Vet Pathol Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McEntee, M. F.
Right arrow Articles by Neilsen, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McEntee, M. F.
Right arrow Articles by Neilsen, N.
Vet Pathol 39:428-436 (2002)
© 2002 American College of Veterinary Pathologists

Cyclooxygenase-2 Expression in Spontaneous Intestinal Neoplasia of Domestic Dogs

M. F. McEntee, J. M. Cates and N. Neilsen

Department of Pathology, University of Tennessee, Knoxville, TN


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Cyclooxygenase-2 (Cox-2) is commonly upregulated during human colorectal tumorigenesis, and its contribution to this process has been clearly demonstrated in genetic mouse models. The only other species that naturally develops intestinal cancer with any frequency is the domestic dog. Intestinal carcinogenesis in humans has been strongly linked to environmental factors such as diet, which may be shared by household pets. We have previously reported that ß-catenin is overexpressed in the neoplastic epithelium of canine colorectal polyps, as it is in humans and rodents. We now show that Cox-2 is also upregulated in the majority of these lesions. Thirteen out of 20 colorectal adenomas (65%) contained immunohistochemically detectable Cox-2 protein restricted to the nonneoplastic tumor stroma, including myofibroblasts and {alpha}-smooth muscle actin–negative mesenchymal cells morphologically consistent with macrophages and/or fibroblasts. In contrast to benign polyps, seven of 15 adenocarcinomas (47%) also expressed Cox-2 in the neoplastic epithelium. These changes duplicate molecular changes in human intestinal tumorigenesis and substantiate a fundamental role for both ß-catenin and Cox-2 in intestinal neoplasia.

Key words: Adenoma; colorectal tumorigenesis; Cox-2; cyclooxygenase; dogs; immunohistochemistry; NSAIDs; tumor.

Colorectal cancer is a significant cause of morbidity and mortality in the United States, with diagnosis of approximately 130,000 new cases expected in 1999.24 This form of cancer has been a prototype in defining genetic and epigenetic molecular changes associated with initiation of early dysplastic changes, development of adenomas, and progression to malignancy (carcinogenesis).1,7,37 Numerous laboratories have contributed to the characterization of these molecular changes in inherited and sporadic forms of human colorectal cancer.21,35 In vitro and in vivo studies with laboratory animal models have provided a better understanding of the specific abnormal gene function relationships that contribute to intestinal neoplastic cell growth and behavior.8,14,16,45 From this body of work, it has become clear that mutations resulting in aberrant upregulation of cytoplasmic and nuclear ß-catenin play a key role in human colorectal carcinogenesis.35

Carcinogenesis in several human organ systems has been strongly associated with environmental factors, and diet is often singled out as being particularly important in this regard, especially in the case of colorectal cancer.26 One potential molecular link to dietary factors is cyclooxygenase-2 (Cox-2), which is commonly overexpressed during the early stages of intestinal tumor development.6,13,52 Dietary lipids can significantly modify tissue levels of arachidonate, which is the substrate for Cox-2, thereby affecting the generation of protumorigenic prostaglandins produced by this enzyme.12 Although the mechanisms responsible for upregulation of Cox-2 in neoplastic tissues are still unclear, the enzyme clearly contributes to intestinal tumorigenesis in rodent models and is a potential target for nonsteroidal anti-inflammatory drugs (NSAIDs), which cause regression of colorectal adenomas in both humans and laboratory animals.4,19,33 Cox-2 is an inducible enzyme that is localized around the nuclear envelope and produces eicosanoids, such as prostaglandin (PG) E2, which contribute to neoplastic cell growth.6,31,42,43,50,52 Dietary factors (e.g., n-3 fatty acids found in fish oil) can significantly decrease PGE2 production and inhibit intestinal tumor growth in vivo, presumably by altering substrate availability for the Cox-2 enzyme.12,25,26 Despite direct and indirect evidence indicating the importance of Cox-2 during intestinal carcinogenesis, there have been relatively few and sometimes conflicting reports describing the distribution of the protein in neoplastic tissues of humans and mice.3,13,14,18,32,33,38–41,44,51

Other than humans, the only species that naturally develops intestinal cancer with any frequency is the domestic dog, which shares our environment and in some households our diet.5,15,34,48 Canine colorectal adenomas may progress to malignancy, as in humans, but little is known about the molecular basis for tumorigenesis in these animals.9,48,54 As in human and mouse intestinal polyps, ß-catenin is strongly overexpressed in the cytoplasm and nucleus of neoplastic canine colorectal epithelial cells, suggesting conservation of early intestinal carcinogenic mechanisms between species.28 Because of this analogy, we were interested in determining whether Cox-2 is also overexpressed in canine intestinal tumors and, if so, in what populations of cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Thirty-one of the 37 cases of colorectal adenoma reported in the current study have been previously characterized with respect to aberrant expression of ß-catenin, E-cadherin, and p53;28 six new cases of colorectal adenoma have been added to this original collection. Tissues were from privately owned dogs and were obtained either as a result of surgical biopsy (n = 34) to diagnose and treat the clinical disease or after euthanasia (n = 3) sanctioned by the owner. More than 15 breeds of dogs were represented and none of the animals were related. The mean age was 8.4 years (range, 1–16 years) with 19 males (two castrated) and 18 females (15 spayed). Dogs for whom tissues were obtained at necropsy were selected based on a minimal postmortem period before tissue collection. All samples were routinely fixed for 18–72 hours in 10% neutral buffered formalin (NBF) before processing into paraffin for histologic sectioning. Normal canine kidney, used as a positive control for Cox-2, was placed in NBF within minutes of euthanasia and fixed for 48 hours before processing.

