Integrating network pharmacology and experimental validation to elucidate the mechanism by which 2-Dihydroailanthone induces HCT116 cell death through suppressing autophagy via the AMPK/mTOR pathway.

BACKGROUND AND

objectiveColorectal cancer (CRC) is a highly prevalent and challenging malignancy worldwide, with chemotherapy resistance and tumor recurrence posing significant clinical hurdles. Previous studies have indicated that activation of protective autophagy under tumor microenvironment stress plays a key role in cancer cell survival and drug resistance. Consequently, inhibiting protective autophagy has emerged as a promising anti-CRC strategy. This study aims to investigate whether 2-Dihydroailanthone, an active constituent isolated from the bark of Ailanthus altissima (a traditional Chinese medicinal plant), induces CRC cell death by suppressing protective autophagy and to elucidate the underlying molecular mechanisms.

methodsNetwork pharmacology was initially employed to predict the potential targets of 2-Dihydroailanthone and to perform pathway enrichment analyses. Molecular docking evaluated its binding interactions with the core targets AMPK and mTOR. Human CRC HCT116 cells were used as an experimental model to assess cell viability, migration, and apoptosis via CCK-8 assay, wound healing assay, and flow cytometry, respectively. Ultrastructural changes and autophagosome formation were examined through transmission electron microscopy (TEM). Protein expression of AMPK/mTOR pathway components and autophagy markers (LC3B-II and p62) were analyzed by Western blotting. Pharmacological gain- and loss-of-function experiments employed the AMPK activator GSK621 and the inhibitor Compound C to validate pathway involvement.

resultsNetwork pharmacology revealed significant enrichment of 2-Dihydroailanthone’s potential targets in autophagy- and AMPK/mTOR-related pathways. Molecular docking demonstrated high-affinity binding of 2-Dihydroailanthone to the active sites of AMPK and mTOR. Functional assays showed that 2-Dihydroailanthone suppressed HCT116 cell viability and migration in a dose-dependent manner and notably induced apoptosis. TEM analyses revealed mitochondrial damage accompanied by reduced autophagosome formation. Correspondingly, Western blot analyses showed decreased LC3B-II levels and accumulation of p62, indicating suppressed autophagic flux. Mechanistically, 2-Dihydroailanthone inhibited AMPK phosphorylation and activated mTOR signaling. These effects were partially reversed by GSK621 and enhanced synergistically by Compound C.

conclusionThis study demonstrates, for the first time, that 2-Dihydroailanthone suppresses protective autophagy and induces apoptosis in HCT116 cells by modulating the AMPK/mTOR pathway. These findings suggest the potential of 2-Dihydroailanthone as a candidate anti-CRC agent and provide new molecular insights for CRC therapies targeting autophagy regulation.