Human Urothelial Cell Lines Research Articles

Osmotic regulation of UT-B urea transporters in the RT4 human urothelial cell line.

Facilitative UT‐B urea transporters play important physiological roles in numerous tissues, including the urino‐genital tract. Previous studies have shown that urothelial UT‐B transporters are crucial to bladder function in a variety of mammalian species. Using the RT4 bladder urothelial cell line, this study investigated the potential osmotic regulation of human UT‐B transporters. Initial end‐point PCR experiments confirmed expression of both UT‐B1 and UT‐B2 transcripts in RT4 cells. Western blotting analysis revealed glycosylated UT‐B protein to be highly abundant and immunolocalization experiments showed it was predominantly located on the plasma membrane. Further PCR experiments suggested that a 48 hr, NaCl‐induced raise in external osmolality increased expression of UT‐B transcripts. Importantly, these NaCl‐induced changes also significantly increased UT‐B protein abundance (p < .01, n = 7, ANOVA), whereas mannitol‐induced changes in external osmolality had no effect (NS, n = 4, ANOVA). Finally, similar increases in both UT‐B RNA expression and protein abundance were observed with urea‐induced changes to external osmolality (p < .05, n = 4, ANOVA). In conclusion, these findings strongly suggest that increases in external osmolality, via either NaCl or urea, can regulate human urothelial UT‐B transporters.

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.

Gene expression and DNA methylation regulation of arsenic in mouse bladder tissues and in human urothelial cells.

According to a report of the International Agency for Research on Cancer, arsenic and inorganic arsenic compounds are classified into Group 1 carcinogens with regard to human health. Epidemiological studies indicate that arsenic is one of the main risk factors for the development of bladder cancer. In the present study, arsenic-altered gene expression in mouse bladder tissues and in human urothelial cells was compared. In the mouse model, sodium arsenite-induced mouse urothelial hyperplasia and intracellular inclusions were present. Following DNA array analysis, four genes with differential expression were selected for quantitative real-time PCR assay. The genes were the following: Cystathionine β-synthase (CBS), adenosine A1 receptor (ADORA1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and Wnt inhibitory factor 1 (Wif1). The results indicated a significant increase in the levels of Cbs and Adora1. The analysis of the DNA CpG methylation levels of the mouse Cbs and Adora1 genes revealed no significant change. In contrast to these observations, the four genes were further analyzed in the human normal urothelial cell line SV-HUC1. The data indicated that WIF1 gene expression was decreased by sodium arsenite, whereas this was not noted for CBS, MALAT1 and ADORA1. Sodium arsenite decreased mRNA and protein expression levels of the WIF1 gene. In addition, the methylation levels of the WIF1 gene were increased. Sodium arsenite inhibited cell proliferation and promoted cell migration as demonstrated in cell functional assays. The gene status was compared in 8 human urothelial cell lines, and WIF1 mRNA expression levels were determined to be higher, whereas DNA CpG methylation levels were lower in SV-HUC1 cells compared with those noted in the other 7 bladder cancer cell lines. In summary, the data indicated that sodium arsenite decreased WIF1 gene expression and promoted cell migration. The increased methylation levels of WIF1 DNA CpG could be a potential biomarker for bladder cancer.

Pharmacokinetics of ciclopirox prodrug, a novel agent for the treatment of bladder cancer, in animals and humans.

