Role Of Ca～(2+) In Diallyl Trisulfide-induced Apoptotic Cell Death Of MGC-803 Cells

Objective: To investigate the growth inhibition and apoptotic induction effects of diallyl trisulfide(DATS) on MGC-803 cell line and the relationship between apoptosis and Ca²⁺ disruption, intracellular level of reactive oxygen species(ROS) and Caspase-3 activity.Methods: Inverted microscope, light microscope, MTT assay, Flow cytometry, Westen-blot were used to observe the effects of DATS on the cell growth inhibition, apoptosis, the change of intracellular Ca²⁺, ROS and Caspase-3.Result: MTT assay showed that apparently growth inhibition in MGC-803 cell line treated with DATS could be seen and exhibited a dose-dependent model, Adding 4, 8, 12, 16, 24 mg.L¹ DATS for 72h suppressed MGC-803 cells growth by 0.231±0.037 , 0.305±0.036 , 0.455±0.029 , 0.607±0.058,0.751±0.019 (P<0.05). After MGC-803 cells were exposed to DATS, the typical apoptotic morphological changes were observed under the light microscope. Flow cytometry analysis revealed that treated MGC-803 cell with 4, 8, 12, 16mg.L^-1 DATS for 24h, the percentage of apoptotic cells were 3.8%, 10.6%, 23.0%, 24.2% respectively, these results showed that DATS could induce apoptosis of MGC-803 cells in a dose-dependent manner. Moreover, treated MGC-803 cells with DATS increased the percentage of cells in G1 phase. Pretreatment of cells with 1,2-bis (2-aminophenoxye-thane) -N,N,N-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), a cellular Ca²⁺ chelator, thiol-containing antioxidant NAC or Caspase-3 inhibitor AC-DEVD-CHO for 1h, then treated the cells with DATS for 24h, the percentage of apoptotic cells were 13.2%, 10.5%, 5.1% respectively, There was distinctively difference compared with thecells treated with 12mgL''DATS alone. These results showed that BAPTA-AM, NAC, AC-DEVD-CHO may inhibit DATS inducing apoptosis of MGC-803 cells.Treated MGC-803 cells with DATS at the concentration of 4, 8, 12, 16mg-L'' for 4h, the Fluo-3 fluorescence intensity were 4.0±0.5, 8.8±0.3, 15.4±0.4, 16.0±0.5, except of 4 mg-I/'group, the fluorescence intensity of treated group were elevated than the control group. These results showed that DATS could elevate the level of intracellular Ca2+ in a dose-dependent manner. Treated MGC-803 cells with DATS at the concentration of 4, 8, 12, 16 mg-L'1 for 5h, DCFH-DA fluorescence intensity were 11.00±1018, 25.40±1.73, 45.87±2.12, 56.87±1.57, except of 4 mg-l/'group, the fluorescence intensity of treated group were elevated than the control group. Pretreatment of cells with BAPTA-AM, the fluorescence intensity was 13.47±0.95, there was distinctively difference compared with the cells treated with DATS alone. These results showed that DATS could elevate the level of intracellular ROS in a dose-dependent manner. Pretreatment of cells with BAPTA-AM, resulted in a blockage of production of ROS. Treated MGC-803 cells with 12 mg-L"1 DATS for 4h, 8 h, 12 h, 24h, the proportion of cells that expressed actived Caspase-3 was 3%, 7.27%, 14.39%, 27.65% respectively, the results showed that DATS could active Caspase-3 in a time-dependent manner(/?<0.01), Pretreatment of cells with BAPTA-AM or NAC, the expression rate of actived Caspase-3 were 6%, 5.71% respectively. There was distinctively difference compared with the cells treated with DATS alone. These result showed that BAPTA-AM and NAC may inhibit the activation of Caspase-3. Western blot analysis revealed that treated MGC-803 cells with DATS at the concentration of 12 and 16mg-L"' for 24h, pro-Caspase3 was decreased and cleaved product was detected. These result showed that DATS may accelerate the activation of pro-Caspase3. Conclusion:1. DATS could inhibit the growth of MGC-803 cells.