Dynorphin A Impairs Mitochondrial Biogenesis in Osteosarcoma Cells by Increasing SP-1

Mitochondria are vital for energy generation, Apoptosis control, and cellular metabolism. As a result, they represent an attractive therapeutic target in Cancer treatment, particularly osteosarcoma (OS). Despite evidence indicating that Dynorphin A exhibits anti-tumor characteristics via multiple mechanisms, its influence on the physiology of osteosarcoma (OS) has not been thoroughly investigated. In this study, we explore the impacts of Dynorphin A on mitochondrial function and biogenesis within human OS U2OS cells. Human osteosarcoma (U2OS) cells were treated with Dynorphin A at varying concentrations for 48 h. Cell viability and cytotoxicity were assessed using the Cell Counting Kit-8 (CCK-8) and LDH assay, respectively. Mitochondrial function was evaluated by measuring complex IV activity, oxygen consumption rate (OCR), and ATP production, while mitochondrial biogenesis was analyzed by determining the mtDNA/nDNA ratio, mitochondrial protein expression (NDUFB8 and MTCO₂), and mitochondrial mass (MitoTracker Red staining). The expression of key mitochondrial regulators (PGC-1α, Nrf1, TFAM) and SP-1 was quantified using real-time RT-PCR and Western blot analysis. Our findings reveal that Dynorphin A notably decreases cell viability and enhances the release of Lactate Dehydrogenase (LDH)., indicating cytotoxicity. It also impaired mitochondrial function, as evidenced by a decrease in complex IV activity, oxygen consumption, and ATP production. Additionally, Dynorphin A suppressed mitochondrial biogenesis, shown by a reduced mtDNA/nDNA ratio, lower expression of mitochondrial proteins (NDUFB8 and MTCO2), and decreased mitochondrial mass. Furthermore, Dynorphin A downregulated key mitochondrial regulators, including PGC-1α, Nrf1, and TFAM. Notably, Dynorphin A also upregulated SP-1 expression, and silencing SP-1 reversed its effects on mitochondrial function and biogenesis. These findings suggest that Dynorphin A exerts antitumor effects by disrupting mitochondrial function and biogenesis in OS cells.

Dynorphin A; PGC‐1α; SP‐1; mitochondrial biogenesis; osteosarcoma.