Normal Bladder Urothelial Cells Research Articles

Abstract B026: APOBEC3 promotes squamous differentiation via IL1A/AP-1 signaling

Abstract The APOBEC family of cytidine deaminase enzymes catalyze the conversion of cytosine to uracil in ssDNA and RNA substrates. The resulting C to U transition has been noted in the innate immune response to viral infection, and somatic hypermutation. However, APOBEC family members APOBEC3A (hA3A) and APOBEC3B (hA3B) have been implicated as drivers of the APOBEC3 mutational signatures in numerous cancer types. Notably, approximately 70% of single nucleotide variant mutations in bladder cancer can be attributed to APOBEC3 induced mutagenesis. Studies have implicated various APOBEC3 family members to be drivers of tumorigenesis, disease progression, and therapeutic resistance. To investigate APOBEC3’s role in bladder cancer, we have generated a novel, cre-inducible, pten and p53 knockout, to overexpress mouse Apobec3(mA3) in Upk3a+ cells (UPPA), compared to a model that does not overexpress mA3 (UPP). Apobec3 expression was shown to accelerate bladder tumor formation, indicated by a shorter median time of tumor latency in the UPPA model (43.3 weeks) compared to the UPP model (53.6 weeks). While both tumor models had presence of squamous differentiation, the extensiveness was more profound in the UPPA tumors. Transcriptional analysis of UPP and UPPA tumors, via scRNA sequencing, revealed an enrichment of squamous gene expression in the UPPA tumors. Utilizing mouse syngeneic bladder cancer cell lines and normal bladder urothelial cells, we overexpressed mA3 in a doxycycline inducible manner. Overexpression of mA3 was shown to prevent luminal differentiation. Additionally, mA3 overexpression resulted in the increase of IL-1a production. IL-1a production was sufficient to drive squamous differentiation in BBN963 and normal bladder urothelial cells. Blocking cFOS and IL-1R, individually, prevented expression of squamous genes. These results suggest that DNA damage induced by mA3 is sufficient to drive squamous differentiation via signaling through IL-1a resulting in AP-1 transcription factor activation. We then analyzed bladder cancer samples from The Cancer Genome Atlas and found that hA3A was the only APOBEC family member to be significantly associated with squamous gene expression. Interestingly, hA3A expression was enriched in the basal/squamous consensus subtype. To further interrogate the potential of hA3A being a driver of squamous differentiation in bladder cancer, we have also generated doxycycline inducible expression of hA3A and hA3B in syngeneic bladder cancer cell lines. Targeting IL-1a in various autoimmune diseases has resulted in FDA approval of antibodies targeting IL-1a and IL-1R. Given the clinical utility of targeting IL-1a, it is plausible to target this signaling axis for therapeutic benefit in bladder cancers with ongoing hA3A activity. In aggregate, our work demonstrates the promotion of an aggressive tumor state, driven by APOBEC mutagenesis and implicates hA3A as a possible driver of squamous differentiation in urothelial carcinoma. Citation Format: Andrew S. Truong, Michael S. Sturdivant, Mi Zhou, Wolfgang A. Beckabir, John Raupp, Ujjawal Manocha, Elliot D. Toomer, Ibardo A. Zambrano, Hung-Jui Tan, Marc A. Bjurlin, Angela B. Smith, Tracy L. Rose, Matthew I. Milowsky, Sara E. Wobker, Kathryn H. Gessner, Jeffrey S. Damrauer, William Y. Kim. APOBEC3 promotes squamous differentiation via IL1A/AP-1 signaling [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B026.

Musashi-2 promotes migration and invasion in bladder cancer via activation of the JAK2/STAT3 pathway

