNCaP cells did not have any appreciable effect on the cell death caused by DATS. The DATS treatment caused a marked decrease in protein levels of XIAP in LNCaP cells, and the HCT116 derived XIAP-/- cells were relatively more sensitive to growth inhibition and apoptosis induction by DATS compared with wild-type HCT116 cells. The DATS treatment resulted in generation of ROS in LNCaP cells, which was attenuated by pretreatment with antioxidant N-acetylcysteine (NAC). The NAC pretreatment conferred near complete protection against DATS-mediated disruption of the mitochondrial membrane potential and apoptosis in LNCaP cells. The DATS-induced apoptosis was not affected by either p53 protein knockdown in LNCaP cells or overexpression of wild-type p53 in PC-3 cells. In conclusion, the present study reveals that the DATS-induced apoptosis is initiated by ROS and regulated by Bax/Bak and XIAP, but independent of Bcl-2, Bcl-xL, Bid and p53. This study was supported by NCI grant CA113363.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA