Normal Human Urothelial Cell Line Research Articles

Effect of Arsenate and p-Phenylenediamine on the Expression of Aquaporins in Cultured Human Urothelial Cells.

Exposure to arsenic (As) orp‑phenylenediamine (PPD) can lead to dysfunction, or even cancer, in various types of organs, including the urinary bladder, yet the underlying mechanisms remain unclear. Aquaporins (AQPs) are widely expressed small water channel proteins that provide the major route for the transport of water and other small molecules across plasma membranes in diverse cell types. Altered expression of AQPs has been associated with pathologies in all major organs, including the urinary bladder. The presentin vitrostudy was performed as a first step towards exploring the possible involvement of AQPs in As- and PPD‑induced bladder diseases. An immortalized normal human urothelial cell line was employed. Cells were exposed to different concentrations of sodium arsenate (0‑20 μM) or PPD (0‑200 μM) for 48 h. Cell viability was subsequently assessed. The mRNA and protein expression levels of AQPs (specifically, AQP3, 4, 7, 9, and 11) were analyzed using reverse transcription‑quantitative polymerase chain reaction and Western blot analyses, respectively. The viability of the cells was decreased in a concentration-dependent manner upon exposure to arsenate. The mRNA and protein expression levels of AQP3, 4, 7, and 9 were substantially reduced, whereas the expression of AQP11 was largely unchanged. As for the experiments with PPD, treatment with increasing concentrations of PPD induced a gradual decrease in cell viability. The mRNA and protein expression levels of AQP3, 4, and 11 were generally unaltered; however, a marked reduction in the expression levels of AQP7 was observed, contrasting with a gradual concentration-dependent decrease in the expression of AQP9. The importance of the differential expression profiles of the AQPs induced by arsenate and PPD requires further investigation; nevertheless, the findings of the present study suggest that AQPs have a role in As‑ and PPD‑induced bladder diseases.

Deglycosylation Does Not Affect the Cytotoxic Action of Adenoviral Interferon α‐Induced Bystander Proteins

We previously reported an indirect effect of adenoviral interferon α (Ad‐IFNα) that is mediated by secreting bystander proteins (BSPs) and causing cytotoxic responses in both interferon α (IFNα)‐sensitive and –resistant cancer cells. Here, we investigated whether glycosylation of BSPs is necessary for the inhibition of IFNα‐resistant human bladder cancer cell line (KU7). The condition medium (CM) from the normal human urothelial cell line, TERT‐NHUC, was collected as described previously and incubated with and without N‐glycosidase F at 37°C (overnight). The growth inhibition of KU7 was compared between the control and experimental conditions. The proliferation of KU7 cells treated with interferon a protein (Intron A) is comparable to the cells treated with control TERT‐NHUC‐CM (without Ad‐IFNα infection) after 72 hr incubation. The growth of KU7 was significantly inhibited by the CM from Ad‐IFNα–infected TERT‐NHUC culture. N‐glycosidase F incubation effectively deglycosylated the BSPs in the CM (Ad‐IFNα TERT‐NHUC) as demonstrated by the band shifting in the PAGE gel verification. The deglycosylated CM (Ad‐IFNα TERT‐NHUC) produced a same degree of inhibition on the proliferation of KU7 cells (Fig 1). In conclusion, the cytotoxic effects of BSPs is independent of its status of glycosylation.

The Cytotoxicity Effect Study of Fosfomycin and Mecillinam Antibiotic on Multi-Drug Resistant Klebsiella pneumoniae from Infected Urothelial Tissue