Musashi-2 (Msi2) is considered to have a crucial role in regulating various key cellular functions. However, the clinical significance and biological role of Msi2 in bladder cancer remains unknown. We examined the expression of Msi2 in bladder cancer cell lines in 167 clinical samples and the biological role of Msi2 in bladder cancer cells. Western blotting was used to investigate the possible mechanism of Msi2-induced migration and invasion in bladder cancer. Msi2 was significantly upregulated in bladder cancer cells and tissues compared with normal bladder urothelial cells and tissues. Immunohistochemical analysis revealed high expression of Msi2 in 57 of 167 (34.1%) bladder cancer specimens. Statistical analysis showed a significant correlation of Msi2 expression with advanced clinical stage, lymph node metastasis, and poor prognosis. Overexpression and ablation of Msi2 promoted and inhibited, respectively, the migration and invasion of bladder cancer cells. Furthermore, we found that Msi2 activated the JAK2/STAT3 pathway and promoted expression of genes downstream of JAK2/STAT3 in bladder cancer. This study demonstrates that Msi2 can induce bladder cancer cell migration and invasion by activating the JAK2/STAT3 pathway, and that Msi2 may be a valuable prognostic biomarker for bladder cancer patients.

MP45-08 STABILIZATION OF INVADOPODIA BY PLECTIN- MEDIATED CONJUNCTION TO VIMENTIN INTERMEDIATE FILAMENT IS A CRITICAL MOLECULAR STEP OF INVASION AND EXTRAVASATION FOR METASTASIS IN BLADDER CANCER.

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2016MP45-08 STABILIZATION OF INVADOPODIA BY PLECTIN- MEDIATED CONJUNCTION TO VIMENTIN INTERMEDIATE FILAMENT IS A CRITICAL MOLECULAR STEP OF INVASION AND EXTRAVASATION FOR METASTASIS IN BLADDER CANCER. Mihoko Sutoh Yoneyama, Shingo Hatakeyama, Tomonori Habuchi, Takamitsu Inoue, Toshiya Nakamura, Tomihisa Funyu, Gerhard Wiche, Shigeru Tsuboi, and Chikara Ohyama Mihoko Sutoh YoneyamaMihoko Sutoh Yoneyama More articles by this author , Shingo HatakeyamaShingo Hatakeyama More articles by this author , Tomonori HabuchiTomonori Habuchi More articles by this author , Takamitsu InoueTakamitsu Inoue More articles by this author , Toshiya NakamuraToshiya Nakamura More articles by this author , Tomihisa FunyuTomihisa Funyu More articles by this author , Gerhard WicheGerhard Wiche More articles by this author , Shigeru TsuboiShigeru Tsuboi More articles by this author , and Chikara OhyamaChikara Ohyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.285AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To investigate the molecular mechanisms of bladder cancer (BCa) metastasis, we previously isolated a high-metastatic BCa cell subpopulation from a low-metastatic cell line by using an in vivo selection system (Sugiyama et al Oncol. Res. 20, 289-295). The high-metastatic BCa cell subpopulation showed a high ability to form invadopodia, the filamentous (F-) actin-based membrane protrusions that play an essential role in cancer cell invasion. Analysis of gene expression profiles revealed that the expression of an intermediate filament (IF) protein, vimentin and a cytoskeletal linker protein, plectin was up-regulated in the high-metastatic BCa cells compared with the low-metastatic BCa cell line. Here we report a novel molecular mechanism of invasive bladder cancer cell extravasation using vimentin IF and plectin. METHODS Six human BCa cell lines (RT-4, 5637, KK-47, T24, BOY and YTS-1) and primary culture of BCa cells from three genetically independent patients were used. The expression of vimentin and plectin in BCa cells was assessed by immunoblotting. The interaction between vimentin and plectin was examined by immunoprecipitation. The spatial correlation between vimentin, plectin and F-actin in BCa cells was analyzed by confocal microscopy. To evaluate the functions of invadopodia, cells were assayed for extracellular matrix (ECM) degradation, Matrigel invasion, transendothelial invasion and lung metastasis. RESULTS In invasive BCa cells, the expression levels of vimentin and plectin were elevated and the vimentin IF-plectin-invadopodia F-actin link was formed. Disruption of this link severely impaired invadopodia formation, reducing the capacities of ECM degradation, Matrigel invasion, transendothelial invasion and metastasis. In addition, the vimentin assembly into the filaments was required for invadepodia formation. CONCLUSIONS Our results strongly suggest that plectin anchoring invadopodia to vimentin IF scaffolds and stabilizes invadopodia, which is a critical molecular step for BCa cell invasion and extravasation for metastasis. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e610-e611 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Mihoko Sutoh Yoneyama More articles by this author Shingo Hatakeyama More articles by this author Tomonori Habuchi More articles by this author Takamitsu Inoue More articles by this author Toshiya Nakamura More articles by this author Tomihisa Funyu More articles by this author Gerhard Wiche More articles by this author Shigeru Tsuboi More articles by this author Chikara Ohyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Ibuprofen regulates the expression and function of membrane-associated serine proteases prostasin and matriptase.

