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Abstract: Objective: To determine histone deacetylase (HDAC) isoenzyme expression patterns in urothelial cancer tissues and cell lines and investigate their potential to predict the efficacy of the HDAC inhibitor vorinostat. Materials and methods: Expression of HDAC mRNAs was determined by quantitative RT-PCR in 18 urothelial cancer cell lines (UCC), normal uroepithelial controls (NUC), 24 urothelial cancer tissues, and 12 benign controls. Results were compared with published microarray data. Effects of pan-HDAC inhibitor vorinostat and on UCCs were determined by viability and apoptosis assays, cell cycle analysis, and measurements of p21CIP1, thymidylate synthase (TS), and EZH2. In addition, protein expression levels of HDACs were investigated in UCCs. Results: Prominent changes in UCCs were HDAC2 and/or HDAC8 up-regulation in 11 of 18 cell lines and decreased expression of HDAC4, HDAC5, and/or HDAC7 mRNA in 15 of 18 cell lines. In cancer tissues, HDAC8 was likewise significantly up-regulated (P = 0.002), whereas HDAC2 up-regulation was detected only in a subset of tumors (9/24, P = 0.085). Overexpression of HDAC2 and HDAC8 mRNA did not correspond with the protein level. Vorinostat induced G2/M arrest, an increase in the sub-G1 fraction, up-regulation of p21, and down-regulation of TS in all UCC. Effects on EZH2 and PARP cleavage as well as activation of caspase 3/7 differed between cell lines. Associations between the overall sensitivity to the pan-HDACi vorinostat and overexpression of HDAC2 and HDAC8 mRNA were not observed. Conclusions: In urothelial cancer, up-regulation of HDAC2 and HDAC8 and down-regulation of HDAC4, HDAC5, and HDAC7 mRNA are common findings. The treatment effect of the pan-HDAC inhibitor vorinostat was variable in UCCs and up-regulation of HDAC2 and HDAC8 was not predictive for treatment response. Whether selective targeting of HDAC2, HDAC8, or other HDACs deregulated in urothelial cancer (e.g., HDAC4, HDAC5, and HDAC7) result in a more consistent treatment response needs further investigation. [Copyright &y& Elsevier]

• First multicenter study comparing urine-based rapid tests for bladder cancer. • Highest sensitivities for HGNMI bladder cancer for BTA stat® and UBC® Rapid Test. • Cytology should mostly be used in specialized centers. • Use of urinary-based rapid tests in management of bladder cancer should be discussed. BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, and CancerCheck® UBC® rapid VISUAL are urinary-based rapid tests. This multicenter study is the first study comparing all available rapid tests on a large cohort of bladder cancer patients and healthy controls in one setting. In total 732 urine samples (second morning urine) in a real-world assessment have been analyzed. We evaluated clinical samples from 464 patients with histologically confirmed urothelial tumors of the urinary bladder (17 solitary CIS, 189 low-grade, 187 high-grade nonmuscle invasive, 71 high-grade muscle invasive), 77 patients with No Evidence of Disease (NED), and from 191 healthy controls. Urine samples were analyzed by the BTA stat®, NMP22® BladderChek®, UBC® Rapid Test point-of-care (POC) system using the concile Omega 100 POC reader, and CancerCheck® UBC® rapid VISUAL. Sensitivities and specificities were calculated by contingency analyses. All investigated urinary markers detected more pathological concentrations in urine of bladder cancer patients compared to tumor-free patients. The calculated diagnostic sensitivities for BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, CancerCheck® UBC® rapid VISUAL, and cytology were 62.4%, 13.4%, 58.2%, 28.6%, 36.2% for low-grade, 83.4%, 49.5%, 84.5%, 63.1%, 71.2% for high-grade nonmuscle invasive, and 95.8%, 35.2%, 76.1%, 50.7%, 67.7% for high-grade muscle-invasive bladder cancer. The specificity was 67.9%, 95.5%, 79.4%, 94.4%, and 83.7%, respectively. The area under the curve (AUC) after receiver operating characteristics (ROC) analysis for high-grade non–muscle-invasive tumors was 0.757, 0.725, 0.819, 0.787, and 0.774, respectively. The analysis of more than 700 urine samples offers an objective view on urine-based rapid diagnostics. Elevated pathological concentrations of markers in urine of bladder cancer patients were detected in all investigated tests. The highest sensitivities for high-grade non–muscle-invasive tumors were calculated for BTA stat® and UBC® Rapid Test, whereas NMP22® BladderChek®, and cytology showed the highest specificities. BTA stat® and UBC® Rapid Test have the potential to be used as a clinical valuable urinary protein biomarker for the detection of high-grade non–muscle-invasive bladder cancer patients and could be included in the management of these tumors. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Carcinoma of the bladder is the fifth most common cancer whose incidence continues to rise. MTDH/AEG-1 is associated with the initiation and progression of many cancers including breast, hepatocellular, ovarian, and colorectal carcinomas. However, the expression and functional importance of MTDH/AEG-1 in bladder cancer remains unknown. The present study was aimed at exploring the functional role of MTDH/AEG-1 in selected bladder cancer cell lines. Methods and materials: The relative expression of MTDH/AEG-1 was assessed by real-time quantitative reverse transcription-polymerase chain reaction in several human bladder cancer cell lines as well as cancerous and benign bladder tissues. Then, expression of MTDH/AEG-1 in RT112 and 647V bladder cancer cell lines was knocked down by an RNA interference strategy. Cell viability and apoptosis were determined after treatment with specific interfering RNA. Potential effects of MTDG/AEG-1 specific interfering RNA on the cell cycle were investigated by flow cytometry. We also performed anchorage-independent growth and wound-healing assays to study MTDH/AEG-1 function. Results: Down-regulation of MTDH/AEG-1 did not significantly affect the cell cycle distribution but rather reduced cell viability via apoptosis, as evidenced by increased annexin V staining and caspase 3/7 activities as well as mitochondrial potential disruption. Of note, serum starvation did not exacerbate the effects of MTDH/AEG-1 knockdown. Furthermore, MTDH/AEG-1 down-regulation significantly decreased anchorage-independent growth and migration of bladder carcinoma cells. Conclusion: Overexpression of MTDH/AEG-1 contributes to the neoplastic phenotype of bladder cancer cells by promoting survival, clonogenicity, and migration. [Copyright &y& Elsevier]

