Abstract

Osteosarcoma is an aggressive malignancy characterized by rapid proliferation and a propensity for metastasis. The endoplasmic reticulum (ER) chaperone GRP78, a critical regulator of osteosarcoma progression, represents a promising therapeutic target. In this study, we identified the natural compound ginkgetin (Gink) as a novel GRP78 inhibitor. Cellular thermal shift assays, surface plasmon resonance, and mutagenesis studies revealed that Gink directly binds to GRP78, with K296 serving as a key interaction site. In vitro, Gink suppressed osteosarcoma cell proliferation, migration, and invasion while inducing apoptosis and autophagy by activating the PERK-eIF2α-ATF4 pathway. Co-immunoprecipitation assays showed that Gink competitively disrupted GRP78-PERK interaction. In orthotopic and patient-derived xenograft models, Gink treatment markedly attenuated tumor growth and metastasis. Gink also reprogrammed the tumor immune microenvironment by reducing M2 macrophage polarization and synergizing with anti-PD1 therapy to enhance CD8 + T-cell activity. Accordingly, Gink could be developed as a GRP78-targeting agent that triggers ER stress and immune activation, offering a dual-pronged therapeutic strategy against osteosarcoma. Ginkgetin (Gink) directly binds to GRP78 in a competitive manner, disrupting the interaction between GRP78 and PERK. This leads to PERK activation and phosphorylation, which in turn phosphorylates eIF2α to trigger ATF4 transcription. Ultimately, this cascade induces apoptosis and autophagy, inhibiting cancer progression. Additionally, Gink suppresses M2 macrophage polarization and enhances CD8 + T cell cytotoxicity, both of which contribute to the prevention of cancer development.