DAPK1 overcomes vincristine resistance in osteosarcoma by inhibiting ER-phagy through the ERK signaling pathway.

BACKGROUND: The treatment of osteosarcoma still relies on surgery combined with chemotherapy. However, the development of chemotherapy resistance severely weakens treatment efficacy and leads to poor prognosis. Endoplasmic reticulum‑selective autophagy (ER-phagy) can maintain intracellular homeostasis by clearing ER fragments, and has been reported to contribute to chemotherapy resistance. However, the role of ER-phagy in chemotherapy resistance in osteosarcoma remains unclear. This study aims to investigate the potential role and molecular mechanisms of death-associated protein kinase 1 (DAPK1) in vincristine (VCR) resistance in osteosarcoma. METHODS: VCR-resistant osteosarcoma cell lines, U2OS/VCR and MG63/VCR, were constructed. Effect of DAPK1 on VCR resistance was evaluated using qRT-PCR, Western blot, CCK-8, colony formation, and immunofluorescence. Furthermore, pathway activation experiments explored the involvement of the ERK pathway in the molecular mechanism of VCR resistance. Finally, a xenograft mouse model was established to validate the role of DAPK1 in vivo. RESULTS: DAPK1 expression was significantly reduced in VCR-resistant osteosarcoma cell lines. DAPK1 overexpression markedly suppressed MG63/VCR proliferation and enhanced its sensitivity to VCR. Mechanistically, DAPK1 overexpression downregulated p-ERK levels, thereby inhibiting ER-phagy, activating ER stress, and promoting apoptosis. Further analysis revealed that the ERK pathway activation partially reversed the inhibitory effect of DAPK1 overexpression on ER-phagy and weakened cellular sensitivity to VCR. Additionally, DAPK1 overexpression rendered MG63/VCR xenografts more sensitive to VCR treatment in vivo. CONCLUSION: We conclude that DAPK1 enhances osteosarcoma cell sensitivity to VCR by inhibiting ERK-mediated ER-phagy, which may represent a promising therapeutic strategy to overcome drug resistance.