Human Urothelial Cell Lines Research Articles

Abstract 5298: Regulation of urothelial bladder cancer by nicotine and stress neurotransmitters

Abstract Urothelial bladder cancer (UBC) is the 7th most common cancer in men and the 17th most common cancer in women. Smoking is an established risk factor for UBC. However, the mechanisms how smoking induces bladder cancer are poorly understood. Using two established human urothelial bladder cancer cell lines, our data show that nicotine significantly increased the proliferation of both cell lines. Both cell lines produced the stress neurotransmitters norepinephrine and epinephrine and nicotine further enhanced this activity. The broad-spectrum beta-blocker propranolol strongly inhibited base level and nicotine-induced proliferation in both cell lines, identifying beta-adrenergic receptors as mediators. Proliferation in response to exogenous addition of epinephrine, norepinephrine or the selective β-adrenergic agonist isoproterenol and complete blockage of these responses by propranolol support this interpretation. Treatment with the inhibitory neurotransmitter γ-aminobutyric acid (GABA), that inhibits beta-adrenergic receptor-mediated proliferation of other cancer types via inhibition of cAMP formation, was less effective. These findings suggest that urothelial bladder cancer cells are stimulated in their growth via beta-adrenergic receptor signaling independent of cAMP by nicotine or psychological stress. Additional investigations are underway to further dissect the signal transduction pathways involved. Supported by the State of Tennessee Center of Excellence in Livestock Diseases and Human Health. Citation Format: Arokya M. Papu John, Jheelam Banerjee, Hildegard M. Schuller. Regulation of urothelial bladder cancer by nicotine and stress neurotransmitters. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5298. doi:10.1158/1538-7445.AM2014-5298

Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors.

While DNA damage response pathways are well characterized in cancer cells, much less is known about their status in normal cells. These pathways protect tumour cells from DNA damage and replication stress and consequently present potential therapeutic targets. Here we characterize the response of human telomerase reverse transcriptase (hTERT)-immortalized normal human urothelial (NHU) and bladder cancer cell lines to agents that disrupt the DNA damage response. Effects of replication and DNA damage response inhibitors on cell cycle progression, checkpoint induction and apoptosis were analysed in hTERT-NHU and bladder cancer cell lines. The primary signalling cascade responding to replication stress in malignant cells (ataxia telangiectasia-mutated (ATM) and Rad3-related-checkpoint kinase 1 (ATR-CHK1)) is not activated in hTERT-NHU cells after treatment with a replication inhibitor and these cells do not depend upon CHK1 for protection from apoptosis during replication stress. Instead, ATM signalling is rapidly activated under these conditions. Intriguingly, an ATM inhibitor suppressed S-phase checkpoint activation after exposure to replication inhibitors and stopped entry of cells into S-phase indicating G1 checkpoint activation. Consistent with this, hTERT-NHU cells treated with the ATM inhibitor showed increased levels of cyclin-dependent kinase inhibitor p19(INK4D), reduced levels of cyclin D1 and CDK4, and reduced phosphorylation of the retinoblastoma protein. In contrast, a bladder cancer cell line cotreated with ATM and replication inhibitors progressed more slowly through S phase and showed a marked increase in apoptosis. Taken together, our findings suggest that ATM and CHK1 signalling cascades have different roles in tumour and normal epithelial cells, confirming these as promising therapeutic targets.

Human urothelial cell lines as potential models for studying cannabinoid and excitatory receptor interactions in the urinary bladder

