| Lung cancer is a common malignancy,with morbidity and mortality increasing in recent years,which has become China's largest cancer.According to the National Cancer Registry in 2014 statistics found that,in the new malignant tumors,lung cancer has occupied the first place,accounting for about of all tumors,the mortality rate accounted for about 24.87%of all deaths.Non-small cell lung cancer accounts for about 80?85%of lung cancer.Most patients were found,more in the late,lost the chance of surgery,therefore,the majority of patients with lung cancer need chemotherapy,chemotherapy for platinum based two drug combination program.However,the clinical application of traditional chemotherapy drugs is limited due to its short survival time,poor specificity,side effects,and poor prognosis.The discovery of targeted drugs for the treatment of non-small cell lung cancer dawn.Epidermal growth factor receptor tyrosine kinase inhibitors as a combined factor receptor tyrosine kinase domain of adenosine and epidermal growth is the most widely used treatment of targeted molecular drugs(gefitinib,erlotinib),the drug can be activated and transfer the relevant signal,the cancer cells proliferation was inhibited,open cell apoptosis.The high expression of EGFR is the basis of application of EGFR-TKI,and the efficacy of TKI is closely related to EGFR mutation.Study on EGFR-TKI sensitive non-small cell lung cancer patients often have EGFR21,19,18 exon gene mutation,and these mutations are more common in female,Asian,non smoking and histological types of adenocarcinoma patients with non-small cell lung cancer,tumor cells harboring EGFR activating mutations in patients with EGFR-TKI treatment efficiency can reach more than 70%,However,the presence of EGFR-TKI or drug resistance,leading to the failure of drugs,treatment failure,the mechanism is not very clear.EGFR two mutation(T790M)and MET gene amplification is the two major molecular mechanisms of acquired resistance to EGFR-TKI at present,other possible mechanisms,tyrosine phosphatase gene deletion,over expression of insulin-like growth factor 1 receptor protein,hepatocyte growth factor(hepatocyte growth,factor,HGF)high expression.HGF is a fibroblast derived factor,which is significantly increased in the serum of patients with non-small cell lung cancer,and is closely related to the invasion of the tumor.HGF plays an important role in the binding of c-Met,and the abnormal HGF/c-Met signaling pathway is related to tumor growth,adhesion,metastasis and apoptosis,HGF induces resistance to EGFR-TKI in non-small cell lung cancer and may be related to activation of c-Met.So this study ideas first in vitro to investigate whether c-Met and its downstream signaling pathway involved in HGF induced by different genotypes of non-small cell lung cancer cells to erlotinib resistance.Followed by the establishment of nude mice model induced by HGF in vivo,explore different genotypes of non-small cell lung cancer cells to erlotinib resistance and resistance mechanisms.Therefore,this study is divided into the following two parts:Part oneThe Mechanism of Erlotinib Resistance Induced by Hepatocyte Growth Factorin Sensitive Non-small Cell Lung Cancer Cells in Vitro ObjectiveTo explore whether HGF induce erlotinib resistace in different EGFR gene types of NSCLC in vitro and the mechanism of HGF-induce erlotinib resistance in different EGFR gene types of NSCLC cells.MethodsEGFR-mutated NSCLC PC9,PC9/Rcells and EGFR-wild type NSCLC H292,A549 cells were chosen and treated with HGF,erlotinibor HGF plus erlotinib.They were divided into four groups:no drug control group(C group),HGF group(H group),erlotinib treatment group(E group),HGF+ plus erlotinib treatment group(HE group).Cell viability was assessed by MTT assays,cell appotosis and cell cycle progression by flow cytometyand protein contents of c-Met and its downstream pathway by Western blotting.RNA infection of different NSCLC cells by lentivirus packaging.The infected lung cancer cells were collected and lysed,and total RNA was extracted.The expression of c-Met-mRNA and protein was detected by RT-PCR.ResultsIt showed that the c-Met and its phosphorylated protein of PC9 