title: Regulation of Intrinsic and Extrinsic Apoptotic Pathways in Osteosarcoma Cells Following Oleandrin Treatment.
abstract: Our previous study has reported the anti-tumor effect of oleandrin on osteosarcoma (OS) cells. In the current study, we mainly explored its potential regulation on intrinsic and extrinsic apoptotic pathway in OS cells. Cells apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected using fluorescence staining and flow cytometry. Caspase-3 activity was detected using a commercial kit. The levels of cytoplasmic cytochrome c, mitochondrial cytochrome c, bcl-2, bax, caspase-9, Fas, FasL, caspase-8 and caspase-3 were detected by Western blotting. z-VAD-fmk was applied to block both intrinsic and extrinsic apoptosis pathways, and cells apoptosis was also tested. Furthermore, we used z-LEHD-fmk and Fas blocking antibody to inhibit intrinsic and extrinsic pathways, separately, and the selectivity of oleandrin on these pathways was explored. Results showed that oleandrin induced the apoptosis of OS cells, which was accompanied by an increase in ROS and a decrease in MMP. Furthermore, cytochrome c level was reduced in mitochondria but elevated in the cytoplasm. Caspase-3 activity was enhanced by oleandrin in a concentration- and time-dependent manner. Oleandrin also down-regulated the expression of bcl-2, but up-regulated bax, caspase-9, Fas, FasL, caspase-8 and caspase-3. In addition, the suppression of both apoptotic pathways by z-VAD-fmk greatly reverted the oleandrin-induced apoptosis. Moreover, the suppression of one pathway by a corresponding inhibitor did not affect the regulation of oleandrin on another pathway. Taken together, we concluded that oleandrin induced apoptosis of OS cells via activating both intrinsic and extrinsic apoptotic pathways.
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, No. 49, North Garden Street, Haidian District, Beijing 100191, China. mayl_pth2012@163.com.
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Ma
authors.affiliation: The Center for Pain Medicine, Peking University Third Hospital, No. 49, North Garden Street, Haidian District, Beijing 100191, China. zhubin_ortho@163.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhu
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, No. 49, North Garden Street, Haidian District, Beijing 100191, China. eathonecho@163.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Yong
authors.affiliation: Department of Anesthesiology, Peking University Third Hospital, No. 49, North Garden Street, Haidian District, Beijing 100191, China. songchunyu0809@163.com.
authors.forename: Chunyu
authors.initials: C
authors.lastname: Song
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, No. 49, North Garden Street, Haidian District, Beijing 100191, China. lx86831@163.com.
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, No. 49, North Garden Street, Haidian District, Beijing 100191, China. yuhuileiah@126.com.
authors.forename: Huilei
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, No. 49, North Garden Street, Haidian District, Beijing 100191, China. wpspine@sina.com.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, No. 49, North Garden Street, Haidian District, Beijing 100191, China. zjlspine@163.com.
authors.forename: Zhongjun
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, No. 49, North Garden Street, Haidian District, Beijing 100191, China. xgliudoctor@163.com.
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Liu
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002298:Cardenolides / Q000494:pharmacology*; D002301:Cardiac Glycosides / Q000494:pharmacology*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053181:Caspase 8 / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D053222:Fas Ligand Protein / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction; D051028:bcl-2-Associated X Protein / Q000819:agonists / Q000235:genetics / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D057134:Antibodies, Neutralizing; D000972:Antineoplastic Agents, Phytogenic; C494826:BAX protein, human; C000595707:BCL2 protein, human; D002298:Cardenolides; D002301:Cardiac Glycosides; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D045304:Cytochromes c; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; C021065:oleandrin
keywords: apoptosis; caspases; oleandrin; osteosarcoma