1,25-Dihydroxyvitamin D3 Induces Mitochondria-dependent Apoptosis In Human Gastric Cancer Cell Line

ObjectiveTo study the effects of 1,25(OH)2D3 on the growth of MGC-803,and investigate whether 1,25(OH)2D3 can inhibit proliferation and induce the apoptosis of gastric cells,and to discuss whether 1,25(OH)2D3 can induce the apoptosis of tumor cells through mitochondrial pathway mediated by p53.Methods1. MTT assay was used to detect the effects of 1,25(OH)2D3 with different concentration at different time on the survival rate of human gastric cells.2. Cell apoptosis rate was detected by FCM after treated by 1,25(OH)2D3 with different concentration for 72h, Annexin V-FITC/PI double staining was used in the analysis of apoptosis.3. Fluorescent probe rhodamine-123 was used to reflect the change of mitochondria membrane potential(MMP) after treated with different concentration of 1,25(OH)2D3 for 72h.4. Fluorescent probe DCFH-DA was used to reflect intracellular ROS formation after treated with different concentration of 1,25(OH)2D3 for 72h.5. RT-PCR was used to detect the change of p53 mRNA after treated with different concentration of 1,25(OH)2D3 for 72h.6. The kit was used to detect the change of Caspase-3、9 after treated with different concentration of 1,25(OH)2D3 for 72h.Results1. MTT assay showed:after cells treated with the low dose of 10-8mol/L of 1,25(OH)2D3for different times,cell survival rates were (96.37±5.68)%、(89.29±2.72)%、(75.76±3.48)%、 (76.03±1.22)% orderly;after cells treated with the dose of 10"7mol/L of 1,25(OH)2D3 for different times,cell survival rates were (92.09±7.39)%、(85.26±4.69)%、(65.05±2.41)%、(63.68±1.25)% orderly;after cells treated with the dose of 10-6mol/L of 1,25(OH)2D3 for different times,cell survival rates were (82.34±4.1)%、(73.86±6.48)%、(53.56±1.94)%、(49.58±1.24)% orderly;after cells treated by the high dose of 10-5mol/L of 1,25(OH)2D3 for different times,cell survival rates were (71.99±6.71)%、(59.43±3.57)%、(40.62±1.73)%、(37.06±1.84)% orderly.There was no statistical significances between normal control group and solvent control group (P>0.05).After cells treated with different concentration of 1,25(OH)2D3 for the same time,and compared with solvent control group, cell survival rates of each experimental group were decreased significantly (P<0.05),except cells treated with the low dose of 10-8mol/L for 24h;After cells treated with the same concentration of 1,25(OH)2D3 for different time,and compared with solvent control group, cell survival rates of each experimental group were decreased significantly (P<0.05).The results show that 1,25(OH)2D3 may effect cell activity in a dose-time dependent.2. The changes of cell morphology were observed under microscope after cells treated with 1,25(OH)2D3,nucleus pycnosis was showed and cells became round, the number of cells decreased,and some cells fall off in the solution.FCM showed:after MGC-803 treated with 1,25(OH)2D3 for 72h,the early apoptosis rates of each group were (2.67±0.32)%、(4.2±0.41)%、 (5.17±0.40)%、(11.5±0.66)%、(19.63±0.75)%;the late apoptosis rates of each group were (1.1±0.20)%、(1.17±0.15)%、(1.67±0.38)%、(1.03±0.21)%、(3.30±0.56)%.The early apoptosis rates were increased by the concentration of 1,25(OH)2D3,and compared with solvent control group,there were statistically differences (P<0.01);But there’s no statistically significant(P>0.05) in late apoptosis rates except the 10-5mol/L group.The results show that 1,25(OH)2D3 may induce the early apoptosis of gastric cancer cells in a dose-dependent.3. After MGC-803 treated with different concentration of 1,25(OH)2D3 for 72h,the fluorescence intensity of Rho-123 was measured by FCM.Experimental group from low dose to high were 94%,91%,83%,71%of solvent control group.The results show that 1,25(OH)2D3 may decrease the MPP in a dose-dependent.4. After MGC-803 treated with different concentration of 1,25(OH)2D3 for 72h,the fluorescence intensity of DCFH-DA was measured by inverted fluorescence microscope and FCM,the former showed intracellular fluorescence intensity and concentration of 1,25(OH)2D3 were positively correlated;and FCM showed that,the ROS formation were 1.05 times,1.2 times,2.15 times,2.9 times of solvent control group.The results show that l,25(OH)2D3may induce intracellular ROS formation in a dose-dependent.5. After MGC-803 treated with different concentration of 1,25(OH)2D3 for 72h,the change of p53 mRNA was detected by RT-PCR,p53 mRNA formation were 97%,1.25 times,1.74 times,2.68 times of solvent control group.The results show that l,25(OH)2D3may induce the transcription of intracellular p53 in a dose-dependent.6. After MGC-803 treated with different concentration of 1,25(OH)2D3 for 72h,the concentration of Caspase-3 of each group were (19.08±2.1)、(18.28±1.33)、(28.62±2.16)、 (31.96±2.17)、(40.23±3.06)μmol/L respectively;and the concentration of Caspase-9 were (22.88±2.53)、(25.75±3.53)、(32.53±1.33)、(49.08±2.27)、(79.65±4.23)μmol/L.There were no statistically significants between solvent control group and low dose group;but there were statistically significants in other groups.The results show that 1,25(OH)2D3 may stimulate the production of Caspase-3、9 in a dose-dependent.Conclusions1,25(OH)2D3 can induce the apoptosis of gastric cancer cells and the mechanism ma y be related to the apoptosis in the path of mitochondria mediated by p53.