Increased expression of cyclooxygenase-2 protein in rat urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl) nitrosamine
Wakashi Kitayama,
Ayumi Denda2,
Eijiro Okajima1,
Toshifumi Tsujiuchi and
Yoichi Konishi
Department of Oncological Pathology, Cancer Center and
1 Department of Urology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan
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Abstract
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The anti-inflammatory drugs, aspirin and piroxicam, are known to possess chemopreventive potential against rat superficial urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Recently, we found similar inhibitory effects with a selective cyclooxygenase (COX)-2 inhibitor, nimesulide. In order to clarify the inhibitory mechanisms, we have further studied the expression of COX-2 protein in urinary bladder tumors induced by BBN in Fischer 344 male rats. For comparison, papillomatosis caused by uracil-induced urolithiasis, and normal epithelial cells, were also investigated. Western blot analysis revealed COX-2 protein to be barely expressed in the normal epithelial cells, whereas it was increased 1322-fold in varying sizes of urinary bladder tumors and 7-fold in papillomatosis. Immunohistochemically, COX-2 protein was diffusely expressed in transitional cell carcinomas and nodulo-papillary hyperplasia but weakly expressed only in basal cells in simple hyperplasia and normal-looking surrounding epithelia. In papillomatosis, it was moderately expressed only in endothelial cells in stroma. These results indicate that COX-2 plays important roles in the development of preneoplastic and neoplastic lesions in the rat urinary bladder, and therefore could be a good target for chemoprevention of superficial lesions.
Abbreviations: BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; COX, cyclooxygenase; EGFR, epidermal growth factor receptor; HBSS, Hank's balanced salt solution (Ca2+/Mg2+-free); NIM, nimesulide; NPH, nodulo-papillary hyperplasia; SH, simple hyperplasia; TCC, transitional cell carcinoma.
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Introduction
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Superficial-type human urinary bladder cancers, which are usually low-grade transitional cell carcinomas (TCCs), are endoscopically easily resectable and recur with a high frequency, are found at higher incidences than invasive lesions (1). Such recurrence is occasionally associated with malignant conversion (1). Safer and more effective means than the presently performed post-operative intravesical instillation of chemotherapeutic or immunotherapeutic agents for prophylaxis of recurrence remain to be developed (1). Non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin and piroxicam, which are candidate chemopreventive agents active against development of cancers, particularly in the colon, and possibly also the mammary gland, skin and liver (24), possess chemopreventive potential against rat superficial-type urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) (5,6). The chemopreventive mechanisms of NSAIDs remain to be elucidated in detail, but have been postulated to involve their abilities to inhibit cyclooxygenase (COX) activity, particularly that of the COX-2 isozyme (3). In fact, either double knockout of the COX-2 gene or selective COX-2 inhibitors suppress intestinal polyposis in APC gene knockout mice (7), and up-regulated expression of COX-2 mRNA and protein have been reported in colon, stomach, skin, breast and lung tumor tissues (812). Although both COX-2 and COX-1 are rate-limiting enzymes for producing prostanoids (13), COX-1 is a constitutively expressed house-keeping gene, contributing to normal physiological functions in most tissues. COX-2, in contrast, is an inducible immediateearly gene (13), involved not only in inflammation and cell proliferation (13) but also in differentiation, apoptosis, angiogenesis, metastasis and immunological surveillance (1319), all of which could affect carcinogenesis.
Recently, we found inhibitory effects of a selective COX-2 inhibitor, nimesulide (NIM), on rat urinary bladder carcinogenesis induced by BBN (20). Reportedly, COX-1 rather than COX-2 protein is expressed in normal rat urinary bladders (21). However, it remains unknown whether COX-2 is up-regulated in urinary bladder tumors of either animals or humans. Here, in order to further clarify the inhibitory mechanisms of NIM, we have studied the expression of COX-2 and COX-1 proteins in urinary bladder tumors induced by BBN, using western blot analysis and immunohistochemistry, in comparison with normal epithelia and the proliferative epithelial condition, papillomatosis, caused by uracil-induced urolithiasis (22).
