Metformin reverses nicotine-induced gefitinib resistance by epithelial-mesenchymal transition

Abstract:

Objective：To investigate the molecular basis for nicotine（NIC） 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 divided into 4 groups，namely control group，NIC group，MET group and MET+NIC group. EMT markers were detected by quantificational real-time polymerase chain reaction（qRT-PCR） and Western blot. Cell counting Kit-8（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. Results：Nicotine in－duced 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 μmol/L nicotine for 10 h，so further experi－ments were completed under this condition. CCK-8 displayed reduction of gefitinib sensitivity of PC-9 cells in the NIC group com－pared with the control group，especially with a gefitinib concentration of 0.05 μmol/L（F=82.005，P=0.000，P=0.000）. In invasion as－says，more penetrating cells were observed in the NIC group than in the control group[（15.70±1.53） vs. （32.70±2.08），F=75.406，P=0.000，P=0.000]. Migration assays presented similar results [（102.70±7.02） vs. （18.70±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. Cells in the MET+NIC group displayed higher sensitivity to gifitinib，which is statis－tically significant with a gifitinib concentration of 0.05，0.10，0.50，1.00 and 5.00 μmol/L. Fewer cells penetrated the Matrigel in the NIC+MET group[（17.00±2.00） vs. （32.70±2.08），F=75.406，P=0.000，P=0.000] and migration assays presented similar results [（51.70±5.03） vs. （102.70±7.02），F=204.038，P=0.000，P=0.000]. 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. Conclusion：Nicotine induced EGFR-TKI resistance in PC-9 cells by EMT in non-small cell lung cancer，which could be reversed by metformin.