To characterize human urothelial cell lines' cannabinoid receptor expression and evaluate their possible use for studying signalling interactions with purinergic and muscarinic receptor activation. PCR was used to detect cannabinoid (CB), muscarinic and purinergic receptor transcripts in HCV29 and UROtsa cells, whilst immunofluorescence evaluated protein expression and localization of cannabinoid receptors. The effect of CB1 agonist (ACEA) on carbachol- and ATP-induced changes in intracellular calcium ([Ca(2+)]i) levels was measured using fluorimetry. The ability of ACEA to reduce intracellular cAMP was investigated in HCV29 cells. CB1 and GPR55 receptor transcripts were detected in HCV29 and UROtsa cells, respectively. Immunofluorescence showed positive staining for CB1 in the HCV29 cells. Both cell lines expressed transcript levels for muscarinic receptors, but carbachol did not raise [Ca(2+)]i levels indicating a lack or low expression of G(q)-coupled muscarinic receptors. Transcripts for purinergic receptors were detected; ATP significantly increased [Ca(2+)]i in HCV29 and UROtsa cells by 395 ± 61 and 705 ± 100 nM (mean ± SEM, n = 6), respectively. ACEA did not alter ATP-induced [Ca(2+)]i or cAMP levels in HCV29 cells. Whilst HCV29 cells expressed CB1 and UROtsa cells expressed GPR55 receptors, these were not functionally coupled to the existing purinergic-driven increase in Ca2+ as such they do not represent a good model to study signalling interactions.

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.

Effect of small interfering RNA targeting survivin on human bleed urothelial carcinoma cell line T24

Objective Down regulating the expression of survivin mRNA and survivinprotein in the human bleed urothelial carcinoma cell line (T24) to observe its affect to the proliferation and apoptosis of T24 cells.Methods The eukaryotic expression plasmids targeting survivin [psilencer-survivin-small interfering RNA (siRNA),si-Sl,si-S2,si-S3] and negative control plasmid (psilencer-random target sequence-siRNA,si-GL3) were constructed simultaneously,and transfected these plasmids to T24 cells.The expression of survivin mRNA and survivin protein were detected by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting; the vitality of T24 cells was examined by cell counting kit-8 (CCK-8) assay; the apoptosis of T24 cells were tested by flow cytometry; the Caspase-3 activity was detected by specific colour test.Results In si-S3 treatment,the expression of survivin mRNA and survivin protein decreased significantly 79％ and 80％,cytoactivity declined with depended time and concentration,cell apoptosis increased 20.96％ with increased 85％ Caspase-3 activity,as compared with the control group (every P ＜ 0.05).Conclusion Downregulating survivin could effectively inhibit the proliferation and induce apoptosis of T24cells,which mechanism may be active Caspase-3 in T24 cells. Key words: Small interfere RNA; survivin; Human bleed urothelial carcinoma cell line

The effect of photochemical internalization of bleomycin in the treatment of urothelial carcinoma of the bladder: An in vitro study

ObjectivesIn this in vitro study, we determined whether meso-tetraphenyl chlorin disulphonate (TPCS2a)-based photochemical delivery of bleomycin was able to potentiate the cytotoxicity of bleomycin on bladder cancer cells. Materials and methodsThe human RT4, RT112, 253J, T24, and rat AY-27 urothelial carcinoma cell lines were used. Cells were seeded in 96-well plates. TPCS2a was added to the growth medium and the plates were incubated overnight. Cells were then resuspended in TPCS2a-free culture medium and incubated for 3 hours. Subsequently, cells were treated for 60 minutes with increasing doses of epirubicin, gemcitabine, mitomycin C, or bleomycin followed by illumination for different periods. Cell viability was measured with a colorimetric assay after 72 hours. ResultsFor the single treatments, in all 5 cell lines a dose-dependent inhibition of cell proliferation was observed. This was seen both after treatment with TPCS2a-based photodynamic therapy (PDT), as well as after treatment with either bleomycin or one of the control chemotherapeutic agents.After treatment with PDT (240-s illumination), bleomycin 9.0µM, and the combination of these treatments, relative survival percentages were 89.2±13.0, 70.2±8.9, and 30.5±6.1, respectively, in the T24 cell line. After treatment with PDT (120-s illumination), bleomycin 27µM and the combination of these treatments, relative survival percentages were 93.6±15.7, 74.7±9.6, and 30.0±11.1, respectively, in the AY-27 cell line. In both cell lines, PDT combined with bleomycin showed significantly (P<0.001) higher cell kill than the sum of the single treatments, suggesting a photochemical internalization effect. ConclusionsTPCS2a-based photochemical internalization of bleomycin showed a significant, at least, additive antiproliferative activity against human and rat urothelial carcinoma cells in vitro. Thus, photochemical internalization may have therapeutic potential as an intravesical strategy against bladder cancer. As the effect is heterogeneous, biomarker studies are warranted to be able to predict the effects of a photochemical internalization–based treatment.

