Human UC Research Articles

Predicting novel biomarkers for early diagnosis and dynamic severity monitoring of human ulcerative colitis.

Ulcerative colitis is an emerging global health concern that poses a significant threat to human health and can progress to colorectal cancer if not diagnosed and treated promptly. Currently, the biomarkers used clinically for diagnosis and dynamic severity monitoring lack disease specificity. Mouse models induced with 2%, 2.5%, and 3% DSS were utilized to simulate human UC with varying severities of inflammation. Transcriptome sequencing technology was employed to identify differentially expressed genes (DEGs) between the control group and each treatment group. Functional enrichment analysis of the KEGG database was performed for shared DEGs among the three treatment groups. DEGs that were significantly and strongly correlated with DSS concentrations were identified using Spearman correlation analysis. Human homologous genes of the interested DEGs were searched in the HomoloGene database, and their regulation patterns in UC patients were validated using the GSE224758 dataset. These genes were then submitted to the DisGeNET database to identify their known associations with human diseases. Online tools, including SignalP 6.0 and DeepTMHMM 1.0, were used to predict signal peptides and transmembrane helices in the amino acid sequences of human genes homologous to the DEGs of interest. A total of 1,230, 995, and 2,214 DEGs were identified in the 2%, 2.5%, and 3% DSS-induced groups, respectively, with 668 DEGs common across all three groups. These shared DEGs were primarily associated with signaling transport, pathogenesis, and immune response. Through extensive screening, LGI2 and PRSS22 were identified as potentially novel biomarkers with higher specificity and ease of detection for the early diagnosis and dynamic severity monitoring of human UC, respectively. We have identified two potentially novel biomarkers, LGI2 and PRSS22, which are easy of detection and more specific for human UC. These findings provide new insights into the accurate diagnosis and dynamic monitoring of this persistent disease.

Therapeutic utility of human umbilical cord-derived mesenchymal stem cells-based approaches in pulmonary diseases: Recent advancements and prospects.

Pulmonary diseases across all ages threaten millions of people and have emerged as one of the major public health issues worldwide. For diverse disease conditions, the currently available approaches are focused on alleviating clinical symptoms and delaying disease progression but have not shown significant therapeutic effects in patients with lung diseases. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) isolated from the human UC have the capacity for self-renewal and multilineage differentiation. Moreover, in recent years, these cells have been demonstrated to have unique advantages in the treatment of lung diseases. We searched the Public Clinical Trial Database and found 55 clinical trials involving UC-MSC therapy for pulmonary diseases, including coronavirus disease 2019, acute respiratory distress syndrome, bronchopulmonary dysplasia, chronic obstructive pulmonary disease, and pulmonary fibrosis. In this review, we summarize the characteristics of these registered clinical trials and relevant published results and explore in depth the challenges and opportunitiesfaced in clinical application. Moreover, the underlying molecular mechanisms involved in UC-MSC-based therapy for pulmonary diseases are also analyzed in depth. In brief, this comprehensive review and detailed analysis of these clinical trials can be expected to provide a scientific reference for future large-scale clinical application.

SLC6A14 promotes ulcerative colitis progression by facilitating NLRP3 inflammasome-mediated pyroptosis.

Ulcerative colitis (UC) is an inflammatory condition with frequent relapse and recurrence. Evidence suggests the involvement of SLC6A14 in UC pathogenesis, but the central regulator remains unknown. To explore the role of SLC6A14 in UC-associated pyroptosis. Quantitative real-time polymerase chain reaction (qRT-PCR), immunoblotting, and immunohistochemical were used to assess SLC6A14 in human UC tissues. Lipopolysaccharide (LPS) was used to induce inflammation in FHC and NCM460 cells and model enteritis, and SLC6A14 levels were assessed. Pyroptosis markers were quantified using enzyme-linked immunosorbent assay, Western blotting, and qRT-PCR, and EdU incubation, CCK-8 assays and flow cytometry were used to examine proliferation and apoptosis. Mouse models of UC were used for verification. SLC6A14 was increased and correlated with NLRP3 in UC tissues. LPS-induced FHC and NCM460 cells showed increased SLC6A14 levels. Reducing SLC6A14 increased cell proliferation and suppressed apoptosis. Reducing SLC6A14 decreased pyroptosis-associated proteins (ASC, IL-1β, IL-18, NLRP3). NLRP3 overexpression counteracted the effects of sh-SLC6A14 on LPS-induced FHC and NCM460 cell pyroptosis. SLC6A14 improved the mucosa in mice with dextran sulfate sodium-induced colitis. SLC6A14 promotes UC pyroptosis by regulating NLRP3, suggesting the therapeutic potential of modulating the SLC6A14/NLRP3 axis.

NOX1 is essential for TNFα-induced intestinal epithelial ROS secretion and inhibits M cell signatures

ObjectiveLoss-of-function mutations in genes generating reactive oxygen species (ROS), such as NOX1, are associated with IBD. Mechanisms whereby loss of ROS drive IBD are incompletely defined.DesignROS measurements and single-cell transcriptomics...

Minimal Cube Explant Provides Optimal Isolation Condition of Mesenchymal Stem Cells from Umbilical Cord.

