中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (23): 4888-4898.doi: 10.12307/2025.089

• 肿瘤干细胞 cancer stem cells • 上一篇    下一篇

藤黄酸下调蛋白C受体表达杀伤三阴性乳腺癌干细胞的作用机制

李  溯1，王清华2，达梦婷3，杨  蕊2，陈道桢1，4   

  1. 1南京医科大学附属无锡妇幼保健院优生优育医学遗传研究所，江苏省无锡市   214002；2江南大学附属妇产医院优生优育医学遗传研究所，江苏省无锡市   214002；3青海大学附属医院乳腺疾病诊疗中心，青海省西宁市   810000；4青海省海东市第二人民医院院长办公室，青海省海东市   810600
  • 收稿日期:2023-12-18 接受日期:2024-05-17 出版日期:2025-08-18 发布日期:2024-09-27
  • 通讯作者: 陈道桢，教授，博士生导师，南京医科大学附属无锡妇幼保健院优生优育医学遗传研究所，江苏省无锡市 214002；青海省海东市第二人民医院院长办公室，青海省海东市 810600
  • 作者简介:李溯，男，1996年生，江苏省宿迁市人，汉族，南京医科大学在读硕士，主要从事综合肿瘤治疗研究。
  • 基金资助:
    青海省自然科学基金面上项目(2022-ZJ-912)，项目负责人：陈道桢

Action mechanism by which gambogic acid down-regulates expression of protein C receptor to kill triple negative breast cancer stem cells

Li Su1, Wang Qinghua2, Da Mengting3, Yang Rui2, Chen Daozhen1, 4   

  1. 1Department of Eugenic Genetics Institute, Affiliated Wuxi Maternity and Child Health Care Hospital of Nanjing Medical University, Wuxi 214002, Jiangsu Province, China; 2Eugenic Genetics Institute, Jiangnan University Maternity Hospital, Wuxi 214002, Jiangsu Province, China; 3Breast Disease Treatment Center, Medical College of Qinghai University, Xining 810000, Qinghai Province, China; 4Dean’s Office, Second People’s Hospital of Haidong City, Haidong 810600, Qinghai Province, China
  • Received:2023-12-18 Accepted:2024-05-17 Online:2025-08-18 Published:2024-09-27
  • Contact: Chen Daozhen, Professor, Doctoral supervisor, Department of Eugenic Genetics Institute, Affiliated Wuxi Maternity and Child Health Care Hospital of Nanjing Medical University, Wuxi 214002, Jiangsu Province, China; Dean’s Office, Second People’s Hospital of Haidong City, Haidong 810600, Qinghai Province, China
  • About author:Li Su, Master candidate, Department of Eugenic Genetics Institute, Affiliated Wuxi Maternity and Child Health Care Hospital of Nanjing Medical University, Wuxi 214002, Jiangsu Province, China
  • Supported by:
    Qinghai Provincial Natural Science Foundation (General Project), No. 2022-ZJ-912 (to CDZ)

PDF

16

摘要：

文题释义：

藤黄酸：是从传统中药藤黄中提取的化合物，抗癌作用与一般化疗药不同，对肿瘤细胞的抑制有较高的选择性，能选择性杀伤肿瘤细胞且在有效剂量范围内毒副作用较小，对正常动物造血及免疫系统功能没有影响。
蛋白C受体：是一种内皮Ⅰ型跨膜受体，可增强凝血酶(Ⅱa) -凝血调节蛋白复合物对蛋白C的激活作用。诸多证据表明，蛋白C受体在乳腺干性和人类肿瘤发生中也发挥着作用。最近，蛋白C受体被鉴定为多能性小鼠乳腺干细胞的标记物，同时也被证明在3D培养中对于人乳腺上皮细胞的细胞组织和生长是必需的。

摘要
背景：藤黄酸对乳腺癌具有很强的细胞毒性，可有效杀伤三阴性乳腺癌干细胞，但潜在的机制尚不清楚。
目的：探讨藤黄酸对三阴性乳腺癌干细胞的杀伤作用及可能的机制。
方法：使用PharmMapper数据库预测藤黄酸的靶点蛋白，使用String网站构建各药物靶点蛋白互作网络关系，使用Cytoscape软件构建活性成分-作用靶点网络，通过R语言软件对潜在靶点进行KEGG信号通路富集分析。采用CCK-8法检测不同浓度藤黄酸对人乳腺癌细胞株MDA-MB-231活力的影响，筛选适宜的作用浓度。采用细胞球培养法富集MDA-MB-231细胞干细胞，经过不同浓度(0，0.5，1.0，2.0 μmol/L)藤黄酸作用24 h，TUNEL荧光染色与流式细胞仪检测干细胞凋亡，qPCR与Western blot检测蛋白C受体表达，Western blot检测p-PI3K、p-AKT、Caspase-3和Cleaved Caspase-3蛋白表达；将干细胞分4组培养：空白对照组(干细胞不做任何处理)、siRNA-NC组、siRNA-蛋白C受体组和siRNA-蛋白C受体+PI3K激动剂组，培养36 h后，采用Western blot法检测p-PI3K、p-AKT、Caspase-3和Cleaved Caspase-3蛋白表达。

