Bladder cancer displays an aggressive phenotype in the muscle-invasive phase, and is associated with a high mortality rate. Therefore, novel molecular therapeutic targets are needed to improve patient survival. A monoclonal antibody against the extracellular domain of the claudin-4 (CLDN4) tight junction protein was established by immunizing rats with a plasmid vector encoding human CLDN4. A hybridoma clone, producing a rat monoclonal antibody recognizing CLDN4 (clone 4D3), was obtained. Immunohistochemistry by using the 4D3 antibody showed that CLDN4 expression was associated with local invasion, nodal metastasis, distant metastasis, and advanced stage in 86 cases of bladder cancer. The 4D3 antibody inhibited growth, invasion, and survival, associated with abrogation of the intratumoral microenvironment; lowered concentrations of epidermal growth factor and vascular endothelial growth factor were found in three-dimensional cultures of T24 and RT4 cells. In combination with cisplatin therapy, 4D3 enhanced cisplatin cytotoxicity by increasing cellular permeability, leading to increased intracellular cisplatin concentrations. In mouse models of subcutaneous tumors and lung metastasis, 4D3 enhanced tumor growth inhibition, alone and with concurrent cisplatin treatment. The anti-tumor activity of the newly established 4D3 antibody suggests that it may be a powerful tool in CLDN4-targeting therapy, and in combination with chemotherapy.

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Abstract. 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), hypoxiainducible factor (HIF)-1·, HIF-2·, 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-1·, HIF-2·, 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-1·, HIF-2·, 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-1·. In clinical specimens of bladder cancer, high expression of HO-1 was correlated with high expression of HIF-1· (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.

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, prostatespecific 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 þ 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.

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.