Thromboxane synthase (TXAS) is one of the enzymes downstream from cyclooxygenase-2 and catalyzes the synthesis of thromboxane A2 (TXA2). TXAS was among the genes we identified based on its overexpression in invasive bladder tumors. TXAS is overexpressed in common forms of bladder tumors: 69 of 97 (71.1%) transitional cell carcinoma (TCC), 38 of 53 (71.6%) squamous cell carcinoma, and 5 of 11 (45.5%) adenocarcinoma relative to nontumor tissue. Overall, 112 of 161 (69.5%) invasive tumors exhibited elevated expression. Significantly, patients with tumors having >4-fold levels of TXAS expression showed significant statistical evidence of lower overall survival expressed by the estimated hazard ratio of 2.74 with P = 0.009 in Cox's regression analysis. TXAS mRNA expression was found to be an independent prognostic marker for patients with bladder cancer. Treatment of bladder cancer cell lines (T24 and TCC-SUP) with TXAS inhibitors and TXA2 (TP) receptor antagonists reduced cell growth, migration, and invasion, whereas TP agonists stimulated cell migration and invasion. The positive correlation between elevated TXAS expression and shorter patient survival supports a potential role for TXAS-regulated pathways in tumor invasion and metastases and suggests that modulation of the TXAS pathway may offer a novel therapeutic approach. (Cancer Res 2005; 65(24): 11581-7)

In the United States, bladder cancer is the fifth most common cancer, accounting for ∼3% of all cancer-related deaths (1). Approximately 60,000 persons in the United States develop bladder cancer each year, 25% of whom will die of the disease (2). In western countries, transitional cell carcinoma (TCC) comprises 90% of primary malignant tumors of the urinary bladder, whereas in areas where Schistosoma is endemic (Africa, Egypt, and the Middle East) both TCC and squamous cell carcinoma (SCC) are common. The clinical course in urinary bladder cancer has been difficult to predict based on current conventional disease variables. TCC displays a great variability in morphologic and biological behavior (3, 4). Of bladder cancers, 80% to 85% are papillary lesions, which are commonly superficial at initial presentation. Recurrence rates after initial treatment are 50% to 80%, with progression to muscle invasive tumors in 10% to 25% of those patients. The remaining nonpapillary muscle invasive bladder cancers represent a much worse prognosis, with a 50% risk of distant metastases (5). Radical cystectomy is one treatment for patients with muscle invasive bladder cancer. Five-year survival is dependent on the pathologic stage and nodal status (610). Unfortunately, recurrence, invasion, and metastasis, even after a seemingly successful treatment, are characteristic of bladder cancer. Additional studies directed toward elucidation of the factors involved in the progression of bladder cancer will facilitate the design of molecularly based diagnostic and therapeutic approaches.

The proposed molecular mechanisms underlying bladder cancer progression include overexpression of oncogenes, such as ras (11) and myc (12, 13), or loss of tumor suppressor genes, such as p53 (14), Rb (14), p16 (15, 16), and PTEN (17, 18). In general, the frequency of oncogene overexpression or tumor suppressor gene loss is greater in high-grade tumors. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-8, and matrix metalloproteinase-9 expression is increased in muscle invasive compared with superficial papillary tumors (19). More recent analyses of global changes in the transcriptome (20, 21) of bladder cancer cells have identified additional alterations associated with progression. Indeed, recent molecular classification of bladder cancer patients by gene expression profiling may lead to the identification of specific therapeutic targets (21).

Our own genomic and transcriptome studies have identified potential prognostic and therapeutic targets (22). Thromboxane synthase (TXAS) was among the genes identified by its overexpression in bladder cancer. TXAS is downstream of cyclooxygenase (COX) 1 and 2 in the metabolism of arachidonic acid. COX-2 is the inducible rate-limiting enzyme that catalyzes the conversion of arachidonic acid to prostaglandin H2 (PGH2), which is rapidly converted to bioactive eicosanoids, including additional prostaglandins and thromboxanes. TXAS is responsible for the synthesis of thromboxane A2 (TXA2) from PGH2, and TXA2 has been found to have mitogenic potential (23). In this study, we determined that TXAS is overexpressed in most bladder cancers and found a significant correlation between TXAS overexpression and clinical outcome. Furthermore, we show that TXAS inhibitors and TXAS (TP) receptor antagonists and agonists modulate bladder cancer cell growth, migration and invasion in vitro.

Patients and tumor specimens. Samples from 165 tumors were obtained from untreated patients who underwent surgery for bladder cancer at the Urology and Nephrology Center (Mansoura, Egypt) between August 1998 and April 2000. The bladder cancer tissue bank used in this study was established in 1992 and contains clinical data on all patients presenting with bladder cancer. Tumor stage and grade were defined according to International Union Against Cancer and WHO classification as described previously (24). In all cases, tumor and adjacent nonneoplastic bladder tissues were available for the study. Before surgery at the center, all patients provided written informed consent to allow any excess tissue for research studies.

Cell culture and chemicals. Bladder cancer cell lines T24, TSU-PR1, TCC-SUP, UM-UC-3, SW780, HT-1376, 5637, J82, SCaBER, and RT4 were obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 with 10% fetal bovine serum (FBS). The SV-HUC immortalized, nontransformed urothelial cell line was kindly provided Dr. Santhanam Swaminathan (University of Wisconsin, Madison, WI) and cultured in Ham's F-12 (Life Technologies/Invitrogen, Carlsbad, CA) supplemented with 1% FBS. All cell lines were propagated at 37°C in an atmosphere containing 5% CO2. TXAS inhibitors (Furegrelate and Ozagrel), TP receptor antagonists [Pinane TXA2 (PTXA2) and SQ29,548], and TP receptor agonist (U46619) were obtained from Cayman Chemical (Ann Arbor, MI). Indomethacin was obtained from Sigma (St. Louis, MO).

