Effect Of Triptolide On Cells Proliferation,Apoptosis And Autophagy In Human Colon Cancer Cell Line HCT116

Background: The incidence of colon cancer are increasing worldwidely.Although some progress has been achieved with adjuvant chemotherapy,it is still limited by severe adverse effects and lack of ideal therapeutic effect.Therefore,extracting effective active ingredients from Chinese herbal medicine for researching and developing new drugs with anti-cancer activity has become a new trend.Triptolide(TP),which is the most effective monomer extracts from the clinical commonly used Chinese herb Tripterygium wilfordii Hook.F.(GTW),can possess a widespread pharmacological action.It has been used in immune regulation and anti-inflammatory in clinical.In recent years,some studies have confirmed it has an outstanding antitumor effect,but the mechanism remains obscure.Objective: To investigate the influence of TP on human colorectal cancer HCT116 cell proliferation,and explore the potential mechanism of TP in treating colon cancer in three ways: cell cycle,cell apoptosis and autophagy.We performed this research to find a new way and provide theoretical bases for treatment of colon cancer with Chinese medicine monomer.Methods: 1.The effects of TP on HCT116 cells:(1)Confirm the effective concentration of TP on HCT116 cell proliferation inhibition by MTT assay.(2)Flow cytometry(FCM)was applied to testing the effects of TP on cell cycle and cell apoptosis.(3)DAPI and MDC staining were used for detecting cell morphology changes after TP intervenes.(4)Western blot(WB)was used for testing the proteins expression level related to cell cycle,cell apoptosis signal pathway and autophagy molecular marker.(5)After apoptosis inhibitor,autophagy inhibitor and autophagy revulsant in combination with TP respectively used for treating with HCT116 cells.Annexin V-FITC/PI double stainings were used for detecting the impact on the rate of HCT116 cell apoptosis level induced by them.2.The relationship between HCT116 cells apoptosis and autophagy induced by TP:(1)After apoptosis inhibitor z-VAD-FMK in combination with TP in treating HCT116 cells.We set up five groups including control,TP,TP+z-VAD-FMK and z-VAD-FMK.Then detect the changes of apoptosis rate with Annexin V-FITC/PI double stainings by FCM in each group.(2)Autophagy inhibitor 3-MA was used with TP in treating HCT116 cells.We set up five groups including control,TP,TP+3-MA and 3-MA groups.Then detect the changes of apoptosis rate with Annexin V-FITC/PI double stainings by FCM in each group.(3)After autophagy inhibitor RAPA in combination with TP in treating HCT116 cells.We set up five groups including control,TP,TP+RAPA and RAPA.Then we detected the changes of apoptosis rates with Annexin V-FITC/PI double stainings by FCM in each group.Results:(1)Effects of TP on HCT116 cells proliferation and cell cycle:TP can significantly reduce the HCT116 cells survival rate in time and dose-dependent manner,and inhibit the HCT116 cells proliferation.TP can induce S phase arrest in HCT116 cells,and suppress the expression of cell cycle protein CDK4 and CyclinD1.(2)Effects of TP on HCT116 cells apoptosis and its mechanism: TP can can induce apoptosis in HCT116 cells,and with the increase of TP concentration,the HCT116 cells apoptosis rate increased gradually.The expression of Caspase3,Caspase9 and Bcl2 in HCT116 cells were down regulated by TP in dose-dependent,but the expression of cleaved Caspase3 and cleaved Caspase9 were up regulated by TP.TP had no influence on the expression of Caspase8.TP can also enhance the ability of Bax transfered to mitochondria,and promote Cytochrome c transferred to cytoplasm.(3)Effects of TP on HCT116 cell autophagy: TP can promote the generation of autophagy vesicle in HCT116 cells.And increase the expression of LC3 related to autophagy in time and dose-dependent.(4)The relationship between autophagy and apoptosis induced by TP in HCT116 cells: HCT116 cells apoptosis rate in the autophagy revulsant(RAPA)combined with TP group was significantly higher than TP group.HCT116 cells apoptosis rate in the autophagy inhibitor(3-MA)combined with TP group and apoptosis inhibitor(z-VAD-FMK)jointed with TP group are both significantly lower than TP group.Conclusion:(1)TP can inhibit the human colon cancer HCT116 cells proliferation and survival by inducing cell cycle retardation and cell apoptosis;(2)TP can induce apoptosis in HCT116 cells via mitochondria pathway;(3)TP can induce autophagy in HCT116 cells,and the cell autophagy can promote HCT116 cells apoptosis induced by TP.