Ouabain Induces Apoptotic Cell Death Through Caspase- And Mitochondria-Dependent Pathways In Human Osteosarcoma U-2 Os Cells

Background/Aim: Ouabain, a plant-derived product/substance with Na+/K+-ATPase inhibiting properties, has been shown to exert anti-cancer activity on human cancer cells. This is the first study to investigate the effect of ouabain on apoptotic cell death of human osteosarcoma-derived U-2 OS cells. Materials and Methods: Flow cytometry was used to examine cell viability, cell cycle, and reactive oxygen species (ROS), Ca2+, mitochondrial membrane potential (MMP) and caspase activity. Morphological changes were examined by contrast-phase microscopy, while apoptosis-associated protein levels were analyzed by western blot. Results: Ouabain, at concentrations of 5-60 mu M, significantly decreased the total viable cells and induced cell morphological changes in a time-dependent manner. It also time-dependently decreased G(0)/G(1) phase and increased S and G(2)/M phase in U-2 OS cells. The production of ROS and the levels of MMPs (Delta Psi(m)) were inhibited, while Ca2+ production in U-2 OS cells was increased. Regarding cell apoptosis, flow cytometry assay revealed increased caspase-3, -8, and -9 activities in U-2 OS cells. Moreover, western blot results showed that ouabain increased the expression of proapoptotic protein Bax and decreased the expression of antiapoptotic protein Bcl-2 in U-2 OS cells. Furthermore, results also showed that ouabain increased cytochrome c release, apoptosis-inducing factor (AIF) and endonuclease (Endo) G that is associated with apoptosis through caspase-dependent and -independent pathway in U-2 OS cells. Conclusion: Our findings provide important insight into the cytotoxic effects of ouabain on U-2 OS cells, in vitro, which are mediated at least partly via cell apoptosis induction.