Loss of Bacillus Calmette-Guérin Viability Adversely Affects the Direct Response of Urothelial Carcinoma Cells to Bacillus Calmette-Guérin Exposure

The attenuated mycobacterium bacillus Calmette-Guérin is widely used as intravesical immunotherapy for nonmuscle invasive urothelial carcinoma. Previous studies demonstrated that in the laboratory and clinical settings bacillus Calmette-Guérin viability is a variable that correlates with antitumor efficacy. We evaluated how loss of viability impacted a number of molecular and phenotypic intermediate end points that characterize and/or contribute to the direct effect of bacillus Calmette-Guérin on urothelial carcinoma cells. We studied the effect of loss of bacillus Calmette-Guérin viability on the tumor cell response to the treatment in 2 human urothelial carcinoma cell lines. The cellular response to bacillus Calmette-Guérin rendered nonviable by heat killing was compared to the response to viable bacillus. Response end points included the induction of oxidative stress, activation of intracellular signaling pathways, gene transactivation and phenotypic changes. Loss of viability resulted in a quantitative decrease in the tumor cell response relative to that of viable bacillus Calmette-Guérin for all measured end points. The decrease in response varied by cell line, ranging from 15% to 100% of the response to viable bacillus. While responses were quantitatively different, nonviable bacillus continued to induce responses that were qualitatively similar to those of bacillus Calmette-Guérin relative to that in untreated controls. Bacillus Calmette-Guérin viability is an important variable influencing the direct tumor cell response to the treatment. Although the magnitude of its effects are attenuated, heat killed bacillus Calmette-Guérin remains active for the induction of bacillus Calmette-Guérin responsive biological end points.

Dual role of TGFBR3 in bladder cancer

Bladder cancer is one of the most common genitourinary malignant diseases worldwide. More than 90% of bladder cancer cases are bladder urothelial carcinoma (BUC). Although transforming growth factor-β III receptor (TGFBR3) has been suggested to play a dual role in cancer progression, little is known about TGFBR3 in bladder cancer. In the present study, fresh tumor and the corresponding paracarcinoma tissue specimens were collected from 56 bladder urothelial carcinoma patients. TGFBR3 expression in these tissues was determined by western blotting. TGFBR3 was also detected in the human normal urothelial cell line SV-HUC-1, the human superficial urothelial bladder cancer cell line 5637, and the human invasive bladder cancer cell line T24 using western blotting and quantitative PCR. Cell growth, motility and invasion were also analyzed in the control and the TGFBR3 gene-silenced T24 cells. As a result, the expression of TGFBR3 was reduced (18/30) in most superficial bladder urothelial carcinoma tissues compared to the corresponding normal tissues, whereas TGFBR3 expression was more enhanced (19/26) in the invasive samples. Similarly, an increase of TGFBR3 expression was found in T24 cells, but a decrease was observed in 5637 cells. Knockdown of TGFBR3 in T24 cells resulted in decreased cell growth, motility and invasion. In conclusion, these findings suggest that TGFBR3 may play a dichotomous role in human bladder cancer, acting as both a tumor suppressor and as a tumor promoter.

