Xanthohumol Regulates Mitophagy in Osteosarcoma Cells via AMPK‐ULK1‐FUNDC1 Signaling Pathway

ABSTRACT Osteosarcoma (OS) is the most common primary bone malignancy. The therapeutic efficacy for OS patients has remained stagnant in recent decades. Xanthohumol (XN), a flavonoid naturally found in hops, has demonstrated significant anticancer properties in lung and breast cancer. However, its effect on OS and the underlying molecular mechanisms remains uncertain. Therefore, the purpose of this study is to explore the relationship between XN and OS. Firstly, we assessed the impact of XN on OS cell proliferation and migration using CCK‐8, wound‐healing, transwell, and clonogenicity assays. Subsequently, we examined the effect of XN on mitophagy in OS cells through flow cytometry, immunofluorescence, transmission electron microscopy, and western blot analysis. Finally, we constructed siRNA targeting AMPK to validate the pathway. In vitro, we demonstrated that XN inhibited the proliferation and migration of OS cells in a concentration‐ and time‐dependent manner. Furthermore, XN induced mitochondrial damage in OS cells and increased reactive oxygen species (ROS) levels. RNA‐seq analysis suggested a potential mitophagy pathway, which we confirmed experimentally by showing that XN reduced ATP levels, altered mitochondrial membrane potential, and increased the expression of Atg5, Beclin‐1, and LC3 proteins. Interestingly, the mitophagy inhibitor Mdivi‐1 reversed the damage caused by XN to OS cells. Furthermore, we found that XN induced mitophagy and exerted anti‐OS effects through the activation of the AMPK‐ULK1‐FUNDC1 signaling pathway, which was effectively reversed after AMPK knockdown. In vivo, we demonstrated the therapeutic potential of XN using a subcutaneous OS nude mouse model without any organ toxicity. XN emerges as a promising pharmaceutical agent for targeting OS.