Potential multi-target inhibition of the PI3K-Akt pathway by Fraxetin suppresses colorectal cancer and restores 5-FU sensitivity in 5-FU-resistant CRC cells: An integrated study combining network pharmacology, molecular simulation, and in vitro validation.

Abstract

Colorectal cancer (CRC) is a malignancy with high global incidence and significant therapeutic challenges, where chemotherapy resistance is a key factor leading to treatment failure. Aberrant activation of the PI3K-Akt signaling pathway plays a central role in CRC progression and drug resistance. Fraxetin, a major active coumarin component of the traditional Chinese medicine Cortex Fraxini, possesses various biological activities, but its anti-CRC effects and underlying mechanisms remain unclear. This study aimed to systematically elucidate the mechanism of action of Fraxetin against CRC, particularly its regulation of the PI3K-Akt pathway and potential to reverse drug resistance, by integrating network pharmacology, computational simulation, and in vitro experiments. Network pharmacology screening identified 108 common targets of Fraxetin and CRC. Protein-protein interaction (PPI) analysis pinpointed 10 core targets, including TNF, AKT1, and EGFR. KEGG enrichment analysis suggested the PI3K-Akt pathway as one of the primary pathways involved. Molecular docking and dynamics simulations confirmed that Fraxetin could stably bind to core targets such as EGFR and ERBB2. In vitro experiments demonstrated that Fraxetin dose-dependently inhibited the proliferation of HCT116 and HT-29 cells, induced reactive oxygen species (ROS) generation, and significantly downregulated the phosphorylation levels of key PI3K-Akt pathway proteins, p-PI3K and p-Akt. Furthermore, Fraxetin combined with 5-fluorouracil (5-FU) or irinotecan exhibited synergistic antiproliferative effects and significantly restored the sensitivity of 5-FU-resistant cells to chemotherapeutic agents. This study systematically demonstrate that Fraxetin exerts multi-faceted effects against colorectal cancer, including anti-cancer activity, synergy with chemotherapy, and restoration of 5-FU sensitivity in 5-FU-resistant CRC cells in vitro, through multi-target inhibition of the PI3K-Akt signaling pathway.