Osteosarcoma is an aggressive malignancy marked by a high incidence of local recurrence and distant metastasis, leading to poor outcomes in advanced stages. While current therapies offer long-term survival primarily for patients with localized disease, effective treatments for metastatic cases remain elusive. Addressing this critical gap, the present study explores, for the first time, the antimetastatic potential of urolithin A (UA), a naturally derived polyphenol, against osteosarcoma cells. Interactome mapping, gene enrichment profiling, target gene expression assessment, and molecular docking coupled with dynamics simulations were performed to elucidate the mechanistic basis of UA action. For experimental studies, UA was synthesized and its effects on osteosarcoma cell viability, apoptosis, migration, adhesion, invasion, and MMP activity were evaluated using alamarBlue assay, flow cytometry, scratch assay, fibronectin-based adhesion assay, Boyden chamber assay, and gelatin zymography, respectively. Results identified AKT1, EGFR, and MMP9 as potential targets of UA associated with osteosarcoma progression. Further analyses revealed critical interactions among these hub targets, with significant upregulation of AKT1 observed in osteosarcoma tissue samples. Molecular docking and dynamics simulations demonstrated strong and stable binding of UA to the kinase domain of AKT1 and the active site of EGFR. Experimental validation showed that treatment with UA significantly inhibited the migration and invasion of osteosarcoma cells, while notably enhancing cell adhesion. This anti-metastatic effect was closely linked to a marked reduction in enzymatic activity of MMP2 and MMP9, key mediators of metastatic dissemination. These findings position UA as a promising therapeutic candidate for targeting osteosarcoma metastasis.

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