A Study Of Acquired Resistance Mechanism Of HCC827-TR EGFR Mutant Cell

Backgroud and Object:As the chemical treatment effect of lung cancer has reached the platform,the molecular target treatment of lung carcinoma is becoming the research hot spot,especially the research to EGFR gene.Most research found that the EGFR is overexpress in more than 80%lung cancer.There are many EGFR target therapy drugs,among them the small molecular tyrosine kinase inhibitor(EGFR tyrosine kinase inhibitors,EGFR-TKI)is most impotant.In clinical research find that EGFR-TKIs are highly effective in lung cinitially ancer patients who harbor active mutations in the EGFR gene.Especilly the sensitive mutation including the 5 amino acid deletions occur ing from codon 746to 750 in exon 19 or a point mutation in leucine(L)858 to arginine(R)(L858R)in kinase domain of EGFR.However,patients who are sensitive to EGFR-TKIs eventually relapse within few years.To date,several major mechanisms of acquired resistance,such as secondary mutation of the EGFR gene,amplification of the MET gene,the two mechanisms possess 70%.30-40%acquired resistant mechanisms are obscure,including IGF-1R high expression,HGF high expression,loss of PTEN expression,et al.In order to study the EGFR-TKI acquired resistant mechanisms,we use the Erlotinib to induce the resistant cells HCC827-TR,use the MTS detecting the drug sensitive,use gene chip selecting different gene associated with drug resistance,use different trays to detect the knowned acquired resistant mechanisms.Methods:1.Culting HCC827-P cells in vitro and using the Erlotinib mutag to induce the drug-resistant cell line HCC827-TR,applying the MTS cytotoxicity test drug sensitivity to Erlotinib and Gefitinib of HCC827-P and HCC827-TR.2.Extracting the total RNA,handing over Affymetrix gene chip to make gene analysis,we analyse the signaling pathways and genetic variations associated with lung cancer by MAS3.0 software.3.Extracting genome DNA,using Realtime PCR to detect the MET gene copy number in the cells HCC827-TR and HCC827-P.4.Extracting total RNA and reverse transcribed into cDNA,Realtime PCR detects the MET,PTEN expression level in HCC827-P and HCC827-TR,PCR for EGFR amplification and sequencing.5.Western blot detect the expression level of p-AKT,p-ERK in the HCC827-P and HCC827-TR cells.Results:1.Successfully induced Erlotinib resistant cell lines HCC827-TR,the MTS results show that when the Erlotinib and Gefitinib concentration is 100 nm,the cell growth inhibition rate of HCC827-P and HCC827-TR cell growth inhibition rate were 57.7%,52.9%and 3.0%,0.1%,HCC827-TR and HCC827-P is characterized by obvious resistance.2.The results of gene chip,1481 differential genes between HCC827-TR and HCC827-P cells,727 up regulation,754 downregulation,73 differential genes involved 11 signal pathways associated with the lung cancerr,and the differential gene associated with lung cancer are maintaining the upregulated gene MET,TGFB2,VEGF,MAPK,AKT3,and the downregulated gene EGFR,IGF1,PI3R3.3.Gene sequencing results show that HCC827-TR and HCC827-P cells have EGFR19 exon sensitive mutation,but not T790M mutation.4.The MET gene copy number and the MET gene express level are in the same level,the same as the PTEN expression.5.Erlotinib can inhibit the EGFR signaling protein(p-AKT,p-ERK)in HCC827-P cell not in HCC827-TR cell,Conclution:1.Successfully induced Erlotinib resistant cell lines HCC827-TR.2.The results of the gene chip provide the good direction for the further study3.The resistance to Erlotinib of HCC827-TR has nothing to do with EGFR T790M mutation and PTEN loss;MET gene amplification relationship is unclear,needed further discussion.