醋酸甲羟孕酮诱导结肠癌SW480细胞凋亡及其差异表达蛋白的分析

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。

Medroxyprogesterone Acetate-Induced Colon Cancer SW480 Cell Apoptosis and Differentially Expressed Protein

Objective To analyze the differentially expressed protein during colon cancer SW480 cell apoptosis induced by medroxyprogesterone acetate(MPA).Methods SW480 cells were treated with MPA at various concentrations(25,50 and 75 μmol/L)for 24,48 and 72 h separately,and determined for proliferation level by MTT method.The cells were treated with MPA at the above-mentioned concentrations for 48 h respectively,and analyzed for apoptosis rate and the percentages of cells at various phases of cell cycle.The cells were treated with 50 μmol/L MPA for 48 h and observed for change of structure by transmission electron microscopy.The total protein of SW480 cells was isolated by two-dimensional electrophoresis(2-DE),and the 2-DE image was analyzed by PDQUEST8.0 software.The differentially expressed proteins were identified by Agilent 1100 HPLC-Chip/MS system and by searching UniProtKB/SWISS-PORT,Homo Sapiens(Human)database.Results Obviously morphological change of apoptosis was observed by transmission electron microscopy in the SW480 cells treated with MPA.The MPA at various concentrations inhibited the proliferation and induced the apoptosis of SW480 cells significantly,each in a dosage-dependent mode.The cell apoptosis rate reached 56.15% at most.The analysis on cell cycle showed that the induction of colon cancer cell apoptosis with MPA might be related to the dosage-dependent arrest of cell cycle.A total of 7 kinds of proteins related to the apoptosis of SW480 cells,i.e.ubiquitin,LIM domain only protein3(LMO3),heat shock protein(HSP)10,soluble carrier family 22 member 11,UCHL2,NDPK-A and Laminin receptor 1,were identified.Conclusions MPA induced the apoptosis of SW480 cells,and the apoptosis-related proteins might be served as the potential biomarkers of MPA against colon cancer.