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

doi:10.12307/2025.089

Abstract

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

CLC Number:

Li Su, Wang Qinghua, Da Mengting, Yang Rui, Chen Daozhen. Action mechanism by which gambogic acid down-regulates expression of protein C receptor to kill triple negative breast cancer stem cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(23): 4888-4898.

Figures/Tables 7

2.2 藤黄酸诱导三阴性乳腺癌干细胞的凋亡作用 TUNEL染色结果显示，藤黄酸以剂量依赖方式诱导三阴性乳腺癌干细胞凋亡，见图3A，表明藤黄酸能够有效杀伤三阴性乳腺癌干细胞。为进一步定量评估藤黄酸诱导三阴性乳腺癌干细胞的凋亡杀伤作用，采用Annexin V-FITC/PI双染法进行流式细胞分析，见图3B，C所示，伴随藤黄酸浓度的增加，三阴性乳腺癌干细胞的凋亡率从30%上升至约80%(P < 0.001)。进一步通过Western blot实验验证凋亡相关蛋白Caspase-3和Cleaved Caspase-3的表达，结果显示，与对照组比较，各浓度藤黄酸干预后三阴性乳腺癌干细胞的凋亡前体蛋白Caspase-3表达降低，活化凋亡蛋白Cleaved Caspase-3升高；与0.5 μmol/L藤黄酸组比较，1.0，2.0 μmol/L藤黄酸组Caspase-3蛋白表达降低，Cleaved Caspase-3蛋白表达升高，具体的组间统计学差异见图3D-F。 2.3 网络药理学证实蛋白C受体是藤黄酸的作用靶点之一依据藤黄酸反向寻靶PharmMapper(http://lilab-ecust.cn/pharmmapper/)数据库筛选结果，预测和藤黄酸作用的靶点蛋白，根据匹配度排序，筛选出前10个潜在分子靶点(表1)，结果表明，收纳阴性乳腺癌干细胞标记物蛋白C受体是藤黄酸的作用靶点之一且匹配度较高，为藤黄酸靶向杀伤三阴性乳腺癌细胞提供可能。 2.4 藤黄酸药物靶点蛋白相互作用网络与蛋白质相互作用利用String在线分析各药物靶点蛋白相互作用网络，筛选阈值0.98并去除离散的靶点蛋白，得到靶点蛋白相互作用图，见图4A。此外，进一步将藤黄酸与靶点蛋白信息导入Cytoscape 3.7.1软件构建活性成分-作用靶点网络，见图4B，C所示，共涉及148个节点、397个边。经计算蛋白相互作用网络的2个关键拓扑参数度和介数确认网络中的核心节点，其中蛋白激酶B、生长因子受体结合蛋白2、非受体酪氨酸激酶、热休克蛋白90α家族A类成员1和蛋白酪氨酸磷酸酶非受体11型等蛋白度值≥10(表2)，表明该节点在网络中越重要。从中可看出藤黄酸药效团可作用于多个靶点基因，充分体现其多靶点的作用特点。"

2.5 藤黄酸的KEGG通路富集分析基于KEGG富集分析藤黄酸作用的138个信号通路，建立了基因通路网络、相应的靶基因，并筛选出P值显著富集的前20个信号通路，如图5显示，包括细胞凋亡、上皮生长因子受体、RAS和PI3K-AKT信号通路等，与癌变信号通路关联的占比约50%；此外，PI3K-AKT信号通路对藤黄酸杀伤作用的贡献最大。 2.6 藤黄酸下调三阴性乳腺癌干细胞中蛋白C受体mRNA和蛋白表达 qPCR检测结果显示，与对照组相比，0.5 μmol/L藤黄酸组蛋白C受体mRNA表达无明显变化(P > 0.05)，1.0，2.0 μmol/L藤黄酸组蛋白C受体mRNA表达降低(P < 0.01，P < 0.001)；0.5，1.0 μmol/L藤黄酸组蛋白C受体mRNA表达高于2.0 μmol/L藤黄酸组(P < 0.01，P < 0.05)，见图6A，表明藤黄酸可能通过下调蛋白C受体mRNA表达实现对三阴性乳腺癌干细胞的杀伤作用。此外，利用Western blot检测验证藤黄酸作用干细胞后蛋白C受体蛋白表达情况，结果显示：与对照组相比，0.5，1.0，2.0 μmol/L藤黄酸组蛋白C受体的蛋白表达降低(P < 0.05，P < 0.01，P < 0.001)；0.5，1.0 μmol/L藤黄酸组蛋白C受体蛋白表达高于2.0 μmol/L藤黄酸组(P < 0.01，P < 0.05)，见图6B，C。 2.7 藤黄酸对三阴性乳腺癌干细胞PI3K/AKT信号通路磷酸化蛋白表达的影响根据此前网络药理学的预测结果，PI3K/AKT信号通路在藤黄酸发挥杀伤作用中起到重要作用，因此采用Western blot检测该相关通路磷酸化蛋白表达情况。 Western blot检测结果显示，与对照组相比，各浓度藤黄酸组p-PI3K和p-AKT蛋白表达都呈现了不同程度的下调趋势，见图7A。定量分析结果显示，与对照组相比，0.5 μmol/L藤黄酸组p-PI3K和p-AKT蛋白表达无明显变化(P > 0.05)，1.0 μmol/L藤黄酸组p-PI3K蛋白表达降低(P < 0.01)、p-AKT蛋白表达无明显变化(P > 0.05)，2.0 μmol/L藤黄酸组p-PI3K蛋白与p-AKT蛋白表达降低(P < 0.001， P < 0.01)；2.0 μmol/L藤黄酸组p-PI3K、p-AKT蛋白表达均低于0.5 μmol/L藤黄酸组(P < 0.001，P < 0.05)，1 μmol/L藤黄酸组p-PI3K蛋白表达低于0.5 μmol/L藤黄酸组(P < 0.05)、高于2.0 μmol/L藤黄酸组(P < 0.05)，见图7B，C。"

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