Primary antibodies included a rabbit polyclonal antibody raised against a mouse Cox-2 peptide (No. 160116; Cayman Chemical, Ann Arbor, MI), a mouse monoclonal antibody against the rat Cox-2 protein (No. C22420, clone 33; Transduction Laboratories, Lexington, KY), and a monoclonal antibody that binds {alpha}-smooth muscle actin ({alpha}-SMA; No. A2547, clone 1A4; Sigma, St. Louis, MO). The six new colorectal polyps not included in our previous report were also stained for ß-catenin (Transduction Laboratories monoclonal antibody) as previously described.28 Deparaffinized sections were pretreated in a 10 mM solution of citrate (pH 6.0) heated at or just below a boil for 10 minutes in a microwave oven and cooled to room temperature for 20 minutes. All sections were treated with 3% H2O2 in phosphate-buffered saline (PBS) followed by normal goat serum block (BioGenex, San Ramon, CA) and incubated overnight at 4 C with primary antibodies diluted 1:40 (6.3 µg/ml monoclonal Cox-2), 1:8,000 (polyclonal Cox-2), or 1:1,000 (6.5 µg/ml monoclonal {alpha}-SMA) in PBS (pH 7.6) containing 1% bovine albumin and 0.1% Tween-20. Sections were then incubated with biotin-conjugated goat anti-mouse or anti-rabbit immunoglobulin (50 minutes, 20 C; BioGenex) followed by streptavidin–peroxidase link (40 minutes, 20 C; BioGenex). Localized peroxidase conjugates were visualized with 3,3'-diaminobenzidine in sections lightly counter stained with hematoxylin.

Cox-2 staining in intestinal tumors was differentiated by location: 1) neoplastic epithelial cells; 2) nonneoplastic stromal cells just below the luminal surface of the tumor; 3) nonneoplastic cells deep within the stroma of the tumor; and 4) nonneoplastic cells immediately underneath or adjacent to the tumor but not part of the tumor mass (e.g., muscularis mucosa, submucosal fibroblasts, adjacent smooth muscle of tunics).

Staining for Cox-2 was scored in individual tumors according to the following criteria. For Cox-2 in tumor (nonneoplastic) stroma (including fibroblasts, myofibroblasts, macrophages, and smooth muscle fibers), scoring was as follows: 0 = negative; 1 = moderately intense staining of infrequent to scattered cells; 2 = multifocal to regionally strong staining (>= macula densa control) in >=15% of stromal cells; 3 = strong staining in most cells. For Cox-2 in tumor (neoplastic) epithelium, scoring was as follows: 0 = negative; 1 = rare positive epithelial cells; 2 = <5% of cells; 3 = 5–10% of cells; 4 = >10% of cells.

Stromal cells were identified as fibroblasts or macrophages based on histologic cell morphology; myofibroblasts were identified by immunohistochemical staining for {alpha}-SMA. Tumors were considered positive for Cox-2 if >5% of neoplastic epithelial cells (i.e., score of >=3) and/or >15–20% of nonneoplastic stromal cells (score of >=2) were immunohistochemically labeled.

Madin–Darby canine kidney cells (MDCK; American Type Culture Collection, Rockville MD) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and gentamicin (50 µg/ml). Cultures at 70% confluence in 100-mm plates were rinsed with sterile buffered saline and incubated 24 hours with DMEM containing 0.1% FBS, followed by 24 hours incubation with fresh medium (0.1% FBS) with or without 10 nM phorbol myristate acetate (PMA; Sigma). Duplicate PMA-stimulated and unstimulated cell cultures were then either lysed for western blot analysis as previously described30 or fixed in NBF. Fixed cells were gently scraped, pelleted, and suspended in 100 µl 1% high melting point agar before routine processing into paraffin for sectioning and immunohistochemistry.

For western blot analysis, total protein content of supernatants from MDCK cell lysates was determined by BCA protein assay according to the manufacturer's instructions (Pierce Chemical, Rockford, IL). Fifty micrograms of total protein from PMA-treated and untreated MDCK cultures, 10 µg total protein of an activated macrophage culture lysate (Cox-2 control; Transduction Laboratories), and molecular weight markers (Kaleidoscope Prestained Standards; BioRad, Cambridge, MA) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Membranes were incubated overnight at 4 C with Cox-2 antibodies diluted 1:1,000 (polyclonal) or 1:100 (2.5 µg/ml, monoclonal) in PBS containing 0.1% Tween-20 and 10% nonfat skim milk powder, followed by a 1-hour incubation at room temperature with peroxidase-conjugated anti-rabbit (1:5,000; Southern Biotechnology Associates, Birmingham, AL) or anti-mouse (1:80,000; Sigma) antibodies. Membranes were then incubated for 1 minute with freshly prepared ECL detection solution (No. RPN2209; Amersham, Piscataway, NJ) and exposed for <30 seconds to Hyperfilm ECL (Amersham), which was routinely developed and electronically scanned to generate a positive image of labeled protein bands.