Long-Term Exposure to Cigarette Smoke Extract Induces Hypomethylation at the RUNX3 and IGF2-H19 Loci in Immortalized Human Urothelial Cells

Cigarette smoking is the single most important epidemiological risk factor for bladder cancer but it is not known whether exposure of urothelial cells to the systemic soluble contents of cigarette smoke is directly causative to bladder cancer and the associated epigenetic changes such as tumor suppressor gene hypermethylation. We undertook this study to investigate if long-term treatment of human urothelial cells with cigarette smoke extract (CSE) results in tumor suppressor gene hypermethylation, a phenotype that was previously associated with long-term constant CSE treatment of airway epithelial cells. We chronically treated an immortalized human urothelial cell line UROtsa with CSE using a cyclic daily regimen but the cells were cultured in CSE-free medium between daily treatments. Bisulfite sequencing and real-time PCR array-based methylation profiling were employed to evaluate methylation changes at tumor suppressor gene loci in the chronically CSE-treated cells versus the passage-matched untreated control cells. The RUNX3 tumor suppressor gene promoter was hypomethylated with a significant increase in proportion of the completely unmethylated haplotype after the long-term CSE treatment; whereas RUNX3 promoter hypermethylation was previously reported for bladder cancers of smokers. Hypomethylation induced by the long-term CSE treatment was also observed for the IGF2-H19 locus. The methylation status at the PRSS8/prostasin and 16 additional loci however, was unaffected by the chronic CSE treatment. Transient CSE treatment over 1 daily regimen resulted in transcriptional down-regulation of RUNX3 and H19, but only the H19 transcription was down-regulated in the chronically CSE-treated urothelial cells. Transcription of a key enzyme in one-carbon metabolism, dihydrofolate reductase (DHFR) was greatly reduced by the long-term CSE treatment, potentially serving as a mechanism for the hypomethylation phenotype via a reduced supply of methyl donor. In conclusion, chronic cyclic CSE treatment of urothelial cells induced hypomethylation rather than hypermethylation at specific loci.

Cross-contamination of a UROtsa stock with T24 cells--molecular comparison of different cell lines and stocks.

BackgroundUROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity.MethodsUROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied.ResultsAll tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly.ConclusionsIt is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.

Inhibition of presenilins attenuates proliferation and invasion in bladder cancer cells through multiple pathways

ObjectivePresenilin (PS)/γ-secretase is a key protease that initiates various biological processes. We investigated the effect of PS/γ-secretase on the expression and inhibition of urothelial cell carcinoma of bladder (UCB) as a potential alternative therapeutic target for UCB. Materials and methodsPS-1 and PS-2 were identified in normal and malignant human bladder transitional cells by immunohistochemistry. We blocked PSs using a PS/γ-secretase inhibitor N-(N-[3,5-difluorophenacetyl]-L-alanyl)-S-phenylglycine-t-butylester (DAPT), and the proliferative and invasive potential of UCB cells SW780, BIU-87, 5637, and T24, and human normal urothelial cell line SV-HUC-1 were analyzed using Western blot, cell viability test, flow cytometry, and transwell assay. All experiments were repeated at least 3 times. ResultsHuman bladder samples of UCB, SW780, BIU-87, 5637, and T24 cells expressed higher PS-1 compared with normal ones. Cell vitality test demonstrated that DAPT attenuated UCB cell proliferation more than SV-HUC-1. Flow cytometry and transwell assay showed that T24 cells were arrested at G1/S checkpoint and its invasive ability was impaired. Western blot assay markedly showed that protein levels of CD44-intracellular domain, insulinlike growth factor-1Rβ, extracellular regulated protein kinase 1/2, cyclin D1, proliferating cell nuclear antigen, and matrix metalloproteinase-9 were downregulated by DAPT, whereas vascular endothelial growth factor receptor-2 and vascular endothelial growth factor-165 were upregulated. ConclusionsOur study revealed that PS-1 might be implicated in the proliferation and invasion of UCB, and that it may serve as a potential therapeutic target for UCB, but further studies are warranted to verify the effects of inhibition of PS/γ-secretase on angiogenesis.

Abstract 1960: Implication of miR-34a in radioresistance of bladder cancer .

