Bladder Urothelial Cells Research Articles

Urine microbiota and bladder cancer

Urine analysis data obtained using modern microbiological methods and 16S rRNA gene sequencing technology indicate that the urinary system has its own microbial ecosystem. Individual microbiota members can play a key role in the development of cancer. Certain bacterial taxa have been revealed in bladder urothelial carcinoma cells that can affect carcinogenesis, treatment response, and the development of relapses through various mechanisms. The studies are conducted to use not only vaccine strains, but also probiotic strains and oncolytic bacteria for the treatment and prevention of relapses.

Comprehensive Genomic Analysis of Puerarin in Inhibiting Bladder Urothelial Carcinoma Cell Proliferation and Migration.

Bladder urothelial carcinoma (BUC) ranks second in the incidence of urogenital system tumors, and the treatment of BUC needs to be improved. Puerarin, a traditional Chinese medicine (TCM), has been shown to have various effects such as anti-cancer effects, the promotion of angiogenesis, and anti-inflammation. This study investigates the effects of puerarin on BUC and its molecular mechanisms. Through GeneChip experiments, we obtained differentially expressed genes (DEGs) and analyzed these DEGs using the Ingenuity® Pathway Analysis (IPA®), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway enrichment analyses. The Cell Counting Kit 8 (CCK8) assay was used to verify the inhibitory effect of puerarin on the proliferation of BUC T24 cells. String combined with Cytoscape® was used to create the Protein-Protein Interaction (PPI) network, and the MCC algorithm in cytoHubba plugin was used to screen key genes. Gene Set Enrichment Analysis (GSEA®) was used to verify the correlation between key genes and cell proliferation. A total of 1617 DEGs were obtained by GeneChip. Based on the DEGs, the IPA® and pathway enrichment analysis showed they were mainly enriched in cancer cell proliferation and migration. CCK8 experiments proved that puerarin inhibited the proliferation of BUC T24 cells, and its IC50 at 48 hours was 218μmol/L. Through PPI and related algorithms, 7 key genes were obtained: ITGA1, LAMA3, LAMB3, LAMA4, PAK2, DMD, and UTRN. GSEA showed that these key genes were highly correlated with BUC cell proliferation. Survival curves showed that ITGA1 upregulation was associated with poor prognosis of BUC patients. Our findings support the potential antitumor activity of puerarin in BUC. To the best of our knowledge, bioinformatics investigation suggests that puerarin demonstrates anticancer mechanisms via the upregulation of ITGA1, LAMA3 and 4, LAMB3, PAK2, DMD, and UTRN, all of which are involved in the proliferation and migration of bladder urothelial cancer cells.

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

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

Portable and Recyclable Luminescent Lanthanide Coordination Polymer Film Sensors for Adenosine Triphosphate in Urine.

Adenosine triphosphate (ATP) is a small molecule that is released to the urine from bladder urothelial cells and the bladder mucosal band of the human body. In certain cases, ATP can serve as a biomarker in bladder disease. For the practical applicability of luminescent sensors for ATP in urine, it is significant to find a new strategy for making the detection progress simple and available for in-field urine analysis. Here, a novel luminescent lanthanide coordination polymer (Tb-BPA) was designed and synthesized for quick and sensitive detection of ATP through luminescence quenching with a quenching constant of 4.90 × 103 M-1 and a detection limit of 0.55 × 10-6 M. Besides, Tb-BPA has excellent anti-interference ability and can detect ATP in simulated urine with a small relative standard deviation (<4%). Moreover, the luminescent polyacrylonitrile nanofiber films modified by Tb-BPA were prepared by electrospinning and were used for ATP visual detection. Notably, this film is easy to recover and reuse, and maintains good detection performance after at least 7 cycles.

G protein-coupled receptor 55 activated by palmitoylethanolamide is associated with the development of nocturia associated with circadian rhythm disorders

