BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, and CancerCheck® UBC® rapid VISUAL as urinary marker for bladder cancer: Final results of a German multicenter study.

INTRODUCTION AND OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

3D Tumor Models in Urology.

Three-dimensional tumor models have become established in both basic and clinical research. As multicellular systems consisting of tumor and tumor-associated cells, they can better represent tumor characteristics than monocellular 2D cultures. In this review, we highlight the potential applications of tumor spheroids and organoids in the field of urology. Further, we illustrate the generation and characteristics of standardized organoids as well as membrane-based 3D in vitro models in bladder cancer research. We discuss the technical aspects and review the initial successes of molecular analyses in the three major urologic tumor entities: urinary bladder carcinoma (BCa), prostate carcinoma (PCa), and renal cell carcinoma (RCC).

Free Fatty Acids Induce Lipid Accumulation, Autophagy, and Apoptosis in Human Sebocytes.

BACKGROUND: A disruption of sebocyte differentiation and lipogenesis has fatal consequences and can cause a wide spectrum of skin diseases, from acne vulgaris to sebaceous carcinoma, however, the relevant molecular mechanisms have not been fully clarified. OBJECTIVES: The induction of autophagy and apoptosis in human sebocytes in response to biologically relevant fatty acids was investigated. METHODS: Free fatty acids (arachidonic acid, linoleic acid, palmitic acid, and palmitoleic acid) and the pan-caspase inhibitor QVD-Oph were added to the supernatant of cultured human SZ95 sebocytes. Individual relevant proteins were analyzed by Western blotting. Apoptosis and cell viability were determined, and typical autophagy structures were detected through electron microscopy. To obtain cell growth curves, cell confluence was continuously monitored by real-time cell analysis. RESULTS: Fatty acids induced the development of intracellular lipid droplets with subsequent apoptosis, whereas arachidonic acid caused the most rapid effect. Cleavage products of caspase-3 were only detected in arachidonic acid-induced apoptosis. The high basal apoptotic rate of cultured SZ95 sebocytes was strongly suppressed by QVD-Oph. Fatty acid-induced apoptosis was also markedly inhibited by QVD-Oph, whereas intracellular lipid droplets further accumulated. While cell viability after incubation with linoleic acid, palmitic acid, or palmitoleic acid and QVD-Oph was comparable with that of non-treated controls, arachidonic acid significantly reduced cell viability and cell density despite the concomitant pan-caspase inhibitor treatment. Using electron microscopy, typical autophagy structures were detected, such as autophagosomes and autolysosomes, at the basal level, which became more pronounced after treatment with fatty acids. CONCLUSIONS: Our findings contribute to a better understanding of the inflammation-associated mechanisms of lipogenesis and cell death induction in human sebocytes and may help to unveil the effects of fatty acid-rich human nutrition.

Prediction of Response to Cisplatin-Based Neoadjuvant Chemotherapy of Muscle-Invasive Bladder Cancer Patients by Molecular Subtyping including KRT and FGFR Target Gene Assessment.

