Targeting SLC1A5 blocks cell proliferation through inhibition of mTORC1 in arsenite-treated human uroepithelial cells.

Arsenic is an environmental contaminant, which is widely distributed in soil, air, and water. There is sufficient evidence to indicate that arsenic increases the risk of bladder cancer in humans. However, its underlying mechanisms remain elusive. Glutamine (Gln) has multiple functions that promote carcinogenesis. Indeed, Gln transporters on cancer cells surface are often upregulated. Elevated expression levels of Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) have been reported in many types of human tumors. This study characterized the role of SLC1A5 in cell proliferation in arsenite-treated cells. In short-term experiments, SV-40 immortalized human uroepithelial (SV-HUC-1) cells were treated with Sodium arsenite (NaAsO2) (0, 0.5, 1, 2, 4, 8 µM) for 24 h. In long-term experiments, SV-HUC-1 cells were exposed to 0.5 µM NaAsO2 for 40 weeks. In both short-term and long-term experiments, arsenite increased expression of SLC1A5 by 1.89-fold and 2.25-fold, respectively. Arsenite increased Gln consumption of SV-HUC-1 cells, and Gln starvation inhibited cell proliferation in long-term arsenite-treated cells. Importantly, inhibiting SLC1A5 blocked cell proliferation by downregulating mTORC1 in long-term arsenite-treated cells. Moreover, SLC1A5 regulated mTORC1 in an αKG-dependent manner. Our results suggest that SLC1A5 plays an important role in cell proliferation of arsenite-treated SV-HUC-1 cells.

CDK7 inhibition by THZ1 suppresses cancer stemness in both chemonaïve and chemoresistant urothelial carcinoma via the hedgehog signaling pathway.

Urothelial carcinoma (UC) is the most common type of bladder cancer, with a 5-year survival rate of only 4.6% in metastatic UC. Despite the advances related to immune-checkpoint inhibitor therapy, chemotherapy remains the standard of care for metastatic diseases, with a 50% response rate. The covalent cyclin-dependent kinase 7 (CDK7) inhibitor THZ1 interferes with transcription machinery and is reported to be effective in cancers without targetable mutations. Therefore, we investigated the therapeutic effect of THZ1 on UC and examined possible mechanisms underlying its effects in both chemonaïve and chemosensitive cancers. CDK7 expression is increased in bladder cancer tissues, especially in patients with chemoresistance. THZ1 induced apoptosis and decreased viability in RT4, BFTC905, HT1376, T24, and T24/R UC cell lines. RNA-sequencing, immunoblotting, and sphere-formation assays confirmed that THZ1 suppressed cancer stemness. In the mouse xenograft model, THZ1 suppressed both chemonaïve and chemoresistant tumors. These results indicate that CDK7 inhibition-related cancer stemness suppression is a potential therapeutic strategy for both chemonaïve and chemoresistant UC.

Cell proliferation of rat bladder urothelium induced by nicotine is suppressed by the NADPH oxidase inhibitor, apocynin.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. In a previous study, nicotine enhanced rat urinary bladder carcinogenesis in a two-stage carcinogenesis model. Nicotine also induced cytotoxicity in the bladder urothelium in a short-term study. In the present study, male rats were treated with nicotine (40 ppm) in drinking water co-administered with the NADPH oxidase inhibitor, apocynin (0, 250 or 750 mg/kg) in diet for 4 weeks. The apocynin treatment induced no clinical toxic effects. Reduction of reactive oxygen species (ROS) by apocynin was confirmed by immunohistochemistry of 8-OHdG in the bladder urothelium. Incidences of simple hyperplasia, cell proliferation and apoptosis were reduced by apocynin treatment in the bladder urothelium. However, despite reduction of cell proliferation (labeling index), apocynin did not affect the incidence of simple hyperplasia, apoptosis, or ROS generation in the kidney pelvis urothelium, in addition to 8-OHdG positivity induced by nicotine being lower. In vitro, apocynin (500 µM) reduced ROS generation, but induced cell proliferation in bladder cancer cell lines (T24 and UMUC3 cells). These data suggest that oxidative stress may play a role in the cell proliferation of the bladder urothelium induced by nicotine.

Expression and prognostic significance of pyruvate dehydrogenase kinase 1 in bladder urothelial carcinoma.

