多柔比星对乳腺癌细胞核内丝切蛋白相互作用蛋白影响的比较蛋白质组学研究

Effects of doxorubicin on cofilin interacting proteins in nucleus of breast cancer cells through comparative proteomics

目的采用比较蛋白质组学的方法,检测多柔比星对乳腺癌细胞核内丝切蛋白(cofilin)相互作用蛋白的影响。方法采用Western blot检测多柔比星对细胞核内cofilin表达的影响;免疫共沉淀后,采用比较蛋白质组学方法,检测对照组与多柔比星处理组细胞核内cofilin相互作用蛋白的差异;采用流式细胞仪检测多柔比星对细胞周期的影响。结果多柔比星处理MDA-MB-231细胞后,细胞核内cofilin表达增加,呈明显量效和时效关系;与对照组比较,多柔比星处理组核内与cofilin相互作用的蛋白结合蛋白、结构分子激活蛋白、细胞骨架蛋白、核酸结合蛋白、核糖体蛋白的种类明显减少(109、60、45、76、34 vs 79、32、20、56、16);多柔比星处理细胞后,可引起细胞周期阻滞在S/G2期,并呈明显量效关系,G1期细胞由(45.92±4.57)%降至(10.50±0.83)%(P<0.01)。结论多柔比星处理细胞后可增加cofilin核聚集,减少细胞核内与cofilin相互作用蛋白的种类,最终使细胞周期阻滞在S/G2期。

Objective To determine the effects of doxorubicin on cofilin interacting proteins in the nucleus of breast cancer cells. Methods The expression of cofilin in the nucleus was evaluated by Western blotting. After immunoprecipitation,the differences of cofilin interacting proteins in the nucleus between the controls and doxorubicin-treated cells were determined by comparative proteomics. The effects of doxorubicin on cell cycle were determined by flow cytometry. Results Doxorubicin treatment resulted in an increase of cofilin level in the nucleus of MDA-MB-231 cells in dose-and time-dependent manner. Compared with the controls,doxorubicin treatment also resulted in a decreases of the types of cofilin interacting proteins including protein binding proteins,structural molecule activity proteins,cytoskeleton proteins,nucleic acid binding proteins,and ribosome proteins（ 109,60,45,76,and 34 vs 79,32,20,56,and 16,respectively）.Furthermore,doxorubicin treatment led to cell cycle arrest at S/G2 phase in a dose-dependent manner and the percentage of cells at G1 phase was declined from（ 45. 92 ± 4. 57） % to（ 10. 50 ± 0. 83） %（ P〈0. 01）.Conclusion Doxorubicin treatment increases the accumulation of cofilin in the nuclear,decreases the types of cofilin interacting proteins,and finally leads to cell cycle arrest at S/G2 phase.