Selective cytotoxicity of anhydroicaritin in ER-positive breast cancer via ESR1-mediated MAPK and apoptotic signaling.

Anhydroicaritin (AHI), a chemically characterized prenylated flavonoid, exhibits strong and selective cytotoxicity against estrogen receptor-positive (ER+) breast cancer cells. In this study, we aimed to elucidate its molecular and cellular toxicological mechanisms using an integrated strategy consisting of chemoinformatics, machine learning-based target prioritization, Mendelian randomization (MR) causal inference, and in vitro mechanistic assays. Network pharmacology analysis revealed that ESR1 (estrogen receptor 1) was ranked as the top hit hub gene and was further supported as a functional mediator of AHI action by machine learning models and MR analysis. Molecular docking and 100-ns molecular dynamics simulations demonstrated that AHI could form a stable and energetically preferred interaction with estrogen receptor 1 (ESR1). Subsequent mechanistic experiments in MCF-7 and ZR-75-1 cells revealed that AHI could attenuate the activity of MAPK signaling pathway and induce apoptosis by downregulating ESR1 mRNA expression through reducing ERα phosphorylation. Notably, AHI exhibited weak cytotoxicity against normal mammary epithelial cells, suggesting that it might exhibit selective toxicity toward malignant phenotypes. Our findings offered mechanistic evidence for the dual-level (post-translational and transcriptional) regulation of ESR1 signaling by AHI in ER+ breast cancer and suggested that AHI might be a subtype-specific chemotherapeutic lead compound.