J. Vet. Med.

A 54, 247249 (2007) 2007 The Authors Journal compilation 2007 Blackwell Verlag ISSN 0931184X

Short Communication
Department of Pathology and Animal health, General Pathology and Anatomic Pathology Division, Naples, Italy

Immunohistochemical Expression of Cyclooxygenase-2 in Canine Ovarian Carcinomas

G. Borzacchiello1,3, V. Russo1 and M. Russo2
Addresses of authors: 1Department of Pathology and Animal Health, General Pathology and Anatomic Pathology Division, Faculty of Veterinary Medicine, University of Naples Federico II Via F. Delpino, 1-80137 Naples, Italy; 2Department of Veterinary Clinical Sciences, Obstetrics and Gynaecology Division, Faculty of Veterinary Medicine, University of Naples Federico II Via F. Delpino, 1-80137 Naples, Italy; 3Corresponding author: Tel.: +390 81 2536467; fax: +390 81 2536186; E-mail: borzacch@unina.it With 2 gures and 1 table Received for publication July 26, 2006

Summary
Ovarian tumours among domestic animals are frequently encountered in bitch. Cyclooxygenase-2 (COX-2) expression has been evaluated in dierent kind of canine primary epithelial neoplasms. Eleven canine ovarian carcinomas and two normal samples were evaluated immunohistochemically for COX-2 expression. Nine of 11 carcinoma samples (81%) expressed COX-2 enzyme isoform. The immunoreactivity was intracytoplasmically recorded and the intensity ranged from faint to strong. Our results show that COX-2 is expressed in canine ovarian carcinoma, suggesting a potential role of COX2 in canine ovarian carcinogenesis.

` 2002; Dore et al., 2003; Borzacchiello et al., 2004) as well as in bovine urothelial carcinomas (Borzacchiello et al., 2003). The aim of the present study was to investigate COX-2 expression by immunohistochemical methods in canine ovarian carcinomas.

Materials and Methods

Eleven samples of ovarian carcinomas were evaluated. Histological diagnosis was assessed on 5 lm thickened HaematoxylinEosin-stained sections according to the World Health Organization International Histological Classication of Tumors of the Genital System of Domestic Animals (Kennedy et al., 1998). Two normal ovarian samples were also included in this study. Briey, paran sections were de-waxed, blocked for endogenous peroxidase in 0.3% H2O2 in methanol, then a microwave antigen retrieval and a subsequent streptavidin-biotin-peroxidase (LSAB Kit; Dakocytomation, Glostrup, Denmark) method were performed. The primary antibody was a rabbit polyclonal anti-COX-2 (Cayman Chemical, Co., Ann Arbor, MI, USA), which was applied overnight at room temperature at 1:200 dilution in phosphate-buered saline. Sections were counterstained with Mayers haematoxylin. In the corresponding negative control section, the primary antibody was either omitted or replaced with normal rabbit serum in place of the polyclonal. The immunoreactivity was scored by two observers (GB and VR). The intensity of the immunosignal was scored in each specimen as: 0, absent; +, weak; ++, moderate and +++, strong.

Introduction
Primary neoplasms of the ovary are reported in dierent animal species (Jones et al., 1997). Epithelial ovarian tumours are considered the most common neoplasm in bitch with adenocarcinoma being most common than benign counterpart (Patnaik and Greenlee, 1987). Frequently this kind of neoplasm involve both ovary and the median age of occurrence of this kind of tumour is 10 years (Patnaik and Greenlee, 1987). Non-steroidal anti-inammatory drugs (NSAIDs) are one of the most used therapeutic agents in the treatment of pain, fever and inammation (Kismet et al., 2004). However, NSAIDs are recently considered for cancer prevention and treatment (Subbaramaiah and Dannenberg, 2003). NSAIDs act by reducing prostaglandin synthesis through the inhibition of cyclooxygenase enzyme. This enzyme exists as two isoforms: cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) (Williams and Dubois, 1996). While COX-1 is considered as the constitutive form, COX-2 expression can be induced by inammatory and mitogenic stimuli in neoplastic and inamed tissues upregulating prostaglandins synthesis (Smith et al., 2000; Wadleigh et al., 2000). Other than in several human cancers, COX-2 overexpression has been documented in many dierent canine primary epithelial neoplasms (Tremblay et al., 1999; Khan et al., 2000, 2001; Pestili de Almeida et al., 2001; McEntee et al.,

Results
Cyclooxygenase-2 immunoreactivity was barely detected as faint cytoplasmic staining in three normal ovarian samples (Fig. 1). The staining pattern was superimposable in all the three normal samples. Nine of eleven tumour samples were COX-2 positive. The immunosignal was always recorded intracytoplasmically www.blackwell-synergy.com

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G. Borzacchiello et al. (Fig. 2). The immunoreactivity intensity ranged from faint to strong (Table 1).