BackgroundThe glycosylphosphatidylinositol-anchored extracellular membrane serine protease prostasin is expressed in normal bladder urothelial cells. Bladder inflammation reduces prostasin expression and a loss of prostasin expression is associated with epithelial-mesenchymal transition (EMT) in human bladder transitional cell carcinomas. Non-steroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of various cancers including bladder cancer, but the molecular mechanisms underlying the anticancer effect of NSAIDs are not fully understood.MethodsThe normal human bladder urothelial cell line UROtsa, the normal human trophoblast cell line B6Tert-1, human bladder transitional cell carcinoma cell lines UM-UC-5 and UM-UC-9, and the human breast cancer cell line JIMT-1 were used for the study. Expression changes of the serine proteases prostasin and matriptase, and cyclooxygenases (COX-1 and COX-2) in these cells following ibuprofen treatments were analyzed by means of reverse-transcription/quantitative polymerase chain reaction (RT-qPCR) and immunoblotting. The functional role of the ibuprofen-regulated prostasin in epithelial tight junction formation and maintenance was assessed by measuring the transepithelial electrical resistance (TEER) and epithelial permeability in the B6Tert-1 cells. Prostasin’s effects on tight junctions were also evaluated in B6Tert-1 cells over-expressing a recombinant human prostasin, silenced for prostasin expression, or treated with a functionally-blocking prostasin antibody. Matriptase zymogen activation was examined in cells over-expressing prostasin.ResultsIbuprofen increased prostasin expression in the UROtsa and the B6Tert-1 cells. Cyclooxygenase-2 (COX-2) expression was up-regulated at both the mRNA and the protein levels in the UROtsa cells by ibuprofen in a dose-dependent manner, but was not a requisite for up-regulating prostasin expression. The ibuprofen-induced prostasin contributed to the formation and maintenance of the epithelial tight junctions in the B6Tert-1 cells. The matriptase zymogen was down-regulated in the UROtsa cells by ibuprofen possibly as a result of the increased prostasin expression because over-expressing prostasin leads to matriptase activation and zymogen down-regulation in the UROtsa, JIMT-1, and B6Tert-1 cells. The expression of prostasin and matriptase was differentially regulated by ibuprofen in the bladder cancer cells.ConclusionsIbuprofen has been suggested for use in treating bladder cancer. Our results bring the epithelial extracellular membrane serine proteases prostasin and matriptase into the potential molecular mechanisms of the anticancer effect of NSAIDs.

A novel gene expression system for detecting viable bladder cancer cells

A novel transcriptional system was developed that can robustly enhance cancer-specific gene expression. In the system, hTERT promoter-driven gene expression was enhanced by an advanced two-step transcriptional amplification (TSTA). This construct was used to develop a novel system for detection of bladder cancer cells. The current study evaluated the advanced TSTA system by examining the cancer-specific gene transcription in various bladder cancer cell lines. The system significantly enhanced cancer-specific luciferase gene expression in the bladder cancer cell lines in comparison to the previous expression system of one-step or conventional TSTA. The fold gain of the enhancement was significantly correlated to the telomerase activity of the cell lines. A green fluorescent protein (GFP) gene encoding plasmid vector was constructed where hTERT promoter-driving transcription is enhanced by the advanced TSTA to utilize the system for the imaging and detection of viable bladder cancer cells. The advanced TSTA-hTERT-GFP plasmid successfully induced cancer-specific gene expression, showing robust GFP expression in human bladder cancer cell lines, but no visible GFP expression in normal bladder urothelial cells. The control GFP plasmid with a CMV promoter yielded GFP expression in both normal bladder cells and cancer cells. The advanced TSTA-hTERT-GFP plasmid allowed selective visualization of viable human bladder cancer cells in mixed cell culture containing 10- and 100-fold more normal bladder urothelial cells. These findings indicate that the advanced TSTA-hTERT expressional system is a valuable tool for detecting viable bladder cancer cells. The current system can be applied for in vitro detection of bladder cancer cells in urine and other types of cancer cells disseminated in vivo.

