Background One of the important characteristics of tumor is abnormal metabolism. As a metabolite, lactic acid also participates in metabolism.
Objective To investigate the effects of lactic acid on glycolysis pressure, proliferation, migration and cycle of A549 non-small cell lung cancer cells (NSCLCs).
Methods Human non-small cell lung cancer cell A549 was cultured and divided into control group, 5 mmol/L and 10 mmol/L lactic acid treatment groups. The control group did not receive any treatment. After 24 hours of lactic acid treatment in the other two groups, the glycolytic stress and mitochondrial substrate selection in A549 cells after lactate treatment were analyzed by Seahorse Cell Metabolism Analyzer. The mRNA level of glycolysis related genes (HK3, LDHA, LDHB, PKM) and cell cycle and migration related genes (CCNE1, CCND1, CCNB1, CDK2, CDH1, TWIST1, SNAI2) were detected by qPCR, Western blotting was used to detect the level of glycolytic related proteins (G6PD, PKM2, HK1 and LDHA). Real time cellular analysis (RTCA) and flow cytometry were used to analyze the effects of lactic acid on proliferation, migration, cycle and other biological characteristics of NSCLC A549 cells.
Results Compared with the control group, the glycolysis of the two lactic acid treatment groups decreased, the maximum capacity of glycolysis and glycolysis reserve decreased, and the reduction of the 10 mmol/L lactic acid treatment group was more significant than that of the 5 mmol/L group (P<0.05). The key enzymes of glycolysis were down-regulated at the level of mRNA and protein. Compared with the control group, the 10 mmol/L lactic acid treatment group was more dependent on fatty acids, and its oxidation ability to glutamine was decreased (P<0.05); RTCA results showed that the cell migration and proliferation ability of the two lactic acid treatment groups were lower than those of the control group, and the proliferation and migration ability of the 10 mmol/L lactic acid treatment group was significantly lower than that of the 5 mmol/L group (P<0.05). The mRNA levels of CCNE1, CCND1, CCNB1 and TWIST1 genes were down-regulated and the mRNA levels of CDK2 and CDH1 genes were up-regulated in the two lactic acid treatment groups. Flow cytometry showed that the number of G0/G1 phase cells increased (P<0.01), while the number of S phase cells and G2/M phase cells decreased (P<0.05, respectively).
Conclusion After adding exogenous lactic acid treatment, the glycolysis ability of A549 cells decreases, and it is negatively correlated with the concentration of lactic acid. The change of the oxidation substrate of the cells decreases significantly with the increase of the concentration of lactic acid, which suggests that adding exogenous lactic acid can inhibit A549 cells in G1 phase through the analysis of cell cycle results.
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