Discussion
This is the rst study, to the best of our knowledge, to document the expression of COX-2 in canine ovarian carcinomas. Particularly, we show that 80% of the carcinoma samples were COX-2 immunopositive. Our results are in accordance with similar human studies showing that COX-2 is likely involved in canine ovarian carcinogenesis (Klimp et al., 2001; Matsumoto et al., 2001). Although mounting evidence suggests that COX-2 plays a role in dierent animal and human cancers (Taketo, 1998), the mechanisms by which this enzyme is involved in carcinogenesis are still poorly understood. However, recent studies indicate that COX-2 expression in human ovarian carcinoma is most probably to be signicantly correlated with angiogenesis indicating a possible role for COX-2 in the production of angiogenic factors (Ali-Fehmi et al., 2003; Raspollini et al., 2004). The exact implication of COX-2 in canine ovarian cancer is still to be elucidated. Ovarian tumours are reported to be more common, among animal species, in bitch; epithelial neoplasms are dicult to diagnose as they are asymptomatic until clinical signs referable to a space-occupying mass occur (Herron, 1983). Often medical attention is prompted when the disease is highly advanced and incurable. These facts make ovarian cancer a candidate target for prevention. Surgery remains the mainstay of treatment for ovarian tumours, although, cisplatin treatment can be also considered (Moore et al., 1991). In this light, some studies have demonstrated that COX-2 inhibitory drugs have anti-tumour and chemopreventive eects on some spontaneous canine neoplasms (Knapp et al., 1992) and the association of the COX inhibitor, piroxicam, with cisplatin results in signicant growth decrease of naturally occurring canine transitional cell carcinoma (Mohammed et al., 2003). Moreover, in a recent report by RodriguezBurford et al. (2002) selective COX-2 inhibitors have shown to inhibit the growth of ovarian cancer cell culture, thus suggesting the use of such drugs as chemopreventive for ovarian cancer. Finally, our study report the expression of COX-2 in a subset of canine ovarian carcinoma.

Fig. 1. Canine ovarian normal epithelium. Cyclooxygenase-2 (COX-2) positive normal epithelial cells are seen (arrowheads). Streptavidinperoxidase method. Mayers haematoxylin counterstain (240).

Fig. 2. Canine ovarian papillary carcinoma. Grade II. (Tumour sample no. 6). Cyclooxygenase-2 (COX-2) immunoreactivity is evident as a cytoplasmic immunosignal in almost all neoplastic cells (arrow). Streptavidin-peroxidase method. Mayers haematoxylin counterstain (120).

Table 1. COX-2 expression in canine ovarian tumours Tumour no. 1 2 3 4 5 6 7 8 9 10 11 Age (years) 7 10 6 3 7 6 Unknown 8 9 6 5 Breed Neapolitan masti Yorkshire terrier Mixed Pug Dobermann Poodle Yorkshire terrier Fox terrier Labrador retriever Cocker Spaniel Mixed Histological type of tumour Papillary carcinoma grade I Papillary carcinoma grade II Cystadenocarcinoma grade I Papillary carcinoma grade II Cystadenocarcinoma grade I Papillary carcinoma grade II Papillary carcinoma grade I Cystadenocarcinoma grade III Papillary carcinoma grade I Cystadenocarcinoma grade III Cystadenocarcinoma grade I COX-2 labelling intensity (0, +, ++,+++) ++ + ++ 0 ++ +++ ++ +++ 0 +++ +

Immunohistochemical staining scoring: 0, no staining; +, weak staining; ++, moderate staining; +++, strong staining.

Expression of COX-2 in Canine Ovarian Carcinomas Additional studies are needed to establish whether selective COX-2 inhibitory drugs may be helpful for treatment and prevention of canine epithelial ovarian cancer too.

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Acknowledgements
We thank Dr Marielda Cataldi for her excellent technical assistance.

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