Patients with muscle-invasive urothelial carcinoma achieving pathological complete response (pCR) upon neoadjuvant chemotherapy (NAC) have improved prognosis. Molecular subtypes of bladder cancer differ markedly regarding sensitivity to cisplatin-based chemotherapy and harbor FGFR treatment targets to various content. The objective of the present study was to evaluate whether preoperative assessment of molecular subtype as well as FGFR target gene expression is predictive for therapeutic outcome­rate of ypT0 status­to justify subsequent prospective validation within the "BladderBRIDGister". Formalin-fixed paraffin-embedded (FFPE) tissue specimens from transurethral bladder tumor resections (TUR) prior to neoadjuvant chemotherapy and corresponding radical cystectomy samples after chemotherapy of 36 patients were retrospectively collected. RNA from FFPE tissues were extracted by commercial kits, Relative gene expression of subtyping markers (e.g., KRT5, KRT20) and target genes (FGFR1, FGFR3) was analyzed by standardized RT-qPCR systems (STRATIFYER Molecular Pathology GmbH, Cologne). Spearman correlation, Kruskal−Wallis, Mann−Whitney and sensitivity/specificity tests were performed by JMP 9.0.0 (SAS software). The neoadjuvant cohort consisted of 36 patients (median age: 69, male 83% vs. female 17%) with 92% of patients being node-negative during radical cystectomy after 1 to 4 cycles of NAC. When comparing pretreatment with post-treatment samples, the median expression of KRT20 dropped most significantly from DCT 37.38 to 30.65, which compares with a 128-fold decrease. The reduction in gene expression was modest for other luminal marker genes (GATA3 6.8-fold, ERBB2 6.3-fold). In contrast, FGFR1 mRNA expression increased from 33.28 to 35.88 (~6.8-fold increase). Spearman correlation revealed positive association of pretreatment KRT20 mRNA levels with achieving pCR (r = 0.3072: p = 0.0684), whereas pretreatment FGFR1 mRNA was associated with resistance to chemotherapy (r = −0.6418: p < 0.0001). Hierarchical clustering identified luminal tumors of high KRT20 mRNA expression being associated with high pCR rate (10/16; 63%), while the double-negative subgroup with high FGFR1 expression did not respond with pCR (0/9; 0%). Molecular subtyping distinguishes patients with high probability of response from tumors as resistant to neoadjuvant chemotherapy. Targeting FGFR1 in less-differentiated bladder cancer subgroups may sensitize tumors for adopted treatments or subsequent chemotherapy.

Endocytosis-Mediated Replenishment of Amino Acids Favors Cancer Cell Proliferation and Survival in Chromophobe Renal Cell Carcinoma.

Chromophobe renal cell carcinoma (chRCC) accounts for approximately 5% of all renal cancers and around 30% of chRCC cases have mutations in TP53. chRCC is poorly supported by microvessels and has markably lower glucose uptake than clear cell RCC and papillary RCC. Currently, the metabolic status and mechanisms by which this tumor adapts to nutrient-poor microenvironments remain to be investigated. In this study, we performed proteome and metabolome profiling of chRCC tumors and adjacent kidney tissues and identified major metabolic alterations in chRCC tumors, including the classical Warburg effect, the downregulation of gluconeogenesis and amino acid metabolism, and the upregulation of protein degradation and endocytosis. chRCC cells depended on extracellular macromolecules as an amino acid source by activating endocytosis to sustain cell proliferation and survival. Inhibition of the phospholipase C gamma 2 (PLCG2)/inositol 1,4,5-trisphosphate (IP3)/Ca2+/protein kinase C (PKC) pathway significantly impaired the activation of endocytosis for amino acid uptakes into chRCC cells. In chRCC, whole-exome sequencing revealed that TP53 mutations were not related to expression of PLCG2 and activation of endocytosis. Our study provides novel perspectives on metabolic rewiring in chRCC and identifies the PLCG2/IP3/Ca2+/PKC axis as a potential therapeutic target in patients with chRCC. SIGNIFICANCE: This study reveals macropinocytosis as an important process utilized by chRCC to gain extracellular nutrients in a p53-independent manner.

Prognostic Role of Survivin and Macrophage Infiltration Quantified on Protein and mRNA Level in Molecular Subtypes Determined by RT-qPCR of KRT5, KRT20, and ERBB2 in Muscle-Invasive Bladder Cancer Treated by Adjuvant Chemotherapy.

