Deciphering the Molecular Mechanisms of Gentisic Acid in Breast Cancer: Combining Network Pharmacology, Molecular Modelling, and Experimental Approaches

Abstract Gentisic acid (GA), a naturally occurring phenolic compound identified in , has demonstrated diverse pharmacological activities; however, its molecular mechanisms against breast cancer (BC) remain poorly understood. This study investigated the anti-BC potential of GA using an integrated systems pharmacology, computational, and experimental approach. The presence of GA in the ethanolic leaf extract of was confirmed by high-resolution mass spectrometry. Network pharmacology identified 604 overlapping genes between GA-predicted targets and BC-associated genes, with PIK3CA, PIK3R1, PTPN11, JAK2, and EGFR emerging as key hub genes. Functional enrichment highlighted EGFR- and SHP2-associated signalling as important regulatory pathways. Molecular docking demonstrated interactions of GA with the identified hub proteins, while molecular dynamics and MM-PBSA analyses supported the stability of the GA–EGFR complex. In vitro studies using MCF-7 cells showed that GA inhibited cell viability in a concentration-dependent manner, with an IC₅₀ of 40 µM, and suppressed cell migration and clonogenic survival. GA treatment also increased intracellular reactive oxygen species, reduced mitochondrial membrane potential, and promoted apoptosis, accompanied by increased Bax and caspase-3 and decreased Bcl-2 expression. Collectively, these findings suggest that GA exerts anti-BC effects through modulation of EGFR-associated signalling, oxidative stress, mitochondrial dysfunction, and apoptotic pathways, providing mechanistic support for further investigation of GA as a potential natural anti-cancer candidate.