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BACKGROUND The purpose of this study was to explore the effects of the Na+/K+ ATPase inhibitor ouabain in regulating osteosarcoma (OS) cell stemness. MATERIAL AND METHODS Western blot, qPCR, sphere-forming analysis, DNA methylation analysis, and Ca²⁺ concentration detection were performed to evaluate the stem-like traits of cells and ouabain-induced effects and related mechanisms on OS cell stemness. Cell viability assessment was performed to evaluate the effect of ouabain on OS cell chemosensitivity. RESULTS Ouabain reduced the ALDH1 activity, the expression of critical stemness regulators, sphere size and number, and migration, invasion, and adhesion ability, but had little effects on cell viability. Additionally, the intracellular Ca²⁺ concentration and methylation level of the critical stemness regulators were higher in OS cells than in spheres formed by OS cells. Mechanistic studies revealed that ouabain leads to DNA methylation of stemness markers through increasing intracellular Ca²⁺ concentration. Notably, inhibition of Ca²⁺ channel or DNA methylation rescued the inhibition of ouabain on OS cell stemness. Additionally, ouabain enhances cisplatin sensitivity of OS cells, which is involved in Ca²⁺ channel and DNA methylation. CONCLUSIONS This work provides a potential compound for treating OS patients, especially OS patients with chemoresistance.

The Na+/K+ ATPase Inhibitor Ouabain Attenuates Stemness and Chemoresistance of Osteosarcoma Cells