Enzymatic digestion and explant method have been widely used for isolating umbilical cord-derived mesenchymal stem cells (UC MSCs), although there is still a strong need for robust protocols for optimal isolation for large-scale stem cell banks. This study aims to establish an explant method for clinical scale production of MSCs from human UC tissue and to characterize UC MSCs isolated and cultured with the explant method. UC MSCs were isolated by enzymatic digestion, minimal cube explant (MCE) 1-2, MCE 2-4, and MCE 10 and cultured, respectively. Also, human antibody array and basic fibroblast growth factor (bFGF) secretion in conditioned medium (CM) was analyzed. The cells were evaluated initial cell number, colony forming unit-fibroblast (CFU-F), proliferation capacity, CD marker expression, and multi-lineage differentiation. SA-β-gal assay as well as expression of p16, p21 and p53 was performed by RT-PCR. MCE 2-4 is the most optimized method for isolation of small umbilical cord-derived fast proliferating cells (smumf cells) with the greatest number. MCE 2-4 had the highest secretion of various bioactive factors including bFGF. The MCE 2-4 provided significantly higher CD146 expression than enzymatic digestion, and that expression was maintained until P20. The gene expression of p16, p21, and p53 of smumf cells did not change until P10 and SA-β-gal activity did not increase until P14. This study demonstrated that MCE 2-4 provided an optimal environment to isolate MSCs with quantity and quality from human whole UC tissue through secretion of various bioactive factors inherent to UC.

NDRG2 regulates adherens junction integrity to restrict colitis and tumourigenesis.

BackgroundParacellular barriers play an important role in the pathogenesis of Inflammatory bowel disease (IBD) and maintain gut homeostasis. N-myc downstream-regulated gene 2 (NDRG2) has been reported to be a tumour suppressor gene and to inhibit colorectal cancer metastasis. However, whether NDRG2 affects colitis initiation and colitis-associated colorectal cancer is unclear.MethodsIntestine-specific Ndrg2 deficiency mice (Ndrg2ΔIEC) were subjected to DSS- or TNBS-induced colitis, and AOM-DSS-induced colitis-associated tumour. HT29 cells, Caco2 cells, primary intestinal epithelial cells (IECs) from Ndrg2ΔIEC mice, mouse embryo fibroblasts (MEFs) from systemic Ndrg2 knockout mice, HEK293 cells and human UC and DC specimens were used to investigate NDRG2 function in colitis and colitis-associated tumour.FindingsNdrg2 loss led to adherens junction (AJ) structure destruction via E-cadherin expression attenuation, resulting in diminished epithelial barrier function and increased intestinal epithelial permeability. Mechanistically, NDRG2 enhanced the interaction of E3 ligase FBXO11 with Snail, the repressor of E-cadherin, to promote Snail degradation by ubiquitination and maintained E-cadherin expression. In human ulcerative colitis patients, reduced NDRG2 expression is positively correlated with severe inflammation.InterpretationThese findings demonstrate that NDRG2 is an essential colonic epithelial barrier regulator and plays an important role in gut homeostasis maintenance and colitis-associated tumour development.FundingNational Natural Science Foundation of China (No. 81770523, 31571437, 81672751), Creative Research Groups of China (No. 81421003), State Key Laboratory of Cancer Biology Project (CBSKL2019ZZ11, CBSKL201406, CBSKL2017Z08 and CBSKL2017Z11), Fund for Distinguished Young Scholars of ShaanXi province (2019JC-22).

Abstract 3703: Development of oncopig urothelial carcinoma cell lines

Abstract Urothelial carcinoma (UC), more commonly known as bladder cancer, affects approximately 81,000 people a year in the United States and is the fourth most common type of cancer among men. Age also plays a tremendous factor, as 90% of people affected by bladder cancer are older than 55, and if the cancer has metastasized the 5-year survival rate is 35%. This requires the advancement of early detection and treatment methods for bladder cancer. The development of clinically relevant systems to serve as a bridge between preclinical murine studies and human clinical practice is of vital importance. The Oncopig Cancer Model (OCM) is a novel transgenic swine platform that recapitulates human cancer through development of site/cell specific tumors after Cre recombinase induced expression of heterozygous KRASG12D and TP53R167H transgene. In this study, we standardized a protocol for the development and growth of Oncopig UC cell lines that faithfully recapitulates in vitro and in vivo features of human bladder cancer. The focus of this preliminary research was to successfully create OCM UC cell lines that mimic features of human UC. OCM urothelial cell lines (n=7) were isolated from whole bladders collected from male Oncopigs at euthanasia. OCM urothelial cell lines were exposed to Cre recombinase 48 hours post isolation to induce expression of KRASG12D and TP53R167H transgenes, resulting in development of OCM UC cell lines. Differences in migration rates were observed between OCM urothelial and UC cell lines, confirming cellular invasiveness. RT-PCR was performed after several passages to confirm KRASG12D and TP53R167H gene expression, which was observed in OCM UC but not urothelial cell lines. Uroplakin II and pan-Cytokeratin (PCK-26) are markers for UC and urothelial cells, respectively, and are used to diagnose UC clinically. OCM UC cells stained positively for Uroplakin II and PCK-26, confirming their identity as malignant UC cells. Future work is needed to confirm OCM and human UC display similar histological and molecular features, and to develop strategies for in vivo OCM UC tumor development. Citation Format: Aisha Qazi, Faith M. Thomas, Sulalita Chaki, Noah Robertson, Shovik Patel, Kyle M. Schachtschneider, Lawrence B. Schook. Development of oncopig urothelial carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3703.

Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway.