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Materials and methods
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Chemicals
BBN and uracil were obtained from Tokyo Kasei Kogyo (Tokyo, Japan), and NIM from Helsinn Healthcare SA (Pambio Noranco, Switzerland). Mouse monoclonal antibodies against rat COX-2 (C-terminal protein fragment corresponding to amino acids 368604) were obtained from Transduction Laboratories (Lexington, KY) (# C22420), and rabbit polyclonal antibodies against rat COX-1 (C448 synthetic peptide) from IBL (Gunma, Japan) (# 18521). Rabbit and goat polyclonal antibodies against synthetic peptides corresponding to C-terminal sequences of murine and human COX-2 were also obtained from Oxford Biomedical Research (Oxford, MI) (# PG26) and Santa Cruz Biotechnology (Santa Cruz, CA) (# SC-1747), respectively. Hank's balanced salt solution (Ca2+/Mg2+-free; HBSS) was purchased from Cosmo Bio (Tokyo, Japan), trypsin from Difco (Detroit, MI) and collagenase and dispase from Boehringer Mannheim (Tokyo, Japan).
Animals, diet and drinking water
Fisher 344 male rats (Japan SLC, Hamamatsu, Japan), 6 weeks old at the commencement, were used for the administration of BBN and uracil. Normal bladder epithelial cells were prepared from animals at 1520 weeks of age. The rats were fed CE-2 basal diet (Japan Clea, Tokyo, Japan) and given tap water ad libitum. Drinking water containing 0.05% BBN was prepared by dissolving the carcinogen in distilled water with the aid of Tween-80 (300 µl/l). The separate diets containing 400 p.p.m. NIM and 3% uracil were prepared by mixing the chemicals with CE-2 powdered basal diet. Animals were killed under ether anesthesia.
Rat urinary bladder tissue samples
For western blot analysis, normal bladder epithelial cells were obtained according to the method of Saeki et al. (23). Briefly, the urinary bladders were everted, washed with HBSS (pH 7.2) and treated with 5 ml of HBSS containing 0.25% trypsin, 0.2 U collagenase/ml and 1.6 U dispase/ml at 37°C for 90 min with shaking (120 strokes/min) in disposable tubes. After removing partly dissociated epithelial cells by centrifuging the medium at 800 g for 5 min, the bladders were further treated with 5 ml of HBSS containing 2 mM EDTA at 37°C for 10 min. Dissociated epithelial cells were again removed by centrifugation, combined with the previous cells and washed twice in cold HBSS. Proliferative epithelial lesions, papillomatosis, which were induced by feeding of 3% uracil for 10 weeks were exfoliated with the aid of a scalpel. Bladder tumors were induced by the administration of 0.05% BBN for 20 weeks and resected. All the samples were stored at 80°C until use.
Urinary bladder tissues obtained in a previous carcinogenesis experiment (20) were used for the immmunohistochemical analysis. Briefly, animals were given 0.05% BBN for 8 weeks, then received either basal diet or a diet containing 400 p.p.m. NIM, for 12 weeks, and were killed 20 weeks after the commencement. Some papillomatosis tissues that were submitted to western blot analysis, were also used for immunohistochemical analysis. In all cases the urinary bladders were inflated, fixed in 10% phosphate-buffered formalin, and routinely embedded in paraffin.
Western blot analysis
Particulate fractions were obtained from the urinary bladder samples basically according to the method of Liu and Rose (11). Briefly, the frozen tissues were homogenized in ice-cold homogenization buffer [50 mM TrisHCl (pH 8.0), 2 mM octyl glucoside, 10 mM EDTA, 1 mM diethyldithiocarbamic acid, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na vanadate, 60 µg/ml soybean trypsin inhibitor, 2 µg/ml leupeptin and 2 µg/ml pepstatin], all from Sigma (St Louis, MO), and then centrifuged at 100 000 g for 1 h at 4°C using a Beckman TLA-100.2 rotor (Beckman Instruments, Palo Alto, CA). The resultant crude pellets were further homogenized in the same buffer as mentioned above (except 20 mM Tris, 45 mM octyl glucoside, 50 mM EDTA and 0.1 mM diethyldithiocarbamic acid) and sonicated for 20 s several times using a ultrasonic cell disruptor (Heat Systems Ultrasonics, Farmingdale, NY). The sonicates were centrifuged at 13 000 g at 4°C and the resultant supernatants were stored at 80°C until use. Protein concentrations were determined using Coomassie brilliant blue G-250 solution (Nacalai Tesque, Kyoto, Japan).
