Human Urothelial Cancer Cell Lines Research Articles

Trichostatin C Synergistically Interacts with DNMT Inhibitor to Induce Antineoplastic Effect via Inhibition of Axl in Bladder and Lung Cancer Cells.

Aberrant epigenetic modifications are fundamental contributors to the pathogenesis of various cancers. Consequently, targeting these aberrations with small molecules, such as histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors, presents a viable strategy for cancer therapy. The objective of this study is to assess the anti-cancer efficacy of trichostatin C (TSC), an analogue of trichostatin A sourced from the fermentation of Streptomyces sp. CPCC 203909. Our investigations reveal that TSC demonstrates potent activity against both human lung cancer and urothelial bladder cancer cell lines, with IC50 values in the low micromolar range. Moreover, TSC induces apoptosis mediated by caspase 3/7 and arrests the cell cycle at the G2/M phase. When combined with the DNMT inhibitor decitabine, TSC exhibits a synergistic anti-cancer effect. Additionally, protein analysis elucidates a significant reduction in the expression of the tyrosine kinase receptor Axl. Notably, elevated concentrations of TSC correlate with the up-regulation of the transcription factor forkhead box class O1 (FoxO1) and increased levels of the proapoptotic proteins Bim and p21. In conclusion, our findings suggest TSC as a promising anti-cancer agent with HDAC inhibitory activity. Furthermore, our results highlight the potential utility of TSC in combination with DNMT inhibitors for cancer treatment.

HUS1 as a Potential Therapeutic Target in Urothelial Cancer.

Platinum-based chemotherapy is the standard of care with concern to first-line systemic therapy for metastatic disease in urothelial cancer (UC). Resistance to chemotherapy despite an initial response is linked with the ability to remove platinum-based DNA adducts and to repair chemotherapy-induced DNA lesions by various DNA repair proteins. The Rad9-Rad1-HUS1 complex that is loaded onto DNA at sites of damage is involved in checkpoint activation as well as DNA repair. Here, we addressed for the first time the potential influence of HUS1 expression in urothelial carcinogenesis (using two human basal urothelial cancer cell lines UM-UC-3 and HT1197) and its role as a potential therapeutic target for predicting responses to platinum-based chemotherapy. Specific inhibition of HUS1 expression in both cell lines was achieved by specific siRNA and validated by Western blot. In order to define the possible importance of HUS1 in the regulation of cellular proliferation, parental and resistant cells were treated with increasing concentrations of either control or HUS1 siRNA. HUS1 protein expression was observed in both human basal urothelial cancer cell lines UM-UC-3 and HT1197. In cisplatin-sensitive cells, knock-down of HUS1 inhibited cellular proliferation in the presence of cisplatin. On the contrary, knock-down of HUS1 in resistant cells did not result in a re-sensitization to cisplatin. Finally, RNAseq data from the Cancer Genome Atlas provided evidence that HUS1 expression is a significant prognostic factor for poor survival in UC patients. In summary, HUS1 may acts as an oncogene in UC and might be a key determinant of the cellular response to cisplatin-based chemotherapy.

In-Vitro Identification and In-Vivo Confirmation of DNA Methylation Biomarkers for Urothelial Cancer.

We identified DNA methylation targets specific for urothelial cancer (UC) by genome-wide methylation difference analysis of human urothelial (RT4, J82, 5637), prostate (LNCAP, DU-145, PC3) and renal (RCC-KP, CAKI-2, CAL-54) cancer cell lines with their respective primary epithelial cells. A large overlap of differentially methylated targets between all organs was observed and 40 Cytosine-phosphate-Guanine motifs (CpGs) were only specific for UC cells. Of those sites, two also showed high methylation differences (≥47%) in vivo when we further compared our data to those previously obtained in our array-based analyses of urine samples in 12 UC patients and 12 controls. Using mass spectrometry, we finally assessed seven CpG sites in this “bladder-specific” region of interest in urine samples of patients with urothelial (n = 293), prostate (n = 75) and renal (n = 23) cancer, and 143 controls. DNA methylation was significantly increased in UC compared to non-UC individuals. The differences were more pronounced for males rather than females. Male UC cases could be distinguished from non-UC individuals with >30% sensitivity at 95% specificity (Area under the curve (AUC) 0.85). In summary, methylation sites highly specific in UC cell lines were also specific in urine samples of UC patients showing that in-vitro data can be successfully used to identify biomarker candidates of in-vivo relevance.