Alterations in Expression of HIF-1α, HIF-2α, and VEGF by Idiopathic Overactive Bladder Urothelial Cells During Stretch Suggest Role for Hypoxia

To test the hypothesis that hypoxia plays a role in overactive bladder (OAB) symptoms by studying how the in vitro stretch of primary cultured bladder urothelial cells (BUCs) from those with OAB and asymptomatic subjects altered the expression of angiogenic factors. The angiogenic factors included hypoxia-inducible factor-1 alpha subunit (HIF-1α), HIF-2 alpha subunit (HIF-2α), and vascular endothelial growth factor (VEGF). HIF-1α, HIF-2α, and VEGF mRNA expression were analyzed using real-time quantitative polymerase chain reaction. Fluorescence-activated cell sorting was used to measure the protein expression. The release of VEGF in the supernatant of stretched OAB and normal BUCs was measured using enzyme-linked immunosorbent assay. Stretching of OAB BUCs increased the expression of mRNA for HIF-1α, HIF-2α, and VEGF by 1.5-fold (P < .01), 1.5-fold (P < .01), and 3.5-fold (P < .001) compared with unstretched OAB BUCs. This augmentation was not detected when comparing stretched normal BUCs with unstretched normal BUCs. Using fluorescence-activated cell sorting quantitation, only HIF-2α was significantly increased (P < .01). Measuring VEGF in the supernatant revealed that stretched OAB BUCs released significantly more VEGF than nonstretched OAB BUCs at multiple points. In contrast, stretched normal BUCs did not release VEGF. OAB BUCs responded to stretch by expressing increased angiogenic markers, HIF-1α, HIF-2α, and/or VEGF, measured at the transcript and protein levels. This suggests that OAB BUCs respond as if they were primed by hypoxia. This knowledge adds to the pathophysiologic understanding of OAB.

Abstract 1897: Isothiocyanates and their metabolites in the plasma of mice fed broccoli sprouts isothiocyanate extract, broccoli sprouts, sulforaphane or erucin: A potential role in bladder cancer prevention

Abstract Background: Epidemiologic evidence suggests that cruciferous vegetables, particularly broccoli, may reduce bladder cancer risk. Our objectives were to characterize glucosinolates (GLUs) and their metabolites, isothiocyanates (ITCs), found in broccoli and broccoli sprouts, define bioactivity in bladder cancer cell lines in vitro, and to determine absorption and bioavailability in mice. Methods: Broccoli and broccoli sprouts GLUs and ITCs were characterized by HPLC-MS-MS and cyclocondensation assay. In vitro effects of non-hydrolyzed (GLUs) and hydrolyzed broccoli and broccoli sprouts (ITCs), and pure ITCs sulforaphane (SFN), erucin (ECN), iberin and allyl ITC, were studied on normal bladder urothelial cells (NBC) and a panel of human bladder cancer cell lines (BCC), representing the spectrum of bladder cancer biology (RT4: non-invasive; J82 and UMUC3: invasive). Cell viability (MTS and SRB assays), apoptosis (caspase-3/7 activity and PARP cleavage) and cell cycle analysis (flow cytometry) were performed. We defined absorption in control female mice and those fed diet containing 4% freeze-dried broccoli sprouts, or 2% freeze-dried broccoli sprouts extract; or gavaged daily with 295 µmol/kg sulforaphane or 295 µmol/kg erucin; for two weeks (n=16/group). Plasma mercapturic acid pathway metabolites of sulforaphane and erucin were extracted and then quantified by UPLC-MS/MS. Results: We observed significant dose-dependent decreases in cell viability of bladder cancer cell lines by broccoli or broccoli sprouts extracts as well as by pure ITCs. Broccoli sprout ITCs and SFN (IC50 = 2.52 µM) followed by ECN (IC50 = 2.24 µM) were the most potent inhibitors. Normal bladder urothelial cells were less sensitive than cancer cells. SFN and ECN resulted in a dose-dependent induction of apoptosis and modulation of the cell cycle in BCC. Metabolites of sulforaphane and erucin were absent in control mice and present in plasma of all treated groups, with dietary and pure phytochemical treatments, resulting in similar plasma metabolite concentrations (micromolar range). N-acetyl cysteine conjugates were found at highest concentrations, followed by cysteinyl and glutathione conjugates and small amounts of detectable cysteinyl glycine conjugates and free SFN. Evidence of inter-conversion of sulforaphane and erucin was seen. Conclusion: Broccoli extracts and pure ITCs inhibit cell viability, induce apoptosis and cell cycle arrest in non-invasive and invasive human bladder cancer cell lines. In vivo, dietary and gavaged ITCs are readily absorbed and metabolized to various conjugates. The distribution to bladder cancer xenografts will also be discussed. 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 1897.

