ARTICLE |
Correspondence to: Jean Sirois, Département de Biomédecine Vétérinaire, Université de Montréal, CP 5000, St-Hyacinthe, Québec, Canada J2S 7C6.
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Summary |
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Prostaglandin endoperoxide synthase (PGHS) is a key rate-limiting enzyme in prostaglandin biosynthesis. PGHS has recently been shown to be expressed in human colorectal cancers and in experimental cutaneous papillomas and carcinomas. However, PGHS expression has not been investigated in ovarian cancers. The objectives of this study were to determine whether PGHS isoenzymes are expressed in human ovarian cancer and, if so, to identify which isoform is involved (PGHS-1 and/or PGHS-2) and to characterize its cellular localization. Sixteen human ovarian adenocarcinomas were studied by immunohistochemistry using specific antibodies recognizing PGHS-1 or PGHS-2. Results showed that all adenocarcinomas demonstrated the presence of tumor cells expressing PGHS-1 but not PGHS-2. Patterns of staining of tumor cells varied among different types of adenocarcinomas, with cells presenting either a mostly diffuse cytoplasmic immunoreactivity or, alternatively, a staining mainly concentrated around the nucleus. No correlation between the intensity of the immunostaining and the degree of malignancy of tumors could be established (r 5 20.03; p>0.05). Immunoblot analysis with PGHS-1-selective antibodies of cell extracts from adenocarcinomas revealed the presence of a characteristic 72,000 Mr immunoreactive band. Therefore, these results show for the first time that PGHS-1 is expressed in human ovarian adenocarcinomas. (J Histochem Cytochem 46:77-84, 1998)
Key Words: prostaglandin endoperoxide synthase, cyclo-oxygenase, ovary, adenocarcinomas, prostaglandins
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Introduction |
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Prostaglandins are important derivatives of the metabolism of arachidonic acid involved in a wide spectrum of biological activities, including reproductive, renal, gastrointestinal, and cardiovascular functions, as well as in hemostasis and inflammation (
Prostaglandin endoperoxide synthase (PGHS) is the first rate-limiting enzyme in the biosynthetic pathway of prostaglandins from arachidonic acid (
Recent studies have shown that PGHS-2 is elevated in colon cancers and might be responsible for the elevated levels of prostaglandins in these tumors (
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Materials and Methods |
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Tissue Samples
Sixteen cases of ovarian tumors surgically resected from women were studied. The patients' ages ranged from 22 to 82 years, and all tumors included in the study were diagnosed as either malignant tumors or as tumors of borderline malignancy (carcinomas of low malignant potential) (Table 1) according to the "Histologic Classification of Ovarian Tumors by the World Health Organization" (
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Antibodies
Affinity-purified polyclonal antibodies 9181 and 8223 were raised in rabbits against ovine PGHS-1 (
Immunohistochemistry
Immunohistochemical staining was performed using the Vectastain ABC kit (Vector Laboratories; Burlingame, CA), as previously described (
Solubilized Cell Extracts and Immunoblot Analysis
Tissues from ovarian carcinomas were homogenized on ice in TED buffer [50 mM Tris (pH 8.0), 10 mM EDTA, and 1 mM DEDTC] containing 0.1% Tween-20 and centrifuged at 30,000 x g for 1 hr at 4C. The crude pellets (membranes, nuclei, and mitochondria) were sonicated (8 sec/cycle; three cycles) in TED sonication buffer [20 mM Tris (pH 8.0), 50 mM EDTA, and 0.1 mM DEDTC] containing 1% Tween-20. The sonicates were centrifuged at 16,000 x g for 15 min at 4C. The recovered supernatant (solubilized cell extract) was stored at -70C until electrophoretic analyses were performed. The protein concentration was determined by the method of Bradford (Bio-Rad Protein Assay; Richmond, CA). Proteins (100 µg) were resolved by one-dimensional SDS-PAGE and electrophoretically transferred to nitrocellulose filters, as previously described (
Statistical Analysis
The coefficient of correlation between the intensity of PGHS-1 staining and the degree of malignancy (grade of adenocarcinomas) was determined to measure the strength of the relationship between the two variables. The coefficient was considered significant when the null hypothesis was rejected at p<0.05 (Student's t distribution). In addition, an analysis of variance was used to test the effect of grade of adenocarcinoma on PGHS-1 staining intensity. All values are given as mean ± SEM.
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Results |
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Expression of PGHS Isoforms in Normal Human Ovaries
To determine if PGHS (PGHS-1 and/or PGHS-2) is expressed under physiological conditions, immunohistochemical staining was performed on normal human ovaries (n = 3). Results of the immunostaining showed that PGHS expression was restricted to two cell types in normal human ovaries. The first type was the surface epithelial cells covering the ovary, which displayed cytoplasmic staining (Figure 1A). Epithelial cells lining crypt-like structures also showed strong PGHS expression (Figure 1C). The second type of cells immunoreactive for PGHS were endothelial cells of some blood vessels that showed cytoplasmic staining. The isoform expressed in these two cell types was determined to be PGHS-1. No PGHS-2 expression was detected in normal human ovaries (Figure 1B and Figure 1D).