Muscular infiltrating bladder urothelial carcinoma (MIBC) is a highly malignant disease with a poor prognosis. Radical cystectomy is the standard treatment. However, due to surgery and postoperative complications, the quality of life of patients is seriously affected. Therefore, it is increasingly important to find prognostic markers and new therapeutic targets for MIBC. Here, we investigated the expression of PDK1, a key regulator of glucose metabolism, in bladder urothelial carcinoma (BLCA) and its effect on prognosis. The expression pattern of PDK1 was examined by bioinformatics analysis and immunohistochemistry. A total of 101 cases of BLCA were selected for tissue microarrays (TMAs) that contained both tumour and paired normal tissues. We demonstrated that PDK1 expression was correlated with tumour grade and Ki67expression in our TMA cohort (all p values < 0.05). Kaplan-Meier survival analysis showed that patients with MIBC with high PDK1 expression had a worse prognosis than patients with low PDK1 expression (p = 0.016). Multifactor risk analysis showed that increased PDK1 expression was an independent prognostic factor affecting the overall survival of MIBC patients. GSEA showed that the mTOR pathway, HIF pathway, glycolysis, PI3K/AKT/mTOR signalling, etc. were differentially enriched in the PDK1 high expression phenotype. Hence, PDK1 may be a prognostic and therapeutic target for MIBC.

Lipoxygenase-5 Expression in Canine Urinary Bladder: Normal Urothelium, Cystitis and Transitional Cell Carcinoma.

Transitional cell carcinoma (TCC) is the most common canine urinary tract tumour and mimics human invasive TCC. Human TCCs overexpress lipoxygenase (LOX)-5 and the use of target inhibitors has proven effective in inhibiting neoplastic growth. In this study, we investigated the immunohistochemical expression of LOX-5 in normal canine urinary bladder, cystitis and TCC. The comparative expression of LOX-5, cyclo-oxygenase (COX)-1 and COX-2 among the three tissue groups was also examined. Biopsy samples from cases of cystitis and TCC were reviewed from 2012 to 2016; samples of histologically normal bladder were used as controls. Dogs were excluded if they had received glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs) and/or chemotherapy prior to tissue collection. LOX-5 was expressed in 95% of TCCs, 23% of cases of cystitis and 10% of controls. LOX-5 and COX-2 immunohistochemistry scores were significantly (P <0.01) higher in TCCs versus cystitis and normal bladders. Results of this study support the rationale for further investigation of the use of NSAIDs with dual anti COX-2 and LOX-5 effect for the treatment of canine TCC.

Increased COX-2 Immunostaining in Urothelial Carcinoma of the Urinary Bladder Is Associated with Invasiveness and Poor Prognosis.

BACKGROUND: Urothelial carcinoma of the urinary bladder (UCB) is the commonest bladder tumor. Cyclooxygenase-2 (COX-2) mediates angiogenesis, cell survival/proliferation, and apoptosis. This study investigates the relation of COX-2 immunostaining in UCB to clinicopathological parameters in Saudi Arabia. METHODS: The study population includes 123 UCB and 25 urothelial mucosae adjacent to UCB. UCB samples were collected before any local or systemic therapy. Tissue microarrays were designed and constructed, and TMA blocks were sliced for further immunohistochemical staining. Immunohistochemical staining was done using a mouse anti-human COX-2 monoclonal antibody. A cutoff point of 10% was chosen as the threshold to determine low and high COX-2 immunostaining. RESULTS: COX-2 immunostaining is higher in UCB than in the adjacent urothelium (p = 0.033). High COX-2 immunostaining is associated with high-grade UCB (p = 0.013), distant metastasis (p = 0.031), lymphovascular invasion (p = 0.008), positive muscle invasion (p = 0.017), pT2 and above (p = 0.003), and high anatomical stages (stage II and above). High COX-2 immunostaining is an independent predictor of higher tumor grade (p < 0.001), muscle invasion (p = 0.015), advanced pathological T (p = 0.014), lymphovascular invasion (p = 0.011), and distant metastasis (p = 0.039). High COX-2 immunostaining is associated with lower overall survival rate (p = 0.019). CONCLUSION: COX-2 immunostaining is associated with the invasiveness of UCB which may be used as an independent prognostic marker. COX-2 may be a significant molecule in the initiation and progression of UCB. Molecular and clinical investigations are required to explore the molecular downstream of COX-2 in UCB and effectiveness of COX-2 inhibitors as adjuvant therapy along with traditional chemotherapy.