Enterobacteriaceae bacteria are clinically important and lead to various diseases associated with high mortality and morbidity, including urinary tract infections, yet their treatment is a challenge due to increasing antibiotic resistance. This study examined the cytotoxicity effects and viability-inducing abilities of mecillinam and fosfomycin antibiotics in the extended-spectrum beta-lactamase Klebsiella pneumoniae strain. The immortalized normal human urothelial cell line was used and the urothelial cell line and bacteria using Klebsiella pneumoniae cultures were prepared. The bacterial association assays with the urothelial cell line were then conducted. The cytotoxic effects were examined using cytotoxicity assays of Klebsiella pneumoniae, cefotaxime-Munich, ampicillinase C, oxacillinase and transmission electron microscopy on the immortalized normal human urothelial cell line. The cytotoxicity of fosfomycin and mecillinam on cell line cytotoxic assays were measured by lactate dehydrogenase, a cell death marker and visualized by hematoxylin. Klebsiella pneumoniae strains showed significantly reduced association with urothelial cells than cefotaxime-Munich strains. The cefotaxime-Munich and ampicillinase C had a higher association with the urothelial cells than the oxacillinase and transmission electron microscopy strains. The cytotoxicity assays indicated that all Klebsiella pneumoniae strains had increased cytotoxicity after 24 or 12 h of incubation. Antibiotic resistance is increased by the bacterial strains that have genes encoding resistance, such as cefotaxime-Munich, ampicillinase C, transmission electron microscopy and oxacillinase.

Quantitative Proteomics of Urinary Bladder Cancer Cell Lines Identify UAP1 as a Potential Therapeutic Target.

Bladder carcinoma (BC) incidence and mortality rates are increasing worldwide. The development of novel therapeutic strategies is required to improve clinical management of this cancer. Aberrant protein expression may lead to cancer initiation and progression. Therefore, the identification of these potential protein targets and limiting their expression levels would provide alternative treatment options. In this study, we utilized a liquid-chromatography tandem mass spectrometry-based global proteomics approach to identify differentially expressed proteins in bladder cancer cell lines. A total of 3913 proteins were identified in this study, of which 479 proteins were overexpressed and 141 proteins were downregulated in 4 out of 6 BC cell lines when compared with normal human urothelial cell line (TERT-NHUC). We evaluated the role of UDP-N-acetylhexosamine pyrophosphorylase (UAP1) in bladder cancer pathogenesis. The silencing of UAP1 led to reduction in proliferation, invasion, colony formation and migration capability of bladder cancer cell lines. Thus, our study reveals UAP1 as a promising therapeutic target for bladder cancer.

Circular RNA SMARCA5 is overexpressed and promotes cell proliferation, migration as well as invasion while inhibits cell apoptosis in bladder cancer

Background: This study aimed to investigate the function of circular RNA SMARCA5 (circ-SMARCA5) on cell proliferation, apoptosis, migration and invasion in bladder cancer. Methods: Ten pairs of human bladder cancer tissue and adjacent tissue, five human bladder cancer cell lines (including TCCSUP, 5637, J82, UM-UC-3 and T-24) and normal human urothelial cell line SV-HUC-1, were obtained for the detection of circ-SMARCA5. Control overexpression and ShRNA, circ-SMARCA5 overexpression and ShRNA were constructed and transfected into UM-UC-3 cells as Control(+), Control(−), Circ-SMARCA5(+) and Circ-SMARCA5(−) groups. The role of circ-SMARCA5 was investigated in terms of cellular proliferation, apoptosis, migration, and invasion. Results: Circ-SMARCA5 was overexpressed in tumor tissue compared to paired adjacent tissue and it was also overexpressed in TCCSUP, 5637, J82 and UM-UC-3 cells compared to normal human urothelial cell line SV-HUC-1. In UM-UC-3 cells, cell proliferation ability, migration rate and invasion cell count were increased in Circ-SMARCA5(+) group compared to Control(+) group, while reduced in Circ-SMARCA5(−) group compared to Control(−) group. Regarding the cell apoptosis, apoptosis rate and apoptotic protein C-Caspase 3 expression were decreased in Circ-SMARCA5(+) group than those in Control(+) group but raised in Circ-SMARCA5(−) group compared to Control(−) group, meanwhile, the anti-apoptotic protein Bcl-2 expression was elevated in Circ-SMARCA5(+) group than that in Control(+) group but reduced in Circ-SMARCA5(−) group compared to Control(−) group. Conclusions: Circ-SMARCA5 is overexpressed, and promotes cell proliferation, migration and invasion, but represses apoptosis in bladder cancer.

Circular RNA SMARCA5 is overexpressed and promotes cell proliferation, migration as well as invasion while inhibits cell apoptosis in bladder cancer.