Inhibition of Heparanase Expression Results in Suppression of Invasion, Migration and Adhesion Abilities of Bladder Cancer Cells.

Heparan sulfate proteoglycan syndecan-1, CD138, is known to be associated with cell proliferation, adhesion, and migration in malignancies. We previously reported that syndecan-1 (CD138) may contribute to urothelial carcinoma cell survival and progression. We investigated the role of heparanase, an enzyme activated by syndecan-1 in human urothelial carcinoma. Using human urothelial cancer cell lines, MGH-U3 and T24, heparanase expression was reduced with siRNA and RK-682, a heparanase inhibitor, to examine changes in cell proliferation activity, induction of apoptosis, invasion ability of cells, and its relationship to autophagy. A bladder cancer development mouse model was treated with RK-682 and the bladder tissues were examined using immunohistochemical analysis for Ki-67, E-cadherin, LC3, and CD31 expressions. Heparanase inhibition suppressed cellular growth by approximately 40% and induced apoptosis. The heparanase inhibitor decreased cell activity in a concentration-dependent manner and suppressed invasion ability by 40%. Inhibition of heparanase was found to suppress autophagy. In N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer mice, treatment with heparanase inhibitor suppressed the progression of cancer by 40%, compared to controls. Immunohistochemistry analysis showed that heparanase inhibitor suppressed cell growth, and autophagy. In conclusion, heparanase suppresses apoptosis and promotes invasion and autophagy in urothelial cancer.

PTRF independently predicts progression and survival in multiracial upper tract urothelial carcinoma following radical nephroureterectomy

ObjectivesPolymerase I and transcript release factor (PTRF) has been implicated in cancer biology but its role in upper tract urothelial carcinoma (UTUC) is unknown. From a pilot transcriptome, we identified PTRF was significantly upregulated in high stage UTUC. Bladder cancer transcriptome from The Cancer Genome Atlas (TCGA) supported our finding and high PTRF level also predicted poor survival. We, therefore, investigated the correlation of PTRF with patients’ clinicopathologic characteristics and outcomes in a multiracial UTUC cohort. Materials and methodsBy immunohistochemical staining, PTRF expression was determined using H-score. PTRF expression of 575 UTUCs from 8 institutions, including 118 Asians and 457 Caucasians, was compared with various clinicopathologic parameters. Human urothelial cancer cell lines were used to evaluate the level of PTRF protein and mRNA expression, and PTRF transcript level was assessed in fresh samples from 12 cases of the cohort. The impact of PTRF expression on disease progression, cancer-specific death and overall mortality was also examined. ResultsHigh PTRF expression was significantly associated with multifocality (P = 0.023), high pathologic tumor stage (P < 0.00001), nonurothelial differentiation (P = 0.035), lymphovascular invasion (P = 0.003) and lymph node metastasis (P = 0.031). PTRF mRNA expression was also markedly increased in advanced stage UTUC (P = 0.0003). High PTRF expressing patients had consistently worse outcomes than patients with low PTRF expression regardless of demographic variation (all P < 0.005). In multivariate analysis, high PTRF expression was an independent predictor for progression-free survival (hazard ratio [HR] 1.70, 95% confidence interval [CI] 1.07–2.69, P = 0.025), cancer-specific survival (HR 2.09, 95% CI 1.28–3.42, P = 0.003), and overall survival (HR 2.04, 95% CI 1.33–3.14, P = 0.001). ConclusionsResults indicate that PTRF is a predictive biomarker for progression and survival and an independent prognosticator of UTUC. Elevated PTRF could probably propel clinically aggressive disease and serve as a potential therapeutic target for UTUC.

