Integrating network pharmacology to investigate the mechanism of esculetin from Nyctanthes arbor-tristis leaves against breast cancer.

UNLABELLED: The development of efficient treatment medicines with multitarget modes of action is necessary because breast cancer continues to be one of the leading causes of cancer-related mortality among women worldwide. Although esculetin (6,7-dihydroxycoumarin), a naturally occurring phenolic compound, has demonstrated promising anticancer activity, its molecular mechanism against breast cancer remains poorly understood. Therefore, this study aimed to investigate the therapeutic potential and underlying molecular mechanisms of esculetin against breast cancer using an integrated computational and experimental approach. We employed an integrated strategy that combined network pharmacology, density functional theory (DFT), molecular docking, molecular dynamics (MD) simulations, and experimental validation on MCF-7 cells. Firstly, we retrieved potential esculetin targets and breast cancer targets from multiple databases to identify overlapping targets. Later, we performed protein-protein interaction (PPI) network analysis and pathway enrichment analyses to identify key targets and associated signaling pathways. The electronic properties of esculetin were evaluated using DFT analysis. At the same time, molecular docking and simulations were conducted to assess protein-ligand interactions and complex stability, followed by MM-PBSA to determine the total energy. Lastly, experimental validation was performed in MCF-7 breast cancer cells using cell viability, live/dead, and reactive oxygen species (ROS) assays. A total of 591 overlapping targets were identified between esculetin and breast cancer. PPI network analysis highlighted EGFR, ERBB2, JAK2, PIK3CA, and PTPN11 as key hub targets. Pathway enrichment analyses indicated significant involvement of ERBB2-associated signaling pathways, including PI3K/AKT, SHP2, MAPK, and EGFR. DFT analysis demonstrated favorable electronic properties, supporting the molecular reactivity of esculetin. Molecular docking revealed that esculetin exhibits moderate binding affinities toward the identified hub protein ERBB2, while molecular simulation showed the structural stability of the esculetin-ERBB2 complex throughout the simulation. In vitro studies showed that esculetin reduced cell viability at 160 μM in MCF-7 cells, induced intracellular ROS accumulation, and promoted cell death as confirmed by AO/PI staining. Overall, this study provides a computationally informed framework for understanding the potential anticancer activity of esculetin in breast cancer. The predicted interaction between ERBB2 and the associated EGFR/ERBB2-PI3K/MAPK signaling network suggests a putative mechanism that warrants experimental validation. The findings, therefore, provide a basis for future target-specific and in vivo studies to establish the molecular relevance and therapeutic potential of esculetin.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s40203-026-00765-7.