Yasushi Nakai

Heme oxygenase-1 promotes angiogenesis in urothelial carcinoma of the urinary bladder

Angiogenesis is necessary for the growth, invasion, and metastasis of solid tumors. Previous studies have shown that heme oxygenase-1 (HO-1) plays an important role in angiogenesis in both normal and cancerous cells, such as vascular endothelial cells and pancreatic cancer cells, respectively. In this study, we analyzed the role of HO-1 and other angiogenic factors in urothelial carcinoma of the bladder. Specifically, we used real-time reverse transcription polymerase chain reaction (PCR) and Western blotting to investigate the upregulation of 7 angiogenic factors, namely, HO-1, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α, HIF-2α, cyclooxygenase-2 (COX-2), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) under hypoxic conditions in the T24 urothelial carcinoma cell line. We also used enzyme-linked immunosorbent assay (ELISA) to measure the amount of VEGF secreted into the growth media. In addition, we administered an HO-1 inhibitor, zinc protoporphyrin IX, to mice with subcutaneous T24 tumors to assess the modulation of angiogenesis in solid tumors in vivo. We also performed immunohistochemical analyses of 23 primary bladder cancer specimens with high-grade tumors infiltrating into the stroma (pT1) for expression of HO-1, VEGF, HIF-1α, HIF-2α, COX-2, and CD31. Image analysis of CD31 staining was performed to estimate microvessel density (MVD), a measure of angiogenesis. Hypoxic conditions induced upregulation of HO-1, VEGF, HIF-1α, HIF-2α, and COX-2 in T24 cells and increased VEGF secretion, which could be suppressed by zinc protoporphyrin IX. In vivo, inhibition of HO-1 decreased tumor growth and MVD by suppressing angiogenic factors, particularly VEGF and HIF-1α. In clinical specimens of bladder cancer, high expression of HO-1 was correlated with high expression of HIF-1α (P=0.027) and high MVD (P=0.005), but not with VEGF expression (P=0.19). In conclusion, since overexpression of HO-1 promotes angiogenesis in urothelial carcinoma cells, HO-1 inhibitors could be used as novel therapeutics for urothelial carcinoma of the urinary bladder.

Clinical Significance of Heme Oxygenase-1 Expression in Non-Muscle-Invasive Bladder Cancer

Introduction: In several malignant diseases, elevated heme oxygenase-1 (HO-1) is associated with progression or resistance to chemotherapy. We evaluated the clinical significance of HO-1 expression in non-muscle-invasive bladder cancer. Patients and Methods: We examined 109 patients with non-muscle-invasive bladder cancer. The immunoexpression of HO-1, p53, and Ki-67 was analyzed using paraffin-embedded tissue from transurethral resection in comparison with the clinicopathological variables. Results: Positive expression of HO-1 was found in 66 of 109 tumors (61%), and the positivity of HO-1 correlated significantly with high tumor grade and the altered expression patterns of p53 and Ki-67. In our analysis of 16 cases treated by intravesical administration of anthracyclines, the positive expression of HO-1 correlated with poor disease-free survival (p = 0.015). In in vitro experiments using urothelial cancer cell lines, HO-1 upregulation was observed by exposure to doxorubicin. Moreover, siRNA-mediated suppression of HO-1 upregulation sensitized the urothelial cancer cells to doxorubicin. Conclusions: Our findings suggested that resistance against anthracyclines correlated with HO-1 and expression analysis of HO-1 may be a useful predictive marker for intravesical administration of anthracyclines.

Exploration of risk factors predicting outcomes for primary T1 high‐grade bladder cancer and validation of the Spanish Urological Club for Oncological Treatment scoring model: Long‐term follow‐up experience at a single institute

To determine the prognostic factors of primary T1 high‐grade bladder cancer and to validate the Spanish Urological Club for Oncological Treatment model in Japanese patients with T1 high‐grade bladder cancer treated at a single institution.

