Bladder Urothelial Cells Research Articles

Role of a Kinesin Motor in Cancer Cell Mechanics.

Molecular motors play important roles in force generation, migration, and intracellular trafficking. Changes in specific motor activities are altered in numerous diseases. KIF20A, a motor protein of the kinesin-6 family, is overexpressed in bladder cancer, and KIF20A levels correlate negatively with clinical outcomes. We report here a new role for the KIF20A kinesin motor protein in intracellular mechanics. Using optical tweezers to probe intracellular mechanics and surface AFM to probe cortical mechanics, we first confirm that bladder urothelial cells soften with an increasing cancer grade. We then show that inhibiting KIF20A makes the intracellular environment softer for both high- and low-grade bladder cancer cells. Upon inhibition of KIF20A, cortical stiffness also decreases in lower grade cells, while it surprisingly increases in higher grade malignant cells. Changes in cortical stiffness correlate with the interaction of KIF20A with myosin IIA. Moreover, KIF20A inhibition negatively regulates bladder cancer cell motility irrespective of the underlying substrate stiffness. Our results reveal a central role for a microtubule motor in cell mechanics and migration in the context of bladder cancer.

Pyroptosis engagement and bladder urothelial cell-derived exosomes recruit mast cells and induce barrier dysfunction of bladder urothelium after uropathogenic E. coli infection.

The specific regulatory mechanism of bladder urothelial barrier dysfunction after infection with uropathogenic Escherichia coli (UPEC) is still unclear. The cross talk between bladder urothelial cells and mast cells may play an important role during UPEC infection. In this study, the pyroptosis of urothelial cells was investigated after UPEC infection both in vivo and in vitro. The levels of IL-1β and IL-18 in exosomes derived from bladder urothelial cells after UPEC infection were detected. The role of these processes in the recruitment and activation of mast cells was measured. The mechanism of mast cell-induced disruption of bladder epithelial barrier function was also assessed. We found that UPEC infection induced pyroptosis of bladder urothelial cells and led to the release of IL-1β and IL-18 in the form of exosomes, which promoted the migration of mast cells. Tryptase secreted by mast cells aggravated the damage to the barrier function of the bladder urothelium by acting on protease-activated receptor 2 (PAR2). Inhibition of pyroptosis or the tryptase-PAR2 axis reduced the disruption of bladder urothelial barrier function and decreased the bacterial burden. The present study supports a novel mechanism by which pyroptosis-dependent release of exosomes from bladder urothelial cells activates mast cells and regulates bladder urothelial barrier function during UPEC infection.

Collagen hollow structure for bladder tissue engineering

Bladder is affected by numerous pathologies which require augmentation or replacement of the organ. Currently, the gold standard is enterocystoplasty which causes many complications. Bioengineering techniques propose options to overcome these issues. The innovative and very simple tissue engineered three-dimensional spherical bladder model reported here mimics the bladder natural shape using collagen-derived scaffold. Bladder mesenchymal cells were embedded inside the scaffold and epithelial cells seeded at its surface.Therefore, the bladder mesenchymal and urothelial cells seeded in the model were subjected to tensions similar to what is found in the native tissue. Both cell types organize themselves simultaneously within a culture period of 15 days. Our spherical model was able to demonstrate characteristics of highly advanced urothelial maturity. Hematoxylin eosin staining, the uroplakins immunodetection and electron microscopy analysis showed the impressive degree of urothelial organization. In addition, collagen remodeling was observed and smooth muscle cells, expressing myosin, presented a tendency to realign parallel to the luminal surface.With properties comparable to native tissue, our three-dimensional spherical bladder model could offer the possibility to produce tissue-engineered bladder implants. This technique could be efficient for partial replacement of pathologic bladder sites.

Long non-coding RNA MEG3 suppresses the development of bladder urothelial carcinoma by regulating miR-96 and TPM1