RNA isolation and Northern blot analysis. Tumor and normal bladder materials were snap frozen in liquid nitrogen within 30 minutes after the surgery. Frozen sections were stained with H&E, and samples identified that had at least 70% tumor cells were selected for further studies. RNA was isolated from 250 mg frozen tissue using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Total RNA (20 μg) was fractionated on 1% agarose gels containing 0.66 mol/L formaldehyde. RNA quality was assessed by ethidium bromide visualization of 28S and 18S rRNA. RNA was transferred to nylon membranes (Duralon, Stratagene, La Jolla, CA) in 0.1 mol/L sodium phosphate buffer (pH 6.8), UV cross-linked, and hybridized for 2 hours at 65°C in QuikHyb (Stratagene). TXAS and TP receptor [α-32P]dCTP-labeled probes were prepared by random-primed synthesis using PrimeIt (Stratagene). Washed membranes were exposed to X-ray film for autoradiography. The Kodak Digital Science 1D version 2.0.2 software running on a PowerMac G3 computer was used to calculate the mean intensity of each ethidium bromide–stained rRNA band or the sum intensity of bands on autoradiograms. The relative intensity of RNA was compared after normalization to 28S rRNA levels.

Western blot analysis. Pulverized tissue powders were lysed in radioimmunoprecipitation assay buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.0), 1% Triton X-100, 0.1% SDS, 1% deoxycholate, and protease inhibitor cocktail (Complete protease inhibitors, Roche, Indianapolis, IN)] for 15 minutes on ice. Equal amounts of total protein (20 μg) were resolved by 12% SDS-PAGE and subjected to Western blot analyses using enhanced chemiluminescence system (Amersham-Pharmacia, Piscataway, NJ). Western blots were probed with rabbit polyclonal antibodies against TXAS (Cayman Chemical) and α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA). The TP receptor polyclonal antipeptide antibody (PH4-2) was generated against amino acids 221 to 231 (25).

Radioligand binding and RIA. Equilibrium radioligand-binding assays were done on crude membranes using previously described procedures (26) and [125I]BOP (27) as the radioligand. Data were analyzed via the method of Scatchard using the Ligand computer program. Media were collected for the assay of thromboxane B2 (TXB2) using a RIA procedure described previously (28).

Migration and invasion assays. Cell migration experiments were carried out using 8-μm pore size migration chambers (Falcon/Becton Dickinson, Franklin Lakes, NJ) precoated at 4°C overnight with fibronectin (Becton Dickinson) at a concentration of 5 μg/cm2 in PBS. The following day, the fibronectin solution was aspirated and the migration chambers were rinsed once with water and allowed to air dry before the migration experiment. Cell invasion experiments were carried out using rehydrated 8-μm pore size invasion chambers precoated with Matrigel (Becton Dickinson). Cells were treated for 24 hours with the indicated TXAS inhibitor or TP receptor antagonist (medium was changed twice daily). After treatment, cells were trypsinized, harvested, and counted. For each condition, cells were seeded at 25,000 per well in 500-μL serum-free medium and then added to each migration and invasion chamber. Medium (750 μL) containing 10% serum was used as a chemoattractant in the lower chamber. Both upper and lower chambers contained the indicated compound. Cells were allowed to migrate for 8 hours or invade for 24 hours at 37°C in the presence of 5% CO2. Cells that did not migrate or invade were removed by wiping the top of the membrane with a cotton swab, and the migrating and invading cells were fixed and stained with DiffQuik according to the manufacturer's protocol (Dade Behring, Newark, DE). Migrating and invading cells in 10 high-power fields in each chamber were counted and the mean cell number was calculated.

Cell growth assay. T24 cells were seeded at 5,000 per well in 96-well plates and then treated with either compound or vehicle (ethanol) alone. To measure the number of viable cells at 1, 2, 3, and 5 days, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was done in quadruplicate according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO). Absorbance was measured at a wavelength of 550 nm with reference wavelength of 690 nm.

Statistical analysis. Kaplan-Meier survival curves were constructed for overall survival for two groups with and without overexpressed TXAS mRNA. A Cox's proportional hazards regression model was used to estimate the prognostic values of the TXAS mRNA overexpression and other relevant explanatory variables, such as stage, gender, age, grade, etc. The statistical evidence for significant effects of TXAS mRNA expression and other explanatory variables were evaluated based on the partial likelihood-based analysis (29). A χ2 test was used to compare the distributions of each explanatory categorical variable in two TXAS mRNA expression groups. For the in vitro studies, PROC GENMOD (in SAS system) was used to determine the statistical significance of differences between groups. Analysis was done using the generalized linear model procedure. Statistical analyses for survival were done using PROC PHREG in SAS software and SPSS software (for PC, version 11.5) for construction of survival curves. For all statistical tests, a P < 0.05 was used to reject the null hypothesis.

Characteristics of the patients. The median age of the study population was 56 years (range, 30-47; Table 1). The majority (88%) had stage pT2, pT3, and pT4 bladder cancer. After a median follow-up period of 32 months (range, 4-60), 74 of the 165 (45%) patients died of bladder cancer.

Table 1.