BackgroundThis study aimed to investigate the function of circular RNA SMARCA5 (circ-SMARCA5) on cell proliferation, apoptosis, migration and invasion in bladder cancer.MethodsTen pairs of human bladder cancer tissue and adjacent tissue, five human bladder cancer cell lines (including TCCSUP, 5637, J82, UM-UC-3 and T-24) and normal human urothelial cell line SV-HUC-1, were obtained for the detection of circ-SMARCA5. Control overexpression and ShRNA, circ-SMARCA5 overexpression and ShRNA were constructed and transfected into UM-UC-3 cells as Control(+), Control(−), Circ-SMARCA5(+) and Circ-SMARCA5(−) groups. The role of circ-SMARCA5 was investigated in terms of cellular proliferation, apoptosis, migration, and invasion.ResultsCirc-SMARCA5 was overexpressed in tumor tissue compared to paired adjacent tissue and it was also overexpressed in TCCSUP, 5637, J82 and UM-UC-3 cells compared to normal human urothelial cell line SV-HUC-1. In UM-UC-3 cells, cell proliferation ability, migration rate and invasion cell count were increased in Circ-SMARCA5(+) group compared to Control(+) group, while reduced in Circ-SMARCA5(−) group compared to Control(−) group. Regarding the cell apoptosis, apoptosis rate and apoptotic protein C-Caspase 3 expression were decreased in Circ-SMARCA5(+) group than those in Control(+) group but raised in Circ-SMARCA5(−) group compared to Control(−) group, meanwhile, the anti-apoptotic protein Bcl-2 expression was elevated in Circ-SMARCA5(+) group than that in Control(+) group but reduced in Circ-SMARCA5(−) group compared to Control(−) group.ConclusionsCirc-SMARCA5 is overexpressed, and promotes cell proliferation, migration and invasion, but represses apoptosis in bladder cancer.

Lipoxygenase Protein Expression and Its Effect on Oxidative Stress Caused by Benzidine in Normal Human Urothelial Cell Lines.

Metabolic activation of indirect-acting carcinogens in the target organ is an effective mechanism of carcinogenesis. Lipoxygenase (LOX) can co-oxidize the bladder carcinogen benzidine (BZ). However, it is not entirely clear whether BZ is activated and which enzyme is involved in its activation in bladder epithelial cells. Our results showed that BZ induced 5-LOX protein expression but had no significant influence on the expression of 15-LOX-2, CYP1B1, and CYP2E1 in SV-40 immortalized human uroepithelial SV-HUC-1 cells. BZ induced oxidative stress in SV-HUC-1 cells by increasing reactive oxygen species (ROS) and malondialdehyde levels significantly in the 100 and 200 μmol/L-BZ-treated groups and decreased the level of the antioxidant reduced glutathione significantly at 200 μmol/L BZ. Concurrently, the activity of catalase was increased, while the activity of superoxide dismutase was increased at 50 μmol/L BZ but gradually decreased with increasing concentrations of BZ ( P < 0.05). However, the oxidative stress and damage in SV-HUC-1 cells caused by BZ were effectively inhibited by the 5-LOX-specific inhibitor AA861 at 10 μmol/L. Thus, 5-LOX is probably the major LOX isozyme to co-oxidize exogenous chemicals in SV-HUC-1 cells. AA861 has a protective effect on the oxidative stress and damage induced by BZ in SV-HUC-1 cells. We conclude that BZ can be activated by 5-LOX to produce ROS and oxidative stress, which may be associated with bladder cancer caused by BZ.

Unique signalling connectivity of FGFR3-TACC3 oncoprotein revealed by quantitative phosphoproteomics and differential network analysis.

The FGFR3-TACC3 fusion is an oncogenic driver in diverse malignancies, including bladder cancer, characterized by upregulated tyrosine kinase activity. To gain insights into distinct properties of FGFR3-TACC3 down-stream signalling, we utilised telomerase-immortalised normal human urothelial cell lines expressing either the fusion or wild-type FGFR3 (isoform IIIb) for subsequent quantitative proteomics and network analysis. Cellular lysates were chemically labelled with isobaric tandem mass tag reagents and, after phosphopeptide enrichment, liquid chromatography-high mass accuracy tandem mass spectrometry (LC-MS/MS) was used for peptide identification and quantification. Comparison of data from the two cell lines under non-stimulated and FGF1 stimulated conditions and of data representing physiological stimulation of FGFR3 identified about 200 regulated phosphosites. The identified phosphoproteins and quantified phosphosites were further analysed in the context of functional biological networks by inferring kinase-substrate interactions, mapping these to a comprehensive human signalling interaction network, filtering based on tissue-expression profiles and applying disease module detection and pathway enrichment methods. Analysis of our phosphoproteomics data using these bioinformatics methods combined into a new protocol—Disease Relevant Analysis of Genes On Networks (DRAGON)—allowed us to tease apart pathways differentially involved in FGFR3-TACC3 signalling in comparison to wild-type FGFR3 and to investigate their local phospho-signalling context. We highlight 9 pathways significantly regulated only in the cell line expressing FGFR3-TACC3 fusion and 5 pathways regulated only by stimulation of the wild-type FGFR3. Pathways differentially linked to FGFR3-TACC3 fusion include those related to chaperone activation and stress response and to regulation of TP53 expression and degradation that could contribute to development and maintenance of the cancer phenotype.

