Methodology of Establishing and Identifying NCI-H2228/Crizotinib-resistant Cell LinesIn Vitro
10.3779/j.issn.1009-3419.2015.06.02
- VernacularTitle:体外诱导建立NCI-H2228/Crizotinib耐药细胞株的方法学探讨及鉴定分析
- Author:
WU DI
1
;
JIN GUIHUA
;
ZHA0 DAWEI
;
ZHANG YUE
;
ZHA0 JING
;
YU HONG
Author Information
1. 吉林大学第一医院肿瘤中心
- Keywords:
Lung neoplasms;
Small-molecule-targeted drugs;
Crizotinib;
Drug resistance;
EML4-ALK
- From:
Chinese Journal of Lung Cancer
2015;(6):330-339
- CountryChina
- Language:Chinese
-
Abstract:
Background and objectivehTe mechanisms of small molecule targeting drug resistance and ways to overcome resistance are now both urgent need to improve the clinical effcacy. hTis 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.MethodshTe study utilized stepwise increase of drug concentrations and chemical mutagen to induce Crizotinib-resistant NCI-H2228 cells. hTe drug 50% inhibitory concentration (IC50) values of parental and resistant cells and the population doubling time were determined by MTT assay. hTe 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 theEML4-ALK genes in the parent and drug-resistant cells and analyze the mechanisms of drug resistance.Results hTe method of gradually increasing drug concentration to induce Crizotinib-resistant NCI-H2228 cells was time-consuming because the cell growth recovery was extremely slow. hTus, this method was considered invalid. However, chemical mutagen ENU can effectively induce NCI-H2228 cells resistant to crizo-tinib in a short time [IC50]= (3.810±1.100) μmol/L,P=0.002,9vs parental cells]. Furthermore, the gene mutation frequency of EML4-ALK in the resistant cells was signiifcantly higher than that in the parent cells.ConclusionChemical 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 resis-tance were also provided.
