Characterization of cyclooxygenase-2 acetylation and prostanoid inhibition by aspirin in cellular systems

Abstract

The most recognized mechanism of aspirin (acetylsalicylic acid, ASA) action, at therapeutic dosing, is the inhibition of prostanoid biosynthesis through the acetylation of cyclooxygenase (COX)-isozymes (COX-1 at serine-529 and COX-2 at serine-516). Whether aspirin, also when given at the low-doses recommended for cardiovascular prevention, reduces the risk of colorectal cancer by affecting COX-2 activity in colorectal adenomatous lesions is still debated. We aimed to develop a direct biomarker of aspirin action on COX-2 by assessing the extent of acetylation of COX-2 at serine-516 using the AQUA strategy, enabling absolute protein quantitation by liquid chromatography-mass spectrometry. We compared the extent of acetylation and the inhibition of prostanoid biosynthesis by ASA using human recombinant COX-2 (hu-COX-2), the human colon cancer cell line HCA-7, isolated human monocytes stimulated with LPS (lipopolysaccharide) or human intestinal epithelial cells stimulated with interleukin (IL)-1β. Hu-COX-2 exposed in vitro to an excess of ASA was acetylated by approximately 40–50% associated with the inhibition of COX-2 activity by 80–90%. In the three cell-types expressing COX-2, the extent of COX-2 acetylation and reduction of prostaglandin (PG) E2 biosynthesis by ASA was concentration-dependent with comparable EC50 values (in the low μM range). The maximal % acetylation of COX-2 averaged 80%, at ASA 1000 μM, and was associated with a virtually complete reduction of PGE2 biosynthesis (97%). In conclusion, we have developed a proteomic assay to evaluate the extent of acetylation of COX-2 at serine-516 by aspirin; its use in clinical studies will allow clarifying the mechanism of action of aspirin as anticancer agent.