Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Zebularine induces chemosensitization to methotrexate and efficiently decreases AhR gene methylation in childhood acute lymphoblastic leukemia cells Augusto F. Andrade a , Kleiton S. Borges a , Angel M. Castro-Gamero a , Vanessa S. Silveira b , Veridiana K. Suazo b , Jaqueline C. Oliveira a , Daniel A. Moreno a , Rosane G. de Paula Queiroz b , Carlos A. Scrideli b and Luiz G. Tone a,b Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in childhood. Despite the advances in treatment, about 20% of patients relapse and/or die, indicating the need for different therapies for this group. Zebularine (ZB) is a potent DNA methyltransferase (DNMT) inhibitor and has been associated with gene demethylation and enhancement of tumor chemosensitivity. This study aimed to evaluate the effects of ZB, alone or combined with chemotherapeutics (methotrexate and vincristine), on childhood ALL cell lines. Cell proliferation, apoptosis, and clonogenic capacity were studied in Jurkat and ReH cell lines. Bisulfite modification, followed by methylation-specific PCR was carried out to evaluate aryl hydrocarbon receptor (AhR) methylation status. Gene expression of DNMT1, DNMT3a, DNMT3b, and AhR was assessed using qRT-PCR. Both cell cultures were sensitive to ZB, showing a dose-dependent and time-dependent response (P < 0.05). ZB induced apoptosis and decreased clonogenic capacity in both cell lines. Combination with methotrexate resulted in a strong synergistic effect, whereas combination with vincristine led to an antagonistic response in both cell lines. ZB treatment decreased gene expression of the three DNMTs and induced AhR gene promoter demethylation and its re-expression. These results indicate that ZB may be a promising drug for the adjuvant treatment of ALL, mainly when combined with methotrexate. Anti-Cancer Drugs 00:000–000  c 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins. Anti-Cancer Drugs 2013, 00:000–000 Keywords: acute lymphoblastic leukemia, DNA methyltransferases inhibitor, methylation, zebularine Departments of a Genetics and b Pediatrics, Ribeira ˜ o Preto Medical School, University of Sa ˜ o Paulo (USP), Ribeira ˜o Preto, SP, Brazil Correspondence to Augusto F. Andrade, MSc, Department of Genetics, Ribeira ˜ o Preto Medical School, University of Sa ˜o Paulo (USP), Avenida Bandeirantes 3900, 14048-900 Ribeira ˜o Preto, SP, Brazil Tel: +55 16 3602 2651; fax: +55 16 3602 2270; e-mail: augustofaria@usp.br Received 11 December 2012 Revised form accepted 26 August 2013 Introduction Acute lymphoblastic leukemia (ALL) is the most common malignant disease in childhood and its survival rate reaches about 75–80%. Nevertheless, B20% of affected children relapse and have a poor prognosis because of the development of drug resistance [1]. The enhancement of cure rates, which was less than 50% up to the 1970s, is because of successful application of combination che- motherapy and changes in drug dosing and schedule [2]. Different events are correlated with leukemia, including the methylation status of several genes [3,4]. Abnormal methylation of cytosine at CpG dinucleotides in the promoter of tumor suppressor genes represents an important mechanism for tumorigenesis and gene in- activation in several types of tumors [5]. The pattern of aberrant hypermethylation is specific for the cancer type as it has been shown for both cancer cell lines [6] and primary human tumors [7]. In leukemia cells, hypermethylation is found in several genes such as those involved in DNA repair (MGMT), cell cycle (p16, p14), and apoptosis (DAPK) [8–10]. As abnormal methylation of the promoters is potentially reversible, pharmacologic inhibitors of DNA methylation provide an attractive and rational approach to re-establish the crucial cellular functions that are abnormally silenced by hyper- methylation [11]. The cytosine nucleoside analogs 5-azacytidine (5-aza-C) and 5-aza-2 0 -deoxycytidine (5-aza-dC) are the best-known demethylating agents [12]. These compounds are incorpo- rated into DNA and inhibit the DNA methyltransferases (DNMTs), the enzymes responsible for DNA methyla- tion [13]. Clinically, these agents have already shown good results for the treatment of leukemia and myelodysplastic syndromes [14]. Both DNMT inhibitors (iDNMTs) have been approved by the Food and Drug Administration for the treatment of myelodysplastic syndromes, despite being toxic and orally unstable [12]. Zebularine (1-[b-D-ribofuranosyl]-1,2-dihydropyrimidin- 2-one) (ZB) is a cytidine analog that was initially Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.anti-cancerdrugs.com). Preclinical report 1 0959-4973  c 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/CAD.0000000000000028