OBJECTIVES: Bladder cancer is a heterogeneous malignancy. Therefore, it is difficult to find single predictive markers. Moreover, most studies focus on either the immunohistochemical or molecular assessment of tumor tissues by next-generation sequencing (NGS) or PCR, while a combination of immunohistochemistry (IHC) and PCR for tumor marker assessment might have the strongest impact to predict outcome and select optimal therapies in real-world application. We investigated the role of proliferation survivin/BIRC5 and macrophage infiltration (CD68, MAC387, CLEVER-1) on the basis of molecular subtypes of bladder cancer (KRT5, KRT20, ERBB2) to predict outcomes of adjuvant treated muscle-invasive bladder cancer patients with regard to progression-free survival (PFS) and disease-specific survival (DSS). MATERIALS AND METHODS: We used tissue microarrays (TMA) from n = 50 patients (38 males, 12 female) with muscle-invasive bladder cancer. All patients had been treated with radical cystectomy followed by adjuvant triple chemotherapy. Median follow-up time was 60.5 months. CD68, CLEVER-1, MAC387, and survivin protein were detected by immunostaining and subsequent visual inspection. BIRC5, KRT5, KRT20, ERBB2, and CD68 mRNAs were detected by standardized RT-qPCR after tissue dot RNA extraction using a novel stamp technology. All these markers were evaluated in three different centers of excellence. RESULTS: Nuclear staining rather than cytoplasmic staining of survivin predicted DSS as a single marker with high levels of survivin being associated with better PFS and DSS upon adjuvant chemotherapy (p = 0.0138 and p = 0.001, respectively). These results were validated by the quantitation of BIRC5 mRNA by PCR (p = 0.0004 and p = 0.0508, respectively). Interestingly, nuclear staining of survivin protein was positively associated with BIRC5 mRNA, while cytoplasmic staining was inversely related, indicating that the translocation of survivin protein into the nucleus occurred at a discrete, higher level of its mRNA. Combining survivin/BIRC5 levels based on molecular subtype being assessed by KRT20 expression improved the predictive value, with tumors having low survivin/BIRC5 and KRT20 mRNA levels having the best survival (75% vs. 20% vs. 10% 5-year DSS, p = 0.0005), and these values were independent of grading, node status, and tumor stage in multivariate analysis (p = 0.0167). Macrophage infiltration dominated in basal tumors and was inversely related with the luminal subtype marker gene expression. The presence of macrophages in survivin-positive or ERBB2-positive tumors was associated with worse DSS. CONCLUSIONS: For muscle-invasive bladder cancer patients, the proliferative activity as determined by the nuclear staining of survivin or RT-qPCR on the basis of molecular subtype characteristics outperforms single marker detections and single technology approaches. Infiltration by macrophages detected by IHC or PCR is associated with worse outcome in defined subsets of tumors. The limitations of this study are the retrospective nature and the limited number of patients. However, the number of molecular markers has been restricted and based on predefined assumptions, which resulted in the dissection of muscle-invasive disease into tumor-biological axes of high prognostic relevance, which warrant further investigation and validation.

Decreased Mitochondrial DNA Content Drives OXPHOS Dysregulation in Chromophobe Renal Cell Carcinoma.

Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are closely related, rare kidney tumors. Mutations in complex I (CI)-encoding genes play an important role in dysfunction of the oxidative phosphorylation (OXPHOS) system in renal oncocytoma, but are less frequently observed in chRCC. As such, the relevance of OXPHOS status and role of CI mutations in chRCC remain unknown. To address this issue, we performed proteome and metabolome profiling as well as mitochondrial whole-exome sequencing to detect mitochondrial alterations in chRCC tissue specimens. Multiomic analysis revealed downregulation of electron transport chain (ETC) components in chRCC that differed from the expression profile in renal oncocytoma. A decrease in mitochondrial (mt)DNA content, rather than CI mutations, was the main cause for reduced OXPHOS in chRCC. There was a negative correlation between protein and transcript levels of nuclear DNA- but not mtDNA-encoded ETC complex subunits in chRCC. In addition, the reactive oxygen species scavenger glutathione (GSH) was upregulated in chRCC due to decreased expression of proteins involved in GSH degradation. These results demonstrate that distinct mechanisms of OXPHOS exist in chRCC and renal oncocytoma and that expression levels of ETC complex subunits can serve as a diagnostic marker for this rare malignancy. SIGNIFICANCE: These findings establish potential diagnostic markers to distinguish malignant chRCC from its highly similar but benign counterpart, renal oncocytoma.

