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目的对醋酸甲羟孕酮(Medroxyprogesterone acetate,MPA)诱导结肠癌SW480细胞凋亡时的差异表达蛋白进行分析。方法以不同浓度(25、50、75μmol/L)的MPA作用SW480细胞24、48和72h,MTT法检测细胞增殖水平。以上述浓度的MPA作用SW480细胞48h,流式细胞术分析细胞的凋亡率及细胞周期各时相比例的变化。以50μmol/L MPA作用SW480细胞48h,透射电镜观察细胞结构的变化;应用双向电泳技术分离细胞总蛋白,PDQUEST8.0软件分析凝胶图谱,选择差异表达蛋白,以Agilent1100HPLC-Chip/MS系统进行MS/MS分析,搜索UniProtKB/SWISS-PORT,Homo Sapiens(Human)数据库筛选目标蛋白质。结果透射电镜下观察经MPA作用的SW480细胞可见明显的凋亡形态学变化;不同浓度的MPA对SW480细胞均有明显的抑制增殖和诱导凋亡作用,且呈剂量依赖性,细胞凋亡率最高可达56.15%;细胞周期分析表明,MPA诱导结肠癌细胞凋亡的作用可能与细胞周期的阻滞相关,此阻滞作用具有剂量依赖性;共鉴定出泛素、只含LIM结构域蛋白质3、热休克蛋白10、有机阴离子转运蛋白4、泛素羧基末端水解酶2、核苷二磷酸激酶-A、层黏连蛋白受体1共7种SW480细胞凋亡相关蛋白。结论 MPA具有诱导SW480细胞凋亡的作用,SW480细胞凋亡相关蛋白可能作为MPA抗结肠癌的潜在生物标志物。
Objective To analyze the differentially expressed proteins in the apoptosis of colon cancer SW480 cells induced by Medroxyprogesterone acetate (MPA). Methods SW480 cells were treated with MPA at different concentrations (25, 50 and 75μmol / L) for 24, 48 and 72 hours, respectively. The cell proliferation was measured by MTT assay. SW480 cells were treated with MPA at the above concentration for 48h. The changes of cell apoptosis rate and cell cycle phases were analyzed by flow cytometry. SW480 cells were treated with 50μmol / L MPA for 48h, and the cell structure was observed by transmission electron microscopy. Total cellular protein was separated by two-dimensional electrophoresis. The gels were analyzed by PDQUEST8.0 software. The differentially expressed proteins were selected and analyzed by Agilent1100 HPLC-Chip / MS / MS analysis, search UniProtKB / SWISS-PORT, Homo Sapiens (Human) database to screen for target proteins. Results The morphological changes of apoptosis of SW480 cells treated with MPA were observed under transmission electron microscope. Different concentrations of MPA significantly inhibited the proliferation and induced apoptosis of SW480 cells in a dose-dependent manner, with the highest rate of apoptosis Up to 56.15%; cell cycle analysis showed that the MPA-induced apoptosis in colon cancer cells may be related to cell cycle arrest, the blockade in a dose-dependent manner; identified a total of ubiquitin, containing LIM domain protein 3 , Heat shock protein 10, organic anion transporter 4, ubiquitin carboxyterminal hydrolase 2, nucleoside diphosphate kinase-A and laminin receptor 1. Conclusion MPA can induce the apoptosis of SW480 cells and the apoptosis related protein of SW480 cells may be a potential biomarker for anti-colon cancer of MPA.