Relationship of ß-catenin and Bcl-2 expression to sulindac-induced regression of intestinal tumors in Min mice
Michael F. McEntee1,
Chun-Hung Chiu and
Jay Whelan
Departments of Pathology and Nutrition, University of Tennessee, Knoxville, TN 37901, USA
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Abstract
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Non-steroidal anti-inflammatory drugs (NSAIDs) can cause regression of early intestinal tumors and although this is believed to involve cyclooxygenase-2 and apoptosis, the molecular mechanisms remain unclear. Cytoplasmic and nuclear ß-catenin are overexpressed in many of these lesions and Bcl-2, which inhibits apoptosis, may also be elevated during the course of intestinal tumorigenesis. We recently showed that sulindac causes regression of 7080% of small intestinal tumors in Min/+ mice within 4 days, but does not have the same impact on colonic lesions; after 20 days of treatment the tumor load stabilizes at 1020% of that in untreated animals. The aim of this study was to determine if NSAID-induced regression of intestinal adenomas might be associated with changes in ß-catenin or Bcl-2 expression. Intestinal tumors from Min/+ mice were harvested after treatment with sulindac for 2, 4 or 20 days and evaluated for expression of ß-catenin and Bcl-2 using immunohistochemistry. There was a
50% decrease in ß-catenin (P = 0.001) and diminishing Bcl-2 (P = 0.019) in small intestinal tumors harvested between 2 and 4 days of treatment when compared with untreated controls. In contrast, small intestinal tumors from animals treated for 20 days were not significantly different from untreated controls. Colonic tumors expressed higher levels of Bcl-2 than those from the small intestine and did not show any significant changes in either Bcl-2 or ß-catenin expression after treatment. Results suggest that modulation of aberrant ß-catenin expression occurs during NSAID-induced regression of intestinal adenomas and that Bcl-2 may confer resistance to these effects.
Abbreviations: COX-2, cyclooxygenase-2; NSAIDs, non-steroidal anti-inflammatory drugs; PBS, phosphate-buffered saline.
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Introduction
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Development of colorectal cancer, from dysplastic crypts to metastatic carcinoma, involves a series of genetic mutations, the earliest often involving the APC gene (1,2). Individuals with familial adenomatous polyposis carry a germline mutation in APC and mutational damage or loss of the second wild-type allele initiates intestinal tumor formation (3,4). Somatic mutations resulting in loss of full-length APC protein also occur early in spontaneous forms of the disease (1). The protein is normally up-regulated as cells exit small intestinal crypts or near the luminal surface of the colon and is believed to have a role in controlling cell migration, apoptosis and possibly proliferation (5,6). An important molecular function of APC is regulation of cytoplasmic pools of ß-catenin (5). ß-Catenin is a necessary component of the E-cadherin adhesion complex and a transduction molecule for the Wnt-1 (Wingless in Drosophila) signaling cascade, cooperating with at least one other factor (Lef-1/Tcf) in transcriptional regulation of c-myc (7,8). APC contributes to a degradation process that eliminates cytoplasmic ß-catenin molecules not bound to E-cadherin and thereby down-regulates ß-catenin signaling (5). Although the biological role of this signaling cascade in mammals is not fully understood, loss of APC function results in increased levels of free ß-catenin in the cytoplasm and nucleus where it presumably effects some aspect of neoplastic cell behavior (9).
Intestinal tumors in Min/+ mice and humans commonly overexpress cyclooxygenase-2 (COX-2) (10,11) and non-steroidal anti-inflammatory drugs (NSAIDs) can cause their regression (12,13), but the mechanisms involved are still a matter of speculation. One probable effect of NSAIDs is induction of apoptosis (1419). Bcl-2 is a key antagonist of apoptosis, commonly overexpressed during early stages of intestinal carcinogenesis and inducible by COX-2 (2023). Inhibition of COX-2 reduces Bcl-2 and causes apoptosis in prostatic and intestinal epithelial cells in vitro (23,24) and the NSAID sulindac decreases ß-catenin expression and increases apoptosis in the normal intestinal mucosa of Min/+ mice (16,25), but it is unknown whether similar molecular responses to NSAIDs occur in neoplastic intestinal epithelial cells in vivo.
The Min mouse model has been used by a number of laboratories to investigate the affects of NSAIDs on intestinal tumor biology (16,26,27). As in humans with FAP, Min/+ mice carry a germline mutation in APC and develop multiple intestinal adenomas following spontaneous loss of the wild-type allele (28). Sulindac causes regression of most pre-existing small intestinal tumors in Min/+ mice within 4 days, but a percentage of these tumors remain intact after 20 days treatment and colonic tumors are relatively immune to this treatment (13). In the present study we have examined the distribution of ß-catenin and Bcl-2 in Min/+ mouse tumors before and after sulindac treatment in an effort to correlate molecular changes with tumor behavior and responsiveness to NSAIDs.
