Benzoylaconine suppresses colorectal cancer progression by modulating the CDK2/MMP2 signaling axis

Abstract

Colorectal cancer (CRC) has a high incidence, and both metastasis and drug resistance remain formidable challenges in clinical management. Owing to their multi-target activity and favorable safety profiles, natural products have become a prolific source for anticancer drug discovery. Benzoylaconine (BAC) is a secondary metabolite of aconitine with markedly attenuated toxicity that retains anti-inflammatory, analgesic, and antitumor properties; however, its antitumor effects and the underlying molecular mechanisms in CRC are poorly defined. This study was designed to systematically characterize the impact of BAC on the malignant phenotypes of CRC cells and to identify its potential molecular targets. HCT116 and SW480 cells were used as model systems. Proliferation was assessed by MTT and EdU assays; migration and invasion by wound-healing and Transwell assays, respectively; cell cycle and apoptosis by flow cytometry. Network pharmacology was applied to predict core targets and construct a protein–protein interaction network. Molecular docking and dynamics simulations examined binding; SPR verified direct binding to CDK2 and MMP2. RNA-seq identified differentially expressed genes and enriched pathways. Western blotting validated key protein changes. Patient-derived CRC organoids (PDOs) were established to evaluate BAC efficacy; organoid viability was assessed by CellTiter-Glo® 2.0 assay, and morphology by bright-field microscopy. BAC dose- and time-dependently inhibited CRC cell proliferation and EdU positivity, slowed wound closure, and reduced Transwell invasion, indicating impaired migration and invasion. Flow cytometric analysis revealed that BAC induced G1/S cell cycle arrest and apoptosis. Network pharmacology identified CDK2 and MMP2 as key targets; molecular dynamics and SPR confirmed stable binding (KD ≈ 3.9 and 4.4 μM). RNA-seq showed enrichment of pathways related to cell cycle regulation and tumor invasion. Western blotting revealed that BAC dose-dependently downregulated CDK2, CDK6, MMP2, Cyclin E1, and Cyclin D1, and upregulated p21. BAC also inhibited the proliferation of colorectal cancer organoids. BAC induces G1/S cell cycle arrest in CRC cells, likely through modulation of the CDK2/MMP2 signaling axis, thereby suppressing proliferation, migration, and invasion while promoting apoptosis, and may represent a promising lead for CRC therapy.