Affiliations of authors: C. Tremblay, M. Doré (Département de Pathologie et Microbiologie), J. Sirois (Centre de Recherche en Reproduction Animale), Faculté de Médecine Vétérinaire, Université de Montréal, Québec, Canada; P. N. Bochsler, Department of Pathology, University of Tennessee, Knoxville.
Correspondence to: Jean Sirois, D.V.M., M.Sc., Ph.D., Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000, St-Hyacinthe, Québec, Canada J2S 7C6 (e-mail: siroisje{at}medvet.umontreal.ca).
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ABSTRACT |
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INTRODUCTION |
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Prostaglandins play a role in the regulation of several important physiologic and pathologic processes, and evidence (8-10) suggest that they could be involved in tumor progression. Prostaglandin G/H synthase (PGHS; also known as cyclooxygenase or COX) is the key rate-limiting enzyme in the biosynthetic pathway of prostaglandins from arachidonic acid (11,12). Two forms of PGHS have been characterized and are known as PGHS-1 (or COX-1) and PGHS-2 (or COX-2) (13-16). PGHS-1 is constitutively expressed in various tissues and was originally referred to as the constitutive isoenzyme involved in the synthesis of prostaglandins necessary for normal cellular processes (17,18). In contrast, PGHS-2 is generally undetectable in most tissues, can be induced by different agonists, and was referred to as the inducible isoenzyme involved in inflammation (17,18). However, results from PGHS-1 and PGHS-2 gene-targeting studies in mice (19-21) suggest that both isoenzymes participate in physiologic and inflammatory processes.
Mounting evidence (8-10,18) suggests that PGHS-2 plays a role in cancer progression. Increased expression of PGHS-2 has been demonstrated in human colon, lung, and gastric cancers and in esophageal high-risk noncancerous lesions (22-26). Oshima et al. (27) reported that introduction of a knockout mutation of the PGHS-2 gene in an Apc (adenomatous polyposis coli) knockout mouse (Apc is the mouse homologue of the human APC gene that has been linked to familial adenomatous polyposis) results in a substantial reduction in the number and size of the intestinal polyps. Moreover, the long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs), which act on PGHS, appears to have a protective effect on the incidence of colorectal cancer (8-10). Enhanced expression of PGHS-2 has also been documented following ultraviolet B irradiation of keratinocytes, suggesting its involvement in skin tumor development (28). A potential role for PGHS-2 in human prostate cancer has been proposed by recent studies in vitro with the use of human prostatic cancer cell lines. Exogenous prostaglandins E2 were shown to increase PGHS-2 transcripts (29), whereas a selective COX-2 inhibitor, NS398 (N-[2-cyclohexyloxy-4-nitrophenyl] methanesulfonamide), induced apoptosis (30). A recent epidemiologic study (31) reported that the regular use of NSAIDs may reduce the relative risk of prostate cancer. However, the incidence of PGHS expression in spontaneous prostate cancer has not been reported for any species. Therefore, the objectives of this study were to determine whether PGHS isoenzymes are expressed in canine prostate cancer and, if so, to identify which isoenzyme is involved (PGHS-1 and/or PGHS-2) and to determine its cellular localization.
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MATERIALS AND METHODS |
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Twenty-four cases of canine prostatic adenocarcinomas submitted to the Département de Pathologie et Microbiologie of the Faculté de Médecine Vétérinaire (Université de Montréal) and the Department of Pathology of the College of Veterinary Medicine (University of Tennessee) were included in the study. All cases were confirmed as prostatic adenocarcinomas by examination of hematoxylin-eosin-saffran-stained sections by a veterinary pathologist. The malignant nature of the prostatic tumors was defined by use of criteria such as anaplasia (anisocytosis, pleomorphism, anisokaryosis), loss of normal polarity, anarchic growth, atypical mitotic figures, and local or vascular invasion. The histologic classification used was the World Health Organization International Histological Classification of Tumors of Domestic Animals (32). Normal prostates were obtained from four adult dogs (one Rottweiler, one MÂtin de Naples, and two mixed breed dogs; ages between 3 and 6 years) euthanized for reasons unrelated to health problems. All tissues were fixed in 10% neutral buffered formalin, whereas samples from two normal prostates and two adenocarcinomas were frozen at -70 °C for immunoblot analysis.