e14705 Background: Ciclopirox Prodrug (CPX-POM) is a novel anticancer agent currently being evaluated in patients with advanced solid tumors participating in a First-in-Human, Phase 1 safety, dose tolerance, pharmacokinetics (PK) and pharmacodynamics trial at four US sites. In vitro and in vivo preclinical proof of principle was established in high grade human urothelial cancer cell lines as well as a mouse model of bladder cancer.Methods: A series of in vivo PK studies were conducted in mice, rats and dogs to characterize the absolute bioavailability of CPX following intravenous (IV), subcutaneous (SC) and oral administration of CPX-POM. The single dose and steady-state plasma and urine pharmacokinetics of CPX-POM are also currently being characterized in patients participating in the ongoing Phase 1 trial. Plasma and urine concentrations of the prodrug and metabolites were determined by LC-MS/MS validated in each specie and matrix. Non-parametric pharmacokinetic parameters were generated from resultant plasma and urine drug and metabolite concentration-time data. Results: CPX-POM is rapidly and completely metabolized to CPX in blood via circulating phosphatases in animals and humans. CPX is completely bioavailable following IV CPX-POM administration in mice, rats and dogs. CPX and its major inactive glucuronide metabolite (CPX-G) are extensively eliminated in urine in all animal species. SC administration of CPX-POM demonstrated excellent bioavailability in rats and dogs. Following IV administration of 30-900 mg/m2CPX-POM to patients, the apparent elimination half-life of CPX ranged from 2 to 8 hours, CPX systemic exposure was dose-proportional and time-independent in cancer patients, and a major portion of the dose was eliminated as CPX-G. Conclusions: IV CPX-POM achieves plasma and urine CPX exposures that exceed in vitro IC50 values several-fold at well tolerated doses in animals and humans. CPX pharmacokinetics observed in animals were predictive of human systemic clearance based on allometric scaling. Clinical trial information: NCT03348514.

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.

Quantum dot-based fluorescent probes for targeted imaging of the EJ human bladder urothelial cancer cell line.

QDs are a type of inorganic nanoparticle with unique optical properties. As a fluorescent label, QDs are widely used in biomedical fields. In the present study, fluorescent probes of quantum dots (QDs) conjugated with a prostate stem cell antigen (PSCA) monoclonal antibody (QD-PSCA) were prepared to study the targeted imaging of QD-PSCA probes in EJ human bladder urothelial cancer cells and analyze the feasibility of QD-based non-invasive tumor-targeted imaging in vivo. QDs with an emission wavelength of 605 nm (QD605) were conjugated with PSCA to prepare QD605-PSCA fluorescent probes by chemical covalent coupling. The optical properties of the probes coupled and uncoupled with PSCA were measured and assessed using an ultraviolet spectrophotometer and a fluorescence spectrophotometer. Direct immune-fluorescent labeling was utilized to detect and analyze imaging of the probes for EJ cells. The results revealed that QD605-PSCA probes retained the fluorescent properties of QD605 and the immunogenicity of the PSCA protein. The probes were able to specifically recognize the PSCA protein expressed in bladder cancer cells, while fluorescence was stable and had a long duration. The present study suggests that QD-PSCA fluorescent probes may be useful for specific targeted labeling and imaging in bladder urothelial cancer cells. Furthermore, the probes possess good optical stability and may be useful for research into non-invasive targeted imaging, early diagnosis and targeted in vivo tumor therapy.

Abstract 1333: Inactivation of NBR1 enhances sensitivity to rapamycin on human urothelial cancer cell lines through AMPK/ULK1-mediated autophagy

Abstract Rapamycin has been highly evaluated in clinical therapeutic intervention for cancer patients as a specific inhibitor of the mammalian target of rapamycin (mTOR) kinase. Rapamycin also stimulates autophagy and mitophagy to remove damaged cells as a regulator of general autophagy. Previously, we identified increased expression of mRNA levels of NBR1 (autophagy cargo receptor) by inhibiting mTOR signaling in human urothelial cancer cell lines. Therefore, we investigated whether loss of NBR1 sensitizes human urothelial cancer cells to autophagic stimulation and stress-induced mitochondrial insults by treatment with rapamycin. NBR1-deficient cells exhibited enhanced sensitivity to rapamycin that was associated with increased autophagy and mitochondrial defects. Loss of NBR1 expression altered the cellular response to rapamycin treatment, resulting in impaired ATP homeostasis and an increase in reactive oxygen species. Moreover, rapamycin treatment also induced autophagy activity through AMPK activation in NBR1-deficient cells, which was involved in blocked mTOR signaling and activated unc-51 like autophagy activating kinase 1 (ULK1). NBR1-deficient cells exhibited profound mitochondrial dysfunction in response to rapamycin treatment as evidenced by Δψm collapse, ATP depletion, ROS accumulation and apoptosis activation. Therefore, our findings provide a rationale for rapamycin treatment of NBR1-deficient human urothelial cancer through induction of autophagy activity by regulating the AMPK/mTOR signaling pathway, indicating that NBR1 can be a potential candidate for the treatment of human urothelial cancer. (NRF-2015R1A1A1A0500110 to I.H.C., NRF-2016R1D1A1B03933826 to Y.M.W., and the Korea Health Technology R&amp;D Project HI17C0710 to C.I.H.) Citation Format: Myeong Joo Kim, Min Ji Cho, In Ho Chang, Young Mi Whang. Inactivation of NBR1 enhances sensitivity to rapamycin on human urothelial cancer cell lines through AMPK/ULK1-mediated autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1333.

