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      Inhibitory effects of evodiamine on human osteosarcoma cell proliferation and apoptosis

      research-article
      , , ,
      Oncology Letters
      D.A. Spandidos
      evodiamine, osteosarcoma cells, proliferation, apoptosis

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          Abstract

          Osteosarcoma is a primary malignancy of bone, which is characterized by the proliferation of malignant mesenchymal cells, particularly in children and adolescents. Evodiamine is extracted from a variety of traditional Chinese medicines, which has been reported to induce apoptosis in certain tumors, including cervical, prostate and breast cancer, however, its effect on oestosarcoma cells remains unclear. The aim of the present study was to investigate the effect of evodiamine on osteosarcoma cell proliferation and apoptosis, and explore the associated underlying molecular mechanism. A Cell Counting Kit 8 assay was performed to detect the effects of evodiamine on the proliferation of human osteosarcoma U2OS cells. Annexin V-fluorescein isothiocyanate/propidium iodide staining was performed to analyze the apoptotic rate of the cells. The effect of evodiamine on the protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3 and survivin were detected by performing western blot analysis. Evodiamine inhibited the growth of human osteosarcoma U2OS cells by inhibiting cell proliferation and inducing cell apoptosis. Western blotting demonstrated that evodiamine downregulated the expression of Bcl-2, caspase-3 and survivin, and upregulated the expression of Bax in human osteosarcoma cells. Evodiamine effectively inhibited proliferation and induced apoptosis of osteosarcoma cells in a dose-dependent manner via downregulation of Bcl-2, caspase-3 and survivin protein expression levels and upregulation of Bax protein expression levels.

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          Most cited references33

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          Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.

          Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria. Overexpression of Bcl-2 prevents cells from undergoing apoptosis in response to a variety of stimuli. Cytosolic cytochrome c is necessary for the initiation of the apoptotic program, suggesting a possible connection between Bcl-2 and cytochrome c, which is normally located in the mitochondrial intermembrane space. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria. Overexpression of Bcl-2 prevented the efflux of cytochrome c from the mitochondria and the initiation of apoptosis. Thus, one possible role of Bcl-2 in prevention of apoptosis is to block cytochrome c release from mitochondria.
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            Bcl-2 functions in an antioxidant pathway to prevent apoptosis.

            Bcl-2 inhibits most types of apoptotic cell death, implying a common mechanism of lethality. Bcl-2 is localized to intracellular sites of oxygen free radical generation including mitochondria, endoplasmic reticula, and nuclear membranes. Antioxidants that scavenge peroxides, N-acetylcysteine and glutathione peroxidase, countered apoptotic death, while manganese superoxide dismutase did not. Bcl-2 protected cells from H2O2- and menadione-induced oxidative deaths. Bcl-2 did not prevent the cyanide-resistant oxidative burst generated by menadione. Two model systems of apoptosis showed no increment in cyanide-resistant respiration, and generation of endogenous peroxides continued at an inherent rate that was unaltered by Bcl-2. Following an apoptotic signal, cells sustained progressive lipid peroxidation. Overexpression of Bcl-2 functioned to suppress lipid peroxidation completely. We propose a model in which Bcl-2 regulates an antioxidant pathway at sites of free radical generation.
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              IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs.

              Survivin is a member of the inhibitor of apoptosis protein (IAP) family. We investigated the antiapoptotic mechanism of Survivin, as well as its expression in 60 human tumor cell lines used for the National Cancer Institute's anticancer drug screening program. In cotransfection experiments, cell death induced by Bax or Fas (CD 95) was partially inhibited (mean +/- SD, 65% +/- 8%) by Survivin, whereas XIAP, another IAP family member, almost completely blocked cell death (93% +/- 4%) under the same conditions. Survivin and XIAP also protected 293 cells from apoptosis induced by overexpression of procaspase-3 and -7 and inhibited the processing of these zymogens into active caspases. In vitro binding experiments indicated that, like other IAP-family proteins, Survivin binds specifically to the terminal effector cell death proteases, caspase-3 and -7, but not to the proximal initiator protease caspase-8. Using a cell-free system in which cytosolic extracts were derived from control- or Survivin-transfected cells and where caspases were activated either by addition of cytochrome c and dATP or by adding recombinant active caspase-8, Survivin was able to substantially reduce caspase activity, as measured by cleavage of a tetrapeptide substrate, AspGluValAsp-aminofluorocoumarin. Similar results were obtained in intact cells when Survivin was overexpressed by gene transfection and caspase activation was induced by the anticancer drug etoposide. Survivin was expressed in all 60 cancer cell lines analyzed, with highest levels in breast and lung cancers and lowest levels in renal cancers. These findings indicate that Survivin, which is commonly expressed in human tumor cell lines, can bind the effector cell death proteases caspase-3 and -7 in vitro and inhibits caspase activity and cell death in cells exposed to diverse apoptotic stimuli. Although quantitative differences may exist, these observations suggest commonality in the mechanisms used by IAP-family proteins to suppress apoptosis.
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                Author and article information

                Journal
                Oncol Lett
                Oncol Lett
                OL
                Oncology Letters
                D.A. Spandidos
                1792-1074
                1792-1082
                February 2015
                11 December 2014
                11 December 2014
                : 9
                : 2
                : 801-805
                Affiliations
                Department of Orthopedics, Beijing Friendship Hospital, Capital Medical University, Xicheng, Beijing 100050, P.R. China
                Author notes
                Correspondence to: Dr Ai Guo, Department of Orthopedics, Beijing Friendship Hospital, Capital Medical University, 95 Yong’an Road, Xicheng, Beijing 100050, P.R. China, E-mail: aiguo4ty@ 123456163.com
                Article
                ol-09-02-0801
                10.3892/ol.2014.2791
                4301500
                25621054
                abaec27b-5908-44bb-80d4-1f49fbfd3ad8
                Copyright © 2015, Spandidos Publications

                This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

                History
                : 15 June 2014
                : 06 November 2014
                Categories
                Articles

                Oncology & Radiotherapy
                evodiamine,osteosarcoma cells,proliferation,apoptosis
                Oncology & Radiotherapy
                evodiamine, osteosarcoma cells, proliferation, apoptosis

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