三羟基异黄酮抑制乳腺癌细胞生长的质谱研究

Proteomic study on the inhibitory effects of genistein on human breast cancer cells

目的初步研究低浓度的三羟异黄酮对人乳腺癌细胞系MCF-7的生长抑制作用,并通过蛋白质组学方法探讨其分子机制。方法MCF-7细胞在含25μmol/L三羟异黄酮的培养液中分别培养1～7d后,进行MTT实验,测得生长曲线;采用顺序抽提的方法将细胞总蛋白分级为水溶性和疏水性两个组分,并应用双向电泳方法对其进行分离,以获取蛋白质的表达谱;通过软件分析,找出表达量存在显著差异的蛋白质点;再应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)与生物信息学相结合的方法对差异表达蛋白点进行分析鉴定。结果25μmol/L的三羟异黄酮对MCF-7细胞具有明显的抑制作用,并在4d后观察到细胞凋亡现象的出现;双向电泳图谱分析表明无论是水溶性蛋白质还是疏水性蛋白都取得了很好的分离,并成功鉴定出24种差异表达蛋白,其中5种下调,19种上调。结论这些蛋白质的功能涉及了细胞的生长调节、应激反应、形态和凋亡等多个方面。

Objective To investigate the inhibitory effects of low concentration of genistein on human breast cancer cell line MCF-7 and explore its molecular mechanism using proteomic technologies. Methods Log phase cells were cultured in 96-well plates at a density of 1 × 10^3 cells/well with 25μmol/L genistein for 1 to 7 days respectively to obtain growth curve by MTT assay. Sequential protein extraction was used and proteins were fractionated as soluble or hydrophobic fractions which were then analyzed by 2-DE. Spots were detected and evaluated by 2D platinum Software 5.0 and the spots with different expression level were subjected to MS analysis. Results Genistein of 25 μmol/L obviously inhibited the growth of MCF-7 cells and could induce apoptosis of the cells after 4 days＇ cultivation; 2-DE patterns demonstrated a good resolution either for soluble proteins or for hydrophobic proteins. Moreover, 24 significant proteins were identified, among which 19 were up-regulated and 5 were down-regulated. Conclusion These proteins＇ functions are involved in many aspects of cell physiological functions such as growth regulation, stress response, cell morphogenesis and apoptosis.