1671 STRETCH-INIDUCED EXPRESSION OF HIF-1α HIF-2α AND VEGF IN BLADDER UROTHELIAL CELLS (BUC) FROM SUBJECTS WITH OVERACTIVE BLADDER (OAB)

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Female Urology II1 Apr 20101671 STRETCH-INIDUCED EXPRESSION OF HIF-1α HIF-2α AND VEGF IN BLADDER UROTHELIAL CELLS (BUC) FROM SUBJECTS WITH OVERACTIVE BLADDER (OAB) Charlotte E. Christiaansen, Yan Sun, Yuchao Hsu, and Toby C. Chai Charlotte E. ChristiaansenCharlotte E. Christiaansen Rotterdam, Netherlands More articles by this author , Yan SunYan Sun Baltimore, MD More articles by this author , Yuchao HsuYuchao Hsu Baltimore, MD More articles by this author , and Toby C. ChaiToby C. Chai Baltimore, MD More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1495AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Previous studies have shown that hypoxia can change the working conditions of the bladder and negatively affects its acute and long-term function. It is also known that abnormalities of BUC may play a role in OAB pathophysiology. We hypothesized that OAB may increases bladder oxygen demands due to increased bladder activity (urinary frequency and/or detrusor overactivity) which results in increased stretch of the bladder. We investigated whether OAB BUC react differently than normal BUC when subjected to stretch by measuring expression of angiogenesis factors: hypoxia-inducible factor 1, alpha subunit (HIF-1α), hypoxia-inducible factor 2, alpha subunit (HIF-2α) and vascular endothelial growth factor (VEGF). METHODS Human BUC were cultured from cystoscopic urothelial biopsies. Normal and OAB BUC were stretched using an in vitro stretch device, the Flexcell. HIF-1α, HIF-2α and VEGF mRNA expression were analyzed using quantitative real-time PCR. Fluorescence activated cell sorting (FACS) was used to determine HIF-1α, HIF-2α, and VEGF protein expression. Furthermore, because VEGF is a known cytokine released by cells, quantitation of VEGF level in the supernatant of OAB and normal BUC after stretching was determined with ELISA. RESULTS mRNA levels for HIF-1α, HIF-2α and VEGF were significantly higher in the stretched OAB BUC compared to stretched normal BUC (HIF-1α, p = 0.023; HIF-2α, p = 0.027; VEGF, p = 0.00017). FACS analysis of protein expression of HIF-2α in stretched OAB BUC showed significantly higher levels than in the non-stretched OAB BUC (p= 0.01). The protein expression of HIF-1α and VEGF in stretched OAB BUC were not different compared to non-stretched OAB BUC. During stretch, OAB BUC released significantly greater VEGF than stretched normal BUC at 8hr (p = 0.031), 24 hr (p = 0.031), and 48 hr (p = 0.043) time points respectively. As a control experiment, stretched OAB BUC released significantly more VEGF compared to non-stretched OAB BUC at 48hr (p = 0.007) and 96hr (p = 0.021) time points respectively. CONCLUSIONS These results demonstrated that OAB BUC responded differently compared to normal BUC to stretch by increased expression of mRNA for HIF-1α, HIF-2α and VEGF. Furthermore, OAB BUC secreted more VEGF in response to stretch. These results showed that OAB BUC responded to stretch as if they were in hypoxic conditions. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e645 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Charlotte E. Christiaansen Rotterdam, Netherlands More articles by this author Yan Sun Baltimore, MD More articles by this author Yuchao Hsu Baltimore, MD More articles by this author Toby C. Chai Baltimore, MD More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

