基于网络药理学及体外实验探讨过氧麦角甾醇对乳腺癌MCF-7细胞的作用机制
Mechanism of ergosterol peroxide on MCF-7 breast cancer cells based on network pharmacology and in vitro experiments
罗然 1邓思琦 1赵音旭 1王璐 1任文康 1邹宇 1林宇 1卜明1
作者信息
- 1. 齐齐哈尔医学院药学院,黑龙江齐齐哈尔 161006
- 折叠
摘要
基于网络药理学方法探究过氧麦角甾醇(EP)抗乳腺癌的靶点及作用机制,并通过体外实验验证.采用网络药理学对EP作用靶点进行筛选,构建靶点网络及蛋白-蛋白互作(PPI)网络,对EP抗乳腺癌潜在的作用靶点及相关通路进行预测.采用MTT法检测EP对乳腺癌MCF-7细胞的增殖抑制活性,克隆形成实验检测EP对MCF-7细胞增殖影响;采用流式细胞术和激光共聚焦显微镜检测细胞的凋亡情况、线粒体膜电位及活性氧水平的变化情况;通过Western blot法检测EP对B细胞淋巴瘤(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素C(Cyt C)、半胱氨酸蛋白酶蛋白(caspase)-7、活化的caspase-7(cleaved caspase-7)、磷脂酰肌醇3-激酶(PI3K)、丝氨酸-苏氨酸激酶B(AKT)蛋白表达水平的影响情况.结果显示,经网络药理学预测,得出EP与乳腺癌有173个共同靶点;KEGG富集分析结果显示EP治疗乳腺癌结果主要在癌症通路、PI3K-AKT信号通路、细胞衰老信号通路、病毒致癌作用等信号通路;MTT结果显示,与对照组相比,EP组MCF-7细胞的活性明显降低,呈时间-浓度依赖趋势;EP能够抑制乳腺癌细胞MCF-7集落形成;采用10、20、40 μmol·L-1 的 EP作用MCF-7细胞24 h后,MCF-7细胞总凋亡率均显著升高,线粒体膜电位均显著降低,活性氧水平均显著升高;此外,MCF-7细胞经过EP处理后,细胞内Cyt C、Bax、cleaved caspase-7蛋白的表达水平上升,p-PI3K、p-AKT、Bcl-2蛋白的表达水平下降.研究表明,EP可抑制乳腺癌MCF-7细胞增殖,减少集落形成,其机制可能与PI3K-AKT通路介导线粒体凋亡途径有关.
Abstract
This study investigated the effects of ergosterol peroxide(EP)on the proliferation and apoptosis of MCF-7 breast cancer cells,explored its possible mechanisms of action,and verified the effects and mechanisms by in vitro experiments.Network pharmaco-logy was used to screen the target proteins of EP and construct target networks and protein-protein interaction(PPI)networks to predict the potential target proteins and related pathways involved in EP anti-breast cancer effects.The MTT assay was performed to measure the inhibitory effect of EP on MCF-7 cell proliferation,and the colony formation assay was used to assess the cell cloning ability.Flow cytometry and laser confocal microscopy were employed to evaluate cell apoptosis,mitochondrial membrane potential and reactive oxy-gen species(ROS)levels.Western blot analysis was conducted to examine the expression levels of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),cytochrome C(Cyt C),caspase-7,cleaved caspase-7,phosphatidylinositol 3-kinase(PI3K),and se-rine/threonine kinase B(AKT)in MCF-7 cells treated with EP.The results of network pharmacology prediction yielded 173 common targets between EP and breast cancer;the results of Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that EP treatment for breast cancer mainly affected the signaling pathways such as cancer pathway,PI3K-AKT signaling pathway,cel-lular senescence signaling pathway,and viral carcinogenesis pathway;and the MTT assay results showed that the viability of MCF-7 cells in the EP group was significantly lower than that in the control group,exhibiting a time-and concentration-dependent trend,and EP can inhibit colony formation of MCF-7 breast cancer cells.Treatment with 10,20,and 40 μmol·L-1 EP for 24 h resulted in a sig-nificant increase in the total apoptosis rate of MCF-7 cells,a significant decrease in mitochondrial membrane potential,and a signifi-cant increase in ROS levels.In addition,treatment with EP led to an upregulation of Cyt C,Bax,and cleaved caspase-7 protein ex-pression,and a downregulation of p-PI3K,p-AKT,and Bcl-2 protein expression in MCF-7 cells.Studies have shown that EP inhibits MCF-7 breast cancer cell proliferation and reduces colony formation by a mechanism that may be related to the PI3K-AKT pathway me-diating the mitochondrial apoptotic pathway.
关键词
过氧麦角甾醇/乳腺癌/MCF-7细胞/网络药理学/细胞凋亡/线粒体膜电位/活性氧Key words
ergosterol peroxide/breast cancer/MCF-7 cells/network pharmacology/cell apoptosis/mitochondrial membrane po-tential/reactive oxygen species引用本文复制引用
基金项目
齐齐哈尔医学院研究生创新基金项目(QYYCX2023-36)
黑龙江省自然科学基金联合引导项目(LH2022H113)
齐齐哈尔市科技局联合引导项目(LHYD2021005)
齐齐哈尔市科技局联合引导项目(LHYD2021021)
出版年
2024
NETL
NSTL
万方数据