ABSTRACTWe aimed at investigating effects of long non-coding RNA maternally expressed 3 (MEG3) on the proliferation, cell cycle and apoptosis of bladder urothelial carcinoma cells and regulatory relationships among lncRNA MEG3, miR-96 and α-tropomyosin 1 (TPM1). Human clinical data from The Cancer Genome Atlas (TCGA) which contains bladder urothelial carcinoma tissues and adjacent tissues were used for analysis. The expression profiles of MEG3, miR-96, TPM1, cell cycle-related genes and apoptosis-related genes were examined by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Regulating relationship among MEG3, miR-96 and TPM1 was confirmed by dual luciferase reporter assay. MTT assay and flow cytometry were performed to observe cell proliferation, cell cycle and apoptosis. The effects of lncRNA MEG3 on bladder urothelial carcinoma were confirmed both in vivo and in vitro. The mRNA expression and protein expression of MEG3, TPM1 were down-regulated in carcinoma tissues, whereas miR-96 expression was up-regulated. MEG3 overexpression resulted in miR-96 downregulation along with TPM1 upregulation, which inhibited cell proliferation and cell cycle but promoted cell apoptosis of bladder urothelial carcinoma cells in vitro, and at the same time inhibited tumor growth in vivo. In this process, expressions of apoptosis-related protein BCL2 associated X (Bax), cleaved-caspase 3 was up-regulated, whereas apoptosis regulator protein (Bcl-2) expression was suppressed when MEG3 was overexpressed, and cell cycle-related protein Cyclin D1 was down-regulated. LncRNA MEG3 low-expression promotes the proliferation and inhibits apoptosis of bladder urothelial carcinoma cells by regulating miR-96 along with TPM1.

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.

Fibroblasts from pBOO promote tumorigenesis by secreting TGFbeta1 to induce EMT in bladder urothelial carcinoma cells.

Urinary bladder cancer (UBC) is one of the most common malignancies worldwide. UBC patients at muscle invasive stage have poor clinical outcome, due to high propensity for metastasis. Non-tumor activated fibroblasts, named α-SMA+Fs, is similar to carcinoma-associated fibroblasts (CAFs) which could express α-SMA. However, whether α-SMA+Fs patients could induce UBC cell invasion is unclear. Herein, we found that characterization of primary α-SMA+Fs separated from PBOO (partial bladder outlet obstruction) rats was fell in between normal fibroblasts (α-SMA-Fs) and CAFs. Additionally, the conditional medium from α-SMA+Fs enhanced the NBT-II cell invasion through inducing EMT, and the oncogenic function of mixed supernatant of α-SMA+Fs/CAFs was stronger than that of CAFs. Inhibition of TGF-β1 by TGF-β1 neutralizing antibody decreased the EMT-associated gene expression and NBT-II cell invasion, suggesting that α-SMA+Fs can induce tumor EMT through TGF-β1. Xenograft experiments showed that the tumorigenic effect of α-SMA+Fs in mice was also between CAFs and α-SMA-Fs, and α-SMA+Fs/CAFs also had a strong tumorigenic effect. We preformed rats with PBOO and found that the incidence of invasive bladder cancer in PBOO+BBN group was higher than in BBN group, suggesting the PBOO treatment contributed to tumorigenesis. Thus, α-SMA+Fs promoted tumorigenesis by secreting TGF-β1 to induce EMT.

Tumor Necrosis Factor-α Initiates miRNA-mRNA Signaling Cascades in Obstruction-Induced Bladder Dysfunction

Bladder outlet obstruction (BOO) and the ensuing clinical lower urinary tract dysfunction are common in elderly patients. BOO is accompanied by urodynamic changes in bladder function and leads to organ fibrosis and ultimately loss of contractility. Comprehensive transcriptome analysis of bladder samples from human patients with different urodynamically defined phenotypes of BOO revealed tumor necrosis factor (TNF)-α as the top upstream signaling pathway regulator. Herein, we validated next-generation sequencing and pathway analysis in cell-based models using bladder smooth muscle and urothelial cells exposed to TNF-α. miRNA profiling and transcriptome analysis of TNF-α-treated bladder smooth muscle cells revealed striking similarities with human BOO. Using a comparative approach, TNF-specific and TNF-independent pathways were delineated in human biopsy specimens. Concomitant down-regulation of smooth muscle cell-specific miRNAs and smooth muscle markers after TNF-α treatment was in accordance with the loss of contractility in humans in advanced obstruction-induced bladder remodeling. The expression levels of four abundant TNF-regulated miRNAs were modulated; the compensatory up-regulation of miR-199a-5p reduced NF-κB signaling. Essential hubs of TNF-α signaling pathways mitogen-activated protein kinase kinase kinase (apoptosis signal-regulating kinase 1) and inhibitor of nuclear factor κ B kinase subunit β (IκB kinase β) were targeted by miR-199a-5p. miR-199a-5p might be part of a negative feedback loop, reducing the impact of TNF, whereas its down-regulation in acontractile bladders from BOO patients advances the disease. The compensatory up-regulation of miR-199a-5p together with TNF-α inhibition may be therapeutically beneficial.

