Nanomolar EP4 receptor potency and expression of eicosanoid-related enzymes in normal appearing colonic mucosa from patients with colorectal neoplasia

Abstract

Background: Aberrations in cyclooxygenase and lipoxygenase (LOX) pathways in non-neoplastic, normal appearing mucosa from patients with colorectal neoplasia (CRN), could hypothetically qualify as predisposing CRN-markers.

Methods: To test this hypothesis, biopsies were obtained during colonoscopy from macroscopically normal colonic mucosa from patients with and without CRN. Prostaglandin E2 (PGE₂) receptors, EP1-4, were examined in Ussing-chambers by exposing biopsies to selective EP receptor agonists, antagonists and PGE₂. Furthermore, mRNA expression of EP receptors, prostanoid synthases and LOX enzymes were evaluated with qPCR.

Results: Data suggest that PGE₂ binds to both high and low affinity EP receptors. In particular, PGE₂ demonstrated EP4 receptor potency in the low nanomolar range. Similar results were detected using EP2 and EP4 agonists. In CRN patients, mRNA-levels were higher for EP1 and EP2 receptors and for enzymes prostaglandin-I synthase, 5-LOX, 12-LOX and 15-LOX.

Conclusions: In conclusion, normal appearing colonic mucosa from CRN patients demonstrates deviating expression in eicosanoid pathways, which might indicate a likely predisposition for early CRN development and furthermore that PGE₂ potently activates high affinity EP4 receptor subtypes, supporting relevance of testing EP4 antagonists in colorectal neoplasia management.

Keywords: Colorectal cancer; EP receptors; Lipoxygenase; Short circuit current; mRNA expression.

Fig. 1

Model of the metabolization of arachidonic acid (AA). AA is metabolized by 3 different groups of enzymes: cyclooxygenases (COX), lipoxygenases (LOX) and epoxygenases (cytochrome P450). The COX pathway consists of 2 isozymes: COX-1 and COX-2. Both isozymes metabolize AA into PGG₂ and then into PGH₂, which is further converted to the prostaglandins (PGs) PGD₂, PGE₂, PGF_2α, PGI₂ and thromboxane A₂, (TXA₂) by their respective synthases [3]. Each product binds to its specific membrane receptor. The CYP-450 pathway converts AA by epoxygenases and ω-hydroxylase into other downstream products, not shown. The LOX pathway consists of 3 main enzymes termed 5-LOX, 12-LOX and 15-LOX (isozymes 15-LOX-1 and 15-LOX-2). They metabolize AA into hydroperoxyl-eicosatetraenoic acids (HPETEs), which are further reduced to hydroxyeicosatetraenoic acids (HETEs). The 5-LOX enzyme differs by also metabolizing 5-HPETE into leukotriene A₄ by means of 5-lipoxygenase-activating protein (FLAP). *Enzymes already investigated in our laboratory; data published. Receptors/enzymes investigated in this study are underlined with red

Fig. 2

Dose–response curves of (A) EP2 agonist ONO-AE1-259 and (B) PGE₂ and EP4 agonist, TCS 2510, experiments. X-axis: ligand concentrations scaled logarithmically. Y-axis: changes in SCC. A: Large dots (black) show increases in SCC as a response to increasing EP2 agonist concentrations. The unbroken line in cyan resembles single receptor model (srm) fitting, while the long dotted line in blue resembles two receptor model (trm) fitting. B: Triangles (black) indicate increases in SCC as a response to increasing PGE₂ concentrations. Large dots (black) show increases in SCC as a response to increasing EP4 agonist concentrations. Dotted and long dotted lines (in blue colors) resemble single (srm) and two receptor model (trm) fitting for PGE₂ respectively. The unbroken and the medium dotted lines (in red colors) show trm and srm respectively for EP4 agonist. The trm fits data points more closely

Fig. 3

Calculated mean EC₅₀ values of PGE₂ and EP receptor agonists using (A) single receptor model (srm) equations and of (B) high and (C) low affinity receptors following PGE₂ and EP receptor agonists stimulation using two receptor model equations (trm). Numbers under the graph show N/n, N = number of patients, n = number of biopsies, NA = not applicable due to insufficient N/n. Data are presented as means ± SEM. *p < 0.05

Fig. 4

Calculated mean R_Max values displayed as µA·cm⁻² from (A) single receptor models and (B) two receptor models upon biopsy stimulation with PGE₂ or a selective EP receptor agonist. Numbers under the graph show N/n, N = number of patients, n = number of biopsies, NA = not applicable due to insufficient N/n. Data are presented as means ± SEM. *p < 0.05

Fig. 5

Dose–response curves of PGE₂ stimulation with and without EP4 antagonist GW627368X (GW-X) and calculated mean EC₅₀ values. A: X-axis: PGE₂ concentrations scaled logarithmically. Y-axis: changes in SCC. Triangles (black) show increases in SCC as a response to PGE₂ doses without the addition of GW-X. Big dots (black) show increases in SCC in the presence of EP4 antagonist GW-X followed by PGE₂ stimulation. The small dotted and the unbroken line (blue colors) resemble single (srm) and two receptor model (trm) fitting. Long dotted line (red) shows srm for experiments with GW-X, trm could not be calculated. B: Mean EC₅₀ (nM) values of PGE₂ and EP4 agonist TCS 2510 following inhibition with GW627368X (GW-X), calculated from single receptor model (srm) equations. Numbers under the graph show N/n, N = number of patients, n = number of biopsies. Data are presented as means ± SEM

Fig. 6

Expression levels of investigated enzymes and receptors. Expression of EP1, EP2 5-LOX, 12-LOX, 15-LOX as well as PTGIS are significantly higher in CRN patients. Expression levels are relative to ß-actin and GAPDH. Data are presented as means ± SEM. *p < 0.05 and **p < 0.01