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.

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.

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

Collagen type IV alpha 1 (COL4A1) and collagen type XIII alpha 1 (COL13A1) produced in cancer cells promote tumor budding at the invasion front in human urothelial carcinoma of the bladder.

Current knowledge of the molecular mechanism driving tumor budding is limited. Here, we focused on elucidating the detailed mechanism underlying tumor budding in urothelial cancer of the bladder. Invasive urothelial cancer was pathologically classified into three groups as follows: nodular, trabecular, and infiltrative (tumor budding). Pathohistological analysis of the orthotopic tumor model revealed that human urothelial cancer cell lines MGH-U3, UM-UC-14, and UM-UC-3 displayed typical nodular, trabecular, and infiltrative patterns, respectively. Based on the results of comprehensive gene expression analysis using microarray (25 K Human Oligo chip), we identified two collagens, COL4A1 and COL13A1, which may contribute to the formation of the infiltrative pattern. Visualization of protein interaction networks revealed that proteins associated with connective tissue disorders, epithelial-mesenchymal transition, growth hormone, and estrogen were pivotal factors in tumor cells. To evaluate the invasion pattern of tumor cells in vitro, 3-D collective cell invasion assay using Matrigel was performed. Invadopodial formation was evaluated using Gelatin Invadopodia Assay. Knockdown of collagens with siRNA led to dramatic changes in invasion patterns and a decrease in invasion capability through decreased invadopodia. The in vivo orthotopic experimental model of bladder tumors showed that intravesical treatment with siRNA targeting COL4A1 and COL13A1 inhibited the formation of the infiltrative pattern. COL4A1 and COL13A1 production by cancer cells plays a pivotal role in tumor invasion through the induction of tumor budding. Blocking of these collagens may be an attractive therapeutic approach for treatment of human urothelial cancer of the bladder.

Loss of DAB2IP expression in human urothelial carcinoma is associated with poorer recurrence-free survival.

The purpose of this study is to investigate the clinical relevance of deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP) expression in human urothelial carcinoma (UC). We studied DAB2IP protein expression by immunohistochemistry in 130 UCs (90 of the bladder and 40 of the upper urinary tract) and 79 adjacent normal tissues and assessed its prognostic value in terms of recurrence-free and progression-free survival in superficial bladder UC. Twelve human UC cell lines were examined for DAB2IP messenger RNA (mRNA) and protein expression using quantitative RT-PCR and western blotting. Selected cell lines were used to study the effect of treatment with chromatin-modifying agents (5-aza-2'-deoxycytidine, Trichostatin A, or both) on DAB2IP expression. Of 90 bladder tumors, 50 (56%) and, of 40 upper tract UC, 11 (28%) were positive for DAB2IP immunostaining (bladder cancer versus upper tract UC, p=0.003). In 65 superficial cases of bladder cancer loss of DAB2IP, expression was significantly associated with decreased recurrence-free survival (p=0.046), but not with progression-free survival. Most human urothelial cancer cell lines consistently express DAB2IP mRNA and protein, without any relation to S-phase kinase protein expression. After treatment with either 5-aza-2'-deoxycytidine or Trichostatin A or both, the low DAB2IP-expressing bladder cancer cell lines BFTC905 and BFTC909 showed increased DAB2IP mRNA expression. DAB2IP protein levels are higher in bladder cancer than in upper tract UC and in superficial bladder cancer. This is associated with longer recurrence-free survival. Epigenetic regulation of DAB2IP protein appears to play an important role in human urothelial carcinoma.

Cancer. TERT promoter mutations and telomerase reactivation in urothelial cancer.

Reactivation of telomerase, the chromosome end-replicating enzyme, drives human cell immortality and cancer. Point mutations in the telomerase reverse transcriptase (TERT) gene promoter occur at high frequency in multiple cancers, including urothelial cancer (UC), but their effect on telomerase function has been unclear. In a study of 23 human UC cell lines, we show that these promoter mutations correlate with higher levels of TERT messenger RNA (mRNA), TERT protein, telomerase enzymatic activity, and telomere length. Although previous studies found no relation between TERT promoter mutations and UC patient outcome, we find that elevated TERT mRNA expression strongly correlates with reduced disease-specific survival in two independent UC patient cohorts (n = 35; n = 87). These results suggest that high telomerase activity may be a better marker of aggressive UC tumors than TERT promoter mutations alone.

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.

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...

Ciprofloxacin and Epirubicin Synergistically Induce Apoptosis in Human Urothelial Cancer Cell Lines

Introduction: Few published in vitro studies have shown antitumor drug action or possible synergistic effects of fluoroquinolones. To assess the potential role of combination therapy, cytotoxic effects of ciprofloxacin and epirubicin alone and in combination were determined. Material and Methods: Human urothelial cancer cell lines HT1197 and HT1376 were exposed in vitro for 1 h to different concentrations of epirubicin (0.02–2 mg/ml) and for 72 h to ciprofloxacin (0.004–0.8 mg/ml). Cytotoxicity was determined using the microculture tetrazolium assay and flow cytometry. Synergistic cytotoxic effects were determined by calculating combination indices. Results: Median effect concentrations of epirubicin for HT1376 and HT1197 cells were as low as 124 and 117 µg/ml, respectively. Ciprofloxacin-treated cells exhibited profound cytotoxic effects at concentrations of 50–100 µg/ml, which is far below the intravesical concentration reached by standard oral application. In addition, a pronounced synergistic effect was found when the two treatments were combined. Conclusions: This study provides evidence that ciprofloxacin and epirubicin exhibit synergistic cytotoxic effects in vitro. After confirmatory animal experiments, future clinical studies of adjuvant chemotherapy after transurethral bladder resection may include treatment arms with combinations of fluoroquinolones based on the observed synergistic effects to reduce both side effects and costs.

Stress-activated kinase pathway alteration is a frequent event in bladder cancer

ObjectivesThe stress-activated MAP kinases (SAPK) signaling pathways play a critical role in the cellular response to toxins and physical stress, mediate inflammation, and modulate carcinogenesis and tumor metastasis. The stress-activated MAP kinases (MAPK) c-Jun N-terminal kinase (JNK) and p38 are activated upon phosphorylation by a widely expressed and conserved family of upstream MAP kinase kinases (MAP2K). Signaling mediated by p38 and JNK has well-established importance in cancer, yet the contribution of this pathway in urothelial bladder cancer is not understood. This study evaluated stress-activated MAP kinase pathway expression in cell lines derived from human urothelial carcinomas. Materials and methodsTotal protein lysates from a panel of human urothelial bladder cancer cell lines (RT4, T24, UMUC-3, J82, 5637, 253J, and 253J-BV) were analyzed by immunoblotting for the JNK and p38 MAPKs, as well as MKK3, MKK4, MKK6, and MKK7. Quantitative real time PCR was utilized to determine mRNA expression levels of the MAP2Ks. Stress stimuli (sorbitol, hydrogen peroxide, and UV irradiation) were used to active p38, which was measured by phospho-antibody. ResultsAlthough protein levels were variable, all cell lines expressed p38 and JNK. On the other hand, with the exception of the well-differentiated cell line RT4, each cell line had a reduction or absence of expression of one or more MAP2K. 253J and 253J-BV exhibited no expression of MKK6, even when an excess of protein was queried. mRNA levels indicated that both transcriptional and post-transcriptional mechanisms are involved in the regulation of MAP2Ks. Decreased MAP2K expression correlated with decreased ability to activate p38 in response to stress stimuli. ConclusionsAberrant MAP2K protein expression indicates that altered cellular signal transduction mediated via JNK and p38 may be common in bladder cancer. Down-regulation of MAP2Ks likely occurs at both the transcriptional and post-transcriptional levels. Consistent with the known function of p38 and JNK in apoptosis, defects in normal pathway function caused by decreased expression of upstream MAP2Ks may provide a survival advantage to bladder cancer cells. Further investigations should focus on identifying a functional role for these pathways in bladder cancer development.

1151 DIRECT ANTITUMOR EFFECT OF BACILLUS CALMETTE-GUERIN IN HIGH GRADE UROTHELIAL CANCER CELLS WHICH EXPRESS VLA5 (ALPHA 5 BETA 1 INTEGRIN)

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20101151 DIRECT ANTITUMOR EFFECT OF BACILLUS CALMETTE-GUERIN IN HIGH GRADE UROTHELIAL CANCER CELLS WHICH EXPRESS VLA5 (ALPHA 5 BETA 1 INTEGRIN) Tomoyuki Kato, Mayu Yagi, Atsushi Yamagishi, Noriyuki Hosoya, Toshihiko Sakurai, Hayato Nishida, Sei Naito, Masaaki Tsukigi, Hisashi Kawazoe, Akinori Muto, Akira Nagaoka, and Yoshihiko Tomita Tomoyuki KatoTomoyuki Kato More articles by this author , Mayu YagiMayu Yagi More articles by this author , Atsushi YamagishiAtsushi Yamagishi More articles by this author , Noriyuki HosoyaNoriyuki Hosoya More articles by this author , Toshihiko SakuraiToshihiko Sakurai More articles by this author , Hayato NishidaHayato Nishida More articles by this author , Sei NaitoSei Naito More articles by this author , Masaaki TsukigiMasaaki Tsukigi More articles by this author , Hisashi KawazoeHisashi Kawazoe More articles by this author , Akinori MutoAkinori Muto More articles by this author , Akira NagaokaAkira Nagaoka More articles by this author , and Yoshihiko TomitaYoshihiko Tomita More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.650AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical instillation of Bacillus Calmette-Guerin (BCG) is an effective treatment for superficial high grade bladder cancer. Although both immunological and direct effects on tumor cells have been proposed, the complete mechanisms for its effect are not fully understood. Presence of fibronectin has been shown to be necessary for binding and internalization of BCG into tumor cells to exert direct effect on them. We have previously demonstrated the correlation between high expression of fibronectin receptor VLA5 (alpha 5 beta 1 integrin) and high malignant potential of urothelial cancer (UC). Here, we assessed the direct antitumor effects of BCG for UC cell lines which beard various amount of VLA5 on the cell surface. METHODS The human UC cell lines (T24, HT1376 and RT4) and Tokyo 172 BCG strain (Immunobladder, Japan BCG Laboratory), BCG cell wall skeleton (BCG-CWS SMP105, Dainippon Sumitomo Pharma) were used in this study. Viability of cell lines was evaluated with crystal violet stainig assay. Fluorescence of apoptotic nuclei stained with Propidium Iodide (PI) was measured with a FACScan. DNA synthesis was evaluated with BrDU incorporation assay. RESULTS First, expression of VLA5 on the surface of cell lines was checked. T24 and HT1376, high grade UC, beard large amount of VLA5 on the cell surface and RT4, a low grade UC, had traces of VLA5. Treatment with BCG resulted in dose dependent decrease of relative viability in T24 (p<0.0001) and HT1376 (p<0.0001). This effect was partially reversed by coculturing cells with VLA5 blocking antibody. In RT4, viability was not altered by treatment with BCG. Sub-G1 fraction of PI stained T24 increased from less than 1% (untreated) to more than 4% (1 mg/ml of BCG) after exposure to BCG and it suppressed BrDU incorporation (p=0.004) in T24 cells. Autoclaved killed BCG also demonstrated cell growth retardation effect in T24 (p<0.0001) and HT1376 (p<0.0001). BCG-CWS SMP105 also delayed T24 and HT1376 cell growth (p<0.0001). CONCLUSIONS These findings suggest that BCG exerts direct antitumor effect on UC cells via VLA5 with induction of apoptosis and inhibition of DNA synthesis. Moreover, killed BCG and BCG cell wall skeleton were able to exert similar effect making this approach attractive for clinical application where potentially serious adverse effect of viable BCG can be avoided while maintaining antitumor effect using killed BCG or cell extracts. Yamagata, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e445-e446 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tomoyuki Kato More articles by this author Mayu Yagi More articles by this author Atsushi Yamagishi More articles by this author Noriyuki Hosoya More articles by this author Toshihiko Sakurai More articles by this author Hayato Nishida More articles by this author Sei Naito More articles by this author Masaaki Tsukigi More articles by this author Hisashi Kawazoe More articles by this author Akinori Muto More articles by this author Akira Nagaoka More articles by this author Yoshihiko Tomita More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Role of epithelial-to-mesenchymal transition (EMT) in drug sensitivity and metastasis in bladder cancer

