Action mechanism by which gambogic acid down-regulates expression of protein C receptor to kill triple negative breast cancer stem cells
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.
triple negative breast cancerstem cellnetwork pharmacologygambogic acidmolecular mechanismsignaling pathway
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