REPORTS

Induction of Prostaglandin G/H Synthase-2 in a Canine Model of Spontaneous Prostatic Adenocarcinoma

Claudine Tremblay, Monique Doré, Philip N. Bochsler, Jean Sirois

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).


    ABSTRACT
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
BACKGROUND: Prostate cancer is the most frequently occurring cancer in men in the United States, with an estimated 179 300 new cases in 1999. The induction of prostaglandin G/H synthase (PGHS), a key rate-limiting enzyme in prostaglandin biosynthesis, has been implicated in various cancers, most notably in colorectal cancers; however, the induction of PGHS expression in prostate cancer in vivo has not been reported for any species. The dog is the only nonhuman species that frequently develops spontaneous cancer of the prostate with increasing age, and the objective of this study was to determine whether PGHS isoenzymes were expressed in canine prostatic adenocarcinomas. METHODS: Four normal canine prostatic tissues and 24 canine prostatic adenocarcinomas were studied by means of immunohistochemistry and immunoblot analysis, using polyclonal antibodies specific for each of the two PGHS isoenzymes, PGHS-1 and PGHS-2. All P values were obtained by use of two-sided Fisher's exact tests. RESULTS: PGHS-1 immunostaining was localized to stromal fibroblasts and vascular endothelium in normal and cancerous prostates. PGHS-2 was not detected in normal prostates, but it was expressed by epithelial tumor cells in 18 (75%) of the 24 adenocarcinomas (P = .01). Immunoblot analysis confirmed the presence of PGHS-1 (69 000 molecular weight) in normal and cancerous tissues and the expression of PGHS-2 (72 000- to 74 000-molecular-weight doublet) only in prostatic adenocarcinomas. CONCLUSION: To our knowledge, these results demonstrate for the first time that PGHS-2 is induced in the majority of canine spontaneous prostatic adenocarcinomas and suggest that its expression may be involved in prostate cancer.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Prostate cancer is the most frequent cancer of men in the United States, with an estimated 179 300 new cases in 1999 (1). The dog represents the only nonhuman species that frequently develops spontaneous cancer of the prostate with increasing age (2). Several aspects of canine prostate cancer are similar to the human disease, and the relevance of the dog as an animal model to study prostatic carcinogenesis was recently highlighted (3). In both species, adenocarcinoma of the prostate affects older subjects and is a locally invasive and insidious disease that metastasizes to distant tissues (3,4). Also, prostatic intraepithelial neoplasia, the most likely precursor of some human prostate cancers, occurs spontaneously in the prostate of elderly, sexually intact dogs, and the majority of dogs with prostate cancer present high-grade prostatic intraepithelial neoplasia (5-7).

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.


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Tissue Samples and Platelet Isolation

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).


    RESULTS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Characteristics of dogs with prostatic adenocarcinomas. The age of the dogs with prostatic adenocarcinoma ranged from 5.5 to 15 years old, with a mean of 9.6 years. Of the 24 case animals, 13 (54%) were castrated, five (21%) were intact, and the neuter status of six animals was unavailable. No breed predilection was observed (Table 1)Go. At the time of diagnosis, a high percentage of dogs (14 [58%] of 24) had metastases to various organs, most frequently to the abdominal serosa, lungs, and lymph nodes (Table 1)Go.


View this table:
[in this window]
[in a new window]
 
Table 1. Characteristics of specimens from dogs with prostatic adenocarcinomas*

 
PGHS-1 expression in normal canine prostates. To determine whether PGHS-1 and/or PGHS-2 were expressed under physiologic conditions, we performed immunohistochemical staining on normal canine prostates (n = 4). Results showed that PGHS-1 was present in fibroblastic cells of the conjunctive stroma and in endothelial cells of some blood vessels (Fig. 1Go, A and B) in all four normal prostates. However, no PGHS staining was observed in prostatic epithelial cells (Fig. 1Go, C), and no PGHS-2 expression was detected in normal canine prostates (Fig. 1Go, D).