All data were expressed as the mean ± SEM and were analyzed by Fisher's exact test (two tailed) or Welch's one way analysis of variance, as appropriate, using JMP software (SAS Institute, Cary, NC). Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Cox-2 is constitutively expressed in the macula densa of normal canine kidneys, whereas Cox-1 is not.20 Two primary anti-Cox-2 antibodies were tested on fixed sections of canine kidney to determine immunohistochemical specificity for the canine protein. Both antibodies clearly stained the nuclear envelope and circumferential perinuclear cytoplasm of epithelium forming the macula densa (Fig. 1), and both stained epithelial cells lining select tubules in the medulla and cortex with a fainter, more diffuse cytoplasmic pattern; the monoclonal antibody also stained endothelial cells lining parenchymal arterioles. To further establish specificity,2 canine cells (MDCK) were stimulated in vitro with the mitogen PMA to induce Cox-2 expression and were stained as fixed intact cells (i.e., processed in the same manner as tissue samples) or analyzed by western blot. Cox-2 protein is concentrated around the nuclear envelope,31 and there was relatively weak staining of the nuclear membrane in scattered untreated canine epithelial cells, with a dramatic increase in reactivity following PMA stimulation (Fig. 2). Staining was completely eliminated when the primary antibody was replaced by nonspecific murine IgG or rabbit serum (not shown). In two separate experiments, both antibodies produced a single band in western blots that migrated at a relative molecular mass of approximately 70 kd. This band was distinctly larger in lysates from the PMA-induced canine protein (Fig. 3) and migrated at the exact same relative molecular mass as did murine Cox-2, as previously reported.22 From these experiments, we concluded that both antibodies bound the induced canine Cox-2 isoform in formalin-fixed tissue sections. Although they produced the same pattern of specific staining in individual tumor samples (not shown), the Transduction Laboratories monoclonal anti-Cox-2 antibody was selected for immunohistochemical characterization of all canine intestinal tumors because of its greater specificity over background in stained sections; all figures represent results from use of this monoclonal antibody in immunohistochemical and western blot procedures.



View larger version (87K):
[in this window]
[in a new window]
  		Fig. 1. Kidney; dog. Cox-2 expression in epithelium of macula densa. Avidin–biotin–peroxidase complex method, Mayer's hematoxylin counterstain. Bar = 25 µm.
Fig. 2. Epithelial cell line (MDCK); dog. Fig. 2A. Formalin-fixed untreated control cells. Arrow indicates relatively infrequent and weak staining around nuclear envelope. Fig. 2B. Cells from a parallel culture treated with the mitogen PMA to induce Cox-2 expression. Avidin–biotin–peroxidase complex method, Mayer's hematoxylin counterstain. Bars = 25 µm.
Fig. 3. Western blot. Epithelial cell line (MDCK); dog. Cell lysates separated by polyacrylamide gel electrophoresis and stained to detect Cox-2 (70 kd) in untreated (Ctrl) and PMA-stimulated cultures of MDCK cells. Immunoperoxidase chemiluminescence.

 
Normal colonic and small intestinal mucosa did not stain for Cox-2. One animal with colitis had variable staining of superficial mucosal fibroblasts and macrophages throughout the large intestine. In these cell types staining was most intense immediately around the nuclear envelope. This pattern was also seen with Cox-2 staining in neoplastic and nonneoplastic cells of the intestinal tumors, except where particularly strong staining extended well into the perinuclear cytoplasm (Fig. 4), as in some PMA-treated MDCK cells. Immunoreactive Cox-2 protein was most common in colorectal polyps and small intestinal carcinomas (Table 1). A greater proportion of adenomas than carcinomas were positive for Cox-2, but this difference was not significant (P = 0.32). Ten of 13 Cox-2–positive large intestinal polyps were from the distal colon or colorectal junction. Three dogs had coincident adenomas and carcinomas (one colorectal, two small intestinal), but none of these were positive for Cox-2.



View larger version (148K):
[in this window]
[in a new window]
  		Fig. 4. Colorectal adenoma; dog. Strong expression of Cox-2 in stromal cells between neoplastic epithelial cells (asterisks) forming glandular structures. Avidin–biotin–peroxidase complex method, Mayer's hematoxylin counterstain. Bar = 25 µm.

 

View this table:
[in this window]
[in a new window]
  		Table 1 Cox-2 and ß-catenin expression in canine intestinal tumors by location and diagnosis.

 
Cox-2 expression in colorectal adenomas was essentially limited to nonneoplastic stromal cells, which morphologically included fibroblasts/myofibroblasts and, less often, macrophages (Figs. 4–6 ). Vasculature was not labeled. Although less dysplastic tubulovillous polyps were most often positive for Cox-2, there was no significant difference in Cox-2 expression based on tumor morphology (not shown). Rare Cox-2–positive neoplastic epithelial cells were scattered in two colonic polyps, which also showed significant stromal staining (score, >=2); none of the other adenomas had any evidence of Cox-2 expression in the epithelium. Stromal staining in colorectal polyps was most common in the superficial luminal aspects of the tumor mass (N = 13; mean score = 2.7 ± 0.13), but Cox-2 was also expressed by deeper stromal cells in 10 of these tumors (2.2 ± 0.23) (Fig. 6). Superficial staining of stromal cells was sometimes associated with and was more intense in areas of erosion where the overlying epithelium had been lost (Fig. 6). Nondegenerate neutrophils in adherent inflammatory exudates covering eroded tumor surfaces also stained for Cox-2 in some instances.