andA549 cells were low or no expression in normal condition,and the expression of c-Met and phosphorylated protein in H292 and PC/R cells was high.With erlotinib concentration increased,H292,PC9 and cell growth inhibition is also enhanced.The concentration-survival curve notably shifted to the right when induced by HGF.In H292,PC9,A549 cells studied,the apoptosis rate in HE group was lower than that in E group(P<0.05),the proportion of cells at GO/G1 phase was significantly higher in E group than in C group(P<0.05).There was no significant difference in the proportion of G0/G1 phase of PC9 and PC9/-R cells between HE group and E group(P>0.05).The proportion of G0/G1 redused significantly(P<0.05)in the H group of A549 than control group and the the ratio of S increased significantly.(P<0.05).The proportion of G0/G1 phase in HE group of H292 cell was significantly lower than that in group E,and the ratio of S and G2/M increased significantly(P<0.05).It showed that HGF could stimulate the phosphorylation of c-Met and the expression of downstream channel protein in PC9,H292,PC9/R and A549 cells.In PC9,H292,A549cells the expression of p-Met,p-Akt,p-Stat3 and p-Erkl/2 in HE group were higher than those in E group,while in PC9/R cell,there was no significant increasing.The expression of c-Met-mRNA and c-Met protein was inhibited in PC9 and H292 infected with virus.Induced by HGF infected PC9 and H292 cells,the erlotinib drug concentration cell survival curve can be restored to the original level.It showed that PC9 and H292,which were infected by virus,did not show the activation of c-Met,Stat3,Akt,Erkl/2 phosphorylation protein in the cells after HGF stimulation.Compared with E group,Stat3,Akt and Erkl/2 phosphorylated proteins in HE group and H292 group were significantly inhibited by PC9.Conclusion1.HGF induces erlotinib resistance in different genetypes of NSCLC cell in vitro.2.HGF-induced phosphorylation of met and its downstream signaling pathway may be an important mechanism of erlotinib resistance in different genetypes of NSCLC cell.3.HGF/c-Met system can be used as a target site for treatment of EGFR-TKI resistant NSCLC and offer the clinical application of EGFR-TKI combined with c-Met inhibitor or HGF antagonist to effectively overcome the resistance of different EGFR genetype NSCLC to EGFR-TKI.Part two The Mechanism of Erlotinib Resistance Induced by Hepatocyte Growth Factor in Sensitive Non-small Cell Lung Cancer Cells in vivo ObjectiveTo explore whether HGF induce erlotinib resistance and whether c-Met and its downstream signaling pathways participate in erlotinib resisitance induced by HGF in different EGFR gene types of NSCLC cells in vivo.MethodsNSCLC cell lines with different EGFR genes(PC9:EGFR-Mutation type,H292:EGFR-Wild type)and Human embryonic lung fibroblasts(MRC-5)were selected.The HGF secreted by H292,PC9 and MRC-5,cells in cell culture supernatants was quantified by ELISA.PC9 and H292 cells were induced by MRC-5 cell culture supernatant,the expression of c-Met and downstream signaling proteins were examined by Western blotting.56 female and SPF BALB/c nude mice were randomly divided into 8 groups,with each group containing 7.In PC9 cell induced by the model,the control group(C group)and Erlotinib treatment group(group E)inoculated subcutaneously in PC9 cell suspension and MRC-5 induced group(H group),MRC-5 and Erlotinib treatment group(group HE)inoculated subcutaneously in PC9 + MR C-5 cell suspension.When the tumor diameter reached 4 mm,Group C and group H were treated with 0.9%Sodium Chloride Solution while E group and HE group were treated with erlotinib5 days a week for a period of 4 weeks.In H292 cell model,C group,E group were inoculated subcutane-ously with H292 cell in nude mice while Group H and group HE were inoculated subcutaneously with H292+MRC-5 cells in nude mice.Tumor volume was measured every 3-4 days and mice were euthanized at after dosing.To compare the weight of transplanted tumor in PC9 and H292 cell model and the tumor inhibition rate was calculated.The expressions of c-Met and downstream signaling proteins in nude mice graft tumor tissues were determined via immunohistochemistry.ResultsHGF was not detected in PC9 and H292 cells culture supernatants and the concentration of HGF in the supernatant of MRC-5 cells was(1262.07±89.78)pg/ml.Western blotting method results showed that HGF in the MRC-5 cell culture supernatant could stimulate the expression of p-Met,p-Akt,p-Stat3 andp-Erkl/2.In PC9 cell induced model:there was no significant difference in the tumor volume between 4th and 7th day.