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation.

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma.

Pomegranate (Punica granatum L.) fruit has been demonstrated to have the inhibitory activities to various tumors. In this study, we try to uncover the molecular mechanism underlying the inhibitory capability of Taiwanese local pomegranate fruit to urinary bladder urothelial carcinoma. The results collected from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated that the ethanol extract of pomegranate peel exhibited better inhibitory activity to human urinary bladder urothelial carcinoma T24 and J82 cells than that of pulp. Furthermore, the ethylacetate layer of peel ethanol extract was observed to have the best inhibitory activity against urinary bladder urothelial carcinoma cells. One of the eight fractions (PEPE2 fraction) collected from the ethylacetate layer with Diaion HP-20 column chromatography demonstrated the highest inhibitory activity in urinary bladder urothelial carcinoma cells. The results of the flow cytometry and apoptotic pathway studies suggested that the inhibitory activity of PEPE2 fraction were attributed to the UBUC cell apoptosis. To confirm the above results, our results of xenograft-induced bladder tumor in nude mice showed that the oral consumption of the ethylacetate layer (2, 5, 10 and 100 mg/kg) could decrease the volume and weight of T24 tumors and caused the apoptosis in the xenografted tumors, which was observed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay. This study provided the likelihood that the traditionally non-edible pomegranate peel waste is re-utilized to make an affordable and promising chemopreventive product to prevent UBUC incidence or recurrence.

Role of neurogenic inflammation in local communication in the visceral mucosa.

Intense research has focused on the involvement of the nervous system in regard to cellular mechanisms underlying neurogenic inflammation in the pelvic viscera. Evidence supports the neural release of inflammatory factors, trophic factors, and neuropeptides in the initiation of inflammation. However, more recently, non-neuronal cells including epithelia, endothelial, mast cells, and paraneurons are likely important participants in nervous system functions. For example, the urinary bladder urothelial cells are emerging as key elements in the detection and transmission of both physiological and nociceptive stimuli in the lower urinary tract. There is mounting evidence that these cells are involved in sensory mechanisms and can release mediators. Further, localization of afferent nerves next to the urothelium suggests these cells may be targets for transmitters released from bladder nerves and that chemicals released by urothelial cells may alter afferent excitability. Modifications of this type of communication in a number of pathological conditions can result in altered release of epithelial-derived mediators, which can activate local sensory nerves. Taken together, these and other findings highlighted in this review suggest that neurogenic inflammation involves complex anatomical and physiological interactions among a number of cell types in the bladder wall. The specific factors and pathways that mediate inflammatory responses in both acute and chronic conditions are not well understood and need to be further examined. Elucidation of mechanisms impacting on these pathways may provide insights into the pathology of various types of disorders involving the pelvic viscera.

Tadalafil attenuates hypotonicity-induced Ca2+ influx via TRPV2 and TRPV4 in primary rat bladder urothelial cell cultures.

To investigate the localization of phosphodiesterase 5 (PDE5) and the molecular mechanism underlying the effect of the PDE5 inhibitor tadalafil in signal transduction in the bladder urothelium. PDE5 expression in rat bladder tissues and cultured primary rat bladder urothelial cells was evaluated using immunochemistry and western blot assays. Ca2+ influx in cells exposed to isotonic solution, hypotonic solution, a selective transient receptor potential vanilloid 2 (TRPV2) channel agonist (cannabidiol), a selective TRPV4 channel agonist (GSK1016790A), a TRP cation channel melastatin 7 (TRPM7) channel agonist (PIP2), or a purinergic receptor agonist (ATP) in the presence or absence of 10 µM tadalafil was evaluated using calcium imaging techniques. We also evaluated stretch-induced changes in ATP concentration in the mouse bladder in the presence or absence of 100 µM tadalafil. Immunochemistry and western blot analyses demonstrated that PDE5 is abundantly expressed in the bladder urothelium and in primary rat urothelial cells. Ca2+ influx induced by hypotonic stimulation, GSK1016790A, or cannabidiol was significantly inhibited by tadalafil, whereas ATP-induced Ca2+ influx was unaffected by tadalafil. PIP2 did not induce Ca2+ influx. ATP release in tadalafil-pretreated bladders significantly decreased compared to control bladders. Tadalafil attenuates Ca2+ influx via TRPV4 and TRPV2, and inhibits ATP release in the bladder urothelium. These findings indicate that tadalafil functions as an inhibitor of urothelial signal transduction.

