Wen-Hsiang Chou1,2,3, Ko-Lin Liu4, Yung-Luen Shih4,5, Ying-Ying Chuang6, Jason Chou7, Hsu-Feng Lu6,8, Herng-Woei Jair6, Ming-Zhe Lee6, Man-Kuan Au9, Jing-Gung Chung10,11. 1. School of Medicine, National Defense Medical Center, Taipei, Taiwan, R.O.C. 2. Department of Orthopedics, Cheng Hsin General Hospital, Taipei, Taiwan, R.O.C. 3. Department of Information Management, St. Mary's Junior College of Medicine, Nursing and Management, Yilan, Taiwan, R.O.C. 4. Department of Pathology and Laboratory Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, R.O.C. 5. School of Medicine, College of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan, R.O.C. 6. Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei, Taiwan, R.O.C. 7. Department of Anatomic Pathology, Cheng Hsin General Hospital, Taipei, Taiwan, R.O.C. 8. Department of Restaurant, Hotel and Institutional Management, Fu-Jen Catholic University, New Taipei City, Taiwan, R.O.C. 9. Department of Orthopedics, Cheng Hsin General Hospital, Taipei, Taiwan, R.O.C. jgchung@mail.cmu.edu.tw ch1835@chgh.org.tw. 10. Department of Biological Science and Technology, China Medical University, Taichung, Taiwan, R.O.C. jgchung@mail.cmu.edu.tw ch1835@chgh.org.tw. 11. Department of Biotechnology, Asia University, Taichung, Taiwan, R.O.C.
Abstract
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 μM, significantly decreased the total viable cells and induced cell morphological changes in a time-dependent manner. It also time-dependently decreased G0/G1 phase and increased S and G2/M phase in U-2 OS cells. The production of ROS and the levels of MMPs (ΔΨm) 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 pro-apoptotic protein Bax and decreased the expression of anti-apoptotic 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. Copyright
BACKGROUND/AIM: Ouabain, a plant-derived product/substance with Na+/K+-ATPase inhibiting properties, has been shown to exert anti-cancer activity on humancancer cells. This is the first study to investigate the effect of ouabain on apoptotic cell death of humanosteosarcoma-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 μM, significantly decreased the total viable cells and induced cell morphological changes in a time-dependent manner. It also time-dependently decreased G0/G1 phase and increased S and G2/M phase in U-2 OS cells. The production of ROS and the levels of MMPs (ΔΨm) 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 pro-apoptotic protein Bax and decreased the expression of anti-apoptotic 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. Copyright
Authors: Marco Túlio C Pessôa; Jéssica M M Valadares; Sayonarah C Rocha; Simone C Silva; Jeff P McDermott; Gladis Sánchez; Fernando P Varotti; Cristóforo Scavone; Rosy I M A Ribeiro; José A F P Villar; Gustavo Blanco; Leandro A Barbosa Journal: Steroids Date: 2019-12-06 Impact factor: 2.668