View larger version (581K):
[in this window]
[in a new window]
 
Fig. 1. Expression of prostaglandin G/H synthase-1 (PGHS-1) by normal canine prostates. Immunohistochemistry was performed on formalin-fixed sections of normal canine prostates, as described in the "Materials and Methods" section. Immunostaining with antibody 8223 (selective for PGHS-1) demonstrated the presence of PGHS-1 in the stroma and in the endothelium of blood vessels (A and B) but not in epithelial cells (C). Immunostaining with antibody MF243 (selective for PGHS-2) showed no reactivity in the stroma or in epithelial cells (D) (original magnifications x200 [A] and x400 [B-D].

 
Induction of PGHS-2 in canine prostatic adenocarcinomas. A different staining pattern was observed in prostatic adenocarcinomas. In contrast to normal prostates where no PGHS-2 was detected, 18 (75%) of the 24 adenocarcinomas studied expressed PGHS-2 (P = .01; Table 1Go). PGHS-2 was observed in 12 of 13 castrated dogs and in four of five intact dogs. PGHS-2 expression was localized to epithelial tumor cells (Fig. 2Go, A-D). The intensity of the reaction varied among tumors (Table 1)Go, ranging from a diffuse pale cytoplasmic positive staining to a very strong reactivity filling the cell. In some cells, the PGHS-2 staining formed a discrete perinuclear halo (Fig. 2Go, C and D). As observed in normal prostates, PGHS-1 was detected in the majority of adenocarcinomas (22 of 24 case animals) in stromal fibroblasts and/or vascular endothelial cells. Also, a faint PGHS-1 signal was observed in epithelial cells in some tumors (Fig. 2Go, E). No staining was detected in control sections with the use of normal rabbit serum (Fig. 2Go, F) or PBS (data not shown).








View larger version (927K):
[in this window]
[in a new window]
 
Fig. 2. Expression of prostaglandin G/H synthase (PGHS)-1 and PGHS-2 by canine prostatic adenocarcinomas. Immunohistochemistry was performed on formalin-fixed sections of canine prostatic adenocarcinomas, as described in the "Materials and Methods" section. Immunostaining with antibody MF243 (selective for PGHS-2) revealed the presence of PGHS-2 in epithelial tumor cells (A-D). Three different adenocarcinomas are shown (A and B are from case animal 2, while C and D are from case animals 1 and 4, respectively, as listed in Table 1Go). In some cells, the PGHS-2 staining formed a discrete perinuclear halo (C and D). Immunostaining with antibody 8223 (selective for PGHS-1) showed some reactivity in some epithelial tumor cells in few tumors (E). Control staining with normal rabbit serum was negative (F) (original magnifications x400 [A-F]).

 
Immunoblotting of PGHS isoenzymes in canine prostate tissues. To document the specificity of anti-PGHS antibodies on canine tissues and characterize each PGHS isoenzyme in the dog, we prepared solubilized cell extracts from normal prostates, prostatic adenocarcinomas, and platelets, and we analyzed proteins by western blotting. When a selective anti-ovine PGHS-1 antibody was used, a 69 000-molecular-weight (Mr) band was detected in normal prostates and in prostatic adenocarcinomas (Fig. 3Go, A). A band of identical molecular weight was detected in canine platelets (Fig. 3Go, A) and thus corresponded to canine PGHS-1. In contrast, when a selective anti-ovine PGHS-2 antibody was used, no signal was detected in normal prostates, but PGHS immunoreactivity was observed in prostatic adenocarcinomas (Fig. 3Go, B). Canine PGHS-2 appeared as a 72 000- to 74 000-Mr doublet and a small 62 000-Mr band (Fig. 3Go, B) believed to correspond to a proteolytic fragment, as previously observed in other species (16,34,35). The absence of detectable PGHS-2 in canine platelets is in keeping with reports in other species (18). The precise nature of smaller and weaker immunoreactive bands detected in Fig. 1Go, A and B, is unknown, but it is thought to represent fragments of PGHS proteins.



