β-脱水淫羊藿素通过ROS介导的线粒体凋亡途径诱导MCF-7细胞凋亡

β-anhydroicaritin Induces Apoptosis of MCF-7 Cells through ROS-Mediated Mitochondrial Apoptosis Pathway

该文旨在探究β-脱水淫羊藿素对人非小细胞肺癌细胞A549、人乳腺癌细胞MCF-7、人前列腺癌细胞PC-3M、人子宫颈鳞癌细胞SiHa和人胰腺癌细胞PANC-1的抗肿瘤活性,并对筛选出抑制活性最佳的细胞株初步探讨其抗肿瘤作用机制。采用MTT法检测不同浓度β-脱水淫羊藿素对5种肿瘤细胞增殖的影响,并以IC_(50)作为标准,筛选出β-脱水淫羊藿素抑制作用较为突出的乳腺癌MCF-7细胞进行后续实验;DAPI染色和流式细胞术检测细胞凋亡情况;DCFH-DA法检测细胞的活性氧水平;Calcein AM探针检测线粒体通透性转换孔开放情况;JC-1探针检测线粒体膜电位变化;分子对接技术预测β-脱水淫羊藿素与活性氧相关蛋白SIRT3之间的结合能力;Western blot检测细胞中线粒体凋亡途径相关蛋白SIRT3、Cytochorme C、Cleaved caspase-3、Cleaved caspase-9、PARP、Cleaved PARP的表达水平。结果显示:β-脱水淫羊藿素能够通过调控SIRT3蛋白表达水平刺激肿瘤细胞活性氧水平升高,使得MCF-7细胞线粒体功能发生障碍,进而刺激线粒体通透性转换孔异常开放,使得膜电位大幅度降低,最终诱导MCF-7细胞凋亡,且这些影响可能是通过活性氧介导的线粒体凋亡途径实现的。

The aim of this study is to investigate the anti-tumor effect ofβ-anhydroicaritin on human non-small cell lung cancer A549,breast cancer MCF-7,prostate cancer PC-3M,cervical squamous cell carcinoma SiHa and pancreatic cancer PANC-1 cells,and to screen out the cell lines with the best inhibitory activity to ex-plore the mechanism of anti-tumor effect.MTT assay was used to detect the effects of different concentrations ofβ-anhydroicaritin on the proliferation of five kinds of tumor cells,and the breast cancer MCF-7 cells with more prominent inhibitory effect ofβ-anhydroicaritin were selected for subsequent experiments based on IC_(50);DAPI staining and flow cytometry were used to detect cell apoptosis;DCFH-DA method was used to detect the level of reactive oxygen species in cells;the opening of mitochondrial permeability transition pore was detected by using Calcein AM probe;JC-1 probe was used to detect mitochondrial membrane potential;molecular docking technology forecastedβ-anhydroicaritin element combination ability with proteins SIRT3 associated with reactive oxy-gen species;Western blot was used to detect the expression levels of SIRT3,Cytochorme C,Cleaved caspase-3,Cleaved caspase-9,PARP,and Cleaved PARP in the cells.The results showed thatβ-anhydroicaritin could stimulate the level of reactive oxygen species in tumor cells by regulating the expression level of SIRT3 protein,which leads to mitochondrial dysfunction in MCF-7 cells,and then stimulated the abnormal opening of mitochondrial perme-ability transition pore,greatly reduced the membrane potential,and finally induced apoptosis of MCF-7 cells.These effects may be mediated by ROS(reactive oxygen species)-mediated mitochondrial apoptosis.