The Mechanism Of Gefitinib Resistance Induced By Hepatocyte Growth Factor In Sensitive Non-small Lung Cancer Cells

Lung cancer is one of the most prevalen tmalignancies and theleading cause of cancer-related death worldwide.Non-small-cell lung cancer (NSCLC) accounts for nearly 80% of lung cancer cases.The median survival of metastatic NSCLC is 8-10months when treated with the most active combination of conventional chemotherapeutic agents.Recent therapeutic strategies for NSCLC have focused on the development of molecular targeted therapies.Epidermal growth factor receptor (EGFR) is a member of a family of closely related growth factor receptor tyrosine kinases.As EGFR is expressed in the majority of cases of EGFR is an attractive target for the development of therapeutic agents.The small-molecule EGFR tyrosine kinase inhibitors (gefitinib) have been evaluated in clinical trials for patients with NSCLC.Gefitinib is a antitumour drug which targeted EGFR, is small molecular EGFR tyrosine kinase inhibitor(TKI.), sensitive patients with gefitinib was EGFR-activating mutation,It has satisfactory therapeutic effects in clinical practice with treatment patients of non-small lung cancer (NSCLC). lung cancer with epidermal growth factor receptor (EGFR)-activating mutations responds favorably to the EGFR tyrosin kinase inhibitors gefitinib. However, not all the patients with EGFR-activating mutation was sure to sensitive to gefitinib,25%-30% of patients with EGFR-activating mutations show intrinsic resistance,and the responders invariably acquire resistance to gefitinib.To overcome the intrinsic and acquired resistance to EGFR tyrosine kinase inhibitors,it is necessary to clarify the molecular mechanisms of the resistance.The mechanism of gefitinib resistance of these patients with EGFR-activating mutation is not cleared.Recently,amplificationof MET proto-oncogene,which contribute to acquired resistance to EGFR tyrosine kinase inhibitors have been reported. On the otherhand,MET amplification has been shown to restore the phosphatidylinositol. MET amplification are found in 20%-50% of patients acquiring resistance to EGFR tyrosine kinase inhibitors.However,the mechanis of intrinsic resistance and acquired resistance are still unknown.The MET gene encodes a high-affinity receptor for hepatocyte growth factor.originally,cloned as a mitogenic protein for hepatocytes, specifically activates MET receptor tyrosine kinase and induces pleiotropic biological effects in a wide variety of cells, including mitogenic, motogenic, mophogenic, and anti apoptotic activities。Hepatocyte growth factor (HGF) is a multifunctional agent from stromal secrete which promote proliferation and movement of multy cells in vitro.Tthe document reported tissue of lung cancer can secret HGF and HGF can promote proliferation and metastasis and invasion of tumor cells. As HGF expressed in lung cancer cells and stromal cells,we investigated Whether HGF is involved in gefitinib resistance of lung adenocacinoma cells with EGFR-activating mutations.commonly,there ware two kinds of cells to gefitinb-resistance, respectively EGFR-activating mutation and EGFR-wild type.we selected PC-9 with EGFR-activating mutation and H292 with wild type EGFR, these cells don't have relative factors of EGFR-T970M mutation or MET amplification or KRAS-activating mutation et al. we induced two cells with hepatocyte growth factor (HGF) and observed cell activity,cell apoptosis,cell cycle, express of EGFR protein and HGF-receptor protein of encoded by MET, research the other one-mechanism of gefitinib-resistance in NSCLC. it may confirm the systerm of HGF/c-Met as important target site to therapy NSCLC, provide rationale basis to possibly treat NSCLC of gefitinib-resistance in clinically by combination with c-Met inhibitor or HGF antagon explore mechanism of gefitinib resistance by examine expression of proteins.The proliferation of PC-9 and H292 cells