Adult Tissue Extracellular Matrix Determines Tissue Specification of Human iPSC-Derived Embryonic Stage Mesodermal Precursor Cells.

The selection of pluripotent stem cell (PSC)-derived cells for tissue modeling and cell therapy will be influenced by their response to the tissue environment, including the extracellular matrix (ECM). Whether and how instructive memory is imprinted in adult ECM and able to impact on the tissue specific determination of human PSC-derived developmentally fetal mesodermal precursor (P-meso) cells is investigated. Decellularized ECM (dECM) is generated from human heart, kidney, and lung tissues and recellularized with P-meso cells in a medium not containing any differentiation inducing components. While P-meso cells on kidney dECM differentiate exclusively into nephronal cells, only beating clusters containing mature and immature cardiac cells form on heart dECM. No tissue-specific differentiation of P-meso cells is observed on endoderm-derived lung dECM. P-meso-derived endothelial cells, however, are found on all dECM preparations independent of tissue origin. Clearance of heparan-sulfate proteoglycans (HSPG) from dECM abolishes induction of tissue-specific differentiation. It is concluded that HSPG-bound factors on adult tissue-derived ECM are essential and sufficient to induce tissue-specific specification of uncommitted fetal stage precursor cells.

Limited utility of qPCR-based detection of tumor-specific circulating mRNAs in whole blood from clear cell renal cell carcinoma patients.

BACKGROUND: RNA sequencing data is providing abundant information about the levels of dysregulation of genes in various tumors. These data, as well as data based on older microarray technologies have enabled the identification of many genes which are upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched normal tissue. Here we use RNA sequencing data in order to construct a panel of highly overexpressed genes in ccRCC so as to evaluate their RNA levels in whole blood and determine any diagnostic potential of these levels for renal cell carcinoma patients. METHODS: A bioinformatics analysis with Python was performed using TCGA, GEO and other databases to identify genes which are upregulated in ccRCC while being absent in the blood of healthy individuals. Quantitative Real Time PCR (RT-qPCR) was subsequently used to measure the levels of candidate genes in whole blood (PAX gene) of 16 ccRCC patients versus 11 healthy individuals. PCR results were processed in qBase and GraphPadPrism and statistics was done with Mann-Whitney U test. RESULTS: While most analyzed genes were either undetectable or did not show any dysregulated expression, two genes, CDK18 and CCND1, were paradoxically downregulated in the blood of ccRCC patients compared to healthy controls. Furthermore, LOX showed a tendency towards upregulation in metastatic ccRCC samples compared to non-metastatic. CONCLUSIONS: This analysis illustrates the difficulty of detecting tumor regulated genes in blood and the possible influence of interference from expression in blood cells even for genes conditionally absent in normal blood. Testing in plasma samples indicated that tumor specific mRNAs were not detectable. While CDK18, CCND1 and LOX mRNAs might carry biomarker potential, this would require validation in an independent, larger patient cohort.

Papillary Renal Cell Carcinomas Rewire Glutathione Metabolism and Are Deficient in Both Anabolic Glucose Synthesis and Oxidative Phosphorylation.

