Comparison of Absolute Expression and Turnover Number of COX-1 and COX-2 in Human and Rodent Cells and Tissues

We aim to quantify the absolute protein expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in various cells and tissues to determine the relative contribution of COX-1 and COX-2 to PGE2 production.An LC-MS method was developed and validated, then used for quantifying the absolute amounts of COX-1 and COX-2 in recombinant human COX-1 and COX-2, lysates from different cells, tissue microsomes of rodents and humans, Pirc rat colonic polyps, and biopsy specimens from squamous cell carcinoma (SCC) patients. The COX-1 and COX-2 turnover numbers were subsequently calculated based on apparent formation rates of PGE2.A robust LC-MS method for quantification of COX-1 and COX-2 was developed and validated and then used to calculate their apparent turnover numbers. The results showed that COX-1 expression levels were much higher than that of COX-2 in all the tested tissues including the colonic epithelium of F344 (28-fold) and Pirc rats (20-fold), colonic polyps of Pirc rats (8-fold), and biopsy specimens of SCC patients (11-17-fold). In addition, both COX-1 and COX-2 were higher in polyps when compared to adjacent mucosa of Pirc rats. The turnover number of recombinant human COX-2 was 14-fold higher than that of recombinant human COX-1. LPS stimulation increased COX-2 protein expression in three cell lines (Raw 264.7, SCC9 and EOMA) as expected but unexpectedly increased COX-1 protein expression (13.8-fold) in EOMA cells.In human oral cancer tissues and cells as well as Pirc rat colon, COX-1 plays an unexpectedly but more important role than COX-2 in abnormal PGE2 production since COX-1 expression is much higher than COX-2. In addition, COX-1 expression levels are inducible in cells, and higher in polyps than surrounding mucosa in Pirc rat colon. These results indicate that targeted suppression of local COX-1 should be considered to reduce colon-specific PGE2-mediated inflammation.