Ganoderma microsporum immunomodulatory protein induces apoptosis and potentiates mitomycin C-induced apoptosis in urinary bladder urothelial carcinoma cells.

Current chemotherapy and immunotherapy treatments followed by transurethral resection for urinary bladder urothelial carcinoma (UC) usually suffer from poor prognosis and high recurrence rate. Design and modification of current formulation with the novel adjuvants are needed. A recombinant protein derived from Ganoderma microsporum named as Ganoderma microsporum immunomodulatory protein (GMIP) was used to treat UC cells. We found GMIP elicits a dose-dependent and time-dependent anti-UC cell proliferation effect, with a half-maximal inhibition concentration (IC50 ) comparable to mitomycin C (MMC), a commonly used chemotherapy agent. After GMIP treatment, UC cells showed apoptotic phenomenon including cell cycle arrest in the G1 phase, elevated sub-G1 population, mitochondrial membrane potential loss, up-regulated p21 expression, p21 nuclear translocation, caspase activation, and PARP cleavage in a p53-independent but p21-mediated pathways. Unlike lung cancer cells, GMIP treated UC cells showed no autophagic scheme including Beclin-1, an autophagy to apoptosis switch marker, was not cleaved by caspase 3 and slight LC3B-II accumulation. Also, the classic autophagic inhibitor, chloroquine had no effect in GMIP-mediated cell death made us conclude that GMIP induced apoptosis through caspase activation but not autophagy in UC cells. Additionally, GMIP showed synergistic effects with MMC in killing UC cells and thus decreased the concentration of MMC usage to reach the comparable apoptotic effects. Our results delineate novel strategies for treatment of UC by GMIP alone or in combination with MMC application and provide a promising therapeutic cocktail for better treatment of urinary bladder urothelial carcinoma.

MiR-99a-5p acts as tumor suppressor via targeting to mTOR and enhances RAD001-induced apoptosis in human urinary bladder urothelial carcinoma cells.

IntroductionmiR-99a-5p, known to play an important role in mammalian target of rapamycin (mTOR) regulation, is downregulated in human bladder cancer. The study aimed to investigate the anticancer activity of miR-99a-5p and the possible mechanism associated with mTOR in bladder cancer cells.Materials and methodsVectors expressing miR-99a-5p were transfected into human urinary bladder urothelial carcinoma (5637 and T24) cells. The level of miR-99a-5p was monitored by microRNA (miRNA) quantitative polymerase chain reaction (QPCR). Luciferase reporter assays were performed to verify the direct binding of miR-99a-5p to mTOR transcripts. The mTOR transcripts and protein levels were measured by QPCR and Western blot, respectively. Cell viability of miR-99a-5p-transfected cells was detected by tetrazolium salt (WST-1). Inhibition of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) signaling was detected by the phosphorylation of mTOR and AKT using Western blot. The ability of miR-99a-5p to enhance RAD001-induced apoptosis was determined as the expression of cleaved caspase 3 and levels of DNA fragmentation.ResultsTransfection of miR-99a-5p-expressing vector elevated the expression level of miR-99a-5p up to sixfold compared to vector-only controls. The results from luciferase assay verified that miR-99a-5p directly binds to the predicted sequence in the 3′ untranslated region (3′-UTR) of mTOR. The levels of mTOR RNA and protein were decreased in miR-99a-5p-transfected cells. Dual inhibition of mTORC1 and mTORC2 by miR-99a-5p was confirmed by the decreased phosphorylation of mTOR (at Ser2448 and Ser2481), phospho-rpS6 and phospho-4EBP1. The phosphorylation of AKT was significantly inhibited in miR-99a-5p-transfected cells upon RAD001 treatment. Enforced expression of miR-99a-5p potentiated RAD001-induced apoptosis in these cells.ConclusionThis is the first study showing that miR-99a-5p markedly inhibits the growth of bladder cancer cells via dual inhibition of mTORC1 and mTORC2. Our data demonstrated that forced expression of miR-99a-5p inhibits the feedback of AKT survival pathway and enhances the induction of apoptosis in RAD001-treated bladder cancer cells.

Co-culturing porcine normal urothelial cells, urinary bladder fibroblasts and smooth muscle cells for tissue engineering research.