Abstract 811: Induction of stem-like cells with malignant properties by chronic exposure of immortalized normal human urothelial cell line to arsenic

Abstract Although there is extensive epidemiologic evidence of increased risk for the development of urothelial carcinoma (UC) associated with arsenic exposure, the mechanisms by which arsenic participates in tumorigenesis are not well understood. To gain mechanistic insights, we recently developed an in vitro arsenic model in an immortalized normal human bladder cell line (HUC1) and in this study we performed extended phenotypic and molecular characterization of the arsenic exposed in vitro model. To assess the impact of arsenic exposure at a time progressive manner, we performed MTT assay using the arsenic exposed and unexposed HUC1 cells. The growth of the HUC1 cells was increased in a time dependent manner after arsenic exposure. In soft agar assay, colonies were observed only in arsenic exposed cells. Similarly, invaded cells in invasion assay were observed only in arsenic exposed cells. To identify whether arsenic exposure induce self-renewal properties, we performed spheres forming assay and our data showed arsenic exposed cells formed more spheres under serum-free medium and non-adherence condition, and the number gradually increased with longer periods of exposure. The sphere forming ability was maintained through two subsequent serial passages, indicating of self-renewal ability. The spheroid cells showed differentiated morphology when cultured in serum containing medium. Finally, to identify epigenetic events associated with the later phenotypic alterations, we performed illumina methylation array and expression array and determined a panel of genes that are epigenetically altered that may be related to arsenic-induced tumorigenesis. Among 29 genes that exhibited reduction of promoter methylation and up-regulation of the expression, 6 genes (EDNRA, GJB2, KRT17, GDF15, S100P, and LCN2) were reported as the oncogenes. Furthermore 2 genes (GJB2 and KRT17) were maintained the alteration even in withdrawal of arsenic for 2.5 months. While, among 13 genes that exhibited induction of promoter methylation and down-regulation of the expression, one gene (TSPYL5) as a tumor suppressor gene was maintained the alteration in withdrawal of arsenic for 2.5 months. In conclusion, chronic arsenic exposure induces malignant phenotype with the acquisition of stem-like activity, and epigenetic alteration might be partial mechanism of tumorigenesis. Validation and detail molecular understanding of later genes are ongoing to determine the arsenic associated pathway alterations in the genesis of UC. Citation Format: Akira Oki, Shahnaz Begum, Mariana Brait, David Sidransky, Mohammad Obaidul Hoque. Induction of stem-like cells with malignant properties by chronic exposure of immortalized normal human urothelial cell line to arsenic. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 811. doi:10.1158/1538-7445.AM2015-811

MiR-34a inhibits proliferation and invasion of bladder cancer cells by targeting orphan nuclear receptor HNF4G.

miR-34a is a member of the miR-34 family and acts as a tumor suppressor in bladder cancer. This study explored the regulative role of miR-34a on an orphan nuclear receptor HNF4G, which has a well-confirmed role in bladder tumor growth and invasion. qRT-PCR analysis was applied to measure miR-34a expression in two tumorigenic bladder cancer cell lines 5637 and T24 and one normal human urothelial cell line SV-HUC-1. Luciferase assay was performed to verify the putative binding between miR-34a and HNF4G. The influence of miR-34a-HNF4G axis on cell viability, colony formation, and invasion was assessed with loss- and gain-of-function analysis. This study observed that the miR-34a expressions in 5637 and T24 cells were significantly lower than in SV-HUC-1, while the muscle invasive cell sublines 5637-M and T24-M had even lower miR-34a expression than in the nonmuscle invasive sublines. HNF4G has a 3′-UTR binding site with miR-34a and is a direct downstream target of miR-34a. miR-34a can directly downregulate the expression of HNF4G and thus inhibit tumor cell viability, colony formation, and invasion. Therefore, miR-34a-HNF4G axis is an important pathway modulating cell viability, proliferation, and invasion of bladder cancer cells.

