【作者】 安昌善；

【导师】 金元哲；

【作者基本信息】 延边大学 ， 生理学， 2010， 博士

【摘要】 吉非替尼是一种靶向抑制表皮生长因子受体(Epidermel Growth Factor Receptor, EGFR)的抗癌药物,为临床上应用的小分子EGFR酪氨酸激酶抑制剂(tyrosine kinase inhibitor, TKI),可以靶向抑制非小细胞肺癌(non-small lung cancer, NSCLC)表达的EGFR,在治疗NSCLC的临床实践中取得了较满意的疗效。由于吉非替尼对特殊患者的选择性,实际上少部分NSCLC患者对吉非替尼有明显的疗效,大部分患者则无效或疗效差。吉非替尼敏感的患者主要是存在EGFR基因的突变,但EGFR基因突变的患者中不一定都对吉非替尼敏感。这是由于约25%-30%的EGFR基因突变者对吉非替尼原发性或获得性耐药,这种EGFR基因突变型患者对吉非替尼的耐药机制不十分清楚。肝细胞生长因子(hepatocyte growth factor, HGF)是一种由间质细胞分泌的多功能因子,在体外促进多种细胞增殖和运动。部分肿瘤组织能表达HGF,与肿瘤细胞增殖、转移、侵袭有关。新近研究显示,肺癌组织中肿瘤编码基因MET扩增与对吉非替尼获得性耐药有关。有报道HGF诱导乳腺癌细胞对抗吉非替尼的生长抑制作用。对吉非替尼耐药的NSCLC细胞的EGFR基因型分两类,分别为EGFR突变型和EGFR野生型。我们选择了不存在EGFRT970M突变或MET扩增或KRAS突变等吉非替尼耐药相关因素,而且对吉非替尼敏感的NSCLC细胞EGFR突变型PC-9和EGFR野生型H292进行了研究。这两株细胞不存在EGFRT970M突变或MET扩增或KRAS突变等吉非替尼耐药相关因素。用HGF诱导这两株细胞,通过检测细胞增殖、周期及凋亡,观察是否对吉非替尼耐药,以及通过免疫印迹检测EGFR蛋白及MET编码的HGF的受体蛋白的表达,研究吉非替尼对非小细胞肺癌耐药的另一个途径的耐药机制,证实HGF/c-Met系统可作为治疗吉非替尼耐药的NSCLC的靶向位点,为临床提供吉非替尼联合c-Met抑制剂或HGF拮抗剂可能有效治疗耐药的NSCLC的理论依据。MTT法检测PC-9和H292细胞增殖,作图法计算出药物半数抑制浓度(IC50); MTT法检测HGF诱导的PC-9和H292细胞增殖,利用exel文档画出药物浓度-生长曲线；实验分为4组：对照组(O)、肝细胞生长因子组(H)、吉非替尼组(G)、肝细胞生长因子+吉非替尼组(HG)。MTT法检测四组细胞存活率；细胞凋亡试剂盒(Annexin V/PE)染色和细胞周期试剂盒(PI)染色,利用流式细胞仪检测细胞凋亡和周期以及最后利用Multicycler 3.0软件分析细胞周期；比色法(Bradford法)为蛋白定量,利用蛋白质免疫印迹(Western bloting)技术检测细胞中c-Met、p-Met、p-EGFR蛋白的表达。通过上述方法得到如下实验结果：1、PC-9和H292均为吉非替尼敏感株,IC50分别为0.05±0.01μmol/L和0.17±0.05μmol/L。吉非替尼对PC-9和H292的生长抑制作用呈浓度依赖性,HGF (40ng/ml)诱导的吉非替尼浓度-存活率曲线均往右移,以H292的表现最为明显;2、HGF(20ng/ml或40ng/ml)没有促进PC-9和H292增殖,但HGF(40ng/ml)和吉非替尼处理组(HG)与单纯吉非替尼处理组(G)相比有更高的存活率(P<0.05),而且低浓度的HGF(20ng/ml)也能明显提高细胞的存活率(P<0.05)；3、HGF (20ng/ml或40ng/ml)没有降低PC-9的凋亡率,而且吉非替尼促进HGF诱导(HG)的凋亡率与单用吉非替尼(G)比较没有差异(P>0.05)。HGF (20ng/ml)能促进PC-9和H292的有丝分裂,但吉非替尼阻滞HGF诱导(HG)的G1期细胞周期比例与单用吉非替尼(G)比较没有差异(P>0.05)；4、HGF (40ng/ml)诱导PC-9和H292增加表达c-Met的磷酸化。吉非替尼处理HGF (40ng/ml)诱导的PC-9和H292,H292有明显的p-Met的表达。吉非替尼能明显抑制HGF (40ng/ml)诱导PC-9增加表达的p-Met,但不能抑制HGF(40ng/ml)诱导H292增加表达的p-Met;5、HGF (40ng/ml)诱导PC-9和H292增加表达EGFR的磷酸化。吉非替尼处理HGF (40ng/ml)诱导的PC-9和H292,H292仍有p-EGFR的表达,而PC-9没有p-EGFR的表达。吉非替尼能抑制HGF (40ng/ml)诱导PC-9增加表达的p-EGFR,但不能完全抑制HGF (40ng/ml)诱导H292增加表达的p-EGFR。提示：1、HGF可诱导敏感肺癌细胞PC-9和H292对吉非替尼耐药；2、HGF诱导细胞凋亡及细胞周期影响可能与HGF诱导吉非替尼耐药无关；3、HGF诱导PC-9和H292表达c-Met磷酸化和反式激活EGFR的磷酸化,可能是敏感肺癌细胞对吉非替尼耐药的重要机制；4、吉非替尼能抑制HGF诱导EGFR突变型细胞表达的c-Met的磷酸化,不能抑制HGF诱导EGFR野生型细胞表达的c-Met的磷酸化。吉非替尼抑制HGF诱导c-Met的磷酸化可能与肺癌细胞基因类型相关；5、吉非替尼完全抑制HGF诱导EGFR突变型细胞EGFR的磷酸化,不完全抑制EGFR野生型细胞EGFR的磷酸化。吉非替尼抑制HGF诱导EGFR的磷酸化可能与肺癌细胞基因类型有一定相关性；6、HGF主要通过激活c-Met磷酸化诱导耐药,所以HGF/c-Met系统可作为治疗吉非替尼耐药的NSCLC的重要靶向位点；7、HGF诱导c-Met磷酸化可能与吉非替尼原发性耐药相关,对EGFR突变的细胞来说吉非替尼能够阻断HGF诱导的EGFR和c-Met的磷酸化,所以同时应用HGF拮抗剂可能效果更佳；而对EGFR野生型的细胞来说吉非替尼不能阻断HGF诱导的c-Met的磷酸化,所以最好同时应用c-Met抑制剂或HGF拮抗剂可能带来更好的治疗效果,这为临床指导治疗NSCLC提供理论参考。更多还原

【Abstract】 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.更多还原