Abstract 2018: Rescue of ΔNp63α inhibits urothelial cell invasion by attenuating epithelial-to-mesenchymal transition (EMT)

Abstract Background: Loss of the transcription factor, p63 is associated with poor prognosis in muscle-invasive bladder cancers. The current study aimed to investigate the tumor-suppressive role of p63 in urothelial cancers. Methods: A 3-dimensional (3D) organotypic raft model of urothelial cancer was established by culturing human urothelial cancer cell lines (HT1376, T24) on human bladder fibroblast-embedded collagen-I. Control rafts contained normal primary human urothelial cells (HUC). Cells were also cultured as monolayers on collagen-I coated dishes before harvesting total RNA and protein for qPCR and Western blot analyses respectively. Results: Non-invasive HUC and HT1376 had an epithelial phenotype, characterized by compact and cuboidal morphology whereas invasive T24 cells were elongated with a spindle morphology, typical of mesenchymal cells and indicative of epithelial-to-mesenchymal transition (EMT), which is necessary for invasion. The number of invasive incidents in 3D rafts established with T24 cells was higher compared to those with HUC or HT1376 cells (N=3, p&amp;lt;0.05). Consistent with this, the invasive cell line had reduced mRNA and protein expression of total p63 (N=3), a differentiation marker, and the epithelial marker E-cadherin (N=3), in addition to upregulated expression of the mesenchymal markers N-cadherin (N=3) and fibronectin (N=3) and pERK-Y204 (N=2) compared to the non-invasive cells. Treatment of invasive T24 cells with histone deacetylase (HDAC) inhibitors (vorinostat (pan) or entinostat (HDAC1,3)) attenuated the number of invasive incidents (N=3, p&amp;lt;0.05), and restored protein expression of total p63 (N=3) and E-cadherin (N=2) while normalizing pERK-Y204 (N=2 for T24). The tumor-suppressive properties of the ΔNp63α isoform were studied by adenoviral-mediated overexpression of ΔNp63α in T24 cells, which mimicked the effects of HDAC inhibitors and diminished EMT by rescuing E-cadherin expression while depleting fibronectin (N=2). Overexpression of ΔNp63α also reduced the number of invasive incidents in 3D models established with T24 cells (N=2). Conclusion: Invasive T24 cells exhibited an EMT phenotype, which coincided with depleted p63 expression. Rescue of ΔNp63α expression by treatment with HDAC inhibitors or through experimental overexpression attenuated the EMT phenotype and cell invasion. Citation Format: Kirtiman Srivastava, Victoria Smith, Eric A. Fernandez, Conor Breen, Karen D. McCloskey. Rescue of ΔNp63α inhibits urothelial cell invasion by attenuating epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2018.

Differentiation Between Normal and Cancerous Human Urothelial Cell Lines Using Micro-Electrical Impedance Spectroscopy at Multiple Frequencies

The present study aims to investigate differences in impedance between normal (SV-HUC-1) and cancerous (TCCSUP) human urothelial cell lines at multiple frequencies by using a micro-electrical impedance spectroscopy (µEIS) device. A µEIS device was designed to measure the impedance of SV-HUC-1 and TCCSUP cells as the cells passed the sensing electrodes in the microfluidic channel. The cellular impedance was measured at frequencies of 10, 50, 100, 500 kHz, and 1 MHz, and the impedance values were compared at each frequency to investigate differences between the two cell lines. Additionally, the linear correlation between impedance and frequency was analyzed. The number of SV-HUC-1 and TCCSUP cells measured was 13 and 9, 21 and 10, 16 and 24, 8 and 8, and 11 and 22 cells at the frequencies of 10, 50, 100, 500 kHz, and 1 MHz, respectively. TCCSUP had significantly smaller amplitudes than SV-HUC-1 at 10, 50, and 100 kHz (p = 0.030, p = 0.031, and p < 0.001, respectively). Moreover, significant differences in phase angle were observed between cell lines at 100, 500 kHz, and 1 MHz (p < 0.001 in all). A significant difference between cell lines in terms of amplitude and phase angle was observed concurrently at 100 kHz. The amplitude of both the cell lines was negatively correlated to frequency, while the phase angle presented no correlation. In conclusion, the µEIS device could effectively differentiate between SV-HUC-1 and TCCSUP on the basis of their impedance.