Characteristics of bladder cancer patients and association with TXAS mRNA expression

CharacteristicsPatient no.TXAS overexpression
P
YesNo
Sex     
    Male 116 86 30 0.1 
    Female 45 28 17  
Mean age (y)     
    56     
Histologic type*     
    TCC 97 69 28 0.2 
    SCC 53 38 15  
    Adenocarcinoma 11  
Histologic grade     
    1 20 12 0.025 
    2 72 42 30  
    3 43 36  
Stage     
    pTa 0.001 
    pT1 12  
    pT2 26 17  
    pT3 45 29 16  
    pT4 42 37  
Lymph node     
    Yes 44 40 <0.0001 
    No 91 50 41  
Schistosomiasis     
    Positive 86 24 62 0.42 
    Negative 75 23 52  
CharacteristicsPatient no.TXAS overexpression
P
YesNo
Sex     
    Male 116 86 30 0.1 
    Female 45 28 17  
Mean age (y)     
    56     
Histologic type*     
    TCC 97 69 28 0.2 
    SCC 53 38 15  
    Adenocarcinoma 11  
Histologic grade     
    1 20 12 0.025 
    2 72 42 30  
    3 43 36  
Stage     
    pTa 0.001 
    pT1 12  
    pT2 26 17  
    pT3 45 29 16  
    pT4 42 37  
Lymph node     
    Yes 44 40 <0.0001 
    No 91 50 41  
Schistosomiasis     
    Positive 86 24 62 0.42 
    Negative 75 23 52  

NOTE: Analyses were limited to patients for whom data were available (151 of 165 patients). Clinicopathologic characteristics of the study group and their relation to the TXAS mRNA expression. Among the 165 tumors, 20 were classified histologically as grade 1, 72 as grade 2, and 43 as grade 3. Four tumors were classified as stage Ta (noninvasive papillary tumors), 12 as stage T1, 26 as stage T2, 45 as stage T3, and 42 as stage T4. Forty-four patients were found on pathologic examination to have metastatic disease in the pelvic lymph nodes. No significant relationship was found between TXAS expression sex, histologic type, and schistosomiasis. Tumor grade, stage, and lymph node involvement had a significant association with TXAS overexpression.

*

Although TXAS overexpression is not correlated with specific histologic type (P = 0.2), the percentage of overexpression is higher in both TCC (71.1%) and SCC (71.7%) compared with adenocarcinoma (45.5%).

Thromboxane synthase overexpression is associated with poor outcome for patients with invasive bladder cancer. TXAS (2.2-kb mRNA) expression was generally low in normal bladder tissues and elevated in tumor tissue relative to normal (adjacent nontumor) bladder tissue (Fig. 1A). The level of expression of TXAS in tumor specimens was quantified relative to matched nontumor bladder specimens by densitometric measurement of the mRNA signals on Northern blots. A total 112 of 161 (70%) invasive tumors exhibited elevated expression defined as at least >4-fold higher signal compared the average expression of 10 matched nontumor bladder tissues. This cut point was selected based only on the overall distribution of patient expression (Fig. 2A) and without consideration of statistical analysis of patient outcome. Selection of a more stringent cutoff of 5.0 would not affect patient distribution and the result of the statistical analysis of patient outcome is unaffected (data not shown), suggesting that our conclusions are fairly robust to the chosen cutoff for the overexpression. For the 112 samples showing such overexpression, the median score of expression in the carcinoma was 11.6-fold higher than that of the nontumor tissue (range, 5.0-18.2). The results of the analysis of association between various clinical factors and TXAS mRNA expression are shown in Table 1. There was a significant association between TXAS overexpression and both tumor grade (P = 0.025) and stage (P = 0.001). Furthermore, there is a strong statistically significant association between node status and TXAS overexpression (P <0.001). There was no significant association with histologic type or schistosomiasis. The difference between the distributions of overall survival for two mRNA expression levels was expressed via the Kaplan-Meier survival curves for two groups (Fig. 2B). Cox's partial likelihood-based analysis showed that overexpression of TXAS was a significant predictor of overall survival even in presence of other relevant predictors, such as age, tumor histologic type, grade, stage, and lymph node status (Table 2). This multiple regression analysis indicated that TXAS is an independent prognostic marker for patients with bladder cancer with an estimated increased hazard ratio of 2.74 and a P of 0.009. This implies an estimated 174% increase in hazard function of a patient attributed to having elevated TXAS expression. The indicator of TXAS expression is found to have highly statistically significant effect on survival (P < 0.01). Only two other variables (indicators of stage 4 and grade 3) are found to have highly statistically significant (P < 0.05) effect on survival, and there is moderate statistical evidence of effect of stage 2 on the survival (P between 0.05 and 0.10). The variables lymph node status and TXAS overexpression are statistically significantly associated (P < 0.0001). The Cox's regression analysis includes both these variables within the explanatory/prognostic variables. Even when lymph node status is removed from the model, the estimates of the hazard ratio and the P corresponding to TXAS overexpression do not change significantly. The Martingale residual analysis for each prognostic variable shows that the Cox's regression model is appropriate for analyzing the overall survival behavior in this study.

Figure 1.

Representative TXAS and TP receptor expression in human bladder cancer. A, total RNA and protein was isolated from bladder cancer (T) and matched nontumor bladder (N) tissue. RNA was resolved on a 1.2% agarose gel, transferred to a Nylon membrane, and hybridized using [α-32P]dCTP-labeled TXAS and TP specific probes as indicated. TXAS expression (2.2-kb mRNA) is elevated in tumor tissue relative to normal bladder tissue. TP receptor mRNA was expressed in both normal and tumor bladder tissues. Ethidium bromide staining of 28S and 18S rRNA is provided as loading control. Representative data are provided (5 of 165 patients). B, TXAS and TP receptor protein levels were examined by Western blot. α-Tubulin protein was used as a loading control. Representative data are provided (3 of 20 patients).

Figure 1.

Representative TXAS and TP receptor expression in human bladder cancer. A, total RNA and protein was isolated from bladder cancer (T) and matched nontumor bladder (N) tissue. RNA was resolved on a 1.2% agarose gel, transferred to a Nylon membrane, and hybridized using [α-32P]dCTP-labeled TXAS and TP specific probes as indicated. TXAS expression (2.2-kb mRNA) is elevated in tumor tissue relative to normal bladder tissue. TP receptor mRNA was expressed in both normal and tumor bladder tissues. Ethidium bromide staining of 28S and 18S rRNA is provided as loading control. Representative data are provided (5 of 165 patients). B, TXAS and TP receptor protein levels were examined by Western blot. α-Tubulin protein was used as a loading control. Representative data are provided (3 of 20 patients).

