Tetra-O-methyl nordihydroguaiaretic acid, an inhibitor of Sp1-mediated survivin transcription, induces apoptosis and acts synergistically with chemo-radiotherapy in glioblastoma cells
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Glioblastoma (GBM), one of the most malignant human neoplasias, responds poorly to current treatment modalities, with temozolomide (TMZ) being the drug most frequently used for its treatment. Tetra-O-methyl Nordihydroguaiaretic Acid (M4N) is a global transcriptional repressor of genes dependent on the Sp1 transcription factor, such as Survivin and Cdk1. In the present study we evaluated the gene expression of Survivin, its spliced variants and Cdk1 in GBM samples and cell lines. Moreover, we investigated the effects of M4N combined or not with TMZ and/or radiation on GBM primary cultures and cell lines. qRT-PCR assays were performed to determine the Survivin-spliced variants and Cdk1 gene mRNA expression in GBM tumor samples and cell lines. Cell proliferation was measured by XTT assay and cell cycle and apoptosis were determined by flow cytometry. Drug combination analyses using different schedules of administration (simultaneous and sequential) were performed on GBM cell lines and primary cultures based on the Chou-Talalay method. For clonogenic survival, doses of 2, 4, and 6 Gy of gamma radiation. were used. All Survivin-spliced variants and the Cdk1 gene were expressed in GBM samples (n = 16) and cell lines (n = 6), except the Survivin-2B variant that was only expressed in GBM cell lines. M4N treatment down regulated the expression of Cdk1, Survivin and the Survivin-ΔEx3 variant, while the Survivin-2B variant was up-regulated. M4N decreased the cell proliferation separately and synergistically with TMZ, and enhanced the effects of radiation, mainly when associated with TMZ. M4N also induced apoptotic cell death, decreased the mitotic index and arrested the cell cycle mainly in the G2/M phase. Our results suggest a potential clinical application of M4N in combination with TMZ and radiation for GB treatment.
KeywordsCdk1 gene Drug combination Glioblastoma M4N Temozolomide Survivin gene
We would like to thank Augusto Faria Andrade, Department of Genetics, School of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, Brazil for assistance with manuscript editing. We also thank Patrícia Vianna Bonini Palma, Camila Cristina de Oliveira Menezes Bonaldo and Daiane Fernanda dos Santos, Hemocentro-FMRP-USP, Ribeirão Preto, Brazil, for assistance with flow cytometry. Research supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, process number 2009/50118-2), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and Fundação de Apoio ao Ensino, Pesquisa e Assistência, Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo.
The authors declare that they have no competing interests.
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