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The DNA methyltransferase inhibitor zebularine exerts antitumor effects and reveals BATF2 as a poor prognostic marker for childhood medulloblastoma

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An Erratum to this article was published on 29 November 2016

Summary

Medulloblastoma (MB) is the most common solid tumor among pediatric patients and corresponds to 20 % of all pediatric intracranial tumors in this age group. Its treatment currently involves significant side effects. Epigenetic changes such as DNA methylation may contribute to its development and progression. DNA methyltransferase (DNMT) inhibitors have shown promising anticancer effects. The agent Zebularine acts as an inhibitor of DNA methylation and shows low toxicity and high efficacy, being a promising adjuvant agent for anti-cancer chemotherapy. Several studies have reported its effects on different types of tumors; however, there are no studies reporting its effects on MB. We analyzed its potential anticancer effects in four pediatric MB cell lines. The treatment inhibited proliferation and clonogenicity, increased the apoptosis rate and the number of cells in the S phase (p < 0.05), as well as the expression of p53, p21, and Bax, and decreased cyclin A, Survivin and Bcl-2 proteins. In addition, the combination of zebularine with the chemotherapeutic agents vincristine and cisplatin resulted in synergism and antagonism, respectively. Zebularine also modulated the activation of the SHH pathway, reducing SMO and GLI1 levels and one of its targets, PTCH1, without changing SUFU levels. A microarray analysis revealed different pathways modulated by the drug, including the Toll-Like Receptor pathway and high levels of the BATF2 gene. The low expression of this gene was associated with a worse prognosis in MB. Taken together, these data suggest that Zebularine may be a potential drug for further in vivo studies of MB treatment.

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Acknowledgments

We would like to thank Patrícia Vianna Bonini Palma, Camila Cristina de Oliveira Menezes Bonaldo, Daiane Fernanda dos Santos, Blood Center - USP, and Prof. Dr. Elza Tiemi Sakamoto Hojo, Departament of Genetics, Ribeirão Preto, Brazil, for their assistance with flow cytometry.

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Authors and Affiliations

  1. Department of Genetics, Ribeirão Preto Medical School, University of São Paulo (USP), Avenida Bandeirantes 3900, 14048-900, Ribeirão Preto, SP, Brazil

    Augusto Faria Andrade, Lenisa Geron & Luiz Gonzaga Tone

  2. Department of Pediatrics – Ribeirão Preto Medical School, University of São Paulo (USP), São Paulo, Brazil

    Kleiton Silva Borges, Veridiana Kiill Suazo, Carolina Alves Pereira Corrêa, Angel Mauricio Castro-Gamero, Carlos Alberto Scrideli & Luiz Gonzaga Tone

  3. Department of Biochemistry, Chemistry Institute, USP, São Paulo, Brazil

    Elton José Rosas de Vasconcelos

  4. Department of Surgery and Anatomy, Ribeirão Preto Medical School, USP, São Paulo, Brazil

    Ricardo Santos de Oliveira

  5. Department of Pathology, Ribeirão Preto Medical School, USP, São Paulo, Brazil

    Luciano Neder

  6. Centro Infantil Boldrini & FCM/Unicamp, Campinas, Brazil

    José Andres Yunes & Simone dos Santos Aguiar

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  1. Augusto Faria Andrade
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Correspondence to Augusto Faria Andrade.

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Conflict of interest

Author Augusto Faria Andrade declares that he has no conflict of interest. Author Kleiton Silva Borges declares that he has no conflict of interest. Author Veridiana Kiill Suazo declares that she has no conflict of interest. Author Lenisa Geron declares that she has no conflict of interest. Author Carolina Alves Pereira Corrêa declares that she has no conflict of interest. Author Angel Mauricio Castro-Gamero declares that he has no conflict of interest. Elton José Rosas de Vasconcelos declares that he has no conflict of interest. Author Ricardo Santos de Oliveira declares that he has no conflict of interest. Author Luciano Neder declares that he has no conflict of interest. Author José Andres Yunes declares that he has no conflict of interest. Author Simone dos Santos Aguiar declares that she has no conflict of interest. Author Carlos Alberto Scrideli declares that he has no conflict of interest. Author Luiz Gonzaga Tone declares that he has no conflict of interest.

Funding

This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant number 2011/22440-7), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number 478974/2013-2). Fundação de Apoio ao Ensino, Pesquisa e Assistência (FAEPA) do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo is also acknowledged.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all participants included in the study.

Additional information

The original version of this article was revised: Due to a formatting error, the name of one of the authors, Simone dos Santos Aguiar, appeared as Simone Santos dos Aguiar.

An erratum to this article is available at http://dx.doi.org/10.1007/s10637-016-0412-1.

Electronic supplementary material

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Supplementary Figure 1

Exposure of MB cells, DAOY and ONS-76, to CIS (A) and VCR (B) after 72 h incubation, measured by the XTT assay. C) Values that represent 50% of inhibition (IC50) were used in the combination assays. D) Different doses of zebularine, VCR and CIS used in combination assays. The drugs were added in simultaneous exposure for 72 h. (JPG 38 kb)

High resolution image (TIFF 1496 kb)

Supplementary Figure 2

BATF2 expression in MB cell lines ONS-76, UW402 and UW473 after treatment with Zebularine. BATF2 gene expression levels were analyzed by qRT-PCR after treatment with different doses of Zebularine (0–200 μM) and 72 h of incubation. The charts show the relative expression compared to control. (JPG 22 kb)

High resolution image (TIFF 1431 kb)

Supplementary Figure 3

BATF2 expression with the clinical status of MB patients. A) The level of expression of BATF2 was measured by qRT-PCR in MB samples (patients). BATF2 levels were also measured in non-neoplastic cerebellums (NC) and MB cell lines (MB lines). B) and C) Mann–Whitney analysis demonstrated that low BATF2 levels were more commonly detected in high risk patients (p=0.016) and that low BATF2 expression was correlated with MB relapse (p=0.042). (JPG 29 kb)

High resolution image (TIFF 1496 kb)

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Andrade, A.F., Borges, K.S., Suazo, V.K. et al. The DNA methyltransferase inhibitor zebularine exerts antitumor effects and reveals BATF2 as a poor prognostic marker for childhood medulloblastoma. Invest New Drugs 35, 26–36 (2017). https://doi.org/10.1007/s10637-016-0401-4

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