title: Bufalin Induces Apoptosis of Human Osteosarcoma U-2 OS Cells through Endoplasmic Reticulum Stress, Caspase- and Mitochondria-Dependent Signaling Pathways.
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.
authors.affiliation: Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli Country 356, Taiwan. heecs@ms38.hinet.net.
authors.forename: Ching-Hsiao
authors.initials: CH
authors.lastname: Lee
authors.affiliation: Department of Pathology and Laboratory Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 111, Taiwan. t005524@ms.skh.org.tw.|School of Medical Laboratory Science and Biotechnology, Taipei Medical University, Taipei 110, Taiwan. t005524@ms.skh.org.tw.|School of Medicine, College of Medicine, Fu-Jen Catholic University, New Taipei City 242, Taiwan. t005524@ms.skh.org.tw.
authors.forename: Yung-Luen
authors.initials: YL
authors.lastname: Shih
authors.affiliation: Department of Genetic Counseling Center, Changhua Christian Hospital, Changhua 500, Taiwan. 29561@cch.org.tw.
authors.forename: Mei-Hui
authors.initials: MH
authors.lastname: Lee
authors.affiliation: Department of Orthopedics, Cheng Hsin General Hospital, Taipei 112, Taiwan. ch6215@chgh.org.tw.
authors.forename: Man-Kuan
authors.initials: MK
authors.lastname: Au
authors.affiliation: Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu 300, Taiwan. yunliang@mail2000.com.tw.
authors.forename: Yung-Liang
authors.initials: YL
authors.lastname: Chen
authors.affiliation: Restaurant, Hotel and Institutional Management, Fu-Jen Catholic University, New Taipei City 242, Taiwan. ch1835@chgh.org.tw.|Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei 112, Taiwan. ch1835@chgh.org.tw.
authors.forename: Hsu-Feng
authors.initials: HF
authors.lastname: Lu
authors.affiliation: Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan. jgchung@mail.cmu.edu.tw.|Department of Biotechnology, Asia University, Wufeng, Taichung 413, Taiwan. jgchung@mail.cmu.edu.tw.
authors.forename: Jing-Gung
authors.initials: JG
authors.lastname: Chung
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001859:Bone Neoplasms / Q000378:metabolism; D002018:Bufanolides / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D051017:Apoptosis Regulatory Proteins; D002018:Bufanolides; D017382:Reactive Oxygen Species; D020169:Caspases; D002118:Calcium; C022777:bufalin
keywords: bufalin; caspase-dependent signaling pathways; endoplasmic reticulum stress-dependent signaling pathways; human osteosarcoma U-2 OS cells; mitochondria-dependent signaling pathways