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Figure 2.

Expression of TXAS and cumulative survival in 161 patients with bladder cancer. A, distribution of TXAS overexpression. Expression was quantified by densitometry of Northern blots. The level of TXAS expression was categorized as overexpressed or normal, with overexpression defined as being at least 4-fold greater expression compared with the average expression of 10 matched nontumor adjacent tissues. B, survival of patients whose tumors displayed TXAS overexpression (dashed line) was compared with those with normal expression (solid line). Overexpression of TXAS correlated with lower survival in patients with invasive bladder cancer (P = 0.0005). Kaplan-Meier survival analysis was done using the SPSS statistics software package (SPSS, Inc., Chicago, IL). Ps were determined by the log-rank test. The median follow-up among the surviving patients was 32 months (range, 4-60) with 74 of 165 (44.9%) patients dying of bladder cancer.

Figure 2.

Expression of TXAS and cumulative survival in 161 patients with bladder cancer. A, distribution of TXAS overexpression. Expression was quantified by densitometry of Northern blots. The level of TXAS expression was categorized as overexpressed or normal, with overexpression defined as being at least 4-fold greater expression compared with the average expression of 10 matched nontumor adjacent tissues. B, survival of patients whose tumors displayed TXAS overexpression (dashed line) was compared with those with normal expression (solid line). Overexpression of TXAS correlated with lower survival in patients with invasive bladder cancer (P = 0.0005). Kaplan-Meier survival analysis was done using the SPSS statistics software package (SPSS, Inc., Chicago, IL). Ps were determined by the log-rank test. The median follow-up among the surviving patients was 32 months (range, 4-60) with 74 of 165 (44.9%) patients dying of bladder cancer.

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Table 2.

Analysis of maximum likelihood estimates under Cox's model

Variable estimateSEHazard ratioχ2P
Age −0.014 0.01 0.99 0.89 0.34 
Sex 0.34 0.37 1.41 0.84 0.35 
Stage pT1 Baseline     
Stage pT2 −0.85 0.51 0.43 2.72 0.09 
Stage pT3 −0.35 0.42 0.70 0.70 0.40 
Stage pT4 −0.98 0.48 0.37 4.10 0.04 
Grade 1 Baseline     
Grade 2 0.006 0.46 1.00 0.00 0.98 
Grade 3 1.142 0.54 3.14 4.33 0.03 
Node status 0.048 0.31 1.05 0.02 0.87 
TXAS expression 1.007 0.38 2.74 6.77 0.009 
Variable estimateSEHazard ratioχ2P
Age −0.014 0.01 0.99 0.89 0.34 
Sex 0.34 0.37 1.41 0.84 0.35 
Stage pT1 Baseline     
Stage pT2 −0.85 0.51 0.43 2.72 0.09 
Stage pT3 −0.35 0.42 0.70 0.70 0.40 
Stage pT4 −0.98 0.48 0.37 4.10 0.04 
Grade 1 Baseline     
Grade 2 0.006 0.46 1.00 0.00 0.98 
Grade 3 1.142 0.54 3.14 4.33 0.03 
Node status 0.048 0.31 1.05 0.02 0.87 
TXAS expression 1.007 0.38 2.74 6.77 0.009 

NOTE: Analyses were limited to patients for whom data were available on the prognostic factors (151 of 165 patients). Cox's maximum likelihood-based analysis showed that overexpression of TXAS is a significant predictor of overall survival even in presence of other relevant predictors, such as age, tumor histologic type, grade, stage, and lymph node status. This multiple regression analysis indicated that TXAS is an independent prognostic marker for patients with bladder cancer.

TXAS protein level was determined by Western blot analysis. Consistent with the mRNA data, TXAS protein expression was significantly higher in tumor samples relative to that observed in matched nontumor tissues (Fig. 1B).

The thromboxane A2 receptor is expressed in human bladder tissue. The presence of the TP receptor in bladder tissues was examined by Northern and Western blot analyses. TP receptor mRNA was expressed in both normal and tumor tissue (Fig. 1A). However, whereas the TP receptor mRNA level was expressed at similar levels in tumor and normal tissues, the protein level detected by Western blot was significantly elevated in tumor tissue (Fig. 1B).

Thromboxane synthase inhibitors and TP receptor antagonists reduce the growth, migration, and invasion of bladder cancer cells. TXAS and TP receptor expression was determined in a series of immortalized and transformed bladder cell lines (Fig. 3). TXAS mRNA level was increased in the majority of bladder cancer–derived cell lines compared with that seen in an immortalized nontransformed bladder cell line (SV-HUC) or that observed in normal bladder tissue. All cell lines tested expressed TP receptor mRNA, and all but SCaBER expressed the TP receptor protein.

Figure 3.

TXAS and TP receptor expression in human bladder cancer cell lines. Total RNA and protein was isolated from bladder cancer cell lines. A, Northern blot analysis of TXAS (top) and TP receptor (middle) mRNA expression in cell lines. Ethidium bromide–stained 28S and 18S rRNA as loading control (bottom). B, Western blotting analysis for TXAS (top) and TP receptor (middle) protein levels in bladder cancer cell lines. α-Tubulin was used as loading control.

Figure 3.

TXAS and TP receptor expression in human bladder cancer cell lines. Total RNA and protein was isolated from bladder cancer cell lines. A, Northern blot analysis of TXAS (top) and TP receptor (middle) mRNA expression in cell lines. Ethidium bromide–stained 28S and 18S rRNA as loading control (bottom). B, Western blotting analysis for TXAS (top) and TP receptor (middle) protein levels in bladder cancer cell lines. α-Tubulin was used as loading control.

