Clinical implications of expression of cyclooxygenase-2 related to angiogenesis in uterine endometrial cancers

H. Toyoki, J. Fujimoto*, E. Sato, H. Sakaguchi and T. Tamaya

Department of Obstetrics and Gynecology, Gifu University School of Medicine, 1-1 Yanagido, Gifu City 501-1194, Japan

* Correspondence to: Dr J. Fujimoto, Department of Obstetrics and Gynecology, Gifu University School of Medicine, 1-1 Yanagido, Gifu City 501-1194, Japan. Tel: +81-58-230-6349; Fax: +81-58-230-6348; Email: jf{at}cc.gifu-u.ac.jp


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background: Angiogenesis is essential for development, growth and advancement of solid tumors. Cyclooxygenase (cox)-2 is recognized as an angiogenic factor in various tumors. This prompted us to study the clinical implications of cox-2 expression and angiogenesis in uterine endometrial cancers.

Patients and methods: Fifty patients underwent curative resection for uterine endometrial cancers. In uterine endometrial cancers, cox-2 levels were determined by enzyme immunoassay, and the localization and counts of microvessels were determined by immunohistochemistry.

Results: There was a significant correlation between microvessel counts and cox-2 levels in uterine endometrial cancers. Cox-2 localized in the cancer cells, but not in the stromal cells of uterine endometrial cancer tissues. Cox-2 levels decreased with the advancement. Furthermore, cox-2 levels significantly correlated with VEGF levels in uterine endometrial cancers.

Conclusions: VEGF associated with cox-2 might work on angiogenesis at an early status in growth. Therefore, long-term administration of cox-2 inhibitors might be effective in the suppression of recurrent initiation of uterine endometrial cancers after curative resection.

Key words: angiogenesis, cox-2, uterine endometrial cancer, VEGF


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Angiogenesis is essential for development, growth and advancement of solid tumors [1Go]. Angiogenic factors from tumors induce and activate matrix metalloproteinase, plasminogen activator, collagenase and other enzymes in endothelial cells. The enzymes dissolve the basement membrane of endothelial cells, after which the endothelial cells proliferate and migrate under the influence of angiogenic factors. Angiogenic factors induce production of integrins in the endothelial cells. The endothelial cells then form immature capillary tubes. Specific angiogenic factors show specific angiogenesis in each tumor. The angiogenic factors vascular endothelial growth factor (VEGF), thymidine phosphorylase (TP) identified with platelet-derived endothelial cell growth factor, basic fibroblast growth factor (bFGF) and interleukin (IL)-8 work on angiogenesis in uterine cancers [2Go–11Go]. The expression of VEGF, particularly its VEGF165 and VEGF121 isomers, decreased with advancement of clinical stage and with dedifferentiation in uterine endometrial adenocarcinomas [3Go]. TP expression was significantly higher in well-differentiated adenocarcinomas (G1) than in moderately differentiated adenocarcinomas (G2) and poorly differentiated adenocarcinomas (G3) [7Go]. Conversely, bFGF expression increased with advancement of clinical stage and with dedifferentiation [9Go]. VEGF expression in a well-differentiated endometrial cancer cell line was sensitively regulated by ovarian steroids [12Go], and TP expression in uterine endometrium was also sensitively regulated by ovarian steroids [13Go]. Integrally, each angiogenic factor works on angiogenesis specific to various states of uterine endometrial cancers.