Curcumin as Treatment for Bladder Cancer: A Preclinical Study of Cyclodextrin-Curcumin Complex and BCG as Intravesical Treatment in an Orthotopic Bladder Cancer Rat Model.

Objective To evaluate the antitumor effect of cyclodextrin-curcumin complex (CDC) on human and rat urothelial carcinoma cells in vitro and to evaluate the effect of intravesical instillations of CDC, BCG, and the combination in vivo in the AY-F344 orthotopic bladder cancer rat model. Curcumin has anticarcinogenic activity on urothelial carcinoma and is therefore under investigation for the treatment of non-muscle invasive bladder cancer. Curcumin and BCG share immunomodulating pathways against urothelial carcinoma. Methods Curcumin was complexed with cyclodextrin to improve solubility. Four human urothelial carcinoma cell lines and the AY-27 rat cell line were exposed to various concentrations of CDC in vitro. For the in vivo experiment, the AY-27 orthotopic bladder cancer F344 rat model was used. Rats were treated with consecutive intravesical instillations of CDC, BCG, the combination of CDC+BCG, or NaCl as control. Results CDC showed a dose-dependent antiproliferative effect on all human urothelial carcinoma cell lines tested and the rat AY-27 urothelial carcinoma cell line. Moreover, intravesical treatment with CDC and CDC+BCG results in a lower percentage of tumors (60% and 68%, respectively) compared to BCG (75%) or control (85%). This difference with placebo was not statistically significant (p=0.078 and 0.199, respectively). However, tumors present in the placebo and BCG-treated rats were generally of higher stage. Conclusions Cyclodextrin-curcumin complex showed an antiproliferative effect on human and rat urothelial carcinoma cell lines in vitro. In the aggressive orthotopic bladder cancer rat model, we observed a promising effect of CDC treatment and CDC in combination with BCG.

Hazard assessment of three haloacetic acids, as byproducts of water disinfection, in human urothelial cells

Disinfection by-products (DBPs) are compounds produced in the raw water disinfection processes. Although increased cancer incidence has been associated with exposure to this complex mixture, the carcinogenic potential of individual DBPs remains not well known; thus, further studies are required. Haloacetic acids (HAAs) constitute an important group among DBPs. In this study, we have assessed the in vitro carcinogenic potential of three HAAs namely chloro-, bromo-, and iodoacetic acids. Using a long-term (8 weeks) and sub-toxic doses exposure scenario, different in vitro transformation markers were evaluated using a human urothelial cell line (T24). Our results indicate that long-term exposure to low doses of HAAs did not reproduce the genotoxic effects observed in acute treatments, where oxidative DNA damage was induced. No changes in the transformation endpoints analyzed were observed, as implied by the absence of significant morphological, cell growth rate and anchorage-independent cell growth pattern modifications. Interestingly, HAA-long-term exposed cells developed resistance to oxidative stress damage, what would explain the observed differences between acute and long-term exposure conditions. Accordingly, data obtained under long-term exposure to sub-toxic doses of HAAs could be more accurate, in terms of risk assessment, than under acute exposure scenarios.

