高内涵法探讨土槿皮乙酸对MCF-7细胞抑制作用的机制

Explore The Mechanism of Inhibitory Effects of Pseudolaric Acid B on MCF-7 Cells by High Content Analysis

土槿皮乙酸(pseudolaric acid B,PAB)是土槿皮(金钱松根皮)的主要生物活性成分,对多种人肿瘤细胞有细胞毒性作用.高内涵分析(high content analysis,HCA)是一种基于荧光显微技术的新技术,它以细胞为研究对象,可以同时对多个荧光靶点的荧光强度、分布,以及细胞形态进行自动化定量分析.利用高内涵分析、流式细胞术研究PAB对人乳腺癌MCF-7细胞抑制作用的机制.磺酰罗丹明实验显示,PAB抑制MCF-7细胞增殖,且呈现出剂量和时间依赖性,72 h IC50为(1.80±0.33)μmol/L.流式细胞术碘化丙锭(PI)单染显示PAB作用24 h,可致MCF-7细胞G2/M期比例增至93%以上,annexin V-FITC和PI双染显示PAB促进MCF-7细胞凋亡.高内涵分析显示:PAB作用16 h,MCF-7细胞有丝分裂指数可达40%左右,伴有cyclin B1含量增加;PAB促进微管解聚,干扰有丝分裂二极纺锤体形成;PAB引起线粒体增生;PAB导致"葡萄串样"细胞核形成,提示有丝分裂滑脱.结果表明,PAB抑制MCF-7细胞增殖、促进MCF-7细胞凋亡,这些作用可能与其促进微管蛋白解聚、干扰二极纺锤体形成、阻滞有丝分裂、促进线粒体增生有关.

Pseudolaric acid B（PAB）,a major biologically active component of ＂TuJinPi＂（the root bark of Pseudolarix kaemferi Gordon）,exhibited cytotoxicity in many human tumor cell lines.High content analysis（HCA） is a fluorescence microscopy-based automated technology used for quantitative analysis of multiple targets in cells.HCA could yield rich information about the temporal-spatial dynamics of the fluorescence-labeled cell constituents.The mechanism of inhibitory effects of pseudolaric acid B on human breast cancer MCF-7 cells was explored by high content analysis and flow cytometry.As shown by sulforhodamine B assay,PAB inhibited the proliferation of MCF-7 cells in a dose-dependent and time-dependent manner,and the 50% inhibition concentration（IC50） for 72 h was（1.80±0.33） μmol/L.Flow cytometry（propidium iodide staining） showed that,after treatment with PAB for 24 h,the proportion of MCF-7 cells at G2/M phase could increase to about 93%.Flow cytometry（annexin V-FITC and propidium iodide staining） showed that,PAB induced apoptosis of MCF-7 cells.High content analysis showed that： after treatment with PAB for 16 h,the mitotic index of MCF-7 could increase to about 40%,and cyclin B1 was upregulated;PAB caused dose-dependent disassembly of microtubules and inhibited the formation of mitotic bipolar spindles;PAB induced increase of mitochondrial mass;PAB induced grape-like giant nuclei indicating mitotic slippage in MCF-7 cells.These results suggest that PAB inhibits MCF-7 cell proliferation and induces apoptosis,these inhibitory effects may be related to disassembly of microtubules,spindle abnormalities,mitotic arrest and increase of mitochondrial mass.