Recently, it has been reported that cox-2 works on angiogenesis associated with tumor growth and advancement of various tumors as follows: advanced ovarian serous carcinomas [14Go], breast cancers [15Go], gastric cancers [16Go], renal cell carcinomas [17Go], head and neck squamous cell carcinoma [18Go] and colon cancers [19Go]. Colon cancer cell line HCA-7 cells with overexpressed cox-2 co-express VEGF, bFGF, transforming growth factor (TGF)-ß and platelet derived growth factor (PDGF) [19Go]. Metabolites of cox-2, prostaglandin (PG) E1 and PGE2 have weak angiogenic activity [20Go], and PGE1 and PGE2 induce VEGF production in osteoblasts, synovial fibroblasts and macrophages [21Go–23Go]. This prompted us to study the protein expressions of cox-2 and various angiogenic factors in patients with various clinical backgrounds to determine the clinical implications of cox-2 in uterine endometrial cancers.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Prior informed consent for the following studies was obtained from all patients and the Research Committee for Human Subjects, Gifu University School of Medicine. Fifty patients ranging from 35 to 79 years of age with endometrioid adenocarcinomas of the uterine endometrium [stage I, 25 cases; stage II, 15 cases; and stage III, 10 cases; and well-differentiated adenocarcinoma (G1), 28 cases; moderately differentiated adenocarcinoma (G2), 13 cases; and poorly differentiated adenocarcinoma (G3), 9 cases] underwent curative surgery at the Department of Obstetrics and Gynecology, Gifu University School of Medicine, between December 1999 and January 2002. None of the patients had received any therapy before the uterine endometrial cancer tissue was taken. A part of each tissue of uterine endometrial cancers was snap-frozen in liquid nitrogen to determine cox-2, IL-1{alpha}, IL-1ß, TNF-{alpha}, IL-8, bFGF, VEGF and TP levels, and a neighboring part of the tissues was submitted for histopathological study including immunohistochemical staining for cox-2 and factor-VIII related antigen. The clinical stage of uterine endometrial cancers was determined by the International Federation of Obstetrics and Gynecology (FIGO) classification [24Go].

Immunohistochemistry
Four-micrometer sections of formalin-fixed paraffin-embedded tissues of uterine endometrial cancers were cut with a microtome and dried overnight at 37°C on a silanized slide (Dako, Carpinteria, CA, USA). Samples were deparaffinized in xylene at room temperature for 80 min and washed with a graded ethanol/water mixture and then with distilled water. Immunohistochemical staining for factor VIII-related antigen, which is synthesized by vascular endothelial cells, is specific for the endothelial cells of blood vessels [25Go] and is useful for detecting tumor angiogenesis [26Go]. The samples for cox-2 antigen were soaked in a citrate buffer, and then microwaved at 100°C for 10 min, and those for factor VIII-related antigen and CD34 were treated with 0.3 µg/ml trypsin in phosphate buffer at room temperature for 20 min. The protocol for a DAKO LSAB2 Kit, Peroxidase (Dako) was followed for each sample. In the described procedures, rabbit anti-human cox-2 (Cayman Chemical, Ann Arbor, MI, USA), rabbit anti-factor VIII-related antigen (Zymed, San Francisco, CA, USA) and mouse CD34 (Dako) were used at dilutions of 1:25, 1:2 and 1:40, respectively, as the first antibodies. The addition of a pre-immune corresponding animal serum (rabbit serum, Kojin Bio, Sakado, Saitama, Japan; and mouse serum, Chemicon, Temecula, CA, USA) instead of the first antibody, rabbit anti-human cox-2, rabbit anti-factor VIII-related antigen or mouse CD34, was carried out in the protocols for negative controls of cox-2, factor VIII-related antigen or CD34, respectively.

Vessels were counted in the five highest density areas at 200x magnification (using a combination of 20x objective and 10x ocular, 0.785 mm2 per field) by two blinded investigators. Microvessel counts (MVC) were expressed as the mean numbers of vessels in these areas [27Go]. Microvessel density was evaluated by the counting of microvessels.

Enzyme immunoassay for determination of cox-2, IL-{alpha}, IL-ß, TNF-{alpha}, IL-8, bFGF, VEGF and TP antigens
All steps were carried out at 4°C. Tissues of uterine endometrial cancers (wet weight: 10–20 mg) were homogenized in HG buffer (5 mM Tris–HCl, pH 7.4, 5 mM NaCl, 1 mM CaCl2, 2 mM ethyleneglycol-bis-ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mM MgCl2, 2 mM dithiothreitol, 25 µg/ml aprotinin, and 25 µg/ml leupeptin) with a Polytron homogenizer (Kinematics, Lucerne, Switzerland). This suspension was centrifuged in a microfuge at 12 000 r.p.m. (10 000 g) for 3 min to obtain the supernatant. The protein concentration of samples was measured by the method of Bradford [28Go] to standardize cox-2, IL-1{alpha}, IL-1ß, TNF-{alpha}, IL-8, bFGF, VEGF and TP antigen levels.

Cox-2, IL-1{alpha}, IL-1ß, TNF-{alpha}, IL-8, bFGF and VEGF antigen levels in the samples were determined by a sandwich enzyme immunoassay using a Human Cox-2 EIA Kit (Immuno-Biological Laboratories, Gunma, Japan), a Human IL-1{alpha} Quantikine (R & D System, Minneapolis, MN, USA), a Human IL-1ß Quantikine (R & D System), a Human TNF-{alpha} Quantikine (R & D System), a Human IL-8 Quantikine (R & D System), a Human bFGF Quantikine (R & D System) and a Human VEGF Assay Kit-IBL (Immuno-Biological Laboratories), respectively, and TP antigen levels were determined by the method described by Nishida et al. [29Go]. The levels of cox-2, IL-1{alpha}, IL-1ß, TNF-{alpha}, IL-8, bFGF, VEGF and TP were standardized with the corresponding cellular protein concentrations.

Statistics
Cox-2, IL-1{alpha}, IL-1ß, TNF-{alpha}, IL-8, bFGF, VEGF and TP levels were measured from three parts of the same tissue in triplicate. Statistical analysis was carried out with the Student's t-test. Differences were considered significant when the P-value was less than 0.05. Correlation evaluations between microvessel counts and cox-2 levels and between VEGF and cox-2 levels were analyzed by Pearson's product-moment correlation coefficient. Positive correlation was considered significant when P < 0.05.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
There was a significant correlation between microvessel counts by immunohistochemical staining for factor VIII-related antigen (MVC-F8) and by that for CD34 (MVC-CD34) and cox-2 levels (r=0.655, P < 0.01 and r=0.565, P < 0.01, respectively, as shown in Figure 1 and Table 1), and also between MVC-F8/MVC-CD34 and IL-8 (r=0.813, P < 0.01/r=0.591, P < 0.01, respectively), between MVC-F8/MVC-CD34 and bFGF (r=0.553, P < 0.01/r=0.464, P < 0.01, respectively), between MVC-F8/MVC-CD34 and VEGF (r=0.563, P < 0.01/r=0.551, P < 0.01, respectively) and between MVC-F8/MVC-CD-34 and TP levels (r=0.671, P < 0.01/r=0.526, P < 0.01, respectively), but not between MVC and IL-1{alpha}, IL-1ß or TNF-{alpha} levels (data not shown) in uterine endometrial cancers (Table 1). Cox-2 was diffusely located in the cancer cells, but not in the stromal cells of uterine endometrial cancer tissues in all cases given, as shown in Figure 2 for a representative case (54-year-old, stage Ic and well-differentiated endometrioid adenocarcinoma).



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Figure 1. Correlation between microvessel count and cox-2 in uterine endometrial cancers.

 

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Table 1. Correlation between microvessel counts and various angiogenic factors

 


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Figure 2. Immunohistochemical staining for cox-2 in uterine endometrial cancers (original magnification, x100). A representative case of Ic well-differentiated endometrioid adenocarcinoma of the uterine endometrium. Rabbit anti-human cox-2 (Cayman Chemicals, Ann Arbor, MI, USA) was used at a dilution of 1:25 as the first antibody. Dark brown staining represents positive for cox-2 antigen.

 
Cox-2 levels significantly decreased with advancement (between stage I and stage II, P < 0.001, between stage II and stage III, P < 0.05, and between stage I and stage III, P < 0.001) as shown in Figure 3. In differentiation status, cox-2 levels were significantly decreased with dedifferentiation (between stage G1 and stage G2, P < 0.001, between G2 and G3, P < 0.05 and between G1 and G3, P < 0.001) as shown in Figure 4. Furthermore, cox-2 levels significantly (r=0.521, P < 0.001) correlated with VEGF levels in uterine endometrial cancers, as shown in Figure 5, but not between cox-2 levels and IL-8, bFGF, TP, IL-1{alpha}, IL-1ß or TNF-{alpha} levels (data not shown) in uterine endometrial cancers.



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Figure 3. Levels of cox-2 in uterine endometrial cancers classified according to clinical stage. Clinical stages of uterine endometrial cancer were determined according to FIGO guidelines. Each level is the mean of nine determinations. *P < 0.05, **P < 0.001.

 


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Figure 4. Levels of cox-2 in uterine endometrial cancers classified according to differentiated grade. Differentiated grades of uterine endometrial cancer were determined according to FIGO guidelines. Each level is the mean of nine determinations. *P < 0.05, **P < 0.001.

 


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Figure 5. Correlation between cox-2 and VEGF in uterine endometrial cancers.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although it is not very difficult to accomplish curative resection in most cases of uterine endometrial cancers, recurrences and metastases occur at some rate. Therefore, in order to obtain better patient prognosis, a critical strategy for suppression of recurrence is needed. Long-term treatment to suppress initial angiogenesis necessary for initial recurrence might be more effective than suppression of later, aggressive angiogenesis in massive recurrence that is easy to detect clinically.

Based on our previous studies, VEGF and TP are induced at the early status of endometrial cancers. Although there is no significant difference in VEGF levels classified according to grades of myometrial invasion, VEGF is down-regulated during cancer progression with dedifferentiation. Namely, VEGF contributes to the early process of advancement via angiogenic activity regardless of myometrial invasion [3Go]. TP transiently accelerates angiogenic activity in the process of invasion of well-differentiated uterine endometrial cancers [7Go]. Therefore, VEGF might work on angiogenesis before myometrial invasion.

It has been reported that semi-quantitative cox-2 mRNA levels did not correlate with any clinicopathological factor, even with early status [30Go]. However, because protein expression is determined by translation of the mRNA and by the stability of the protein, the mRNA expression does not always reflect the protein expression and function, and the protein expression could be much more available than the mRNA expression to postulate the protein function. Therefore, the down-regulated cox-2 protein levels might reflect more appropriately angiogenic potential with advancement. In this study, we precisely determined cox-2 protein levels using ELISA. Positive correlation of cox-2 with microvessel density indicates that cox-2 might be a candidate for the role of angiogenic mediator in uterine endometrial cancers. Positive correlation of VEGF with cox-2 indicates that angiogenic potential by VEGF might be supported by cox-2 in cancer cells. Cox-2 expressions remained high at the early status, and was down-regulated after myometrial invasion. In addition, there was no significant difference in the expression of alpha-actin-smooth muscle as a stroma marker with advancement of endometrial cancers, although there seems to be an increase in stromal cell population during the late phases of tumor progression (data not shown). Therefore, cox-2 expressed in cancer cells can be available to analyze fairly the clinical implication of cox-2 in cancer tissue. These indicate that VEGF interacting with cox-2 might work on angiogenesis at an early status in the growth of uterine endometrial cancers. Endometrial cancer recurrence occurs without any macroscopic lesion after curative resection of the cancer, and the size of cluster of cancer cells in the recurrent lesion is smaller than 2 mm in diameter. Hence, the initiation manner of angiogenesis in the recurrent lesion might be similar to that in the primary tumor, and the angiogenic status in the initiation of recurrence might mimic the early status in growth. Therefore, inhibition of cox-2 might lead to suppression of VEGF expression in the initial tumor of recurrence. Actually, a cox inhibitor, ibuprofen, inhibited angiogenesis in rat glioma [31Go]. A selective cox-2 inhibitor and aspirin inhibited the production of VEGF, bFGF, TGF-beta and PDGF, and the stimulation of endothelial migration and tube formation in cox-2 overexpressing cells [19Go]. Therefore, long-term administration of cox-2 inhibitors might be effective in suppressing the initiation of recurrence of uterine endometrial cancers after curative resection.


    Acknowledgements
 
We wish to thank John Cole for proofreading the English of this manuscript. This study was supported in part by funds from the following Ministry of Health and Welfare programs of the Japanese Government: Grant for Scientific Research Expenses for Health and Welfare Programs, Foundation for Promotion of Cancer Research, and Grant-in-Aid for the Second Term Comprehensive 10-year Strategy for Cancer Control.

Received for publication February 16, 2004. Revision received July 29, 2004. Accepted for publication September 2, 2004.


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 Discussion
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