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

Li Li; Rongjin Sun; Joseph Zenga; Heather Himburg; Lu Wang; Shengnan Duan; Jingwen Liu; Dinh Bui; Zuoxu Xie; Ting Du; Lijun Xie; Taijun Yin; Stu Wong; Song Gao; Ming Hu

doi:10.2147/JIR.S365842

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

J Inflamm Res. 2022 Aug 4:15:4435-4447. doi: 10.2147/JIR.S365842. eCollection 2022.

Authors

Li Li¹, Rongjin Sun¹, Joseph Zenga², Heather Himburg³, Lu Wang¹, Shengnan Duan¹, Jingwen Liu¹, Dinh Bui¹, Zuoxu Xie¹, Ting Du⁴, Lijun Xie¹, Taijun Yin¹, Stu Wong², Song Gao⁴, Ming Hu¹

Affiliations

¹ Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, USA.
² Department of Otolaryngology and Communication Sciences, Medical College of Wisconsin, Milwaukee, WI, USA.
³ Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI, USA.
⁴ Department of Pharmaceutical Science, College of Pharmacy and Health Sciences, Texas Southern University, Houston, TX, USA.

Abstract

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: absolute quantification; cyclooxygenase-1; cyclooxygenase-2; inflammation; liquid chromatography with tandem mass spectrometry; turnover number.