The Mechanism Of Gefitinib Resistance Induced By Cigarette Smoke Extract In Lung Adenocarcinoma Cell Line PC-9 And Exploration Of Strategy In Overcoming Resistance

Objective The study aimed to explore the molecular basis of EGFR-TKI resistance induced by cigarette smoke extract and whether NAC could overcome CSE inducing gefitinib resistance in lung adenocarcinoma cell line PC-9 with EGFR mutation.MethodsPC-9 cells were exposed to different concentration of CSE for various times, and EMT makers were detected to screen the proper exposure time and working concentration of CSE to do the next experiments. Observing the impacts of CSE exposure(CSE group),CSE exposure after Src inhibitor PP2 pretreatment(PP2+10%CSE group) or CSE exposure after NAC pretreatment(NAC+10%CSE group) on the protein and m RNA expression of EMT makers and EMT associated signaling pathway, gefitinib sensitivity, the morphology, the invasion and migration ability of PC-9 cells.q RT-PCR was applied to detect the changes of E-cadherin and Vimentin m RNA expression levels. Src, phosphorylation of Src at Tyr416, E-cadherin and Vimentin protein expression levels were determined by Western blot. Immunofluorescence staining was done to detect the morphology, the location and semiquantitative expression of E-cadherin and Vimentin. CCK-8 assays were used to evaluate the sensitivity of different cell lines to gefitinib and the non-toxic dosage of PC-9 cells to NAC. The invasion ability and migration ability of PC-9 cells under different treatment were assessed by matrigel invasion assays and wound healing assays, respectively.Resultsq RT-PCR showed CSE down-regulated the m RNA expression of E-cadherin and up-regulated that of Vimentin in PC-9 cells in time and concentration dependent manners, so 10%CSE and 72 h were picked to do the following study. Compared with the control group, the m RNA expression of E-cadherin was significantly decreased(p<0.001) and that of Vimentin was significantly increased(p<0.001) in the 10%CSE group; compared with the 10%CSE group, the m RNA expression of E-cadherin in the PP2+10%CSE group(p<0.01) and NAC+10%CSE group(p<0.01) were significantly increased, and that of Vimentin in the PP2+10%CSE group(p<0.001) and NAC+10%CSE group(p<0.001) was significantly decreased.Western blot demonstrated similar results with q RT-PCR about the protein expression of E-cadherin and Vimentin; and 10%CSE increased Src phosphorylation, which was supressed by PP2 or NAC pretreatment.Immunofluoresence showed similar results with q RT-PCR and Western blot about the expression of E-cadherin and Vimentin. Vimentin was expressed in the cytoplasm and E-cadherin was expressed in the cytomembrane. Meanwhile, PC-9 cells transformed from the epithelial shape to the spindle-shape upon CSE exposure; the spindle-shape of CSE-exposure PC-9 cells restored their epithelial shape when adopting PP2 or NAC pretreatment.CCK-8 assays displayed that the non-toxic dosage of PC-9 cells to NAC was 5m M. The IC50 value of PC-9 cells for gefitinib in the control group, 10%CSE group, PP2+10%CSE group, NAC+10%CSE group was(0.0566±0.0060)μM,(0.8340±0.2780)μM,(0.1380±0.0878)μM,(0.1650±0.0242)μM, respectively. The IC50 value of PC-9 cells for gefitinib in the 10%CSE group was much higher than that of the control group(p<0.01), which were much lower in the PP2+10%CSE group(p<0.05) and NAC+10%CSE group(p<0.05) than that of the 10%CSE group.Transwell assays showed the number of penetrated cells in the 10%CSE group was significantly higher than that of the control group(p<0.0001), which were significantly lower in the PP2+10%CSE group(p<0.0001) and NAC+10%CSE group(p<0.0001)than that of the 10%CSE group.Wound healing assays showed the area of wounded region lacking cells in the 10%CSE group is smaller than that of the control group;and the scrape wound recovered significantly slower in the PP2+10%CSE and NAC+10%CSE group than that of the 10%CSE group.Conclusion1. CSE exposure induced EMT in PC-9 cells in time and concentration dependent manners;2. CSE exposure induced gefitinib resistance in PC-9 cells;3. Src activation plays an critical role in CSE inducing EMT of PC-9 cells;4. EMT and Src activation are the main molecular basis of gefitinib resistance induced by CSE in PC-9 cells;5. NAC may alleviate smoking induced gefitinib resistance through inhibiting Src activation and EMT reversal.