Canine platelets were isolated from whole blood collected by venipuncture from a healthy adult dog in anticoagulant (citrate phosphate dextrose solution; Abbott Laboratories, Chicago, IL). Platelet-rich plasma was isolated by successive centrifugations at room temperature of the citrated blood for 3 minutes at decreasing speed (700g, 650g, and 600g), as previously described (33). Platelets were recovered from the platelet-rich plasma by centrifugation at 16 000g for 10 minutes at room temperature and were stored at -70 °C. All animal procedures were approved by the institutional animal care and use committee of the Université de Montréal.
Anti-PGHS Antibodies
Two anti-PGHS antibodies (antibodies 8223 and MF243) were used. Affinity-purified polyclonal antibody 8223 was raised in rabbits against ovine PGHS-1 and was shown to be selective for PGHS-1 in various species (34,35). Antibody MF243 was provided by Drs. Jilly F. Evans and Stacia Kargman (Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Québec). MF243 was raised in rabbits against ovine placental PGHS-2, and its selectivity for PGHS-2 has previously been characterized (22).
Immunohistochemistry
Immunohistochemical staining was performed with the use of the Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA), as previously described (36). Briefly, formalin-fixed tissues were paraffin embedded, and 3-µm-thick sections were prepared and deparaffinized through graded alcohol series. Endogenous peroxidase was quenched by incubating the slides in 0.3% hydrogen peroxide in methanol for 30 minutes. After being rinsed in phosphate-buffered saline (PBS) for 15 minutes, sections were incubated with diluted normal goat serum for 20 minutes at room temperature. Primary antibodies diluted in PBS were applied (8223 at 1 : 100 dilution and MF243 at 1 : 7500 dilution), and sections were incubated overnight at 4 °C. Control sections were incubated with PBS or with nonimmune rabbit serum. After sections were rinsed in PBS for 10 minutes, a biotinylated goat anti-rabbit antibody (1 : 222 dilution) was applied, and sections were incubated for 45 minutes at room temperature. Sections were washed in PBS for 10 minutes and incubated with the avidin DH-biotinylated horseradish peroxidase H reagents for 45 minutes at room temperature. After the PBS wash for 10 minutes, the reaction was revealed with the use of diaminobenzidine tetrahydrochloride as the peroxidase substrate. Sections were counterstained with Gill's hematoxylin stain and mounted. Immunoreactivity was evaluated in a blinded fashion by two independent observers using a grading system where 0 = 0% of positive cells, 1 = less than 10% of positive cells, 2 = 10%-30% of positive cells, 3 = 31%-60% of positive cells, and 4 = greater than 60% of positive cells. Also, the intensity of PGHS-2 immunoreactivity was graded as - = no staining, + = weak staining, ++ = moderate staining, and +++ = strong staining.
Solubilized Cell Extracts and Immunoblot Analysis
Solubilized cell extracts were prepared as previously described (35). Briefly, tissues were homogenized on ice in TED buffer (50 mM Tris [pH 8.0], 10 mM ethylenediaminetetraacetic acid [EDTA], and 1 mM diethyldithiocarbamic acid [DEDTC]) containing 2 mM octyl glucoside and centrifuged at 30 000g for 1 hour at 4 °C. The crude pellets (membranes, nuclei, and mitochondria) were sonicated (8 seconds per cycle; three cycles) in TED sonication buffer (20 mM Tris [pH 8.0], 50 mM EDTA, and 0.1 mM DEDTC) containing 32 mM octyl glucoside. The sonicates were centrifuged at 16 000g for 15 minutes at 4 °C. The recovered supernatant (solubilized cell extract) was stored at -70 °C until electrophoretic analyses were performed. The protein concentration was determined by the method of Bradford (37) (Bio-Rad Protein Assay). Proteins were resolved by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose filters, as described (35). Filters were incubated with anti-PGHS antibodies diluted in TBS (10 mM Tris-buffered saline [pH 7.5]) containing 2% nonfat dry milk. [125I]Protein-A (1 x 106 cpm/mL in TBS containing 2% milk) was used to visualize immunopositive proteins. Filters were washed three times (10 minutes per wash) in TBS containing 0.05% Tween 20 and exposed to Kodak X-OMAT AR film at -70 °C.
Statistical Analysis
Fisher's exact test was used to compare the frequency of PGHS-2 expression between normal prostates and prostatic adenocarcinomas. All P values reported are two-sided. Statistical analyses were performed with the use of the JMP Software (SAS Institute, Inc., Cary, NC).
