Preliminary Study Of Energy Metabolism Of Resistant A549 Cells In Lung Adenocarcinoma

Objective1 To clarify the metabolic state and the major energy-generating pathways of resistant A549 cells in lung adenocarcinoma.2 To explore the effect of drug targeting the major energy-generating pathways on the antitumor effect of chemotherapeutics.Methods1 The establishment of resistant cell line in lung adenocarcinoma1.1 The cisplatin-resistant A549 cell line(A549/CR) was induced from their parental A549 cells by gradually increasing the concentration of cisplatin.1.2 Verify the resistant characteristic of A549/CR cells using the MTS assays.2 The energetic state and the major energy-generating pathway of resistant A549 cells2.1 we measured the cellular ATP contents of the A549/CR cells and A549 cells using the ATP-based Cell Titer-Glo Luminescent Cell Viability kit.2.2 We analyzed the glucose uptake of the A549/CR cells and A549 cells using the fluorescent 2-deoxy-glucose(2-DG) analog 2-NBDG.2.3 Lactate production of the A549/CR cells and A549 cells were detected using the L-lactate assay kit.2.4 To further explore the major energy-generating pathway of A549/CR cells, we detected the effect of glycolytic inhibitor and oxidative phosphorylation inhibitor on the cellular ATP content in A549/CR cells and A549 cells.3 The effect of metabolic inhibitor on survival of resistant cells.3.1 We compared the effect of 0?mol/m L?10?mol/m L ? 20?mol/mL 2-DG on the survival of A549/CR cells.3.2 Treat A549/CR cells with combined 2-DG and cisplatin in different concentrations and test the cell viability of A549/CR cells using MTS assays.Results1 Establish the A549/CR cell line successfully1.1 After three months' continuous administration of cisplatin, A549/CR cells can maintain growth in medium containing higher concentration of cisplatin(5?g/m L).1.2 MTS assays showed that the viability of A549/CR cells is significantly higher than A549 cells when the concentration of cisplatin is 40?g/m L.2 Resistant A549 cells exhibit higher energetic state and mainly rely on glycolysis2.1 The ATP content was higher in A549/CR cells compared to A549 cells.2.2 The fluorescence intensity is enhanced in A549/CR cells compared to A549 cells, indicating the more 2-NBDG they uptaked.2.3 Lactate production of A549/CR cells was significantly increased than that of A549 cells2.4 ATP depletion assays showed that A549/CR cells were more sensitive to 2-DG than to oligomycin. However, A549 cells were more sensitive to oligomycin. 2-DG induced ATP depletion in both subpopulations, with more potent effect on A549/CR cells. By contrast, oligomycin affected the A549 cells more intensively compared with A549/CR cells.3 The effect of 2-DG on proliferative capacities of resistant A549 cells.3.1 the low concentration of 2-DG didn't affect proliferation of A549/CR cells as measured in MTS assays(n=3,P>0.05).3.2 the combination of 2-DGand cisplatin in a lower concentration induced a significant cell death in resistant A549 cells.(n=3,P<0.01).Conclusion1 Resistant lung carcinoma A549 cells exhibit higher ATP levels and rely more on glycolysis to meet their energy demands.2 Inhibitor of glycolysis reversed the sensitivity of resistant A549 cells to cisplatin.In summary, inhibition of glycolysis enhanced the antitumor effect of cisplatin, implying that strategies targeting the metabolism of resistant cells may eliminate the resistant cells in lung adenocarcinoma.