Expression of survivin in bladder cancer cell lines using quantitative real-time polymerase chain reaction

ObjectivePrevious studies have reported that survivin expression is significantly associated with various malignancies including bladder cancer. However, the relationship between the expression of survivin and the tumor stage and grade of bladder cancer still require further study. MethodsTo determine whether survivin plays a role in the differentiation of bladder cancer cells, we conducted a preliminary study to examine the expression of survivin in bladder cancer cell lines. ResultsIn this study, we observed that the gene expression fold changes of survivin ranged from 3.2 to 16.7 in various tumor grades (G1–G4) of bladder cancer cell lines, which were higher than that in normal human urothelial cell line. With the worse differentiation of bladder cancer cell lines, the gene expression fold changes of survivin increased significantly (3.2-fold in RT4, 5.8-fold in 5637, 6.6-fold in T24, and 16.7-fold in HT1197). In addition, we observed different genotypes among various cell lines (C/C in HUC4449, C/G in RT4, C/G in 5637, G/G in T24, and C/C in HT1197). The relationship between survivin −31 C/G polymorphism and various bladder cancer cell lines was non-significant. However, the overexpression of survivin may be associated with aggressive features of bladder cancer. ConclusionOur findings suggest that survivin could be a potential therapeutic target through the inhibition of cell proliferation in bladder cancer.

Direct gene transfer of adenoviral-mediated interferon α into human bladder cancer cells but not the bystander factors produced induces endoplasmic reticulum stress-related cytotoxicity

We have previously shown that adenoviral-mediated interferon α (Ad-IFNα) is cytotoxic both to cells that are sensitive to recombinant interferon α (IFNα) and to cells which are resistant to IFNα. The cancer cell-specific cytotoxic effects of Ad-IFNα involve three different mechanisms: 1. The direct effect of IFNα production causing cancer cell death in IFNα sensitive cells (1); 2. The direct effect of Ad-IFNα infection and high levels of IFNα expression in IFNα resistant cancer cells (2); and 3. The indirect effect of the Ad-IFNα bystander factors produced (2-4). After Ad-IFNα infection, the cells produce a large amount of perinuclear localized IFN protein. This protein over-load could be a major factor in the direct cancer cell death of those cells infected with Ad-IFNα compared with the indirect cytotoxic effects of the bystander factors produced. Here, we investigated whether a component of Ad-IFN-induced cell death involves protein overload-induced endoplasmic reticulum (ER) stress, using an IFNα-resistant human bladder cancer cell line (KU7), and the normal human urothelial cell line, TERT-NHUC, as preclinical models. We found that the two ER stress response pathways examined were activated in KU7 cells. In contrast, following treatment of the normal TERT-NHUC cells with Ad-IFNα, no ER stress signals were observed. In addition, no ER stress related changes were seen, when KU7 cells were exposed to conditioned medium from Ad-IFNα-treated KU7 cells, indicating that bystander produced cytotoxicity did not involve ER stress. After 24 h of Ad-IFNα infection, the KU7 cancer cells produced spliced X-box binding protein 1 and activating transcription factor 6 protein (ATF6), evoking an ER stress response that could contribute to Ad-IFNα induced apoptosis in these cancer cells. In addition, GADD153/CHOP, GADD34 and BAX were also subsequently modified following activation of the ER stress pathways, thereby signaling downstream effectors in a pro-apoptotic manner.