View larger version (210K):
[in this window]
[in a new window]
  		Fig. 5. Colorectal adenoma; dog. Fig. 5A. Overexpression of ß-catenin within the neoplastic epithelium. Fig. 5B. Serial section demonstrating upregulation of Cox-2 within nonneoplastic stromal cells. Avidin–biotin–peroxidase complex method, Mayer's hematoxylin counterstain. Bars = 50 µm.
Fig. 6. Colorectal adenoma; dog. Cox-2 expression is most prominent immediately below the tumor surface (arrowhead), but Cox-2–positive spindle-shaped cells are also scattered deeper in the tumor mass (arrow), closely apposed to and more distant from neoplastic epithelial cells. Avidin–biotin–peroxidase complex method, Mayer's hematoxylin counterstain. Bar = 100 µm.
Fig. 7. Colorectal adenoma; dog. Stromal spindle-shaped cells immediately adjacent to neoplastic epithelium (arrow) often express {alpha}-SMA, indicating a myofibroblast phenotype. Avidin–biotin–peroxidase complex method, Mayer's hematoxylin counterstain. Bar = 50 µm.
Fig. 8. Colorectal adenocarcinoma; dog. Neoplastic epithelial cells in malignancies often express Cox-2, as in this dog, whereas Cox-2 is restricted to the nonneoplastic stroma in adenomas. Avidin–biotin–peroxidase complex method, Mayer's hematoxylin counterstain. Bar = 25 µm.
Fig. 9. Colorectal adenocarcinoma; dog. Smooth muscle cells adjacent to infiltrating malignant epithelium (asterisk) express Cox-2 (arrow, cells at lower right). Avidin–biotin–peroxidase complex method, Mayer's hematoxylin counterstain. Bar = 25 µm.

 
In both the normal mucosa and neoplastic tissues, there was often a layer of spindle-shaped {alpha}-SMA–positive cells bordering crypts and tubular–glandular structures, consistent with the myofibroblast cellular phenotype.36 {alpha}-SMA was also expressed by some mesenchymal cells between and subjacent to tubular–glandular structures in the adenomas (Fig. 7) and by smooth muscle cells of arteriolar walls throughout all sections. The number and distribution of {alpha}-SMA–positive stromal cells differed among and within tumors, and these cells were usually more plump (viz hypertrophied) than those in the normal mucosa. In direct comparisons of serial sections of colorectal polyps stained for {alpha}-SMA and Cox-2, only 40–60% of Cox-2–positive cells were myofibroblasts. Conversely, not all myofibroblasts in these tumors were Cox-2 positive, and none of the {alpha}-SMA–positive cells in the adjacent normal mucosa expressed detectable levels of Cox-2. Cox-2–positive cells that did not express {alpha}-SMA were plump, angular to elongate, or stellate in outline (Fig. 4) and could represent either fibroblasts or macrophages based on morphology alone, although lack of phagocytic activity and elongate shape would be more typical of fibroblasts. Eight of the 13 Cox-2–positive colorectal polyps and four of those that did not contain significant Cox-2 reactivity in the tumor mass had some Cox-2 staining in subjacent cells, including smooth muscle cells of the muscularis mucosa, submucosal fibroblasts/myofibroblasts, and scattered mononuclear inflammatory cells (macrophages and/or mast cells).

In contrast to adenomas, Cox-2 staining was significantly more common in the neoplastic epithelium of carcinomas (mean score of 2.2 ± 0.29 versus 0.15 ± 0.10; P = 0.0013) and less common in tumor stromal cells (mean score = 1.3 ± 0.41), although the latter was not significantly different from adenomas (P = 0.08). Five carcinomas had a Cox-2 score of >=3 for epithelial staining (all small intestinal tumors), and four others had a score between 1 and 2 (i.e., <5% of cells; two small intestine and two large intestine tumors). Although most Cox-2–positive carcinomas contained a relatively small percentage of stained epithelial cells (5–10%), >40% of tumor cells strongly expressed the enzyme in one tumor (Fig. 8). Staining was heterogenous within individual tumors, but positive neoplastic cells were generally localized to relatively discrete regions rather than evenly dispersed throughout the mass. Although fibroblasts in the immediate vicinity of individual neoplastic cell infiltrates (including those in regions of desmoplasia) usually did not stain for Cox-2, the nuclear envelope and adjacent cytoplasm of smooth muscle cells frequently did stain in areas where the muscularis mucosa or intestinal tunics had been disrupted by infiltrating cancer cells (Fig. 9). Perinuclear Cox-2 staining was also variably present in widely scattered round cells within the connective tissues adjacent to vessels distant from the tumors. In serial sections stained with hematoxylin and eosin or toluidine blue, these cells corresponded to perivascular mast cells (not shown). Cox-2–positive round cells were also identified within several other tumors but could not be clearly distinguished as mast cells, macrophages, or other mononuclear inflammatory cells in these locations.

Six colorectal polyps included in this study had not been previously examined for ß-catenin expression.28 All six of the adenomas had the same pattern of cytoplasmic and nuclear ß-catenin reactivity as previously reported (Fig. 5).28 There was no significant association between increased cytoplasmic/nuclear ß-catenin staining and upregulation of Cox-2 in individual tumors (Tables 1, 2; P = 0.73). Staining for ß-catenin was confined to lateral cell membranes (viz normal distribution) in the six carcinomas with the greatest Cox-2 expression in the neoplastic epithelium, and Cox-2 was restricted to the nonneoplastic stroma in 13 of the 20 colorectal polyps where ß-catenin was upregulated in neoplastic cells (Fig. 5).


