Study On The Effect Of Paris Total Saponin On Proliferation And Radiosensitivity Of Human Hepatocellular Carcinoma HepG2 Cells

Part ? Study on the inhibitory effect of total RPTS on proliferation of human hepatocellular carcinoma Hep G2 cellsObjective: To observe the effect of RPTS on human hepatocellular carcinoma Hep G2 cell proliferation effect;Methods: Human hepatocellular carcinoma(Hep G2)cells were treated with different concentrations of RPTS and cultured for different time.The proliferation of human hepatoma Hep G2 cells was detected by CCK8 assay;Results:CCK8 assay was used to detect the proliferation of human hepatoma Hep G2 cells treated with different concentrations of RPTS.The results showed that the proliferation of human hepatoma Hep G2 cells was inhibited by the RPTS.Moreover,in the range of drug concentration,with the increase of drug concentration and the prolongation of drug action time,the proliferation inhibition rate of human hepatoma Hep G2 cells increased gradually.The IC50 value(half inhibitory concentration)after treated with RPTS of 24 h,48h and 72 h were(90.38±0.27)ug/ml?(59.84±0.38)ug/ml?(48.62±0.32)ug/ml respectively;Conclusion:Within the drug concentration range set up in the experiment,the RPTS could inhibit the proliferation of human hepatoma Hep G2 cells,and its inhibition rate increased with the increase of drug concentration and duration,showing a concentration and time dependent.Part ? Study on the effects of RPTS on radiosensitivity of Hep G2 cells of human hepatocellular carcinomaObjective: To investigate the effects of RPTS on human hepatocellular carcinoma radiosensitivity of Hep G2 cell and its possible mechanism;Methods: 1.The human hepatoma Hep G2 cells with logarithmic growth phase were divided into four groups: blank control group,simple drug group,simple irradiation group,irradiation plus drug group after cleaning and digestion.Blank control group: the normal culture medium to culture 48h;simple drug group: containing certain concentration of RPTS(IC20=25ug/ml)liquid medium to culture 48h;irradiation group: the normal culture medium to culture 48 h,and then used 6MV X-ray raying cells,irradiation dose is set to 8GY;irradiation+drug group: first with the added concentration of RPTS(IC20=25ug/ml)cultured in vitro after 48 h,and then used 6MV X-ray raying cells,irradiation dose is set to 8GY.After the irradiation,the cells in each group were further cultured at 12 h,after the operation was completed,the cell apoptosis and the cell cycle distribution were detected by flow cytometry.2.The human hepatoma Hep G2 cells with logarithmic growth phase were divided into four groups: blank control group,simple drug group,simple irradiation group,irradiation plus drug group after cleaning and digestion.Blank control group: the normal culture medium were cultured tumor cells 48 h,72h;simple drug group: containing certain concentration of RPTS(IC20=25ug/ml)liquid medium were cultured tumor cells 48 h,72h;irradiation group: first the normal culture medium were cultured tumor cells 48 h,72h,then using 6MV X-ray raying cells,irradiation dose is set to 8GY;irradiation + drug group: first with the added concentration of RPTS(IC20=25ug/ml)liquid medium were cultured tumor cells 48 h,72h,and then using 6MV X-ray raying cells,irradiation dose is set to 8GY.After the irradiation,the cells in each group were further cultured at 12 h,after completion of operation,the expression level of MUC-1 protein in hepatocellular carcinoma cells was detected by Western blot,and the expression of MUC-1 m RNA in hepatocellular carcinoma cells was detected by real-time fluorescence quantitative PCR(RT-PCR).Results: 1.Flow cytometry was used to detect the distribution of cell cycle showed that RPTS of low cell toxicity concentration(25ug/ul)of human hepatocellular carcinoma Hep G2 cells after 48 h,tumor cell cycle distribution changes,G0/G1 phase cells decreased and S cells increased,the differences were statistically significant(P < 0.05);at the same time,the amount of RPTS can also increase G2/M cell distribution,but the results showed no significant difference(P > 0.05);after completion of X ray irradiation,the Hep G2 level of G2/M phase was significantly reduced,the statistical results were different(P < 0.05),while the G0/G1 and S phases increased to varying degrees,but the statistical results showed no significant difference(P > 0.05).2.Flow cytometry was used to detect cell apoptosis results show that in the low cytotoxicity of Paris total saponin concentration(25ug/ul)of human hepatocellular carcinoma Hep G2 cells after 48 h,early and late apoptosis cells were significantly increased,the difference has statistical significance(P < 0.001);and low cytotoxicity of Paris saponins(25ug/ul)further joint X-ray induced tumor cell apoptosis,the apoptotic rate increased significantly,with statistical significance(P < 0.05).3.The results of Western Blot detection of the expression of MUC-1 protein showed that,The expression of MUC-1 protein in human hepatoma Hep G2 cells was inhibited in a time-dependent manner by low cytotoxic concentration of RPTS(25ug/ul)and X-ray,and the results were statistically significant(P < 0.001);the expression of MUC-1 protein in human hepatoma Hep G2 cells was further inhibited by the combination of RPTS and X-ray,and the results were statistically significant(P < 0.001),which indicated that the RPTS could inhibit the expression of MUC-1 protein in combination with X-ray.4.The expression of MUC-1m RNA was detected by RT-PCR.The results showed that the low cytotoxic concentration of RPTS(25ug/ul)and X-ray could inhibit the expression of MUC-1m RNA in human hepatoma Hep G2 cells in a time-dependent manner,and the results were statistically significant(P < 0.001);the expression of MUC-1 m RNA in human hepatoma Hep G2 cells was further inhibited by the combination of total saponins and X-ray.The results were statistically significant(P < 0.001),which indicated that the total saponins could inhibit the expression of MUC-1 m RNA in combination with X ray,and the results were consistent with the results of Western Blot assay for the expression of MUC-1 protein.Conclusion: The low cytotoxic concentration of RPTS(25ug/ul)has a certain radiosensitization effect on human hepatoma Hep G2 cells.The mechanism may be related to inhibiting the proliferation of tumor cells,inducing apoptosis of tumor cells,and reducing the expression of MUC-1 protein in Hep G2 cells of human liver cancer.However,there may be no obvious relationship with the regulation of tumor cell cycle changes.