RESUMO
AIM:To investigate the effects of 17-AAG on apoptosis and cell cycle of HCT-15 cells and to clar-ify the related mechanisms .METHODS: MTT method was employed to evaluate the inhibitory effects of 17-AAG with Aifferent time and different doses on the proliferation of HCT-15 cells.The cells were stained with Annexin V-FITC/propid-iumiodide and measured by flow cytometry .The expression of STAT3, cyclin D1, Cyt C, caspase 9 and caspase 3 at mR-NA and protein levels was determined by RT-PCR and Western blotting .RESULTS:Treatment with 17-AAG at concentra-tion of 1.25~20 mg/L for 24 h and 48 h significantly inhibited the activity of HCT-15 cells at both time-and concentra-tion-dependent manners .Treatment with 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h significantly in-duced apoptosis and cell cycle arrest of HCT-15 cells.The exposure of 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h to the HCT-15 cells significantly down-regulated the expression of STAT 3 and cyclin D1 at mRNA and pro-tein levels, but up-regulated Cyt C, caspase 9 and caspase 3 mRNA and protein in a concentration-dependent manner . CONCLUSION:17-AAG inhibits the cell activity , induces apoptosis and G 1 arrest by down-regulating the expression of cyclin D1, and promoting the mitochondria apoptosis through STAT 3 pathway.
RESUMO
Diallyl disulfide (DADS) induced apoptosis through the caspase-3 dependent pathway in leukemia cells was earlier reported from this laboratory. In this study, we investigated the involvement of Ca(2+) in DADS-induced apoptotic cell death of HCT-15, human colon cancer cell line. DADS induced the elevation of cytosolic Ca(2+) by biphasic pattern; rapid Ca(2+) peak at 3 min and following slow and sustained elevation till 3 h after the addition of DADS. Production of H(2)O(2) was also observed with its peak value at 4 h. Apoptotic pathways including the sequence of caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation by DADS were completely blocked by various inhibitors such as specific caspase-3 inhibitor, free radical scavenger, and intracellular Ca(2+) chelator. N-acetylcystein and catalase treatment prevented the accumulation of H2O2 and later caspase-3 dependent apoptotic pathway. However, these radical scavengers did not block the elevation of intracellular Ca(2+). Treatment of cells with 1, 2-bis (2-aminophenoxyethane)-N, N, N-tetraacetic acid tetrakis -acetoxymethyl ester (BAPTA-AM), cellular Ca(2+) chelator, resulted in a complete blockage of the caspase-3 dependent apoptotic pathway of HCT-15 cells. It abolished the elevation of intracellular Ca(2+), and furthermore, completely inhibited the production of H(2)O(2). These results indicate that cytosolic Ca(2+) elevation is an earlier signaling event in apoptosis of HCT-15 cells. Collectively, our data demonstrate that DADS can induce apoptosis in HCT-15 cells through the sequential mechanism of Ca(2+) homeostasis disruption, accumulation of H(2)O(2), and resulting caspase-3 activation.