Papillary renal cell carcinoma (pRCC) is a malignant kidney cancer with a prevalence of 7-20% of all renal tumors. Proteome and metabolome profiles of 19 pRCC and patient-matched healthy kidney controls were used to elucidate the regulation of metabolic pathways and the underlying molecular mechanisms. Glutathione (GSH), a main reactive oxygen species (ROS) scavenger, was highly increased and can be regarded as a new hallmark in this malignancy. Isotope tracing of pRCC derived cell lines revealed an increased de novo synthesis rate of GSH, based on glutamine consumption. Furthermore, profound downregulation of gluconeogenesis and oxidative phosphorylation was observed at the protein level. In contrast, analysis of the The Cancer Genome Atlas (TCGA) papillary RCC cohort revealed no significant change in transcripts encoding oxidative phosphorylation compared to normal kidney tissue, highlighting the importance of proteomic profiling. The molecular characteristics of pRCC are increased GSH synthesis to cope with ROS stress, deficient anabolic glucose synthesis, and compromised oxidative phosphorylation, which could potentially be exploited in innovative anti-cancer strategies.

VEGF - Supplemented extracellular matrix is sufficient to induce endothelial differentiation of human iPSC.

Extracellular matrix (ECM) provides a scaffold for cells and tissues, but also supports organogenesis and tissue remodeling. The required instructive properties of the ECM to interact with cells depend on matrix architecture, structural proteins and functional matrix components such as growth factors, providing spatial, chemical and functional cues. Decellularized ECM (dECM) has been proposed as an instructive material that promotes tissue regeneration. We investigated the instructive ECM elements preserved in dECM and necessary to promote endothelial differentiation of human induced pluripotent stem cells (hiPSC). We show that detergent-decellularized human kidney ECM remains structurally intact and carries a number of heparin-binding growth factors, including FGF2, VEGF, BMP2, HGF, EGF, PDGF-BB and TGFß, albeit at reduced levels compared to native tissues. Clearance of these heparin-binding factors, or heparan-sulfate proteoclycans from ECM resulted in massively reduced differentiation of hiPSC, suggesting that remaining structural dECM proteins such as laminin, collagen or fibronectin alone are not instructive. In contrast, replenishing dECM with VEGF replaced medium-supplemented VEGF and resulted in more efficient differentiation of hiPSC into endothelial cells, and even in the absence of other culture-supplemented differentiation factors dECM alone was superior to geltrex. In conclusion, conditioning of dECM with specific growth factors acting as functional cues may allow to generate functional niches by selective promotion of cell attachment, survival and differentiation.

Karyopherin Alpha 2 Is an Adverse Prognostic Factor in Clear-Cell and Papillary Renal-Cell Carcinoma.

BACKGROUND: Karyopherin α2 (KPNA2) is involved in the nucleocytoplasmic transport system and is functionally involved in the pathogenesis of various solid tumors by the translocation of cancer associated cargo proteins. However, the role of KPNA2 in renal-cell carcinoma (RCC) is still unknown. The aim of the present study was to investigate the protein expression of KPNA2 in cancerous and healthy renal tissues to evaluate its prognostic value in RCC. PATIENTS AND METHODS: We assessed KPNA2 protein expression via immunohistochemistry in a well-characterized cohort of 240 RCC patients by using a quantitative image analysis software. In addition, we analyzed publicly available gene expression data from The Cancer Genome Atlas (TCGA). RESULTS: A subgroup of clear-cell RCC (ccRCC) showed elevated protein expression levels of KPNA2. Most remarkably, we detected a correlation between high KPNA2 protein expression and shorter overall survival times as well as higher tumor stage and International Society of Urologic Pathology grade in ccRCC. However, the prognostic value of KPNA2 was not confirmed by multivariate Cox regression analysis when tested together with strong prognostic factors like tumor stage, lymph node metastasis, International Society of Urologic Pathology grade, and resection status. The results of the TCGA gene expression data analysis confirmed the prognostic value of KPNA2 in ccRCC. Additionally, KPNA2 expression was identified as an adverse factor in papillary RCC at the transcript level. CONCLUSION: KPNA2 appears to be involved in the carcinogenesis of RCC and functions as a novel prognostic indicator.

Diagnostic and Prognostic Potential of MicroRNA Maturation Regulators Drosha, AGO1 and AGO2 in Urothelial Carcinomas of the Bladder.

Bladder cancer still requires improvements in diagnosis and prognosis, because many of the cases will recur and/or metastasize with bad outcomes. Despite ongoing research on bladder biomarkers, the clinicopathological impact and diagnostic function of miRNA maturation regulators Drosha and Argonaute proteins AGO1 and AGO2 in urothelial bladder carcinoma remain unclear. Therefore, we conducted immunohistochemical investigations of a tissue microarray composed of 112 urothelial bladder carcinomas from therapy-naïve patients who underwent radical cystectomy or transurethral resection and compared the staining signal with adjacent normal bladder tissue. The correlations of protein expression of Drosha, AGO1 and AGO2 with sex, age, tumor stage, histological grading and overall survival were evaluated in order to identify their diagnostic and prognostic potential in urothelial cancer. Our results show an upregulation of AGO1, AGO2 and Drosha in non-muscle-invasive bladder carcinomas, while there was increased protein expression of only AGO2 in muscle-invasive bladder carcinomas. Moreover, we were able to differentiate between non-muscle-invasive and muscle-invasive bladder carcinoma according to AGO1 and Drosha expression. Finally, despite Drosha being a discriminating factor that can predict the probability of overall survival in the Kaplan⁻Meier analysis, AGO1 turned out to be independent of all clinicopathological parameters according to Cox regression. In conclusion, we assumed that the miRNA processing factors have clinical relevance as potential diagnostic and prognostic tools for bladder cancer.

Circular RNAs: a new class of biomarkers as a rising interest in laboratory medicine.

Circular RNAs (circRNAs) are a distinct family of RNAs derived from the non-regular process of alternative splicing. CircRNAs have recently gained interest in transcriptome research due to their potential regulatory functions during gene expression. CircRNAs can act as microRNA sponges and affect transcription through their complex involvement in regular transcriptional processes. Some early studies also suggested significant roles for circRNAs in human diseases, especially cancer, as biomarkers and potential clinical targets. Therefore, there is a great need for laboratory scientists to translate these findings into clinical tools to advance testing for human diseases. To facilitate a better understanding of the promise of circRNAs, we focus this review on selected basic aspects of circRNA research, specifically biogenesis, function, analytical issues regarding identification and validation and examples of expression data in relation to human diseases. We further emphasize the unique challenges facing laboratory medicine with regard to circRNA research, particularly in the development of robust assays for circRNA detection in different body fluids and the need to collaborate with clinicians in the design of clinical studies.

Comprehensive Evaluation of Prostate Specific Membrane Antigen Expression in the Vasculature of Renal Tumors: Implications for Imaging Studies and Prognostic Role.

PURPOSE: Prostate specific membrane antigen is expressed by the endothelium of many tumors. The aim of the study was to find a rationale for prostate specific membrane antigen based imaging and investigate the prognostic role of vascular prostate specific membrane antigen expression in patients with renal cell carcinoma. MATERIALS AND METHODS: A total of 257 patients with renal cell carcinoma were included in study with a median followup exceeding 10.0 years. Prostate specific membrane antigen expression on tumor vessels was detected by immunohistochemistry. Vascular expression of FOLH1 gene (prostate specific membrane antigen) mRNA was investigated in clear cell carcinoma and papillary renal cell carcinoma using TCGA (The Cancer Genome Atlas) data. RESULTS: Endothelial prostate specific membrane antigen protein expression was higher in clear cell than in papillary and chromophobe renal cell carcinoma. Higher grade and stage, metastatic and lethal clear cell renal cell carcinoma showed higher prostate specific membrane antigen expression in tumor vessels. On univariate and multivariate analysis the intensity of positive vs negative endothelial prostate specific membrane antigen protein expression was significantly associated with overall survival. TCGA based analyses confirmed the prognostic role of vascular expression of FOLH1 mRNA. The analyses also supported the usefulness of prostate specific membrane antigen based imaging in cases of clear cell but not papillary renal cell carcinoma. CONCLUSIONS: We provide a rationale for further development of prostate specific membrane antigen targeted imaging in patients with clear cell renal cell carcinoma. The prognostic role of prostate specific membrane antigen was determined at the protein level in clear cell renal cell carcinoma and at the mRNA level in clear cell and papillary renal cell carcinoma.

Punicalagin, a polyphenol from pomegranate fruit, induces growth inhibition and apoptosis in human PC-3 and LNCaP cells.

Prostate cancer (PCa) is an international health problem and search for its effective treatment is in progress. Punicalagin (PN), polyphenol from pomegranate fruit, is known to exhibit potent anticancer activity in lung, breast and cervical cells. However, there is paucity of information on its effect in PCa. This study evaluated anti-proliferative effects of PN and its effects on extrinsic pathway of apoptosis in PCa cells, and angiogenesis in chicken chorioallantoic membrane (CAM). Antioxidant activities of PN were determined by 2,2-diphenyl-1-picryhydrazyl (DPPH) radical scavenging and inhibition of lipid peroxidation (LPO) methods. PCa (PC-3 and LNCaP) and normal prostate (BPH-1) cells were cultured and treated with PN (10, 50 and 100 µM). Cytotoxicity and viability effects of PN were determined by lactate dehydrogenase (LDH) and XTT assays, respectively. Antiangiogenic effects were measured using CAM assay, while apoptosis was assessed by DNA fragmentation, enrichment factor by Cell Death Detection ELISA kit and expressions of caspases-3 and -8. Results showed that PN (10-200 µM) significantly scavenged DPPH and inhibited LPO in a concentration-dependent manner. Furthermore, PN (10-100 µM) concentration-dependently inhibited viability in PC-3 and LNCaP, while viability in BPH-1 was insignificantly affected. PN had low toxicity on cells in vitro at concentrations tested. Also, PN (100 µM) increased enrichment factor in PC-3 (2.34 ± 0.05) and LNCaP (2.31 ± 0.26) relative to control (1.00 ± 0.00). In addition, PN (50 µM) decreased the network of vessels in CAM, suggesting its anti-angiogenic effect. Moreso, PN increased the expressions of caspases-3 and -8 in PC-3. Overall, PN exerts anti-proliferative activity in PCa cells via induction of apoptosis and anti-angiogenic effect.

Antioxidant and antiproliferative potentials of methanol extract of Xylopia aethiopica (Dunal) A. Rich in PC-3 and LNCaP cells.

BACKGROUND: Our previous studies showed that fruit methanol extract from Xylopia aethiopica (MEXA) exhibited antiproliferative activity in human cervical cancer cells via the induction of apoptosis. The present study was designed to assess the antiproliferative, antiangiogenic and antioxidant effects of MEXA on prostate cancer (PCa) cells (PC-3 and LNCaP). METHODS: PC-3 and LNCaP cells were cultured and treated with MEXA (10, 50 and 100 µg/mL). The sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) and lactate dehydrogenase (LDH) assays were used to evaluate cell viability and cytotoxicity, respectively. DNA fragmentation was determined by cell death detection ELISA plus, and angiogenesis was assessed by chicken chorioallantoic membrane (CAM) assay. The antioxidant activities of MEXA were determined by DPPH and hydroxyl (OH) radicals' scavenging methods as well as through the inhibition of lipid peroxidation (LPO) in rats' liver homogenate. RESULTS: MEXA at 100, 250 and 500 µg/mL scavenged DPPH by 48%, 62%, 70% and OH radical by 39%, 58%, 67%, respectively. MEXA significantly (p<0.05) inhibited LPO in a concentration-dependent manner. In addition, MEXA had antiproliferative effects on PC-3 and LNCaP with IC50 of 62.1 and 73.6 µg/mL, respectively, at 96 h. The LDH assay showed that MEXA had low toxicity in vitro at its IC50 values. The extent of DNA fragmentation by MEXA showed higher values in PC-3 and LNCaP, suggesting the possible induction of apoptosis. In contrast, MEXA did not affect the network of vessels in CAM, thus lacking anti-angiogenic property. CONCLUSIONS: These findings suggest that MEXA induces antiproliferative activity in PCa cells through a mechanism that involves apoptosis. Therefore, MEXA may be a potential therapeutic agent for PCa.