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Equilibrium radioligand-binding assays were done using the crude membrane preparations from T24 and SCaBER cells. Although no binding could be detected in SCaBER cells, we were able to measure ligand binding in T24 cells (Kd, 5.3 nmol/L; Bmax, 0.36 pmol/mg membrane protein). Furthermore, T24 and TCC-SUP cells each synthesized TXB2, the stable metabolite of TXA2 (946 and 689 pg/mL, respectively). The addition of arachidonic acid increased production to 7,498 and 2,554 pg/mL, respectively. T24 and TCC-SUP human bladder cancer cell lines, which express both TXAS and the TP receptor, were selected as models to examine possible effects of either TXAS inhibitors (Furegrelate and Ozagrel), TP receptor antagonists (PTXA2 and SQ29,548), or TP receptor agonist (U46619). Anchorage-dependent growth was monitored by MTT assay. Cultures treated with either TXAS inhibitors or receptor antagonists showed a reduced time-dependent increase in cell number compared with vehicle-treated cultures with the most significant effects observed after treatment with either Furegrelate or PTXA2 (Fig. 4A and B). The effect of these agents on T24 and TCC-SUP cell migration and invasion in vitro was determined by measuring the ability of treated cells to migrate through filters coated with fibronectin or invade through Matrigel, a reconstituted basement membrane, respectively. Addition of the TXAS inhibitors Furegrelate and Ozagrel to the growth medium resulted in 52% and 56% (P < 0.0001) reduction in T24 cell migration and 49% and 31% (P < 0.0001) reduction in cell invasion, respectively (Fig. 4C and E). Treatment of TCC-SUP with Furegrelate and Ozagrel resulted in 19% and 32% reduction in cell migration and 26% and 28% reduction in cell invasion, respectively (P < 0.0001; Fig. 4D and F). The receptor antagonists SQ29,548 and PTXA2 reduced T24 cell migration by 48% and 60% (P < 0.0001) and T24 cell invasion by 40% and 69% (P < 0.0001) at the indicated concentrations. These antagonists also reduced TCC-SUP migration (63% and 34%; P < 0.0001) and invasion (44% and 32%; P < 0.0001).

Figure 4.

Effects of TXAS inhibitors and TP receptor antagonists and agonists on cell growth, migration, and invasion. A and B, effects of TXAS inhibitors or TP receptor antagonists on the growth of T24 (A) and TCC-SUP (B) human bladder cancer cells. Cells were seeded at 5 × 104 per well in 96-well plates in RPMI 1640 supplemented with 10% FBS and allowed to attach overnight. After overnight incubation, the medium was aspirated and fresh RPMI 1640 without or with either TXAS inhibitors was added. Compounds (2 mmol/L Furegrelate or 1 mmol/L Ozagrel) or TXAS receptor antagonists (1 μmol/L PTXA2 or 1 μmol/L SQ29,548) were added twice daily for a period of 0, 1, 2, 3, and 5 days. Cell growth was monitored by MTT colorimetric assay. C and D, effect of TXAS-specific inhibitors TP receptor antagonists or TP receptor agonists on bladder cancer cell migration. E and F, modulation of bladder cancer cell invasion by TXAS inhibitors, TP receptor antagonists, and agonists. Columns, mean of three independent experiments; bars, SD. P < 0.0001, effects of all compounds compared with vehicle control were statistically significant. Indo, indomethacin.

Figure 4.

Effects of TXAS inhibitors and TP receptor antagonists and agonists on cell growth, migration, and invasion. A and B, effects of TXAS inhibitors or TP receptor antagonists on the growth of T24 (A) and TCC-SUP (B) human bladder cancer cells. Cells were seeded at 5 × 104 per well in 96-well plates in RPMI 1640 supplemented with 10% FBS and allowed to attach overnight. After overnight incubation, the medium was aspirated and fresh RPMI 1640 without or with either TXAS inhibitors was added. Compounds (2 mmol/L Furegrelate or 1 mmol/L Ozagrel) or TXAS receptor antagonists (1 μmol/L PTXA2 or 1 μmol/L SQ29,548) were added twice daily for a period of 0, 1, 2, 3, and 5 days. Cell growth was monitored by MTT colorimetric assay. C and D, effect of TXAS-specific inhibitors TP receptor antagonists or TP receptor agonists on bladder cancer cell migration. E and F, modulation of bladder cancer cell invasion by TXAS inhibitors, TP receptor antagonists, and agonists. Columns, mean of three independent experiments; bars, SD. P < 0.0001, effects of all compounds compared with vehicle control were statistically significant. Indo, indomethacin.

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In contrast, treatment with the receptor agonist U46619 significantly stimulated cell migration and invasion (P < 0.0001). Consistent with the presence of a functional arachidonate pathway in these cells, indomethacin treatment reduced the ability of treated cells to migrate and invade (P < 0.0001). Addition of U46619 enhanced (T24) or restored (TCC-SUP) cell migration and invasion when combined with indomethacin (Fig. 4C-F).

In this report, we show that TXAS and TP receptor are overexpressed in invasive forms of SCC and TCC, which represent the most common forms of bladder cancer. TXAS is similarly elevated in patients with or without schistosomiasis. Significantly, elevated expression was associated with patients with higher grade and stage and associated with poor patient survival. The current data suggest that TXAS mRNA is not overexpressed in noninvasive TCC; however, the limited sample size of pTa cases (four cases) does not allow us to infer anything conclusively about TXAS mRNA expression in noninvasive TCC. In contrast to the correlation between TXAS mRNA and protein expression levels, TP receptor protein elevation without significant mRNA change indicates that post-transcriptional control of TP receptor in cancer cells may be different than that in normal cells. We recognize that the epithelial cell content of nontumor bladder tissue is less than that typically found in cancer samples, as it also includes fibrous connective tissue and smooth muscle. Future immunohistochemical studies will examine this issue in greater detail. We also showed that TXAS-specific inhibitors reduced bladder cancer cell growth, migration, and invasion. Consistent with the presence of a functional TP receptor, we found that treatment with receptor antagonists affected several cellular functions, including growth, migration, and invasion. Additional data show that the TP receptor agonist increased cell migration and invasion. Whereas indomethacin-mediated inhibition of the COX pathway resulted in reduced T24 and TCC-SUP migration and invasion, cotreatment with TP agonist restored or enhanced cell migration and invasion. We also detected formation of TXB2 in conditioned medium from bladder cancer cells in the basal state and this was augmented by the addition of arachidonic acid. Thus, it seems that TXAS and thus TXA2 contribute to the pathogenesis of bladder cancer cells.