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Materials and methods
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Tissue samples were obtained during the course of a previously reported study and details of the experimental protocol are presented therein (13). In brief, heterozygous Min/+ mice (Min) were obtained from Jackson Laboratories (Bar Harbor, ME) at ~35 days of age and immediately started on a powder diet (AIN-93G; Dyets, Bethlehem, PA). Sulindac (Sigma, St Louis, MO) was added to the diet at 320 p.p.m. for the treatment groups. Mice were randomly assigned to four different groups: (1) controls (no sulindac supplementation) killed at 80 days of age; (2) sulindac starting at 78 days of age, killed at 80 days (2 days on sulindac); (3) sulindac starting at 78 days of age, killed at 82 days (4 days on sulindac); (4) sulindac starting at 78 days of age, killed at 98 days (20 days on sulindac).
All animal procedures were approved by the University of Tennessee Animal Care and Use Committee and were in accord with the NIH Guide for the Care and Use of Laboratory Animals (NRC, 1985). At death, the entire intestinal tract was removed, flushed with cold (4°C) phosphate-buffered saline (PBS) and opened longitudinally. Tissue samples were harvested under a dissecting microscope and immediately fixed in 10% neutral buffered formalin.
Four micrometer paraffin-embedded sections were stained for ß-catenin and Bcl-2 by indirect peroxidase biotinstreptavidin immunohistochemistry. Sections stained for Bcl-2 were pretreated with a microwave antigen retrieval procedure using 10 mM citrate buffer, pH 6 (10 min). All sections were pre-blocked with 3% H2O2 in PBS followed by normal goat serum. Sections were then incubated for 1 h at room temperature with anti-ß-catenin (0.5 µg/ml; Transduction Laboratories, Lexington, KY) or overnight at 4°C with anti-mouse Bcl-2 (1:300; PharMingen, San Diego, CA) followed by biotinylated anti-mouse or anti-rabbit immunoglobulin (50 min, 20°C) and streptavidinperoxidase link (40 min, 20°C; BioGenex, San Ramon, CA). Localized peroxidase conjugates were visualized with 3,3'-diaminobenzidine in sections lightly counterstained with hematoxylin. Internal positive controls included normal mucosal epithelial cells and lymphocytes (for Bcl-2). Primary antibody was eliminated or an irrelevant antibody substituted in its place as negative controls for each set.
Staining was analyzed subjectively and, for ß-catenin, objectively by morphometry on coded slides without knowledge of the treatment group. Since the protocol for any single protein was identical for all samples, it was possible to directly compare the relative amount (staining intensity) and distribution of each individual protein in various tumors and the normal mucosa. Subjective evaluation of both proteins entailed direct visual comparison of staining in tumor cells with components of the adjacent normal mucosa and in neoplastic and non-neoplastic tissues harvested from the colon and small intestine. For Bcl-2, a scoring scheme was based on comparison with normal crypt epithelial cells in immediately adjacent areas of small intestinal or colonic mucosa: no staining = 0; a staining gradient from positive deep in the tumor (at the level of adjacent crypts) to negative in upper regions = 1 (i.e. `normal' distribution pattern); staining in both the deeper and upper regions of the tumor (above the level of the adjacent crypts) that was equal to or greater than that of the crypts focally/multifocally = 2 and diffusely = 3. Since the intensity of Bcl-2 staining in neoplastic epithelial cells was equal to or only slightly greater than non-neoplastic crypt epithelium, this variable was not scored for individual tumors. The relative staining intensity of tissues in different regions of the intestinal tract (i.e. small intestine versus colon) was ascertained by direct visual comparison.
All morphometric measurements on ß-catenin stained sections were done at a fixed magnification (4x objective) and illumination using BioQuant/TCW Image Analysis software (version 2.2; R & M Biometrics, Nashville, TN). Each individual tumor was manually outlined on the screen to give a total area expressed in pixels. Abnormal ß-catenin staining was measured by empirically adjusting the color `threshold' setting for a typical untreated small intestinal tumor to delimit only regions with increased cytoplasmic and/or nuclear staining and then all other tumors were evaluated at the same setting. The demarcated areas were summed to give a total area representing increased staining within each individual tumor and divided into the total tumor area. ß-Catenin data therefore represent the percentage of each tumor overexpressing ß-catenin in abnormal intracellular locations as determined by a uniformly applied protocol of tissue handling, staining and analysis.