Xanthine oxidase inhibitory terpenoids of Amentotaxus formosana protect cisplatin-induced cell death by reducing reactive oxygen species (ROS) in normal human urothelial and bladder cancer cells

The diterpenoids (+)-ferruginol ( 1), ent-kaur-16-en-15-one ( 2), ent-8(14),15-sandaracopimaradiene-2α,18-diol ( 3), 8(14),15-sandaracopimaradiene-2α,18,19-triol ( 4), and (+)-sugiol ( 5) and the triterpenoids 3 β-methoxycycloartan-24(24 1)-ene ( 6), 3 β,23 β-dimethoxycycloartan-24(24 1)-ene ( 7), 3 β,23 β-dimethoxy-5α-lanosta-24(24 1)-ene ( 8), and 23( S)-23-methoxy-24-methylenelanosta-8-en-3-one ( 9), isolated from Amentotaxus formosana, showed inhibitory effects on xanthine oxidase (XO). Of the compounds tested, compound 5 was a potent inhibitor of XO activity, with an IC 50 value of 6.8 ± 0.4 μM, while displaying weak ABTS radical cation scavenging activity. Treatment of the bladder cancer cell line, NTUB1, with 3–10 μM of compound 5 and 10 μM cisplatin, and immortalized normal human urothelial cell line, SV-HUC1, with 0.3–1 μM and 10–50 μM of compound 5 and 10 μM cisplatin, respectively, resulted in increased viability of cells compared with cytotoxicity induced by cisplatin. Treatment of NTUB1 with 20 μM cisplatin and 10 or 30 μM of compound 5 resulted in decreased ROS production compared with ROS production induced by cisplatin. These results indicate that 10 or 30 μM of compound 5 in NTUB1 cells may mediate through the suppression of XO activity and reduction of reactive oxygen species (ROS) induced by compound 5 cotreated with 20 μM cisplatin and protection of subsequent cell death.

Abstract 1502: Adenoviral-mediated interferon α induces endoplasmic reticulum stress related cytotoxicity in human cancer but not normal cells

Abstract We have previously shown that adenoviral-mediated interferon α (Ad-IFNα) treatment is specifically cytotoxic to various cancer cells, not only to the cancer cells sensitive to the interferon protein (IFNα), but also to the cells which are resistant to IFNα. We also found that there is very little cytotoxic effect of Ad-IFNα on non-cancer cells. The cancer cell specific toxic effects of Ad-IFNα can be classified into three different mechanisms: 1. Expression and secretion of interferon α protein that results in kill of IFNα sensitive cancer cells; 2. Direct kill of IFNα resistant cancer cells by Ad-IFNα and 3. Cancer cell kill by Ad-IFNα produced bystander factors. After Ad-IFNα infection, the host cells produce a large amount of IFN protein. We hypothesized that this protein over-load is cytotoxic to cancer cells and may be a major reason for Ad-IFNα direct effect. We now report that Ad-IFNα infection induces ER stress and ER stress related apoptosis in human cancer but not normal urothelial cells. In addition we found that the Ad-IFNα produced bystander cytotoxicity was not related to ER stress. To investigate whether Ad-IFNα induced cancer specific cytotoxicity was correlated with the activation of ER stress pathways, the expression of several markers of ER stress was studied. Experiments utilized the interferon resistant human bladder cancer cell line, KU7, and the normal human urothelial cell line, TERT-NHU, for the studies. We found that three ER stress response pathways examined were activated in KU7 cells. In contrast, the ER stress response pathways remained silent in the normal TERT-NHU cells or in KU7 cells treated with conditioned medium from Ad-IFNα treated KU7 cells (to test the effect of bystander factor produced cytotoxicity) the ER stress response pathways remained silent. After 24hr of Ad-IFNα exposure, KU7 cancer cells produced spliced X-box binding protein 1 (sXBP1), activating transcription factor 4 (ATF4) and activating transcription factor (ATF6) protein, and evoked an ER stress response that contribute to Ad-IFNα induced apoptosis in cancer cells. In addition, we found GADD153/CHOP and GADD34 were also upregulated following the activation of the three ER stress pathways, thereby signaling downstream effectors in a pro-apoptotic manner. Ad-IFNα recently has been used in Phase I clinical trial for patient with superficial bladder cancer. The results presented here could provide a mechanistic basis for Ad-IFNα action and support the positive clinical results obtained to date. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1502.

Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion

Purpose Testis specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, is essential for the growth of spermatocytes and cancer cells. We investigated the association of HSP70-2 expression with clinical behaviour and progression of urothelial carcinoma of bladder. Experimental design We assessed the HSP70-2 expression by RT-PCR and HSP70-2 protein expression by immunofluorescence, flow cytometry, immunohistochemistry and Western blotting in urothelial carcinoma patient specimens and HTB-1, UMUC-3, HTB-9, HTB-2 and normal human urothelial cell lines. Further, to investigate the role of HSP70-2 in bladder tumour development, HSP70-2 was silenced in the high-grade invasive HTB-1 and UMUC-3 cells. The malignant properties of urothelial carcinoma cells were examined using colony formation, migration assay, invasion assay in vitro and tumour growth in vivo. Results Our RT-PCR analysis and immunohistochemistry analysis revealed that HSP70-2 was expressed in both moderate to well-differentiated and high-grade invasive urothelial carcinoma cell lines studied and not in normal human urothelial cells. In consistence with these results, HSP70-2 expression was also observed in superficially invasive (70%) and muscle-invasive (90%) patient’s tumours. Furthermore, HSP70-2 knockdown significantly suppressed cellular motility and invasion ability. An in vivo xenograft study showed that inhibition of HSP70-2 significantly suppressed tumour growth. Conclusions In conclusion, our data suggest that the HSP70-2 expression is associated with early spread and progression of urothelial carcinoma of bladder cancer and that HSP70-2 can be the potential therapeutic target for bladder urothelial carcinoma.

Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT)

BackgroundThe glycosylphosphatidylinositol (GPI)-anchored epithelial extracellular membrane serine protease prostasin (PRSS8) is expressed abundantly in normal epithelia and essential for terminal epithelial differentiation, but down-regulated in human prostate, breast, and gastric cancers and invasive cancer cell lines. Prostasin is involved in the extracellular proteolytic modulation of the epidermal growth factor receptor (EGFR) and is an invasion suppressor. The aim of this study was to evaluate prostasin expression states in the transitional cell carcinomas (TCC) of the human bladder and in human TCC cell lines.MethodsNormal human bladder tissues and TCC on a bladder cancer tissue microarray (TMA) were evaluated for prostasin expression by means of immunohistochemistry. A panel of 16 urothelial and TCC cell lines were evaluated for prostasin and E-cadherin expression by western blot and quantitative PCR, and for prostasin gene promoter region CpG methylation by methylation-specific PCR (MSP).ResultsProstasin is expressed in the normal human urothelium and in a normal human urothelial cell line, but is significantly down-regulated in high-grade TCC and lost in 9 (of 15) TCC cell lines. Loss of prostasin expression in the TCC cell lines correlated with loss of or reduced E-cadherin expression, loss of epithelial morphology, and promoter DNA hypermethylation. Prostasin expression could be reactivated by demethylation or inhibition of histone deacetylase. Re-expression of prostasin or a serine protease-inactive variant resulted in transcriptional up-regulation of E-cadherin.ConclusionLoss of prostasin expression in bladder transitional cell carcinomas is associated with epithelial-mesenchymal transition (EMT), and may have functional implications in tumor invasion and resistance to chemotherapy.

Adenovirus-mediated gene therapy for bladder cancer: efficient gene delivery to normal and malignant human urothelial cells in vitro and ex vivo.

Existing local therapies for superficial transitional cell carcinoma (TCC) of the bladder have limited success in preventing progression to life-threatening, muscle-invasive disease, and novel therapies are needed. Recent studies have raised doubts concerning the feasibility of adenovirus-mediated gene therapy for bladder cancer. We have therefore investigated adenoviral transduction of normal and malignant human urothelial cells, both as primary cultures and in intact epithelium. All 15 primary normal human urothelial cell lines tested were transduced in vitro by Adv-cmv-beta-gal at high efficiency, and better than most human TCC cell lines. Eight primary human TCC explants were also successfully transduced. In contrast, in intact normal urothelium, transduction efficiency was lower, and occurred only in superficial epithelial layers. Expression of the hCAR adenovirus receptor, however, occurred throughout the full thickness of urothelium. Transduction of human TCC biopsy specimens was at least as efficient as intact normal urothelium.We demonstrate for the first time that adenoviral transduction of both normal and malignant human urothelial cells is feasible. A physical barrier, rather than hCAR status, may be the main determinant of transduction of intact epithelium. Clinical trials of adenovirus-mediated gene therapy for superficial bladder cancer are warranted.