was mesured by MTT assay, and IC50 of drug was calculated by graphic method; the proliferation of PC-9 and H292 cells was mesured by MTT assay and the curve of drug concentration-growth in exel document were painted.PC-9 and H292 cells were devided into 4 group,controul group, hepatocyte growth factor (HGF) group, gfitinib group and combine group (HGF+gefitinib).The survival of four cell's group was mesured by MTT assay; the cells stain by Annexin V-PE Apoptosis Detection Kit and Flow Cytometry Analysis Of Cell Kit, cell cycle distribution and apoptosis by flow cytometry, finally analyses of cell cycle by Multicycler 3.0 soft; quantitate the protein by Bradford method.The expression of c-Met,p-Met,p-EGFR protein were examined by western blotting.Get the as follows that experimental result by said methods:1. Both PC-9 and H292 cells were sensitive to gefitinib,the IC50 is 0.05±0.01μmol/L and 0.17±0.05μmol/L, respectively. gefitinib inhibited cell growth of PC-9 and H292 in a dose-dependent manner, both the inhibition ratio-concentration curve of gefitinib shifts right when induced with HGF (40ng/ml)2. HGF did not affect proliferation of PC-9 and H292 cells,higher rate of survival was present when treated with HGF (40ng/ml) and gefitinib than treated with gefitinib alone (P＜0.05);In addition,low concentration of HGF (20ng/ml) could enhance rate of survival (P＜0.05)3. HGF (40ng/m,20ng/ml) alone had no effect on induction of apoptosis of PC-9 cells and the rate of apoptosis showed no diferrence between HG group (40ng/m,20ng/ml) and G group (P>0.05).Gefitinib HGF (20ng/ml) alone could enhanced mitosis of PC-9 and H292 cells. However, the ratio of inhibing cell cycle (G1 phase) showed no diferrence between HG group and G group (P>0.05)4. HGF (40ng/ml) stimulated phosphorylation of Met in PC-9. In the presence of gefitinib,HGF slitely restored phosphorylation of Met in PC-9; HGF (40ng/ml) stimulated phosphorylation of EGFR in H292.Even in the presence of gefitinib, HGF evidently restored phosphorylation of EGFR in H292;5. HGF (40ng/ml) stimulated phosphorylation of Met in PC-9 and H292. In the presence of gefitinib,HGF slitely restored phosphorylation of EGFR in H292.Hint:1. HGF induced gefitinib resistance of PC-9 with EGFR-activating mutation and H292 cells with EGFR-wild type; 2. Cell apoptosis and cell cycle effection induced by HGF cell might not be related with the gefitinib resistance of of NSCLC with EGFR-activating mutation or with EGFR-wild type;3. HGF induced PC-9 and H292 cells enhancing phosphorylation of EGFR and Met,which may be inportent mechanism of gefitinib resistance.of of NSCLC with EGFR-activating mutation or with EGFR-wild type;4. HGF-induced phosphorylation of c-Met was inhibited by gefitinib in mutant EGFR cell line, but not down-regulated by gefitinib in wild type EGFR cell line. It might be related with EGFR gene type of NSCLC that gefitinib inhibited HGF-induced phosphorylation of c-Met;5. HGF-induced phosphorylation of EGFR was inhibited by gefitinib in mutant EGFR cell line, but not completely down-regulated by gefitinib in wild type EGFR cell line.It might be related with EGFR gene type of NSCLC that gefitinib inhibited HGF-induced phosphorylation of EGFR;6. HGF induced gefitinib resistance by activating phosphorylation of c-Met mainly, so systerm of HGF/c-Met might be target sites of treating NSCLC with EGFR;7. HGF-induced phosphorylation of c-Met is related with gefitinib-resistance, HGF-induced phosphorylation of c-Met was evidently inhibited by gefitinib in mutant EGFR cell line, so gefitinib combining with HGF antagonist may be more effective in treating NSCLC with mutant EGFR; but HGF-induced phosphorylation of c-Met wasn't inhibited byefitinib in wild type EGFR cell line, so gefitinib combining with c-Met inhibitor may be more effective in treating NSCLC with wild type EGFR.These provide rationale reference to guide therapy of NSCLC in clinically.