COX-1 and COX-2 catalyze the conversion of arachidonic acid to PGH2, which is rapidly converted to bioactive prostanoids, including prostaglandin E2 (PGE2), prostaglandin D2, TXA2, and prostaglandin I2. Many cancers show an up-regulation of COX-2. More than 80% of human colorectal cancers show higher expression of COX-2 relative to normal mucosa and this overexpression is associated with tumorigenesis and angiogenesis of colorectal cancers (30). Such observations provided a rationale to test the ability of COX-2 inhibitors [e.g., nonsteroidal anti-inflammatory drugs (NSAID)] to protect against the development of precancerous colon polyps.

Bladder cancer also shows an up-regulation of COX-2 (31) and increased urinary PGE2 production, and COX-2 protein expression correlates with bladder cancer, urinary tract infections, and inflammatory processes (32). RT4, 5637, and T24 bladder cancer cell lines expressed high levels of COX-2 mRNA, whereas COX-1 mRNA expression was detected only in T24 cells (33). Studies to evaluate whether COX expression is correlated with disease progression have yielded different conclusions. In one study, no correlation was detected between COX-1 or COX-2 expression and tumor differentiation and stage. Although COX-2 is expressed in a high percentage of patients with carcinoma in situ and superficial bladder cancer (stage T1 TCC), expression was not correlated with progression (34). In another study of a pathologically homogeneous group of patients with pT1 and grade 3 TCC of the bladder, the expression of COX-2 was correlated with recurrence and progression. Thus, patients with COX-2-positive superficial bladder cancer may need to be followed up more vigorously (35). Collectively, these results indicate that COX-2 is widely expressed in human bladder carcinomas and COX-2 inhibition may allow chemopreventive intervention. A population-based case-control study found that intake of most nonselective NSAIDs was negatively associated with bladder cancer risk, and regular analgesic users were at a decreased risk of bladder cancer overall (36). The preventive value of NSAIDS has been attributed to inhibition of COX-2 enzymatic activity.

Although previous studies have suggested that COX-2 contributes to tumor associated blood vessel development, subsequent investigations have shown that TXAS also has a role in endothelial cell migration, angiogenesis, and tumor metastasis. TXAS catalyzes the conversion of PGH2 to TXA2. TXA2 is secreted from activated platelets, monocytes, and damaged vessels and induces platelet aggregation and vascular smooth muscle contraction (vasoconstriction) and hypertrophy. TXA2 and its receptors are well characterized as mediators of coronary disease, acute myocardial infarction, reocculsion after coronary thrombolysis, and ischemia of multiple organs. Angiogenic growth factors, bFGF and VEGF, stimulate TXA2 synthesis in endothelial cells. In addition, in this study, another TXA2 mimetic, U46619, stimulated endothelial cell migration. Conversely, inhibition of TXA2 synthesis reduced human umbilical vascular endothelial cell migration. Furthermore, a TXA2 receptor antagonist, SQ29,548, inhibited cell migration. Significantly, TXA2 inhibition reduced bFGF-induced angiogenesis and development of lung metastasis in vivo (37).

Possible roles for TXA2 in epithelial cancer cells have received limited attention, but current data support the model that TXA2 promotes migration and survival of cancer cells. TXAS is expressed in glioma cell lines. Glioma cell lines have a wide range of TXB2 levels and the expression of TXB2 is correlated with migration rate. Consistent with a promigration effect, glioma cell migration was effectively blocked by specific inhibitors of TXAS (38). TXAS was found to be overexpressed in human glioma cells selected for migration on a glioma-derived extracellular matrix (39). In addition, TXAS inhibitors have been shown to induce apoptosis in glioma cell lines, without inducing apoptosis in normal astrocytes or fibroblasts (40, 41). Inhibition of synthase activity and receptor functions has been shown to induce apoptosis in several cancer cells, including prostate (42), astrocytoma (39), glioma (40, 41), and pituitary adenomas and carcinomas (43). A positive association between TXAS expression level and frequency of tumor metastasis has been described (44). Retroviral expression of TXAS in murine colon adenocarcinoma cells that lack endogenous TXAS led to increased tumor growth rate and vessel density in vivo. These effects were attributed to altered endothelial cell migration and angiogenesis. The growth rate of murine colon cells transduced with TXAS was unaffected (45), although possible autocrine effects or presence of the TP receptor were not analyzed.

Significantly, inhibition of COX-2 and TXAS have different effects on the migration and apoptotic response of glioma (40) and endothelial (46) cells, suggesting that TXAS and thus TXA2 may have effects that are independent of prostaglandin synthesis controlled by COX (38). Therefore, the profile of downstream COX metabolites rather than the levels of COX activity may be relevant in the regulation of tumor progression and angiogenesis (46). Collectively, these studies support the model that downstream metabolites of COX may be determinants of tumor progression, affecting cell migration, growth and/or survival, migration and/or invasion, and angiogenesis.

