Ejecución del prototipo

mCTR1 immunoreactivity was detected in all colonic tissues of these mice but this staining was limited to the colonic crypts of Winnie mice and C56BL/6 mice. The overall percentage of positive mCTR1 immunostaining for all these samples was measured as 66 ± 13%, which was significantly higher than that of normal tissues (66 ± 13% vs 50 ± 7.9, P < 0.05). In Winnie

mice exposed to DSS, mCTR1 staining was detected in all these colon samples (Fig. 6-3C). Strikingly, the overall staining level for mCTR1 was significantly reduced by 3 folds in DSS- treated Winnie mice compared to that of untreated Winnie mice (23 ± 12% vs 66 ± 13, P < 0.05) (Fig. 6-3A and Tab 6-1).

Next, we analyzed the expression of ATP7A in colonic tissues collected from Winnie mice of colitis, DSS-treated Winnie mice and wild-type C57BL/6 mice using the same method used to quantify mCTR1 expression. Overall, ATP7A immunoreactivity was detected in all colonic tissues analyzed. The overall percentage of positive ATP7A immunostaining of all ten samples from DSS-treated mice was measured as 51 ± 9.7%, which was similar to that of normal colon samples (50 ± 15%). In Winnie mice, ATP7A staining was detectable in all these colon samples despite overall weaker expression intensity. The overall level of ATP7A expression was significantly reduced compared to that of untreated Winnie mice (20 ± 5.3% vs 51 ± 9.7%, P

< 0.05) (Fig. 6-3B and Tab 6-1).

Subsequently, we analyzed the expression of ATP7B in colonic tissues collected from Winnie

mice of colitis, DSS-treated Winnie mice and wild-type C57BL/6 mice with the same methods used to quantify mCTR1 and ATP7A. Overall, ATP7B immunoreactivity was detected in all colonic tissues of mice. The overall percentage of positive ATP7B immunostaining of all ten samples was measured as 64 ± 9.5%, which was similar to that of normal colon samples (66 ± 4.9%). Immunohistochemistry was also performed on paraffin-embedded sections of dysplastic colon samples from ten Winnie mice exposed to DSS. ATP7B staining was detectable in all these colon samples despite weak expression. The overall level for ATP7B staining significantly reduced compared to untreated Winnie mice (11 ± 3% vs 64 ± 9.5%, P < 0.05) (Fig. 6-3C and Tab 6-1)

Figure 6-3. Semi-quantitative analysis of representative DAB-stained images showing immunostaining of mCTR1, ATP7A and ATP7B in colonic tissues

A. Immunostaining of mCTR1 in colonic tissues of normal mice, Winnie mice with chronic colitis and DSS-treated Winnie mice with colonic dysplasia. B. Immunostaining of ATP7A in colonic tissues of normal mice, Winnie mice with chronic colitis and DSS-treated Winnie mice with colonic dysplasia. C. Immunostaining of ATP7B in colonic tissues of normal mice, Winnie mice with chronic colitis and DSS- treated Winnie mice with colonic dysplasia.Standard DAB immunohistochemistry was performed on paraffin-embedded colonic tissue sections using specific antibodies as mentioned in Section 2.1. Sections were counterstained with haematoxylin. DAB-IHC images were deconvoluted and analyzed using a semi-quantitative method to acquire the percentage of pixel intensity and counts of immunoreactivity. Mag. 40x.

Table 6-1. The expression percentage of copper transporters in colonic tissues of mice

*, P < 0.05 compared to values of normal mice; +, P < 0.05 compared to values of mCTR1.

Group Percentage expression of copper transporters (Mean ±S.D, n=10) mCTR1 ATP7A ATP7B Winnie + DSS 23 ± 12 * _{20 ± 5.3} * _{11 ± 3}*,+

Winnie 66 ± 13 * 51 ± 9.7 + 64 ± 9.5 Normal 50 ± 8 50 ± 15 66 ± 4.9 +

6.4 Discussion

Colitis-associated colorectal cancer is a chronic disease that normally takes 15-20 years to develop from inflammatory bowel disease over colonic dysplasia to adenocarcinoma [218]. The Winnie mouse model provides an opportunity to study the expression of copper transporters in colon tissues within a shorter time period. For the first time, this study observed that mCTR1 expression is elevated in colon tissues of Winnie mice compared to normal C57BL/6 mice, which supports our hypothesis that copper transporters play a role in the development of inflammatory bowel disease.

Histological examination evaluated the severity of inflammation and the presence of neoplasia in the colon of Winnie and DSS-treated Winnie mice. Typical inflammation associated histological or structural changes were detected in all colon samples of

Winnie mice, suggesting that this chronic colitis model recapitulates the main pathological features of the human disease [151]. Upon challenging with DSS, all examined samples showed low to medium-level dysplasia as well as signs of exacerbated inflammation of the colon wall. These results are consistent with another report that characterizes histological inflammation and dysplasia-related pathology in these two models [148].

Following confirmation of inflammation and dysplasia in colon samples, the expression of copper transporters was examined. mCTR1 expression was up regulated in colon tissues of Winnie mice but reduced in the precancerous colon tissues compared to the normal tissue samples. Our first result is in line with previous reports that CTR1 expression is elevated in the eyes of patients with Eales disease, an idiopathic inflammatory disease [219] or chemical induced inflammation in rat liver [220]. A complete understanding of the mechanisms behind this phenomenon is beyond the

scope of this study, however, due to their wide involvement in several inflammation processes, copper or copper containing enzymes may be the key to explain this result. The second finding that mCTR1 was down regulated in dysplastic colon tissue is contradictory to the expected role of hCTR1 in the carcinogenesis of colorectal cancer as discussed in Chapter 5. Considering that copper can be pro-inflammatory, mCTR1 down-regulation may be the result of the mucosa’s response to acute DSS-induced inflammation. On the other hand, there is evidence that DSS can suppress the transcription of a multitude of genes by directly inhibiting DNA polymerase activity [221]. Consequently, this down regulation could be a consequence of DSS itself and might have nothing to do with the carcinogenic process per se. Therefore, one should be cautious to extrapolate this finding to the human condition.

Next, we compared the expression of the copper efflux transporters, ATP7A and ATP7B in colonic samples of Winnie mice with that of normal C57BL/6 mice. However, the overall expression of ATP7A or ATP7B was similar in colitis compared to normal tissue. The roles of copper efflux transporters in colon tissues under inflammatory conditions have not been studied yet. However, there is some evidence suggesting that copper efflux transporters might be altered under inflammatory conditions. Increased expression of ATP7A and trafficking of these proteins into cytoplasmic vesicles is observed in cultured BV-2 microglial cells in the presence of the pro-inflammatory agent interferon-gamma [222]. Mutation of ATP7B in Wilson disease is a well-known cause of severe inflammation in liver [127]. Perhaps copper efflux transporters are different from mCTR1 in the roles they play in the inflammation of colon.

Given the numerous reports that ATP7A and ATP7B are up regulated in many human cancers [103], [63], we expected to see increased expression. However, we observed a

consistent reduction of AP7A or ATP7B in colonic samples of colonic dysplasia, similar to the response of mCTR1 in the same tissues. Consequently, the same limitations of DSS treatment postulated for mCTR1 expression could also apply for the expression levels Cu-efflux transporters. Therefore, these results should be interpreted with caution, especially when comparing them to the human condition.