| Aims:Despite a dramatic initial response to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) Gefitinib and Erlotinib, the majority of patients with non-small cell lung cancer (NSCLC) with EGFR-activating mutations develop resistance, usually within one year of treatment. Therefore, there is an urgent need to elucidate the underlying mechanisms of resistance in NSCLC to overcome this obstacle. Recent studies suggest that mechanisms of resistance to EGFR-TKIs are occurrence of T790M, the mutation of PI3K, KRAS, BRAF gene in the EGFR downstream pathway, c-Met amplification, HGF expression, EMT and the SCLC type conversion. However, although the causes of resistance have been investigated, in more than 30% of patients with resistance to EGFR-TKI treatment, the mechanisms remain unknown. Malignant tumor cells exhibit considerably different metabolism compared to normal cells. However, the mechanism responsible for EGFR-TKI on the metabolic level remains unclear.Methods:In this study, we chose three NSCLC cell lines with different EGFR status, A549 (Wild type), PC-9 (sensitive mutant 19DEL), H1975 (L858R and T790M secondary mutant). The A549, PC-9 and H1975 state of EGFR mutations were confirmed by DNA direct sequencing. Drug-resistant strains, A549/IR, PC-9/IR and H1975/IR, were induced by domestic EGFR-TKI (Icotinib) in small doses increasing concentration gradient method in vitro. Drug resistance and resistant stability was detected by MTT assay; the changes of morphology were observed by light microscopy; the T790M mutation of PC-9/IR cell lines was detected by DNA sequencing; the expression of EGFR and its bypass signal molecules were evaluated by RT-PCR and Western blot. Mice transplanted tumor model was performed to investigate its proliferation ability in vivo. The samples of parental cells and resistant cells (A549, PC-9, and H1975; A549/IR, PC-9/IR and H1975/IR) were also collected and different metabolites between parental cells and resistant cells were analyzed using nuclear magnetic resonance analysis. The different metabolites were analyzed by PCA analysis and PLS-DA analysis to the distinguish parent cells from resistance cells and significant metabolites were analyzed with VIP scores. Analyze and conclude the different metabolites to screen the new drug target. Then to test the impact of new drug target for PC-9/IR cells growth and proliferation and the expression of proteins associated with new drug target in PC-9/IR cells were detected.Results:A549/IR and H1975/IR were acquired through 8 months and PC-9/IR cell line was developed after about 6 months. The drug resistance of A549/IR, H1975/IR and PC-9/IR were detected by MTT. The IC50 of PC-9/IR was 21.49μmol/L, compared to 1.32μmol/L of its parental cell line, which showed an increased resistance to Icotinib.There was no T790M mutation occurred to PC-9/IR cell lines. Furthermore, the relative expression of EGFR、PI3K、AKT、STAT3 and c-Met in the PC-9/IR cells was 12.09、5.73、3.72、7.41 and 5.31 folds higher than that in the parental cells. The phosphorylation of AKT and STAT3 can’t be blocked in the PC-9/IR cells by Icotinib and the expression of the muti-drug resistance protein ABCG2 is higher in PC-9/IR cells; In consider of the higher expression of c-Met and downstream molecules in PC-9/IR resistant cells and the c-Met inhibitor (Crizotinib) can inhibit the proliferation of PC-9/IR cells in vitro and in vivo effectively, we presume that c-Met bypass signaling pathway was activated in the PC-9/IR cells and responsible for the Icotinib resistance.The drug resistancy was stable in 2μmol/L Icotinib for four months. The metabolic footprints of A549/IR, H1975/IR, PC-9/IR, A549, PC-9 and H1975 were obtained by nuclear magnetic resonance (NMR) analysis. We had detected six major categories including 62 metabolites and their concentration. These metabolites included amino acids and their derivatives, organic acids, sugars, alcohols, nucleotides components and other types of metabolites. The parental cells and resistant cells can be separated, and its metabolic profile are different. Differences in metabolic traits between parent cells and resistant cells were observed using PCA and PLS-DA multivariate statistical analysis, and fifteen significant differences metabolites (VIP value>1) were found. The different metabolites between PC-9 and PC-9/IR cells is glutamate, acetate, uric acid, choline and alanine; the different metabolites between A549 and A549/IR cells is glutamic acid, glutamine acid, lactic acid, aspartic acid and glutathione; The different metabolites between H1975 and H1975/IR cells is lactic acid, glutamic acid, taurine, acetate and proline.The growth of PC-9/IR cells was depended on glutamine, and the glutamine inhibitor Compound 968 can inhibit the proliferation of PC-9/IR cells in a dose dependent manner. The c-Myc protein, which can promote the expression of glutaminase, was expressed higher in PC-9/IR cells and Icotinib couldn’t inhibit the expression of c-Myc in PC-9/IR cells.Conclusions:We established three Icotinib resistant cell lines A549/IR, H1975/IR and PC9/IR; the activation of c-Met pathway, the increased expression of ABCG2 and the changes of glutamine metabolism are associated with the resistance of PC-9/IR. Glutaminase inhibitors can inhibit the proliferation of PC-9/IR in a dose dependent manner, suggested that Glutamine metabolic pathway maybe a potential target for the EGFR-TKI resistance cells. |
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