BackgroundUlcerative Colitis (UC) is an Inflammatory Bowel Disease (IBD) characterized by uncontrolled immune response, diarrhoea, weight loss and bloody stools, where sustained remission is not currently achievable. Dextran Sulphate Sodium (DSS)-induced colitis is an animal model that closely mimics human UC. Ultrasound (US) has been shown to prevent experimental acute kidney injury through vagus nerve (VN) stimulation and activation of the cholinergic anti-inflammatory pathway (CAIP). Since IBD patients may present dysfunctional VN activity, our aim was to determine the effects of therapeutic ultrasound (TUS) in DSS-induced colitis.MethodsAcute colitis was induced by 2% DSS in drinking water for 7 days and TUS was administered to the abdominal area for 7 min/day from days 4–10. Clinical symptoms were analysed, and biological samples were collected for proteomics, macroscopic and microscopic analysis, flow cytometry and immunohistochemistry.FindingsTUS attenuated colitis by reducing clinical scores, colon shortening and histological damage, inducing proteomic tolerogenic response in the gut during the injury phase and early recovery of experimental colitis. TUS did not improve clinical and pathological outcomes in splenectomised mice, while α7nAChR (α7 nicotinic acetylcholine receptor - indicator of CAIP involvement) knockout animals presented with disease worsening. Increased levels of colonic F4/80+α7nAChR+ macrophages in wild type mice suggest CAIP activation.InterpretationThese results indicate TUS improved DSS-induced colitis through stimulation of the splenic nerve along with possible contribution by VN with CAIP activation.FundIntramural Research Programs of the Clinical Centre, the National Institute of Biomedical Imaging and Bioengineering at the NIH and CAPES/Brazil.

DNA methylome of human neonatal umbilical cord: Enrichment of differentially methylated regions compared to umbilical cord blood DNA at transcription factor genes involved in body patterning and effects of maternal folate deficiency or children's sex.

The DOHaD (developmental origins of health and disease) hypothesis claims that fetal malnutrition or exposure to environmental pollutants may affect their lifelong health. Epigenetic changes may play significant roles in DOHaD; however, access to human fetuses for research has ethical and technical hurdles. Umbilical cord blood (CB) has been commonly used as an epigenetic surrogate of fetuses, but it does not provide direct evidence of fetal exposure to pollutants. Here, we propose umbilical cord tissue (UC), which accumulates substances delivered to fetuses during gestation, as an alternative surrogate for epigenetic studies on fetuses. To explore the feasibility to examine UC epigenome by deep sequencing, we determined CpG methylation profiles of human postnatal UC by reduced representation bisulfite sequencing. Principal component analysis clearly separated the DNA methylomes of UC and CB pairs isolated from the same newborn (n = 10). Although all UC chromosomes were modestly hypomethylated compared to CB chromosomes, GO analysis revealed strong enrichment of differentially methylated regions (DMRs) at promoter-associated CpG islands in the HOX gene clusters and other genes encoding transcription factors involved in determination of the body pattern. DNA methylomes of UC autosomes were largely comparable between males and females. Deficiency of folate during pregnancy has been suggested to affect fetal DNA methylation to cause congenital anomalies. Whereas DNA methylome of UC was not significantly affected by early-gestational (12 weeks) low levels of maternal plasma folate (< 8 ng/ml, n = 10) compared to controls (>19 ng/mL, n = 10), two specific loci of LTR12C endogenous retroviruses in chromosome 12 were significantly hypermethylated in the low-folate group. Our study suggests that UC is useful as an alternative surrogate for studying environmental effects on DNA methylation in human fetuses, compensating CB by providing additional information about epigenetic regulation of genes involved in developmental body patterning and endogenous retroviruses.

Genetic Mouse Models with Intestinal-Specific Tight Junction Deletion Resemble an Ulcerative Colitis Phenotype.

Background and AimsA key pathogenetic feature of ulcerative colitis [UC] is an intrinsic low mucus phosphatidylcholine[PC] content. Recently, a paracellular transport for PC across tight junctions[TJs] was described, suggesting TJ disturbance as a cause of diminished luminal PC transport. Therefore, we aimed to generate mutant mice with TJ deletion to evaluate whether a UC phenotype developed.MethodsCL57BL/6 control wild-type mice were compared to mutant mice with tamoxifen-induced villin-Cre-dependent intestinal deletion of kindlin 1 and 2.ResultsElectron microscopy of mucosal biopsies obtained from both mutants before overt inflammation following only 2 days of tamoxifen exposure revealed a defective TJ morphology with extended paracellular space and, by light microscopy, expanded mucosal crypt lumina. PC secretion into mucus was reduced by >65% and the mucus PC content dropped by >50%, causing a >50 % decrease of mucus hydrophobicity in both mutants. Consequently, the microbiota was able to penetrate the submucosa. After 3 days of tamoxifen exposure, intestinal inflammation was present in both mutants, with loose bloody stools as well as macroscopic and histological features of colitis. Oral PC supplementation was able to suppress inflammation. By analogy, colonic biopsies obtained from patients with UC in remission also showed a defective epithelium with widened intercellular clefts, and enlarged crypt luminal diameters with functionally impaired luminal PC secretion.ConclusionsGenetic mouse models with intestinal deletion of kindlin 1 and 2 resulted in TJ deletion and revealed pathophysiological features of impaired PC secretion to the mucus leading to mucosal inflammation compatible with human UC.