Epithelial-mesenchymal transition-associated microRNA/mRNA signature is linked to metastasis and prognosis in clear-cell renal cell carcinoma.

Clear-cell renal cell carcinomas (ccRCCs) are genetically heterogeneous tumors presenting diverse clinical courses. Epithelial-mesenchymal transition (EMT) is a crucial process involved in initiation of metastatic cascade. The aim of our study was to identify an integrated miRNA/mRNA signature associated with metastasis and prognosis in ccRCC through targeted approach based on analysis of miRNAs/mRNAs associated with EMT. A cohort of 230 ccRCC was included in our study and further divided into discovery, training and validation cohorts. EMT markers were evaluated in ccRCC tumor samples, which were grouped accordingly to EMT status. By use of large-scale miRNA/mRNA expression profiling, we identified miRNA/mRNA with significantly different expression in EMT-positive tumors and selected 41 miRNAs/mRNAs for training phase of the study to evaluate their diagnostic and prognostic potential. Fifteen miRNAs/mRNAs were analyzed in the validation phase, where all evaluated miRNA/mRNA candidates were confirmed to be significantly deregulated in tumor tissue. Some of them significantly differed in metastatic tumors, correlated with clinical stage, with Fuhrman grade and with overall survival. Further, we established an EMT-based stage-independent prognostic scoring system enabling identification of ccRCC patients at high-risk of cancer-related death. Finally, we confirmed involvement of miR-429 in EMT regulation in RCC cells in vitro.

Cooperative Effect of miR-141-3p and miR-145-5p in the Regulation of Targets in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Due to the poor prognosis for advanced renal cell carcinoma (RCC), there is an urgent need for new therapeutic targets and for prognostic markers to identify high risk tumors. MicroRNAs (miRNAs) are frequently dysregulated in tumors, play a crucial role during carcinogenesis and therefore might be promising new biomarkers. In previous studies, we identified miR-141-3p and miR-145-5p to be downregulated in clear cell RCC (ccRCC). Our objective was to investigate the functional association of these miRNAs, focusing on the cooperative regulation of new specific targets and their role in ccRCC progression. METHODS: The effect of miR-141-3p and miR-145-5p on cell migration was examined by overexpression in 786-O cells. New targets of both miRNAs were identified by miRWalk, validated in 786-O and ACHN cells and additionally characterized in ccRCC tissue on mRNA and protein level. RESULTS: In functional analysis, a tumor suppressive effect of miR-141-3p and miR-145-5p by decreasing migration and invasion of RCC cells could be shown. Furthermore, co-overexpression of the miRNAs seemed to result in an increased inhibition of cell migration. Both miRNAs were recognized as post-transcriptional regulators of the targets EAPP, HS6ST2, LOX, TGFB2 and VRK2. Additionally, they showed a cooperative effect again as demonstrated by a significantly increased inhibition of HS6ST2 and LOX expression after simultaneous overexpression of both miRNAs. In ccRCC tissue, LOX mRNA expression was strongly increased compared to normal tissue, allowing also to distinguish between non-metastatic and already metastasized primary tumors. Finally, in subsequent tissue microarray analysis LOX protein expression showed a prognostic relevance for the overall survival of ccRCC patients. CONCLUSION: These results illustrate a jointly strengthening effect of the dysregulated miR-141-3p and miR-145-5p in various tumor associated processes. Focusing on the cooperative effect of miRNAs provides new opportunities for the development of therapeutic strategies and offers novel prognostic and diagnostic capabilities.