CXCL1-Mediated Interaction of Cancer Cells with Tumor-Associated Macrophages and Cancer-Associated Fibroblasts Promotes Tumor Progression in Human Bladder Cancer

Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are reported to be associated with poor prognosis, depending on their pro-tumoral roles. Current knowledge of TAMs and CAFs in the tumor microenvironment of urothelial cancer of the bladder (UCB) is limited. Therefore, we investigated the paracrine effect induced by TAMs and CAFs in the tumor microenvironment of human UCB. For this, we first carried out immunohistochemical analysis for CXCL1, CD204 (TAM marker), αSMA (CAF marker), E-cadherin, and MMP2 using 155 UBC tissue samples. Next, CXCL1-overexpressing clones of THP-1-derived TAMs and NIH3T3-derived CAFs were developed by lentiviral vector infection. The immunohistochemical study showed high CXCL1 levels in UCB cells to be associated with enhanced recruitment of TAMs/CAFs, higher metastatic potential, and poor prognosis. Three-dimensional (3D) co-culture of UCB cells and TAMs/CAFs suggested that CXCL1 production in TAMs/CAFs play an important role in cell-to-cell adhesion and interaction among cancer cells and these stromal cells. CXCL1-expressing TAMs/CAFs enhanced tumor growth of subcutaneous UCB tumors in nude mice when injected together. In addition, an experiment using the orthotopic bladder cancer model revealed that CXCL1 production in TAMs/CAFs supported tumor implantation into the murine bladder wall and UCB growth when injected together, which was confirmed by clinical data of patients with bladder cancer. Thus, CXCL1 signaling in the tumor microenvironment is highly responsible for repeated intravesical recurrence, disease progression, and drug resistance through enhanced invasion ability. In conclusion, disrupting CXCL1 signaling to dysregulate this chemokine is a promising therapeutic approach for human UCB.

Bone Scan Can Be Spared in Asymptomatic Prostate Cancer Patients with PSA of ≤20 ng/ml and Gleason Score of ≤6 at the Initial Stage of Diagnosis

OBJECTIVE According to several guidelines, it is acceptable to spare a bone scan in the patients who are newly diagnosed with low-risk prostate cancer. Our aim is to clarify a suitable group whereby a bone scan could be spared at the initial staging of prostate cancer. METHODS Consecutive 857 patients who were newly diagnosed from 2004 through 2009 and received bone scans using technetium 99m methylene diphosphonate at the initial staging were enrolled. The proportion of positive bone metastases by age distribution, prostate-specific antigen level at diagnosis, Gleason score and clinical T stage were evaluated. Univariate and multivariate logistic regression analyses were performed to identify the predictors of positive bone metastases. RESULTS Of all 857 patients, 40 patients (4.7%) showed bone metastases. Patients with higher age, prostate-specific antigen level, clinical stage and Gleason score showed significantly higher rate of bone metastases (P < 0.001). In univariate logistic regression analyses, age, prostate-specific antigen level, clinical stage and Gleason score were independent predictors of bone metastasis. The multivariate analysis showed that both the prostate-specific antigen level >50 ng/ml and the Gleason score ≥4 + 3 were independent predictors of bone metastases. CONCLUSIONS The incidences of bone metastases in patients with a prostate-specific antigen level of ≤20 ng/ml and Gleason score of ≤6 were reasonably low. Collectively, a bone scan is not necessary as a routine examination for these patients at their initial staging of prostate cancer.

Neutrophil-to-lymphocyte ratio as a detection marker of tumor recurrence in patients with muscle-invasive bladder cancer after radical cystectomy.

