Inhibition of the PI3K signaling pathway in cancer cells by Agrimonia eupatoria L. ethanolic extract: identification of tricoumaroyl spermidine as a potential PI3K inhibitor.

BACKGROUND: Cancer remains one of the most significant global health challenges, requiring continuous efforts to identify novel anticancer agents. Agrimonia eupatoria L. (AE), is a perennial herb with diverse therapeutic properties, showing promise in preclinical studies for its anticancer potential. The aim of this study was to investigate the inhibitory effect of the AE extract on cancer cells in vitro and assess its impact on the phosphoinositide 3-kinase (PI3K) signaling pathway, a key regulator of cancer-related processes and one of its potential targets. METHODS: Metabolomic profiling of the AE ethanol extract composition was done using an advanced LC-Q-Orbitrap HRMS technique. The MTT assay was used to assess the cytotoxicity of the AE extract against four human cancer cell lines (MCF-7, HT-29, A549, HeLa). PI3K signaling pathway was elucidated with an In-Cell ELISA assay, WB, ICC/IF, and molecular docking identified potential PI3K inhibitors. RESULTS: MTT results showed significant cytotoxicity of the AE extract across all tested cell lines. A cellular antioxidant activity assay revealed a pro-oxidant effect in cancer cells, a process linked to PI3K/Akt regulation. The AE extract reduced both total and phosphorylated PI3K, and indicating inhibition of the PI3K/Akt/mTOR/COX-2/MMP-2/HIF1a axis. Molecular docking identified tricoumaroyl spermidine as a potential PI3K inhibitor with high binding affinity. CONCLUSION: These findings support the potential of AE as a source of novel anticancer agents. However, this study has limitations, being confined to in vitro and in silico models; the extract’s overall biological activity is likely due to the synergistic effects of multiple constituents. Future in vivo validation and pharmacokinetic studies are necessary. Despite these challenges, tricoumaroyl spermidine was identified as a promising lead compound for further development as a PI3K inhibitor.