Abstract Purpose : Investigate resistance of radiation via miR-34a expression in human urothelial carcinoma Methods : Human urothelial carcinoma cell lines derived from well-differentiated superficial bladder tumors as well as from high-grade invasive tumors were screened for their sensitivity to radiation. Growth inhibition was monitored by clonogenic assays. qRT-PCR analysis was used to evaluate expression of miR-34a among the human urothelial carcinoma cell lines. Methylation-specific polymerase chain reaction (MSP) was performed to determine CpG methylation status of miR-34a gene promoter among the cell lines. Association between miR-34a and sensitivity to radiation was also investigated by ectopic expression of miR-34a. Western blot was performed to evaluate protein expression. Results: Remarkable differences in sensitivity to radiation have been shown among those cell lines in vitro. Of interest, miR-34a expression correlates with human bladder cancer cell response to radiation, with resistant cell lines showing lower expression of miR-34a and that is due to CpG methylation of its promoter. Additionally, an inverse correlation was observed between miR-34a and expression of sirtuin1 (SIRT1). Ectopic expression of miR-34a in cell lines resistant to radiation showed a positive effect of miR-34a to the response therapy. Growth inhibition observed after treatment could be explained, in part, by the increased expression of the cell cycle inhibitor p21. Conclusion: Preliminary results suggest that detection of miR-34a expression may potentially be used as a predictor of therapeutic efficacy, and its regulation represent an attractive mechanism for the management of bladder cancer therapy. Citation Format: Jose J. Mansure, Roland Nassim, Wassim Kassouf. Implication of miR-34a in radioresistance of bladder cancer . [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 1960. doi:10.1158/1538-7445.AM2013-1960

Contributors to HMGB1 Release by Urothelial Carcinoma Cells in Response to Bacillus Calmette-Guérin

Our group previously noted that HMGB1 release by urothelial carcinoma cells occurs as a consequence of bacillus Calmette-Guérin induced nonapoptotic cell death. Additional studies demonstrated that HMGB1 release in response to bacillus Calmette-Guérin is required for the invivo tumor response to bacillus Calmette-Guérin. We evaluated the steps required for HMGB1 release by human urothelial carcinoma cells in response to bacillus Calmette-Guérin exposure. We used the T24 and 253J human urothelial carcinoma cell lines. HMGB1 concentrations in cell culture supernatant with and without bacillus Calmette-Guérin treatment served as the principal end point to assess the role of potentially involved variables. Specific techniques were used to determine the role of α5β1 antigen receptor cross-linking, TLR signaling, inducible nitric oxide synthase expression/nitric oxide production, p21 expression, and bacillus Calmette-Guérin adherence, internalization and viability. Cross-linking of α5β1 integrin or signaling through TLR2/4 did not contribute to HMGB1 release. Optimal HMGB1 release required bacillus Calmette-Guérin adherence and internalization. Bacillus Calmette-Guérin viability correlated with the magnitude of HMGB1 release. Inhibition of oxidative stress and p21 expression in response to bacillus Calmette-Guérin decreased the magnitude of HMGB1 release. Bacillus Calmette-Guérin induced nonapoptotic cell death and HMGB1 release occur as a consequence of a complex multistep process. Understanding the steps and mechanisms involved in the induced HMGB1 release would provide an opportunity for targeted strategies to improve bacillus Calmette-Guérin treatment efficacy.

IGFBP-5 overexpression as a poor prognostic factor in patients with urothelial carcinomas of upper urinary tracts and urinary bladder

BackgroundUrothelial carcinoma (UC) is prevalent worldwide. Dysregulation of cell growth is a critical event of tumorigenesis and has not been assessed systemically in UC. We thus assessed the published transcriptome...

