Role And Mechanism Of Mcl-1 In Bile Salt (GCDA)-induced Survival And Chemoresistance Of Hepatocellular Carcinoma Cells

ObjectiveTo study expression and clinical significance of Mcl-1 in hepatocellular carcinoma(HCC) tissues,adjacent liver tissues and normal liver tissues.Identify the effects of Mcl-1 down-regulated by RNA interference on survival and drug sensitivity of HCC cells.Investigate the effects and mechanisms of bile salt(GCDA) on apoptosis and chemotherapeutic drug sensitivity of HCC cells.Then on this basis, clarify the role of Mcl-1 in hepatocellular carcinogenesis and its anti-apoptotic mechanism regulated by GCDA.This foundation will provide a combined target to Mcl-1 with a new strategy and explore new method in HCC therapy and create a new way in molecular targeted therapy of tumors.Methods1.Expression of Mcl-1 were detected in 51 HCC tissues,49 adjacent liver tissues and 25 normal liver tissues by Strepavidin-peroxidase immunohistochemistry staining(SP method) and analyzed with clinical pathological characteristics.2.Changes of protein expression and subcellular localization were detected by immunofluorescence method after GCDA treatment in HCC cells.3.Human HCC cell line HepG2 was cultured in vitro and transfected with Mcl-1 siRNA by Lipofectamine^TM 2000 which interfering the expression of Mcl-1.4.HepG2 cells were treated with GCDA±chemotherapeutic drug or chemotherapeutic drug±Mcl-1 siRNA.Cell viability and proliferation were detected by MTT assay.5.Cells were treated with chemotherapeutic drugs,GCDA or Mcl-1 siRNA.Cell cycle was assayed by Flow Cytometry(FCM).6.HepG2 cells were treated with GCDA±chemotherapeutic drug or chemotherapeutic drug±Mcl-1 siRNA and dyed with Annexin V-FITC and PI.Cell apoptosis morphology was observed by fluorescence microscope or electron microscope.Cell apoptotic rate was assayed by Flow Cytometry(FCM).7.The interaction of Mcl-1 and pro-apoptotic proteins was assessed by immunoprecipitation(IP) and Western Blotting(WB) after GCDA treatment.8.Total protein of HCC cells was extracted,and the concentration was measured by BCA method.Expressions of apoptosis-regulating proteins were assessed by Western Blotting.The reference gene wasβ-Tubulin.Gray scale values of proteins were analyzed by BandScan software. 9.DNA strand damage after GCDA treatment was assessed by Comet Assay in single cell level.Results1.Mcl-1 was expressed in HCC tissues and adjacent liver tissues with the positive rate of 68.63%and 58.10%,respectively.Compared to 8%of normal liver tissues,they had markedly significance of difference (p＜0.01).Intracellular location of Mcl- 1 was mainly in cytoplasm,partly in nuclei.The expression of Mcl-1 had no relationship with clinical pathological characteristic such as gender,age,HBV infection,tumor size and pathology grade(p＞0.05).However,the positive rate in well-differentiated HCC tissues was high to 67.86%.2.The results of RNA interference indicated expression of Mcl-1 protein was down-regulated obviously after Mcl-1 siRNA was transfected to HepG2 cells about 24h,48h,72h.The level of Mcl-1 protein had significance of difference(p＜0.01) to control group,especially after 48h, which the gray scale value was 27.70%of control group.After Mcl-1 was down-regulated in HepG2 cells,expression of anti-apoptotic protein such as Bcl-xl decreased while pro-apoptotic proteins such as Bak,Bim,Bax increased slightly.3.The results of MTT indicated GCDA had proliferation effect to HepG2 cells within short time,especially 10-20rain(p＜0.05),or with concentration lower than 200μM,especially 50～200μM(p＜0.05).With the treatment of chemotherapeutic drugs in HepG2 cells,the survival rate after Mcl-1 siRNA was significantly lower than single drug group(P＜0.05).4.FCM results indicated GCDA or Lipofectamine^TM 2000 had no effect to cell cycle and apoptosis(p＞0.05).With the treatment of chemotherapeutic drugs in HepG2 cells,cell apoptotic rate after Mcl-1 down-regulation(33.17%) was markedly higher than single drug group (17.60%)(P＜0.05).5.Immunofluorescence results indicated GCDA strengthened cellular expression of Mcl-1 and pERK,especially in nuclei.Expression of Mcl-1 and pERK weakened slightly after treatment of Irinotecan, especially in nuclei.6.Immunoprecipitation results indicated the binding of Bak/Bim reduced after GCDA treatment while Bak/Mcl-1 and Bim/Mcl-1 bingding increased.7.Western Blotting results indicated GCDA increased the expression of anti-apoptotic protein Mcl-1 and decreased the expression of pro-apoptotic proteins such as Bim,Bak and Bax.Though protein synthesis inhibitor made Mcl-1 degradation in 2 to 3 hours,the time was last to over 6 hours at the co-presence of GCDA.The expressions of pERK and Mcl-1 were inhibited after treatment of Irinotecan and revived at the co-presence of GCDA.8.Comet Assay results indicated DNA strand damage of HepG2 cells was enhanced gradually when the concentration of GCDA increased. Western Blotting results indicated DNA repair protein PARP was increased obviously.Conclusions1.Anti-apoptotic protein Mcl-1 is over-expression in HCC tissues.It has a progressive tendency in tumorigenesis.It has a high positive rate in well-differentiated HCC tissues.This indicates that anti-apoptosis function of Mcl-1 is related to HCC genesis.2.RNA interference can reduce expression level of Mcl-1 protein in HCC efficiently.Specific down-regulation of Mcl-1 protein increases apoptotic rate of HCC cells with the treatment of chemotherapeutic drugs and enhances drug sensitivity of HCC cells.When Mcl-1 protein level is down-regulated,the function of its homologous anti-apoptotic members in Bcl-2 family is inhibited partly and the pro-apoptotic proteins such as Bax,Bim,Bak are activated to induce cell apoptosis.3.GCDA can induce cell survival and chemoresistance of HCC cells through up-regulating expression level of anti-apoptotic proteins and down-regulating expression level of pro-apoptotic proteins.4.GCDA can increase expression of Mcl-1 protein in HCC cells and prevent its degradation,enhance its stability and apoptotic function in cells.The mechanism is that GCDA activate MAPK/ERK1/2 pathway to increase ERK phosphorylation and translocation to nuclei,which activate the target protein Mcl-1 and enhance its anti-apoptotic function.GCDA can promote the binding of Mcl-1/Bim and Mcl-1/Bak,and induce inactivation of pro-apoptotic proteins.It can also reduce the binding of Bim/Bak and prevent cytochrome release and prohibit cell apoptosis.5.GCDA can induce occurrence of DNA damage and repair,which is a good foundation of further study.