Bioinformatics characteristics of lncRNA-uc.167 and its temporal and spatial expression pattern for mouse embryonic development

Objective To explore the basic biological characteristics of lncRNA-uc.167, and its spatial distribution, temporal expression pattern during the mouse embryonic development. Methods The UCSC genome browser of ENCODE was used to analyze preliminary bioinformatics of lncRNAs.Real-time (RT)-PCR was applied to detect the expression of uc.167 and neighboring genes in the embryonic mouse heart in different stages (P7.5, P11.5, P14.5, P18.5). Dimethyl sulphoxide was used to induce P19 cell differentiation into the cardiomyocytes.RT-PCR was applied to detect the expression changes in uc.167 and neighboring genes on differential day 0, 4, 6, 8 and 10. Results Full-length of human uc.167 was 201 bp, and human uc.167 was located in the genome 5q14.3 (chr5: 88179623-88179824, GRCh37/hg19). uc.167 mainly expressed in the ventricular muscle tissue.The expression of uc.167 was gradually decreased in the mouse embryonic heart development process(P7.5: 1.000±0.100, P11.5: 0.714±0.107, P14.5: 0.393±0.043, P18.5: 0.125±0.013), while the expression of its neighboring Mef2c gene was gradually increased(P7.5: 1.081±0.118, P11.5: 2.340±0.351, P14.5: 3.958±0.542, P18.5: 9.361±0.722), which showed an opposite trend.The expression of uc.167 during P19 cell differentiation into cardiomyocytes showed a an increase at first and then a decrease pattern, and the highest level expression of uc.167 was on differential day 4(d0: 1.071±0.117, d4: 4.714±0.501, d6: 3.572±0.414, d8: 2.550±0.314, d10: 0.786±0.085). The expression of neighboring gene Mef2c was in an opposite trend(d0: 1.012±0.041, d4: 0.353±0.037, d6: 2.470±0.329, d8: 6.706±0.682, d10: 7.765±0.705). Conclusions It is suggested that uc.167 may take part in the process of embryonic heart development and may play a role through negatively regulating its neighboring gene Mef2c. Key words: Bioinformatics analysis; Cardiac development; LncRNA

MP83-07 ROLE OF HINT3 IN P21 INDUCED NECROTIC CELL DEATH UPON BCG EXPOSURE IN UROTHELIAL CARCINOMA CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP83-07 ROLE OF HINT3 IN P21 INDUCED NECROTIC CELL DEATH UPON BCG EXPOSURE IN UROTHELIAL CARCINOMA CELLS Gopitkumar Shah, Fanghong Chen, GuangJian Zhang, and William See Gopitkumar ShahGopitkumar Shah More articles by this author , Fanghong ChenFanghong Chen More articles by this author , GuangJian ZhangGuangJian Zhang More articles by this author , and William SeeWilliam See More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2189AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Exposure of urothelial carcinoma cells (UCC) to BCG results in activation of p21. Previously we have shown that p21 is sufficient and necessary for the full antiapoptotic effect of BCG. p21 expression alone is necessary for HMGB1 release which is required for direct cytotoxic effect of BCG in vivo. Microarray of p21 overexpressing cell lines showed induction of hint3 gene, however its role has not been elucidated. The objective of the current study is to characterize the role of hint3 in antiapoptotic effects of BCG. METHODS The blockade of hint3 expression in response to BCG was established in the human UC line T24 using the stable transfection of a siRNA construct targeting hint3 mRNA. The effect of inhibiting hint3 expression on tumor phenotype was assessed using assays for viability, LDH and HMGB1 release and caspase activation. RESULTS Exposure of parental UC cell lines to BCG resulted in significant increase in hint3 expression. Blockade of hint3 expression using shRNA decreased its expression by 3-5 folds in BCG exposed cells (p < 0.05). LDH and HMGB1 release in response to BCG was significantly lower in hint3 mutant cell line compared to the parental cell line and scrambled control cell lines (p < 0.01 for LDH; p < 0.05 for HMGB1). There was no significant decrease in BCG cytotoxicity as measured by decrease in viability amongst the parental and mutant cell line ( p = 0.12). Hint3 mutant cell lines showed significant increase in caspase activity in response to BCG exposure compared to parental and control cell lines p < 0.05). CONCLUSIONS BCG induces hint3 expression in p21 dependent manner. Hint3 mutation results in decreased HMGB1 release indicating the p21 acts via hint3 for induction of HMGB1 release. Hint3 mutation also induces caspase 3 activity indicating changes in mechanism of cell death from necrosis to apoptosis. As HMGB1 is critical for in vivo cytotoxicity of BCG, characterizing the pathways for HMGB1 induction would provide a novel target to improve BCG efficacy. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1083 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Gopitkumar Shah More articles by this author Fanghong Chen More articles by this author GuangJian Zhang More articles by this author William See More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MicroRNA 429 Regulates Mucin Gene Expression and Secretion in Murine Model of Colitis.

