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[Methodology of Establishing and Identifying NCI-H2228/Crizotinib-resistant Cell Lines In Vitro].

ABSTRACT: The mechanisms of small molecule targeting drug resistance and ways to overcome resistance are now both urgent need to improve the clinical efficacy. This study aimed to investigate the feasibility of using different methods to establish the crizotinib-resistant non-small cell lung cancer NCI-H2228/Crizotinib cell lines and to clarify the mechanisms of resistance to small molecule targeting drug, thus providing experimental and theoretical bases for further studies to overcome the mechanisms of Crizotinib resistance.The study utilized stepwise increase of drug concentrations and chemical mutagen to induce Crizotinib-resistant NCI-H2228 cells. The drug 50% inhibitory concentration (IC50) values of parental and resistant cells and the population doubling time were determined by MTT assay. The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) expression was evaluated by RT-PCR and Western blot. Full-length sequencing method was used to compare the EML4-ALK genes in the parent and drug-resistant cells and analyze the mechanisms of drug resistance.The method of gradually increasing drug concentration to induce Crizotinib-resistant NCI-H2228 cells was time-consuming because the cell growth recovery was extremely slow. Thus, this method was considered invalid. However, chemical mutagen ENU can effectively induce NCI-H2228 cells resistant to crizotinib in a short time [IC50]= (3.810±1.100) ?mol/L, P=0.002,9 vs parental cells]. Furthermore, the gene mutation frequency of EML4-ALK in the resistant cells was significantly higher than that in the parent cells.Chemical mutagen-induced cell resistance was easily operated and had effectively shortened the experimental process. Preliminary technical methods and experimental evidence for in-depth study of drug resistance mechanisms and approaches to overcome the targeted drug resistance were also provided.?.


PROVIDER: S-EPMC5999905 | BioStudies | 2015-01-01

REPOSITORIES: biostudies

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