Luo Ran, Deng Si-Qi, Zhao Yin-Xu, Wang Lu, Ren Wen-Kang, Zou Yu, Lin Yu, Bu Ming
College of Pharmacy, Qiqihar Medical University Qiqihar 161006, China.
Zhongguo Zhong Yao Za Zhi. 2024 Jul;49(13):3627-3635. doi: 10.19540/j.cnki.cjcmm.20240202.705.
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 oxygen 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, cellular 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 significant increase in the total apoptosis rate of MCF-7 cells, a significant decrease in mitochondrial membrane potential, and a significant increase in ROS levels. In addition, treatment with EP led to an upregulation of Cyt C, Bax, and cleaved caspase-7 protein expression, 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 mediating the mitochondrial apoptotic pathway.
本研究探讨了过氧化麦角甾醇（EP）对MCF-7乳腺癌细胞增殖和凋亡的影响，探究其可能的作用机制，并通过体外实验进行验证。运用网络药理学筛选EP的靶蛋白，构建靶标网络和蛋白质-蛋白质相互作用（PPI）网络，以预测EP抗乳腺癌作用涉及的潜在靶蛋白和相关通路。采用MTT法检测EP对MCF-7细胞增殖的抑制作用，利用集落形成实验评估细胞克隆能力。通过流式细胞术和激光共聚焦显微镜评估细胞凋亡、线粒体膜电位和活性氧（ROS）水平。采用蛋白质免疫印迹分析检测经EP处理的MCF-7细胞中B细胞淋巴瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）、细胞色素C（Cyt C）、半胱天冬酶-7、裂解的半胱天冬酶-7、磷脂酰肌醇3激酶（PI3K）和丝氨酸/苏氨酸激酶B（AKT）的表达水平。网络药理学预测结果显示，EP与乳腺癌之间存在173个共同靶点；京都基因与基因组百科全书（KEGG）富集分析结果表明，EP治疗乳腺癌主要影响癌症通路、PI3K-AKT信号通路、细胞衰老信号通路和病毒致癌通路等信号通路；MTT实验结果显示，EP组MCF-7细胞活力显著低于对照组，呈时间和浓度依赖性，且EP可抑制MCF-7乳腺癌细胞的集落形成。用10、20和40 μmol·L⁻¹ EP处理24 h可导致MCF-7细胞总凋亡率显著升高、线粒体膜电位显著降低以及ROS水平显著升高。此外，EP处理导致MCF-7细胞中Cyt C、Bax和裂解的半胱天冬酶-7蛋白表达上调，p-PI3K、p-AKT和Bcl-2蛋白表达下调。研究表明，EP可能通过PI3K-AKT途径介导线粒体凋亡途径来抑制MCF-7乳腺癌细胞增殖并减少集落形成。