MP65-09 NEAR INFRARED PHOTOIMMUNOTHERAPY USING ANTI-CD47 ANTIBODY FOR HUMAN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2018MP65-09 NEAR INFRARED PHOTOIMMUNOTHERAPY USING ANTI-CD47 ANTIBODY FOR HUMAN BLADDER CANCER Bernhard Kiss, Kelly McKenna, Robert Ertsey, Nynke Sjoerdtje Van den Berg, Kathleen Mach, Eben Rosenthal, and Joseph Liao Bernhard KissBernhard Kiss More articles by this author , Kelly McKennaKelly McKenna More articles by this author , Robert ErtseyRobert Ertsey More articles by this author , Nynke Sjoerdtje Van den BergNynke Sjoerdtje Van den Berg More articles by this author , Kathleen MachKathleen Mach More articles by this author , Eben RosenthalEben Rosenthal More articles by this author , and Joseph LiaoJoseph Liao More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2075AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Near infrared photoimmunotherapy (PIT) is a new molecular-targeted cancer therapy that uses a specific photosensitizer based on a near-infrared (NIR) phthalocyanine dye, IRDye700DX, conjugated to a monoclonal antibody. CD47 is a cell surface protein that elicits a ″don′t eat me signal″ to prevent macrophage engulfment and is expressed on over 80% of bladder cancer tumors but absent on the luminal cell layer of normal bladder urothelium. Thus, targeting CD47 for PIT has the potential to selectively induce cell death in CD47 expressing tumor cells as well as increase phagocytosis of tumor cells. METHODS The anti-CD47 antibody (B6H12) was conjugated to IRDye700DX NHS ester and purified using a dialysis spin column. Anti-CD47-IRDye700 concentration was determined with a Coomassie Plus protein assay. The molecular weight of the conjugated antibody was verified by SDS-page and IRDye700 conjugation verified using the 700nm fluorescence channel of a PEARL imager. Binding of anti-CD47-IRDye700DX to CD47 was verified by ELISA. Three different human urothelial cell lines (UMUC3, HT-1376 and 639V) were used for in vitro NIR-PIT experiments. 639V cells were stably transfected with GFP using a lentiviral vector. After plating in a 96well plate and incubation with 10 µg/ml of anti-CD47-IRDye700 or unconjugated anti-CD47 antibody (1h on ice), cells were irradiated with a red light-emitting diode (LED) at 670-710nm wavelength at different energy levels. The cytotoxic effects of NIR-PIT with anti-CD47-IRDye700DX were determined by propidium iodide staining and flow cytometry. To evaluate macrophage activity, a phagocytosis assay using human macrophages and analyzed by flow cytometry was performed. RESULTS Cancer-cell death significantly increased with anti-CD47-IRDye700DX in a light-dose dependent manner in all 3 human bladder cancer cell lines. Significant cancer-cell death was first observed with 2J exposure and when exposed to 40J of NIR light over 90% of 639V and UMUC3 cells and over 50% of HT1376 died. Phagocytosis of cancer-cells incubated with anti-CD47-IRDye700DX and irradiated with 8J was significantly increased compared to anti-CD47-antibody alone (p=0.0002). There was no cytotoxicity associated with anti-CD47 antibody alone with NIR light, with NIR light alone or with anti-CD47-IRDye700DX without NIR light. CONCLUSIONS NIR-PIT with anti-CD47-IRDye700 shows significantly increased cancer-cell cytotoxicity and significantly increased cancer-cell phagocytosis making this combination a highly promising novel approach for molecular targeted bladder cancer therapy. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e862 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Bernhard Kiss More articles by this author Kelly McKenna More articles by this author Robert Ertsey More articles by this author Nynke Sjoerdtje Van den Berg More articles by this author Kathleen Mach More articles by this author Eben Rosenthal More articles by this author Joseph Liao More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP65-04 LONG NON-CODING RNA GAS5 MODULATES GEMCITABINE EFFICACY IN BLADDER CANCER VIA REGULATION OF DEOXYCYTIDINE KINASE EXPRESSION

