The Mechanism Of AZD-8055 Combined With Chloroquine Inhibit The Proliferation And Migration In Cholangiocarcinoma Cells

Objective:To exploe the effects and mechanisms of proliferation,migration and the process of EMT in cholangiocarcinoma cells after co-treament with AZD-8055 and CQ.Methods:The effects of AZD-8055(50?M,100nM,200nM)and CQ(5?M?10?M,20?M)on the proliferation of cholangiocarcinoma cells HuCCT1 and RBE were detected by MTT assay.The effects of AZD-8055,rapamycin and everolimus on the proliferation of HuCCTl and RBE cells at concentration of 100nM was detect by MTT assay.The effects of AZD-8055 and CQ on the proliferation of human normal bile duct epithelial cells HIBEpiC and cholangiocarcinoma cells HuCCT1 and RBE were examed by MTT assay.The value of combination index(CI)in HuCCT1 and RBE cell was detected by Chou-Talalay method.MTT assay was used to detect the effect of AZD-8055 and CQ on the proliferation of cholangiocarcinoma cells HuCCT1 and RBE.The effect of AZD-8055 and CQ alone or combination on the proliferation of human cholangiocarcinoma cells HuCCT1 and RBE were detected by plate cloning assay,EdU cell proliferation assay.Besides,the formation of autophagosomes in cholangiocarcinoma cells after AZD-8055 and CQ co-treament was observe by immunofluorescence staining.The number of apoptotic bodies in cholangiocarcinoma cells was detected by hoechst33342 staining.What's more,the apoptotic rates of cholangiocarcinoma cell lines HuCCT1 and RBE after treament with AZD-8055 and CQ was detected by flow cytometry.In addition,the lateral and longitudinal migration ability of HuCCT1 and RBE cells was observe by scratch healing and migration experiments.Furthermore,the expression of autophagy-related proteins LC3,p62,p-ULK1,ULK1,apoptosis-related proteins Bcl-2,Bax,Akt/mTOR signaling pathway and p38/MAPK signaling pathway-related proteins in cholangiocarcinoma cells were detect by western blotting.Results:The results of MTT showed that the inhibition rates of AZD-8055 50nM,100nM and 200nM on cholangiocarcinoma cells HuCCT1 were 22%,31%and 47%,and the inhibition rates of RBE were 22%,27%and 44%.CQ inhibited the proliferation of cholangiocarcinoma cells at the concentration of 20 ?M(P<0.05).The results of MTT showed that AZD-8055 had the most significant inhibitory on cholangiocarcinoma cells at 100 nM concentration compared with rapamycin and everolimus(P<0.05).The combination of AZD-8055 and CQ had no significant inhibitory effect on human bile duct epithelial cell line HIBEpiC,but had inhibitory on cholangiocarcinoma cells(P<0.05).The results of Chou-Talalay test showed that the CI value of HuCCTl and RBE in cholangiocarcinoma cells was less than 1(P<0.05).MTT results showed that AZD-8055 combined with CQ had a stronger inhibitory effect on the proliferation of cholangiocarcinoma cells than that of single drug group(P<0.05).The results of plate cloning and EdU cell proliferation test showed that AZD-8055 combined with CQ had stronger inhibitory effect on the proliferation of cholangiocarcinoma cells than that of single drug group(P<0.05).The results of immunofluorescence staining showed that the expression of LC3 in cytoplasm was increased in the combination group.The results of hoechst33342 staining showed that the number of apoptotic bodies was significantly increased in the combination group compared with the single agent.The results of flow cytometry showed that the apoptosis rate of the combination group was significantly higher than that of the single agent group.The results of scratch healing test and migration experiment showed that compared with the single agent,the ability of lateral and longitudinal migration was significantly inhibited in the combination group.The results of western blotting showed that in the combination group,the expression of autophagy-related proteins LC3II,p-62,p-ULK1 was significantly up-regulated,while the expression of ULK1 was down-regulated and the ratio of Bax/Bcl2 was significantly increased.The expression of p-Akt,p-S6 and p-4EBP1,which is related to the signal pathway of PI3K/Akt/mTOR,was down-regulated,and the experssion of total proteins was no significant changes.The expression of p-p38,which is related to the signal pathway of p38/MAPK,was increased in the combination group,and the expression of p38 was not obvious.Conclusions:1.The combination of AZD8055 and CQ can inhibit the ability of proliferation in cholangiocarcinoma cells,which may be closely related to the inhibition of autophagic flow in cholangiocarcinoma cells and the promotion of apoptosis in cholangiocarcinoma cells after the co-treament of AZD8055 and CQ.2.The combination of AZD8055 and CQ can inhibit the ability of migration and the process of EMT in cholangiocarcinoma cells,which is related to the regulation of Akt/mTOR signaling pathway and p38/MAPK signaling pathway.