The cytosine analogues 5-azacytosine (azacytidine) and 2 0 -deoxy-5-azacytidine (decitabine) are the currently most advanced drugs for epigenetic cancer therapies. These compounds function as DNA methyltransferase inhibitors and have shown substantial potency in reactivating epigenetically silenced tumor suppressor genes in vitro. However, it has been difficult to define the mode of action of these drugs in patients and it appears that clinical responses are influenced both by epigenetic alterations and by apoptosis induction. To maximize the clinical efficacy of azacytidine and decitabine it will be important to understand the molecular changes induced by these drugs. In this review, we examine the pharmacological properties of azanucleosides and their interactions with various cellular pathways. Because azacytidine and decitabine are prodrugs, an understanding of the cellular mechanisms mediating transmembrane transport and metabolic activation will be critically important for optimizing patient responses. We also discuss the mechanism of DNA methyltransferase inhibition and emphasize the need for the identification of predictive biomarkers for the further advancement of epigenetic therapies. ' 2008 Wiley-Liss, Inc.Key words: azacytidine; decitabine; DNA methyltransferase; DNA methylation; cancer More than 40 years ago, the azanucleosides 5-azacytidine (azacytidine) and 2 0 -deoxy-5-azacytidine (decitabine) were developed as classical cytostatic agents. 1 Several years later, it was shown that these compounds inhibit DNA methylation in human cell lines, which provided a mechanistic explanation for their differentiation-modulating activity. 2 In addition, this observation also initiated the development of azanucleosides as epigenetic drugs. After substantial refinements in their clinical dosing schedules, both azacytidine and decitabine have now shown significant clinical benefits in the treatment of myelodysplastic syndrome (MDS), a preleukemic bone marrow disorder. 3,4 As a consequence, these drugs have now received FDA approval for the treatment of MDS. There are substantial ongoing efforts to identify and develop novel DNA methyltransferase inhibitors. 5,6 However, the currently available compounds appear to have weaker gene reactivation potencies than azanucleosides 7,8 and none of the candidate drugs has reached an advanced clinical testing stage for epigenetic indications yet. This has established azacytidine and decitabine as archetypal drugs for epigenetic cancer therapies. 9 Despite the renewed interest in azacytidine and decitabine, surprisingly little is known about the molecular mode of action of these drugs. It is clear that azanucleosides have cytotoxic effects and that they can cause DNA demethylation, but the relationships between these characteristics and their respective significance for clinical responses has not been established yet. A comprehensive understanding of drug characteristics will be critically important for defining their modes of action and to further advance their clinical de...

MicroRNAs (miRNAs) are small noncoding RNAs that repress their target mRNAs by complementary base pairing and induction of the RNA interference pathway. It has been shown that miRNA expression can be regulated by DNA methylation and it has been suggested that altered miRNA gene methylation might contribute to human tumorigenesis. In this study, we show that the human let-7a-3 gene on chromosome 22q13.31 is associated with a CpG island. Let-7a-3 belongs to the archetypal let-7 miRNA gene family and was found to be methylated by the DNA methyltransferases DNMT1 and DNMT3B. The gene was heavily methylated in normal human tissues but hypomethylated in some lung adenocarcinomas. Let-7a-3 hypomethylation facilitated epigenetic reactivation of the gene and elevated expression of let-7a-3 in a human lung cancer cell line resulted in enhanced tumor phenotypes and oncogenic changes in transcription profiles. Our results thus identify let-7a-3 as an epigenetically regulated miRNA gene with oncogenic function and suggest that aberrant miRNA gene methylation might contribute to the human cancer epigenome.

DNA methyltransferase inhibitors represent promising new drugs for cancer therapies. The first of these compounds (5-azacytidine, Vidaza) has recently been approved as an antitumor agent, and others are presently in various stages of their preclinical or clinical development. Most of the archetypal inhibitors have been established and characterized in different experimental systems, which has thus far precluded their direct comparison. We have now established defined experimental conditions that allowed a comparative analysis of the six most widely known DNA methyltransferase inhibitors: 5-azacytidine (5-aza-CR), 5-aza-2V-deoxycytidine (5-aza-CdR), zebularine, procaine, (À)-epigallocatechin-3-gallate (EGCG), and RG108. Of these, 5-aza-CR, 5-aza-CdR, zebularine, and EGCG were found to exhibit significant cytotoxicity in human cancer cell lines. 5-aza-CdR and EGCG were also found to be genotoxic, as evidenced by the induction of micronuclei. In addition, 5-aza-CR, 5-aza-CdR, zebularine, and RG108 caused concentration-dependent demethylation of genomic DNA, whereas procaine and EGCG failed to induce significant effects. Finally, the experiments in cancer cell lines were complemented by a cell-free in vitro assay with purified recombinant DNA methyltransferase, which indicated that RG108 is the only drug capable of direct enzyme inhibition. These results show a substantial diversity in the molecular activities of DNA methyltransferase inhibitors and provide valuable insights into the developmental potential of individual drugs. (Cancer Res 2006; 66(5): 2794-800)