吉非替尼耐药细胞株的筛选和基因谱表达研究

Selection and establishment of gefitinib-resistant PC9 cell line and its gene expression profile

目的:从对吉非替尼(gefitinib)敏感的肺腺癌细胞株PC9中筛选并建立了6株gefitinib耐药细胞亚克隆并研究其生物学特性。方法:采用N-甲基-N’-硝基-N-亚硝基胍(MNNG)诱变筛选和有限稀释法进行耐药性亚克隆的单细胞培养,MTT法检测肿瘤细胞对gefitinib的敏感性和IC50,DNA微列阵检测耐药细胞株与野生型PC9细胞的基因表达差异,免疫组织化学法测定细胞外间质成分fibronectin和collagenⅣ的表达差异,FCM法进行细胞周期分析。结果:获得了对gefitinib具有较高耐药性的2个细胞株:PC9/G2和PC9/G4,尤其PC9/G2的IC50为7～10μmol/L,较野生型PC9细胞(IC50:0.03～0.05μmol/L)提高耐受性160～260倍。PC9的细胞倍增时间为(16.2±3.5)h,PC9/G2的倍增时间为(36.5±4.8)h。PC9/G2同PC9的基因表达谱存在明显差异,耐药细胞株PC9/G2的脂肪酸代谢和氧化磷酸化基因表达下降,糖酵解基因表达上升,核糖体各亚基蛋白表达下调,高尔基体和囊泡、笼形蛋白衣被小泡等相关基因表达上调,细胞蛋白分泌功能加强;泛素-蛋白酶体循环相关基因上调;DNA修复基因和解旋酶类基因表达上调;具有蛋白激酶活性的30个基因表达上调,11个基因表达下调;15个磷酸化蛋白磷酸酶活性的基因表达上调;具有GTP结合活性的基因21个表达上调,4个下调;涉及细胞骨架和细胞运动功能的8个基因上调;整合素β1亚基、类胰岛素受体、NFκB级联反应的正向调节因子表达上调。结论:成功建立了6株PC9细胞的gefitinib耐药细胞株,初步研究表明对gefitinib中度耐药可能与细胞外基质组分改变及其黏附信号通路有关,对gefitinib高度耐药则可能涉及替代性信号通道的激活和信号通道的下游激活。

Objective：To establish 6 subclones of gefitinib-resistant cells from gefitinib-sensitive lung adenocarcinoma cell line PC9 and study their biological characteristics. Methods：The PC9 cells were induced to mutate by a mutagen MNNG and selected. The subclone of gefitinib-resistant cell was obtained by limited dilution. The sensitivity and IC50 for gefitinib of various cell lines were determined by MTT assay. The different profile of gene expression between the wildtype PC9 cell and the gefitinib-resistant cells were determined by DNA microarray. Fibronectin and collagen Ⅳ expressions in various PC9 cell lines were checked by immunohistochemistry. Cell cycle was analyzed by flow cytometry. Results： Two resistant cell lines to gefitinib were obtained, named PC9/G2 and PC9/G4, respectively, and the IC5o for the gefitinib was 7-10 μmol/L , about 160 to 260 folds higher compared with wildtype PC9 （IC50：0.03- 0.05 μmol/L）. The doubling time of PC9 cells and PC9/G2 cells were （ 16.2± 3.5 ） h and （36.5 ± 4.8 ） h, respectively. The gene expression profile had significant difference between wildtype PC9 cells and resistant PC9/G2 cells. Gene function annotation and grouping showed that in PCg/G2 cells fatty acid metabolism- and oxidative phosphorylation-related genes were down-regulated, while glycolysisgenes were up-regulated ; the expressions of ribosomal subproteins were down-regulated, and the gene groups located in Golgi Stack, cisternae, clathrin-coated vesicle were up-regulated; the protein-secreting function was enhanced and ubiquitin-proteosome system was activated ; DNA repair and helicase genes were also up-regulated. For the genes with protein kinase activity, 30 of them were up-regulated, while 11 of them were down-regulated; 15 genes with phosphoprotein phosphatase activity were up-regulated. For the genes with GTP binding function, 21 of them were up-regulated, while 4 of them were down-regulated; 8 genes related to cytoskeletal movement and cell adhesion were up-regulated; integrin betal subunit, insulin-like receptor and positive regulative factors for NFκB cascade were up-regulated. Conclusion： Six lines of gefitinib-resistant PC9 cells were successfully established. Our results suggested that moderate resistance to gefitinid might be related with extracellular matrix changes and adhesion signaling cascade, while high resistance may be due to activation of instead signaling pathway and the downstream molecules of signaling pathway.