All data were tested for normality. ß-Catenin data were log transformed and analyzed by analysis of variance (ANOVA) using JMP software (SAS Institute, Cary, NC). Dunnett's pairwise comparison test was used to determine differences (P < 0.05) between the experimental groups and untreated controls. The Bcl-2 data consisted of discrete numeric values and could not be normalized. Bcl-2 data were therefore analyzed using a KruskalWallis non-parametric test for individual pairwise comparisons, followed by a Bonferroni's adjustment for significance. Spearman's correlation and Kendall's
-b tests were applied to the Bcl-2 data for analysis of trend and significance was established at P < 0.05.
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Results
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ß-Catenin
Normal mucosal epithelial cells of the small intestine and colon were weakly positive to negative under the described staining protocol. More uniform and darker staining of cell membranes could be achieved by increasing the concentration or incubation time of anti-ß-catenin antibody with the tissues. Staining was always restricted to the lateral cell membranes and there was no consistent difference in reactivity between the colon and small intestine or between the crypt and surface epithelium. The staining intensity of neoplastic cells in the small intestine and colon was distinctly increased, in comparison with adjacent normal epithelium, and concentrated in the cytoplasm and/or nucleus (Figure 1
, insert) with little evidence of membrane staining. In most small intestinal tumors, the greatest staining was restricted to the deeper and lateral aspects of the mass (Figure 1
). In the smallest lesions, consisting of single cystic expansions of the crypt (Figure 2
) or lesions that had not yet progressed much beyond the involvement of one or two villi, staining was typically more widespread with a substantially greater percentage of positive cells than the majority of small intestinal tumors evaluated (mean 61.8 versus 23.1%, P < 0.0001). In comparison with lesions of the small intestine, colonic tumors had significantly greater ß-catenin staining (Table I
; P = 0.0009) with a more uniform distribution from the base of the mass to the surface (Figure 3
). The relative intensity and intracellular pattern of staining in neoplastic colonic epithelial cells was similar to that of cells forming small intestinal lesions.

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Fig. 1. Tumor from the small intestine of an untreated Min mouse, stained for ß-catenin. Staining was generally most intense in deeper aspects of small intestinal tumors where the cytoplasm and nucleus of neoplastic cells were strongly reactive (insert). ß-Catenin staining in adjacent normal epithelial cells was less intense and restricted to the intercellular membranes (lower left of insert).
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Fig. 2. Early cystic lesion from the small intestine of an untreated Min mouse with overexpression of cytoplasmic and nuclear ß-catenin.
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Table I. Mean percent ß-catenin staining and Bcl-2 scores for small intestinal (SI) and colonic tumors from untreated Min mice and those on sulindac for 2, 4 and 20 days
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As shown in Table I
, ß-catenin staining decreased significantly in small intestinal tumors within 2 days of sulindac treatment (P < 0.0009). Although fewer tumors were available for evaluation, this decrease was also evident after 4 days of sulindac treatment (P = 0.001). In contrast, ß-catenin in tumors from animals maintained on sulindac for 20 days was not significantly different from untreated controls (P = 0.782). The pattern of staining in tumors from all treated animals resembled untreated controls in that reactivity was predominantly in the deep to lateral aspects of the lesions (Figure 4
). The cytoplasm and/or nucleus stained, as in untreated tumors, without an increase in cytoplasmic membrane reactivity. The mean area of small intestinal tumors analyzed from all treatment groups was less than that of untreated controls and this difference was significant (P < 0.05) for the 2 day treatment group (data not shown).

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Fig. 4. Small intestinal tumor from a Min mouse treated with sulindac for 4 days. ß-Catenin staining is limited to scattered positive nuclei at the base of the tumor (arrows).
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ß-Catenin staining in colonic tumors treated with sulindac for 2, 4 or 20 days tended to be slightly less than in untreated tumors (Table I
) but differences were not statistically significant (all P-values
0.30). There were no discernible differences in ß-catenin staining intensity or distribution. There were also no significant differences in the size of colonic tumors evaluated from the different treatment groups (data not shown).
Bcl-2
Many lymphocytes in the lamina propria were positive for Bcl-2 and intraepithelial lymphocytes were often strongly reactive. The pattern of staining for Bcl-2 in normal epithelial cells of both the small intestine and colon was similar, but quantitatively distinct. In the small intestine, there was a faint homogeneous to granular staining of the cytoplasm in normal crypt epithelial cells that was absent in epithelium above the cryptvillus junction (Figure 5
). This reactivity for Bcl-2 was not as strong as that seen in deep crypt cells of the colon, where staining also rapidly decreased to negative in cells higher up the crypt and on the luminal surface. Neither the colonic nor small intestinal crypt epithelium stained as intensely as normal intraepithelial lymphocytes.