Supernatant samples containing 100 µg protein, were mixed 1:1 with sample buffer (4% SDS, 20% glycerol, 12% ß-mercaptoethanol, 0.05% bromphenol blue and 100 mM TrisHCl pH 6.8), boiled for 5 min, electrophoresed using a 4.75% stack and a 10% running polyacrylamide gel, and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% non-fat dry milk in 0.05M Tris-buffered saline (pH 7.6) containing 0.1% Tween-20 (TBS-T) and incubated with primary antibodies to COX-2 and COX-1 for 1 h at a dilution of 1:1000 (# C22420 and # PG26), 1:200 (# SC-1747) and 1:50 (# 18521) in TBS-T. Secondary horseradish peroxidase-linked sheep anti-mouse, donkey anti-rabbit (Amersham) and donkey anti-goat (Santa Cruz Biotechnology) IgG antibodies were then employed and the membranes were analyzed by the ECL detection system (Amersham). Mouse macrophage lysate prepared from the RAW 264.7 cell line activated by interferon-
and lipopolysaccharide (Transduction Laboratories), and sheep COX-1 protein (Cayman Chemical Company, Ann Arbor, MI), respectively, were used as COX-2 and COX-1 positive controls.
Immunohistochemical and histological analysis
For the immunohistochemical analyses, paraffin sections cut at 34 µm thickness were deparaffinized, antigens were retrieved by microwaving for 50 min in 0.01 M citrate buffer (pH 6.0) for the COX-1 analysis, and endogenous peroxidase activity was blocked by incubation in 0.3% H2O2 in methanol for 45 min. After blocking non-specific binding with 5% normal horse or goat serum in TBS0.25% Triton for 20 min, the tissues were incubated with a primary antibody to COX-2 (1:100 dilution in TBS0.25% Triton) or COX-1 (1:60 dilution) for 2 h followed by a biotinylated secondary horse anti-mouse or goat anti-rabbit IgG antibody for 30 min at room temperature. Immunoreactivity was detected using a Vectastain Elite ABC kit (PK-6102 or PK-6101; Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine hydrochloride (Sigma) followed by counterstaining with Mayer's hematoxylin. A non-immune serum, mouse IgG1 or rabbit IgG (Dako Japan, Kyoto, Japan) was used as a control for the primary antibody. Urinary bladder lesions were histologically diagnosed in H&E stained sections basically according to the criteria of Oyasu et al., as described previously (20).
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Results
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Western blot analysis for COX-2 and COX-1 protein expression in rat urinary bladder epithelial cells, tumors and papillomatosis
A representative western blot for COX-2 (#C22420 antibody used) and COX-1 protein expression in the normal epithelial cells, tumors induced by BBN and papillomatosis induced by uracil, in rat urinary bladders, is shown in Figure 1A
. The density of each band was quantified using NIH image, and the results are presented in Figure 1B
. Normal urinary bladder epithelial cells to be submitted to western blot analysis, were enzymatically separated since they were unable to be exfoliated by scalpel with clean separation from submucosa and muscle layers. Their appearance with WrightGiemsa staining is illustrated in Figure 2A
. The normal epithelial cells barely expressed COX-2 protein (Figure 1A
). Epithelial papillomatosis lesions induced by uracil, shown in Figure 2B
, could be exfoliated by a scalpel, and exhibited a substantial expression of COX-2 protein, ~7-fold that of normal epithelial cells (Figure 1A and B
). All sizes of resected bladder tumors, ranging from 1 to >10 mm in diameter, exhibited strong expression of COX-2 protein, 1322-fold of the normal epithelial cells, with a slight dependency on the tumor size (Figure 1A and B
). Similar western blot findings were observed when other anti-COX-2 antibodies (#PG26 and sc-1747) were used (data not shown). In contrast, COX-1 protein was highly and almost equally expressed in the normal epithelial cells, varying sizes of tumors, and the papillomatosis (Figure 1A and B
).

