Prevention of COX-2 Derived DNA and Histone Modifications in Cancer

 描述(由申请人提供)：环氧合酶(COX)酶的高活性代谢物有助于COX依赖的癌症生长的假设将在拟议的研究中进行检验。一系列一致的证据表明，COX有助于恶性肿瘤的进化，它包括流行病学研究和受控临床试验以及动物模型和细胞调查的结果。诱导型COX-2亚型在许多上皮细胞肿瘤中都有表达，包括结肠、肺、乳腺和胰腺肿瘤。Coxs的产物是前列腺素H2(PGH2)，它经过细胞特异性的酶生物转化为前列腺素D2、E2、F2a、I2和血栓素A2。目前所有关于Cox促进癌症的机制的研究都涉及这些酶衍生的前列腺素的作用。然而，PGH2也进行了非酶重排，生成了高活性的-酮醛、左旋糖苷(LG)E_2和D_2，占总产物的20%。LGs几乎瞬间反应形成伯胺的共价加合物。我们已经证明了LGE2与DNA中的脱氧胞苷碱基形成共价加合物。加合物的稳定形式被表征为吡咯，我们利用稳定同位素稀释法建立了对它的高效液相色谱/串联质谱分析。这使得可以在两种癌细胞系中鉴定出依赖于COX-2的LG-DNA加合物的形成。这种由COX衍生的大体积共价加合物的形成表明，它可能会导致突变和/或染色体不稳定。除了DNA的这种修饰外，LG还与赖氨酸的epsilon-amine反应，形成具有内酰胺结构的稳定加合物。在人类肺癌细胞系中也发现了COX-2衍生的LG-组蛋白加合物，这增加了驱动基因或肿瘤抑制基因转录的表观遗传学改变的可能性。我们已经开发出一种有效的LGS清除剂，乙基水杨胺，它可以防止DNA和组蛋白的LG加合物的形成，但不抑制Coxs。它还清除其他反应性的羰基，如丙二醛，这是环氧合酶和脂质过氧化的产物。这项拟议的研究将使用乙基水杨胺作为工具，解决以下假设：DNA和组蛋白被反应性羰基修饰，如LG和丙二醛，有助于COX-2依赖型癌症的生长。这些DNA和组蛋白修饰在体内癌症中的存在以及乙基水杨胺减少这些修饰的存在将被确定，以验证药物干预的效果。COX-2与高度恶性的癌症有关，仅在美国每年就有20万人死于这种癌症。因此，了解它如何促进恶性肿瘤的进化是很重要的。

 DESCRIPTION (provided by applicant): The hypothesis that highly reactive metabolites of the cyclooxygenase (COX) enzymes contribute to the COX-dependent growth of cancers will be examined in the proposed research. A concerted body of evidence indicates that the COXs contribute to the evolution of malignant neoplasms, and it includes findings from epidemiological studies and controlled clinical trials as well as investigations in animal models and cells. The inducible COX-2 isoform is expressed in many epithelial cell tumors, including those of the colon, lung, breast, and pancreas. The product of the COXs is prostaglandin H2 (PGH2) which undergoes cell specific enzymatic biotransformation to the prostaglandins D2, E2, F2a, I2, and thromboxane A2. All current research on the mechanism by which the COXs contribute to cancer addresses the actions of these enzymatically derived prostanoids. However, PGH2 also undergoes non-enzymatic rearrangement yielding the highly reactive -ketoaldehydes, levuglandins (LG) E2 and D2 as 20% of the total products. The LGs react almost instantaneously to form covalent adducts of primary amines. We have demonstrated that LGE2 forms a covalent adduct with deoxycytidine bases in DNA. The stable form of the adduct has been characterized as a pyrrole, and we have developed an HPLC/tandem mass spectrometric analysis for it, employing stable isotope dilution. This has permitted identification of COX-2 dependent formation of the LG-DNA adduct in two cancer cell lines. The formation of this bulky COX-derived covalent adduct suggests that it could engender mutagenesis and/or chromosomal instability. In addition to this modification of DNA, LG reacts with the epsilon-amine of lysine to form stable adducts with a lactam structure. COX-2 derived LG-histone adducts also have been identified in a human lung cancer cell line, raising the possibility of epigenetic alteration of the transcription of driver genes or tumor suppressor genes. We have developed a potent scavenger of LGs, ethyl-salicylamine, that prevents formation of LG adducts of DNA and histones but does not inhibit the COXs. It also scavenges other reactive carbonyls such as malondialdehyde which is a product of the cyclooxygenases and of lipid peroxidation. The proposed research will employ ethyl-salicylamine as a tool with which to address the hypothesis that modification of DNA and histone by reactive carbonyls such as LG and malondialdehyde contribute to the growth of COX-2 dependent cancers. The presence of these DNA and histone modifications in in vivo cancers as well as reduction of these modifications by ethyl-salicylamine will be determined to verify the efficacy of the pharmacologic intervention. COX-2 is associated with highly malignant cancers that result in >200,000 deaths annually in the US alone. Accordingly, understanding how it contributes to the evolution of malignancy is important.