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2018MP65-04 LONG NON-CODING RNA GAS5 MODULATES GEMCITABINE EFFICACY IN BLADDER CANCER VIA REGULATION OF DEOXYCYTIDINE KINASE EXPRESSION Wei Chen, Junlong Zhuang, Wenmin Cao, Wenli Diao, and Hongqian Guo Wei ChenWei Chen More articles by this author , Junlong ZhuangJunlong Zhuang More articles by this author , Wenmin CaoWenmin Cao More articles by this author , Wenli DiaoWenli Diao More articles by this author , and Hongqian GuoHongqian Guo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2070AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Gemcitabine is a common used agent for therapy in bladder cancer. Our objective was to characterize the ability of Long non-coding RNA GAS5 to modulate the gemcitabine efficacy. METHODS Expression of GAS5 was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR) in 30 patients following radical cystectomy and gemcitabine treatment. All subjects signed written informed consent. The study was approved by the Hospital Ethics Boards prior to initiation. The correlation between GAS5 and deoxycytidine kinase (DCK) was further examined by evaluating DCK expression in cells that either overexpressed or knocked down GAS5. Gemcitabine efficacy was identified by CCK-8 assays. DCK and HuR were analyzed by western blotting and immunofluorescence. RESULTS GAS5 was significantly downregulated in bladder cancer tissues compared with the paired adjacent non-tumorous tissues. Lower GAS5 expression levels were associated with advanced pathological stages and poor overall survival (Fig. 1). LncRNA GAS5 expression is also lower in T24, 5637 and SW780 compared with the human normal urothelial cell line (Sv-Huc-1). GAS5 level was well correlated with the sensitivity of the cancer cells toward gemcitabine. Gemcitabine can dose-dependently increase GAS5 level in bladder cancer cells. Of note, pretreated gemcitabine attenuated GAS5-induced with a second dose of gemcitabine. GAS5-overexpressing cells displayed an elevated level of cell death induced by gemcitabine. Accordingly, knockdown of GAS5 in bladder cancer cells by small interfering RNA conferred tolerance to gemcitabine-induced cytotoxicity (Figure 3C, 3D). DCK is the rate-limiting enzyme responsible for conversion of gemcitabine to active antimetabolite. By knocking down GAS5 in bladder cancer cells, we experimentally confirmed that GAS5 regulates DCK expression. Furthermore, we revealed that proapoptotic function of GAS5 was correlated with promoting HuR nuclear export and the biological consequence of deoxycytidine kinase expression (Fig. 2). CONCLUSIONS This study shows that long non-coding RNA GAS5 modulates HuR-mediated DCK expression, and GAS5 low expression confers gemcitabine tolerance. The expression of GAS5 may represent a novel companion diagnostic and target to undermine chemotherapy resistance. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e859-e860 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Wei Chen More articles by this author Junlong Zhuang More articles by this author Wenmin Cao More articles by this author Wenli Diao More articles by this author Hongqian Guo More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cholinergic regulation of proliferation of the urothelium in response to E. coli lipopolysaccharide exposition

How the proliferation of the urothelium is regulated is known to a little degree. E. coli lipopolysaccharide (LPS) activates the innate immune response of the urinary bladder via the Toll-like receptor 4 (TLR4) on the urothelium but induces also urothelial proliferation. We wanted to assess whether muscarinic receptors are involved in the regulation of urothelial proliferation triggered by LPS stimulation. Female Fischer 344 rats were instilled with LPS or saline (control) in the urinary bladder in the absence or presence of muscarinic receptor blockade with atropine and regeneration of the urothelium was assessed 4h and 24h later. In the Fischer 344 bladder, urothelial thinning and urothelial caspase 3 up-regulation occurred at 4h after LPS urinary bladder instillation, which were totally blocked in rats pre-treated with atropine. TLR4 was only expressed in blood vessels in the Fischer 344 bladder, while it was also expressed in umbrella cells in the Sprague-Dawley bladder. Proliferation (Ki67 incorporation) of the human urothelial cell line UROtsa was reduced in the presence of the muscarinic receptor antagonists methoctramine (M2/M4-selective) and pirenzepine (M1/M4-selective), while proliferation instead was enhanced in the presence of atropine. In UROtsa cells exposed to LPS for 24h, 4-DAMP (M3/M1/M5-selective) inhibited instead proliferation. In conclusion, muscarinic receptors regulate urothelial proliferation and LPS may induce urothelial apoptosis via muscarinic receptor-dependent pathways. Our findings also suggest that species differences exist in the expressional pattern of TLR4 in the urothelium.

Perilla extract improves frequent urination in spontaneously hypertensive rats with enhancement of the urothelial presence and anti-inflammatory effects.