New strategies for culturing and co-culturing of the main types of urinary bladder cells are essential for successful establishment of biomimetic in vitro models, which could be applied for research into, and management of, diverse urological disorders. Porcine normal urothelial cells are available in nearly unlimited amounts and have many properties equivalent to human urothelial cells. In the present study, we established normal differentiated porcine urothelial cells in co-cultures with porcine urinary bladder normal fibroblasts and/or smooth muscle cells. The optimal culture medium for establishment of differentiated urothelial cells, demonstrated by positive immunofluorescence of uroplakins, cytokeratins (CK 7, CK 20), zonula occludens 1 (ZO-1), claudin 4, claudin 8, and E-cadherin, was the medium composed of equal parts of Advanced Dulbecco's modified Eagle's medium (A-DMEM) and MCDB 153 medium with physiological calcium concentration of 2.5 mM and without fetal bovine serum, named UroM (+Ca2+ - S). This medium was also proven to be suitable for culturing of bladder fibroblasts and smooth muscle cells and co-culturing of urothelial cells with these mesenchymal cells. Urothelial cell differentiation was optimal in UroM (+Ca2+ - S) medium in all co-culture conditions and when compared to all conditioned-media combinations. To summarize, these strategies for culturing and co-culturing of urinary bladder urothelial cells with mesenchymal cells could be used as new in vitro models for future basic and applicable research of the urinary bladder and thus potentially also for translational tissue engineering studies.

A Versatile Assay for Detection of Aberrant DNA Methylation in Bladder Cancer.

Urothelial carcinoma of the bladder is one of the most common malignancies in the industrialized world, mainly caused by smoking and occupational exposure to chemicals. The favorable prognosis of early stage bladder cancer underscores the importance of early detection for the treatment of this disease. The high recurrence rate of this malignancy also highlights the need for close post-diagnosis monitoring of bladder cancer patients. As for other malignancies, aberrant DNA methylation has been shown to play a crucial role in the initiation and progression of bladder cancer, and thus holds great promise as a diagnostic and prognostic biological marker. Here, we describe a protocol for a versatile DNA methylation enrichment method, the Methylated CpG Island Recovery Assay (MIRA), which enables analysis of the DNA methylation status in individual genes or across the entire genome. MIRA is based on the ability of the methyl-binding domain (MBD) proteins, the MBD2B/MBD3L1 complex, to specifically bind methylated CpG dinucleotides. This easy-to-perform method can be used to analyze the methylome of bladder cancer or urothelial cells shed in the urine to elucidate the evolution of bladder carcinogenesis and/or identify epigenetic signatures of chemicals known to cause this malignancy.

Chitosan hydrochloride has no detrimental effect on bladder urothelial cancer cells

Bladder cancer is among the most common and aggressive human malignant carcinomas, thus targeting and removal of bladder cancer cells is still a challenge. Although it is well known that chitosan hydrochloride (CH-HCl) causes desquamation of normal urothelial cells, its effect on cancer urothelial cells has not been recognized yet.In this in vitro study, we analyzed the cytotoxicity of 0.05% CH-HCl on three urothelial models: two cancer urothelial models, i.e. invasive and papillary urothelial neoplasms, and a normal urothelial model. The cytotoxicity of CH-HCl was evaluated with viability tests, transepithelial resistance (TER) measurements, and electron microscopy.TER measurements showed that 15-minute treatment with CH-HCl caused no reduction in TER of the cancer models, whereas the TER of the normal urothelial model significantly decreased. Furthermore, after CH-HCl treatment, the viability of cancer cells was reduced by only 5%, whereas the viability of normal cells was reduced by 30%. Ultrastructural analysis revealed necrotic cell death in all cases. We have demonstrated that although CH-HCl increases the mortality of cancer urothelial cells, it increases the mortality of normal urothelial cells even more so. However, shorter 2-minute CH-HCl treatment only temporarily increases the permeability of normal urothelial model, i.e. disrupts tight junctions and reduces TER without comprising cell viability, and enables the complete recovery of the permeability barrier after 24h.Overall, our results suggest that CH-HCl cannot be used as a self-sufficient anticancer agent for urothelial bladder cancer treatment; nevertheless a possibility of its use as an enhancer of cytostatic treatment is discussed.