View larger version (72K):
[in this window]
[in a new window]
 
Fig. 3. Immunoblot analysis of prostaglandin G/H synthase (PGHS)-1 and PGHS-2 isoenzymes in canine prostatic tissues. Solubilized cell extracts were prepared from normal prostates, prostatic adenocarcinomas (case animals 1 and 2; Table 1Go), and canine platelets and were analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, as described in the "Materials and Methods" section. Duplicate blots were probed with antibody 8223 selective for PGHS-1 (A) or antibody MF243 selective for PGHS-2 (B). Markers on the right indicate migration of molecular weight (Mr) standards. Equal amounts of platelets (20 µg), normal prostates (100 µg), and adenocarcinomas (100 µg) were loaded in each panel. [125I]Protein-A was used to visualize immunopositive proteins. Filters A and B were exposed to Kodak X-OMAT AR film at -70 °C for 16 hours.

 

    DISCUSSION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
To our knowledge, this study demonstrates for the first time the induction of PGHS-2 in a high percentage of cancerous prostatic tumors. Whereas no PGHS-2 was detected in normal prostates, 75% of prostatic adenocarcinomas contained epithelial tumor cells expressing PGHS-2. PGHS is the key rate-limiting enzyme in the biosynthetic pathway of prostaglandins from arachidonic acid, and increasing evidence (38-42) supports a role for prostaglandins in prostate carcinogenesis. The administration of indomethacin, a potent PGHS inhibitor, to rats bearing subcutaneous implants of an androgen-insensitive prostate adenocarcinoma was shown to reduce tumor size, metastatic rate, and mortality (38). Inhibitors of eicosanoid biosynthesis have also been reported to inhibit cellular proliferation of some human prostate cancer cell lines (39,40). It has been shown that malignant prostatic tissues have lower concentrations of arachidonic acid and produce more prostaglandin E2 in vitro when compared with benign prostatic tissues (41). Faas et al. (42) showed that phospholipase A2 activity and fatty acyl-CoA lysophosphatidylcholine acyltransferase activity are increased in prostate cancer and proposed that increased prostaglandin synthesis may be important for the growth of malignant prostatic tissues.

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.


    NOTES
 
Supported by grants from the Medical Research Council of Canada (J. Sirois) and the Fonds du Centenaire de l'Université de Montréal (M. Doré and J. Sirois).

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.


    REFERENCES
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 

1 Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics 1999. CA Cancer J Clin 1999;49:8-31.[Abstract/Free Full Text]

2 Maini A, Archer C, Wang CY, Haas GP. Comparative pathology of benign prostatic hyperplasia and prostate cancer. In Vivo 1997;11:293-9.[Medline]

3 Waters DJ, Sakr WA, Hayden DW, Lang CM, McKinney L, Murphy GP, et al. Workgroup 4: spontaneous prostate carcinoma in dogs and nonhuman primates. Prostate 1998;36:64-7.[Medline]

4 Bell FW, Klausner JS, Hayden DW, Feeney DA, Johnston SD. Clinical and pathologic features of prostatic adenocarcinoma in sexually intact and castrated dogs: 31 cases (1970-1987). J Am Vet Med Assoc 1991;199:1623-30.[Medline]

5 Waters DJ, Bostwick DG. Prostatic intraepithelial neoplasia occurs spontaneously in the canine prostate. J Urol 1997;157:713-6.[Medline]

6 Waters DJ, Hayden DW, Bell FW, Klausner JS, Qian J, Bostwick DG. Prostatic intraepithelial neoplasia in dogs with spontaneous prostate cancer. Prostate 1997;30:92-7.[Medline]

7 Davidson D, Bostwick DG, Qian J, Wollan P, Oesterling JE, Rudders RA, et al. Prostatic intraepithelial neoplasia is a risk factor for adenocarcinoma: predictive accuracy in needle biopsies. J Urol 1995;154:1295-9.[Medline]

8 Marnett LJ. Aspirin and the potential role of prostaglandins in colon cancer. Cancer Res 1992;52:5575-89.[Medline]

9 Thun MJ, Namboodiri MM, Heath CW Jr. Aspirin use and reduced risk of fatal colon cancer. N Engl J Med 1991;325:1593-6.[Abstract]

10 Taketo MM. Cyclooxygenase-2 inhibitors in tumorigenesis (part 1). J Natl Cancer Inst 1998;90:1529-36.[Abstract/Free Full Text]

