Establishment Of The Third Generation EGFR-TKI AZD9291 Drug-resistance NSCLC Cell Lines In Vitro

Background & AimsLung cancer has become the highest incidence and mortality rate among cancers worldwide,and NSCLC accounts for 80% of lung carcinoma,and can be further divided into adenocarcinoma,squamous cell carcinoma,large cell carcinoma and so on.Previous studies on gene mutation of lung adenocarcinoma in Europe and America showed that K-ras was the most common gene mutation,followed by epidermal growth factor receptor(EGFR)mutation,while in Asian NSCLC population,the highest mutation rate of EGFR was 50%.Recently,the application of EGFR-TKIs are gradually increased and became a first-line therapy choice for advanced NSCLC who carry EGFR mutations.In recent years,the use of inhibitors for EGFR sensitive mutations,such as erlotinib and gefitinib,as well as afatinib of the second generation,has been increasing.However,the majority of patients with advanced NSCLC who used EGFR tyrosine kinase inhibitor(EGFR-TKI)developed acquired drug resistance approximately at 9 to 11 months.At present,the drug resistance mechanism of the first and second generation TKIs for EGFR sensitive mutation has been more thoroughly studied,and T790 M mutation is the most important drug resistance mechanism.Osimertininb(AZD9291),the representative drug of the third generation EGFR-TKI which aimed at T790 M mutation,has just been put into first-line clinical use in China,therefore we still do not know well about the possible mechanism of drug resistance and the situation of the subsequent use of chemotherapeutic agents.Therefore,we intend to further study the mechanism of multidrug resistance after first-line use of AZD9291 then in combination with chemotherapeutic drugs.In this study,AZD9291-resistance H1975 cells and PC-9 cells were established.It will lay a foundation for the further analysis of its biological characteristics and the study of the anti-tumor effect and drug resistance mechanism of the third generation EGFR-TKI in the first line in combination with chemotherapy of NSCLC,and provide the basis for guiding the clinical application of NSCLC targeted therapy.Materials & methodsEstablishment of multidrug-resistant NSCLC cell linesWe selected non-small cell lung cancer(A549 cell line),NCI-H1975 cell line(L858R / T790 M alternative mutation)and PC-9 cell(exon 19 deletion).We first used AZD9291 to treat the above three lung cancer cells.Then two groups of different methods of AZD9291 combined with chemotherapeutic were used to induce multidrug resistant cell lines.The first group was treated with platinum-based double chemotherapeutic drugs to impact NSCLC cells that were already resistant to AZD9291;the second group was treated alternately with AZD9291 combined with platinum based double chemotherapeutic drugs on NSCLC cells.Establishment of AZD9291-resistant NSCLC cell linesWe selected A549 cells,NCI-H1975 cells and PC-9 cells of non-small cell lung cancer to induce the establishment of AZD9291 resistant strains.IC50 and drug resistance index(RI)obtained from MTT assay confirmed the successful construction of drug-resistant cell line.Cell function assays and apoptosis assay were used to compare the changes before and after drug resistance.Statistical analysis was performed using Graphpad Prism 6.0 and SPSS 23.0 to analyze the associations of cell viabity and IC50 value between drug-resist cells andtheir parental cells by nonlinear analysis and t test.A two-tailed p value of less than 0.05 was considered statistically significant.ResultsAfter we treated three lung cancer cells with AZD9291 in the first line,we used two different groups of AZD9291 combined with chemotherapeutic agents to induce three drugresistant NACLC cell lines.After 13 months of induction,no multidrug resistance to AZD9291 and chemotherapeutic agents was found in the cells.After verifing A549 cell,H1975 cell and PC-9 cell are harbored thrir own mutate,we used high concentration AZD9291 medium method to continuously culture these three cells respectively for 6 months.Finally,we obtained a AZD9291-resist NCI-H1975 cell lines (H1975/AR).H1975/AR culture with 200 n M AZD9291 grew normally.After 2 weeks the cells cultured without AZD9291 medium,we performed some experiments to access the drug resistance level.MTT proliferation inhibition assay showed that AZD9291 inhibited H1975/AR cells significantly lower than parental cells(P < 0.05),while IC50 was 3.354±0.1 M(P = 0.002),drug resistance index was 17.7.The ability of colony formation as well as adhesion /invasion and anti-apoptosis of H1975/AR were significantly higher than those of parental cells(P < 0.05).After 6 months induction of continuous high concentration method,we obtained AZD9291-resistant PC-9/AR cell line,which could be cultured maintenance at 200 n M AZD9291 medium and cells grew normally.However,after 2 weeks PC-9/AR cell cultured without AZD9291 medium,it was suggested that PC-9/AR was a unstable resistant cell strain.MTT proliferation inhibition assay told us that the same condition of AZD9291 medium,the inhibitoon of PC-9/AR was much lower than parental cells,but there was no statistical significance(P < 0.0.0576).IC50 was 2.816±0.556 M for PC-9 / AR,and the drug resistance index was 7.24.The anti-apoptotic ability,colony formation ability and invasion ability of PC-9/AR were much higher than those of parental cells,but no obvious difference was observed in cell adhesion experiment.Conclusion1.We successfully obtained AZD9291-resistant H1975/AR stable cell line by continuously using high concentration induction method;2.NCI-H1975 cells with T790 M mutant were more sensitive to AZD9291 comparison with PC-9 cells which with EGFR sensitive mutations,and the former was much easier to established stable AZD9291-resistant cell lines.3.The ability of H1975/AR and PC-9/AR resistant cells to anti-apoptosis,growth and invasion was significantly enhanced.