细胞培养稳定同位素标记定量分析食管癌顺铂耐药相关蛋白

Differential proteins in esophageal squamous cell line EC9706/CDDP identified by SILAC quantitative proteomic approach

多药耐药性(multidrug resistance,MDR)是临床肿瘤化疗失败的主要原因之一,定量分析与鉴定食管鳞癌顺铂(cis-diamminedichloroplatinum,CDDP)耐药相关蛋白对阐明食管癌耐药的分子机制具有重要理论意义。本研究采用浓度递增法建立食管癌CDDP耐药细胞系EC9706/CDDP,细胞培养稳定同位素标记(stable isotope labeling with amino acids in cell culture,SILAC)和高效液相-电喷雾串联质谱检测、生物信息学定量分析并鉴定EC9706/CDDP及其母细胞系EC9706的差异蛋白表达谱。EC9706/CDDP细胞呈贴壁性生长但增殖缓慢,多形性、异形性明显,耐药指数为3.23。74种差异表达的蛋白质分子主要包括细胞骨架相关蛋白(20%)、能量代谢相关蛋白(11%)、转录调控及DNA修复相关蛋白(11%)、氧化还原内稳态维持类蛋白(9.5%)、蛋白合成及参与mRNA处理类蛋白(12%)、核糖体结构类蛋白(8.1%)、分子伴侣蛋白(8.1%)、免疫/炎症反应相关蛋白(5.4%)、细胞内转运功能相关蛋白(5.4%)、核组装相关蛋白(2.7%)等。表明食管癌顺铂耐药的发生是多分子参与、多代谢通路失调的复杂过程,差异表达的蛋白分子将有助于深入理解MDR发生的分子机制,并为MDR表型逆转的新型药物设计提供有价值的分子靶点。

Multidrug resistance（MDR） is one of the main causes leading to the failure in cancer treatment.Differential proteins between esophageal squamous cell carcinoma（ESCC） cell line EC9706 and its cis-diamminedichloroplatinum（CDDP）-resistant subline EC9706/CDDP revealed by quantitative analysis may provide deeper insights into the molecular mechanisms of MDR implicated in ESCC.EC9706/CDDP was generated by exposure of its parental sensitive EC9706 to a step-wise increase of CDDP concentration during EC9706 cultivation.The stable isotope labeling with amino acids in cell culture（SILAC） was used to label EC9706 and EC9706/CDDP with heavy and light medium,separately.Mixed peptides derived from EC9706 and EC9706/CDDP were analyzed by high performance liquid chromatography-electrospray ionization-mass spectrometry（HPLC-ESI-MS/MS） and subsequently subjected to bioinformatics analysis to identify differential proteins between EC9706 and EC9706/CDDP.Compared to parental EC9706,EC9706/CDDP manifested phenotypes of slow proliferation,cell pleomorphology,atypia and increased resistant-index 3.23.Seventy-four differential proteins identified in the present study belongs to various families with multiple functions,such as cytoskeleton（20%）,energy metabolism（11%）,transcription regulation and DNA repair（11%）,redox homeostasis（9.5%）,protein biosynthesis and mRNA processing（12%）,ribosome constituent（8.1%）,molecular chaperone（8.1%）,immunity/inflammation（5.4%）,intracellular transport（5.4%） and nucleosome assembly（2.7%）,which indicated that development of MDR is a complicated process involving dysregulation of multiple molecules and pathways.The data is of great value for in-depth elucidation of molecular mechanisms of the MDR implicated in ESCC and may represent potential molecular targets for future therapeutic development.