11 DeWitt DL, Meade EA, Smith WL. PGH synthase isoenzyme selectivity: the potential for safer nonsteroidal antiinflammatory drugs. Am J Med 1993;95(2A):40S-44S.[Medline]

12 Funk CD. Molecular biology in the eicosanoids field. Prog Nucleic Acids Res Mol Biol 1993;45:67-98.[Medline]

13 DeWitt DL, Smith WL. Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from complementary DNA sequence [published erratum appears in Proc Natl Acad Sci U S A 1988;85:5056]. Proc Natl Acad Sci U S A 1988;85:1412-6.[Abstract]

14 Xie W, Chipman JG, Robertson DL, Erikson RL, Simmons DL. Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci U S A 1991;88:2692-6.[Abstract]

15 Kujubu DA, Fletcher BS, Varnum BC, Lim RW, Herschman HR. TIS10, a phorbol ester tumor promotor-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem 1991;266:12866-72.[Abstract/Free Full Text]

16 Sirois J, Richards JS. Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles. J Biol Chem 1992;267:6382-8.[Abstract/Free Full Text]

17 Herschman HR. Regulation of prostaglandin synthase-1 and prostaglandin synthase-2. Cancer Metastasis Rev 1994;13:241-56.[Medline]

18 Dubois RN, Abramson SB, Crofford L, Gupta RA, Simon LS, Van De Putte LB, et al. Cyclooxygenase in biology and disease. FASEB J 1998;12:1063-73.[Abstract/Free Full Text]

19 Dinchuk JE, Car BD, Focht RJ, Johnston JJ, Jaffee BD, Covington MB, et al. Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II. Nature 1995;378:406-9.[Medline]

20 Langenbach R, Morham SG, Tiano HF, Loftin CD, Ghanayem BI, Chulada PC, et al. Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration. Cell 1995;83:483-92.[Medline]

21 Morham SG, Langenbach R, Loftin CD, Tiano HF, Vouloumanos N, Jennette JC, et al. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell 1995;83:473-82.[Medline]

22 Kargman SL, O'Neill GP, Vickers PJ, Evans JF, Mancini JA, Jothy S. Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer. Cancer Res 1995;55:2556-9.[Abstract]

23 Sano H, Kawahito Y, Wilder RL, Hashiramoto A, Mukai S, Asai K, et al. Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res 1995;55:3785-9.[Abstract]

24 Hida T, Yatabe Y, Achiwa H, Muramatsu H, Kozaki K, Nakamura S, et al. Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas. Cancer Res 1998;58:3761-4.[Abstract]

25 Wilson KT, Fu S, Ramanujam KS, Meltzer SJ. Increased expression of inducible nitric oxide synthase and cyclooxygenase-2 in Barrett's esophagus and associated adenocarcinomas. Cancer Res 1998;58:2929-34.[Abstract]

26 Ristimaki A, Honkanen N, Jankala H, Sipponen P, Harkonen M. Expression of cyclooxygenase-2 in human gastric carcinoma. Cancer Res 1997;57:1276-80.[Abstract]

27 Oshima M, Dinchuk JE, Kargman SL, Oshima H, Hancock B, Kwong E, et al. Suppression of intestinal polyposis in Apc delta 716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 1996;87:803-9.[Medline]

28 Buckman SY, Gresham A, Hale P, Hruza G, Anast J, Masferrer J, et al. COX-2 expression is induced by UVB exposure in human skin: implications for the development of skin cancer. Carcinogenesis 1998;19:723-9.[Abstract]

29 Tjandrawinata RR, Dahiya R, Hughes-Fulford M. Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells. Br J Cancer 1997;75:1111-8.[Medline]

30 Liu XH, Yao S, Kirschenbaum A, Levine AC. NS398, a selective cyclooxygenase-2 inhibitor, induces apoptosis and down-regulates bcl-2 expression in LNCaP cells. Cancer Res 1998;58:4245-9.[Abstract]

31 Norrish AE, Jackson RT, McRae CU. Non-steroidal anti-inflammatory drugs and prostate cancer progression. Int J Cancer 1998;77:511-5.[Medline]

32 Hall WC, Nielsen SW, McEntee K. Tumours of the prostate and penis. In: Bulletin of The World Health Organization, International Histological Classification of Tumors of Domestic Animals. Vol. 53. Geneva (Switzerland); 1976. p. 247-56.