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Fig. 5. Cystic lesion (same as Figure 2 ) from the small intestine of an untreated Min mouse stained for Bcl-2. Crypt cells and the cystic lesion are weakly positive for Bcl-2 whereas epithelial cells above the cryptvillus junction are negative. Intraepithelial lymphocytes are strongly positive for Bcl-2 (arrow).
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Small intestinal tumors showed several different patterns of staining for Bcl-2. Reactivity varied from diffusely less than that of normal crypt epithelium (negative), to diffusely positive (comparable with or slightly greater than that of crypt epithelium) (Figure 6
), to an intermediate pattern with staining in deeper tumor cells but none above the level of adjacent crypts (i.e. distribution similar to the normal mucosa). The mean Bcl-2 staining score for untreated small intestinal tumors was 2.34 (Table I
), with a score of 1 indicating essentially the same reactivity as the normal mucosa. Cells in some of the smallest lesions stained for Bcl-2 above the level of the adjacent crypts (Figure 5
). In some serially sectioned small intestinal tumors, cytoplasmic reactivity for Bcl-2 co-localized with cytoplasmic/nuclear ß-catenin. Colonic tumors were more strongly positive for Bcl-2 than their small intestinal counterparts with reactivity throughout most lesions and little evidence of a staining gradient. Staining of most colonic tumor cells was equal to or greater than that of the adjacent normal crypt epithelium and in some instances showed increased reactivity around nuclear membranes (Figure 7
).

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Fig. 6. Edge of small intestinal tumor from untreated Min mouse stained for Bc-2. Most of the tumor cells are positive for Bcl-2 whereas the mature villus epithelium of the adjacent mucosa is negative.
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Fig. 7. Bcl-2 staining in colonic tumor cells distinguishes them from the adjacent non-neoplastic epithelium (arrow). Bcl-2 reactivity in tumor cells often outlines the nuclear envelope.
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In small intestinal tumors there was a sequential decrease in the mean Bcl-2 score after 2 and 4 days of sulindac treatment (Table I
). There was no difference between Bcl-2 staining in control tumors and those from animals on sulindac for 20 days (P > 0.5). Although comparison of the 4 day group with untreated controls yielded a low P-value (0.0425), this was not statistically significant after applying Bonferroni's adjustment, which indicated significance only at P < 0.017. However, the downward trend in Bcl-2 scores from untreated controls to the 2 and 4 day treatment groups was statistically significant (P = 0.019). All colonic tumors from treated and untreated animals stained similarly for Bcl-2 with a score of 3 out of 3.
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Discussion
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The earliest molecular event that heralds the appearance of most intestinal tumors is an APC mutation(s) which results in loss of the full-length protein and an accumulation of ß-catenin in the cytoplasm and nucleus of affected cells (1,5,9). We have shown that Min mouse tumors and cystic crypts in the small intestine, previously described as the earliest stage of tumor growth (29), are strongly reactive for nuclear and cytoplasmic ß-catenin, clearly overexpressing this protein in relation to the adjacent normal epithelium where staining is restricted to cell membranes. This loss of ß-catenin regulation is consonant with the somatic APC mutations which have been shown to initiate essentially all Min mouse tumors (4).
We found that ß-catenin significantly decreases in small intestinal tumors from animals actively experiencing NSAID-induced tumor regression. Staining for ß-catenin in small intestinal tumors from animals treated with sulindac for 2 days was ~50% of that in untreated tumors and was further reduced after 4 days of treatment. This decrease does not involve a change in the intracellular distribution of the protein (i.e. no increase in membrane staining), but may be due to a decrease in total ß-catenin levels such that fewer cells are immunohistochemically reactive. Decreased ß-catenin in tumor cells undergoing NSAID-induced regression suggests a causeeffect relationship, but it remains to be proven that this is a primary effect of the drug.
The non-neoplastic intestinal epithelium of Min mice contains more ß-catenin than that of wild-type mice and sulindac treatment reduces ß-catenin levels while increasing apoptosis and the relatively slow rate of cell migration out of the crypt (16,25). If NSAIDs directly cause a reduction in ß-catenin in tumor cells, the tumorigenic defect initiated with loss of full-length APC protein could be at least partially corrected with re-establishment of more normal cellular behavior (i.e. migration, apoptosis, etc.), perhaps through a reduction in c-myc expression (8). Modulation of ß-catenin in tumor cells that lack full-length APC might involve some other constituent of the normal post-transcriptional degradation pathway that compensates for the lack of APC. Conductin was recently identified as a novel component of the cytoplasmic APCGSK3ß complex and an important contributor in ß-catenin degradation (30). Behrens et al. also showed that overexpression of conductin in SW480 cells decreased levels of cytoplasmic and nuclear ß-catenin even though these cells lack full-length APC protein (30). It is therefore possible that sulindac may up-regulate conductin expression in Min mouse tumors to effect a decrease in ß-catenin levels.