miRNAs are non-coding RNAs that play important roles in the pathogenesis of human diseases by regulating target gene expression in specific cells or tissues. We aimed to detect miRNAs related to ulcerative colitis [UC], identify their target molecules, and analyse the correlation between the miRNAs and their target genes in colorectal cells and dextran sulphate sodium [DSS]-induced mouse colitis. UC-associated miRNAs were identified by miRNA microarray analysis using DSS-induced colitis and normal colon tissues. The results were validated by quantitative real-time polymerase chain reaction [RT-PCR]. We identified target genes of MIR429, a colitis-associated miRNA, from our screen by comparing the mRNA microarray analysis in MIR429-overexpressed cells with predicted candidate target genes. We constructed luciferase reporter plasmids to confirm the effect of MIR429 on target gene expression. The protein expression of the target genes was measured by western blot,enzyme-linked immunosorbent assay [ELISA] analysis, or immunohistochemistry. We identified 37 DSS-induced colitis associated miRNAs. We investigated MIR429 that is down-regulated in DSS-induced colitis, and identified 41 target genes of MIR429. We show that the myristoylated alanine-rich protein kinase C substrate [MARCKS] is a direct target of MIR429. MARCKS mRNA and protein expression levels are down-regulated by MIR429, and MIR429 regulates the expression of MARCKS and MARCKS-mediated mucin secretion in colorectal cells and DSS-induced colitis. In addition, anti-MIR429 up-regulates MARCKS expression in colorectal cell lines. Our findings suggest that MIR429 modulates mucin secretion in human colorectal cells and mouse colitis tissues by up-regulating of MARCKS expression, thereby making MIR429 a candidate for anti-colitis therapy in human UC.

Tu1707 Endogenous Opioids From Human IBD Supernatants Modulate TRPV1 on DRG Neurons During Chronic Colitis

Our previous mouse studies have shown that endogenous opioid signaling is enhanced during the evolution of colonic inflammation, altering pain signaling. The mechanisms underlying this action are unknown but given that TRPV1 channels are important in mechano and chemosensitive nociceptive signaling, and that TRPV1 is co-expressed with opioid receptors on dorsal root ganglia (DRG) neurons, we postulated that TRPV1 signaling may be involved. Therefore, in the present work we sought to investigate if the endogenous opioids released during the chronic inflammation model regulate TRPV1 activity. The 2% chronic DSS (cDSS) colitis model was employed in C57/BL6 mice. Human colonic biopsies from patients with chronic ulcerative colitis, crohn's desease or healthy controls and colonic tissue from cDSS colitis were used to generate supernatants (sup). DRG neurons were incubated overnight with mouse or human supernatants or μ and δ opioid agonists DAMGO (10 nM) and DADLE (1 μM) respectively and with or without the opioid receptor antagonist naloxone (10 μM). For Ca2+ imaging 1 μM Fura-2 AM and capsaicin (250 nM) was used as a TRPV1 agonist. T-test or one way ANOVA were used to analyze the data obtained, n= number of neurons. The capsaicin-evoked Ca2+ signal was inhibited 66% by cDSS sup compared to control sup (F340/380: Ctrl=2.08±0.33 n=19, cDSS=0.70±0.14 n=16, P= <0.01). This effect was blocked by naloxone 10 μM (F340/380: cDSS+NLX= 2.32±0.65, n=6, P= <0.05), demonstrating that endogenous opioids were involved. Moreover, the supernatant effect was mimicked by the use of low concentration DAMGO (10 nM) or DADLE (1 μM) alone. (F340/380: Ctrl=5.66±0.52 n=26, DAMGO=2.42±0.55 n=19, DADLE=3.45±50, P= <0.001), DRG neurons incubated with the human UC sup showed a similar effect with a 58% reduction of the Ca2+ signal (F340/380: Ctrl=5.51±0.51 n=18, UC sup=2.30±0.67 n= 9, P= 0.0011). In contrast, in a symptomatic IBD patient where inflammation had resolved (post-inflammatory IBS) there was no difference in the Ca2+ signal compared to control, thus suggesting the endogenous opioid effect was lost (F340/380: Ctrl=4.59±0.58 n=15, Crohn's sup=3.95±0.79 n=11). These results suggest that endogenous opioids released during chronic inflammation have an important role in inhibiting TRPV1 channels in nociceptive neurons. Following resolution of the inflammation in some patients however, the pain persists but the endogenous opioid system appears to be inactive.

OP1-12 REDUCED TRAP1 EXPRESSION IS ASSOCIATED WITH POOR PROGNOSIS IN UPPER URINARY TRACT UROTHELIAL CARCINOMA PATIENTS: ITS IMPLICATIONS FOR TUMOR INVASION