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Fig. 2. (A) Enzymatically separated normal bladder epithelial cells, WrightGiemsa staining; x50. (BF and H) Immunohistochemical localization of COX-2 (BF) and COX-1 (H) protein in papillomatosis induced by uracil [(B) x66], and in normal-looking epithelial mucosa [(C and H) x80], NPH [(D) x16], TCCs classified as T1, grade 1 [(E) x13.2] and T1, grade 2 [(F) x50], in rats given 0.05% BBN for 8 weeks followed by a basal diet for 12 weeks. (G) The same NPH as (D) but stained with non-immune mouse IgG1 as the primary antibody; x16.
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Immunohistochemical analysis of COX-2 and COX-1 protein in the urinary bladder lesions induced by BBN and/or NIM
Results for COX-2 immunohistochemical stainability of rat urinary bladder lesions, induced by the administration of 0.05% BBN for 8 weeks followed by either basal diet or a diet containing 400 p.p.m. NIM for 12 weeks, are summarized in Table I
. Details for the incidence and multiplicity of the bladder lesions and their histological findings in the treated animals, with NIM inhibition of development, have already been reported (20). In accordance, numbers of lesions, simple hyperplasia (SH), nodulo-papillary hyperplasia (NPH) and TCC, examined in the BBN + 400 p.p.m. NIM group in the present study, were lower than for the BBN alone group (Table I
). However, NIM did not significantly affect the stainability of the lesions with COX-2 antibody (Table I
). Only basal cells frequently exhibited a slight expression of COX-2 protein in the normal-looking surrounding bladder epithelia (Figure 2C
) and SH (data not shown). In contrast, the majority of NPH and all TCCs examined, highly and diffusely expressed COX-2 protein (Figure 2DF
; Table I
). COX-2 protein was localized in the nuclear membrane and cytoplasm of both normal basal cells and tumor cells. Non-immune serum showed negative staining (Figure 2G
). All of TCCs classified as T1, grade 1, as well as Ta, grades 1 and 2, exhibited a strong (++) stainability with COX-2 antibody, whereas ~30% of TCCs classified as T1, grade 2, showed rather reduced stainability (+), where COX-2 protein was preferentially localized in the nuclear membrane rather than cytoplasm (Figure 2F
). In papillomatosis, COX-2 protein was moderately expressed only in the endothelial cells in stroma (Figure 2B
). Muscle cells and, occasionally endothelial cells, infiltrating macrophage- and fibroblast-like cells, were stained slightly positive for COX-2 antibody. On the other hand, COX-1 protein was diffusely expressed in the normal-looking surrounding bladder epithelia (Figure 2H
), SH, NPH, TCC and papillomatosis (data not shown), and localized in the cytoplasm and frequently in the nuclear membrane. Endothelial cells, infiltrating macrophage-like cells and muscle cells were also stained diffusely positive for COX-1 antibody. Non-immune serum showed negative staining.
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Table I. COX-2 immunohistochemical stainability of urinary bladder lesions in rats given 0.05% BBN for 8 weeks followed by a basal diet or a diet containing 400 p.p.m. NIM for 12 weeks
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Discussion
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In the present study, western blot analysis and immunohistochemistry revealed COX-2 protein to be barely expressed in normal urinary bladder epithelium, only weakly positive in the basal cells, and in very early SH, whereas it was highly and diffusely expressed in the majority of NPH, the preneoplastic lesions and all of TCCs examined. In contrast, in papillomatosis caused by uracil-induced urolithiasis, COX-2 protein was substantially expressed but only in the endothelial cells in stroma. Western blot analysis and immunohistochemistry revealed that COX-1 protein, on the other hand, was clearly and almost equally expressed in normal epithelial cells, tumors and papillomatosis. These results, together with our previous findings of an inhibitory effect of post-initiation treatment of a selective COX-2 inhibitor NIM on the development of TCCs induced by BBN (20), clearly indicate possible important roles for COX-2 in the development of preneoplastic and neoplastic lesions induced by BBN in the rat urinary bladder.