1143 INTRAVESICAL ADMINISTRATION OF HEAT SHOCK PROTEIN 90-SURVIVIN COMPLEX INHIBITOR SUPPRESSES THE GROWTH OF NON-INVASIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20101143 INTRAVESICAL ADMINISTRATION OF HEAT SHOCK PROTEIN 90-SURVIVIN COMPLEX INHIBITOR SUPPRESSES THE GROWTH OF NON-INVASIVE BLADDER CANCER Akihiro Yano, Yasuhisa Fujii, Hitoshi Masuda, Satoru Kawakami, and Kazunori Kihara Akihiro YanoAkihiro Yano More articles by this author , Yasuhisa FujiiYasuhisa Fujii More articles by this author , Hitoshi MasudaHitoshi Masuda More articles by this author , Satoru KawakamiSatoru Kawakami More articles by this author , and Kazunori KiharaKazunori Kihara More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.642AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Heat shock protein (Hsp) 90 is a molecular chaperone that is involved in signaling pathways for cell proliferation and/or survival of cancer cells. Survivin, one of the Hsp90 client proteins, plays important roles in regulation of cellular survival. Survivin is selectively expressed in most solid cancers and is rarely detected in normal differentiated tissues. Recently, shepherdin, a novel anticancer peptidomimetic modeled on the Hsp90-survivin binding interface was generated and shown to destabilize survivin plus additional Hsp90 client proteins, causing massive killing of cancer cells. In the present study, we investigated whether shepherdin can be a cancer-specific molecular targeting agent for non-invasive bladder cancer by intravesical administration using an orthotopic xenograft model. METHODS The effects of shepherdin on bladder cancer cell proliferation and client protein expression were examined by MTS assay and Western blotting, respectively. The effects of shepherdin by intravesical administration on the growth of UM-UC-3 orthotopic xenograft tumors were determined by IVIS real-time in vivo imaging system using UM-UC-3-luciferase cells. RESULTS Treatment of the bladder cancer cell lines with shepherdin resulted in dose-dependent cell killing as compared with untreated or control scrambled peptide-treated cells. UM-UC-3 cells treated with shepherdin exhibited loss of expression of Hsp90 client proteins, including survivin and Akt, and increase in the level of cytochrome c in the cytosol. The scrambled peptide did not modulate client protein expression. In vivo, intravesical administration of shepherdin abolished the growth of UM-UC-3 orthotopic xenograft tumors (P < 0.01). Shepherdin treatment attenuated expression of survivin and Akt compared with the control group, and did not show any toxicity to normal bladder urothelial cells in the histological specimens. CONCLUSIONS This study is the first to show that intravesical administration of Hsp90-, especially survivin-targeting agent can be a potent and promising therapy for non-invasive bladder cancer minimizing damage to normal urothelial cells. Optimal cancer therapy is a fine balance between effective cancer cell killing and at the same time avoiding damage to the surrounding normal tissue. Intravesical administration of molecular targeting agent, which minimizes toxicity to healthy tissues, such as shepherdin may be the treatment of choice for patients with non-invasive bladder cancer in clinical setting. Tokyo, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e442 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akihiro Yano More articles by this author Yasuhisa Fujii More articles by this author Hitoshi Masuda More articles by this author Satoru Kawakami More articles by this author Kazunori Kihara More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Augmented bladder urothelial polyamine signaling and block of BK channel in the pathophysiology of overactive bladder syndrome

