The Mechanism Of Erlotinib Resistence Induced By Hepatocyte Growth Factor In Non-small Cell Lung Cancer Cells Line In Vivo

Objective:The previous studies have shown that sensitive non-small cell lung cancer(NSCLC)cells can produce drug resistance on EGFR-TKIs,which were induced by hepatocyte growth factor(HGF)in vitro.C-Met can be stimulated by HGF,and also be induced its phosphorylation.The aim of this study was to investigate the drug resistance and intrinsic mechanism HGF induced in NSCLC models(sensitive type)constructed in nude mice mediated by Erlotinib.Methods:Both the NSCLC cells line PC9 which was sensitive and EGFR mutated,and MRC-5(human embryonic lung fibroblasts)were selected as experimental cells in our study in vitro.The concentration of HGF was measured by ELISA,and the distribution of HGF in MRC-5 cells and PC9 was compared in the cells test.IC50 of PC9 cells under the influence of erlotinib was measured,Then added PC9 cells to MRC-5 medium containing HGF.MTT colorimetric method was used to calculate the concentration of PC9 cells in the presence of erlotinib PC9 cell viability,while calculating the survival rate of viable cells.In addition,PC9 cells cultivated alone would compare with its' combination of MRC-5 supernatant which contained HGF,and the C-Met series channels were examined by Western Blot.Every 7 PC9 transplanted nude mice tumor models were chosen radomly and divided into four different groups.Both of them were females,4 weeks in size.This 4 groups called H group(HGF),C group(control),E group(erlotinib),and HE group(erlotinib+HGF).Observing the tumor tissue changes Regularly.Depicting the corresponding growth curve,measuring the weight of the tumor,calculating the tumor inhibition rate according to the corresponding formula.Finally,the changes of C-Met related pathways were observed by immunohistochemistry.Results:ELISA showed no HGF secretion in PC9 cell alone,and the concentration of HGF in MRC-5 supernatant was 1183.62±86.53 pg/mL.MTT showed that proliferation rate of PC9 with HGF was 78.92%under the influence of Erlotinib in ICso.Western Blot showed that HGF(secreted by MRC-5)could affect the activity of p-Met,p-Stat3,p-Erk1/2,p-Akt in PC9 when MRC-5 was mixed with PC9.And it convinced that HGF can be secreted by human embryonic lung f broblasts cells(MRC-5)in vitro,which can increase the survival rate of PC9 cells mediated by erlotinib,It can also stimulate the protein expression of C-Met series channels in this cells.In the model of PC9 transplanted tumor,the tumor volume in each group showed no significant difference in C and H group(P>0.05),but in E and HE group(P<0.01).The inhibition rates of Erlotinib on E and HE were 84.34%and 48.29%.The expression levels of P-met,p-Erk1/2,p-Akt and p-Star3 in PC9 were lower than those in group C and E(P<0.05)under immunohistochemical analysis.That means HGF produced by MRC-5 promoted the development of tumor tissues under the influence of erlotinib in the PC9 and MRC-5 cells co-inoculated nude mice models.HGF can also stimulated the phosphorylation of the C-Met protein.Conclusion:The sensitive lung cancer PC9 models constructed in nude mice can induce drug resistance on Erlotinib caused by HGF MRC-5 cells produced,and its potential mechanism for Erlotinib drug resistance is considered to be related to HGF's activation of phosphorylation in C-Met series proteins.