33 Catafalmo JL, Dodds WJ. Isolation of platelets from laboratory animals. In: Hawiger J, editor. Methods in enzymology. Vol 169. San Diego (CA): Academic Press; 1989. p. 27-34.

34 Sirois J. Induction of prostaglandin endoperoxide synthase-2 by human chorionic gonadotropin in bovine preovulatory follicles in vivo. Endocrinology 1994;135:841-8.[Abstract]

35 Sirois J, Dore M. The late induction of prostaglandin G/H synthase-2 in equine preovulatory follicles supports its role as a determinant of the ovulatory process. Endocrinology 1997;138:4427-34.[Abstract/Free Full Text]

36 Dore M, Hawkins HK, Entman ML, Smith CW. Production of a monoclonal antibody against canine GMP-140 (P-selectin) and studies of its vascular distribution in canine tissues. Vet Pathol 1993;30:213-22.[Abstract]

37 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72: 248-54.[Medline]

38 Drago JR, Al-Mondhiry AB. The effect of prostaglandin modulators on prostate tumor growth and metastasis. Anticancer Res 1984;4:391-4.[Medline]

39 Rose DP, Connolly JM. Effects of fatty acids and eicosanoid synthesis inhibitors on the growth of two human prostate cancer cell lines. Prostate1991 ;18:243-54.[Medline]

40 Viljoen TC, van Aswegen CH, du Plessis DJ. Influence of acetylsalicylic acid and metabolites on DU-145 prostatic cancer cell proliferation. Oncology 1995;52:465-9.[Medline]

41 Chaudry AA, Wahle KW, McClinton S, Moffat LE. Arachidonic acid metabolism in benign and malignant prostatic tissue in vitro: effects of fatty acids and cyclooxygenase inhibitors. Int J Cancer 1994;57:176-80.[Medline]

42 Faas FH, Dang AQ, Pollard M, Hong XM, Fan K, Luckert PH, et al. Increased phospholipid fatty acid remodeling in human and rat prostatic adenocarcinoma tissues. J Urology 1996;156:243-8.[Medline]

43 Hwang D, Scollard D, Byrne J, Levine E. Expression of cyclooxygenase-1 and cyclooxygenase-2 in human breast cancer. J Natl Cancer Inst1998 ;90:455-60.[Abstract/Free Full Text]

44 Giovannucci E, Egan KM, Hunter DJ, Stampfer MJ, Colditz GA, Willett WC, et al. Aspirin and the risk of colorectal cancer in women. N Engl J Med 1995;333:609-14.[Abstract/Free Full Text]

45 Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol 1971;231:232-5.[Medline]

46 Paganini-Hill A, Chao A, Ross RK, Henderson BE. Aspirin use and chronic diseases: a cohort study of the elderly. BMJ 1989;299:1247-50.[Medline]

47 Schreinemachers DM, Everson RB. Aspirin use and lung, colon, and breast cancer incidence in a prospective study. Epidemiology 1994;5:138-46.[Medline]

48 Tsujii M, DuBois RN. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 1995;83:493-501.[Medline]

49 Tsuji M, Kawano S, DuBois RN. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci U S A 1997;94:3336-40.[Abstract/Free Full Text]

50 Tsuji M, Kawano S, Tsuji S, Sawaoka H, Hori M, Dubois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998;93:705-16.[Medline]

51 Young MR. Eicosanoids and the immunology of cancer. Cancer Metastasis Rev 1994;13:337-48.[Medline]

52 Rhim JS, Kung HF. Human prostate carcinogenesis. Crit Rev Oncog 1997;8:305-28.[Medline]

53 Waters DJ, Patronek GJ, Bostwick DG, Glickman LT. Comparing the age at prostate cancer diagnosis in humans and dogs. J Natl Cancer Inst 1996;88:1686-7.

Manuscript received December 28, 1998; revised June 11, 1999; accepted June 22, 1999.


This article has been cited by other articles in HighWire Press-hosted journals:


             
Copyright © 1999 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement