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Quercetin, a dietary flavonoid found in fruits and vegetables, has been described as a substance with many anti-cancer properties in a variety of preclinical investigations. In the present study, we demonstrate that 2D and 3D melanoma models exhibit not only different sensitivities to quercetin, but also opposite, cancer-promoting effects when metastatic melanoma spheroids are treated with quercetin. Higher concentrations of quercetin reduce melanoma growth in three tested cell lines, whereas low concentrations induce the opposite effect in metastatic melanoma spheroids but not in the non-metastatic cell line. High (&gt, 12.5 &micro, M) or low (&lt, 6.3 &micro, M) quercetin concentrations decrease or enhance cell viability, spheroid size, and cell proliferation, respectively. Additionally, melanoma cells cultivated in 2D already show significant caspase 3 activity at very low concentrations (&gt, 0.4 &micro, M), whereas in 3D spheroids apoptotic cells, caspase 3 activity can only be detected in concentrations &ge, M. Further, we show that the tumor promoting or repressing effect in the 3D metastatic melanoma spheroids are likely to be elicited by a precisely controlled regulation of Nrf2/ARE-mediated cytoprotective genes, as well as ERK and NF-&kappa, B phosphorylation. According to the results obtained here, further studies are needed to better characterize the mechanisms of action underlying the pro- and anti-carcinogenic effects of quercetin on human melanomas.

Specific gene promoter DNA methylation is becoming a powerful epigenetic biomarker in cancer diagnostics. Five genes (CDH1, CDKN2Ap16, RASSF1A, TERT, and WT1) were selected based on their frequently published potential as epigenetic markers. Diagnostic promoter methylation assays were generated based on bisulfite-converted DNA pyrosequencing. The methylation patterns of 144 non-small-cell lung cancer (NSCLC) and 7 healthy control formalin-fixed paraffin-embedded (FFPE) samples were analyzed to evaluate the applicability of the putative diagnostic markers. Statistically significant changes in methylation levels are shown for TERT and WT1. Furthermore, 12 NSCLC and two benign lung cell lines were characterized for promoter methylation. The in vitro tests involved a comparison of promoter methylation in 2D and 3D cultures, as well as therapeutic tests investigating the impact of CDH1/CDKN2Ap16/RASSF1A/TERT/WT1 promoter methylation on sensitivity to tyrosine kinase inhibitor (TKI) and DNA methyl-transferase inhibitor (DNMTI) treatments. We conclude that the selected markers have potential and putative impacts as diagnostic or even predictive marker genes, although a closer examination of the resulting protein expression and pathway regulation is needed.

// Nico Jacobi 1 , Rita Seeboeck 2 , Elisabeth Hofmann 2 , Helmut Schweiger 1 , Veronika Smolinska 1 , Thomas Mohr 3 , Alexandra Boyer 1 , Wolfgang Sommergruber 4 , Peter Lechner 5 , Corina Pichler-Huebschmann 5 , Kamil Onder 6, 7 , Harald Hundsberger 2 , Christoph Wiesner 2 and Andreas Eger 1, 2 1 IMC University of Applied Sciences Krems, Department Life Sciences, Research Institute for Applied Bioanalytics and Drug Development, A-3500 Krems, Austria 2 IMC University of Applied Sciences Krems, Department Life Sciences, Institute of Medical and Pharmaceutical Biotechnology, A-3500 Krems, Austria 3 Medical University of Vienna, Institute for Cancer Research, A-1090 Vienna, Austria 4 Boehringer Ingelheim, RCV GmbH and Co KG, A-1121 Vienna, Austria 5 University Clinic Tulln, Department Surgery, A-3430 Tulln, Austria 6 Research Program for Rational Drug Design in Dermatology and Rheumatology, Department of Dermatology, Paracelsus Medical University of Salzburg, A-5020 Salzburg, Austria 7 ProComCure Biotech, A-5081 Anif, Austria Correspondence to: Andreas Eger, email: andreas.eger@fh-krems.ac.at Keywords: drug discovery; 3D models; ERBB signaling; targeted drugs; oncogene addiction Received: August 25, 2017 Accepted: October 27, 2017 Published: November 17, 2017 ABSTRACT Complex three-dimensional (3D) in vitro models that recapitulate human tumor biology are essential to understand the pathophysiology of the disease and to aid in the discovery of novel anti-cancer therapies. 3D organotypic cultures exhibit intercellular communication, nutrient and oxygen gradients, and cell polarity that is lacking in two-dimensional (2D) monolayer cultures. In the present study, we demonstrate that 2D and 3D cancer models exhibit different drug sensitivities towards both targeted inhibitors of EGFR signaling and broad acting cytotoxic agents. Changes in the kinase activities of ErbB family members and differential expression of apoptosis- and survival-associated genes before and after drug treatment may account for the differential drug sensitivities. Importantly, EGFR oncoprotein addiction was evident only in the 3D cultures mirroring the effect of EGFR inhibition in the clinic. Furthermore, targeted drug efficacy was strongly increased when incorporating cancer-associated fibroblasts into the 3D cultures. Taken together, we provide conclusive evidence that complex 3D cultures are more predictive of the clinical outcome than their 2D counterparts. In the future, 3D cultures will be instrumental for understanding the mode of action of drugs, identifying genotype-drug response relationships and developing patient-specific and personalized cancer treatments.

Background: Malignant melanoma is an aggressive type of skin cancer with high risk for metastasis and chemoresistance. Disruption of tightly regulated processes such as cell cycle, cell adhesion, cell differentiation and cell death are predominant in melanoma development. So far, conventional treatment options have been insufficient to treat metastatic melanoma and survival rates are poor. Anthraquinone compounds have been reported to have anti-tumorigenic potential by DNA-interaction, promotion of apoptosis and suppression of proliferation in various cancer cells. Methods: In the current study, the racemic tetrahydroanthraquinone derivative (±)-4-deoxyaustrocortilutein (4-DACL) was synthesized and the cytotoxic activity against melanoma cells and melanoma spheroids determined by CellTiter-Blue viability Assay and phase contrast microscopy. Generation of reactive oxygen species (ROS) was determined with CellROX Green and Deep Red Reagent kit and microplate-based fluorometry. Luciferase reporter gene assays for nuclear factor kappa B (NF-κB) and p53 activities and western blotting analysis were carried out to detect the expression of anti-proliferative or pro-apoptotic (p53, p21, p27, MDM2, and GADD45M) and anti-apoptotic (p65, IκB-α, IKK) proteins. Cell cycle distribution and apoptosis rate were detected by flow cytometry, the morphological changes visualized by fluorescence microscopy and the activation of different caspase cascades distinguished by Caspase Glo 3/7, 8 and 9 Assays. Results: We demonstrated that 4-DACL displayed high activity against different malignant melanoma cells and melanoma spheroids and only low toxicity to melanocytes and other primary cells. In particular, 4-DACL treatment induced mitochondrial ROS, reduced NF-κB signaling activity and increased up-regulation of the cell cycle inhibitors cyclin-dependent kinase inhibitor p21 (p21WAF1/Cip1) and the tumor suppressor protein p53 in a dose-dependent manner, which was accompanied by decreased cell proliferation and apoptosis via the intrinsic pathway. Conclusion: According to these results, we suggest that 4-DACL may be a promising therapeutic agent for the treatment of malignant melanoma.

In bladder cancer (BCa) patients undergoing radical cystoprostatectomy (RCPx), concomitant prostate cancer (PCa) is a common finding. Up to now there is no clear evidence to suggest that concomitant PCa is a predictor of outcome in these patients. Aim of this study was to assess incidence and clinicopathologic characteristics of concomitant PCa in RCPx specimen and correlate it to survival parameters from a single-centre material over two decades. All men who had undergone RCPx for BCa at our institution between 1994 and 2013 were included in this study. Clinicopathologic parameters for BCa and PCa were evaluated and correlated with outcome parameters. Survival analysis was performed for the subgroup of nonmetastatic organ-confined BCa to evaluate the role of concomitant Gleason Score (GS) ≥7 PCa. Of 945 men who had undergone RCPx for BCa, concomitant PCa was present in 237 patients (25.1 %). There was a significant increase in PCa incidence from 18.9 to 32.3 % between 1994 and 2013 (p = 0.009). Concomitant PCa represented a more aggressive phenotype at the end of the study (p = 0.037). In nonmetastatic organ-confined BCa, concomitant GS ≥7 PCa (HR 3.09; p = 0.0001) and age > 68 (HR 1.80; p = 0.0004) were independent negative predictors for overall survival. Concomitant PCa in RCPx specimen of BCa patients is a common finding. The incidence of concomitant PCa has significantly increased within 2 decades, presenting a more aggressive phenotype. Age and in particular concomitant GS ≥7 PCa are independent prognosticators for poor survival in patients with nonmetastatic organ-confined BCa.

Introduction: To analyze the primary stone free rate (pSFR) of flexible ureterorenoscopy (fURS) in the treatment of renal stones and to identify clinical predictors for the primary freedom from renal stones. Materials and Methods: Two hundred and seventy five patients, who underwent fURS for kidney stones were analyzed. Results: Index stone size was 6 mm. The stone was located in the lower calyx in 48%. Ureteral access sheath was used in 97%. Operation time was 35 min and primary stone clearance was 83%. pSFR increased from 74% in 2012 to 83% in 2013 and 90% in 2014 (p = 0.001). Preoperative stenting, index stone size, cumulative stone size, lithotripsy, ureteral access sheath and operation time were significantly correlated with the pSFR by univariate analysis. Multivariate regression analysis showed index stone size, cumulative stone size, ureteral access sheath and operation time as independent parameters for pSFR. Conclusions: fURS for kidney stones is safe with a high pSFR. Clinical parameters for pSFR are stone size, use of ureteral access sheath and operation time. In future, the effective use of fURS for the removal of kidney stones needs to be checked by prospective randomized trials.

The methanol extract of the Vietnamese freshwater cyanobacterium Nostoc sp. CAVN2 exhibited cytotoxic effects against MCF-7 and 5637 cancer cell lines as well as against nontumorigenic FL and HaCaT cells and was active against methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae. High-resolution mass spectrometric analysis indicated the presence of over 60 putative cyclophane-like compounds in an antimicrobially active methanol extract fraction. A paracyclophanes-focusing extraction and separation methodology led to the isolation of 5 new carbamidocyclophanes (1-5) and 11 known paracyclophanes (6-16). The structures and their stereochemical configurations were elucidated by a combination of spectrometric and spectroscopic methods including HRMS, 1D and 2D NMR analyses and detailed comparative CD analysis. The newly described monocarbamoylated [7.7]paracyclophanes (1, 2, 4 and 5) differ by a varying degree of chlorination in the side chains. Carbamidocyclophane J (3) is the very first reported carbamidocyclophane bearing a single halogenation in both butyl residues. Based on previous studies a detailed phylogenetic examination of cyclophane-producing cyanobacteria was carried out. The biological evaluation of 1-16 against various clinical pathogens highlighted a remarkable antimicrobial activity against MRSA with MICs of 0.1-1.0 μM, and indicated that the level of antibacterial activity is related to the presence of carbamoyl moieties.

Chronic inflammation is at least partially mediated by the chemokine-mediated attraction and by the adhesion molecule-directed binding of leukocytes to the activated endothelium. Therefore, it is therapeutically important to identify anti-inflammatory compounds able to control the interaction between leukocytes and the endothelial compartments of the micro- and macrocirculation. When testing novel drug candidates, it is, however, of the utmost importance to detect side effects, such as potential cytotoxic and barrier-disruptive activities. Indeed, minor changes in the endothelial monolayer integrity may increase the permeability of small blood vessels and capillaries, which, in extreme cases, can lead to edema development. Here, we describe the development of a high-throughput screening (HTS) platform, based on AlphaLISA technology, able to identify anti-inflammatory nontoxic natural or synthetic compounds capable of reducing tumor necrosis factor (TNF)-induced chemokine (interleukin [IL]-8) and adhesion molecule (ICAM-1) expression in human lung microvascular endothelial cells. Quantification of cell membrane-expressed ICAM-1 and of cell culture supernatant-associated levels of IL-8 was analyzed in HTS. In parallel, we monitored monolayer integrity and endothelial cell viability using the electrical cell substrate impedance sensing method. This platform allowed us to identify natural secondary metabolites from cyanobacteria, capable of reducing ICAM-1 and IL-8 levels in TNF-activated human microvascular endothelial cells in the absence of endothelial monolayer barrier disruption.

Background Chemokines play a critical role in tumor initiation, progression, and metastasis and have been associated with poor prognosis in diverse malignancies. The prognostic impact of chemokines for renal cell cancer (RCC) remains to be defined. Methods Patients diagnosed with RCC and operated between 07/07 and 05/11 were differentially assessed for expression profiles of a series of chemokines and their receptors by RT-qPCR and Western Blot analysis (tumor and adjacent normal tissue, n=37) and by Luminex for corresponding serum expression levels. Results were statistically correlated with clinicopathologic parameters. Results Gene expression of CCL2, CCR7, CXCL12, CXCR3, CXCR5 and CX3CL1 chemokines was significantly down-regulated in tumor compared to normal tissue. The gene profile for CCR6 was positively correlated with tumor size and stage. A positive linear correlation was found between CXCL12 and tumor stage as well as between CX3CR1 and C-reactive protein. In contrast to clear cell RCCs those of a chromophobe type showed a significantly down-regulated gene expression for CCR6, CCL20, and CXCL12. The CXCR7 serum level was significantly increased in patients with tumor-related mortality during postoperative follow-up. Conclusions Chemokines may serve as novel diagnostic and prognostic biomarkers for RCC. Studies on larger collectives are required for further assessment of potential clinical application.

The influence of the bisphosphonate zoledronic acid (ZA) on prostate cancer (PC) growth, adhesion and invasive behavior was investigated.PC-3, DU-145 and LNCaP cells were treated with ZA, and tumor-cell growth was then investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Furthermore, tumor-cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins, as well as migratory properties of the cells, was evaluated. Integrin β subtypes, integrin-dependent signaling, as well as cell-cycle regulating proteins, were analyzed by western blots.ZA dose-dependently reduced tumor-cell growth but did not impair tumor-endothelium and tumor-matrix interaction. However, ZA significantly inhibited tumor migration and invasive activity. Cyclin E was reduced by ZA in LNCaP and DU-145, and p21 was elevated in LNCaP cells. p27 was upregulated in all tumor cell lines, compared with the controls. ZA elevated β1-integrin in PC-3 and diminished β4-integrin in PC-3 and DU-145 cells.ZA inhibits PC growth and motility but does not influence the mechanical contact between tumor cells and the vascular wall.

The growth potential of PC3 prostate cancer cells, sensible (PC3(par)) or resistant (PC3(res)) to the mTOR inhibitor everolimus (RAD001) was investigated. Cell growth and proliferation of PC3(res) was similar to that of PC3(par), and late apoptosis increased in PC3(par) but decreased in PC3(res) following treatment with low dosed everolimus. PC3(res) accumulated in the G2/M-phase, accompanied by cdk1, cdk2 and cyclin B elevation. Knocking down cdk1 or cyclin B distinctly blocked the growth activity of PC3(res). One reason for everolimus resistance may be up-regulation of the cdk1-cyclin B complex in prostate cancer cells, leading to enhanced progression towards G2/M.

Study Type – Diagnostic (exploratory cohort) Level of Evidence 2b What’s known on the subject? and What does the study add? Clinically relevant GSU in the prostatectomy specimen is a common phenomenon. Clinically relevant GSU occurs in one of three patients with clinically ‘very’ low-risk PCa, and a low number of biopsy cores is the key negative predictor. OBJECTIVE • To evaluate clinical predictors for Gleason score upgrade (GSU) in radical prostatectomy (RP) specimen, especially in patients with ‘very’ low risk PCA (T1c and biopsy Gleason score ≤6 and PSA

The concept of molecular tumor targeting might provide new hope in the treatment of advanced prostate cancer. We evaluated metastasis blocking properties of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell lines. RAD001 or VPA were applied to PC-3 or LNCaP cells, either separately or in combination. Adhesion to vascular endothelium or to immobilized collagen, fibronectin or laminin was quantified. Migration and invasion were explored by a modified Boyden chamber assay. Integrin α and β subtypes were analyzed by flow cytometry, western blotting and RT-PCR. Effects of drug treatment on integrin related signaling, Akt and p70S6kinase activation, histone H3 and H4 acetylation were also determined. Separate application of RAD001 or VPA distinctly reduced tumor cell adhesion, migration and invasion, accompanied by elevated Akt activation and p70S6kinase de-activation. Integrin subtype expression was altered significantly by both compounds (VPA > RAD001). When both drugs were used in concert additive effects were observed on the migratory and invasive behavior but not on tumor-endothelium and tumor-matrix interaction. Separate mTOR or HDAC inhibition slows processes related to tumor metastasis. The RAD001-VPA combination showed advantage over VPA monotreatment with particular respect to migration and invasion. Ongoing studies are required to assess the relevance of VPA monotherapy versus VPA-RAD001 combination on tumor cell motility.

Aims To analyze the combined impact of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell growth. Main methods PC-3, DU-145 and LNCaP cells were treated with RAD001, VPA or with an RAD001–VPA combination for 3 or 5 days. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT assay, flow cytometry and Western blotting, respectively. Effects of drug treatment on cell signaling pathways were determined. Key findings Separate application of RAD001 or VPA distinctly reduced tumor cell growth and impaired cell cycle progression. Significant additive effects were evoked when both drugs were used in concert. Particularly, the cell cycle regulating proteins cdk1, cdk2, cdk4 and cyclin B were reduced, whereas p21 and p27 were enhanced by the RAD001–VPA combination. Signaling analysis revealed deactivation of EGFr, ERK1/2 and p70S6k. Phosphorylation of Akt was diminished in DU-145 but elevated in PC-3 and LNCaP cells. Significance The RAD001–VPA combination exerted profound antitumor properties on a panel of prostate cancer cell lines. Therefore, simultaneous blockage of HDAC and mTOR related pathways should be considered when designing novel therapeutic strategies for treating prostate carcinoma.

OBJECTIVE •To analyze the impact of a delayed radical cystectomy (rCx) and clinical variables on cancer-specific survival (CSS) in patients presenting 'high-risk' carcinoma not invading bladder muscle (nmiBCA). PATIENTS AND METHODS •Between 1989 and 2006, 278 patients who presented 'high-risk' nmiBCA finally underwent rCx in our institution. •CSS was correlated with clinical variables such as the number of transurethral resections of the bladder (TURBs), interval between first TURB and rCx, adjuvant therapies, tumour upstaging at rCx, tumour stage and lymph node (LN) status. RESULTS •The overall 5- and 10-year CSS was 82% and 76%, respectively. Significant correlations were found between the 5-year CSS and categorized number of TURBs (≤2 vs >2: 88% vs 71%; P= 0.001), interval between first TURB and rCx (≤4 months vs >4 months: 86% vs 77%; P= 0.04), adjuvant therapies (no vs yes: 86% vs 66%; P= 0.001), tumour upstaging at rCx (no vs yes: 89% vs 67%; P < 0.001), tumour stage at rCx (bladder confined vs non-confined: 88% vs 56%; P < 0.001) and LN status (no vs yes: 88% vs 36%; P < 0.001). •Multivariate analysis identified categorized number of TURBs (hazard ratio, HR, 0.14; 95% CI, 0.07-0.44; P < 0.001), categorized interval between first TURB and rCx (HR, 3.27; 95% CI, 1.24-8.59; P= 0.017), LN status (HR, 0.13; 95% CI, 0.06-0.26; P < 0.001) and tumour stage at rCx (HR, 0.49; 95% CI, 0.26-0.92; P= 0.03) as independent risk factors for CSS. CONCLUSION •Delay of rCx in 'high-risk' nmiBCA deteriorates CSS and should be avoided. The number of TURBs and the interval between first TURB and rCx are causative factors for delayed rCx and are independently correlated with CSS.

Background: Metastasis is associated with poor prognosis for melanoma. The formation of metastases is a multi-step process, in which cancer cells can subsequently acquire the potential to intravasate into the blood or lymph vessels, disseminate through the circulation, extravasate through the endothelium and invade the connective tissue. There is increasing evidence that chemokines have a pivotal role in the dissemination and establishment of melanoma metastasis. Methods: We isolated melanoma cells from melanoma metastasis and performed different migration assays and transendothelial resistance measurements of endothelial monolayers co-cultured with melanoma cells, in order to monitor barrier function and diapedesis and confirmed these results by confocal microscopy. Results: We observed that tumour endothelial cells (ECs) secrete high levels of CXCL9 in all, and CXCL10 in most melanoma metastases. Migration studies revealed that low concentrations of these chemokines induce chemotaxis, whereas high concentrations induce spontaneous migration of melanoma cells (chemokinesis/chemorepulsion) and the disruption of the endothelial barrier, resulting in an accelerated transendothelial migration (TEM). Addition of anti-CXCL9 or anti-CXCR3 antibodies to the co-cultures delayed the TEM of melanoma cells. Conclusion: Our data represent novel mechanisms by which tumour cells in melanoma metastases might use the chemokine-expressing endothelium to leave the tumour and eventually to form additional metastases at distinct sites.

Study Type – Diagnostic (exploratory cohort) Level of Evidence 2b What’s known on the subject? and What does the study add? Bladder cancer is initially diagnosed by white light cystoscopy followed by histopathological evaluation after transurethral resection of tissue suspicious for cancer. Difficulties may occur especially in the assessment of flat lesions or in discrimination between CIS and inflammatory disease. Confocal endomicroscopy during diagnostic colonoscopy has been a valuable tool for in vivo microscopic visualization and detection of colonic neoplasias and has contributed to optimized detection rates of up to 99%. We evaluated resected bladder urothelium of 18 patients by confocal endomicroscopy and correlated microscopic findings with standard histopathology. Typical tumour growth patterns such as altered nuclear to cytoplasmic ratio, pseudopapillar tissue stratification and neoangiogenesis were readily visible. As nuclear and subnuclear architecture of healthy bladder tissue could be discriminated against neoplastic tissue, confocal endomicroscopy is a promising novel tool for the in vivo microscopic visualization of bladder cancer. OBJECTIVE • To evaluate bladder urothelium by confocal laser endomicroscopy (CLE) and correlate microscopic findings with standard histopathology. PATIENTS AND METHODS • Fresh bladder urothelium tissue specimens were topically stained with acriflavine for instantaneous microscopic imaging. • A single-line laser in a handheld CLE probe delivered an excitation wavelength of 488 nm providing a high resolution of 0.7 µm and an adjustable imaging depth of 0–250 µm. • Resection specimens of 18 patients were investigated with 1000-fold magnification and ex vivo findings were compared with targeted histopathology (haematoxylin and eosin staining). RESULTS • Typical tumour growth patterns such as altered nuclear to cytoplasmic ratio, pseudopapillar tissue stratification and neoangiogenesis were readily visible. • Nuclear and subnuclear architecture of healthy bladder tissue could be discriminated against neoplastic tissue. CONCLUSIONS • In addition to white light cystoscopy, CLE is a promising novel tool for the in vivo microscopic visualization of bladder cancer; first results of the present study show its potential to define microscopic characteristics of bladder cancer tissue. • Further in vivo studies are necessary to determine sensitivity and specificity of the technique.

OBJECTIVE To investigate the associations between different overall or topographically restricted lymph node (LN) variables and cancer-specific survival (CSS) after radical cystectomy (RC) and extended LN dissection (LND) with curative intent in patients with LN-positive bladder cancer. PATIENTS AND METHODS Between 2001 and 2006, 152 patients had RC with standardized extended LND for bladder cancer with curative intent. Patients with positive LNs were stratified according to the median of the LN variables (LNs removed, number of positive LNs, LN density). CSS was related to overall and topographically restricted LN variables, e.g. different levels of LND, and relationships were tested by univariate and multivariate analyses. Level 1 LND comprised the regions of the external and internal iliac LNs and of the obturator LNs, level 2 the templates of common iliac and presacral LNs, and level 3 the para-aortic and paracaval LNs up to the inferior mesenteric artery. The mean (range) follow-up was 22 (1–84) months. RESULTS LN metastases were diagnosed in 46 of the 152 patients (30%) with extended LND. In these 46 patients, the median number of removed LNs was 33 (level 1, 15.5; level 2, 9.0; level 3, 7.0), the median number of positive LNs was 3 (1.5, 0.5 and 0.0, respectively) and the median LN density was 0.11 (0.10, 0.02 and 0.0, respectively). The CSS was 76% at 1 year and 23% at 3 years. There were significant correlations between the 3-year CSS and the overall LN density (≤0.11 vs >0.11; 34% vs 8%, P = 0.008), and the total number of positive LNs (≤3 vs >3; 33% vs 8%; P = 0.05). Overall LN density (hazard ratio 0.33, 95% confidence interval 0.15–0.72; P = 0.006) was an independent predictor for CSS in multivariate analysis. CONCLUSIONS Overall LN density is an independent predictor of survival after RC and extended LND with curative intent. Evaluation of topographically restricted LN positivity and density for different regions and levels of LND does not improve the prediction of CSS compared with overall LN positivity and density. A low incidence of level 3 LN positivity questions the clinical relevance of removing para-aortic and paracaval LNs. However, our data need to be confirmed by a prospective randomized trial.

Fur Docetaxel-refraktare Patienten mit metastasiertem hormonrefraktaren Prostatakarzinom (mHRPC) existiert bisher kein etabliertes Therapieregime. Deshalb berichten wir hier uber unsere Erfahrungen mit Mitoxantron plus Prednison (MP) als Zweitlinenchemotherapie. Zehn Docetaxel-refraktare mHRPC-Patienten wurden mit MP behandelt. Untersucht wurden PSA-Abfall, progressionsfreies Intervall und Schmerzstatus unter Chemotherapie. Unter MP zeigten 2 Patienten eine partielle PSA-Remission, 3 ein „stable disease“ und 5 waren progredient. Das progressionsfreie Intervall bei partieller PSA-Remission lag im Mittel bei 8 Monaten und bei „stable disease“ im Median bei 2 Monaten. Eine Schmerzreduktion wurde in 4 von 7 Patienten unter MP beobachtet. Eine Grad-4-Neutropenie trat in nur bei einem Patienten auf. Patienten mit PSA-Abfall unter Erst- und Zweitlinientherapie hatten ein progressionsfreies Intervall im Median von 11,5 Monaten. Zum gegenwartigen Zeitpunkt wird die Indikation fur MP als Zweitlinienchemotherapie fur Docetaxel-refraktare mHRPC-Patienten gesehen, die nicht in eine Studie eingeschlossen werden konnen. Trotz der masigen Rate von partiellen PSA-Remissionen wird bei jedem zweiten Patienten eine Schmerzreduktion erzielt. Ein progressionsfreies Intervall von mehreren Monaten kann erreicht werden. Die therapiebedingten Nebenwirkungen sind vergleichsweise gering.

The tyrosine kinase receptor c-kit and its ligand stem cell factor (SCF) have not been explored in prostate cancer (PC) bone metastasis. Herein, we found that three human PC cell lines and bone marrow stromal cells express a membrane-bound SCF isoform and release a soluble SCF. Bone marrow stromal cells revealed strong expression of c-kit, whereas PC cells showed very low levels of the receptor or did not express it all. Using an experimental model of PC bone metastasis, we found that intraosseous bone tumors formed by otherwise c-kit–negative PC3 cells strongly expressed c-kit, as demonstrated using immunohistochemical and Western blot analyses. Subcuta-neous PC3 tumors were, however, c-kit–negative. Both bone and subcutaneous PC3 tumors were positive for SCF. Immunohistochemical analysis of human specimens revealed that the expression frequency of c-kit in epithelial cells was of 5% in benign prostatic hyperplasia, 14% in primary PC, and 40% in PC bone metastases, suggesting an overall trend of increased c-kit expression in clinical PC progression. Stem cell factor expression frequency was more than 80% in all the cases. Our data suggest that the bone microenvironment up-regulates c-kit expression on PC cells, favoring their intraosseous expansion.

Objective To study clinical and histopathologic parameters after cystectomy and lymphadenectomy in non-muscle-invasive transitional cell carcinoma (TCC) of the bladder and their association with the prevalence of lymph node metastases (N+). Patients and methods Of 866 patients treated with radical cystectomy and lymphadenectomy, 219 had non-muscle-invasive TCC of the bladder. The prevalence of N+ was related to parameters such as gender, age, number of transurethral resections of the bladder (TURBs), intervals between first TURB and cystectomy, adjuvant therapy, maximum histopathologic tumor stage and grade at TURB, and tumor upstaging in the cystectomy specimen by univariate and multivariate analysis. Results A total of 33 patients (15%) had N+. By multivariate analyses, tumor upstaging and the number of TURBs were independent predictors of N+ at cystectomy. The number of TURBs increased the prevalence of N+ from 8% (one TURB) to 24% (two to four TURBs). Tumor upstaging in the cystectomy specimen increased the prevalence of N+ from 4% to 36%. Conclusion Inappropriate delay and staging errors of"high risk" non-muscle-invasive TCC of the bladder contribute to an increased prevalence of N+ and should be avoided. In our series, the number of TURBs and tumor upstaging in the cystectomy specimen were independent predictors for N+ by multivariate analysis.

OBJECTIVE To assess primary tumours of the urethra in males. PATIENTS AND METHODS We retrospectively reviewed our database from 1986 to 2006 for primary tumours of the male urethra; nine patients with primary tumours of the urethra were analysed and follow-up information was obtained. RESULTS Three patients had tumours of the prostatic urethra, two of which had proliferating focal inflammation and one a low-grade, superficial urothelial cancer. All patients were treated successfully with transurethral resection. Six patients had carcinoma of the bulbar or penile urethra, including two with previous local percutaneous radiotherapy for prostate cancer. All had primary surgical excision that was adapted to tumour location and extension. One patient had adjuvant chemotherapy after surgery. All but one patient remain recurrence-free after a median follow-up of 20 months. CONCLUSION Primary carcinoma of the male urethra is a rare entity. Previous radiotherapy might be a predisposing factor. Local surgical tumour control is essential for long-term survival, but the extent of surgery depends on tumour location and stage. Multimodal therapy might be required to obtain an optimum oncological outcome.

Acute lung injury is accompanied by an increased endothelial chemokine production and adhesion molecule expression, which may result in an extensive neutrophil infiltration. Moreover, a destruction of the alveolar epithelium and capillary endothelium may result in permeability edema. As such, the search for novel anti-inflammatory substances, able to downregulate these parameters as well as the tissue damage holds therapeutic promise. We therefore describe here the use of human endothelial cell-based in vitro assays for the detection of anti-inflammatory and wound-healing metabolites from cyanobacteria.

Authors from Mainz, Germany present the long-term follow-up of submucosal tunnel and serosa-lined extramural tunnel ureter implantation in the ileocaecal continent cutaneous urinary diversion first described in that department, called the Mainz-Pouch I. OBJECTIVE To assess upper urinary tract complications and renal function in patients with a submucosal tunnel and serosa-lined extramural tunnel ureter implantation during the long-term follow-up of ileocaecal continent cutaneous urinary diversion (Mainz pouch I). PATIENTS AND METHODS In all, 458 patients who had diversion with the ileocaecal pouch were analysed in a retrospective follow-up study. Uretero-intestinal implantation was done using a submucosal tunnel (ST) in 809 reno-ureteric units (RUs) and by the serosa-lined extramural tunnel (ET) technique in 74 RUs. The median age of the patients at the time of surgery was 47.1 years, and the median follow-up was 89.0 months. RESULTS For the ST, there was anastomotic obstruction in 59 RUs (7.3%) at a median of 16.8 months after diversion; the obstruction-free intervals at 1, 5 and 10 years were 97%, 93% and 91%, respectively. Obstruction rates were 13.9% for previously dilated upper tracts and 17.1% in patients with a neurogenic bladder. Serum creatinine levels were ≤1.6 mg/dL in 97% of the patients at the latest follow-up. For ET, there was anastomotic obstruction in three RUs (4.1%) at a median of 17.2 months after diversion. Obstruction-free intervals at 1, 5 and 10 years were 100%, 96% and 96%. Preoperative dilation of the upper tracts did not reduce the obstruction rate (3.1%), but it was 7.1% in patients with a neurogenic bladder. Serum creatinine levels were ≤1.6 mg/dL in 98% of the patients at the latest follow-up. CONCLUSIONS The ET gives lower obstruction rates than the ST, especially when upper tracts are dilated and in patients with a neurogenic bladder. Renal function remained stable with both techniques in the long term.

Die zunehmende Uberalterung unserer Gesellschaft erhoht kontinuierlich den Anteil alterer und polymorbider Patienten innerhalb der Inkontinenzklientel. Finanzielle und personelle Ressourcenknappheit zwingt zu rationellem und teleologisch orientiertem Umgang mit dem vorhandenen diagnostischen Instrumentarium. Die bei einem alten Menschen mit Harninkontinenz durchzufuhrenden Untersuchungen sollten einem nach Invasivitat gestaffelten Algorithmus folgen und sich auf den fur eine primar konservative Therapieplanung notwendigen Mindestumfang beschranken. Sinnvolle Diagnostik objektiviert beklagte Beschwerden, dient der Subgruppendifferenzierung, der Ursachenforschung und der Schweregradeinteilung, ermoglicht die Beurteilung von Komorbiditaten, hilft bei der Therapieplanung und dokumentiert Therapieverlauf und mogliche Behandlungskomplikationen. Dabei ist sie bei hinreichend detailliert dokumentierten Untersuchungsergebnissen so wenig invasiv und einfach wie moglich. Die Harninkontinenzbasisdiagnostik umfasst Anamnese, korperliche Untersuchung, Urinanalyse, freie Harnstrahlmessung mit sonographischer Restharnbestimmung, Miktionstagebuch und die Bestimmung des mentalen Status. Auf diese Weise ist die Harninkontinenz fast aller alteren Patienten ausreichend genau beurteilbar, um einen konservativen Behandlungsversuch zu beginnen. Nur eine kleine Minderheit mit inkongruenten Untersuchungsergebnissen oder therapierefraktare Rezidivfalle sollten einer invasiveren Zusatzdiagnostik (Zysturethroskopie, Urodynamik, morphologische und funktionelle Rontgendiagnostik, Perineal-/Introitussonographie) unterzogen werden, wenn sich aus ihr therapeutische Konsequenzen ableiten liesen. Ohne diese Therapierelevanz ist eine kostenintensive Spezialdiagnostik bei alteren Menschen auch wegen der nicht unerheblichen Untersuchungsmorbiditat abzulehnen.

Nowadays, male stress urinary incontinence is rare and almost always of iatrogenic origin (radiotherapy, pelvic surgery). However, the prognosis of urinary incontinence following surgery is good and can be improved by pelvic floor muscle exercises in combination with biofeedback systems. For the remaining patient cohort with persistent urinary incontinence, several established surgical treatment options are available. Suburothelial injections of bulking agents can easily be performed in an ambulatory setting. However, regardless of the material used, long-term results are disappointing. Moreover, the residual urethral function deteriorates due to cicatrization of the suburothelial plexus with consequent loss of urethral elasticity. The fascial sling procedure in males has to be performed in preoperated areas and is as technically demanding for the surgeon as it is burdening for the patient. Alloplastic material is not used, thus minimizing risks for arrosion or infection. Since the sling tension can neither be standardized nor postoperatively readjusted, the risk of overcorrection is considerable and the success of the procedure is heavily dependent on the surgeon's experience. Despite wear and high revision rates, the technically mature artificial sphincter produces excellent continence results and has become the gold standard in the therapy of male stress urinary incontinence. The circumferential and continuous urethral compression by the cuff is highly effective, but at the price of an almost inevitable urethral atrophy. To overcome this problem, various surgical techniques have been developed (tandem cuff, cuff downsizing, transcorporal cuff placement). However, the expensive artificial sphincter is not a nostrum for every incontinent man, since it requires certain minimal cognitive and manual capabilities. Therefore, the search for less demanding treatment alternatives seems to be necessary, even if one has to accept lower continence rates.

Matrix metalloproteinases (MMPs) have been associated with initiation, progression and vascularization of a number of tumors. However, clinical trials using MMP inhibitors failed to meet expectations. Previously, we demonstrated the potential importance of MMP-9 activity in experimental prostate cancer bone tumor tissue. However, the particular roles of host- and tumor-derived MMP-9 remains to be defined. Herein, we examined the role of host MMP-9 in subcutaneous and intraosseous growth of the human androgen independent prostate cancer cell line PC3 in MMP-9 deficient mice. In the subcutaneous model, the tumor incidence in the control (RAG-1 ko/ko ) and experimental (RAG-1 ko/ko /MMP-9 ko/ko ) group was 100%, with similar tumor growth kinetics and microvascular densities. In the intraosseous tumor model, the tumor incidence was higher in RAG-1 ko/ko /MMP-9 ko/ko mice than in RAG-1 ko/ko mice (67% and 39%, respectively), though no statistical differences were found. The intraosseous tumor areas were similar in both groups, and the number of tumor-associated osteoclasts did not differ significantly. However, the microvascular density of intraosseous tumors was higher in RAG-1 ko/ko than in RAG-1 ko/ko /MMP-9 ko/ko mice, though no changes in tumor growth could be detected. In an in vitro assay, we found that bone marrow (BM) cells increased the invasiveness of PC3 cells, and that this enhancement was independent of MMP-9 expression by marrow cells. Our results with the RAG-1 model suggest that host-derived MMP-9 is neither necessary nor sufficient for subcutaneous or intraosseous PC3 tumor growth, osteoclastic response, or in vitro invasiveness of tumor cells.

We report the clinical outcome of more than 800 patients, who underwent continent cutaneous urinary diversion with an ileocecal reservoir (Mainz-pouch I) in two urological tertiary referral centers at a mean follow-up of 7.6 years. Complications related to the continence mechanism (intussuscepted ileal nipple vs. submucosally embedded in situ appendix) and the antirefluxive uretero-intestinal anastomosis (submucosal tunnel vs. serosa-lined extramural tunnel) were recorded retrospectively. Stomal stenosis was observed in 23.5% of the patients with appendix stoma and in 15.3% of the patients with intussuscepted ileal nipple. The incidence of calculi was 10.8% in reservoirs with intussuscepted ileal nipple and 5.6% in reservoirs with appendix stoma. Eleven patients (eight with appendix, three with ileal nipple) required reoperation because of ischemic degeneration of the continence mechanism. The overall continence rate (day and night) was 92.8%. Anastomotic strictures of the afferent limb occurred in 6.5% of renal units (RUs) with a submucosal tunnel and in 5.0% of RUs with a serosa-lined extramural tunnel. Continent cutaneous urinary diversion with an ileocecal pouch is a highly satisfactory and safe option for patients, in whom orthotopic urinary diversion is impossible or contraindicated.

The Mainz Pouch II has now been used widely and found to be a valuable addition to the various types of urinary diversion. The authors from Mainz describe how a colonic conduit can be converted into a Mainz Pouch II, and the expected outcome from such a procedure. OBJECTIVE To report our long-term results of conversion from conduit conversion into a continent anal urinary diversion, as after conduit urinary diversion in childhood, some patients wish to have a later conversion to a continent diversion to avoid external appliances and to improve their quality of life. PATIENTS AND METHODS Between 1992 and 2003, 139 patients had a urinary diversion with a recto-sigmoid pouch (Mainz pouch II), of whom four had a conversion from a colonic conduit diversion to a recto-sigmoid pouch. The mean (range) age at conduit diversion was 5.5 (3–14) years and the mean interval between conduit diversion and conversion to a continent anal diversion was 8 (4–18) years. The mean age at conversion into a Mainz pouch II was 13 (8–32) years and the follow-up afterward was 11.5 (1–13) years. The conversion was done by incorporating the pre-existing colonic conduit into the recto-sigmoid pouch with no ureteric reimplantation. RESULTS There were no early complications; one nephrectomy was required 5 years after conversion because of uretero-intestinal obstruction and pyelonephritis. All other reno-ureteric units remained stable and renal function was maintained. All patients are continent day and night; three require substitution with alkali at a base excess of

After failure of conservative treatment of neurogenic bladders (deterioration of the upper urinary tract/incontinence) continent cutaneous diversion has to be considered in those patients with irreparable urethral sphincter defects or those who are unable to perform trans-urethral self-catheterization. In this second part of the study we investigated the long-term safety of using the Mainz pouch I with regard to protecting the upper urinary tracts and to provide urine continence. Between 1985 and 2002, operations to form an ileocaecal pouch with umbilical stoma (Mainz pouch I) were performed on 70 children and adolescents of median age 15.3 years (range 5.7-20 years). During the follow-up period five patients died 2.4-14 years postoperatively of causes not related to urinary diversion. A follow-up period of 8.7 years (0.9-18) was achieved in 65 patients with 118 renal units (RUs). As compared to preoperatively, the upper urinary tracts had remained stable or improved in 113/118 RUs (95.8%) at the latest follow-up. Complete continence was achieved in 97% of patients with a continent cutaneous diversion. Surgical revisions were required for: incontinence of the outlet mechanism in 9%, stoma prolapse in 2%, stoma stenosis in 23%, pouch calculi in 15%, symptomatic reflux in 1%, ureter stenosis in 16% of the RUs with submucosal tunnel and in 3% of the RUs with an extramural tunnel. We conclude that, in patients with irreparable sphincter defect and those who are unable to perform urethral self-catheterization, continent cutaneous urinary diversion with the Mainz pouch I provides a high continence rate with preservation of the upper urinary tracts in the long run. In patients with dilated ureters, the extramural tunnel technique results in a lower complication rate.

Although groups at several institutions have long experience with radical perineal prostatectomy (RPP), only few reports of larger series describe associated complications, mostly without reporting management options in detail. We analyzed specific perioperative and postoperative complications of the perineal approach and management strategies thereof.The medical records of 630 patients who underwent RPP between January 1997 and May 2003 were retrospectively reviewed in regard to complications and their management. Median followup was 8 months (range 1 to 68).Major complications requiring open surgical intervention were noted in 11 patients (1.7%) for a total surgical revision rate of 2.4% (15 of 630). Two patients with persistent urinary fistula required fistula excision and closure. Two patients with a rectocutaneous fistula needed temporary diverting colostomy. Three patients with a combined urinary and fecal fistula were treated with protective colostomy, fistula excision and the interposition of a tunica vaginalis graft. No further morbidity was observed in these patients. In 7 patients a subvesical hematoma was drained surgically, including 3 mentioned in whom a hematoma expanded into the urethral anastomosis. Minor complications, which could be successfully managed conservatively or with endoscopic interventions only, developed in 124 patients for a total rate of 19.7%. In the long term 9% of the patients experienced postoperative de novo changes in stool habits after RPP but only 2.7% reported distressing anal sphincter incompetence.RPP is a safe and reproducible procedure with low major complication and reintervention rates even in a training center setting with many involved surgeons. A subvesical hematoma should be revised early since it can be the origin of subsequent major complications.