(P>0.05);there was significant difference in the tumor volume between 11th,14th,18th,21th,25th day,(P<0.05);thetumor volume in E group was less than C group in 11th,14th,18th,21th,25th day,there was significant difference(P<0.05);the tumor volume in HE group was less than H group in 18th,21th,25th day,there was significant difference(P<0.05);the tumor volume in HE group was large than E group in 11th,14th,18th,21th,25th day,there was significant difference(P<0.05).In H292 cell induced model:there was no significant difference in the tumor volume between 4 and 7 days(P>0.05);there was significant difference in the tumor volume between 11th,14th,18th,21th,25thday.(P<0.05);the tumor volume in E group was less than C group in 11th,14th,18th,21th,25th day,there was signifi cant difference(P<0.05);the tumor volume in HE group was less than H group in 11th,14th,18th,21th,25th day,there was significant difference(P<0.05);the tumor volume in HE group was large than E group in 11th,14th,18th,21th,25th day,there was significant difference(P<0.05).InPC9 cell induced model:C group,H group,E group,HE group transplanted tumor weight were(680.31±373.38)?(804.84±492.36)?(108.89±83.76)?(361.19± 172.08)mg.In H292 cell induced model:C group,H group,E group,HE group transplanted tumor weight were(920.43±121.72)?(979.14±175.65)?(389.29±109.40)?(564.00±176.08)mg.Erlotinib on PC9 cells of E group and HE group,the inhibition rates were 83.99%and 46.91%of H292 cells in E group and HE group the inhibition rates were 57.71%and 38.72%.The weight of transplanted tumor in group E was less than C group(P<0.05);the weight of transplanted tumor in HE group was less than that of H group,which was higher than that in group E(P<0.05)in PC9 cell induced model.The weight of transplanted tumor in group E was less than C group(P<0.05);the weight of transplanted tumor in HE group was less than that of H group,which was higher than that in group E(P<0.05)in H292 cell induced model.c-Met and p-Met were localized in the cell membrane and cytoplasm.In PC9,H292 cell induction model C group,H group,E group,HE group,the expression level of c-Met,the difference was not statistically significant(P>0.05).and the expression level of p-Met in H group and HE group was higher than that in group C and group E(P<0.05).Stat3 was localized in cytoplasm and p-Stat3 was localized in nucleus.In PC9,H292 cell induction model:C group,H group,E group,HE group,the expression level of Stat3,the difference was not statistically significant(P>0.05),the expression level of p-Stat3 in H group and HE group was higher than that in group C and group E(P<0.05).Akt and p-Akt were located in cytoplasm.In PC9,induced H292 cell model:C group,H group,E group,HE group,Akt expression level,there were no significant differences(P>0.05);H group,HE group,p-Akt expression levels were higher than that of C group and E group(P<0.05).Erkl/2 was localized in cytoplasm and p-Erkl/2 was localized in nucleus.In PC9,induced H292 cell model:C group,H group,E group,HE group,Erkl/2 expression level,there were no significant differences(P>0.05);the expression level of p-Erkl/2 in H group and HE group were higher than that of C group and E group(P<0.05).Conclusion1.HGF induce erlotinib resistance in different genetypes of NSCLC cell in vivo.2.Met and its downstream signaling proteins phosphorylation induced by HGF,maybe an important mechanism of erlotinib resistance in different ECGF gene types of NSCLC cells in vivo.3.HGF/c-Met system is another important way of NSCLC resistance to EGFR-TKI,and offer the clinical application of EGFR-TKI combined with c-Met inhibitor or HGF antagonist to effectively overcome the resistance of different EGFR genetype NSCLC to EGFR-TKI. |
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