Epithelial-to-mesenchymal transition (EMT) is a process that plays essential roles in development and wound healing that is characterized by loss of homotypic adhesion and cell polarity and increased invasion and migration. At the molecular level, EMT is characterized by loss of E-cadherin and increased expression of several transcriptional repressors of E-cadherin expression (Zeb-1, Zeb-2, Twist, Snail, and Slug). Early work established that loss of E-cadherin and increased expression of MMP-9 was associated with a poor clinical outcome in patients with urothelial tumors, suggesting that EMT might also be associated with bladder cancer progression and metastasis. More recently, we have used global gene expression profiling to characterize the molecular heterogeneity in human urothelial cancer cell lines (n = 20) and primary patient tumors, and unsupervised clustering analyses revealed that the cells naturally segregate into two discrete "epithelial" and "mesenchymal" subsets, the latter consisting entirely of muscle-invasive tumors. Importantly, sensitivity to inhibitors of the epidermal growth factor receptor (EGFR) or type-3 fibroblast growth factor receptor (FGFR3) was confined to the "epithelial" subset, and sensitivity to EGFR inhibitors could be reestablished by micro-RNA-mediated molecular reversal of EMT. The results suggest that EMT coordinately regulates drug resistance and muscle invasion/metastasis in urothelial cancer and is a dominant feature of overall cancer biology.

Prognostic Relevance of Prothymosin-α Expression in Human Upper Urinary Tract Transitional Cell Carcinoma

To investigate the prognostic role of prothymosin-alpha (PTMA) expression in human upper urinary tract transitional cell carcinoma (UUT-TCC). Paraffin-embedded tissues were collected from 91 patients with UUT-TCC and from 15 paired normal renal cortex and 13 paired urothelial walls. The primary antibody for PTMA (2F11) used was validated in 4 human urothelial cancer cell lines before assessing the surgical specimen. Immunohistochemistry was then conducted to determine the expression intensity of PTMA, the calculation of immunostaining density using imaging analysis, and for immunostaining localization. The correlates with clinicopathologic characteristics and patient survival were explored. The expression intensity of PTMA demonstrated a significant enhancement of PTMA expression in UUT-TCCs compared with both paired normal tissues (P = .0002 and P = .0004 for UUT-TCC vs the urothelial wall and vs the renal cortex, respectively). As for the localization of PTMA immunoreactivity, of the 91 tumor specimens, 33 (36.3%) were cytoplasmic PTMA-expressing, 51 (56.0%) were nuclear PTMA-expressing, and 7 (7.7%) were PTMA-negative tumors. On univariate and multivariate analyses, PTMA expression localization was the sole independent prognostic indicator for recurrence-free survival (hazard ratio 4.90, 95% confidence interval 1.73-13.9; P = .003), although pathologic staging was an independent prognostic indicator for both progression-free survival (hazard ratio 22.6, 95% confidence interval 2.56-198; P = .005) and disease-specific overall survival (hazard ratio 5.60, 95% confidence interval 1.48-21.2; P = .011). The limitations of our study included small patient numbers and short follow-up. The results of our study have shown that PTMA is overexpressed in UUT-TCCs and that cytoplasmic PTMA expression can provide significant prognostic information for subsequent tumor recurrence in the residual urinary tract after nephroureterectomy.