View this table:
[in this window]
[in a new window]
  		Table 2. Number of canine intestinal tumors expressing Cox-2 and/or ß-catenin.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Considerable epidemiologic and laboratory evidence indicates that Cox-2 significantly contributes to early stages of human colorectal tumorigenesis.52 Increased expression of Cox-2 and the beneficial effects of NSAIDs in familial adenomatous polyposis (FAP) patients suggests this may be particularly true when mutations in the adenomatous polyposis coli (APC) gene, which result in upregulation of ß-catenin, initiate tumorigenesis.10,11,23,37,44 NSAIDs also cause regression of intestinal tumors in genetic mouse models of FAP, where expression of Cox-2 has specifically been shown to effect tumor development.4,19,33 Colorectal tumorigenesis in domestic dogs is commonly associated with loss of ß-catenin regulation.28 Neoplastic epithelium in all canine colorectal polyps examined to date (N = 20) overexpress cytoplasmic and nuclear ß-catenin, with loss of full-length immunoreactive Apc protein in at least some of these tumors.28 This finding implicates early somatic mutation of the canine Apc gene (or perhaps ß-catenin) during colorectal tumorigenesis in this species, but more definitive analysis is required for confirmation. The overexpression of ß-catenin in naturally occurring intestinal neoplasms of humans and dogs is consistent with the inherent importance of this pathway during colorectal tumorigenesis. The results of the current study show that Cox-2 is also commonly upregulated in canine intestinal tumors, providing a second molecular link between humans and dogs in the development of this disease.

There has been some inconsistency in the reported localization of Cox-2 expression in intestinal tumors from humans and rodent models.3,13,14,18,32,38–41,44,51 We have found that Cox-2 is expressed almost exclusively by mesenchymal cells in intestinal tumors from MinApc/+ mice (unpublished), consistent with other reports indicating that Cox-2 is upregulated in the nonneoplastic stromal compartment of adenomas from both humans and mouse models.3,14,18,32,40,41 In contrast to expression in adenomatous polyps, COX-2 expression in human colorectal tumors shifts from the stroma to the neoplastic epithelium following progression to malignancy.32 A similar sequence is evident in dogs, with upregulation of Cox-2 in the stroma of adenomas and additional expression in the epithelium of carcinomas. Cox-2 in canine colorectal polyps was essentially restricted to myofibroblasts, fibroblasts, and perhaps macrophages (based on immunohistochemistry and cell morphology). Not all myofibroblasts in the adenomas were Cox-2 positive, and none of the myofibroblasts stained for Cox-2 in the normal mucosa, where they were smaller and presumed to be relatively quiescent. Although stromal Cox-2 expression was not as prominent in the carcinomas, there was additional immunostaining in smooth muscle cells of the muscularis mucosa and outer tunics, suggesting upregulation where the cancer had invaded the underlying tissues. Therefore, a variety of nonneoplastic mesenchymal cells may express Cox-2 in association with benign and malignant canine intestinal neoplasms, where the bioactive lipid products likely contribute to tumorigenesis as established in rodent models.33,53 The pathophysiologic and clinical significance of Cox-2 expression in the malignant epithelial cells remains to be demonstrated.

Numerous factors may activate myofibroblasts, smooth muscle cells, and macrophages to induce expression of Cox-2,6,36,52 but the mechanisms responsible for upregulation of this protein in the stroma or neoplastic epithelium of intestinal tumors is still unclear. Although loss of wild-type Apc may be responsible for increased Cox-2 in neoplastic intestinal epithelium, perhaps through modulation of ß-catenin–mediated transcription,17,29 less direct mechanisms must be responsible for Cox-2 upregulation in the nonneoplastic stroma. Numerous factors can induce Cox-2 including IL-1ß, tumor necrosis factor, transforming growth factor, bile acids, endotoxin, and perhaps other components of bacterial cell walls.6,27,36,49,52,55 Paracrine interactions between neoplastic epithelium and adjacent stromal cells could also result in upregulation of Cox-2, and physical characteristics of the neoplastic lesions causing exposure of stromal tissues to intestinal contents could have the same effect.36,39,52 Damage and loss of gastrointestinal epithelium causes upregulation of Cox-2 in (myo)fibroblasts, forming the ulcer granulation tissue bed where it is believed to promote healing.36 In addition, epithelial tight junctions in intestinal neoplasms may not be as strong as those in the normal mucosa,46 resulting in leakage of luminal bile acids, bacteria, and/or bacterial products into the underlying tissue spaces with direct or indirect (i.e., via IL-1ß) induction of Cox-2. Cox-2 expression in the canine colorectal polyps was frequently most intense just below the luminal surface of the tumor and was associated in particular with erosion of the overlying epithelium. A similar pattern of Cox-2 expression has been reported in intestinal tumors from MinApc/+ mice and humans.3,40 Therefore, superficial stromal induction of Cox-2 may result from damage to or the porous nature of overlying neoplastic epithelium, whereas expression deeper in the mass may be due to paracrine stimulation by factors derived from the neoplastic epithelium.