Overactive bladder syndrome (OAB) is an idiopathic condition characterized by urinary urgency and urge incontinence. Detrusor overactivity has been traditionally described as the physiologic mechanism for OAB. However, the bladder urothelium (BU) may also be involved in the pathophysiology. This study measured polyamine signaling and its downstream effects on membrane conductivity in bladder urothelial cells (BUC) obtained from asymptomatic and OAB subjects. Immunohistofluorescence was used to measure ornithine decarboxylase (ODC) expression in BU. BUC, cultured from BU biopsies, were used for electrophysiologic studies. dl-alpha-Difluoromethylornithine (DFMO), spermine, or spermidine was used to modulate polyamine signaling in BUC. Results showed ODC overexpression in OAB BU. In OAB BUC, whole cell and cell-attached configuration showed significantly decreased currents. Using inside-out patches, outward currents increased significantly, suggesting a cytoplasmic source of the outward current block in OAB BUC. In control BUC, outward currents were mediated by the large-conductance calcium-activated potassium (BK) channel due to calcium dose-dependence and block by iberiotoxin. Spermidine and spermine blocked the outward current in normal BUC in dose-dependent fashion. Conversely, DFMO significantly increased (P < 0.01) outward currents in OAB BUC both in cell-attached and in whole cell configuration. The outward currents in DFMO-treated-OAB BUC could be significantly reduced (P < 0.05) by adding back spermidine and spermine. These data suggest that polyamine signaling is upregulated in OAB urothelium and OAB BUC. Furthermore, polyamines in BUC block the BK channel. Targeting of bladder urothelial polyamine signaling may represent a novel approach for OAB treatment based on pathophysiologic mechanisms.

Changes in Adenosine Triphosphate-stimulated ATP Release Suggest Association Between Cytokine and Purinergic Signaling in Bladder Urothelial Cells

To determine whether antiproliferative factor (APF) or epidermal growth factor (EGF) can induce changes in purinergic signaling in normal bladder urothelial cells (BUCs) and/or whether antagonizing EGF activity or blocking adenosine triphosphate (ATP)-purinergic receptors can induce changes in purinergic signaling in interstitial cystitis (IC) cells. IC and normal BUCs were obtained from patients' bladder biopsy specimens. IC BUCs were treated with genistein, which antagonizes EGF's activity, and normal BUCs were treated with EGF, mock APF, or APF. Suramin, which antagonizes ATP activity, was used to treat the APF-treated normal BUCs. ATP release was determined by stimulating the BUCs with 30 microM ATP and then collecting the supernatant for a 3-hour period. ATP quantification was measured by luciferin-luciferase assay. Purinergic receptor P2X, ligand-gated ion channel, 3 (P2X3) expression on BUCs was determined by fluorescence-activated cell sorting. Genistein treatment of IC BUCs resulted in significantly decreased ATP release, thus reverting IC cells to a normal purinergic signaling phenotype. Conversely, normal BUCs treated with EGF or APF resulted in significantly increased ATP release and P2X3 expression, converting normal BUCs to an IC phenotype. Also, suramin treatment of APF-treated normal BUCs significantly reduced ATP release. Genistein and suramin reversed the augmented ATP release in IC BUCs and APF-treated normal BUCs, respectively, suggesting the possibility of intravesical use of these agents in IC treatment. EGF and APF induced augmented purinergic signaling in normal BUCs, as determined by increased ATP release and increased P2X3 expression. These data suggest an association between cytokines and purinergic signaling in human BUCs that should be explored further.

Defects in Muscarinic Receptor Cell Signaling in Bladder Urothelial Cancer Cell Lines

To explore muscarinic receptor signaling in 4 bladder cancer cell lines, bladder urothelial cells (BUC) have been shown to release and respond to various putative neurotransmitters. Reverse transcription-polymerase chain reaction was used to detect the presence of m1-m5 transcripts in the J82, RT4, T24, and 5637 lines of cancer BUC. Immunofluorescence was used to detect expression of m3 protein. Cancer and normal BUC were stimulated with carbachol (100 microM), a muscarinic agonist. Carbachol-evoked changes in intracellular calcium ([Ca(2+)](i)) levels were measured using fura-2 ratiometric microfluorimetry. Transfection of J82 cells with m3 plasmid was performed, and changes in carbachol-evoked [Ca(2+)](i) were re-examined. None of the cancer cell lines expressed m3 transcripts, unlike normal BUC, which expressed m3. None of the 4 bladder cancer cell lines responded to carbachol. However, 47% of normal BUC responded to carbachol. The m3-transfected J82 cells expressed both m3 transcript and protein. Thirteen percent of m3-transfected J82 cells responded to carbachol. This is the first description of altered muscarinic signaling in cancer BUC. Unlike normal BUC, bladder urothelial cancer cells neither expressed m3 transcript nor responded to carbachol, as measured by changes in [Ca(2+)](i). We could partially reverse this defect in one of the cancer cell lines, J82, by transfecting these cells with the m3 plasmid. Although the effects of muscarinic receptor signaling on urothelial cell are unknown, this signaling pathway may play a role in urothelial cell adhesion similar to that in keratinocytes.