The beneficial effect of NSAIDs in modulating intestinal tumorigenesis is currently believed to involve the induction of apoptosis (6,1419). Bcl-2 inhibits apoptosis under most conditions and increased levels of PGE2 in early intestinal tumors, associated with overexpression of COX-2, may cause increases in Bcl-2 and resistance to apoptosis (16,23,31). Inhibition of prostaglandin biosynthesis in these lesions could therefore promote apoptosis by reducing Bcl-2, as in prostatic cells treated with the COX-2 inhibitor NS398 (24). However, while COX-2 is clearly important to intestinal tumor growth (10,16,32), there is also evidence suggesting that NSAIDs act through mechanisms besides inhibition of prostaglandin biosynthesis (13,17,19,33). Sulindac may induce apoptosis by increasing arachidonic acid and thereby generating ceramide (17). Ceramide induces apoptosis which can be blocked by Bcl-2 (34,35), perhaps through inhibition of arachidonic acid release by phospholipase (36).
We found weak staining for Bcl-2 in the normal crypt epithelial cells of the small intestine and stronger reactivity in normal colonic crypt epithelium. Although others have also described Bcl-2 expression in both small and large intestinal crypts (20,37), some suggest that Bcl-2 is only found in the colonic crypt epithelium (38,39). While this discrepancy might be explained by variation between individual laboratories in application of immunohistochemistry, our findings do indicate that Bcl-2 levels in crypt epithelium of the colon are higher than the small intestine. This distinction is contrary to what might be expected (38), given the relative number of lesions that develop in the large and small intestines of these mice.
In aggregate, Bcl-2 staining in small intestinal tumors from untreated Min mice was interpreted to indicate overexpression of this protein because of frequent staining (
crypt epithelium) in upper regions of the masses where adjacent villus (mature, non-neoplastic) epithelial cells were negative. Colonic tumor cells also stained for Bcl-2 with an intensity comparable with the normal colonic crypt epithelium and were more strongly reactive than tumors in the small intestine. One potential implication of the latter point may be reflected in the apparent insensitivity of Min mouse colonic tumors to NSAID-induced regression (13,27), providing that it occurs through apoptosis. There was no indication that Bcl-2 staining changed in colonic tumors after treatment with sulindac.
In contrast to colonic lesions, small intestinal adenomas showed a significant sequential decrease in Bcl-2 staining after 2 and 4 days of sulindac treatment, which would presumably favor increased rates of apoptosis (14,16,19,23). In contrast, there was no significant reduction in tumors harvested after 20 days of sulindac treatment, at which point the tumor load had reached a relatively stable plateau and appeared to represent a subset of NSAID-resistant small intestinal tumors (13). When taken together, the colonic and small intestinal tumor data indicate that inherent (or pre-treatment) levels of Bcl-2 may explain the different sensitivity of these lesions to sulindac-induced regression and that a reduction in Bcl-2 is associated with regression of small intestinal tumors. It is not yet clear whether sustained levels of Bcl-2 in NSAID-resistant lesions of the colon and small intestine represent a barrier to drug-induced apoptosis or whether the upstream mechanisms responsible for initiation of apoptosis and down-regulation of Bcl-2 are lacking in these cells.
The molecular mechanisms responsible for NSAID-induced regression of intestinal tumors are only starting to be elucidated. APC mutation is a key element in the early steps of intestinal tumorigenesis and regulation of ß-catenin is an important function of the normal APC protein (5). Our results suggest that sulindac may at least partially correct the imbalance in intracellular ß-catenin that results from mutational loss of full-length APC protein. If proved true, this suggests an important mechanism for NSAID-mediated control of intestinal tumorigenesis. Neither ß-catenin nor Bcl-2 levels change significantly in sulindac-resistant tumors, but the regulatory mechanisms that govern the levels of these proteins during sulindac-induced regression and their relationship to the arachidonic acid cascade through COX-2 and prostaglandin or ceramide biosynthesis remain to be defined.
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Acknowledgments
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This work was supported by a grant from the American Institute for Cancer Research.
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Notes
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1 To whom correspondence should be addressed Email: mmcentee{at}utk.edu 
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Received July 31, 1998;
revised October 30, 1998;
accepted December 4, 1998.