PURPOSE High-neutrophil to lymphocyte ratio (NLR) values have been shown to be associated with a poor prognosis in many human malignant tumors. We evaluated the correlation of the NLR with other variables in patients with muscle-invasive bladder cancer after radical cystectomy (RC); in particular, we evaluated chronological changes in the postoperative NLR. METHODS We included the data from a total of 110 patients who underwent RC for muscle-invasive bladder cancer. The NLR was calculated using complete blood counts determined before RC. Kaplan-Meier and Cox proportional regression analyses of recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS) were performed to identify significant prognostic variables. RESULTS The median patient age was 72 years (41-91 years). In univariate analysis, the pretreatment NLR (≥2.6 vs.<2.6) was associated with RFS (hazard ratio [HR] = 2.41, P = 0.008), CSS (HR = 2.89, P = 0.006), and OS (HR = 2.73, P = 0.002). In multivariate analysis, an NLR≥2.6 and an infiltrative growth pattern at the tumor invasion front were significantly associated with RFS (HR = 2.61, P = 0.023), CSS (HR = 2.58, P = 0.08), and OS (HR = 2.77, P = 0.004). Postoperative chronological analysis revealed that the NLR of 68 patients without recurrence remained low during follow-up, whereas the NLR of the remaining 42 patients with recurrence increased significantly in the last visit before recurrence was detected radiographically (P< 0.01). CONCLUSIONS The NLR and tumor growth pattern were strong predictors of prognosis for patients undergoing RC. Our results suggest that an increase in the NLR during follow-up after RC is a potential marker for the early detection of recurrence.

Collagen type IV alpha 1 (COL4A1) and collagen type XIII alpha 1 (COL13A1) produced in cancer cells promote tumor budding at the invasion front in human urothelial carcinoma of the bladder

Current knowledge of the molecular mechanism driving tumor budding is limited. Here, we focused on elucidating the detailed mechanism underlying tumor budding in urothelial cancer of the bladder. Invasive urothelial cancer was pathologically classified into three groups as follows: nodular, trabecular, and infiltrative (tumor budding). Pathohistological analysis of the orthotopic tumor model revealed that human urothelial cancer cell lines MGH-U3, UM-UC-14, and UM-UC-3 displayed typical nodular, trabecular, and infiltrative patterns, respectively. Based on the results of comprehensive gene expression analysis using microarray (25 K Human Oligo chip), we identified two collagens, COL4A1 and COL13A1, which may contribute to the formation of the infiltrative pattern. Visualization of protein interaction networks revealed that proteins associated with connective tissue disorders, epithelial-mesenchymal transition, growth hormone, and estrogen were pivotal factors in tumor cells. To evaluate the invasion pattern of tumor cells in vitro, 3-D collective cell invasion assay using Matrigel was performed. Invadopodial formation was evaluated using Gelatin Invadopodia Assay. Knockdown of collagens with siRNA led to dramatic changes in invasion patterns and a decrease in invasion capability through decreased invadopodia. The in vivo orthotopic experimental model of bladder tumors showed that intravesical treatment with siRNA targeting COL4A1 and COL13A1 inhibited the formation of the infiltrative pattern. COL4A1 and COL13A1 production by cancer cells plays a pivotal role in tumor invasion through the induction of tumor budding. Blocking of these collagens may be an attractive therapeutic approach for treatment of human urothelial cancer of the bladder.

Protoporphyrin IX induced by 5-aminolevulinic acid in bladder cancer cells in voided urine can be extracorporeally quantified using a spectrophotometer.

BACKGROUND We evaluated the feasibility of photodynamic diagnosis of bladder cancer by spectrophotometric analysis of voided urine samples after extracorporeal treatment with 5-aminolevulinic acid (ALA). METHODS Sixty-one patients with bladder cancer, confirmed histologically after the transurethral resection of a bladder tumor, were recruited as the bladder cancer group, and 50 outpatients without history of urothelial carcinoma or cancer-related findings were recruited as the control group. Half of the voided urine sample was incubated with ALA (ALA-treated sample), and the rest was incubated without treatment (ALA-untreated sample). For detecting cellular protoporphyrin IX levels, intensity of the samples at the excitation wavelength of 405 nm was measured using a spectrophotometer. The difference between the intensity of the ALA-treated and ALA-untreated samples at 635 nm was calculated. RESULTS The differences in the bladder cancer group were significantly greater than those in the control group (p < 0.001). These differences were also significantly greater in patients with high-grade tumors than in those with low-grade tumors (p = 0.004), and also in patients with invasive bladder cancer than in those with noninvasive bladder cancer (p = 0.007). The area under the curve was 0.84. Sensitivity and specificity of the method were 82% and 80%, respectively. CONCLUSIONS We demonstrated that protoporphyrin IX levels in urinary cells treated with ALA could be quantitatively detected by spectrophotometer in patients with bladder cancer. Therefore, this cancer detection system has a potential for clinical use.