You have accessJournal of UrologyOutstanding Posters: Research1 Apr 2014OP1-12 REDUCED TRAP1 EXPRESSION IS ASSOCIATED WITH POOR PROGNOSIS IN UPPER URINARY TRACT UROTHELIAL CARCINOMA PATIENTS: ITS IMPLICATIONS FOR TUMOR INVASION Hiroshi Fukushima, Soichiro Yoshida, Fumitaka Koga, Yasukazu Nakanishi, Toshiki Kijima, Junichiro Ishioka, Yoh Matsuoka, Noboru Numao, Kazutaka Saito, Yasuhisa Fujii, and Kazunori Kihara Hiroshi FukushimaHiroshi Fukushima More articles by this author , Soichiro YoshidaSoichiro Yoshida More articles by this author , Fumitaka KogaFumitaka Koga More articles by this author , Yasukazu NakanishiYasukazu Nakanishi More articles by this author , Toshiki KijimaToshiki Kijima More articles by this author , Junichiro IshiokaJunichiro Ishioka More articles by this author , Yoh MatsuokaYoh Matsuoka More articles by this author , Noboru NumaoNoboru Numao More articles by this author , Kazutaka SaitoKazutaka Saito More articles by this author , Yasuhisa FujiiYasuhisa Fujii More articles by this author , and Kazunori KiharaKazunori Kihara More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.620AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a molecular chaperone abundantly expressed in mitochondria. TRAP1 negatively regulates oxidative phosphorylation by regulating the activity of the mitochondrial respiratory chain. TRAP1 also functions as an invasion-suppressor by modulating reactive oxygen species (ROS) in some cancer cell lines. Here, we investigated the functional and prognostic role of TRAP1 in upper tract urothelial carcinoma (UTUC). METHODS To investigate the role of TRAP1 in UC invasion, a loss-of-function and a gain-of-function approach were applied to a human UC cell line (5637 cells). Cell invasion was evaluated with a Matrigel Invasion Assay. Furthermore, TRAP1 expression was evaluated immunohistochemically in tumor specimens obtained from 62 UTUC patients (cTa-4N0M0) treated with radical nephroureterectomy alone. TRAP1 expression was visually scored on a scale of 0 to 3+. We analyzed the association of TRAP1 expression as well as clinicopathological factors including age, gender, pathological T stage, grade and lymph node involvement with cancer-specific survival (CSS). RESULTS We identified endogenous TRAP1 protein in mitochondrial preparations only. Transient TRAP1 knockdown enhanced cell invasion. This increased cell invasion was attenuated by ROS scavenging agent N-acetylcysteine, suggesting that ROS may contribute to enhanced cell invasion introduced through TRAP1 silencing. TRAP1-Flag overexpression hampered cell invasion . The impact of TRAP1 expression on cell invasion suggested an association between aggressiveness of UTUC and reduced TRAP1 expression. Of the total 62 patients, 33 (53%) showed low TRAP1 expression (0 and 1+). During a median follow-up of 60 months, 16 (48%) of 33 with lower TRAP1 expression (0 and 1+) and two (7%) of 29 with higher TRAP1 expression (2+ and 3+) died of cancer (5-y CSS of 52% vs. 95%; p < 0.001). On multivariate analysis, lower TRAP1 expression was an independent predictor of shorter CSS (HR 21.85, p < 0.001), along with grade (p < 0.001) and lymph node involvement (p = 0.014). CONCLUSIONS Reduced TRAP1 expression is associated with increased invasive potential via ROS modulation and poor prognosis in UTUC. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e166-e167 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Hiroshi Fukushima More articles by this author Soichiro Yoshida More articles by this author Fumitaka Koga More articles by this author Yasukazu Nakanishi More articles by this author Toshiki Kijima More articles by this author Junichiro Ishioka More articles by this author Yoh Matsuoka More articles by this author Noboru Numao More articles by this author Kazutaka Saito More articles by this author Yasuhisa Fujii More articles by this author Kazunori Kihara More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

DHCR24 is an Independent Predictor of Progression in Patients with Non-Muscle-Invasive Urothelial Carcinoma, and Its Functional Role is Involved in the Aggressive Properties of Urothelial Carcinoma Cells

The DHCR24 gene that encodes 3b-hydroxysterol Δ24-reductase, an oxidoreductase involved in cholesterol biosynthesis, has been identified as a progression-related gene based on the quantitative real-time PCR (qPCR) gene signature. Here, the functional role of DHCR24 and its clinical relevance in non-muscle-invasive urothelial carcinoma (NMIUC) were investigated. Primary NMIUC tissue specimens (n = 162) were analyzed by qPCR. Immunohistochemical staining was also performed on 63 subsets of NMIUC tissues. The present study was also undertaken in order to verify the effect of DHCR24 on human urothelial carcinoma cells. The mRNA expression levels of DHCR24 were significantly higher for patients in with higher grades of tumors than for those with lower grades of tumors (P = 0.003). Kaplan-Meier estimates revealed significant differences in the time to progression between low- and high-mRNA expression groups (log-rank test, P < 0.001). Multivariate Cox regression analysis revealed that the level of DHCR24 expression is an independent predictor of progression (hazard ratio, 5.464; 95 % confidence interval, 1.746-17.099; P = 0.004). The results of immunohistochemical staining were generally concordant with mRNA expression levels. Enforced expression of DHCR24 caused proliferation, adhesion, and migration, while DHCR24 loss resulted in slower proliferation and a reduction in cell viabilities compared with control cells. DHCR24 was found to be closely associated with progression among patients with NMIUC and showed aggressive properties in human UC cells.

Abstract 4066: Loss of Sh3gl2/endophilin A1 is an early event in urothelial carcinoma that regulates malignant behavior.