Regardless of the mechanisms involved, upregulation of Cox-2 clearly promotes intestinal tumor growth in vivo.33 The bioactive eicosanoids produced by Cox-2 are presumably responsible for this protumorigenic effect, which likely includes angiogenesis, cell proliferation, and inhibition of apoptosis.43,47,53 We have shown that Cox-2 is commonly upregulated in canine intestinal tumors, which occur spontaneously in the pet population. Widespread dysregulation of ß-catenin in these tumors is consistent with somatic mutation of the canine Apc gene or perhaps ß-catenin. The molecular mechanisms responsible for intestinal carcinogenesis in humans and mouse models are therefore conserved in dogs and include upregulation of Cox-2.21,35,37 The beneficial effects of NSAIDs and dietary n-3 fatty acids on colorectal tumorigenesis in laboratory rodents presumably is related, at least in part, to Cox-2 activity, and similar responses could therefore be expected in domestic dogs.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Arends JW: Molecular interactions in the Vogelstein model of colorectal carcinoma. J Pathol 190:412-416, 2000[CrossRef][Medline]
  2. Burry RW: Specificity controls for immunocytochemical methods. J Histochem Cytochem 48:163-166, 2000[Abstract/Free Full Text]
  3. Chapple KS, Cartwright EJ, Hawcroft G, Tisbury A, Bonifer C, Scott N, Windsor AC, Guillou PJ, Markham AF, Coletta PL, Hull MA: Localization of cyclooxygenase-2 in human sporadic colorectal adenomas. Am J Pathol 156:545-553, 2000[Abstract/Free Full Text]
  4. Chiu CH, McEntee MF, Whelan J: Sulindac causes rapid regression of preexisting tumors in Min/+ mice independent of prostaglandin biosynthesis. Cancer Res 57:4267-4273, 1997[Abstract/Free Full Text]
  5. Church EM, Mehlhaff CJ, Patnaik AK: Colorectal adenocarcinoma in dogs: 78 cases (1973–1984). J Am Vet Med Assoc 191:727-730, 1987[Medline]
  6. DuBois RN, Abramson SB, Crofford L, Gupta RA, Simon LS, Van de Putte LBA, Lipsky PE: Cyclooxygenase in biology and disease. Fed Am Soc Exp Biol J 12:1063-1073, 1998
  7. Fearon ER, Vogelstein B: A genetic model for colorectal tumorigenesis. Cell 61:759-767, 1990[CrossRef][Medline]
  8. Fodde R, Smits R, Hofland N, Kielman M, Meera KP: Mechanisms of APC-driven tumorigenesis: lessons from mouse models. Cytogenet Cell Genet 86:105-111, 1999[CrossRef][Medline]
  9. Gamblin RM, Sagartz JE, Couto CG: Overexpression of p53 tumor suppressor protein in spontaneously arising neoplasms of dogs. Am J Vet Res 58:857-863, 1997[Medline]
  10. Giardiello FM, Hamilton SR, Krush AJ, Piantadosi S, Hylind LM, Celano P, Booker SV, Robinson CR, Offerhaus GJ: Treatment of colonic and rectal adenomas with sulindac in familial adenomatous polyposis. N Engl J Med 328:1313-1316, 1993[Abstract/Free Full Text]
  11. Giardiello FM, Offerhaus JA, Tersmette AC, Hylind LM, Krush AJ, Brensinger JD, Booker SV, Hamilton SR: Sulindac induced regression of colorectal adenomas in familial adenomatous polyposis: evaluation of predictive factors. Gut 38:578-581, 1996[Abstract/Free Full Text]
  12. Hansen-Petrik M, McEntee MF, Chiu CH, Whelan J: Antagonism of arachidonic acid is linked to the anti-tumorigenic effect of eicosapentaenoic acid in ApcMin/+ mice. J Nutr 130:1153-1158, 2000[Abstract/Free Full Text]
  13. Hao X, Bishop AE, Wallace M, Wang H, Willcocks TC, Maclouf J, Polak JM, Knight S, Talbot IC: Early expression of cyclooxygenase-2 during sporadic colorectal carcinogenesis. J Pathol 187:295-301, 1999[CrossRef][Medline]
  14. Harada N, Tamai Y, Ishikawa T, Sauer B, Takaku K, Oshima M, Taketo MM: Intestinal polyposis in mice with a dominant stable mutation of the ß-catenin gene. Eur Mol Biol Organ 18:5931-5942, 1999
  15. Head KW: Tumours of the lower alimentary tract. Bull World Health Organ 53:167-186, 1976[Medline]
  16. Heyer J, Yang K, Lipkin M, Edelmann W, Kucherlapati R: Mouse models for colorectal cancer. Oncogene 18:5325-5333, 1999[CrossRef][Medline]
  17. Hsi LC, Angerman-Stewart J, Eling TE: Introduction of full-length APC modulates cyclooxygenase-2 expression in HT-29 human colorectal carcinoma cells at the translational level. Carcinogenesis 20:2045-2049, 1999[Abstract/Free Full Text]
  18. Hull MA, Booth JK, Tisbury A, Scott N, Bonifer C, Markham AF, Coletta PL: Cyclooxygenase 2 is up-regulated and localized to macrophages in the intestine of Min mice. Br J Cancer 79:1399-1405, 1999[CrossRef][Medline]
  19. Jacoby RF, Seibert K, Cole CE, Kelloff G, Lubet RA: The cyclooxygenase-2 inhibitor celecoxib is a potent preventive and therapeutic agent in the Min mouse model of adenomatous polyposis. Cancer Res 60:5040-5044, 2000[Abstract/Free Full Text]
  20. Khan KNM, Venturini CM, Bunch RT, Brassard JA, Kiki AT, Morris DL, Trump BF, Maziasz TJ, Alden CL: Interspecies differences in renal localization of cyclooxygenase isoforms: implications in nonsteroidal antiinflammatory drug-related nephrotoxicity. Toxicol Pathol 26:612-620, 1998[Medline]
  21. Kinzler KW, Vogelstein B: Lessons from hereditary colorectal cancer. Cell 87:159-170, 1996[CrossRef][Medline]
  22. Kraemer SA, Arthur KA, Denison MS, Smith WL, Dewitt DL: Regulation of prostaglandin endoperoxide H synthase-2 expression by 2,3,7,8,-tetrachlorodibenzo-p-dioxin. Arch Biochem Biophys 330:319-328, 1996[CrossRef][Medline]
  23. Labayle D, Fischer D, Vielh P, Drouhin F, Pariente A, Bories C, Duhamel O, Trousset M, Attali P: Sulindac causes regression of rectal polyps in familial adenomatous polyposis. Gastroenterology 101:635-639, 1991[Medline]
  24. Landis SH, Murray T, Bolden S, Wingo PA: Cancer statistics, 1999. CA Cancer J Clin 49:8-31, 1999[Abstract/Free Full Text]
  25. Li B, Birdwell C, Whelan J: Antithetic relationship of dietary arachidonic acid and eicosapentaenoic acid on eicosanoid production in vivo. J Lipid Res 35:1869-1877, 1994[Abstract]
  26. Lipkin M, Reddy B, Newmark H, Lamprecht SA: Dietary factors in human colorectal cancer. Annu Rev Nutr 19:545-586, 1999[CrossRef][Medline]
