人肺腺癌细胞中的耐药相关泛素化蛋白质研究 - 第三军医大学学报.doc

人肺腺癌耐药细胞株A549/CDDP中相关泛素化蛋白质研究 秦 霞1，邱宗荫2，邱 峰1，粟 林1，屈 灿1（400016 重庆，重庆医科大学附属第一医院药学部） 目的 研究肺腺癌中与泛素化或类泛素化蛋白质。从处理和未处理的A549细胞其耐株中分离泛素化类泛素化蛋白质，通过1D SDS和Chip HPLC-MS/MS分析蛋白，Western blot法验证；MTT法检测细胞对顺铂的药性。 鉴定发生类泛素化修饰蛋白质PKM2，并发现其在耐药细胞株中低表达；以蛋白酶体抑制剂MG132干预细胞后，PKM2表达上调；CDDP和MG132联合作用后，细胞耐药性下降。 细胞中PKM2表达水平与肿瘤细胞耐药程度呈负相关PKM2受蛋白质类泛素化修饰SUMO化修饰调节，PKM2可能与肿瘤耐药的发生密切。 A549细胞；A549/CDDP细胞; 丙酮酸激酶M2；肿瘤耐药；泛素化；SUMO化 R73-3 [文献标志码] A Analysis of ubiquitination-associated proteins in Human LungAdenocarcinoma Cisplatin-resistant Cell Strain A549/DDP Qin Xia, Qiu Zongyin, Qiu Feng, Su Lin, Qu Can(1Department of Pharmacy, the First Affiliated Hospital of Chongqing Medical University, Chongqing，400016, ChinaChongqing Medical University, Chongqing，400016, China)[Abstract] Objective Post-translational modification of protein was the most important regulatory mechanism of cell protein in playing biological function. This study was major in researching ubiquitination or neddylation associated proteins in tumor drug resistance. Methods We employed 1D SDS PAGE and Chip HPLC-MS/MS to separate and identify ubiquitinated or neddylated proteins from the lysates of CDDP treated or untreated A549 and A549/CDDP cells. Western blotting and Confocal laser scanning microscope validate those results. The sensitivity of cisplatin to cells was determined by MTT. PKM2 located at cytoplasm and nucleus. Results we Identified a SUMO protein Pyruvate kinase M2 (PKM2), which was located at cytoplasm and nucleus. The expression of PKM2 in the A549/CDDP cell was lower than it in A549 cell, but it was up-regulated when treated A549/CDDP cell with MG132. Drug resistance was down-regulated when treated cells with CDDP combination MG132. Conclusion This study suggests that the expression of PKM2 was inverse correlation with drug resistance, which suggested PKM2 and drug resistance had a closely correlation. The expression of PKM2 was regulated by SUMO which was a neddylation modification and may be