Dysfunction of bladder urothelium and bladder urothelial cells in interstitial cystitis

The human bladder urothelium (BU) and bladder urothelial cells (BUCs) play an important role in the normal functioning of bladder including bladder storage. Current evidence in interstitial cystitis (IC) supports multiple abnormalities in bladder urothelial physiology. These data have come primarily from human studies. The discovery of a novel protein termed the antiproliferative factor (APF) uniquely expressed by IC BUCs is extremely important. APF induces increased permeability of normal BUCs grown in culture. Furthermore, APF regulates expression of other cytokines, including upregulating heparin-binding epidermal growth factor-like growth factor and downregulating epidermal growth factor by BUCs. These cytokine abnormalities were also related to increases in purinergic (adenosine triphosphate) signaling, which could mediate increased bladder sensation. Recent studies of uroplakins, which are specialized proteins expressed only in the apical urothelial cells, suggest that uroplakins play a role in the barrier function of the BU. It is also conceivable that alterations in uroplakins may result in bladder symptoms related to increased permeability or decreased protective function. As the body of knowledge about BU and BUC function increases, novel therapies targeting urothelial cells should become clinically feasible.

EGF and HB-EGF modulate inward potassium current in human bladder urothelial cells from normal and interstitial cystitis patients

Interstitial cystitis (IC) is an idiopathic condition characterized by bladder hyperalgesia. Studies have shown cytokine and purinergic signaling abnormalities in cultured bladder urothelial cells (BUC) from IC patients. We performed single-cell electrophysiological studies in both normal and IC BUC. A strongly inward rectifying potassium current with conductance of the Kir2.1 channel was identified in normal BUC. This current was significantly reduced in IC BUC. Kir2.1 protein and mRNA were detected in both IC and normal BUC. Epidermal growth factor (EGF) caused a dose-dependent decrease in the inward potassium current in normal BUC. EGF is secreted in higher amounts by IC BUC and is known to decrease Kir2.1 conductance by phosphorylation of Kir2.1. Genistein, a nonspecific phosphorylation inhibitor, increased the inward potassium current in IC BUC and blocked the effect of EGF on normal BUC. Treatment of IC BUC with heparin-binding epidermal growth factor-like growth factor (HB-EGF), previously shown to be secreted in lower amounts by IC BUC, significantly increased inward potassium current. These data show that the inward potassium current in BUC can be modulated by EGF and HB-EGF. Changes in BUC membrane potassium conductance caused by altered levels of EGF and HB-EGF may therefore play a role in the pathophysiology of IC.

Monoclonal antibodies for intravesical radioimmunotherapy of human bladder cancer.

Local administration of radioimmunoconjugates may allow successful tumor therapy. Bladder cancer appears well suited to this approach, because of its superficial and multifocal nature, and because it will allow direct intravesical administration of conjugates. Implantation of human bladder cancer cell lines in the bladder wall of nude rats results in tumor formation, providing an excellent model to test this. We have developed two murine monoclonal antibodies (MAbs), BLCA-8, IgG3, and BLCA-38, IgG1, both of which react with malignant cells and shed into voided urine of patients with transitional cell carcinoma (TCC) of the bladder, but not with normal bladder urothelial cells. Radioimmunoconjugates produced with 131Iodine (131I) or 125I have been used for biodistribution studies following administration directly into the bladder. Radioiodinated intact MAbs or Fabs administered intravesically into nontumor-bearing rats did not leak into the systemic circulation and were stable in urine for up to 100 h. Biodistribution studies carried out following intraperitoneal or intravesical administration of radioimmunoconjugates to tumor-bearing nude rats indicate good tumor uptake of both MAbs. Together with immunoreactivity assays, these studies demonstrate that 131I-labeled MAbs have considerable potential for intravesical radioimmunotherapy of human bladder tumors, and further studies are under way.