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RESULTS |
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DISCUSSION |
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Recent studies indicate that PGHS-2, the inducible form of the enzyme, is implicated in the production of prostaglandins by cancerous cells from a variety of tissues. Increased expression of PGHS-2 was first demonstrated in human colorectal cancers but has now been reported in tumor cells from pulmonary, gastric, skin, esophageal, and breast carcinomas (22-26,43). Increased expression of PGHS-2 messenger RNA has been shown in two human prostate cancer cell lines (29), but the expression of PGHS-2 by prostate cancer cells in vivo has not been documented. The potential role played by PGHS in cancer is underscored by the finding that the long-term use of aspirin and other NSAIDs appears to significantly decrease the relative risk for certain types of cancer, notably colorectal cancer (8-10,44). Aspirin is known to acetylate and irreversibly inactivate PGHS enzymes (45). Epidemiologic studies (46,47) investigating aspirin use in relation to various cancers have reported weak inverse associations for prostate cancer risk. A recent population-based, case-control study (31) aimed at specifically investigating a possible association between prostate cancer risk and NSAIDs found an inverse association between the risk of advanced prostate cancers and the regular use of aspirin and other NSAIDs.
The mechanisms by which increased synthesis of PGHS contributes to tumor development are slowly beginning to unravel. Studies in vitro have shown that intestinal epithelial cells overexpressing PGHS-2 exhibit increased adhesion to extracellular matrix and are resistant to induced apoptosis, two phenotypic changes that could enhance their tumorigenic potential (48). It is interesting that Liu et al. (30) recently reported that a selective PGHS-2 inhibitor, NS398, induced apoptosis and caused a decreased synthesis of bcl-2 (an antiapoptotic oncoprotein) in a human prostate cancer cell line. Expression of PGHS-2 could also be associated with increased metastatic potential, as suggested by the increased invasiveness of a human colon cancer cell line transfected with a PGHS-2 expression vector (49). The same group of authors (50) also recently presented evidence that colon cancer cells overexpressing PGHS-2 produce high levels of angiogenic factors that could contribute to tumor angiogenesis, a step essential to tumor growth. They (50) suggested that PGHS-2 modulates production of angiogenic factors by cancer cells, while PGHS-1 in endothelial cells plays a role in endothelial tube formation. Furthermore, prostaglandins are known to contribute to cancer development by acting at different levels of malignant transformation, including stimulation of cell growth, involvement in tumor promotion, and suppression of the immune response (8,51).
Although prostate cancer represents the most common male cancer in the United States, the factors responsible for its high prevalence are poorly understood. In men, prostate cancer is influenced by age, race, and certain environmental, dietary, and familial factors (52). However, very little is known about the molecular mechanisms leading to prostate cancer. For reasons not yet understood, the dog is the only nonhuman species that frequently develops spontaneous prostate cancer with advancing age (2). Of interest, many aspects of canine prostate cancer are similar to the human disease (3,4). For example, prostate cancer is strongly influenced by age in both species. A study (53) has shown that the physiologic age at prostate cancer diagnosis, expressed in human years, was similar between the two species (67 and 70 years for dogs and men, respectively). Also, certain premalignant and malignant changes at the histologic level are similar in both species, including the presence of high-grade prostatic intraepithelial neoplasia in canine prostate (5,6). This premalignant change has been reported in the prostate of normal dogs and in dogs with spontaneous prostate carcinoma (5,6). In normal dogs, the prevalence of high-grade prostatic intraepithelial neoplasia seems to be influenced by age and testicular androgens (5). Moreover, prostate cancer in dogs displays a high incidence of osseous metastases, as observed in men (3). These similarities were underscored in a recent international workshop on animal models of prostate cancer that proposed the dog as a relevant model for the study of spontaneous prostate cancer (3). The induction of PGHS-2 in a majority of canine prostatic adenocarcinomas provides a novel element in our understanding of prostate cancer in dogs. Further study of PGHS-2 may provide insight into human prostate carcinogenesis and progression.
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NOTES |
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We thank Drs. Jilly F. Evans and Stacia Kargman, Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Québec, Canada, for providing the MF243 antibody.
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Manuscript received December 28, 1998; revised June 11, 1999; accepted June 22, 1999.
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