Study On The Effects Of Gentiopicroside On Proliferation And Apoptosis Of HCT116 Cells

Objective:The aim of the present study is to study the effects of GTP which extracts from Gentianella acuta on proliferation and apoptosis of colon cancer cells HCT 116 cells in vitro.And Preliminary explore the underlying mechanisms,provide evidence for the mechanisms of action of this compound,and then to provide a theoretical basis for making full use of GTP in some sense.Methods:1.HCT116 which were purchased from the Shanghai Institute of Cell Biology were cultured in RPMI 1640 supplemented with 10%fetal calf serum,penicillin and streptomycin at 37? in a humidified atmosphere of 5%CO2.morphology change were abserved by microscope.2.Cell viability was monitored by using the CCK-8 method.3.The cell cycle was analyzed by flow cytometry,and apoptotic cells were identified by Annexin V-FITC/PI double staining.4.Measurement of mitochondrial membrane potential by JC-1 staining.5.Intracellular ROS was detected by using a DCFDA fluorescent probe.Results:1.In the microscope,we found that cells morphology changed significantly with concentrations increasing.HCT 116 cells changed from regular shape to ground,and the space of adjacent cells became larger.Nucleus became smaller at the same time.2.GTP inhibit cell proliferation by using CCK-8 method.The results show that HCT 116 cells are sensitive to GTP,different concentration of GTP can inhibit the growth of HCT 116 cells,and the effects are based on the concentration and time.When concentration reach 50ug/ml,the inhibition reached to platform.Meanwhile,we can see that the inhibitory of GTP on HCT 116 cells is apparent when treated for 24 hours,and reached to platform when treated for 48 h.3.Cell cycles analysis:The results of FCM show that after 24 hours treated with GTP,a significant number of cells were arrested at G2/M phase,and the ratio were based on concentration.4.FCM analysis with Annexin V-FITC/PI double stainng indicated that GTP can induce apoptosis in HCT116 in vitro,meanwhile the apoptotic rate by treating with GTP for 24h was Elevated With the increase of the concentration.we can see the total apoptosis of untreated is 2.38%±0.43,while the 6.25ug/ml GTP induced apoptosis is 8.02%±1.05,and the apoptosis reached 14.05%± 1.35 while treated with 12.50ug/ml GTP,the apoptosis is 21.08%±2.32 while treated with 25.00ug/ml,when treated with 50.00ug/ml GTP,we can see the apoptosis get to 39.8 2%±3.76 apparently.5.MMP was detected by FCM with JC-1:After treated with various concentrations of GTP,the MMP level was decreased with the increase of the concentration of GTP.6.GTP induced ROS generation:While treated with GTP for 24h and 48h,we can find that the level of intracellular ROS was relatively higher,compared to the control group.(P<0.05)Conclusions:1.GTP could inhibit the growth of the human colorectal cancer cell line HCT116,and it can induce the apoptosis of HCT116 in a dose-time dependent way and cell cycles cells were arrested at G2/M phase.2.GTP induced apoptosis of HCT116 may be associated with ROS-mediated mitochondrial apoptosis signaling pathway.