DIA Quantitative Proteomic Analysis Reveals Early Proteomic Responses to Honeybee-Derived Melittin in Murine Breast Cancer Cells

Breast cancer remains a major clinical challenge because of its molecular heterogeneity, therapeutic resistance, and disease recurrence. Melittin, the principal bioactive peptide of honeybee venom, exhibits antitumor activity in multiple experimental models; however, its early proteome-wide effects in breast cancer cells remain incompletely characterized. In this study, E0771 murine mammary carcinoma cells were exposed to melittin for 15 min. Colony formation and CCK-8 assays using 0–8 μg/mL melittin showed concentration-dependent reductions in clonogenic capacity and cell viability. For DIA-based quantitative proteomic profiling, E0771 cells were treated with 4 μg/mL melittin for 15 min. Among 9748 quantified protein entries, 339 proteins met the exploratory criteria for candidate differential abundance, including 114 up-regulated and 225 down-regulated candidates. GO and KEGG analyses associated these candidate proteins with cell junctions, cytoskeletal organization, protein kinase-related functions, metabolic processes, and cell-death-associated pathways. A STRING-derived association network containing 132 nodes and 216 edges further prioritized CASP8, RPS27A, ANXA5, and PIK3CA as relatively high-connectivity candidates. Collectively, these findings describe early proteomic responses to short-term melittin exposure and provide candidate proteins and biological processes for subsequent mechanistic validation rather than establishing causal molecular mechanisms.