Mechanistic insights into gallic acid-induced autophagy in triple negative breast cancer through network pharmacology, molecular docking, and in vitro validation.

Gallic acid (GA), a natural polyphenol, has been enormously recognized for its anticancer properties across multiple cancer types, including triple-negative breast cancer (TNBC), yet its precise molecular mechanisms underlying its effects on TNBC, particularly those related to autophagy, remain unclear. This investigation sought to elucidate the autophagy-related targets and pathways modulated by GA in TNBC by integrating network pharmacology, bioinformatics, molecular docking, and in vitro validation. Network pharmacology identified 23 overlapping targets between GA, TNBC, and autophagy-related gene sets, including key genes such as CASP3, TP53, AKT1, PIK3CA, and HIF-1 A. Protein-Protein Interaction network analysis revealed ten hub genes as central nodes in GA's action mechanism. Gene Ontology enrichment highlighted biological processes such as DNA damage response, protein phosphorylation, and regulation of apoptosis and autophagy. KEGG pathway analysis further indicated enrichment in pathways related to EGFR inhibitor resistance, insulin signaling, autophagy, and apoptosis, among others. Molecular docking demonstrated strong binding affinities between GA and the identified autophagy modulatory genes. In vitro experiments showed that GA inhibited the proliferation and migration of BT-549 and HCC1395 TNBC cells while inducing autophagy. Induction of autophagy by GA in the BT-549 and HCC1395 cell lines was proved by acridine orange, MAP1LC3B fluorescence, and western blotting of key autophagy regulators. Taken together, this integrated approach, combining network pharmacology and experimental validation, identified key targets and signaling pathways through which GA regulates autophagy in TNBC. These findings enhance our understanding of GA's anticancer mechanisms and support its plausible development as a therapeutic entity for TNBC.