  27. Maloney CG, Thompson SD, Hill HR, Bohnsack JF, McIntyre TM, Zimmerman GA: Induction of cyclooxygenase-2 by human monocytes exposed to group B streptococci. J Leukoc Biol 67:615-621, 2000[Abstract]
  28. McEntee MF, Brenneman KA: Dysregulation of ß-catenin is common in canine sporadic colorectal tumors. Vet Pathol 36:228-236, 1999[Abstract]
  29. Mei JM, Hord NG, Winterstein DF, Donald SP, Phang JM: Differential expression of prostaglandin endoperoxide H synthase-2 and formation of activated ß-catenin-LEF-1 transcription complex in mouse colonic epithelial cells contrasting in Apc. Carcinogenesis 20:737-740, 1999[Abstract/Free Full Text]
  30. Merryman JI, Neilsen N, Stanton DD: Transforming growth factor-ß enhances the ultraviolet-mediated stress response in p53-/- keratinocytes. Int J Oncol 13:781-789, 1998[Medline]
  31. Morita I, Schindler M, Regier MK, Otto JC, Hori T, Dewitt DL, Smith WL: Different intracellular locations for prostaglandin endoperoxide H synthase-1 and -2. J Biol Chem 270:10902-10908, 1995[Abstract/Free Full Text]
  32. Muller-Decker K, Albert C, Lukanov T, Winde G, Marks F, Furstenberger G: Cellular localization of cyclo-oxygenase isozymes in Crohn's disease and colorectal cancer. Int J Colorectal Dis 14:212-218, 1999[CrossRef][Medline]
  33. Oshima M, Dinchuk JE, Kargman SL, Oshima H, Hancock B, Kwong E, Trzaskos JM, Evans JF, Taketo MM: Suppression of intestinal polyposis in APC{Delta}716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 87:803-809, 1996[CrossRef][Medline]
  34. Patnaik AK, Hurvitz AI, Johnson GF: Canine intestinal adenocarcinoma and carcinoid. Vet Pathol 17:149-163, 1980[Abstract]
  35. Polakis P: The oncogenic activation of ß-catenin. Curr Opin Genet Dev 9:15-21, 1999[CrossRef][Medline]
  36. Powell DW, Mifflin RC, Valentich JD, Crowe SE, Saada JI, West AB: Myofibroblasts. II. Intestinal subepithelial myofibroblasts. Am J Physiol 277:C183-C201, 1999
  37. Prescott SM, White RL: Self-promotion? Intimate connections between APC and prostaglandin H synthase-2. Cell 87:783-786, 1996[CrossRef][Medline]
  38. Sano H, Kawahito Y, Wilder RL, Hashiramoto A, Mukai S, Asai K, Kimura S, Kato H, Kondo M, Hla T: Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res 55:3785-3789, 1995[Abstract/Free Full Text]
  39. Shao J, Sheng H, Aramandla R, Pereira MA, Lubet RA, Hawk E, Grogan L, Kirsch IR, Washington MK, Beauchamp RD, DuBois RN: Coordinate regulation of cyclooxygenase-2 and TGF-ß1 in replication error-positive colon cancer and azoxymethane-induced rat colonic tumors. Carcinogenesis 20:185-191, 1999[Abstract/Free Full Text]
  40. Shattuck-Brandt RL, Lamps LW, Heppner Goss KJ, DuBois RN, Matrisian LM: Differential expression of matrilysin and cyclooxygenase-2 in intestinal and colorectal neoplasms. Mol Carcinog 24:177-187, 1999[CrossRef][Medline]
  41. Shattuck-Brandt RL, Varilek GW, Radhika A, Yang F, Washington MK, DuBois RN: Cyclooxygenase 2 expression is increased in the stroma of colon carcinomas from IL-10-/- mice. Gastroenterology 118:337-345, 2000[CrossRef][Medline]
  42. Sheng H, Shao J, Morrow JD, Beauchamp RD, DuBois RN: Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 58:362-366, 1998[Abstract/Free Full Text]
  43. Sheng H, Shao J, Washington MK, DuBois RN: Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J Biol Chem 276:18075-18081, 2001[Abstract/Free Full Text]
  44. Sinicrope FA, Lemoine M, Xi L, Lynch PM, Cleary KR, Shen Y, Frazier ML: Reduced expression of cyclooxygenase 2 proteins in hereditary nonpolyposis colorectal cancers relative to sporadic cancers. Gastroenterology 117:350-358, 1999[CrossRef][Medline]
  45. Smits R, Kielman MF, Breukel C, Zurcher C, Neufeld K, Jagmohan-Changur S, Hofland N, van Dijk J, White R, Edelmann W, Kucherlapati R, Khan PM, Fodde R: Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli protein involved in tumorigenesis and development. Genes Dev 13:1309-1321, 1999[Abstract/Free Full Text]
  46. Soler AP, Miller RD, Laughlin KV, Carp NZ, Klurfeld DM, Mullin JM: Increased tight junctional permeability is associated with the development of colon cancer. Carcinogenesis 20:1425-1431, 1999[Abstract/Free Full Text]
  47. Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN: Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 93:705-716, 1998[CrossRef][Medline]
  48. Valerius KD, Powers BE, McPherron MA, Hutchison JM, Mann FA, Withrow SJ: Adenomatous polyps and carcinoma in situ of the canine colon and rectum: 34 cases (1982–1994). J Am Anim Hosp Assoc 33:156-160, 1997[Abstract]
  49. van Tol EA, Holt L, Li FL, Kong FM, Rippe R, Yamauchi M, Pucilowska J, Lund PK, Sartor RB: Bacterial cell wall polymers promote intestinal fibrosis by direct stimulation of myofibroblasts. Am J Physiol 277:G245-G255, 1999[Abstract/Free Full Text]
  50. Watanabe K, Kawamori T, Nakatsugi S, Ohta T, Ohuchida S, Yamamoto H, Maruyama T, Kondo K, Ushikubi F, Narumiya S, Sugimura T, Wakabayashi K: Role of the prostaglandin E receptor subtype EP1 in colon carcinogenesis. Cancer Res 59:5093-5096, 1999[Abstract/Free Full Text]
  51. Williams CS, Luongo C, Radhika A, Zhang T, Lamps LW, Nanney LB, Beauchamp RD, DuBois RN: Elevated cyclooxygenase-2 levels in Min mouse adenomas. Gastroenterology 111:1134-1140, 1996[CrossRef][Medline]
  52. Williams CS, Mann M, DuBois RN: The role of cyclooxygenases in inflammation, cancer, and development. Oncogene 18:7908-7916, 1999[CrossRef][Medline]
  53. Williams CS, Tsujii M, Reese J, Dey SK, DuBois RN: Host cyclooxygenase-2 modulates carcinoma growth. J Clin Invest 105:1589-1594, 2000[Medline]
  54. Wolf JC, Ginn PE, Homer B, Fox LE, Kurzman ID: Immunohistochemical detection of p53 tumor suppressor gene protein in canine epithelial colorectal tumors. Vet Pathol 34:394-404, 1997[Abstract]
  55. Zhang F, Altorki NK, Mestre JR, Subbaramaiah K, Dannenberg AJ: Curcumin inhibits cyclooxygenase-2 transcription in bile acid– and phorbol ester–treated human gastrointestinal epithelial cells. Carcinogenesis 20:445-451, 1999[Abstract/Free Full Text]
Request reprints from Dr. M. McEntee, Department of Pathology, PO Box 1071, Knoxville, TN 37901 (USA). E-mail: mmcentee{at}utk.edu.




