Exploring the molecular link between arecoline exposure and prostate cancer-related alterations: integrative evidence from network toxicology, single-cell transcriptomics, molecular simulation, and experimental validation

Arecoline, the major alkaloid of areca nut, is a common exposure in chewing products, but its relationship with prostate cancer (PCa) is unclear. We integrated network toxicology, bulk RNA machine learning, single-cell transcriptomics, molecular simulation, and <i>in vitro</i> validation to prioritize candidate molecular nodes potentially linking arecoline exposure to PCa-related alterations. Potential arecoline targets were intersected with PCa-related genes, followed by protein-protein interaction and enrichment analyses. Candidate genes were prioritized using TCGA-PRAD and external GEO cohorts, then refined in GWAS-relevant epithelial subpopulations from GSE141445. AR was further evaluated by the Human Protein Atlas, molecular docking, molecular dynamics, RT-qPCR, and Western blotting. We identified 97 overlapping targets enriched mainly in apoptosis-, p53-, and MAPK-related pathways. The optimal bulk RNA model showed good external performance (AUC = 0.956). Integrative analyses identified androgen receptor (AR) as the only consensus core gene. AR-high epithelial cells showed increased androgen response, mTORC1 signaling, and MYC targets. Molecular simulation provided computational support for structural compatibility between arecoline and AR, while <i>in vitro</i> experiments showed increased AR mRNA and protein expression after arecoline treatment in LNCaP cells. These findings suggest AR as a plausible candidate molecular node linking arecoline exposure to PCa-related molecular alterations and provide a hypothesis-generating framework for future mechanistic and exposure-focused studies.