In conclusion, we have shown that expression of TXAS is associated with high grade, advanced stage, lymph node involvement, and poor long-term outcome for patients with bladder cancer. Our in vitro studies indicate that blocking the TXAS pathway has anti-invasion effects. Based on our studies and published literature, we hypothesize that TXAS may function in autocrine as well as paracrine pathways that affect cancer cells and endothelial cells, respectively, and may facilitate cancer progression. Although one might propose the use of selective COX-2 inhibitors for preventing and/or treatment of bladder cancer, the recent removal from the market because of increased risk of cardiovascular events precludes their use (47, 48). Thus, inhibition of TXAS function or TP receptor signaling using TXAS-specific inhibitors or TP receptor antagonists may provide new therapeutic targets for the treatment of bladder cancer.

Note: N.K. Bissada is currently at Department of Urology, University of Arkansas (Little Rock, AR).

Grant support: National Cancer Institute grant R21 CA106570 and Department of Defense grant 42153MK-GC-3532/N66001-03 (D.K. Watson).

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.

We thank Dr. Santhanam Swaminathan for providing the SV-HUC cell line, Linda Walker for performing the radioligand-binding assays, and Sarah Aston for the RIA.

1
Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, 2004.
CA Cancer J Clin
2004
;
54
:
8
–29.
2
Silverman DT, Hartge P, Morrison AS, Devesa SS. Epidemiology of bladder cancer.
Hematol Oncol Clin North Am
1992
;
6
:
1
–30.
3
Stein JP, Grossfeld GD, Ginsberg DA, et al. Prognostic markers in bladder cancer: a contemporary review of the literature.
J Urol
1998
;
160
:
645
–59.
4
Hoque MO, Lee CC, Cairns P, Schoenberg M, Sidransky D. Genome-wide genetic characterization of bladder cancer: a comparison of high-density single-nucleotide polymorphism arrays and PCR-based microsatellite analysis.
Cancer Res
2003
;
63
:
2216
–22.
5
de Braud F, Maffezzini M, Vitale V, et al. Bladder cancer.
Crit Rev Oncol Hematol
2002
;
41
:
89
–106.
6
Bassi P, Ferrante GD, Piazza N, et al. Prognostic factors of outcome after radical cystectomy for bladder cancer: a retrospective study of a homogeneous patient cohort.
J Urol
1999
;
161
:
1494
–7.
7
Sternberg CN, Calabro F. Chemotherapy and management of bladder tumours.
BJU Int
2000
;
85
:
599
–610.
8
Stein JP, Lieskovsky G, Cote R, et al. Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1,054 patients.
J Clin Oncol
2001
;
19
:
666
–75.
9
Madersbacher S, Hochreiter W, Burkhard F, et al. Radical cystectomy for bladder cancer today—a homogeneous series without neoadjuvant therapy.
J Clin Oncol
2003
;
21
:
690
–6.
10
Chahal R, Sundaram SK, Iddenden R, Forman DF, Weston PM, Harrison SC. A study of the morbidity, mortality and long-term survival following radical cystectomy and radical radiotherapy in the treatment of invasive bladder cancer in Yorkshire.
Eur Urol
2003
;
43
:
246
–57.
11
Czerniak B, Cohen GL, Etkind P, et al. Concurrent mutations of coding and regulatory sequences of the Ha-ras gene in urinary bladder carcinomas.
Hum Pathol
1992
;
23
:
1199
–204.
12
Lipponen PK. Expression of c-myc protein is related to cell proliferation and expression of growth factor receptors in transitional cell bladder cancer.
J Pathol
1995
;
175
:
203
–10.
13
Schmitz-Drager BJ, Schulz WA, Jurgens B, et al. c-myc in bladder cancer. Clinical findings and analysis of mechanism.
Urol Res
1997
;
25
:
S45
–9.
14
Cordon-Cardo C, Zhang ZF, Dalbagni G, et al. Cooperative effects of p53 and pRB alterations in primary superficial bladder tumors.
Cancer Res
1997
;
57
:
1217
–21.
15
Williamson MP, Elder PA, Shaw ME, Devlin J, Knowles MA. p16 (CDKN2) is a major deletion target at 9p21 in bladder cancer.
Hum Mol Genet
1995
;
4
:
1569
–77.
16
Balazs M, Carroll P, Kerschmann R, Sauter G, Waldman FM. Frequent homozygous deletion of cyclin-dependent kinase inhibitor 2 (MTS1, p16) in superficial bladder cancer detected by fluorescence in situ hybridization.
Genes Chromosomes Cancer
1997
;
19
:
84
–9.
17
Kagan J, Liu J, Stein JD, et al. Cluster of allele losses within a 2.5 cM region of chromosome 10 in high-grade invasive bladder cancer.
Oncogene
1998
;
16
:
909
–13.
18
Tanaka M, Koul D, Davies MA, Liebert M, Steck PA, Grossman HB. MMAC1/PTEN inhibits cell growth and induces chemosensitivity to doxorubicin in human bladder cancer cells.
Oncogene
2000
;
19
:
5406
–12.
19
Izawa JI, Slaton JW, Kedar D, et al. Differential expression of progression-related genes in the evolution of superficial to invasive transitional cell carcinoma of the bladder.
Oncol Rep
2001
;
8
:
9
–15.
20
Sanchez-Carbayo M, Socci ND, Charytonowicz E, et al. Molecular profiling of bladder cancer using cDNA microarrays: defining histogenesis and biological phenotypes.
Cancer Res
2002
;
62
:
6973
–80.
21
Dyrskjot L, Thykjaer T, Kruhoffer M, et al. Identifying distinct classes of bladder carcinoma using microarrays.
