泛素化蛋白PKM2与肿瘤耐药相关性研究.doc

人肺腺癌细胞中的耐药泛素化秦霞 邱宗荫 邱峰 粟林 屈灿（400016 重庆，重庆医科大学附属第一医院药学部） [摘要] 目的 研究瘤耐药中，与泛素化或类泛素化蛋白质。方法 从CDDP处理和未经处理的A549细胞其耐药株中分离泛素化、类泛素化蛋白质，通过1D SDSPAGE和Chip HPLC-MS/MS分析目标蛋白，并以Western blotting法验证；细胞对顺铂的药性以MTT法检测。结果 鉴定了发生类泛素化修饰的蛋白质PKM2，在耐药细胞株中PKM2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。结论 PKM2与肿瘤耐药具有密切关系，其表达水平与肿瘤细胞耐药程度呈负相关；细胞中PKM2的受蛋白质类泛素化修饰SUMO化修饰调节，可能与肿瘤耐药的发生具有密切关系。 [关键词] A549细胞；A549/CDDP细胞; 丙酮酸激酶M2；肿瘤耐药；泛素化；SUMO化 [中图法分类号] [文献标志码] Analysis of ubiquitination-associated proteins in Human Lung Adenocarcinoma Cisplatin-resistant Cell Strain Qin Xia, Zhang Yuan, Qiu Zongyin, Su Lin, Qu Chan, Qiu Feng (Department of Pharmacy, the First Affiliated Hospital of Chongqing 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 closely with the devel