结果与结论：①网络药理学发现，三阴性乳腺癌干细胞标志物蛋白C受体是藤黄酸的作用靶点之一；KEGG富集分析涉及细胞凋亡、上皮生长因子受体、RAS和PI3K-AKT信号通路等；②CCK-8检测结果显示，藤黄酸可抑制MDA-MB-231细胞活力，半数抑制浓度IC50值为(1.18±0.34) μmol/L，因此后续实验选择浓度0.5，1.0，2.0 μmol/L；③TUNEL荧光染色和流式细胞术检测证实，藤黄酸以剂量依赖的方式诱导三阴性乳腺癌干细胞凋亡(P < 0.05)；qPCR和Western blot检测证实，藤黄酸可下调蛋白C受体mRNA和蛋白表达，下调Caspase-3、p-PI3K和p-AKT蛋白表达，上调Cleaved Caspase-3蛋白表达(P < 0.05)；siRNA-蛋白C受体转染实验也进一步证实，敲低三阴性乳腺癌干细胞蛋白C受体表达可提升Cleaved Caspase-3蛋白表达(P < 0.05)、下调PI3K/AKT信号通路磷酸化水平(P < 0.05)，而应用PI3K激动剂740 Y-P能够降低Cleaved Caspase-3蛋白表达(P < 0.05)、提升p-PI3K和p-AKT的磷酸化水平(P < 0.05)，一定程度上改善细胞凋亡情况；④结果表明，藤黄酸可能通过下调蛋白C受体来发挥三阴性乳腺癌及干细胞杀伤及诱导凋亡作用，进一步的分子机制可能和PI3K/AKT信号通路抑制有关。

https://orcid.org/0000-0001-5779-3476 (李溯) 


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 三阴性乳腺癌, 干细胞, 网络药理学, 藤黄酸, 分子机制, 信号通路

Abstract: BACKGROUND: Gambogic acid is highly cytotoxic to breast cancer and can effectively kill triple negative breast cancer stem cells, but the underlying mechanism is still unclear.
OBJECTIVE: To investigate the lethal effect of gambogic acid on triple negative breast cancer stem cells as well as the possible mechanisms. 
METHODS: PharmMapper database was used to predict the target protein of gambogic acid. String website was used to construct the protein interaction network of various drug targets. Active ingredient-target network was constructed by Cytoscape software. KEGG signal pathway enrichment analysis was performed on potential targets by R language software. The effect of different concentrations of gambogic acid on the activity of human breast cancer cell line MDA-MB-231 was detected by CCK-8 assay. The appropriate concentration was screened. MDA-MB-231 stem cells were enriched by cell ball culture method and treated with gambogic acid at different concentrations (0, 0.5, 1.0, and 2.0 μmol/L) for 24 hours. TUNEL fluorescence staining and flow cytometry were used to detect apoptosis of stem cells. qPCR and western blot assay were used to detect protein C receptor expression. The expression levels of p-PI3K, p-AKT, Caspase-3, and cleaved Caspase-3 were detected by western blot assay. Stem cells were cultured in four groups: Blank control group (stem cells were not treated), siRNA-NC group, siRNA-protein C receptor group, and siRNA-protein C receptor + PI3K agonist group. After culture for 36 hours, the expression levels of p-PI3K, p-AKT, Caspase-3, and cleaved Caspase-3 were detected by western blot assay.
RESULTS AND CONCLUSION: (1) Network pharmacology exhibited that the protein C receptor, a marker of triple negative breast cancer stem cells, was one of the targets of gambogic acid. KEGG enrichment analysis involved apoptosis, epithelial growth factor receptor, RAS, and PI3K-AKT signaling pathways. (2) CCK-8 assay results showed that gambogic acid could inhibit the viability of MDA-MB-231 cells, and the median inhibitory concentration IC50 value was (1.18±0.34) μmol/L, so the concentrations of 0.5, 1.0, and 2.0 μmol/L were selected for subsequent experiments. (3) TUNEL fluorescence staining and flow cytometry showed that gambogic acid induced apoptosis of triple negative breast cancer stem cells in a dose-dependent manner (P < 0.05). qPCR and western blot assay confirmed that gambogic acid down-regulated mRNA and protein expression of protein C receptor, down-regulated Caspase-3, p-PI3K, and p-Akt protein expression, and up-regulated cleaved Caspase-3 protein expression (P < 0.05). siRNA-protein C receptor transfection experiments further confirmed that knockdown of protein C receptor expression in triple negative breast cancer stem cells increased cleaved Caspase-3 protein expression (P < 0.05), and down-regulated phosphorylation of PI3K/AKT signaling pathway (P < 0.05). Application of PI3K agonist 740 Y-P decreased cleaved Caspase-3 protein expression (P < 0.05), increased phosphorylation levels of p-PI3K and p-AKT (P < 0.05), and improved apoptosis to a certain extent. (4) The results show that gambogic acid may play a role in killing and inducing apoptosis of triple negative breast cancer stem cells by down-regulating protein C receptor, and the further molecular mechanism may be related to the inhibition of PI3K/AKT signaling pathway.

Key words: triple negative breast cancer, stem cell, network pharmacology, gambogic acid, molecular mechanism, signaling pathway

中图分类号: