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Clinical Cancer Research Vol. 6, 2424-2430, June 2000
© 2000 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Cyclooxygenase-2 Expression Is Up-Regulated in Transitional Cell Carcinoma and Its Preneoplastic Lesions in the Human Urinary Bladder1

Tsutomu Shirahama2

Department of Urology, Faculty of Medicine, Kagoshima University, Kagoshima 890-8520, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenase (COX) is a key enzyme in the synthesis of prostaglandins from arachidonic acid. Much evidence, including that from epidemiological and experimental studies, suggests that the inducible form of COX, COX-2, is increased in colon tumor tissues and is involved in colon cancer tumorigenesis. To determine the significance of COX-2 in tumorigenesis in the urinary bladder, the expression of COX-2 in transitional cell carcinoma and preneoplastic lesions of the bladder was examined. Tumor specificity of COX-2 immunoblotting was 100% in 12 of 35 (34%) tumors, but in 0 of the 10 normal urothelia samples. COX-2 expression was significantly correlated with tumor stage in 9 of 20 (45%) muscle-invasive (pT2–4) tumors and in 3 of 15 (20%) superficially invasive (pT1) tumors (P < 0.05). Immunohistochemical examination revealed that 13 of 14 (93%) samples of carcinoma in situ (CIS), which may be the precursor of muscle-invasive-type tumors, expressed COX-2, whereas 10 of 21 (48%) samples of dysplasia, which may be the precursor of both superficially invasive and muscle-invasive tumors, expressed COX-2. From the expression profile of COX-2 in these various urothelia, it is suggested that COX-2 is involved in the development of transitional cell carcinoma of the urinary bladder, especially that of muscle-invasive tumors via CIS. Furthermore, COX-2 may be a therapeutic target for CIS because of the high expression rate of COX-2 in CIS lesions.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
COX3 catalyzes the conversion of arachidonic acid to prostaglandins by two different COX isoforms, COX-1 and COX-2 (1) . COX-1 is constitutively expressed in most tissues and mediates the synthesis of prostaglandins required for normal physiological functions. COX-2 is not detectable in most normal tissues, but it is induced by cytokines, growth factors, oncogenes, and tumor promoters (2, 3, 4, 5, 6) .

Both epidemiological (7, 8, 9) and animal studies (10 , 11) have suggested that nonsteroidal anti-inflammatory drugs, COX inhibitors, reduce the risk of colorectal cancer. Oshima et al. (12) demonstrated that the inactivation of COX-2 and treatment with a COX-2 inhibitor in APC mutant mice, a model of human familial adenomatous polyposis, significantly reduce the incidence of intestinal polyps. This provided the first direct evidence that COX-2 plays a key role in tumorigenesis. Recently, COX-2 has been found to be overexpressed in tumors in the colon (10 , 11) , stomach (13) , lungs (14) , and pancreas (15) , suggesting an important role for COX-2 in tumorigenesis.

The natural history of the bladder tumor is not well understood, but exposure to carcinogens, including aromatic amines, is considered a major risk factor for the development of the disease (16, 17, 18) . Workers exposed to aromatic amines frequently have a mutation of the p53 gene (19) , a tumor suppressor gene involved in the tumorigenesis of many tumors (20) . COX, which is involved in the activation of carcinogens including aromatic amines (21) , may be responsible for tumorigenesis in the bladder. Previous animal studies supported this idea because experimental tumorigenesis in the bladder was suppressed by both nonselective COX inhibitors (22 , 23) and a selective COX-2 inhibitor (24) . To the best of my knowledge, however, the expression of COX in human bladder tumors has not been investigated. Therefore, the expression status of COX-1 and COX-2 in various urothelial epithelia including transitional cell carcinoma, dysplasia, and CIS lesions was examined (25 , 26) , and I present the first evidence that COX-2 may be involved in the development of muscle-invasive tumors via CIS in the human urinary bladder.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor Samples.
Tumor tissue was obtained from 35 patients with bladder tumors who had undergone radical cystectomy (26 patients) or transurethral resection (9 patients) between January 1995 and May 1998. The tissue was quickly frozen in liquid nitrogen, and the remaining portion of the specimens was fixed in 10% formalin in PBS and embedded in paraffin. Samples included tumors and surrounding nontumorous epithelial tissue with no histological evidence of cancer cells. None of the patients had received prior chemotherapy or irradiation. The median age at surgery was 64.5 years (range, 54–72 years). All experiments were performed after obtaining informed consent according to institutional rules. Histopathological findings were assessed according to the criteria of the Japanese Urological Association (27) . After initial examination of the H&E-stained slides, serial sections from one representative paraffin block were immunostained.

Tissue Samples of Urothelial Dysplasia and CIS.
Dysplasia and CIS specimens were taken transurethrally from patients with or without bladder tumors between January 1990 and May 1998. The specimens included 2 primary and 19 secondary dysplasias and 2 primary and 12 secondary CIS samples. Specimens were fixed in 10% formalin and embedded in paraffin. All specimens fulfilled the diagnostic criteria for urothelial dysplasia proposed by the International Society of Urological Pathology (28) . Dysplasia was not subclassified into mild and moderate categories. Severe dysplasia was regarded as CIS.

Immunoblot Analyses.
Samples were homogenized and lysed in modified radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 1% NP40, 0.1% deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin (29) ]. The lysates were centrifuged at 10,000 x g for 20 min at 4°C, and the supernatants were used for immunoblot analysis. Protein content was determined by the method of Bradford (30) .

Samples containing 50 µg of protein were resolved in 8% SDS-polyacrylamide gels and electrophoretically transferred to a sheet of nitrocellulose. The blots were incubated overnight with goat polyclonal anti-COX-1 antibody (catalogue number sc-1752; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-COX-2 antibody [catalogue number sc-1745; Santa Cruz Biotechnology, Inc. (1:200 dilution)]. The nitrocellulose membrane was incubated with horseradish peroxidase-conjugated antigoat IgG antibody (catalogue number sc-2020; Santa Cruz Biotechnology, Inc.). Localized horseradish peroxidase activities were detected using the enhanced chemiluminescence Western blotting system (Amersham, Bucks, United Kingdom). To confirm antibody activity, anti-COX-1 and COX-2 antibodies were incubated with the respective blocking peptide [10 µg; COX-1 (catalogue number sc-1752p); COX-2 (catalogue number sc-1745p; Santa Cruz Biotechnology Inc.)] before use.

Tissue Staining and Evaluation.
Tissue samples were fixed using 10% formaldehyde in PBS, embedded in paraffin, and cut into three thick sections. The sections were deparaffinized using xylene, dehydrated using 98% ethanol, and microwaved for 10 min at 65°C. Endogenous peroxidase was inactivated by immersing the slides in 0.3% hydrogen peroxide in absolute methanol for 30 min at room temperature. The sections were incubated in 5% skim milk for 30 min at room temperature and then incubated with a goat anti-COX-1 or anti-COX-2 polyclonal antibody diluted 1:200 with 5% skim milk in PBS for 1 h at room temperature. The sections were washed with PBS and incubated for 30 min with biotinylated goat antirabbit IgG at room temperature. After washing, the sections were incubated for 30 min with avidin-biotin-peroxidase complex (Vectastain kit; Vector Laboratories, Inc., Burlingame, CA). Color was developed by 0.005% (v/v) diaminobenzidine (Nakarai Chemicals Ltd., Kyoto, Japan) and 0.008% (v/v) hydrogen peroxide in PBS for 20 min. The sections were counterstained with hematoxylin and mounted on slides. For negative controls, the primary antibody was omitted from samples or preincubated with the blocking peptides as described previously.

Specimens were regarded as COX negative if 5% of the cells were stained and COX positive if 5% of the cells were stained.

Statistical Analysis.
The correlation between COX expression and tumor stage was analyzed statistically using the {chi}2 test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunoblot Analyses of COX in Bladder Tumors and Matched Nontumorous Epithelial Tissues.
Tissue samples from 35 patients who had undergone radical total cystectomy or transurethral resection were analyzed for the expression of COX activity, and the results are summarized in Table 1Citation .


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  		Table 1 Characterization of 35 surgical specimens of transitional cell carcinoma tissue of the bladder and expression of COX as detected by immunoblot and immunohistochemical analysis

 
COX-1 was expressed in both the tumor and the nontumorous epithelial tissue. There was no evidence that the tumor tissue expressed COX-1 more strongly than the corresponding nontumorous epithelial tissue. In contrast, COX-2 was strongly expressed only in tumor tissue. COX-2 expression was also observed in 3 of 15 (20%) pT1 tumors and 9 of 20 (45%) pT2–4 tumors, suggesting a greater expression of COX-2 in advanced-stage tumors (P < 0.05). Results of the immunoblot analyses of COX-1 and COX-2 expression in the representative tumor and matched normal tissue samples are shown in Fig. 1, A and BCitation , respectively. COX-1 and COX-2 antibodies pretreated with the respective blocking peptides did not react with the COX-1 or COX-2 proteins from the tumor samples (Fig. 1C)Citation , demonstrating the specificity of the antibodies used.



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  		Fig. 1. Immunoblot analyses of COX-1 and COX-2 in transitional cell carcinoma tissue in the bladder and matched nontumorous epithelial tissue. A and B, COX-1 (A) and COX-2 (B) expression is shown in three and five representative tumor (T) and normal (N) tissue samples, respectively. Samples 1–5 correspond to patients 1, 16, 25, 8, and 31, respectively, in Table 1Citation . C, preincubation of anti-COX-1 or anti-COX-2 antibody with the respective peptide abolishes reactivity with cell lysates. Lane 1, anti-COX-1 antibody only; Lane 2, anti-COX-1 antibody plus COX-1 peptide; Lane 3, anti-COX-2 antibody only; Lane 4, anti-COX-2 antibody plus COX-2 peptide. Samples 1 and 2 correspond to patient 1 in Table 1Citation , and samples 3 and 4 correspond to patient 31 in Table 1Citation .

 
Immunohistochemical Analyses of COX-1 and COX-2 Expression in Bladder Tumor Tissue.
All tumor tissue and nontumorous epithelial tissue (Table 1)Citation were examined immunohistochemically for COX-1 and COX-2 expression. COX-1 was not localized in either tumor tissue or nontumorous epithelial tissue. COX-1 was strongly expressed in the smooth muscle tissue, and I speculate that cell lysates from both the tumor and nontumorous epithelial tissue contained a substantial amount of COX-1 protein derived from muscle elements (including the muscularis mucosa in the lamina propria and the propria muscle), which could be responsible for the reaction with the anti-COX-1 antibody in the immunoblot analyses. COX-1 was not localized in tumor vessels or neighboring stromal cells. In contrast, COX-2 was expressed exclusively in tumor cells. Thus, immunohistochemical results of COX-2 expression were consistent with those of the immunoblot analysis. The representative COX-2 immunostaining patterns in transitional cell carcinoma tissue are shown in Fig. 2, ACitation (patient 1) and B (patient 31). The pattern of COX-1 expression in transitional cell carcinoma is shown in Fig. 2, CCitation (patient 23) and D (patient 16).



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  		Fig. 2. Immunohistochemical analyses of transitional cell carcinoma tissue in the bladder for COX-1 and COX-2 expression. A, tumor cells (pT1, G2) show no reactivity with anti-COX-2 antibody (patient 1); x200. B, most muscle-invasive tumor cells (pT3, G3) express COX-2 (patient 31); x200. C, muscle tissues (M) are strongly positive for COX-1, whereas both tumor and stromal cells are largely negative for COX-1 (patient 23). Very few stromal cells (arrow) are shown to be positive; x100. D, COX-1 immunoreactivity is limited only in muscle tissues (M; patient 16); x100. E, dysplasia shows positive reactivity for COX-2 (patient 36); x200. F, CIS tissue expresses COX-2 (patient 60); x200.

 
Immunohistochemical Analyses of COX-2 Expression in Dysplasia and CIS.
COX-1 immunostaining was not performed because the tumor cells did not express COX-1. Ten of 21 (48%) dysplasia lesions stained positive for COX-2 expression. The mean percentage of positively stained cells was 45% (range, 10–80%; Table 2Citation ). However, COX-2 was expressed in 13 of 14 (93%) CIS lesions. The mean percentage of positively stained cells was 49% (range, 15–80%; Table 2Citation ). The representative immunostaining patterns in CIS (patient 60) and dysplasia (patient 36) lesions are illustrated in Fig. 2, E and FCitation , respectively. Expression profiles of COX-2 in normal urothelium, transitional cell carcinoma, CIS, and dysplasia lesions are summarized in Table 3Citation .


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  		Table 2 Immunohistochemical detection of COX-2 in dysplasia and CIS lesions in the bladder

 

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  		Table 3 COX-2 expression profiles in normal urothelium, transitional cell carcinoma, CIS, and dysplasia lesions in the urinary bladder

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
I demonstrated that COX-2 was frequently expressed in muscle-invasive transitional cell carcinomas in the urinary bladder and was expressed in the majority of samples of CIS, which is considered to be the precursor for muscle-invasive tumors. This suggests that COX-2 is involved in the development of muscle-invasive bladder tumors via CIS lesions. COX-2 was also frequently expressed in dysplasia lesion as the precursor for superficial and muscle-invasive tumors. However, the relationship between COX-2 expression and dysplasia tumor development remains to be investigated.

Although a specific COX-2 inhibitor suppressed experimental tumorigenesis in the urinary bladder (24) , the involvement of COX-2 in tumorigenesis is poorly understood. The mechanism of elevated COX-2 expression in tumor cells may depend on the activation of oncogenes (31, 32, 33) . Activation of the K-ras oncogene is associated with an elevated expression of COX-2 (31, 32, 33) , and the K-ras oncogene is frequently activated in bladder tumors (34) . This particular mechanism may help explain the level of COX-2 expression found in bladder tumors. COX activates many carcinogens, one of which binds directly to hot spots for mutation in the p53 gene in lung (35) and bladder (19) cancer. Thus, COX may be involved in tumorigenesis by inactivating tumor suppressor genes such as p53.

COX-2-selective inhibitors may provide an alternative approach for the treatment of CIS. COX-2-selective inhibitors suppress colon cancer growth in vitro by inducing apoptosis, dependent (11) and independent (36 , 37) of COX-2 inhibition, and suppress tumorigenesis in experimental models including rat bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (24) . Although the antitumor effects of COX-2-selective inhibitors in bladder cancer cell remain to be determined, the majority of CIS lesions that express COX-2 and the few that do not express COX-2 could both be good targets for the treatment by COX-2-selective inhibitors. It appears worthwhile to investigate whether intravesical instillation therapy using COX-2 inhibitors is safe and effective for the treatment of CIS because intravesical but not oral administration of the agents will allow the use of high concentrations of the agents, which may be sufficient to kill cancer cells.

There is much evidence that the COX-2 gene is involved in features of tumor aggressiveness such as invasiveness and metastasis (29 , 38) . For example, COX-2 increases adhesion to the extracellular matrix and decreases the level of the cell adhesion molecule, E-cadherin, in rat intestinal epithelial cells (29) . Human colon cancer cells transfected with a COX-2 expression vector have increased activity of metalloproteinase-2, which is necessary for the degradation of extracellular matrix, resulting in increased tumor cell migration (38) . However, there is no evidence that COX-2 is associated with invasiveness and metastasis in human tumors. In the present study, COX-2 was expressed in muscle-invasive bladder tumors that are at a more advanced stage than superficial-type tumors. Thus, it would be interesting to determine in additional studies whether COX-2 is a prognostic factor in bladder tumors.

In conclusion, transitional cell carcinoma tissue in the bladder, especially the muscle-invasive type, and dysplasia and CIS lesions frequently express COX-2. Thus, there may be a link between COX-2 expression and the development of transitional cell carcinoma tissue in the bladder. Additional investigations are needed to determine whether COX-2 expression has any prognostic value and whether COX-2 inhibitors are useful for chemoprevention and cancer treatment of bladder tumors.


   ACKNOWLEDGMENTS
 
I thank Y. Yonekura and N. Nishi for laboratory assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by grants-in-aid from the Japanese Ministry of Education, Science and Culture. Back

2 To whom requests for reprints should be addressed, at Department of Urology, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. Phone: 81-99-275-5395; Fax: 81-99-265-9727; E-mail: tsu{at}med5.kufm.kagoshima-u.ac.JP Back

3 The abbreviations used are: COX, cyclooxygenase; CIS, carcinoma in situ. Back

Received 12/23/99; revised 2/28/00; accepted 3/ 2/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Omega-3 Fatty Acids to Augment Cancer Therapy
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 Integr Cancer TherHome page
J. M. Wallace
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S. Zha, W. R. Gage, J. Sauvageot, E. A. Saria, M. J. Putzi, C. M. Ewing, D. A. Faith, W. G. Nelson, A. M. De Marzo, and W. B. Isaacs
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T. Shirahama and C. Sakakura
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A. Ristimaki, O. Nieminen, K. Saukkonen, K. Hotakainen, S. Nordling, and C. Haglund
Expression of Cyclooxygenase-2 in Human Transitional Cell Carcinoma of the Urinary Bladder
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