The Mechanism Of N-Acetyl-L-Cysteine In Reversing EGFR-TKI Resistance Induced By Cigarette Smoke Extract In NSCLC Cell Lines

Objectives: Targeted therapy has been the first-line treatment for non-small cell lung cancer(NSCLC)patients with corresponding mutations.EGFR-mutated NSCLC patients with a smoking history have a poorer prognosis than those never smokers.The aim of this study was to explore the potential mechanism of cigarette smoke extract(CSE)induced gefitinib resistance in NSCLC cell lines,and if N-Acetyl-L-Cysteine(NAC)could confer the CSE-induced gefitinib resistance.MethodsIn the first part,PC-9 and A549 cells were divided into gefitinib control group,10% CSE group.MTT assay was used to detect the gefitinib sensitivity of CSE to PC-9 and A549 cells,the expressions of EGFR / AKT signal pathway proteins were examined by western blot,the effect of CSE on intracellular ROS levels were detected by laser confocal scanning microscopy and flow cytometry,cell apoptosis was detected by flow cytometry.In the second part,we divided PC-9 and A549 cells into the control group,10% CSE group,10% CSE + NAC group,Gefitinib group,10%CSE + Gefitinib group,10% CSE + NAC + Gefitinib group.Gefitinib sensitivity of NAC on CSE-treated PC-9 and A549 cells was detected by MTT assays.Western blot was used to investigate the effect of NAC on the EGFR / AKT phosphorylation induced by CSE.Intracellular ROS levels pre-treated by NAC were observed by laser confocal scanning microscopy and flow cytometry.The effect of NAC on apoptosis of lung adenocarcinoma cells was detected by flow cytometry.ResultsMTT assays presented that the IC50 values of gefitinib control group,10% CSE group and 10% CSE + NAC group were 0.0671 ± 0.0131?M,0.9268 ± 0.2042?M,0.0519 ± 0.0018?M in PC-9 cells,respectively,while22.11 ± 1.13?M,55.51 ± 3.40?M and 31.01 ± 1.25?M in A549 cells,respectively.The IC50 value of 10% CSE group was significantly higher than the control group(P <0.01),while the IC50 value after NAC treatment was significantly lower than that of the 10% CSE group(P <0.01),both in PC-9 and A549 cells.Western blot detected that the expression of p Y1068,pY1173 and p-AKT in 10% CSE group was significantly higher than the control group,and there was no significance between the 10%CSE group and the10%CSE+Gefitinib group.While compared with 10% CSE group and 10%CSE + Gefitinib group,EGFR / AKT phosphorylated protein expressions was significantly reduced in 10%CSE+NAC+Gefitinib group.Laser scanning confocal microscope and flow cytometry showed that the expressions of reactive oxygen species were significantly increased after exposure to tobacco extract,while NAC could decrease the intracellular ROS levels induced by CSE.The apoptosis rates of control group,10% CSE group and 10% CSE +NAC group were(6.24 ± 0.22)%,(7.51 ± 1.11)%,(19.46±3.79)% in PC-9cells while(4.71 ± 1.27)%,(5.34 ± 2.91)%,(8.83 ± 1.54)% in A549 cells,respectively.The apoptotic rate of the control group was not different from the 10% CSE group(P>0.05).Compared with 10% CSE group,the apoptotic rate of 10% CSE +NAC group was significantly increased(P<0.05)in PC-9 cells,while in A549 cells,the two groups were not significantly different(P> 0.05).Conclusion: 1.CSE could induce gefitinib resistance via stimulating ROS production,activating EGFR / AKT signaling pathway.2.NAC could reverse the CSE-induced EGFR-TKI resistance,the specific mechanism might be related to inhibiting the CSE-induced intracellular ROS levels,EGFR-related signaling pathway activation.Alternatively,combined NAC with EGFR-TKIs to treat EGFR mutated NSCLC patients with smoking history may be a potential choice in clinical setting.