Abstract

Background and objective Colorectal 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. Methods Network 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. Results Network 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.