Diagnostic approach for cancer cells in urine sediments by 5-aminolevulinic acid-based photodynamic detection in bladder cancer.

Bladder urothelial carcinoma is diagnosed and followed up after transurethral resection using a combination of cystoscopy, urine cytology and urine biomarkers at regular intervals. However, cystoscopy can overlook flat lesions like carcinoma in situ, and the sensitivity of urinary tests is poor in low‐grade tumors. There is an emergent need for an objective and easy urinary diagnostic test for the management of bladder cancer. In this study, three different modalities for 5‐aminolevulinic acid (ALA)‐based photodynamic diagnostic tests were used. We developed a compact‐size, desktop‐type device quantifying red fluorescence in cell suspensions, named “Cellular Fluorescence Analysis Unit” (CFAU). Urine samples from 58 patients with bladder cancer were centrifuged, and urine sediments were then treated with ALA. ALA‐treated sediments were subjected to three fluorescence detection assays, including the CFAU assay. The overall sensitivities of conventional cytology, BTA, NMP22, fluorescence cytology, fluorescent spectrophotometric assay and CFAU assay were 48%, 33%, 40%, 86%, 86% and 87%, respectively. Three different ALA‐based assays showed high sensitivity and specificity. The ALA‐based assay detected low‐grade and low‐stage bladder urothelial cells at shigher rate (68–80% sensitivity) than conventional urine cytology, BTA and NMP22 (8–20% sensitivity). Our findings demonstrate that the ALA‐based fluorescence detection assay is promising tool for the management of bladder cancer. Development of a rapid and automated device for ALA‐based photodynamic assay is necessary to avoid the variability induced by troublesome steps and low stability of specimens.

Photodynamic diagnosis of shed prostate cancer cells in voided urine treated with 5-aminolevulinic acid

BackgroundPast attempts at detecting prostate cancer (PCa) cells in voided urine by traditional cytology have been impeded by undesirably low sensitivities but high specificities. To improve the sensitivities, we evaluate the feasibility and clinical utility of photodynamic diagnosis (PDD) of prostate cancer by using 5-aminolevulinic acid (5-ALA) to examine shed prostate cancer cells in voided urine samples.MethodsOne hundred thirty-eight patients with an abnormal digital rectal exam (DRE) and/or abnormal prostate-specific antigen (PSA) levels were recruited between April 2009 and December 2010. Voided urine specimens were collected before prostate biopsy. Urine specimens were treated with 5-ALA and imaged by fluorescence microscopy and reported as protoporphyrin IX (PPIX) positive (presence of cells demonstrating simultaneous PPIX fluorescence) or PPIX negative (lack of cells demonstrating fluorescence).ResultsOf the 138 patients, PCa was detected on needle biopsy in 81 patients (58.7%); of these 81 patients with PCa, 60 were PPIX-positive (sensitivity: 74.1%). Although 57 patients did not harbor PCa by conventional diagnostic procedures, 17 of these at-risk patients were found to be PPIX-positive (specificity: 70.2%). PPIX–PDD was more sensitive compared with DRE and transrectal ultrasound and more specific compared with PSA and PSA density. The incidence of PPIX–PDD positivity did not increase with increasing total PSA levels, tumor stage or Gleason score.ConclusionsTo our knowledge, this is the first successful demonstration of PPIX in urine sediments treated with 5-ALA used to detect PCa in a noninvasive yet highly sensitive manner. However, further studies are warranted to determine the role of PPIX–PPD for PCa detection.