This article has been cited by other articles:


Home page
 Vet Rec.Home page
S. Boonsoda and P. Wanikiat
Possible role of cyclooxygenase-2 inhibitors as anticancer agents
Vet Rec., February 2, 2008; 162(5): 159 - 161.
[Full Text] [PDF]

Home page
 jvdiHome page
S. J. Newman and L. Mrkonjich
Cyclooxygenase-2 expression in feline pancreatic adenocarcinomas
J Vet Diagn Invest, November 1, 2006; 18(6): 590 - 593.
[Abstract] [Full Text] [PDF]

Home page
 Vet PatholHome page
J. S. Munday, M. M. Brennan, and M. Kiupel
Altered Expression of {beta}-catenin, E-cadherin, Cycloxygenase-2, and p53 Protein by Ovine Intestinal Adenocarcinoma Cells.
Vet. Pathol., September 1, 2006; 43(5): 613 - 621.
[Abstract] [Full Text] [PDF]

Home page
 Vet PatholHome page
M. Brunelle, E. A. Sartin, L. G. Wolfe, J. Sirois, and M. Dore
Cyclooxygenase-2 expression in normal and neoplastic canine mammary cell lines.
Vet. Pathol., September 1, 2006; 43(5): 656 - 666.
[Abstract] [Full Text] [PDF]

Home page
 Vet PatholHome page
J. A. Landolfi and K. A. Terio
Transitional Cell Carcinoma in Fishing Cats (Prionailurus viverrinus): Pathology and Expression of Cyclooxygenase-1, -2, and p53.
Vet. Pathol., September 1, 2006; 43(5): 674 - 681.
[Abstract] [Full Text] [PDF]

Home page
 Vet PatholHome page
D. A. Heller, C. A. Clifford, M. H. Goldschmidt, D. E. Holt, F. S. Shofer, A. Smith, and K. U. Sorenmo
Cyclooxygenase-2 Expression is Associated with Histologic Tumor Type in Canine Mammary Carcinoma
Vet. Pathol., November 1, 2005; 42(6): 776 - 780.
[Abstract] [Full Text] [PDF]

Home page
 Vet PatholHome page
D. A. Heller, C. A. Clifford, M. H. Goldschmidt, D. E. Holt, M. J. Manfredi, and K. U. Sorenmo
Assessment of Cyclooxygenase-2 Expression in Canine Hemangiosarcoma, Histiocytic Sarcoma, and Mast Cell Tumor
Vet. Pathol., May 1, 2005; 42(3): 350 - 353.
[Abstract] [Full Text] [PDF]

Home page
 Vet PatholHome page
S. L. Beam, K. M. Rassnick, A. S. Moore, and S. P. McDonough
An Immunohistochemical Study of Cyclooxygenase-2 Expression in Various Feline Neoplasms
Vet. Pathol., September 1, 2003; 40(5): 496 - 500.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McEntee, M. F.
Right arrow Articles by Neilsen, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McEntee, M. F.
Right arrow Articles by Neilsen, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS