CdCl₂ Induces Proliferation In Lung Cells Via Autophagy-dependent Glycolysis

Objective:According to the 2018 global cancer epidemiological statistics report,lung cancer accounts for about 11.6%of the 18.1 million new cancer cases,and about18.4%of the 9.6 million cancer deaths are due to lung cancer.Among the risk factors associated with lung cancer,smoking and secondhand smoke are the main causes of lung cancer.Among them,metal cadmium is one of the five cancerous substances in tobacco and tobacco smoke,and occupational cadmium exposure is also emphasized in relation to the occurrence of lung cancer.Long-term continuous exposure to cadmium can lead to pulmonary fibrosis,emphysema and lung cancer.Therefore,it is especially important to understand and explore the lung toxicity of cadmium and its underlying mechanisms.Our previous study found that ATG4B-mediated autophagy plays a key role in cadmium-induced proliferation and invasion of A549 cells.This study was designed to investigate the possible mechanism by which cadmium chloride promotes proliferation of A549 and HELF cells.Methods:In this study,adenocarcinomic human alveolar basal epithelial cells?A549 cells?and human embryonic lung fibroblasts?HELF cells?were used as experimental models.The effects of CdCl₂ on the relative protein expression of autophagy,aerobic glycolysis and cell cycle of A549 and HELF cells were detected by Western blotting.MTT assay was used to detect the effect of CdCl₂ on cell proliferation of A549 and HELF cells,and the effect of glycolysis inhibitor 2-deoxy-D-glucose?2DG?on cell proliferation induced by CdCl₂.The content of glucose and lactic acid in the medium treated with CdCl₂ for different time was determined by the glucose assay kit and the lactic acid assay kit.Meanwhile,ATP content in cells treated with CdCl₂ at different time was determined by ATP assay kit.Autophagy inhibitors 3-Methyladenine?3MA?and Chloroquine?CQ?were used to detect the effects of autophagy on glycolysis and cell cycle by Western blotting.The effects of autophagy-related genes on glycolysis and cell cycle were observed by Western blotting using small interfering RNA?siRNA?and overexpression plasmid ATG4B.Results:After treatment with CdCl2 at 2?M and 0.05?M for 12,24,36 and 48 h,the glucose content of A549 and HELF cells decreased significantly;while the lactate release increased significantly?P<0.05?;and ATP content increased?P<0.05?;glycolysis-related enzymes:glucose transporter?GLUT1?,hexokinase?HK II?,pyruvate kinase?PKM2?and lactate dehydrogenase?LDHA?increased in a time-dependent manner?P<0.05?,suggesting that CdCl2 induced an increase in aerobic glycolysis in A549 and HELF cells;Cyclin D1 and Cyclin E were significantly higher than the baseline level?P<0.05?;LC3 was up-regulated consistently,and P62 decreased significantly during the whole process of CdCl₂ treatment?P<0.05?.After treatment with glycolysis inhibitor 2DG,the growth of A549 and HELF cells induced by low dose CdCl₂ was decreased?P<0.05?,and the expression of Cyclin D1 and Cyclin E induced by CdCl₂ was decreased?P<0.05?.When pretreatment 3MA or co-treatment CQ with CdCl₂ for 48 h,the results showed that the expression of glycolysis-related proteins and cell cycle proteins were lower than that of single CdCl₂ exposure group?P<0.05?,suggesting that autophagy could limit the glycolysis level and cell cycle induced by CdCl₂ in A549 and HELF cells.Compared with CdCl₂ alone,the expression of glycolysis-related proteins was decreased in siATG4B treatment A549 cells?P<0.05?,while the expression of glycolysis-related proteins increased after overexpression of ATG4B?P<0.05?.The results of inhibition of glycolysis by 2DG combined with ATG4B plasmid transfection showed that the expression of cell cyclin proteins was lower than that of the overexpression ATG4B group alone?P<0.05?,suggesting that ATG4B-mediated autophagy decreased the cell cycle process by inhibiting glycolysis by 2DG.Conclusion:ATG4B-mediated autophagy-dependent glycolysis plays an important role in CdCl₂-induced proliferation of A549 and HELF cells.