Evaluation of Sienna Cancer Diagnostics hTERT Antibody on 500 Consecutive Urinary Tract Specimens.

OBJECTIVES: Telomerase activity can be detected in up to 90% of urothelial carcinomas (UC). Telomerase activity can also be detected in urinary tract cytology (UTC) specimens and indicate an increased risk of UC. We evaluated the performance of a commercially available antibody that putatively binds the telomerase reverse transcriptase (hTERT) subunit on 500 UTC specimens. STUDY DESIGN: Unstained CytospinTM preparations were created from residual urine specimens and were stained using the anti-hTERT antibody (SCD-A7). Two algorithms were developed for concatenating the hTERT result and cytologic diagnosis: a "no indeterminates algorithm," in which a negative cytology and positive hTERT result are considered positive, and a "high-specificity algorithm," in which a negative cytology and positive hTERT result are considered indeterminate (and thus negative for comparison to the gold standard). RESULTS: The "no indeterminates algorithm" and "high-specificity algorithm" yielded a sensitivity of 60.6 and 52.1%, a specificity of 70.4 and 90.7%, a positive predictive value of 39.1 and 63.8%, and a negative predictive value of 85.0 and 85.8%, respectively. CONCLUSIONS: A positive hTERT result may identify a subset of patients with an increased risk of high-grade UC (HGUC) who may otherwise not be closely followed, while a negative hTERT immunocytochemistry result is associated with a reduction in risk for HGUC.

Comparison of arsenic methylation capacity and polymorphisms of arsenic methylation genes between bladder cancer and upper tract urothelial carcinoma.

Arsenic exposure is an environmental risk factor for urothelial carcinoma (UC). The natural history of upper tract urothelial carcinoma (UTUC) differs from that of bladder cancer (BC). However, the risk factors of BC and UTUC are not exactly the same and should be discussed separately. The aims of this study were to evaluate 1) the association between arsenic methylation capacity and UTUC and/or BC, separately, and 2) the association between polymorphisms of the arsenic metabolism-related genes AS3MT, GSTOs, and PNP against BC and/or UTUC, separately. We conducted a hospital-based study and collected 216 BC and 212 UTUC cases, and 813 healthy controls, from September 2007 to October 2011. Urinary arsenic profiles were measured using high-performance liquid chromatography-hydride generator-atomic absorption spectrometry. The polymorphisms of AS3MT, GSTO, and PNP were identified using the Sequenom MassARRAY platform with iPLEX Gold chemistry. We found that inefficient arsenic methylation capacity was associated with BC in a significant dose-response relationship, but only found that high urinary total arsenic concentration was related to the risk of UTUC, also in a significant dose-response manner. Those with a total urinary arsenic level of > 30.28 µg/L compared to ≤ 9.78 µg/L, had a odds ratio (OR), and 95% confidence interval (CI) of UTUC, of 4.80 (2.22-10.39). The polymorphisms of AS3MT rs11191438, AS3MT rs10748835, and AS3MT rs1046778 were related to the risk of BC and UTUC, while the polymorphisms of AS3MT rs3740393, AS3MT rs11191453, and AS3MT rs11191454 were associated with arsenic methylation capacity. The AS3MT gene polymorphisms and arsenic methylation capacity appear to independently affect the risk of BC and UTUC.

Implications of TERT promoter mutations and telomerase activity in urothelial carcinogenesis.

Telomerase activity imparts eukaryotic cells with unlimited proliferation capacity, one of the cancer hallmarks. Over 90% of human urothelial carcinoma of the bladder (UCB) tumours are positive for telomerase activity. Telomerase activation can occur through several mechanisms. Mutations in the core promoter region of the human telomerase reverse transcriptase gene (TERT) cause telomerase reactivation in 60-80% of UCBs, whereas the prevalence of these mutations is lower in urothelial cancers of other origins. TERT promoter mutations are the most frequent genetic alteration across all stages of UCB, indicating a strong selection pressure during neoplastic transformation. TERT promoter mutations could arise during regeneration of normal urothelium and, owing to consequential telomerase reactivation, might be the basis of UCB initiation, which represents a new model of urothelial cancer origination. In the future, TERT promoter mutations and telomerase activity might have diagnostic and therapeutic applications in UCB.

The extracellular matrix-degrading protein ADAMTS5 is expressed in the nuclei of urothelial cells in healthy rats.

