Mechanism Of Autophagy Signal Regulation In Cholangiocarcinoma Cells Apoptosis Induced By Norcantharidin

Background: At present,the incidence of cholangiocarcinoma is gradually increasing,besides,the high malignant degree of cholangiocarcinoma and the poor therapeutic effect,it is important to study the pathogenesis of cholangiocarcinoma and the signaling pathways involved in the process of cholangiocarcinoma apoptosis.Objective: This study is enhancing the cholangiocarcinoma apoptosis effect by NTCD via inhibiting autophagy by knock down ATG5,and the mechanism of this process will be further studied.Methods:RNA interference technology and autophagy inhibitor 3MA were used to construct the cholangiocarcinoma autophagy inhibition model,HBSS was used to induce cholangiocarcinoma autophagy model.After that,NCTD was used to induced cell apoptosis and the relationship between autophagy and apoptosis were studied,we further studied the autophagy regulated apoptosis pathway.Then Subcutaneous seeding tumor experiment in nude mice was used to further verify the results of the vitro experiment.Results: This study shows that NCTD can induce cholangiocarcinoma cells apoptosis with time and dose dependent manners,IC50 was 0.16 m M.The apoptosis level was increased with inhibiting autophagy by ATG5 siRNA and 3-MA,the apoptosis level was decreased by the autophagy occurring.We can get four results by the apoptosis pathway detection:1.With the treatment of NCTD,the cytoplasmic cytochrome C was increased by the inhibition of autophagy and mitochondrial cytochrome C was decreased at the same time.Otherwise,the cytoplasmic cytochrome C was decreased by the induction of autophagy and mitochondrial cytochrome C was increased at the same time.2.With the treatment of NCTD,after autophagy was inhibited,Bax expression increased significantly meanwhile the Bcl-2 expression was decreased.3.With the treatment of NCTD,Caspase-9,caspase-3 and PARP were activated while the autophagy was inhibited but the caspase 8 has no changes.4.With the treatment of NCTD,mitochondrial membrane potential collapse increased when LC3-II was down-regulated,but it showed the opposite trend in the autophagy signal induced group.Meanwhile,it showed that the inhibition of autophagy induced the ROS,but autophagy induction reduced the ROS.Finally,the expression of phosphorylated PI3K-Akt protein was decreased in autophagy down-regulation group,while it showed the opposite trend in the autophagy signal induced group.It showed that low expression of ATG5 gene inhibited the subcutaneous tumorigenicity of nude mice,and further promoted the subcutaneous tumorigenicity of nude mice by norcantharidin.The results of related pathway proteins were consistent with those in vitro.Conclusion: Autophagy could regulate the cholangiocarcinoma cell apoptosis,cholangiocarcinoma cell apoptosis could be strengthened when cell in the low autophagy levels.This process induced by mitochondrial apoptosis pathway.Besides,the increase of ROS and the activation of PI3K-AKT also plays a role in this process.