DNA Methylase Inhibitors

在断奶前易患癌症的ApcMin/+ (Min)小鼠的饮用水中持续使用DNA甲基化酶抑制剂zebularine，以确定该药物对正常小鼠发育和癌症预防的影响。在慢性口服治疗后，泽丙林引起雌性Min小鼠小肠和大肠B1短穿插核苷酸元件DNA甲基化的组织特异性降低，但在其他器官中没有。在治疗期间，小鼠的平均体重没有显著差异。此外，对来自雌性的结肠上皮细胞的整体基因表达分析表明，只有3%至6%的基因在其表达中受到影响。肝脏和肠道组织病理分析未发现毒性和异常。最后，我们在Min小鼠身上测试了慢性口服莪术林是否可以预防肿瘤的发生。雌性的平均息肉数从58个减少到1个，而雄性的平均息肉数没有变化，可能是由于醛氧化酶活性的差异。综上所述，我们的研究结果首次表明，长期口服西布拉林可引起肠道肿瘤的性别特异性消除，同时引起组织特异性DNA去甲基化。重要的是，长期使用表观遗传药物治疗小鼠只导致轻微的发育和组织学变化。以前发表的研究并没有涉及到西布拉林的长期毒性，而主要是研究这种药物的抗癌特性。因此，探索甲基化抑制剂长期给药后，整个动物体内的斑马碱的作用是很重要的。慢性DNA甲基化抑制在小鼠中的作用提示是否有可能在人类中进行安全的长期治疗。这篇论文的重要性值得发表在同一期的Perspective (Cancer Prevention Research 2008, 1,219 -222)，其中强调了我们的工作结果，强调现在有强有力的证据表明早期肿瘤的表观遗传成分，并且表观遗传调节的癌症预防特性的数据正在出现。有了临床前的原理证明，挑战是将这些表观遗传学发现转化为可测试的临床假设。在此期间，我们还研究了zebularine作为相应的2'-脱氧-5'-三磷酸的DNA结合，修饰寡核苷酸的杂交特性及其作为模板编码核碱基的作用（核苷核苷酸与核酸2008年，27,131-145和化学与生物多样性，2008年，在出版中）。最后，我们还成功地用2'-脱氧西蓝碱的前药磷酰胺衍生物将DNA甲基化沉默的p16基因重新激活（J. Med. Chem）。手稿提交)。

Preweaned cancer prone ApcMin/+ (Min) mice were treated continuously with the DNA methylase inhibitor,zebularine, in their drinking water to determine the effects of the drug on normal mouse development as well as cancer prevention. Zebularine caused a tissue-specific reduction in DNA methylation at B1 short interspersed nucleotide elements in the small and large intestines of female Min mice but not in other organs examined after chronic oral treatment. No significant difference in the average weights of mice was observed during the treatment. In addition, analysis of global gene expression of colonic epithelial cells from the females indicated that only 3% to 6% of the genes were affected in their expression. We did not detect toxicity and abnormalities from the histopathologic analysis of liver and intestinal tissues. Lastly, we tested whether prevention of tumorigenesis can be achieved with chronic oral administration of zebularine in Min mice. The average number of polyps in Min females decreased from 58 to 1, whereas the average polyp number remained unaffected in Min males possibly due to differential activity of aldehyde oxidase. Taken together, our results show for the first time that long-term oral administration of zebularine causes a gender-specific abrogation of intestinal tumors while causing a tissue specific DNA demethylation. Importantly, prolonged treatment of mice with epigenetic drugs resulted in only minor developmental and histologic changes. Previous published studies have not addressed the long-term toxicity of zebularine and have mainly dealt with the anticancer properties of the drug. Therefore, it is important to explore the effects of zebularine in the entire animal following chronic administration of the methylation inhibitor. The effect of chronic DNA methylation inhibition in mice should suggest whether there is a potential for safe long-term therapy in man. The importance of this paper merited the publication of a Perspective in the same issue (Cancer Prevention Research 2008, 1, 219-222) that highlighted the results obtained in our work emphasizing that there is now strong evidence for an epigenetic component of early neoplasia, and data on the cancer preventive properties of epigenetic modulation are emerging. With preclinical proof of principle at hand, the challenge is to translate these epigenetic findings into testable clinical hypotheses. During this period we have also investigated the DNA incorporation of zebularine as the corresponding 2'-deoxy-5'-triphosphate, the hybridization properties of modified oligos and its role as a template-coding nucleobase (Nucleosides Nucleotides & Nucleic Acids 2008, 27, 131-145 and Chemistry & Biodiversity, 2008, in press). Finally, we have also succeeded in the reactivation of p16 gene silenced by DNA methylation with prodrug phosphoramidite derivatives of 2'-deoxyzebularine (J. Med. Chem. manuscript submitted).