OBJECTIVE: The aim of this study was to investigate whether protein expression of the extracellular matrix-degrading protease ADAMTS5 can be demonstrated in the urinary bladder of healthy rats, and, if so, to determine the localization of this enzyme. MATERIALS AND METHODS: The experiments were conducted with eight inbred male Sprague-Dawley rats. Immunohistochemistry was used to investigate the expression of ADAMTS5 in the urinary bladder. Negative controls were established by either excluding the primary antibody or applying the antibody after it had been preabsorbed with its immunogenic peptide. Confocal microscopy was used to visualize the distribution of ADAMTS5 in the urinary bladder tissue. RESULTS: Immunoreactivity for ADAMTS5 was demonstrated in the urothelium and in the detrusor. This expression was localized not only in the cytoplasm, but also in the nuclei. Confocal microscopy corroborated these findings. CONCLUSION: Expression of ADAMTS5 was demonstrated in the cytoplasm as well as in the nuclei of the urothelium and detrusor cells, suggesting that it may play a role at the transcriptional level.

RAB27B requirement for stretch-induced exocytosis in bladder umbrella cells.

Umbrella cells, which must maintain a tight barrier, modulate their apical surface area during bladder filling by exocytosis of an abundant, subapical pool of discoidal- and/or fusiform-shaped vesicles (DFVs). Despite the importance of this trafficking event for bladder function, the pathways that promote DFV exocytosis remain to be identified. We previously showed that DFV exocytosis depends in part on a RAB11A-RAB8A-MYO5B network, but RAB27B is also reported to be associated with DFVs, and knockout mice lacking RAB27B have fewer DFVs. However, the RAB27B requirements for DFV exocytosis and the relationship between RAB27B and the other umbrella cell-expressed RABs remains unclear. Using a whole bladder preparation, we observed that filling-induced exocytosis of human growth hormone-loaded DFVs was significantly inhibited when RAB27B expression was downregulated using shRNA. RAB27A was also expressed in rat urothelium; however, RAB27A-specific shRNAs did not inhibit exocytosis, and the combination of RAB27A and RAB27B shRNAs did not significantly affect DFV exocytosis more than treatment with RAB27B shRNA alone. RAB27B and RAB11A showed a small degree of overlap when quantified using Squassh segmentation software, and expression of dominant-active or dominant-negative mutants of RAB11A or RAB8A, or expression of a RAB11A-specific shRNA, had no significant effect on the size, number, or intensity of RAB27B-positive DFVs. Likewise, treatment with RAB27B-specific shRNA had no effect on RAB11A-positive DFV parameters. We conclude that RAB27B, but not RAB27A, regulates DFV exocytosis in bladder umbrella cells in a manner that may be parallel to the previously described RAB11A-RAB8A-MYO5B pathway.

Expression of Indoleamine 2,3-Dioxygenase Gene Is a Feature of Poorly Differentiated Non-muscle-invasive Urothelial Cell Bladder Carcinomas.

AIM: To evaluate indoleamine 2,3-dioxygenase (IDO) gene expression in non-muscle-invasive urothelial cell bladder carcinoma (NMIBC). PATIENTS AND METHODS: Seventy-four patients undergoing surgical treatment for NMIBC were enrolled in the study. IDO gene expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: IDO gene expression was detectable significantly more frequently (48/74, 64.86% vs. 5/21, 23.81%, p<0.001) and to significantly higher extents (p=0.01) in cancer tissues than in normal bladder mucosa. IDO gene expression was observed significantly more frequently in large (p=0.02), high-grade (p=0.05) and stage T1 (p=0.03) than in small, low-grade and stage Ta tumors. Expression levels were also significantly higher in large, high-grade and stage T1 tumors (p<0.01, p=0.05 and p=0.03, respectively). A direct positive correlation between IDO gene expression in tumor tissues and tumor size (R=0.24, p=0.04), grade (R=0.23, p=0.05) and stage (R=0.25, p=0.03) was detected. Multivariate analysis suggested a trend (p=0.08) towards longer overall survival in patients bearing tumors that did not express IDO gene. CONCLUSION: These data indicate that IDO gene expression is a feature of aggressive NMIBC, suggesting a potential immunosuppressive role of IDO.

A Functional Genetic Screen Identifies the Phosphoinositide 3-kinase Pathway as a Determinant of Resistance to Fibroblast Growth Factor Receptor Inhibitors in FGFR Mutant Urothelial Cell Carcinoma.