Background: Cisplatin-based regimens are routinely employed for the treatment of urothelial carcinoma. However, therapeutic success is hampered by the primary presence of or the development of cisplatin resistance. This chemoresistance is executed by multiple cellular pathways. In recent years, the cellular process of autophagy has been identified as a prosurvival pathway of cancer cells. On the one hand, autophagy enables cancer cells to survive conditions of low oxygen or nutrient supply, frequently found in tumors. On the other hand, autophagy supports chemoresistance of cancer cells. Here, we aimed at investigating the involvement of autophagy for cisplatin resistance in different urothelial carcinoma cell lines.Materials& Methods: We analyzed the expression levels of different autophagy-related proteins in cisplatin-sensitive and cisplatin-resistant urothelial carcinoma cell lines. Furthermore, we performed cell viability assays and caspase activity assays with cells treated with cisplatin, non-specific or specific autophagy inhibitors (chloroquine, 3-methyladenine, SAR405) or combinations thereof.Results: We found that autophagy-related proteins are up-regulated in different cisplatin-resistant urothelial carcinoma cells compared to the sensitive parental cell lines. Furthermore, inhibition of autophagy, in general, or of the autophagy-inducing class III PtdIns3K complex, in particular, sensitized both sensitive and resistant urothelial carcinoma cells to cisplatin-induced cytotoxic effects.Conclusion: We propose that targeting the autophagic machinery might represent a suitable approach to complement or even increase cisplatin efficacy in order to overcome cisplatin resistance in urothelial carcinoma. [ABSTRACT FROM AUTHOR]

Background: Rapid development of novel treatment options demands valid preclinical screening models for urothelial carcinoma (UC). The translational value of high-throughput drug testing using 2-dimensional (2D) cultures is limited while for xenograft models handling efforts and costs often become prohibitive for larger-scale drug testing. Therefore, we investigated to which extent the chicken chorioallantoic membrane (CAM) assay might provide an alternative model to study antineoplastic treatment approaches for UC.Methods: The ability of 8 human UC cell lines (UCCs) to form tumors after implantation on CAMs was investigated. Epithelial-like RT-112 and mesenchymal-like T-24 UCCs in cell culture or as CAM tumors were treated with cisplatin alone or combined with histone deacetylase inhibitors (HDACi) romidepsin and suberanilohydroxamic acid. Tumor weight, size, and bioluminescence activity were monitored; tumor specimens were analyzed by histology and immunohistochemistry. Western blotting and quantitative real time polymerase chain reaction were used to measure protein and mRNA expression.Results: UCCs were reliably implantable on the CAM, but tumor development varied among cell lines. Expression of differentiation markers (E-cadherin, vimentin, CK5, CK18, and CK20) was similar in CAM tumors and 2D cultures. Cellular phenotypes also remained stable after recultivation of CAM tumors in 2D cultures. Bioluminescence images correlated with tumor weight. Cisplatin and HDACi decreased weight and growth of CAM tumors in a dose-dependent manner, but HDACi treatment acted less efficiently as in 2D cultures, especially on its typically associated molecular markers. Synergistic effects of HDACi and subsequent cisplatin treatment on UCCs were neither detected in 2D cultures nor detected in CAM tumors.Conclusion: Our results demonstrate that the CAM assay is a useful tool for studying tumor growth and response to conventional anticancer drugs under 3D conditions, especially cytotoxic drugs as cisplatin. With some limitations, it might serve as a cost- and time-effective preclinical screening assay for novel therapeutic approaches before further assessment in expensive and cumbersome animal models. [ABSTRACT FROM AUTHOR]