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063 COLITIS-ASSOCIATED ADENOCARCINOMA IN IL10-/- MICE: ASSOCIATION OF iNOS AND EP2 EXPRESSION R Zhang1,2, DM McCafferty1 BACKGROUND: We have previously shown that colitis-associated iNOS acts to limit colonic adenocarcinoma in IL-10-/- mice. A growing body of evidence suggests that COX-2, PGE2 and its receptors (EP1, EP2 and EP4) play key roles in the development of colon cancer. Here we determined the effect of colitis-associated iNOS on COX-2-PGE2 pathway in IL-10-/- mice.
1Gastrointestinal Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta; 2Department of Digestive Disease, 2nd Hospital of Shanxi Medical University, Shanxi, China
METHODS: Wild type (WT), IL-10-/- and IL-10-/- iNOS-/- mice were used at 3 and 6 months old. Colonic mRNA and protein were assessed by using real-time PCR and Western blot respectively. COX-2 activity was determined by measuring PGE2 levels by ELISA in presence or absence of COX-2 inhibitor (NS398).
RESULTS: At 6 months colonic COX-2 mRNA levels were significantly elevated in the absence of iNOS (9.0±4.5 vs 1.6±0.76 in IL-10-/- mice; p<0.05). COX-2 protein level in IL-10-/-/iNOS-/- (1.3±0.3) was significantly increased from WT (0.47±0.2), but not significantly different between the two mutants (1.2±0.3 in IL-10-/-). Colonic PGE2 synthesis (expressed as % of vehicle control) indicated a significant increase in COX-2 activity in absence of iNOS (58% vs 23%; P<0.05). However total PGE2 synthesis was not significantly different in the presence or absence of iNOS (3386±850 vs 3905±555 pg/mg respectively) in IL-10-/- mice. Next, we examined mRNA levels of EP1, EP2 and EP4. EP4 mRNA was significantly increased in IL-10-/- (1.4±0.3) and IL-10-/-iNOS-/- (1.1±0.3) compared with WT (0.04±0.02) however the absence of iNOS did not significantly alter EP4 message. No difference in EP1 mRNA was observed in IL-10-/- iNOS-/- (0.3±0.2) compared with WT(0.25±0.1) mice however a significant increase in message was observed in IL-10-/- (1.2±0.3; P<0.05). No significant increase in EP2 mRNA was observed in IL-10-/- (0.07±0.02) compared with WT (0.04±0.02) however an 8-fold increase in message was observed in IL-10-/-/iNOS-/- (0.69±0.13) mice. A reciprocal increase in EP2 protein was observed by Western blot analysis. To determine if these changes in EP2 message occurred prior to the development of cancer we examined colonic tissue in 3 months old animals. Interestingly, as the mice age a significant increase in EP2 mRNA was observed in IL-10-/-/iNOS-/- (0.3±0.13 at 3 months) however a decrease was observed in IL-10-/- (0.37±0.14 at 3 months) mice. These data illustrate that as IL-10-/- mice age the absence of iNOS results in enhanced message and protein for EP2.
CONCLUSION: Changes in EP2 receptor expression are associated with the development of colitis-associated adenocarcinoma in the absence of iNOS in IL-10-/- mice.
Supported by CIHR Operating Grant and Group Grant