Arsenic treatment increase Aurora-A overexpression through E2F1 activation in bladder cells

BackgroundArsenic is a widely distributed metalloid compound that has biphasic effects on cultured cells. In large doses, arsenic can be toxic enough to trigger cell death. In smaller amounts, non-toxic doses may promote cell proliferation and induces carcinogenesis. Aberration of chromosome is frequently detected in epithelial cells and lymphocytes of individuals from arsenic contaminated areas. Overexpression of Aurora-A, a mitotic kinase, results in chromosomal instability and cell transformation. We have reported that low concentration (≦1 μM) of arsenic treatment increases Aurora-A expression in immortalized bladder urothelial E7 cells. However, how arsenic induces carcinogenesis through Aurora-A activation remaining unclear.MethodsBromodeoxyuridine (BrdU) staining, MTT assay, and flow cytometry assay were conducted to determine cell proliferation. Messenger RNA and protein expression levels of Aurora-A were detected by reverse transcriptional-PCR and Western blotting, respectively.Centrosome of cells was observed by immunofluorescent staining. The transcription factor of Aurora-A was investigated by promoter activity, chromosome immunoprecipitation (ChIP), and small interfering RNA (shRNA) assays. Mouse model was utilized to confirm the relationship between arsenic and Aurora-A.ResultsWe reveal that low dosage of arsenic treatment increased cell proliferation is associated with accumulated cell population at S phase. We also detected increased Aurora-A expression at mRNA and protein levels in immortalized bladder urothelial E7 cells exposed to low doses of arsenic. Arsenic-treated cells displayed increased multiple centrosome which is resulted from overexpressed Aurora-A. Furthermore, the transcription factor, E2F1, is responsible for Aurora-A overexpression after arsenic treatment. We further disclosed that Aurora-A expression and cell proliferation were increased in bladder and uterus tissues of the BALB/c mice after long-term arsenic (1 mg/L) exposure for 2 months.ConclusionWe reveal that low dose of arsenic induced cell proliferation is through Aurora-A overexpression, which is transcriptionally regulated by E2F1 both in vitro and in vivo. Our findings disclose a new possibility that arsenic at low concentration activates Aurora-A to induce carcinogenesis.

Naimi’s Structure and Migration: A Tissue-Specific Pattern of Organization Discovered in Normal Bladder Urothelia

Introduction: Sequential migration and organization are critical events in tissue development. The urothelium, the epithelium lining urologic tissues, is highly specialized regarding the watertight function and adaptation to large and frequent changes in urine volume. To establish the functional state of this tissue, horizontal cells growth is required to allow the vertical development into a multilayered uroepithelium. If this first step is already documented, the mechanism implicated in the switch toward the vertical growth is not yet described. Methods: We had elaborated a three-dimensional bladder model, made of bladder mesenchymal cells (BMC) seeded in a collagen matrix which can promote bladder urothelial cells (BUC) development. Results: This in vitro model allowed us to discover a behavior specific to BUC, resulting in a specific urothelial stratification and differentiation pattern. Cellular alignment into a circular arrangement, named Naimi’s structure, followed by coordinated gyratory cellular movement, named Naimi’s migration, were sequentially observed in our in vitro model and were also confirmed in native bladder Conclusion: We describe here the steps that led to Naimi’s structure and migration. Ultrastructural observations are also provided to document the remarkable organization of urothelial cells and the underlying mesenchymal rearrangement. Keywords: Urinary bladder; Urothelium; Cell migration; Extracellular matrix

Targeting imaging of bladder urothelial cancer cells with quantum dots - prostate stem cell antigen fluorescent probes

Objective To prepare the fluorescent probes of quantum dots conjugated with prostate stem cell antigen (PSCA) monoclonal antibody (QDs-PSCA), study their specific labeling imaging of bladder cancer cells, analyze the feasible of non-invasive tumor-targeted imaging in vivo, provide the experimental basis for non-invasive tumor-targeted imaging in vivo. Methods The method of chemical covalent coupling was used to prepare QD605-PSCA fluorescent probes, then, the absorption spectrum and emission spectrum of probes were measured. Direct immunofluorescence labeling was utilized to detect the targeted imaging of the probes for bladder cancer EJ cells and observe the duration of the fluorescence after labeling. Results The QD605-PSCA probes still had fluorescent properties of QD605. The probes could specifically recognize PSCA protein in bladder cancer cells, the specific fluorescence was mainly expressed in the cell membrane of living cells, the fluorescence was stable and had a longer duration. Conclusion The QDs-PSCA fluorescent probes could have the ability of specific targeted labeling and imaging with bladder cancer cells, and the probes possess good optical stability. This is contributed to the research development of bladder cancer-targeted non-invasive imaging in vivo, tumor early diagnosis and targeted therapy. Key words: Quantum dots; Bladder cancer; Cell labeling; Fluorescent probe

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.