To investigate the effects of perilla extract on urinary symptoms in spontaneously hypertensive rats as a model of spontaneous overactive bladder. Spontaneously hypertensive rats were randomly divided into two groups and fed either a control diet or a perilla extract-containing diet. Cystometry, gene expression and histological analyses were carried out to evaluate the effects of perilla extract after 2-week feeding of either the control or the perilla extract diet. The expression of inflammation-related genes in the human urothelial cell line HT-1376 and the normal human bladder epithelial cell was measured after the treatment with perillaldehyde, the main component of perilla extract, or perillic acid, the final metabolite of perillaldehyde. A significant 27% increase in the micturition interval and decreased expression of nerve growth factor, tumor necrosis factor-α, interleukin-1β and transient receptor potential V1 were observed in the perilla group compared with the control group. The level of uroplakin 3A was 40% higher in the perilla group than in the control group. The urothelium in the control group was thin or defective, but it was almost completely intact in the perilla group. Perillaldehyde and perillic acid suppressed the induction of nerve growth factor and tumor necrosis factor-α by interleukin-1β in HT-1376 and normal human bladder epithelial cells. The present findings suggest that perilla extract improves frequent urination, and this improvement seems to be mediated, at least in part, by enhancement of the urothelial presence and by the anti-inflammatory effects of perilla.

Anti-PD-L1/TGFβR2 (M7824) fusion protein induces immunogenic modulation of human urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis

BackgroundAvelumab has recently been approved by the Food and Drug Administration for the therapy of Merkel cell carcinoma and urothelial carcinoma. M7824 is a novel first-in-class bifunctional fusion protein comprising a monoclonal antibody against programmed death-ligand 1 (PD-L1, avelumab), fused to the extracellular domain of human transforming growth factor beta (TGFβ) receptor 2, which functions as a TGFβ “trap.” Advanced urothelial tumors have been shown to express TGFβ, which possesses immunosuppressive properties that promote cancer progression and metastasis. The rationale for a combined molecule is to block the PD-1/PD-L1 interaction between tumor cells and immune cell infiltrate and simultaneously reduce or eliminate TGFβ from the tumor microenvironment. In this study, we explored the effect of M7824 on invasive urothelial carcinoma cell lines. MethodsHuman urothelial (transitional cell) carcinoma cell lines HTB-4, HTB-1, and HTB-5 were treated with M7824, M7824mut (M7824 that is mutated in the anti-PD-L1 portion of the molecule and thus does not bind PD-L1), anti-PD-L1 (avelumab), or IgG1 isotype control monoclonal antibody, and were assessed for gene expression, cell-surface phenotype, and sensitivity to lysis by TRAIL, antigen-specific cytotoxic T lymphocytes and natural killer cells. ResultsM7824 retains the ability to mediate antibody-dependent cellular cytotoxicity of tumor cells, although in some cases to a lesser extent than anti-PD-L1. However, compared to anti-PD-L1, M7824 increases (A) gene expression of molecules involved in T-cell trafficking in the tumor (e.g., CXCL11), (B) TRAIL-mediated tumor cell lysis, and (C) antigen-specific CD8+ T-cell–mediated lysis of tumor cells. ConclusionsThese studies demonstrate the immunomodulatory properties of M7824 on both tumor cell phenotype and immune-mediated lysis. Compared to anti-PD-L1 or M7824mut, M7824 induces immunogenic modulation of urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis. These findings show the relevance of the dual blockade of PD-L1 and TGFβ in urothelial carcinoma cell lines and thus support the rationale for future clinical studies of M7824 in patients with urothelial cancer.

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.

Dose-Dependent Response to 3-Nitrobenzanthrone Exposure in Human Urothelial Cancer Cells.

A product of incomplete combustion of diesel fuel, 3-nitrobenzanthrone (3-NBA), has been classified as a cancer-causing substance. It first gained attention as a potential urinary bladder carcinogen due to the presence of its metabolite in urine and formation of DNA adducts. The aim of the present study was to characterize the dose-response relationship of 3-NBA in human urothelial cancer cell line (RT4) exposed to concentrations ranging from 0.0003 μM (environmentally relevant) to 80 μM by utilizing toxicological and metabolomic approaches. We observed that the RT4 cells were capable of bioactivation of 3-NBA within 30 min of exposure. Activity measurements of various enzymes involved in the conversion of 3-NBA in RT4 cells demonstrated NAD(P)H:quinone oxidoreductase (NQO1) as the main contributor for its bioactivation. Moreover, cytotoxicity assessment exhibited an initiation of adaptive mechanisms at low dosages, which diminished at higher doses, indicating that the capacity of these mechanisms no longer suffices, resulting in increased levels of intracellular reactive oxygen species, reduced proliferation, and hyperpolarisation of the mitochondrial membrane. To characterize the underlying mechanisms of this cellular response, the metabolism of 3-NBA and metabolomic changes in the cells were analyzed. The metabolomic analysis of the cells (0.0003, 0.01, 0.08, 10, and 80 μM 3-NBA) showed elevated levels of various antioxidants at low concentrations of 3-NBA. However, at higher exposure concentrations, it appeared that the cells reprogrammed their metabolism to maintain the cell homeostasis via activation of pentose phosphate pathway (PPP).

Abstract 3354: Histone deacetylase-1 promotes urothelial cell migration and invasion by modulating p63-pMLC2 signaling

Abstract Background: Altered expression of histone deacetylases (HDAC) and p63 transcription factor are associated with poor prognosis in invasive urothelial tumors. The current study focused on HDAC-evoked epigenetic modification of p63 and its downstream effects. Methods: Primary human urothelial cells (HUC) and urothelial cancer cell lines (HT1376, T24, TCCSUP) were cultured either on human bladder fibroblast-embedded collagen-I to establish 3D-organotypic rafts or as 2D-monolayers. Scratch wound assay was used to study the rate of cell migration before studying the differential mRNA and protein expressions of HDAC1,2,3, p63, p21, p27, Rho-kinase1 (ROCK1), 14-3-3 eta and myosin light chain-2 (MLC2). Results: T24 and TCCSUP cells exhibited invasive incidents on 3D-organotypic rafts, which coincided with their enhanced cell migration capabilities. qPCR and Western blotting analysis demonstrated enhanced expression of HDAC1, HDAC3, p27, p-p27-Thr157 and 14-3-3 eta alongside depleted levels of p63, ROCK1, pMLC2-Ser19, pMLC2-Thr18-Ser19 and p21 in invasive cells compared to normal (N=5, p&amp;lt;0.05). These results coincided with the loss of stress fiber formations visualized by immunofluorescence detection of F-actin in invasive cells indicating that cytoskeletal reorganization may be pivotal for cell migration. Treatment with HDAC inhibitors (vorinostat (pan) and entinostat (HDAC1, 3)) attenuated the number of invasive incidents in T24 and TCCSUP cells, restored expression of p63, p21, pMLC2-Ser19, pMLC2-Thr18-Ser19 (N=5, p&amp;lt;0.05), and normalized expression of 14-3-3 eta and p-p27-Thr157. Reappearance of stress fiber formations traversing across the cell after HDAC inhibitor treatments was supportive of these results, while nuclear enrichment of p27 indicated its enhanced cytoplasm-to-nuclear trafficking. Transient knockdown of HDAC1 in the invasive population mimicked the effect of HDAC inhibitors (N=5, p&amp;lt;0.05), while successive depletion of p63 appeared to abolish the effects of HDAC1 knockdown by upregulating 14-3-3 eta and depleting ROCK1 expression, respectively. Conclusions: Inhibition of HDAC1 activity attenuates urothelial cell migration and invasion by restoring the expression of p63 which, (a) upregulates the expression and activity of ROCK1, and (b) suppresses the expression of 14-3-3 eta, thereby alleviating the cytoplasmic p27 levels and facilitating the pMLC2-mediated cytoskeletal remodeling. These findings provide further support and mechanistic evidence for the potential use of HDAC inhibitors to treat invasive/poor-prognosis bladder cancer. Citation Format: Kirtiman Srivastava, Conor Breen, Karen McCloskey. Histone deacetylase-1 promotes urothelial cell migration and invasion by modulating p63-pMLC2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3354. doi:10.1158/1538-7445.AM2017-3354