Kaempferol targets Src to exert its chemopreventive effects on mammary tumorigenesis via regulation of the PI3K/AKT pathway

Background: Breast cancer (BC) is a prevalent malignancy that poses significant risks to the health of women worldwide. The incidence and mortality rates of BC continue to be high, despite improvements in diagnosis and treatment, indicating a need for novel prevention strategies. Kaempferol (KAM) is a common dietary flavonoid with known antitumour properties, but its role in the chemoprevention of BC and the underlying mechanisms largely unexplored. Purpose: This study aimed to evaluate the chemopreventive effects of KAM on carcinogen-induced BC in vivo and in vitro and to elucidate the underlying molecular mechanisms. Methods: In this study, we used an N-methyl-N-nitrosourea (NMU)-induced rat model of BC and 17(3-oestradiol (E2)-treated MCF-10A cells to evaluate the chemopreventive effects of KAM on mammary tumorigenesis. The antioxidant capacity of KAM was assessed by measuring oxidative damage marker levels and antioxidant enzyme expression. Flow cytometry and Hoechst 33258 staining were utilized to analyse cell cycle distribution and apoptosis. The core target of KAM was identified by network pharmacology and validated by molecular docking, MD simulation, CESTA, and BLI. KEGG enrichment analysis, molecular biology tests and the application of specific protein inhibitors were conducted to elucidate the molecular mechanisms modulated by KAM. Results: In vivo, KAM inhibited the progression of mammary tumours and delayed pathological changes in the morphological structure of mammary gland cells to varying degrees. In vitro, KAM reduced cell viability, migration, and anchorage-independent growth while triggering cell cycle arrest and apoptosis in E2-treated MCF-10A cells. Furthermore, KAM increased cellular antioxidant capacity and attenuated E2-induced oxidative stress. Mechanistically, KAM directly interacted with Src and inhibited its phosphorylation, thus leading to PI3K/AKT pathway inhibition. Notably, the inhibition of E2-induced cell migration and anchorage-independent growth in vitro by Src-or PI3K/AKT pathway-specific inhibitors was not further enhanced when the cells were cultured with KAM. Conclusion: In summary, KAM targets the Src-mediated PI3K/AKT pathway to reduce oxidative stress and facilitate apoptosis and cell cycle arrest, thereby inhibiting mammary tumorigenesis. Our study is the first to identify Src kinase as a direct target of KAM in mammary tumorigenesis. These findings give significant perspectives on the potential application of KAM in BC chemoprevention.