591 THE EFFECT OF PHOTOCHEMICAL INTERNALIZATION OF BLEOMYCIN IN THE TREATMENT OF UROTHELIAL CARCINOMA: AN IN VITRO STUDY

You have accessJournal of UrologyBladder Cancer: Basic Research (I)1 Apr 2013591 THE EFFECT OF PHOTOCHEMICAL INTERNALIZATION OF BLEOMYCIN IN THE TREATMENT OF UROTHELIAL CARCINOMA: AN IN VITRO STUDY Harm C. Arentsen, Jos Falke, Anders Høgset, Egbert Oosterwijk, and J. Alfred Witjes Harm C. ArentsenHarm C. Arentsen Nijmegen, Netherlands More articles by this author , Jos FalkeJos Falke Nijmegen, Netherlands More articles by this author , Anders HøgsetAnders Høgset Oslo, Norway More articles by this author , Egbert OosterwijkEgbert Oosterwijk Nijmegen, Netherlands More articles by this author , and J. Alfred WitjesJ. Alfred Witjes Nijmegen, Netherlands More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1987AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Photochemical internalization (PCI) is a novel light-directed drug delivery system. It triggers the release of endocytosed drugs into the cytosol. Thus the drugs can reach their intracellular target of action, realizing their therapeutic potential, instead of being degraded by lysosomal hydrolases. The PCI effect is achieved with photosensitizing compounds specifically localized in the membranes of endocytic vesicles, destroying these membranes by an oxidative process after illumination. Bleomycin is used in multiple standard cancer chemotherapy regimens. The relatively large and hydrophilic chemical structure limits its ability to penetrate membranes, which causes accumulation in endocytic vesicles. PCI of bleomycin can induce a synergistic inhibition of tumor growth in different animal tumor models. In this in vitro study we determined whether meso-tetraphenyl chlorin disulphonate (TPCS2a)-based photochemical delivery of bleomycin was able to potentiate the cytotoxicity of bleomycin on bladder cancer cell lines. Epirubicin, gemcitabine and mitomycin C were used as controls. METHODS The human RT4, RT112, 253J, T24 and rat AY-27 urothelial carcinoma cell lines were used in this study. Cells were seeded in 96-well plates. TPCS2a was added to the growth medium and the plates were incubated overnight. Cells were then resuspended in TPCS2a-free culture medium and incubated for 3 h. Subsequently, cells were treated for 60 min with increasing doses of epirubicin, gemcitabine, mitomycin C or bleomycin followed by illumination for different periods (0-5 min) to obtain dose-response curves. Cell viability was measured with a colorimetric assay after 72 hours. RESULTS For the single treatments, in all five cell lines a dose dependent inhibition of cell proliferation was observed. This was seen both after treatment with TPCS2a-based PDT alone as well as after treatment with either bleomycin or one of the control chemotherapeutic agents alone. For the combination treatments, a significant (p<0,001) synergistic effect, i.e. photochemical internalization effect, was only observed for PDT combined with bleomycin in the human T24 cell line and the rat AY-27 cell line. CONCLUSIONS TPCS2a-based photochemical internalization of bleomycin showed a significant synergistic antiproliferative activity against human and rat urothelial carcinoma cells in vitro. Thus, PCI may have therapeutic potential as an intravesical strategy against NMIBC. Since the effect is heterogeneous, further animal studies to investigate the in vivo effects seem justified. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e242 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Harm C. Arentsen Nijmegen, Netherlands More articles by this author Jos Falke Nijmegen, Netherlands More articles by this author Anders Høgset Oslo, Norway More articles by this author Egbert Oosterwijk Nijmegen, Netherlands More articles by this author J. Alfred Witjes Nijmegen, Netherlands More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Overexpression of immunoglobulin G prompts cell proliferation and inhibits cell apoptosis in human urothelial carcinoma

Only B lymphocytes can express immunoglobulins according to the traditional immunological theories, and the expression of immunoglobulin G (IgG) messenger RNA (mRNA) and protein was found in certain human cancer cells recently. However, the expression pattern of IgG and its possible role in human urothelial carcinoma are still elusive. In this study, we investigated the expression of IgG in two human urothelial carcinoma cell lines, T24 and BIU-87, and in 56 cases of clinical urothelial carcinoma tissues. The mRNA of IgG was positively detected by in situ hybridization and reverse transcription PCR; furthermore, IgG protein was also positively detected by immunohistochemistry and Western blot. Moreover, blockade of tumor-derived IgG by either antihuman IgG antibody or antisense oligonucleotides increased cell apoptosis and inhibited cell growth in bladder cancer cell lines in vitro, and antihuman IgG antibody could suppress the growth of xenotransplant tumor in vivo. In addition, either antihuman IgG antibody or antisense oligonucleotides enhanced the sensitivity to mitomycin C in bladder cancer cell line T24. Furthermore, blockade of IgG in bladder cancer cell T24 resulted in upregulation of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. Our results indicated that bladder cancer cells were capable of expressing IgG, and blockade of IgG expression induced cell apoptosis through activation of caspase-dependent pathway. A novel potential targeted therapy for bladder cancer will be possibly developed based on these data.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-013-0717-z) contains supplementary material, which is available to authorized users.

