A Bioinformatic Strategy for Investigating the Mechanism of Hispolon in the Treatment of Triple-Negative Breast Cancer Combined with In vitro Experiments.

Abstract

Hispolon, a phenolic compound isolated from the medicinal yellow fungal mulberry, exhibits a strong anti-triple-negative breast cancer (TNBC) effect. However, the antitumor mechanisms of Hispolon have not been fully explored. In this study, we systematically investigated the mechanism of Hispolon against TNBC based on bioinformatics and in vitro experiments. The Hispolon-related targets were first collected from the SwissTarget database. Differential Expression Genes (DEG) were screened between TNBC and normal breast tissue using the Gene Expression Comprehensive (GEO) dataset. The overlapping targets between Hispolon and DEG were analyzed by plotting Venn maps. Protein-protein interaction (PPI) network was constructed to analyze the interactions among these targets. The focus was on mining the core targets of anti-TNBC effects of Hispolon via the Cytohubba and MCODE plugin of Cytoscape 3.7.2 software. We performed survival analysis on these core targets to screen the best-matched targets, including EGFR, KIT, and PLAU. This correlated strongly with our validation of Hispolon by molecular docking. In addition, Gene Ontology (GO) anal-ysis and KEGG pathway analysis were performed using R software (ClusterProfiler package). Finally, in vitro experiments were performed to assess the accuracy of predicted target genes. The ADME results suggested that Hispolon has great potential to develop into a drug. Twenty overlapping targets were screened by matching the 107 targets of Hispolon to the 2,013 targets of TNBC DEG. Seven core targets of Hispolon against TNBC were initially identified, including EGFR, IGFBP3, MMP9, MMP2, MMP1, PLAU, and KIT. GO enrich-ment analysis demonstrated that the biological process of Hispolon acting on TNBC mainly involves lymphocyte activation in immune response and phosphatidylinositol-mediated signal-ing. Additionally, the relaxin signaling pathway, estrogen signaling pathway, proteoglycans in cancer, and others might be the key pathways of Hispolon against TNBC. Furthermore, Hispo-lon inhibited the proliferation of MDA-MB-231 cells in a concentration-dependent manner and regulated the RNA and protein expression of the core targets EGFR, PLAU, and KIT for the treatment of TNBC. In this study, the polygenic pharmacological mechanism of action of Hispolon against TNBC was explored through network pharmacology and in vitro experiments, provid-ing a new insight into the mechanism of TCM monomer against TNBC.