Abstract Urothelial carcinoma (UC) causes substantial morbidity and mortality worldwide. However, the molecular mechanisms underlying urothelial cancer development and tumor progression are still largely unknown. Using informatics analysis we identified Sh3gl2 (endophilin A1) as a transcript and protein highly enriched in bladder epithelium (urothelium). The gene encoding Sh3gl2 is located on chromosome 9p, a genomic region frequently altered in UC. Sh3gl2 is known to regulate endocytosis of receptor tyrosine kinases implicated in oncogenesis, such as the epidermal growth factor receptor (EGFR) and c-Met. However, its role in UC pathogenesis is unknown. Informatics analysis of expression profiles in Oncomine™, as well as immunohistochemical staining of tissue microarrays revealed Sh3gl2 expression to be decreased in UC specimens compared to non-tumor tissues. Loss of Sh3gl2 was associated with increasing tumor grade and with muscle invasion, a reliable predictor of metastatic disease and cancer-derived mortality. Sh3gl2 expression was undetectable in 15 of 16 human UC cell lines, but preserved in the low grade, well- differentiated cell line RT4. In UC cell lines lacking expression of Sh3gl2, treatment with the demethylating agent 5-aza-deoxycytidine led to upregulation of Sh3gl2 mRNA levels. Methylation-specific restriction analysis and PCR of genomic DNA revealed hypermethylation of a CpG island in the Sh3gl2 promoter in cells lacking Sh3gl2 expression, but no promoter methylation in RT4 cells that express Sh3gl2, suggesting that lack of Sh3gl2 expression results, at least in part, from methylation-induced gene silencing. Stable knockdown of Sh3gl2 in RT4 cells by RNA interference (i) enhanced proliferation and colony formation in vitro; (ii) inhibited EGF-induced EGFR internalization and increased EGFR phosphorylation; (iii) stimulated phosphorylation of Src family kinases and STAT3; (iv) altered sensitivity to chemotherapeutic agents, including the dual-specificity EGFR/ERBB2 inhibitor lapatinib; and (v) promoted formation of RT4 xenografts in subrenal capsule tissue recombination experiments. Together, these findings identify loss of Sh3gl2 as an early event in UC development that promotes disease progression. Sh3gl2 status in UC biopsies may provide a potential marker of risk for UC progression or recurrence and may enable determination of which patients require more aggressive intervention in the form of surgery or chemotherapy in order to improve prognosis. Citation Format: Shyama Majumdar, Edward Gong, Dolores Di Vizio, Jonathan Dreyfuss, David DeGraff, Martin Hager, Peter Park, Joaquim Bellmunt, Robert Matusik, Jonathan Rosenberg, Rosalyn Adam. Loss of Sh3gl2/endophilin A1 is an early event in urothelial carcinoma that regulates malignant behavior. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4066. doi:10.1158/1538-7445.AM2013-4066

587 LOSS OF BCG VIABILITY ADVERSELY AFFECTS THE DIRECT RESPONSE OF UROTHELIAL CARCINOMA CELLS TO BCG EXPOSURE

You have accessJournal of UrologyBladder Cancer: Basic Research (I)1 Apr 2013587 LOSS OF BCG VIABILITY ADVERSELY AFFECTS THE DIRECT RESPONSE OF UROTHELIAL CARCINOMA CELLS TO BCG EXPOSURE Guangjian Zhang, Fanghong Chen, Yanli Cao, Jacek Zielonka, Gang Cheng, Gopit Shah, Balaraman Kalyanaraman, and William See Guangjian ZhangGuangjian Zhang Milwaukee, WI More articles by this author , Fanghong ChenFanghong Chen Milwaukee, WI More articles by this author , Yanli CaoYanli Cao Milwaukee, WI More articles by this author , Jacek ZielonkaJacek Zielonka Milwaukee, WI More articles by this author , Gang ChengGang Cheng Milwaukee, WI More articles by this author , Gopit ShahGopit Shah Milwaukee, WI More articles by this author , Balaraman KalyanaramanBalaraman Kalyanaraman Milwaukee, WI More articles by this author , and William SeeWilliam See Milwaukee, WI More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1983AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Previous studies have demonstrated that in both the laboratory and clinical setting BCG viability is a variable that correlates with anti-tumor efficacy. Our group has demonstrated that loss of BCG viability reduces BCG induced cellular necrosis and HMGB1 release, a protein that is required for the in vivo anti-tumor effect of BCG. This study evaluated how loss of BCG viability impacts additional molecular and phenotypic intermediate endpoints that characterize, and/or contribute to, the direct effect of BCG on UC cells. METHODS Two human UC cell lines (253J amd T24) were used to study the effect of loss of BCG viability on the tumor cell response to BCG. The cellular response to BCG rendered non-viable by heat killing (hk) was compared to the response to viable BCG. The response endpoints evaluated included the induction of oxidative stress (iNOS expression), activation of intracellular signaling pathways (NFκB and NRF2), gene transactivation (IL6, IL8, CXCL1, CXCL3, CCL20, and CD54), and cytotoxicity (membrane integrity, LDH release, MTT). RESULTS Loss of viability (hkBCG) resulted in a quantitative decrease in the tumor response, relative to viable BCG, for all of the measured endpoints. The magnitude and statistical significance of the decrease in response varied by cell line and endpoint, ranging from 15% to 100% of the response to viable BCG. The attached table provides average fold changes for specific endpoints, relative to control cells,for the viable and hkBCG treatment groups. While quantitatively different non-viable BCG continued to induce responses that were qualitatively similar to BCG relative to untreated controls. CONCLUSIONS BCG viability is an important variable influencing the direct tumor cell response to BCG. Loss of BCG viability results in quantitative reductions in the magnitude of BCG's direct effects on UC cell biology. However non-viable BCG continues to have activity across the range of biologic endpoints evaluated in this study. Further study will be required to identify the underlying mechanism through which BCG's activity is attenuated as a consequence of loss of viability. Average fold change in tumor cell response endpoint for cells exposed to either viable or heat-killed BCG, relative to controls. iNOS NRF2 NFkB p21 Gene Expression LDH Release Vital Dye Exclusion Viable BCG 6.6 2.0 2.1 2.1 15 3.8 13 Heat Killed BCG 4.5 1.5 1.8 1.7 6 3.6 6 P Value (viable vs HK) = 0.15 < 0.01 = 0.3 < 0.01 < 0.05 = 0.3 < 0.001 © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e240 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Guangjian Zhang Milwaukee, WI More articles by this author Fanghong Chen Milwaukee, WI More articles by this author Yanli Cao Milwaukee, WI More articles by this author Jacek Zielonka Milwaukee, WI More articles by this author Gang Cheng Milwaukee, WI More articles by this author Gopit Shah Milwaukee, WI More articles by this author Balaraman Kalyanaraman Milwaukee, WI More articles by this author William See Milwaukee, WI More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

