Metformin Reverses Nicotine-induced Gefitinib Resistance By Epithelial-mesenchymal Transition

Objective To investigate the molecular basis for nicotine to induce resistance of epithelial growth factor receptor tyrosine kinase inhibitors(EGFR-TKI)in EGFR-mutated PC-9 cells and the effect of the Metformin in the process.Methods PC-9 cells were diviced into different groups,namely control group,NIC,MET group and MET+NICgroup,SB431542 group,SB+NIC group..EMT markers were detected by qRT-PCR and western blot.CCK-8 assays were used to evaluate the sensibility to gefitinib of different group cells.The invasion ability and migration ability of different groups were detected by Transwell assay.ResultsNicotine induced epithelial-mesenchymal transition(EMT)of PC-9 cells in a time and concentration dependent manner.Down-regulation of E-cadherin and up-regulation of vimentin were most significant with a treatment of 10 ?mol/L nicotine for 10 h,so further experiments were completed under this condition.CCK-8 displayed reduction of gefitinib sensitivity of PC-9 cells in the NIC group compared with the control group,especially with a gefitinib concentration of 0.05 ?mol/L(F=82.005,P =0.000,P=0.000).SB431542 show same results.In invasion assays,more penetrating cells were observed in the NIC group than in the control group [(15.7±1.53)vs.(32.7±2.08),F=75.406,P =0.000,P=0.000].Migration assays presented similar results.[(102.7±7.02)vs.(18.7±2.08),F=204.038,P=0.000,P=0.000].E-cadherin mRNA expression decreased [(0.932±0.100)vs.(0.459±0.024),F=25.924,P =0.000,P=0.000)]and Vimentin mRNA expression increased [(1.200±0.200)vs.(1.973±0.129),F=20.998,P =0.000,P=0.000] in the NIC group compared with the control group,and Western blot showed the same results,p-Smad2 expression deceased.SB431542+Nic show same resuits.Cells in the MET+NIC group displayed higher sensitivity to gefitinib,which is statistically significant with a gifitinib concentration of 0.05?mol/L,0.1?mol/L,0.5?mol/L,1?mol/L and 5?mol/L.Cells in the SB431542+Nic group displayed the same sensitivity to gefitinib.Less cells penetrated the Matrigel in the NIC+MET group[(17.0±2.00)vs.(32.7±2.08),F=75.406,P =0.000,P=0.000],and migration assays presented similar results[((51.7±5.03)vs.(102.7±7.02),F=204.038,P =0.000,P=0.000].Less cells penetrated the Matrigel in the SB431542+Nic group compared with the NIC group.E-cadherin mRNA expression was upregulated [(0.459±0.024)vs.(0.838±0.058),F=25.924,P =0.000,P=0.000)],and vimentin mRNA expression was downregulated [(1.973±0.129)vs.(1.467±0.115),F=20.998,P =0.000,P=0.003)] in the NIC+MET group compared with the NIC group.Western blot showed the same results.E-cadherin m RNA was elevated and Vimentin m RNA decreased in the SB431542+NIC group compared with that in the NIC group.ConclusionNicotine induced EGFR-TKI resistance in pc-9 cells by EMT in non-small cell lung cancer,which could be reversed by metformin.and SB431542.