Ca～(2+) Signal Pathway Mediated Apoptosis Induced By Diallyl Trisulfide In Nasopharyngeal Cancer CNE2 Cells

Objective: To investigate the growth inhibition and apoptosis induction effects of diallyl disulfide (DADS) on CNE2 cell line and the relationship between apoptosis and Bcl-2, Bax, Caspase 3 activity, Ca2+ disruption , intracellular level of reactive oxygen species(ROS).Methods: light microscope, MTT assay, flow cytometry, DNA agarose gel electrophoresis, immunocytochemical technique and westen-blot were used to observe the effects of DADS on the CNE2 cells growth inhibition, apoptosis, the protein levels of Bcl-2, Bax and Caspase 3, the change of intracellular Ca2+and ROS.Results: After exposure to DADS, partial cells presented characteristic morphological changes of apoptosis under the light microscopy, including cell rounding, kytoplasm strong acidophily, karyopyknosis and anachromasis, and nuclear chromatin accumulation in the caryotheca. MTT assay showed that 50, 100, 200, 400μmol·L-1DADS significantly inhibited proliferation of CNE2 cells at 48 hours, its inhibition ratio were 3.66%, 14.25%, 29.66% and 61.28%, respectively, in dose-dependent manner (P<0.05). Flow cytometry analysis showed that CNE2 cells treated with 50, 100, 200, 400μmol·L-1 DADS for 24 hours significantly increased the percentage of apoptosis cells, its apoptosis rate were 10.8±1.3%, 14.8±1.1%, 21.7±2.0% and 30.8±2.6%, respectively (P<0.05). Ladder bands occurred after exposure to 200μmol·L-1 and 400μmol·L-1DADS for 24 hours in DNA agarose gel electrophoresis. Immunocytochemistry revealed that the Bcl-2 expression was decreased. Its average optical value decreased from 5.22±0.74 to 1.07±0.08. while the Bax and Caspase 3 expression were increased. Their average optical value increased from 0.81±0.07 to 4.05±0.63 and 0.77±0.07 to 4.23±0.72, respectively (P<0.01). After CNE2 cells treated with 50, 100, 200, 400μmol·L-1 DADS for 24 hours , Western Blot showed that the Bcl-2 expression was decreased, the gray scale value ratio of Bcl-2/β-Actin were 1.92±0.21, 1.21±0.18, 0.89±0.14 and 0.41±0.09, respectively, compare with 2.24±0.26 as a control (P<0.05), while Bax expression were increased , the gray scale value ratio of Bax/β-Actin were 0.78±0.14, 1.12±0.19, 1.54±0.22 and 2.08±0.27, respectively, compare with 0.33±0.08 as a control (P<0.05), Caspase 3 expression were increased, the gray scale value ratio of Caspase 3/β-Actin were 0.33±0.11, 0.72±0.15, 1.39±0.21 and 2.28±0.29, respectively, compare with 0.14±0.06 as a control (P<0.05). They indicated that DADS could down-regulate Bcl-2 protein expression and up-regulate Bax protein expression, result in down-regulating the ratio of Bcl-2/bax, and Caspase 3 activation.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 for 1h, then treated the cells with DADS for 24h. The percentage of apoptotic cells were 10.5±0.8% and 11.9±1.4% respectively. There was distinctively difference compared with the cells treated with 200μmol·L-1DADS alone(P<0.01). These results showed that BAPTA-AM, NAC would inhibit DADS inducing the apoptosis of CNE2 cells. CNE2 cells treated with DADS at the concentration of 50, 100, 200, 400μmol·L-1 for 4h, the Fluo3 fluorescence intensity were 4.10±0.20, 7.37±0.25, 9.73±0.15 and 12.63±0.45, respectively. The fluorescence intensity of the treated group were elevated than the control group(1.67±0.21). These results showed that DADS could elevate the level of intracellular Ca2+ in a dose-dependent manner. CNE2 cells treated with DADS at the concentration of 50, 100, 200, 400μmol·L-1 for 5h, DCFH-DA fluorescence intensity were 18.70±2.43, 33.23±1.53, 41.53±0.78 and 60.90±1.97, repectively. the fluorescence intensity of treated group were elevated compared with the control group(8.27±1.37). Pretreatment of cells with BAPTA-AM, the fluorescence intensity was 26.80±2.69, there was distinctively difference compared with the cells treated with DADS alone. These results showed that DADS 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. Conclusion:1. DADS could inhibit the growth of CNE2 cells and induce its apoptosis.2. The inducing apoptosis effect of DADS on CNE2 cells could down-regulate the ratio of Bcl-2/bax and active Caspase 3.3. The inducing apoptosis effect of DADS on CNE2 cells could through the sequential mechanism of Ca2+ homeostasis disruption and accumulation of ROS.4. DADS induced apoptosis of CNE2 cells through Ca2+ signal pathway.