582 H2O2 GENERATION BY BCG CORRELATES WITH IN VITRO ANTITUMOR ACTIVITY

You have accessJournal of UrologyBladder Cancer: Basic Research (I)1 Apr 2013582 H2O2 GENERATION BY BCG CORRELATES WITH IN VITRO ANTITUMOR ACTIVITY Gopitkumar Shah, Guangjian Zhang, Fanghong Chen, Jacek Zielonka, Balaraman Kalyanaraman, and William See Gopitkumar ShahGopitkumar Shah milwaukee, WI More articles by this author , Guangjian ZhangGuangjian Zhang milwaukee, WI More articles by this author , Fanghong ChenFanghong Chen milwaukee, WI More articles by this author , Jacek ZielonkaJacek Zielonka milwaukee, WI More articles by this author , Balaraman KalyanaramanBalaraman Kalyanaraman milwaukee, WI More articles by this author , and William SeeWilliam See milwaukee, WI More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1978AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prior work has demonstrated that exposure of urothelial carcinoma cells to Bacille calmette Guerin (BCG) alters cellular redox status. The mechanism(s) contributing to these effects remains unclear. This study examined free radical production by BCG and its correlation with BCG viability and direct cytotoxicity. METHODS Global profiling of reactive oxygen (ROS) and nitrogen species (RNS) by BCG was carried out using specific fluorescent probes for H2O2, NO, and superoxide. Non-viable heat killed (hk) BCG was compared with viable BCG for ROS/RNS production. Free radical production by BCG/hkBCG was correlated with cellular response endpoints including gene transactivation, cell viability, LDH and HMGB1 release in two human UC cell lines (253J and T24). RESULTS Viable BCG was observed to produce H2O2 but not NO or superoxide. Loss of viability decreased production of H2O2 by 50% compared to viable BCG (p≤0.05). Live (media-cultured) BCG produced 1.5-fold more H2O2 then lyophilized-reconstituted BCG (p≤0.001). Storage of viable BCG at 4°C for 10 days did not significantly alter H2O2 production. The cellular response to BCG was significantly correlated with H2O2 production for all endpoints. Relative to viable BCG, hkBCG was significantly less effective in activation of 6 immune response genes (T24≤0.05, 253J ≤0.01). Viable BCG significantly increased the number of cells with loss of cytoplasmic membrane integrity compared to hkBCG (P≤0.0001). Viable BCG significantly increased HMGB1 release by 2 fold compared to hkBCG (p≤0.001). There was no significant difference in LDH release between viable and hkBCG (p=0.28). CONCLUSIONS The observation of H2O2 production by BCG is a previously unreported finding. Decreases in H2O2 production associated with loss of BCG viability, and its correlation with a diminished cellular response may explain the known dependence of a clinical treatment response on BCG viability. BCG production of H2O2 may also contribute to the previously reported BCG-induced alterations in cellular redox status. Further studies to mechanistically link BCG H2O2 production to treatment response are warranted. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e238 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gopitkumar Shah milwaukee, WI More articles by this author Guangjian Zhang milwaukee, WI More articles by this author Fanghong Chen milwaukee, WI More articles by this author Jacek Zielonka milwaukee, WI More articles by this author Balaraman Kalyanaraman milwaukee, WI More articles by this author William See milwaukee, WI More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Establishment and validation of a new semi-chronic dextran sulfate sodium-induced model of colitis in mice

BackgroundThe dextran sulfate sodium (DSS)-induced model of colitis is a commonly used model of inflammatory bowel disease (IBD) in animals. However, there were few studies on the therapeutic efficacy of drugs for IBD after the onset of colitis in this model. We established a semi-chronic model of DSS-induced colitis in mice and used it to assess the therapeutic efficacy of agents for IBD. Materials and methodsColitis was induced by administration of 3% DSS in drinking water to mice for 7days followed by 5days of normal drinking water. ResultsUlcerative colitis (UC)-like symptoms including diarrhea, bloody stools and body-weight loss were observed from days 3 to 5, and continued until day 12 after DSS administration. Persistent colitis was associated with sustained local production of cytokines and was characterized by infiltration of inflammatory cells, crypt loss and erosion in the distal colon. These features are similar to those found in patients with UC. In this model, anti-tumor necrosis factor (TNF)-α antibody or anti-interleukin (IL)-12/23p40 antibody significantly ameliorated colitis when administered after the onset of colitis. However, treatment with FK506, prednisolone or sulfasalazine provided limited therapeutic benefit. ConclusionThe DSS-induced colitis established here showed similar symptomatic and histopathological features to those seen in human UC. This model may be available for predicting the clinical efficacy of candidate compounds for UC.