Decreased Tumorigenesis and Mortality from Bladder Cancer in Mice Lacking Urothelial Androgen Receptor

Much fewer mice lacking androgen receptor (AR) in the entire body develop bladder cancer (BCa). However, the role of urothelial AR (Uro-AR) in BCa development remains unclear. In the present study, we generated mice that lacked only Uro-AR (Uro-AR(-/y)) to develop BCa by using the carcinogen BBN [N-butyl-N-(4-hydroxybutyl)-nitrosamine] and found that Uro-AR(-/y) mice had a lower incidence of BCa and a higher survival rate than did their wild-type (WT; Uro-AR(+/y)) littermates. In vitro assay also demonstrated that Uro-AR facilitates the neoplastic transformation of normal urothelial cells to carcinoma. IHC staining exhibited less DNA damage, with much higher expression of p53 and its downstream target protein PNCA in Uro-AR(-/y) than that found in WT urothelium, which suggests that Uro-AR may modulate bladder tumorigenesis through p53-PCNA DNA repair signaling. Indeed, Uro-AR(-/y) mice with the transgene, simian vacuolating virus 40 T (SV40T), in the urothelium (Uro-SV40T-AR(-/y)) had a similar incidence of BCa as did their WT littermates (Uro-SV40T-AR(+/y)), and p53 was inactivated by SV40T in both genotypes. Use of the AR degradation enhancer ASC-J9 led to suppression of bladder tumorigenesis, with few adverse effects in the BBN-induced BCa mouse model. Together, these results provide the first direct in vivo evidence that Uro-AR has an important role in promoting bladder tumorigenesis and BCa progression. Targeting AR with ASC-J9 may provide a novel approach to suppress BCa initiation.

ALKBH2, a novel AlkB homologue, contributes to human bladder cancer progression by regulating MUC1 expression

The ALKBH family of proteins are highly expressed in various types of human cancer where they are involved in tumor growth and progression. However, multiple isoforms of ALKBH exist and the effect of individual isoforms on the development of urinary bladder cancer is unknown, particularly the molecular mechanisms involved in the progression from a noninvasive to invasive phenotype. We examined the role and function of ALKBH2 in human bladder cancer development in vitro and provide the first report that suppression of ALKBH2 in a human urothelial carcinoma cell line, KU7, reduces the expression of the transmembrane mucin protein, MUC1, and induces G1 cell cycle arrest. Moreover, reduction of ALKBH2 suppressed epithelial to mesenchymal transition (EMT) via increasing E-cadherin and decreasing vimentin expression. Transfection of MUC1 siRNA inhibited cell proliferation and EMT to the same extent as ALKBH2 gene silencing in vitro. ALKBH2 knockdown significantly suppressed MUC1 expression and tumor volume of bladder cancers in vivo as assessed in an orthotopic mouse model using ALKBH2 shRNA transfected KU7 cells. Immunohistochemical examination showed high expression levels of ALKBH2 in human urothelial carcinoma samples, especially in high-grade, superficially and deeply invasive carcinomas (pT(1) and >pT(2)), and in carcinoma in situ but not in normal urothelium. This study demonstrates that ALKBH2 is an upstream molecule of the oncoprotein, MUC1, and regulates cell cycle and EMT, resulting in progression of urothelial carcinomas.

