Nobiletin enhances Doxorubicin sensitivity in osteosarcoma through ER stress-induced apoptosis mediated by the PI3K/AKT pathway.

Osteosarcoma (OS) is the most prevalent primary malignant bone tumor, and chemotherapy resistance represents one of the primary challenges in its treatment. Nobiletin (Nob), a natural compound with anti-tumor properties, has an unclear mechanism of action but may increase the sensitivity of tumor cells to chemotherapy agents. This study explores how Nob enhances the effects of Doxorubicin (Dox) on OS cells. Two OS cell lines (143B and U2OS) were used in vitro experiments, where different concentrations of Nob and Dox were combined to treat the cells. The CCK-8 was used to calculate the cell proliferation inhibition rate, and SynergyFinder 3.0 was employed to assess the synergistic concentration of the interventions. Colony formation and cell scratch assays were used to assess the proliferation and migration potential of the cells. Apoptosis levels were identified by flow cytometry, while the expression of proteins related to apoptosis, endoplasmic reticulum stress (ERS), and the PI3K-AKT pathway was detected by Western blotting and immunofluorescence. Moreover, qPCR was employed to measure the expression levels of associated mRNAs. GO and KEGG analyses were used to confirm the differential biological processes and signaling pathways in OS cells. A tumor xenograft model, complemented with H&E and immunohistochemistry staining, were employed to validate the anti-tumor effects and underlying biological mechanisms of the combined treatment with Nob and Dox. The combined treatment of Nob and Dox markedly inhibited both the proliferation and migration of OS cells, exhibiting a pronounced synergistic effect. The combination treatment led to an increased apoptosis rate, characterized by reduced expression of Bcl-2 and elevated levels of Bax and Caspase3. Additionally, the combination group significantly upregulated ERS-related proteins GRP78, CRT, CHOP and ATF6, while the activity of the PI3K-AKT pathway was notably diminished. In a tumor xenograft model, the combination of Nob and Dox significantly inhibited tumor growth, inducing ERS and apoptosis. This study reveals that Nob enhances Dox-induced ERS and apoptosis by inhibiting the PI3K-AKT pathway, thereby increasing the sensitivity of OS cells to Dox. This discovery offers a promising new strategy for the treatment of OS.