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Immunohistochemical Expression of PGHS Isoforms in Human Ovarian Carcinomas
Of the 16 cases of malignant epithelial tumors studied, all showed some immunoreactivity for PGHS (Table 1). Different cell types in the tumors expressed PGHS, including epithelial tumor cells, fibroblasts, vascular endothelial cells, and vascular smooth muscle cells. Immunostaining with anti-PGHS-1 and anti-PGHS-2 antibodies showed that PGHS-1 was the only isoform expressed by the epithelial tumor cells (Figure 2A and Figure 2B) as well as by the other cell types. Patterns of staining of tumor cells varied among different types of adenocarcinomas, with cells presenting either a staining mainly concentrated around the nucleus (Figure 2C) or, alternatively, a mostly diffuse cytoplasmic immunoreactivity (Figure 2A and Figure 2D). Cytoplasmic staining was the most common pattern observed in adenocarcinomas of the serous type (Figure 2D), although some tumor cells also exhibited perinuclear immunoreactivity (Figure 2C). Intense staining concentrated around the nuclear membrane was commonly observed in the mucinous type of adenocarcinoma (Figure 2E). Occasionally, some epithelial cells of mucinous adenocarcinomas also presented a coarsly granular staining distributed throughout their cytoplasm (Figure 2A). The intensity of PGHS expression was heterogeneous (Table 1), being very light in some tumors and strong in others. No significant difference in PGHS-1 staining intensity was present (p> 0.05) among the tumors of different grades, with mean PGHS-1 staining of 2.5 ± 1.3 for tumors of borderline malignancy, 1.8 ± 0.3 for tumors of Grades I and II, and 2.3 ± 1.0 for tumors of Grade III. In addition, no correlation between the intensity of the immunostaining and the degree of malignancy (r = -0.030) of tumors could be established. Interestingly, immunostaining of one benign epithelial ovarian tumor (a serous cystadenoma) showed that the tumor epithelial cells strongly expressed PGHS-1 (Figure 2F). Control staining with normal rabbit serum was always negative (data not shown).
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Immunoblotting of PGHS Isoforms in Human Ovarian Carcinomas
Solubilized cell extracts were prepared from a serous adenocarcinoma and a mucinous adenocarcinoma (Patients 8 and 15, Table 1) and proteins analyzed by Western blotting using anti-PGHS antibodies. In both tumors, a 72,000 Mr band was visualized using antibody 9181 (recognizes both PGHS-1 and PGHS-2) and antibodies 8223 and MF241 (selective for PGHS-1) (Figure 3). No immunoreactive PGHS was detected with antibody MF243 (selective for PGHS-2) (Figure 3). The precise nature of the lower band (Mr 50,000) detected in Figure 3C (and to a lesser extent in Figure 3D) is unknown.
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Discussion |
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Our study demonstrates for the first time that PGHS, a key enzyme in the prostaglandin synthesis pathway, is expressed in human ovarian adenocarcinomas. Using antibodies specific to each isoform of PGHS (PGHS-1 or PGHS-2), we showed that PGHS-1 is present in ovarian adenocarcinomas. By immunohistochemistry, we determined that the enzyme was localized to the tumor epithelial cells and, in some tumors, to fibroblasts, vascular endothelial cells, and vascular smooth muscle cells. Interestingly, this study is first to document that the epithelium covering the surface of the ovary (from which ovarian adenocarcinomas are believed to be derived) expresses abundant amounts of PGHS-1. The physiopathological significance of PGHS-1 expression in normal surface epithelial cells and in ovarian adenocarcinomas is presently unknown.
PGHS-1 and PGHS-2 are two distinct but related enzymes possessing similar activities and generating similar eicosanoid products. Their primary structure is approximately 60% identical, as deduced from amino acid sequences of complementary DNAs (cDNAs) (
In rat and bovine ovarian follicles, there is a selective induction of PGHS-2 after the endogenous surge of luteinizing hormone (
Breast cancer as well as head and neck tumors have been shown to produce PGE2 (
In addition to their effects on cellular adhesion and inhibition of apoptosis, prostaglandins have been implicated as possible modulators of the immune defenses against cancer. Suppression of immune functions by tumor-derived PGE2 and PGE2-producing suppressor macrophages may impair the destruction of tumor cells by the host defense ( (TNF-
) and lymphotoxin (TNF-ß) (
Conversely, some prostaglandins are able to inhibit proliferation of tumor cells (
The significance of PGHS-1 expression by ovarian cancer cells can only be speculative at this time. However, the present study clearly establishes that the surface epithelium of the normal ovary and tumor cells in ovarian adenocarcinomas express large amounts of PGHS-1, a key rate-limiting enzyme in the prostaglandin biosynthetic pathway. Future studies will be needed to unravel the potential role(s) of prostaglandin synthesis in ovarian carcinogenesis.
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Acknowledgments |
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Supported by grants from the Medical Research Council of Canada (JS) and the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (FCAR) (JS and MD).
We thank Drs Jilly F. Evans and Stacia Kargman (Merck Frosst Centre for Therapeutic Research; Pointe-Claire-Dorval, Québec) for kindly supplying antibodies MF241 and MF243.
Received for publication March 24, 1997; accepted July 11, 1997.
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