摘要
目的 通过毛菊苣倍半萜成分抗肿瘤活性初筛,探究其有效活性成分山莴苣苦素的作用靶点及治疗三阴性乳腺癌(Triple negative breast cancer, TNBC)的作用机制。方法 采用CellTiter-Glo发光法,在不同肿瘤细胞株上检测毛菊苣倍半萜成分中山莴苣素和山莴苣苦素的体外抗肿瘤活性。采用细胞克隆实验检测山莴苣苦素对MDA-MB-468细胞克隆形成能力的影响。借助PubChem和SwissTargetPrediction数据库获得山莴苣苦素的作用靶点;通过GeneCards、DisGeNET和OMIM在线数据平台获取TNBC的作用靶点;通过Venny 2.1.0在线工具绘制韦恩图并获取交集靶点;通过Cytoscape 3.9.1软件构建“TNBC-靶点-山莴苣苦素”网络图;通过STRING和Cytoscape 3.9.1软件构建蛋白质-蛋白质互作(Protein-protein interaction, PPI)网络图和核心靶点分析;对交集靶点进行基因本体(Gene ontology, GO)功能富集和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)通路富集分析。通过蛋白印迹法(Western blot)探究山莴苣苦素的作用机制。结果 体外抗肿瘤活性筛选显示,山莴苣苦素对三阴性乳腺癌MDA-MB-468细胞的抑制作用较显著;细胞克隆实验结果显示,山莴苣苦素可抑制MDA-MB-468细胞的增殖和克隆形成能力。经数据库查找获得100个山莴苣苦素相关靶点,3 521个TNBC相关靶点,利用韦恩图获得45个交集靶点,通过PPI网络筛选得到6个核心靶点:EGFR、MAPK1、PARP1、SRC、MAPK8、IGF1R。GO和KEGG富集分析结果显示,PI3K/AKT通路在山莴苣苦素治疗TNBC中发挥关键作用。Western blot结果表明,与对照组相比,山莴苣苦素低、中、高剂量组p-mTOR蛋白表达水平降低;山莴苣苦素极低、低、中剂量组p-AKT蛋白表达水平降低,高剂量组p-AKT蛋白表达水平升高,山莴苣苦素实验组Caspase-3和Bax蛋白表达水平升高,差异均有统计学意义(P<0.05)。结论 山莴苣苦素抑制三阴性乳腺癌MDA-MB-468细胞增殖和克隆形成,通过作用于AKT/mTOR信号通路发挥治疗TNBC的作用。
Objective The anti-tumour activity of Cichorum glandulosum Boiss.et Huet and semiterpene components was preliminarily screened,and the target of its effective active ingredient lactucopicrin and its mechanism of action in the treatment of triple-negative breast cancer(TNBC)were explored.Methods The in vitro antitumor activity of lactucin and lactucopicrin(sesquiterpene components derived from Cichorum glandulosum Boiss.et Huet)was evaluated using the Cell Titer-Glo luminescent assay across multiple tumor cell lines.Aclonogenic assay was employed to assess the inhibitory effect of lactucopicrin on the colony-forming ability of MDA-MB-468 cells.Potential targets of lactucopicrin were predicted via the PubChem and SwissTargetPrediction databases,while TNBC-related targets were retrieved from GeneCards,DisGeNET and OMIM.Intersection targets were identified using a Venn diagram generated by Venny 2.1.0 and further analyzed by constructing a"TNBC-Target-Lactucopicrin"interaction network Cytoscape 3.9.1.A protein-protein interaction(PPI)network was established using STRING database and visualized with Cytoscape 3.9.1 to identify core targets.The intersection targets were subjected to gene ontology(GO)functional enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.The molecular mechanism of lactucopicrin was validated by Western blotting.Results The in vitro antitumor activity screening demonstrated that lactucopicrin exhibited significant inhibitory effects on triple-negative breast cancer(TNBC)MDA-MB-468 cells.Clonogenic assays revealed that lactucopicrin effectively suppressed the proliferation and colony-forming capacity of MDA-MB-468 cells in a dose-dependent manner.Through database mining,the research identified 100 lactucopicrin-related targets and 3521 TNBC-associated targets.Venn diagram analysis yielded 45 overlapping targets,from which protein-protein interaction(PPI)network analysis identified 6 core targets:EGFR,MAPK1,PARP1,SRC,MAPK8 and IGF1R.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses indicated that the PI3K/AKT signaling pathway plays a pivotal role in lactucopicrin's therapeutic action against TNBC.The Western blot results indicated that,compared with the control group,the expression level of p-mTOR protein in the low-dose,medium-dose and high-dose lactucarium groups was decreased.The expression levels of p-AKT protein decreased in the very low,low and medium-dose groups of romaine lettuce,increased in the high-dose group,and increased in the expression levels of Caspase-3 and Bax proteins in the romaine lettuce experimental group.The differences were all statistically significant(P<0.05).Conclusion Lactucopicrin suppresses the proliferation and colony formation of triple-negative breast cancer(TNBC)MDA-MB-468 cells by targeting the AKT/mTOR signaling pathway,thereby exerting therapeutic effects against TNBC.
作者
努尔曼古丽·买买提明
弯东磊
郝锋
康金森
阿吉艾克拜尔·艾萨
杨建
Nuermanguli Maimaitiming;WAN Donglei;HAO Feng;KANG Jinsen;Ajiaikebaier Aisa;YANG Jian(School of Pharmacy,Xinjiang Medical University;Key Laboratory of Active Components,Xinjiang Natural Medicine and Drug Release Technology,Urumqi 830017,China;Kyinno Biotechnology,Beijing 100010,China)
出处
《新疆医科大学学报》
2025年第5期667-674,共8页
Journal of Xinjiang Medical University
基金
国家自然科学基金项目(82360721)
新疆维吾尔自治区重大科技专项子课题(2022A03018-4)。
