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Molecules 2017, 22(3), 437; doi:10.3390/molecules22030437

Bufalin Induces Apoptosis of Human Osteosarcoma U-2 OS Cells through Endoplasmic Reticulum Stress, Caspase- and Mitochondria-Dependent Signaling Pathways

1
Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli Country 356, Taiwan
2
Department of Pathology and Laboratory Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 111, Taiwan
3
School of Medical Laboratory Science and Biotechnology, Taipei Medical University, Taipei 110, Taiwan
4
School of Medicine, College of Medicine, Fu-Jen Catholic University, New Taipei City 242, Taiwan
5
Department of Genetic Counseling Center, Changhua Christian Hospital, Changhua 500, Taiwan
6
Department of Orthopedics, Cheng Hsin General Hospital, Taipei 112, Taiwan
7
Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu 300, Taiwan
8
Restaurant, Hotel and Institutional Management, Fu-Jen Catholic University, New Taipei City 242, Taiwan
9
Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei 112, Taiwan
10
Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan
11
Department of Biotechnology, Asia University, Wufeng, Taichung 413, Taiwan
*
Authors to whom correspondence should be addressed.
Academic Editor: Maurizio Battino
Received: 22 December 2016 / Revised: 6 March 2017 / Accepted: 6 March 2017 / Published: 10 March 2017
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Abstract

Bone cancer is one of the cancer-related diseases, and there are increased numbers of patients with bone cancer worldwide. Therefore the efficacy of treatment of bone cancer is considered extremely vital. Bufalin has been showed to have biological activities including anticancer activities in vitro and in vivo. However, the exact associated mechanisms for bufalin induced apoptosis in human bone cancer cells are still unclear. In the present study, we investigated the effect of bufalin on the cytotoxic effects in U-2 OS human osteosarcoma cells. For examining apoptotic cell deaths, we used flow cytometry assay, Annexin V/PI double staining, and TUNNEL assay. Reactive oxygen species (ROS), Ca2+, mitochondrial membrane potential (ΔΨm), and caspase-8, -9 and -3 activities were measured by flow cytometry assay. Furthermore, western blotting and a confocal laser microscopy examination were used for measuring the alterations of apoptotic associated protein expression and translocation, respectively. The results indicated that bufalin induced cell morphological changes, decreased the viable cell number, induced apoptotic cell death, and increased the apoptotic cell number, and affected apoptotic associated protein expression in U-2 OS cells. Bufalin increased apoptotic proteins such as Bak, and decreased anti-apoptotic proteins such as Bcl-2 and Bcl-x in U-2 OS cells. Furthermore, bufalin increased the protein levels of cytochrome c (Cyto c), AIF (Apoptosis inducing factor) and Endo G (Endonuclease G) in cytoplasm that were also confirmed by confocal microscopy examination. Based on those findings, bufalin induced apoptotic cell death in U-2 OS cells may be via endoplasmic reticulum (ER) stress, caspase-, and mitochondria-dependent pathways; thus, we may suggest that bufalin could be used as an anti-cancer agent for the treatment of osteosarcoma in the future, and further in vivo studies are needed. View Full-Text
Keywords: bufalin; human osteosarcoma U-2 OS cells; endoplasmic reticulum stress-dependent signaling pathways; caspase-dependent signaling pathways; mitochondria-dependent signaling pathways bufalin; human osteosarcoma U-2 OS cells; endoplasmic reticulum stress-dependent signaling pathways; caspase-dependent signaling pathways; mitochondria-dependent signaling pathways
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MDPI and ACS Style

Lee, C.-H.; Shih, Y.-L.; Lee, M.-H.; Au, M.-K.; Chen, Y.-L.; Lu, H.-F.; Chung, J.-G. Bufalin Induces Apoptosis of Human Osteosarcoma U-2 OS Cells through Endoplasmic Reticulum Stress, Caspase- and Mitochondria-Dependent Signaling Pathways. Molecules 2017, 22, 437.

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