Autophagy Inhibitor Chloroquine Increases Sensitivity To Cisplatin In QBC939 Cholangiocarcinoma Cells By Mitochondrial ROS

BackgroundThe histological origin of cholangiocarcinoma is bile duct epithelial cells,which with hepatocellular carcinoma are the top two of the most common malignant tumors in the hepatobiliary system,respectively.Compared with hepatocellular carcinoma,there are many studies have pointed out that the cholangiocarcinoma has obvious primary multidrug resistance,which is generally not sensitive to cisplatin and other chemotherapy drugs.It is inferred that the two different tissue sources of tumor cells— hepatoma cells and cholangiocarcinoma cells — may lead to their own antioxidant capacity due to metabolic differences,and therefore show different sensitivity to cisplatin induced apoptosis.As the close relationship between glucose metabolism and cell antioxidant capacity,and cisplatin increasing the level of intracellular ROS while inducing the apoptosis of tumor cells,especially the mitochondrial ROS,therefore,it is speculated that the resistance of cholangiocarcinoma to cisplatin may be related to its antioxidant capacity.Glucose metabolism provides cells with the raw materials to synthesize biological macromolecule and the energy required for various life activities.As one of the important branches of glucose metabolism,pentose phosphate pathway can not only produce the raw material of nucleotide — ribose,but also produce a lot of NADPH.The latter is an important reducing substrate in cell,which can maintain the reducibility of GSH and participate in the regulation of cell redox equilibrium.Autophagy is a necessary condition for maintaining cellular metabolism,and can also be activated by mitochondrial dysfunction and oxidative stress.The proteins and organelles of the abnormal function can be degradated by molecular chaperone-mediated autophagy or macroautophagy,and the degradation products ofautophagy can participate in the metabolism process,reused to generate biological macromolecule or provide energy.As a classic autophagy inhibitor CQ,it can inhibit the development of lung cancer and colon cancer,and has the potential to treat cancer.The study points out that the autophagy is significantly increased in the level of ROS,due to the close relationship between autophagy and metabolism,it is speculated that autophagy plays the role of maintaining homeostasis of intracellular environment through interaction with metabolic pathway.On the contrary,whether inhibition of autophagy can inhibit cell metabolic activity,and then reduce its antioxidant capacity,increase the sensitivity of tumor cells to chemotherapy drugs,has become the focus of our study.PurposeIn this study,we chose two different kinds of hepatobiliary tumor,hepatocellular carcinoma Hep G2 cells and cholangiocarcinoma QBC939 cells,to compared differences in cisplatin resistance,metabolism and antioxidant capacity,and to investigate the role of autophagy inhibitor CQ in increasing the level of intracellular mt ROS by inhibiting the PPP and promoting the production of intracellular ROS,and further enhancing cisplatin induced QBC939 cells death.Such a strategy may provide a new approach for drug-resistance reversion by exploring the mechanism of reducing the activity of glucose metabolism through inhibiting autophagy-lysosomal pathway to increase the sensitivity of tumor cells to cisplatin.Methods(1)Cell viability and apoptosis rate: MTT assay was used to detect the changes of cell viability after drug treatment.The apoptotic rate was detected by flow cytometry with Annexin V/PI staining.(2)The level of intracellular ROS and mitochondrial membrane potential: the fluorescent probe DCFH-DA was used to observe the changes of the overall level of ROS cells under the inverted fluorescence microscope.Staining on cells by usingfluorescent probe Mito SOX to observe the changes of mitochondrial ROS level under the inverted fluorescence microscope.The cells after staining by JC-1 detected the fluorescence intensity changes of cells by flow cytometry.(3)Detection of basal glucose consumption and lactate release: to detect glucose and lactic acid in the culture medium before and after culturing cells using glucose test kit and lactic acid kit,by calculating the difference between the two is a cell culture medium on glucose consumption and lactate release amount.In order to standardize the data of glucose consumption and lactate release,the Bradford was used to detect the total protein amount of cells cultured in the medium,and the amount of changes in glucose and lactic acid value were divided by the total amount of cells protein.(4)G6PDH activity detection: after collecting the cells and lysis,the lysate was treated with G6 PDH activity detection kit and the activity of G6 PDH was detected by colorimetric method.(5)Detection of GSH/GSSG ratio,NADPH/NADP ratio and hydroxyl radical content: cells were collected and lysed,respectively treated by GSH/GSSG kit,NADPH/NADP kit and hydroxyl radical detection kit and content by colorimetric detection of GSH,GSSG,NADPH,NADP and intracellular hydroxyl radical,respectively.(6)Detection of the expression of autophagy related proteins: after collecting the cells and lysis,the changes of autophagy related proteins LC3 and p62 were detected by Westernblot.Results(1)After CDDP treatment,compared with hepatocellular carcinoma cell line Hep G2,cholangiocarcinoma QBC939 cells showed less decrease in cell vitality,lower apoptosis rate and smaller increase in mitochondrial ROS level.(2)Compared with Hep G2 cells,QBC939 cells showed significantly enhanced the uptake and consumption of glucose,so as the release of lactic acid.The expressionand activity of G6 PDH,the key enzyme of pentose phosphate pathway,and the ratios of NADPH/NADP and GSH/GSSG in QBC939 cells were significantly higher than that of Hep G2 cells.(3)Both 2-DG and DHEA can increase the intracellular ROS level.Compared with Hep G2,the sensitivity of QBC939 to DHEA is low,and the total ROS level and mitochondrial ROS level in QBC939 cells are increased slightly under the treatment of DHEA.(4)The vitality of Hep G2 and QBC939 cells was significantly inhibited by CQ and 3-MA,and the expression levels of LC3 and p62 were significantly increased in the cells,and QBC939 cells were more sensitive to CQ.Compared with Hep G2 cells,general ROS and mt ROS increased significantly in QBC939 cells under the treatment of CQ,and the sensitivity to CDDP induced apoptosis was also significantly increased.(5)Compared with Hep G2 cells,QBC939 cells treated by CQ exhibited G6 PDH activity,GSH/GSSG ratio and NADPH/NADP ratio were decreased,mitochondrial membrane potential decreased significantly,while the hydroxyl radical content of cells increased significantly.Conclusion(1)There is a metabolic pattern difference between hepatocellular carcinoma cell line Hep G2 and cholangiocarcinoma cell line QBC939.(2)Increased activity of pentose phosphate pathway may be one of the mechanisms of low sensitivity of QBC939 cells to cisplatin.(3)Autophagy inhibitor chloroquine may be one of the mechanisms of increasing cisplatin sensitivity in QBC939 cells by inhibiting the activity of the pentose phosphate pathway and reducing the antioxidant capacity.(4)Chloroquine inhibited the clearance of damaged mitochondria and promoted the Fenton reaction,increased the intracellular ROS level and promoted the increase of cell sensitivity to cisplatin.In summary,we use hepatocellular carcinoma Hep G2 cells and cholangiocarcinoma QBC939 cells as the research object,through the comparative study on the relationship between glucose metabolism related antioxidant capacity and cisplatin resistance,and found that chloroquine increased intracellular levels of ROS cells through the inhibition of the pentose phosphate pathway and other ways,increase the sensitivity of cells to cisplatin.It suggested that inhibition of autophagy lysosome pathway might be a new way to inhibit cholangiocarcinoma cells.