Autophagy-dependent Glycolysis Regulated By Endoplasmic Reticulum Stress Plays An Important Role In The Inhibition Of Apoptosis Induced By Cr(?) In A549 Cells

Objective: Hexavalent chromium [Cr(VI)],which is widely found in occupational and industrial environments,is a recognized human carcinogen.In our previous study,we found that autophagy could be induced by Cr(VI),which can induce cell proliferation in A549 cells.This study was to investigate the role of endoplasmic reticulum(ER)stress in apoptosis,autophagy and glycolysis induced by Cr(VI)in A549 cells,and the protective effect of autophagy-induced glycolysis on Cr(VI)-induced apoptosis.It mainly reveals the inhibitory effect of ER stress-mediated autophagy-dependent glycolysis on apoptosis,and provides a new theory for the toxic mechanism of chromium,which provides a new target for the prevention of chromium toxicity.Methods: In this experiment,A549 cells were used as experimental models.Western blot was used to detect the expression of ER stress,apoptosis and autophagy-related proteins in A549 cells treated with Cr(VI)for 2,4,6,12 and 24 hours.The morphological changes of the ER of A549 cells were observed by transmission electron microscopy.Hoechst staining was used to observe the effects of Cr(VI)at different times on the number of nuclear pyknosis,translucent and fragmentation of A549 cells.The effect of Cr(VI)on the autophagosomes of A549 cells was observed by AO staining.Western blot was used to detect the expression of glycolytic-related proteins in different time of Cr(VI)in A549 cells.The Glucose Detection Kit and the Lactic Acid Detection Kit were used to detect the glucose consumption and the production of lactic acid in the medium of the A549 cells treated with Cr(VI)at different times.The ATP Detection Kit was used to detect the ATP production of A549 cells treated with Cr(VI)at different times.The ER stress inhibitor 4-PBA was used to determine the effect of ER stress on Cr(VI)-induced apoptosis,autophagy and glycolysis.The apoptosis inhibitor Z-VAD-FMK was used to determine the role of Cr(VI)-induced apoptosis in autophagy.The autophagy inhibitor 3-MA was used to determine the effect of Cr(VI)-induced autophagy on apoptosis and glycolysis.The glycolysis inhibitor 2-DG was used to determine the effect of Cr(VI)-induced glycolysis on apoptosis.Results: After treating with potassium dichromate Cr(VI)(0.2 ?M)for 2,4,6,12 and 24 hours in A549 cells,ER stress-related protein(p-PERK,GRP78)and autophagyrelated protein LC3-II/LC3-I expression gradually increased,p62 expression gradually decreased;apoptosis-related proteins(cleaved caspase-8,cleaved caspase-9,cleaved caspase-3)gradually increased in 2~12 hours and decreased in 24 hours.Hoechst staining showed that in Cr(VI)-treated A549 cells,the number of nucleus pyknosis,translucent and fragmentation increased significantly at 12 hours.However,the number of nucleus pyknosis,translucent and fragmentation was significantly reduced at 24 to 36 hours.The results of AO staining showed that in Cr(VI)-treated A549 cells,the number of autophagosomes increased gradually from 12 to 24 hours,and decreased at 36 hours.Transmission electron microscopy showed that the ER lumen was significantly expanded after treatment of A549 cells with Cr(VI)for 24 hours.The expression of HK2,GLUT1,PKM2,LDHA was gradually increased after treatment of A549 cells with Cr(VI)for 12,24 and 36 hours.The glucose content in the culture medium showed a gradual decrease after treatment with Cr(VI)for a different period of time,indicating an increase in glucose consumption.The lactic acid content in the culture medium showed a gradual increase after treatment with Cr(VI)for a different period of time,and the production of ATP in the cells also gradually increased.After pretreatment with ER stress inhibitor 4-PBA,the expression of Cr(VI)-induced cleaved caspase-9 and cleaved caspase-3 decreased,but the expression of cleaved caspase-8 did not change significantly.The expression of LC3-II/LC3-I was decreased,and the expression of p62 was increased.The expression of HK2,GLUT1,PKM2 and LDHA was decreased.Hoechst staining showed that the number of nucleus pyknosis,translucent and fragmentation in the Cr(VI)group after 4-PBA pretreatment was significantly reduced compared with the Cr(VI)-treated group.The results of AO staining showed that the number of autophagosomes in the Cr(VI)group was significantly reduced after 4-PBA pretreatment compared with the Cr(VI)-treated group.After pretreatment with the apoptosis inhibitor Z-VAD-FMK,the expression of LC3-II/LC3-I was decreased and the expression of p62 was increased compared with the Cr(VI)-treated group.After pretreatment with autophagy inhibitor 3-MA,the expression of cleaved caspase-9 and cleaved caspase-3 was increased compared with the Cr(VI)-treated group,and the expression of HK2,GLUT1,PKM2 and LDHA is reduced.After pretreatment with the glycolytic inhibitor 2-DG,the expression of cleaved caspase-9 and cleaved caspase-3 was elevated compared with the Cr(VI)-treated group.Conclusion: In our study,the results indicated that ER stress plays an important role in Cr(VI)-induced apoptosis and autophagy in A549 cells,and there is an interaction between apoptosis and autophagy.Cr(VI)-induced apoptosis in the mitochondrial pathway induced autophagy,which increases glycolytic activity,provides energy to cells,and alleviates mitochondrial energy synthesis,thereby reducing apoptosis and making the cells to be allowed to proliferate under Cr(VI)induced stress conditions.