The purpose of this study was to test the hypothesis that renal cell carcinoma (RCC) exhibits an increased intratumoral interstitial fluid pressure (IT-IFP). Therefore, resected tumors from human primary (n = 23) or metastatic RCC (n = 3) were xenografted in SCID mice. The IFP of single tumor nodules (n = 65) and normal mouse tissue (n = 195) was measured by means of the "wick-in-needle" technique. Data demonstrate that the mean IT-IFP at neoplasia was 35 times greater than in normal tissue, and decreased precipitously at the tumor boundary. IT-IFP values tended to increase with the grade of malignancy of the tumor cells and tumor size. The mean IT-IFP of xenografts derived from primary RCC was twice as high as that from metastatic RCC tissue. These findings indicate a biophysical barrier to drug delivery in RCC; this may, in concert with cellular-based drug resistance mechanisms, be an additional explanation for resistance of the tumor to certain blood-borne anticancer therapies.

The influence of the histone deacetylase (HDAC)-inhibitor, valproic acid (VPA), on bladder cancer cell adhesion in vitro was investigated in this paper. TCCSUP and RT-112 bladder cancer cells were treated with VPA (0.5 or 1 mM) twice or thrice weekly for 14 days. Controls remained untreated. Tumour cell interaction with immobilized collagen was evaluated by a flow-based adhesion assay using a shear force of 2 or 4 dyne/cm2. The effects of VPA on the integrin adhesion receptors α3, α5, β1, β3 and β4 were assessed by flow cytometry to determine integrin surface expression and by western blotting to determine the cytoplasmic integrin level. VPA of 0.5 mM and 1 mM significantly prevented binding of both RT-112 and TCCSUP cells to collagen, compared with the untreated controls. Adhesion was reduced to a higher extent when RT-112 (subjected to 2 dyne/cm2) or TCCSUP (subjected to 2 or 4 dyne/cm2) tumour cells were treated with VPA three times a week, compared to the two times a week protocol. VPA caused a significant up-regulation of the integrin α3, α5, β1, β3 and β4 subtypes on the TCCSUP cell surface membrane. In RT-112 cells, only integrin α5 was elevated on the cell surface following VPA exposure. Western blotting revealed an up-regulation of α3, α5, β3 and β4 integrins and down-regulation of the integrin β1 protein by VPA in TCCSUP. VPA also up-regulated α5 and down-regulated β1 integrin in RT-112 cells, but also reduced α3 and β3 in TCCSUP. VPA exerted adhesion-blocking properties on bladder cancer cells under physiologic flow conditions. The effects were accompanied by distinct modifications of the integrin expression profile, which differ depending on the cell lines used. Application of VPA might be an innovative option to prevent bladder cancer dissemination.

Background: Inhibitors of the mammalian target of rapamycin (mTOR) might become a novel tool to treat advanced prostate cancer. However, chronic drug exposure may trigger resistance, limiting the utility of mTOR inhibitors. Methods: Metastatic potential of PC3 prostate cancer cells, susceptible (PC3par) or resistant (PC3res) to the mTOR-inhibitor RAD001 was investigated. Adhesion to vascular endothelium or immobilised collagen, fibronectin and laminin was quantified. Motility, migration and invasion were explored by modified Boyden chamber assay. Integrin α and β subtypes were analysed by flow cytometry, western blotting and real-time PCR. Integrin-related signalling, EGFr, Akt, p70S6kinase and ERK1/2 activation were determined. Results: Adhesion was reduced, whereas motility, migration and invasion were enhanced in PC3res. The α2 and β1 integrin subtypes were dramatically elevated, integrins α1 and α6 were lowered, whereas α5 was nearly lost in PC3res. Activation of the Akt signalling pathway was strongly upregulated in these cells. Treating PC3par cells with RAD001 reduced motility, migration and invasion and deactivated Akt signalling. Blocking studies revealed that α2 and β1 integrins significantly trigger the motile behaviour of the tumour cells. Conclusion: Chronic RAD001 treatment caused resistance development characterised by distinct modification of the integrin-expression profile, driving prostate cancer cells towards high motility.

Metastasis is associated with poor prognosis for melanoma responsible for about 90% of skin cancer-related mortality. To metastasize, melanoma cells must escape keratinocyte control, invade across the basement membrane and survive in the dermis by resisting apoptosis before they can intravasate into the circulation. α-Catulin (CTNNAL1) is a cytoplasmic molecule that integrates the crosstalk between nuclear factor-kappa B and Rho signaling pathways, binds to β-catenin and increases the level of both α-catenin and β-catenin and therefore has potential effects on inflammation, apoptosis and cytoskeletal reorganization. Here, we show that α-catulin is highly expressed in melanoma cells. Expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of expression of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. Knockdown of α-catulin promoted adhesion to and inhibited migration away from keratinocytes in an E-cadherin-dependent manner and decreased the transmigration through a keratinocyte monolayer, as well as in Transwell assays using collagens, laminin and fibronectin coating. Moreover, knockdown promoted homotypic spheroid formation and concomitantly increased E-cadherin expression along with downregulation of transcription factors implicated in its repression (Snail/Slug, Twist and ZEB). Consistent with the molecular changes, α-catulin provoked invasion of melanoma cells in a three-dimensional culture assay by the upregulation of matrix metalloproteinases 2 and 9 and the activation of ROCK/Rho. As such, α-catulin may represent a key driver of the metastatic process, implicating potential for therapeutic interference.

Although increasing evidence suggests a critical role for platelet-derived growth factor (PDGF) receptor β (β-PDGFR) signaling in prostate cancer (PCa) progression, the precise roles of β-PDGFR and PDGF isoform-specific cell signaling have not been delineated. Recently, we identified the PDGF-D isoform as a ligand for β-PDGFR in PCa and showed that PDGF-D is activated by serine protease-mediated proteolytic removal of the CUB domain in a two-step process, yielding first a hemidimer (HD) and then a growth factor domain dimer. Herein, we demonstrate that the expression of PDGF-D in human PCa LNCaP cells leads to enhanced bone tumor growth and bone responses in immunodeficient mice. Histopathological analyses of bone tumors generated by PDGF-D-expressing LNCaP cells (LNCaP-PDGF-D) revealed osteolytic and osteoblastic responses similar to those observed in human PCa bone metastases. Importantly, we discovered a novel function of PDGF-D in the regulation of osteoclast differentiation, independent of the RANKL/RANK signaling axis. Although both PDGF-B and -D were able to activate β-PDGFR, only PDGF-D was able to induce osteoclastic differentiation in vitro, and upregulate the expression and nuclear translocation of nuclear factor of activated T cells 1, a master transcription factor for osteoclastogenesis. Taken together, these results reveal a new function of PDGF-D as a regulator of osteoclastic differentiation, an activity critical for the establishment of skeletal metastatic deposit in PCa patients.

We evaluated whether low-dosed interferon alpha (IFNa) may augment the anti-tumor potential of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on prostate cancer cells in vitro and in vivo. PC-3, DU-145, or LNCaP prostate cancer cells were treated with VPA (1 mM), IFNa (200 U/ml), or with the VPA-IFNa combination. Tumor cell growth, cell cycle progression, and cell cycle regulating proteins were then investigated by the MTT assay, flow cytometry, and western blotting. Tumor cell adhesion to endothelium or to immobilized extracellular matrix proteins, as well as migratory properties of the cells, were evaluated. Integrin α and β adhesion molecules and alterations of cell signaling pathways were analyzed. Finally, effects of the drug treatment on prostate cancer growth in vivo were determined in the NOD/SCID mouse model. VPA reduced tumor cell adhesion, migration, and growth in vitro. A much stronger anti-cancer potential was evoked by the VPA-IFNa combination, although IFNa in itself did not block growth or adhesion. The same effect was seen when tumor growth was evaluated in vivo. Molecular analysis revealed distinct elevation of histone H3 acetylation caused by VPA which was further up-regulated by VPA-IFNa, whereas IFNa alone did not alter H3 acetylation. The combinatorial benefit became obvious in Akt phosphorylation, p21 and p27 and integrin α1, α3, and β1 expression. Application of low-dosed IFNa to a VPA based regimen profoundly boosts the anti-tumor properties of VPA. The combined use of VPA and low-dosed IFNa may therefore be an innovative option in treating advanced prostate cancer. Prostate 72:1719–1735, 2012. © 2012 Wiley Periodicals, Inc.

Our aim was to analyze the impact of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on bladder cancer cell growth in vitro. RT-4, TCCSUP, UMUC-3, and RT-112 bladder cancer cells were treated with VPA (0.125-1 mmol/l) without and with preincubation periods of 3 and 5 days. Controls remained untreated. Tumor cell growth, cell cycle progression, and cell cycle-regulating proteins were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and western blotting, respectively. Effects of VPA on histone H3 and H4 acetylation and HDAC3 and HDAC4 were also determined. Without preincubation, no tumor cell growth reduction was observed with 0.125 and 0.25 mmol/l VPA in TCCSUP, UMUC-3, and RT-112 cells, whereas 0.5 and 1 mmol/l VPA diminished the cell number significantly. VPA (0.25 mmol/l) did exert tumor growth-blocking effects after a 3-day preincubation. To achieve antitumor effects with VPA (0.125 mmol/l), a 5-day preincubation was necessary. A 3-day or 5-day preincubation was also necessary to distinctly delay cell cycle progression, with maximum effects at VPA (1 mmol/l). After the 5-day preincubation, the cell cycle-regulating proteins cdk1, cdk2, cdk4, and cyclins B, D1, and E were reduced, whereas p27 was enhanced. Diminished HDAC3 and 4 expression induced by VPA was accompanied by elevated acetylation of H3 and H4. VPA exerted growth-blocking properties on a panel of bladder cancer cell lines, commensurate with dose and exposure time. Long-term application induced much stronger effects than did shorter application and should be considered when designing therapeutic strategies for treating bladder carcinoma.

OBJECTIVES To analyze the incidence and management of anastomotic strictures (ASs) after radical perineal prostatectomy (RPP) and retropubic prostatectomy (RRP) and to identify possible predisposing factors. METHODS Between 1997 and 2007, we performed 866 RPP and 2052 RRP for localized prostate cancer. Median follow-up was 52 months (12-136). We analyzed preoperative serum prostate-specific antigen, prostate size, clinical and pathologic tumor stage, neoadjuvant hormone deprivation, previous transurethral resection of the prostate, transfusion requirement, anastomotic insufficiency, and acute urinary retention (AUR) and its subsequent management to identify possible predisposing factors for AS formation. RESULTS The rate of AS after RPP and RRP was 3.8% (33/863) and 5.5% (113/2048), respectively (P = .067). In multivariate analysis, RRP was a statistically significant risk factor for AS (P = .0002). On survival analysis, the incidence of AS was lower for RPP as compared with RRP at median follow-up (P = .0229). Primary response to endoscopic AS incision or resection was 94% (31/33) and 72.6% (82/113) after RPP and RRP, respectively. On multivariate logistic regression analysis biopsy Gleason score, previous transurethral resection of the prostate, prostate volume, pathologic tumor stage and grade, transfusion requirement, AUR, and surgical technique were independent risk factors for the development of AS. An AS developed in 45.4% (20/44) and 10.9% (5/46) of the postoperative AUR cases treated with a suprapubic cystostomy tube and a transurethral Foley catheter, respectively (P

OBJECTIVE To evaluate the expression of urokinase-plasminogen-activator receptor (uPA-R) in disseminated tumour cells (DTC) in bone marrow (BM) and peripheral blood (PB) of patients with clinically localized prostate cancer before radical prostatectomy (RP), and to assess the associations with pathological variables and prognosis. PATIENTS AND METHODS In all, 52 patients (47 with clinically localized cancer and five with benign prostatic hyperplasia, BPH, as controls) were prospectively enrolled. BM and PB samples were drawn before surgery. DTC were enriched using a commercial system, cytokeratin (CK) 8/18 was used to detect DTC, and uPA-R expression was detected by dual-immunostaining of the DTC. The final pathology of the RP specimen was compared with the results of immunostaining. Follow-up was initiated to detect tumour relapse (defined as a prostate-specific antigen (PSA) level of ≥0.2 ng/mL). RESULTS Overall, there was expression of ‘CK + uPA-R’ in 60% of the BM and in 19% of the PB specimens. Expression of this marker in BM was most significantly increased in those with unfavourable Gleason scores (P = 0.004), followed by high-risk cancer (P = 0.005). The relative risk for CK + uPA-R expression in the BM was 3.1 times higher in high-risk than in low-risk prostate cancer. No relevant expression rates were detected for PB. In the control group, no patient showed CK or uPA-R expression in BM or PB. The PSA-recurrence free survival was significantly lower in patients with CK + uPA-R-positive BM cells (P = 0.01). CONCLUSION In this pilot study, the preoperative detection rate of CK + uPAR expression in BM of patients with prostate cancer increased with Gleason score and in those with high-risk disease. All patients with a later PSA relapse had had uPA-R expression in their DTC from the BM. DTC with uPA-R expression was an adverse prognostic factor for prostate cancer.

We determined indications for preoperative prostate biopsy in patients undergoing radical cystoprostatectomy for bladder cancer.Of 316 cystoprostatectomy specimens concomitant prostate cancer was diagnosed in 21.5%. Prostate cancer was diagnosed preoperatively in 24% of cases (evident prostate cancer), 32% were suspicious for prostate cancer but no biopsy was done (suspected prostate cancer) and in 44% prostate cancer was incidental. Patients were stratified into probability groups of intermediate/high risk prostate cancer by digital rectal examination and prostate specific antigen. The incidence of unfavorable histopathology was determined in each group.Of prostate cancers 85% were organ confined and the Gleason score was favorable (2-6) in 74%. Of cases of incidental prostate cancer tumors were organ confined in 97%. There were no unfavorable Gleason scores (8-10). In the low probability group 83% of patients had organ confined prostate cancer and only 17% had an unfavorable Gleason score. In the intermediate probability group prostate cancer was organ confined in 73% of patients, 45% had a favorable Gleason score (2-6) and 55% had an intermediate Gleason score (7). In the high probability group 29% of patients had high risk prostate cancer.Most concomitant prostate cancers were organ confined and had a favorable or intermediate Gleason score when digital rectal examination was not suspicious and prostate specific antigen was less than 10 ng/ml. As a consequence, patients with a low/intermediate probability of detecting intermediate/high risk prostate cancer do not require preoperative prostate biopsy unless nerve sparing surgery is planned. In contrast, all patients should undergo preoperative biopsy for prostate cancer when digital rectal examination is suspicious or prostate specific antigen is more than 10 ng/ml because the rate of high risk prostate cancer was 29% in this group.