Combined activity of COX-1 and COX-2 is increased in non-neoplastic colonic mucosa from colorectal neoplasia patients

Abstract

Background: Cyclooxygenase (COX) activity is increased in endoscopic normal colonic mucosa from patients with colorectal neoplasia (CRN). COX-2 is thought to be the predominant COX isozyme involved in neoplasia. Meanwhile, relative contributions of COX-1 and COX-2 isoforms are unknown. Knowledge about their mutual activity in colonic mucosa is important for diagnostics and targeted therapy for CRN. The aim of this study was to assess the relative function, expression and localization of COX-1 and COX-2 enzymes in colonic non-neoplastic human mucosa and thereby to potentially reveal a mucosal disease predisposition for better treatment.

Methods: Biopsies were pinched from normal appearing colonic mucosa in patients undergoing endoscopy. Ussing chamber technique was applied for an indirect assessment of epithelial activity, RT-qPCR for expression and immunohistochemistry for localization of COX-1 and COX-2 enzymes in patients without (ctrls) and with a history of CRN (CRN-pts).

Results: Combined COX-1 and COX-2 activity was higher in CRN-pts, p = 0.036. COX-2 was primarily localized in absorptive cells, while COX-1 appeared to be restricted to nonenteroendocrine tuft cells of the colonic epithelium.

Conclusions: In biopsies from endoscopic normal appearing colonic mucosa, combined activity of COX-1 and COX-2 enzymes is increased in CRN-pts compared with ctrls. This indicates that COX-1 and COX-2 together contribute to an increased proliferation process. Of note, in colonic epithelial cell lining, the COX-1 enzyme seems localized in tuft cells.

Keywords: Biopsy; Carcinogenesis; Cyclooxygenase; Endoscopic; Short circuit current (SCC); Tuft cells.

Fig. 1

A simplified scheme for involvement of COX enzymes in development of CRN. COX-1 and COX-2 convert active arachidonic acid (AAA) into PGE₂, which at the basolateral side is transported out off the cell via an ABCC5-transporter. Through a Gs-protein-coupled EP-4-receptor, PGE₂ mediates conversion of ATP to cAMP, thus inducing mucosal Cl⁻ secretion and cell proliferation. COX isozymes may be inhibited selectively by SC-560 and celecoxib or non-selectively by indomethacin. Pathways with markers are hypothetically increased in CRN, thereby increasing cell proliferation and carcinogenesis. PGE₂: prostaglandin E2, cAMP: cyclic adenosine monophosphate, AMP: adenosine monophosphate, ATP: adenosine triphosphate, COX: cyclooxygenase, CRN: colorectal neoplasia, G_s: stimulatory heterotrimeric G protein, ABCC5: ATP-binding cassette transporter C5, EP4: prostaglandin receptor subtype 4, SC-560: selective COX-1 inhibitor, celecoxib: selective COX-2 inhibitor

Fig. 2

Examples of typical recording in Ussing Chamber experiments on short circuit current (SCC) following exposure to COX-1 (cyclooxygenase) inhibitor SC-560 (a) and COX-2 inhibitor celecoxib (b). Compounds were added to biopsies in the following concentrations: amiloride (20 μM, mucosal side), theophylline (400 μM, both sides), either COX-1 inhibitor (SC-560, 500 nM, both sides) or COX-2 inhibitor (celecoxib, 500 nM, both sides), indomethacin (13 μM, both sides) and prostaglandin (PGE₂, 100 nM, serosal side)

Fig. 3

Immunohistochemical staining of colonic mucosa with cyclooxygenase (COX) subtype specific antibodies in patients with colorectal neoplasia. a COX-2 immunohistochemical staining appears localized to the cytoplasm of absorptive cells (yellow arrows). No staining is seen in goblet cells (red arrows). A pericryptal stromal cell is marked (green arrow). b COX-1 immunohistochemical staining, here with antibody sc-1752, appears to stain morphological appearing tuft cells (red arrows) and in the lamina propria proposedly myofibroblasts (green arrow). c Fluorescence double labeling shows no co-localization between COX-1 (green) and specific markers for endocrine cells (red). Arrows point at COX-1 positive tuft cells. Subfigures 1, 2, 3, 5 and 7 have proportions of 100 μm per centimeter, subfigures 4 and 6 have proportions of 50 μm per centimeter. 1: Glucagon-like peptide-1, 2: Somatostatin, 3: Gastric Inhibitory Polypeptide, 4: Proprotein Convertase-1, 5: Proprotein Convertase-2, 6: Serotonin, 7: Chromogranin A