Awor, please read this study…This is very informative…Zebularine maybe a safer alternative than procainamide. Read Read!
http://jnci.oxfordjournals.org/cgi/content/full/95/5/399
Background: Gene silencing by abnormal methylation of promoter regions of regulatory genes is commonly associated with cancer. Silenced tumor suppressor genes are obvious targets for reactivation by methylation inhibitors such as 5-azacytidine (5-Aza-CR) and 5-aza-2’-deoxycytidine (5-Aza-CdR). However, both compounds are chemically unstable and toxic and neither can be given orally. We characterized a new demethylating agent, zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], which is a chemically stable cytidine analog. Methods: We tested the ability of zebularine to reactivate a silenced Neurospora crassa gene using a hygromycin gene reactivation assay. We then analyzed the ability of zebularine to inhibit DNA methylation in C3H 10T1/2 Cl8 (10T1/2) mouse embryo cells as assayed by induction of a myogenic phenotype and in T24 human bladder carcinoma cells, using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay. We also evaluated the effects of zebularine (administered orally or intraperitoneally) on growth of EJ6 human bladder carcinoma cells grown in BALB/c nu/nu mice (five mice per group) and the in vivo reactivation of a methylated p16 gene in these cells. All statistical tests were two-sided. Results: In N. crassa, zebularine inhibited DNA methylation and reactivated a gene previously silenced by methylation. Zebularine induced the myogenic phenotype in 10T1/2 cells, which is a phenomenon unique to DNA methylation inhibitors. Zebularine reactivated a silenced p16 gene and demethylated its promoter region in T24 bladder carcinoma cells in vitro and in tumors grown in mice. Zebularine was only slightly cytotoxic to T24 cells in vitro (1 mM zebularine for 48 hours decreased plating efficiency by 17% [95% confidence interval (CI) = 12.8% to 21.2%]) and to tumor-bearing mice (average maximal weight change in mice treated with 1000 mg/kg zebularine = 11% [95% CI = 4% to 19%]). Compared with those in control mice, tumor volumes were statistically significantly reduced in mice treated with high-dose zebularine administered by intraperitoneal injection (P<.001) or by oral gavage (P<.001). Conclusions: Zebularine is a stable DNA demethylating agent and the first drug in its class able to reactivate an epigenetically silenced gene by oral administration.
INTRODUCTION
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Notes
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Aberrant de novo DNA methylation is commonly associated with cancer (1), and several studies have shown that de novo methylation of CpG islands in regulatory sequences of tumor suppressor genes can result in their silencing (2–4). Thus, DNA methylation may lead to abnormal growth of cancer cells. Examples of regulatory genes that are commonly hypermethylated in cancer cells include the RB1 gene in retinoblastomas (5,6), the VHL gene in sporadic renal cell carcinomas (7), the H19 gene in Wilms’ tumors (8), the p15 gene in leukemias (9), and the p16 gene in several human cancer cell lines (9,10). Given the high frequency of hypermethylation of genes critical to the control of cell proliferation (4,11), pharmacologic use of DNA methylation inhibitors has been considered to reactivate antiproliferative, apoptotic, and differentiation-inducing genes in cancer cells.
The most well characterized and widely used drugs to inhibit DNA cytosine methylation and reactivate silenced genes are several nucleoside analogs, including 5-azacytidine (5-Aza-CR) and 5-aza-2’-deoxycytidine (5-Aza-CdR) (12,13), and several non-nucleoside drugs, including procainamide (14,15). Both 5-Aza-CR and 5-Aza-CdR have a nitrogen in place of a carbon at position 5 of the pyrimidine ring (Fig. 1). These nucleoside analogs were originally developed as cancer chemotherapeutic agents (16) and are powerful inducers of genes silenced by DNA methylation (17). Although 5-Aza-CR and 5-Aza-CdR are both being tested in international clinical trials, especially for the treatment of acute myeloid leukemia and myelodysplastic syndrome (18–20), their instability in neutral solution has complicated their clinical use. 5-Aza-CR has a short half-life [approximately 90 minutes at 50 °C in phosphate-buffered saline (PBS) at pH 7.4 (21)] and is unstable in neutral aqueous solutions, and the hydrolysis products have been well characterized (22). This chemical instability encouraged the development of other analogs, such as 5,6-dihydro-5-azacytidine (23) and pseudoisocytidine (24), which possess more stable ring systems but have not been clinically useful. 5-Fluoro-2’-deoxycytidine also inhibits DNA methylation and reactivates silenced genes when incorporated into DNA (12) but generates 5-fluorodeoxyuridine and its metabolites, which may be toxic (25). Thus, there is still a need for an effective, stable, and minimally toxic inhibitor of DNA methylation.
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Fig. 1. Structures of cytidine and its analogs, 5-azacytidine and zebularine. 5-azacytidine contains a nitrogen in position 5 and zebularine contains a 2-(1H)-pyrimidinone ring. Both ring systems have been shown to form covalent bonds with DNA methyltransferase enzymes after incorporation into DNA (43,53).
In our search for new DNA methylation inhibitors, we fortuitously came across a cytidine deaminase inhibitor, zebularine. Zebularine is a cytidine analog containing a 2-(1H)-pyrimidinone ring that was originally developed as a cytidine deaminase inhibitor because it lacks an amino group at position 4 of the pyrimidine ring (Fig. 1) (26–29). In addition, unlike aza nucleosides, zebularine is stable in aqueous solution up to a pH of 12 (30,31). In vitro experiments have shown that synthetic oligonucleotides containing zebularine form tight complexes with bacterial methyltransferases (32), leading to a potent inhibition of DNA methylation. To assess the ability of zebularine to inhibit DNA methylation, we took advantage of a simple microbial system consisting of an antibiotic gene (hph) silenced by DNA methylation in the filamentous fungus Neurospora crassa (33,34) and determined the reactivation of this gene by zebularine. We next tested whether zebularine is an effective inhibitor of mammalian DNA methylation in the mouse embryo (C3H 10T1/2 Cl8 [10T1/2]) and human bladder carcinoma (T24) cell lines. Finally, we examined the effects of zebularine on tumor growth in vivo and its ability to reactivate a silenced p16 gene in EJ6 human bladder carcinoma cells grown in BALB/c nu/nu mice.
