17-AAG耐药性的产生促进结肠癌细胞发生EMT改变

Acquired Resistance to 17-AAG Promotes Epithelial-mesenchymal Transition in Colon Cancer Cells

目的研究结肠癌细胞对17-AAG的获得性耐药,以及耐药细胞株的生物学特性改变。方法培养结肠癌细胞SW480、SW620,用SRB法检测其17-AAG的IC50;通过17-AAG浓度递增法诱导培养建立耐药细胞株;用克隆形成实验检查细胞克隆形成能力,用Transwell小室法检测细胞迁移能力;用蛋白质免疫印迹(Western blot)法检测上皮-间质细胞转化(EMT)分子标志物的表达。结果经过6～8周17-AAG诱导培养,得到稳定的耐药细胞株SW480-R;与母细胞相比,SW480-R细胞呈现EMT的表型特征;SW480-R克隆形成和迁移能力显著高于其母细胞(P<0.05);SW480-R细胞中E-钙黏蛋白(E-cadherin)表达下调(P<0.05),而N-钙黏蛋白(N-cadherin)和β-连环素(β-catenin)的表达显著上调(P<0.05)。结论17-AAG可诱导结肠癌细胞产生获得性耐药,耐药细胞株显示浸润转移的特性,其机制可能与耐药细胞株发生EMT有关。

Objective To study the acquired resistance of 17-AAG in colon cancer cell lines,and further to investigate the biological properties in the drug resistant cells.Methods Colon cancer cell lines SW480 and SW620 were cultured and treated continually with 17-AAG to generate the drug resistant cell lines.Cell growth and IC50 of 17-AAG were detected by SRB assay.Cell cloning formation and migration assay were performed to identify the cell biological properties.Epithelial-mesenchymal transition(EMT)biomarkers were detected by Western blot.Results SW480 cells were generated resistance to 17-AAG in 6-8 weeks and named as SW480-R.The SW480-R cells showed obvious morphology changes with EMT-like phenotype,and significant elevating in cloning formation and migration(all P<0.05).Western blot analysis found that the expression of E-cadherin was downregulated,whereas that of N-cadherin and β-catenin upregulated in SW480-R cells(all P<0.05).Conclusion Colon cancer cells could be induced to become resistant to 17-AAG.The drug resistant cells showed a metastatic phenotype,which may be associated with EMT.