Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Authors

  • Marcela Gimenez,

    1. Protein Chemistry Center and Department of Molecular and Cell Biology and Pathogenic Bioagents, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
    2. CEPID-CTC Center for Cell Therapy, Fundação Hemocentro de Ribeirão Preto, Ribeirão Preto, Brazil
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  • Suely K.N. Marie,

    1. Department of Neurology, Medical School of São Paulo, University of São Paulo, São Paulo, Brazil
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  • Sueli M. Oba-Shinjo,

    1. Department of Neurology, Medical School of São Paulo, University of São Paulo, São Paulo, Brazil
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  • Miyuki Uno,

    1. Department of Neurology, Medical School of São Paulo, University of São Paulo, São Paulo, Brazil
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  • Roseli da Silva,

    1. Department of Neurology, Medical School of São Paulo, University of São Paulo, São Paulo, Brazil
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  • Helen Julie Laure,

    1. Protein Chemistry Center and Department of Molecular and Cell Biology and Pathogenic Bioagents, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
    2. CEPID-CTC Center for Cell Therapy, Fundação Hemocentro de Ribeirão Preto, Ribeirão Preto, Brazil
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  • Clarice Izumi,

    1. Protein Chemistry Center and Department of Molecular and Cell Biology and Pathogenic Bioagents, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
    2. CEPID-CTC Center for Cell Therapy, Fundação Hemocentro de Ribeirão Preto, Ribeirão Preto, Brazil
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  • Andreia Otake,

    1. Department of Radiology and Oncology, Medical School of São Paulo, University of São Paulo, São Paulo, Brazil
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  • Roger Chammas,

    1. Department of Radiology and Oncology, Medical School of São Paulo, University of São Paulo, São Paulo, Brazil
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  • Jose Cesar Rosa

    Corresponding author
    1. CEPID-CTC Center for Cell Therapy, Fundação Hemocentro de Ribeirão Preto, Ribeirão Preto, Brazil
    • Protein Chemistry Center and Department of Molecular and Cell Biology and Pathogenic Bioagents, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
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Correspondence: Professor Jose Cesar Rosa, Av. Bandeirantes, 3900, 14049-900-Ribeirão Preto, SP, Brazil

E-mail: jcrosa@fmrp.usp.br

Fax: 55-16-2101-9366

Abstract

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

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