It is worth noting that in contrast to COX-2, ornithine decarboxylase, which is a rate-limiting enzyme of polyamine biosynthesis and postulated to be involved in cell proliferation and tumorigenesis in urinary bladders and skin, is diffusely expressed in uracil-induced papillomatosis (24). This latter is a reversible proliferative lesion, although long term exposure to uracil-induced urolithiasis results in bladder cancers, with secondarily induced gene alterations associated with the increased cell proliferation, plausibly contributing (22). Thus, our present findings, particularly of diffuse COX-2 expression in NPH but not in SH and papillomatosis, suggest that COX-2 might be involved in processes other than merely increased cell proliferation per se. Taking into account that NPH and TCCs, in contrast to the normal bladder epithelia where proliferating cells in the basal layer, differentiate, ascend and undergo apoptosis, exhibit either upward or downward expansive growth, COX-2 might play roles in abnormal differentiation. In fact, overexpression of COX-2 in cultured rat intestinal epithelial cells results in alteration in cellular adhesion and resistance to apoptosis (15).
The present results for COX-2 expression in the preneoplastic NPH contrast with the colon carcinogenesis case, where only interstitial and not epithelial cells of early stage adenomas, are positive (7). COX-2 expressed in interstitial cells is postulated to mediate aberrant reciprocal paracrine cross-talk in epithelialstromal interactions, while its presence in adenocarcinoma cells (8) suggests roles not only in paracrine cross-talk but also autocrine signaling (25). Therefore, in urinary bladder carcinogenesis, COX-2 may act in both autocrine and paracrine fashions even in the early stage. In this context, phenotypic changes observed in cultured rat intestinal cells that overexpress COX-2 are of interest. These include alteration in cellular adhesion, increased resistance to apoptosis due to up-regulation of the bcl-2 gene, increased invasiveness through induction of membrane-type metalloproteinase and activation of metalloproteinase-2, disturbance of immunological surveillance by down-regulation of HLA class II antigen expression, and, particularly, elevated induction of angiogenic factors, which might clearly be involved in the hyper-vascularization associated with superficial bladder tumor development (1519). Similar to the present findings, expression of COX-2 in early preneoplastic lesions, adenomatous hyperplasias, as well as adenomas and adenocarcinomas has recently been reported in human lungs (12).
Normal urine of men and experimental animals contains the growth factor and tumor promotor, epidermal growth factor, probably originating from salivary glands, which is assumed to contribute to recurrence of superficial bladder tumors (26,27). Expression of epidermal growth factor receptor (EGFR) in low- and high-grade TCC cells as well as dysplastic epithelial cells, but only in basal cell layers in normal epithelia, has been reported in human bladders (26), suggesting a close parallel to our present findings for COX-2 expression, although only TCC cells have been reported to express EGFR in the rat bladder (27). Nevertheless, taking into account the evidence that mitogenic signaling through EGFR includes induction of COX-2 and this can be prevented by a selective COX-2 inhibitor (28), the possibility of an involvement of EGFR expression in the present COX-2 expression in bladder lesions warrant further study. Rather than blocking of ligand binding to EGFR, application of selective COX-2 inhibitors might be useful prophylactic approach to prevention of superficial bladder cancer recurrence.
The present findings that 30% of advanced TCCs classified as T1, grade 2, exhibited rather weak stainability with COX-2 antibody, but partly ascribable to its preferential localization in the nuclei rather than cytoplasm, might suggest altered rather than reduced roles in such advanced lesions, since translocation of COX-2 to nuclei has been reported (28,29).
In conclusion, our present and previous findings clearly indicate, for the first time to the authors' knowledge, that COX-2 is highly expressed in preneoplastic lesions NPH as well as TCCs, providing evidence for important roles in superficial-type urinary bladder carcinogenesis induced by BBN in rats.
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Acknowledgments
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This work was supported in part by a Grant-in-Aid 09253104 for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, a Grant-in-Aid 10-36 for Cancer Research from the Ministry of Health and Welfare, Japan, and by the Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation.
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Notes
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2 To whom correspondence should be addressed Email: adenda{at}nmu-gw.cc.naramed-u.ac.jp 
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Received April 28, 1999;
revised September 10, 1999;
accepted September 10, 1999.