Activating mutations and translocations of the FGFR3 gene are commonly seen in urothelial cell carcinoma (UCC) of the bladder and urinary tract. Several fibroblast growth factor receptor (FGFR) inhibitors are currently in clinical development and response rates appear promising for advanced UCC. A common problem with targeted therapeutics is intrinsic or acquired resistance of the cancer cells. To find potential drug targets that can act synergistically with FGFR inhibition, we performed a synthetic lethality screen for the FGFR inhibitor AZD4547 using a short hairpin RNA library targeting the human kinome in the UCC cell line RT112 (FGFR3-TACC3 translocation). We identified multiple members of the phosphoinositide 3-kinase (PI3K) pathway and found that inhibition of PIK3CA acts synergistically with FGFR inhibitors. The PI3K inhibitor BKM120 acted synergistically with inhibition of FGFR in multiple UCC and lung cancer cell lines having FGFR mutations. Consistently, we observed an elevated PI3K-protein kinase B pathway activity resulting from epidermal growth factor receptor or Erb-B2 receptor tyrosine kinase 3 reactivation caused by FGFR inhibition as the underlying molecular mechanism of the synergy. Our data show that feedback pathways activated by FGFR inhibition converge on the PI3K pathway. These findings provide a strong rationale to test FGFR inhibitors in combination with PI3K inhibitors in cancers harboring genetic activation of FGFR genes.

Cigarette smoke induced urocystic epithelial mesenchymal transition via MAPK pathways.

Cigarette smoke has been shown to be a major risk factor for bladder cancer. Epithelial-mesenchymal transition (EMT) is a crucial process in cancer development. The role of MAPK pathways in regulating cigarette smoke-triggered urocystic EMT remains to be elucidated. Human normal urothelial cells and BALB/c mice were used as in vitro and in vivo cigarette smoke exposure models. Exposure of human normal urothelial cells to cigarette smoke induced morphological change, enhanced migratory and invasive capacities, reduced epithelial marker expression and increased mesenchymal marker expression, along with the activation of MAPK pathways. Moreover, we revealed that ERK1/2 and p38 inhibitors, but rather JNK inhibitor, effectively attenuated cigarette smoke-induced urocystic EMT. Importantly, the regulatory function of ERK1/2 and p38 pathways in cigarette smoke-triggered urocystic EMT was further confirmed in mice exposed to CS for 12 weeks. These findings could provide new insight into the molecular mechanisms of cigarette smoke-associated bladder cancer development as well as its potential intervention.

Effects of Cyclooxygenase on the Urothelium of the Urinary Bladder of Mice Exposed to Pelvic Radiation.

OBJECTIVE: To determine the role of cyclooxygenase (COX) expression in the urothelium of the urinary bladder during radiation injury caused by pelvic radiotherapy for cancer therapy. STUDY DESIGN: Twenty-four male Swiss Albino mice were separated into 4 groups. The first group was the control group (Group 1) and the second, third, and fourth groups were euthanized after 24 hours (Group 2), 48 hours (Group 3), and 7 days (Group 4), respectively. A single-fractioned 10 Gy of ionizing radiation was applied to all mice's pelvic zone with Co-60. Bladders were removed completely from the pelvic region. Histochemical analysis using hematoxylin and eosin and immunohistochemical analysis using anti-COX-1 and COX-2 antibodies were performed on tissue samples. The immunoreactivities of the urinary bladder were quantified using H-score measurement, and statistical comparison was performed. RESULTS: In the immunohistochemical examination the COX-1 immunoreactivities were found to be higher in the urothelium of the bladder in the radiation exposed groups than in the normal control group (group 1) (p < 0.005). Additionally, high immunoreactivity of COX-2 molecule was established in groups 2, 3, and 4 of radiation groups as compared to group 1 (p < 0.005) in examination of the urothelium. COX-1 and COX-2 immunoreactivities in the submucosa were detected higher in group 4 than in the other groups (p < 0.005). CONCLUSION: COX-1 and COX-2 expressions in the urothelium and subepithelium of the urinary bladder were investigated in mice during the acute radiation response. The expression of COX-1 and COX-2 in the urothelium seems to prevent bladder damage from radiation, supplying differentiation and restoration of the urothelium.

Patterns and Significance of PIM Kinases in Urothelial Carcinoma.