Bradykinin modulates spontaneous nerve growth factor production and stretch-induced ATP release in human urothelium

The urothelium plays a crucial role in integrating urinary bladder sensory outputs, responding to mechanical stress and chemical stimulation by producing several diffusible mediators, including ATP and, possibly, neurotrophin nerve growth factor (NGF). Such urothelial mediators activate underlying afferents and thus may contribute to normal bladder sensation and possibly to the development of bladder overactivity. The muscle-contracting and pain-inducing peptide bradykinin is produced in various inflammatory and non-inflammatory pathologies associated with bladder overactivity, but the effect of bradykinin on human urothelial function has not yet been characterized.The human urothelial cell line UROtsa expresses mRNA for both B1 and B2 subtypes of bradykinin receptors, as determined by real-time PCR. Bradykinin concentration-dependently (pEC50=8.3, Emax 4434±277nM) increased urothelial intracellular calcium levels and induced phosphorylation of the mitogen-activated protein kinase (MAPK) ERK1/2. Activation of both bradykinin-induced signaling pathways was completely abolished by the B2 antagonist icatibant (1μM), but not the B1 antagonist R715 (1μM). Bradykinin-induced (100nM) B2 receptor activation markedly increased (192±13% of control levels) stretch-induced ATP release from UROtsa in hypotonic medium, the effect being dependent on intracellular calcium elevations. UROtsa cells also expressed mRNA and protein for NGF and spontaneously released NGF to the medium in the course of hours (11.5±1.4pgNGF/mgprotein/h). Bradykinin increased NGF mRNA expression and accelerated urothelial NGF release to 127±5% in a protein kinase C- and ERK1/2-dependent manner. Finally, bradykinin up-regulated mRNA for transient-receptor potential vanilloid (TRPV1) sensory ion channel in UROtsa.In conclusion, we show that bradykinin represents a versatile modulator of human urothelial phenotype, accelerating stretch-induced ATP release, spontaneous release of NGF, as well as expression of sensory ion channel TRPV1. Bradykinin-induced changes in urothelial sensory function might contribute to the development of bladder dysfunction.

HMGB1 Release by Urothelial Carcinoma Cells in Response to Bacillus Calmette-Guérin Functions as a Paracrine Factor to Potentiate the Direct Cellular Effects of Bacillus Calmette-Guérin

Prior study demonstrated that HMGB1 release by urothelial carcinoma cells in response to bacillus Calmette-Guérin is required for an in vivo antitumor effect. We evaluated the direct effects of HMGB1 on the in vitro response of urothelial carcinoma cells to bacillus Calmette-Guérin. Two human urothelial carcinoma cell lines were used to study the effect of exogenous HMGB1 alone and combined with bacillus Calmette-Guérin on the tumor cell response to bacillus Calmette-Guérin. Antibody mediated blockade of receptors for HMGB1 or HMGB1 protein was used to determine the contribution of paracrine HMGB1 release to bacillus Calmette-Guérin biological effects. Response end points evaluated included the activation of intracellular signaling pathways, gene transactivation and cytotoxicity. Urothelial carcinoma cells expressed the receptor for HMGB1 signaling. Antibody blockade of the RAGE receptor confirmed the dependence of signaling in response to HMGB1 on RAGE function. Exogenous HMGB1 activated cell signaling pathways for NFκB, NRF2 and CEBP. Quantitative reverse transcriptase-polymerase chain reaction on a panel of bacillus Calmette-Guérin responsive genes revealed peak expression resulting from the combination of bacillus Calmette-Guérin and HMGB1. Blockade of paracrine HMGB1 released in response to bacillus Calmette-Guérin using HMGB1 and/or RAGE receptor blocking antibodies showed a significant decrease in gene expression relative to that of bacillus Calmette-Guérin alone. HMGB1 potentiated the cytotoxic effects of bacillus Calmette-Guérin. HMGB1 released by urothelial carcinoma cells after bacillus Calmette-Guérin treatment functions as a paracrine factor to potentiate the urothelial carcinoma cell response to bacillus Calmette-Guérin. This paracrine activity likely contributes to the dependence of an in vivo tumor response on HMGB1 release.