| Objective: To provide novel theoretical and experimental bases for elucidating cholangiocarcinoma.The role and mechanism of oblongifolin C on the apoptosis,autophagy and mitochondrial functions of human cholangiocarcinoma QBC939 were investegated.Methods: Different concentrations(5,10,20 and 40 ? M)of OC,3-methyladenine(3-MA)+OC and rapamycin(RP)+OC were incubated with QBC939 cells for 48 hours,respectively.Subsequently,the relative viability was assessed by methylthiazole tetrazolium reduction assay.Flow cytometry with Annexin V/PI staining was used to detect cell apoptosis of different groups.Levels of mitochondrial membrane potential(MMP)and ATP were used to evaluate the mitochondrial dysfunction(Mt D).The protein expression levels of LC3 II,LC3I and p62 were determined by western blot.Results: 1.OC significantly inhibited human cholangiocarcinoma QBC939 cells viability and induced apoptosis in a dose?dependent manner at concentrations(10 to 40 ?M).Compared with control group,OC significantly decreased cell viability and induced apoptosis on human cholangiocarcinoma QBC939 cells(at concentration of 10 ?M,P<0.05).2.Following incubation with different concentrations(5,10,20 and 40 ?M)of OC for 48 h,the ratio of LC3II/LC3 I and p62 levels were increased at 10 ?M OC(P<0.05),compared with the control group.The addition of OC inhibits autophagic flux in human cholangiocarcinoma QBC939 cells in a dose?dependent manner at concentrations from 10 to 40 ?M.3.Following incubation with different concentrations(5,10,20 and 40 ?M)of OC for 48 h,compared with control group,10 ?M OC significantly inhibited the mitochondrial membrane potential(P<0.05),while the ATP concentration was also decreased(P<0.05).This effect of OC existed in a dose-dependent manner at concentrations from 10 to 40 ?M in QBC939 cells.4.Compared with OC group,cell apoptosis was significantly decreased(P<0.05),the ratio of LC3II/LC3 I was increased(P<0.05)and the expression level of p62 protein was decreased(P<0.05)in RP+OC(10 ? M)group.In addition,the concentration of mitochondrial membrane potential and ATP was markedly increased(P<0.05).5.Compared with OC group,cell apoptosis was significantly increased(P<0.05),the level of LC3II/LC3 I was decreased(P<0.05)and the expression of p62 protein was increased(P<0.05)in 3-MA+OC(10 ?M)group.In addition,the concentration of mitochondrial membrane potential and ATP was markedly decreased(P<0.05).Conclusions: The apoptosis of QBC939 cells was induce by OC in a dose-dependent manner.OC induced apoptosis was mediated through the inhibition of autophagy and Mt D in human cholangiocarcinoma QBC939 cells.Objective: To detect the relationship of FOXO1 induced autophagic flux and the apoptosis of BC939 cells.To elucidate the effects of FOXO1 on the oxidative stress,Mt D and cell apoptosis in QBC939 cells.And,in order to provide theoretical and experimental basis for elucidating the role of FOXO1 in cholangiocarcinoma.Methods: Human cholangiocarcinoma QBC939 cells were cultured in vitro under normal conditions or serum starvation(SS).CQ,3-MA and Rap were used to regulated autophagy,respectively.FOXO1-si RNA and Res were used to further elucidate the role of FOXO1 in inducing autophagy.An Methylthiazole tetrazolium(MTT)reduction assay was used as a qualitative index of cell viability.Flow cytometry with Annexin V/PI staining was useed to detect cell apoptosis.2'7'-dichlorofluorescin diacetate(DCFDA)was used to detect renal ROS production.Levels of mitochondrial membrane potential(MMP)and ATP were used to evaluate the mitochondrial dysfunction(Mt D).Western blot analysis was used to monitor autophagic fluctuation by determining the protein expression of LC3II/LC3 I and p62.Results: 1.Compared with control group,FOXO1 levels in SS group and SS+si RNA-Con group were elevated(P<0.05)and seemed to be related to the autophagic process,as evidenced by increased p62 degradation(P<0.05)and LC3-II accumulation(P<0.05);compared with SS group,p62 degradation was blocked and LC3-II was further ac-cumulated in SS+CQ group(P<0.05);compared with SS+si RNA-Con group,the expression of FOXO1 was decreased(P<0.05),while p62 degradation was decreased(P<0.05)and LC3II/LC3 I was decreased(P<0.05)in SS+si RNA-FOXO1 group.2.Compared with control group,no detectable changes in ubiquitylation or phosphorylation of cytosolic FOXO1 were observed in SS group,whereas the level of acetylated FOXO1 was significantly increased in QBC939 cells(P<0.05);in addition,acetylation of FOXO1 and its interaction with Atg7 was also increased(P<0.05).3.Compared with control group,the level of acetylated FOXO1 was decreased(P<0.05)and the interaction between acetylated FOXO1 and Atg7 was also inhibited(P<0.05)in Res(a sirt1 agonist)group,accompanied by impaired autophagic flux as evidenced by increased LC3 and p62 accumulation(P<0.05).4.Compared with control group,cell viability was decreased(P<0.05)and cell apoptosis was increased(P<0.05)in human cholangiocarcinoma QBC939 cells in both Res group and Res+si RNA-con group.Compared with Res group,treating QBC939 cells with 3-MA resulted in an enhancement of QBC939 damage induced by resveratrol as indicated by the increased apoptosis and decreased cell ability(P<0.05).On the contrary,treatment with Rap reduced QBC939 cells damage induced by resveratrol(P<0.05).Compared with Res+si RNA-con group,cell viability was decreased(P<0.05)and cell apoptosis was increased(P<0.05)in Res+si RNA-FOXO1 group.5.Compared with control group,ROS level was significantly increased(P<0.05)and MMP and ATP production were decreased(P<0.05)in both Res group and Res+si RNA-con group.Compared with Res group,RP treatment attenuated the ROS levels and improved the Mt D in QBC939 cells(P<0.05),whereas 3-MA further increased the level of ROS and impaired the Mt D in QBC939 cells(P<0.05).Compared with Res+si RNA-con group,the level of ROS was increased(P<0.05)and MMP and ATP production were decreased(P<0.05)in Res+si RNA-FOXO1 group.Conclusions: FOXO1 exists in autophagy regulation and impairing autophagic flux could lead to oxidative stress,Mt D and apoptosis in human cholangiocarcinoma QBC939 cells.FOXO1 could serve as a potential therapeutic target to cure against human cholangiocarcinoma. |
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