BACKGROUND: The Provirus integrating site Moloney murine leukemia virus (Pim) family are proteins with serine/threonine kinase activity. Studies have demonstrated overexpression of Pims in cancer. To our knowledge, only a single study has examined Pim-1 in urothelial carcinoma. The aim of this investigation was to evaluate Pim-1, Pim-2, and Pim-3 in urothelial carcinoma and assess for expression that may contribute to disease progression and serve as a site for targeted therapy. METHODS: This retrospective study included 137 cases taken from specimens from the University of Utah, Department of Pathology (2008 to 2011). Tissue was stained with antibodies against Pim-1, Pim-2, and Pim-3. Cases were classified into 3 groups, based upon current World Health Organization criteria (invasive high-grade urothelial carcinoma [IHG] [n=84], noninvasive high-grade urothelial carcinoma/carcinoma in situ [n=32], and noninvasive low-grade urothelial carcinoma [NILG] [n=21]). Cases were scored and recorded as positive or negative on the basis of the percentage of cells with cytoplasmic and/or nuclear staining. RESULTS: NILG showed higher expression of Pim-1 (relative expression rate [RER]=2.28; 95% confidence interval [CI], 0.183-0.764) and Pim-3 (RER=3.06; 95% CI, 0.423-0.816) compared with other lesions. IHG had lower expression of Pim-1 (RER=0.31; 95% CI, 0.401-0.844) and Pim-3 (RER=0.354; 95% CI, 0.322-0.816) and noninvasive high-grade urothelial carcinoma (NIHG) demonstrated increased expression of Pim-1 and (RER=2.09; 95% CI, 0.124-0.739) and Pim-2 (RER=1.70; 95% CI, 0.151-0.591). At least 1 Pim kinase protein was expressed at the following rates: 49% in IHG, 66% in NIHG, and 76% in NILG. CONCLUSION: A high percentage of urothelial carcinomas express Pim kinases. Pim expression differs in NILG, NIHG, and IHG lesions.

Carbonic anhydrase IX as a diagnostic urinary marker for urothelial bladder cancer.

UNLABELLED: Urinary biomarkers are needed to improve the management and reduce the cost of urothelial bladder cancer (UBC); however, none have been recommended yet for clinical practice. This study evaluated carbonic anhydrase IX (CAIX) as a diagnostic urinary biomarker for UBC. CAIX was analyzed by quantitative polymerase chain reaction in urine samples of 196 patients with UBC and 123 controls with hematuria. Paired samples from urine and tumor tissue were evaluated in 16 cases. Data were validated in 155 independent samples. The sensitivity and specificity of CAIX for UBC detection were 86.2% and 95.1%, respectively (area under the curve [AUC]: 90.5%). There was a significant association of CAIX expression between the paired urine and tumor specimens (p=0.002). CAIX showed a significantly higher predictive accuracy than urinary cytology (90.5% vs 71.7%), specifically in low-grade tumors (90.0% vs 61.8%). CAIX expression decreased with increasing tumor stage and grade. Analyses in an independent validation cohort confirmed the high accuracy of CAIX for diagnosing UBC (AUC: 88.3%). PATIENT SUMMARY: We evaluated carbonic anhydrase IX (CAIX) as a urinary marker for bladder cancer (BCa) using a large series of patients from a single hospital. We found that urinary CAIX has a high sensitivity and specificity for diagnosing BCa.

Expression and function analysis of indoleamine 2 and 3-dioxygenase in bladder urothelial carcinoma.

Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for tryptophan metabolism inducing immune tolerance of tumors. The purpose of this study is to investigate IDO expression and its prognostic significance in bladder urothelial carcinoma (BUC). In this study, immunohistochemical staining for IDO expression in BUC tissues (n = 84) and normal bladder tissues (n = 22) was performed. The mRNA expression levels of IDO in BUC and normal bladder were analyzed by quantitative RT-PCR. Survival analysis was performed for the correlation of IDO expression and clinicopathological factors with disease-free survival. Positive expression of IDO was found in 48 of 84 cases in BUC tissues and was significantly correlated with histological classification, histological grade and TNM stage. While IDO expression in normal bladder tissues was expressed in only 4 of 22 (18.2%) cases. Moreover, IDO mRNA levels of BUC were significantly higher than that of normal bladder. We also found that IDO, histological grade and TNM stage were closely associated with DFS. These results indicated that IDO was related to the progression of BUC and might be one of the crucial prognostic factors for BUC.