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.
Highly malignant cancers of the colon, lung, breast and pancreas account for more than half of the approximately 560,000 cancer deaths annually. The cyclooxygenase-2 enzyme (COX-2) is present in the majority of these cancers and has been shown to contribute to their highly malignant character. These studies will address the mechanism whereby COX-2 contributes to the malignancy and assess new approaches to cancer prevention that derive from an understanding of this mechanism.
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