Nat Genet
2003
;
33
:
90
–6.
22
Moussa O, Szalai G, Abol-Enein H, Bissada NK, Ghoneim MA, Watson DK. Detection of chromosomal aberrations in transitional cell carcinoma of the bladder by representational difference analysis.
Cancer Genom Proteom
2004
;
1
:
9
–16.
23
Hanasaki K, Nakano T, Arita H. Receptor-mediated mitogenic effect of thromboxane A2 in vascular smooth muscle cells.
Biochem Pharmacol
1990
;
40
:
2535
–42.
24
Muscheck M, Abol-Enein H, Chew K, et al. Comparison of genetic changes in schistosome-related transitional and squamous bladder cancers using comparative genomic hybridization.
Carcinogenesis
2000
;
21
:
1721
–6.
25
Pawate S, Schey KL, Meier GP, Ullian ME, Mais DE, Halushka PV. Expression, characterization, and purification of C-terminally hexahistidine-tagged thromboxane A2 receptors.
J Biol Chem
1998
;
273
:
22753
–60.
26
Allan CJ, Higashiura K, Martin M, et al. Characterization of the cloned HEL cell thromboxane A2 receptor: evidence that the affinity state can be altered by Gα13 and Gαq.
J Pharmacol Exp Ther
1996
;
277
:
1132
–9.
27
Morinelli TA, Oatis JE, Jr., Okwu AK, et al. Characterization of an 125I-labeled thromboxane A2/prostaglandin H2 receptor agonist.
J Pharmacol Exp Ther
1989
;
251
:
557
–62.
28
Wise WC, Cook JA, Eller T, Halushka PV. Ibuprofen improves survival from endotoxic shock in the rat.
J Pharmacol Exp Ther
1980
;
215
:
160
–4.
29
Cox DR. Regression models and life-tables.
J R Stat Soc
1972
;
34
:
187
–220.
30
Williams CS, Mann M, DuBois RN. The role of cyclooxygenases in inflammation, cancer, and development.
Oncogene
1999
;
18
:
7908
–16.
31
Moyad MA. An introduction to aspirin, NSAIDs, and COX-2 inhibitors for the primary prevention of cardiovascular events and cancer and their potential preventive role in bladder carcinogenesis. Part I.
Semin Urol Oncol
2001
;
19
:
294
–305.
32
Wheeler MA, Hausladen DA, Yoon JH, Weiss RM. Prostaglandin E2 production and cyclooxygenase-2 induction in human urinary tract infections and bladder cancer.
J Urol
2002
;
168
:
1568
–73.
33
Bostrom PJ, Aaltonen V, Soderstrom KO, Uotila P, Laato M. Expression of cyclooxygenase-1 and -2 in urinary bladder carcinomas in vivo and in vitro and prostaglandin E2 synthesis in cultured bladder cancer cells.
Pathology
2001
;
33
:
469
–74.
34
Shariat SF, Matsumoto K, Kim J, et al. Correlation of cyclooxygenase-2 expression with molecular markers, pathological features and clinical outcome of transitional cell carcinoma of the bladder.
J Urol
2003
;
170
:
985
–9.
35
Kim SI, Kwon SM, Kim YS, Hong SJ. Association of cyclooxygenase-2 expression with prognosis of stage T1 grade 3 bladder cancer.
Urology
2002
;
60
:
816
–21.
36
Castelao JE, Yuan JM, Gago-Dominguez M, Yu MC, Ross RK. Non-steroidal anti-inflammatory drugs and bladder cancer prevention.
Br J Cancer
2000
;
82
:
1364
–9.
37
Nie D, Lamberti M, Zacharek A, et al. Thromboxane A(2) regulation of endothelial cell migration, angiogenesis, and tumor metastasis.
Biochem Biophys Res Commun
2000
;
267
:
245
–51.
38
Giese A, Hagel C, Kim EL, et al. Thromboxane synthase regulates the migratory phenotype of human glioma cells.
J Neurooncol
1999
;
1
:
3
–13.
39
McDonough W, Tran N, Giese A, Norman SA, Berens ME. Altered gene expression in human astrocytoma cells selected for migration. I. Thromboxane synthase.
J Neuropathol Exp Neurol
1998
;
57
:
449
–55.
40
Kurzel F, Hagel C, Zapf S, Meissner H, Westphal M, Giese A. Cyclo-oxygenase inhibitors and thromboxane synthase inhibitors differentially regulate migration arrest, growth inhibition and apoptosis in human glioma cells.
Acta Neurochir (Wien)
2002
;
144
:
71
–87.
41
Yoshizato K, Zapf S, Westphal M, Berens ME, Giese A. Thromboxane synthase inhibitors induce apoptosis in migration-arrested glioma cells.
Neurosurgery
2002
;
50
:
343
–54.
42
Nie D, Che M, Zacharek A, et al. Differential expression of thromboxane synthase in prostate carcinoma: role in tumor cell motility.
Am J Pathol
2004
;
164
:
429
–39.
43
Onguru O, Scheithauer BW, Kovacs K, et al. Analysis of Cox-2 and thromboxane synthase expression in pituitary adenomas and carcinomas.
Endocr Pathol
2004
;
15
:
17
–27.
44
Nanji AA. Thromboxane synthase and organ preference for metastases.
N Engl J Med
1993
;
329
:
138
–9.
45
Pradono P, Tazawa R, Maemondo M, et al. Gene transfer of thromboxane A(2) synthase and prostaglandin I(2) synthase antithetically altered tumor angiogenesis and tumor growth.
Cancer Res
2002
;
62
:
63
–6.
46
Jantke J, Ladehoff M, Kurzel F, Zapf S, Kim E, Giese A. Inhibition of the arachidonic acid metabolism blocks endothelial cell migration and induces apoptosis.
Acta Neurochir (Wien)
2004
;
146
:
483
–94.
47
Bresalier RS, Sandler RS, Quan H, et al. Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial.
N Engl J Med
2005
;
352
:
1092
–102.
48
Solomon SD, McMurray JJ, Pfeffer MA, et al. Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention.
N Engl J Med
2005
;
352
:
1071
–80.