AimsBladder function is regulated by clock genes and dysregulation of circadian bladder function can cause nocturia. The blood concentration of palmitoylethanolamide (PEA), a fatty acid metabolite, changes with circadian rhythm. Clock gene abnormalities demonstrate the highest PEA levels during the sleep phase. PEA is a GPR55 agonist that influences urination; therefore, increased PEA during the sleep phase may cause nocturia. Herein, we investigated the function of GPR55 to evaluate the relationship between GPR55 and nocturia that evoked higher PEA during the sleep phase in patients with circadian rhythm disorders. Main methodsMale C57BL/6 mice were used. GPR55 localization was evaluated by immunofluorescence staining, qRT-PCR, and western blotting. Variations in PEA-induced intracellular Ca2+ concentrations were measured in primary cultured mouse urothelial cells (UCs) using Ca2+ imaging. PEA-induced NGF and PGI2 release in UCs was measured by ELISA. The micturition reflex pathway after PEA administration was evaluated using immunofluorescence staining. Key findingsGPR55 was predominant in the UC layer. PEA induced release of Ca2+ from the endoplasmic reticulum into the UC cytoplasm. ELISA and immunofluorescence staining revealed that NGF and PGI2 were released from bladder UCs, stimulated the pontine micturition center in mice, and induced nocturia. SignificanceThe loss of regular circadian metabolizing rhythm in fatty acids causes higher blood PEA levels during the sleep phase. Binding of PEA to GPR55 in UC may activate the downstream processes of the micturition reflex, leading to nocturia. These findings suggest a new mechanism for nocturia and its potential as a therapeutic target.

Synthesis and secretion of Nerve Growth Factor is regulated by Nitric Oxide in bladder cells in vitro under a hyperglycemic environment

Urine samples of female patients with overactive bladder (OAB) are characterized by low levels of nerve growth factor (NGF) and elevated concentrations of nitric oxide (NO) compared to healthy controls. We therefore examined how NO might regulate NGF synthesis using rat bladder smooth muscle (SMCs) and urothelial (UROs) cells in culture. In UROs, incubation in hyperglycemic conditions to mimic insulin insensitivity present in the OAB cohort increased secretion of NO and concomitantly decreased NGF, except when the NO synthase inhibitor, l-NAME (1 mM) was present. Sodium nitroprusside (SNP) (300 μM, 24 h), a NO generator, decreased NGF levels and decreased cyclic GMP (cGMP) content, a process validated by the cGMP synthase inhibitor ODQ (100 μM). Alternatively, SNP increased mRNA of both NGF and matrix metalloproteinase-9 (MMP-9). MMP-9 knockout of UROs by Crispr-Cas9 potently decreased the effect of SNP on NGF, implying a dependent role of NO on MMP-9. On the other hand, matrix metalloproteinase-7 (MMP-7) activity was increased by SNP, which taken together with increase in NGF mRNA, suggests a compensatory mechanism. In SMCs, hyperglycemic conditions had the same effect on extracellular content of NO and NGF than in UROs. SNP also decreased NGF secretion but increased cGMP content. Stable permeable analogs of cGMP 8-(4-Chlorophenylthio)-cGMP (1 mM) and N2,2′-O-Dibutyryl-cGMP (3 mM) inhibited NGF release. NGF and MMP-9 mRNA expression was unchanged by SNP. Deletion of MMP-9 in SMCs by Crispr-Cas9 did not alter the effect of SNP. Finally, SNP decreased MMP-7 activity, diminishing the conversion of proNGF to NGF. These results demonstrate that enhanced NO secretion triggered by high glucose decreases NGF secretion through pathways unique for each cell type that involve cGMP and proteases MMP-7 and MMP-9. These results might help to explain our observations from the urine from patients with OAB associated with metabolic syndrome.

Single-cell RNA sequencing reveals sexual diversity in the human bladder and its prospective impacts on bladder cancer and urinary tract infection

BackgroundSome bladder-related diseases, such as bladder urinary tract infection (UTI) and bladder cancer (BCa), have significant six differences in incidence and prognosis. However, the molecular mechanisms underlying these sex differences are still not fully understood. Understanding the sex-biased differences in gene expression in normal bladder cells can help resolve these problems.MethodsWe first collected published single-cell RNA sequencing (scRNA-seq) data of normal human bladders from females and males to map the bladder transcriptomic landscape. Then, Gene Ontology (GO) analysis and gene set enrichment analysis (GSEA) were used to determine the significant pathways that changed in the specific cell populations. The Monocle2 package was performed to reconstruct the differentiation trajectories of fibroblasts. In addition, the scMetabolism package was used to analyze the metabolic activity at the single-cell level, and the SCENIC package was used to analyze the regulatory network.ResultsIn total, 27,437 cells passed stringent quality control, and eight main cell types in human bladder were identified according to classical markers. Sex-based differential gene expression profiles were mainly observed in human bladder urothelial cells, fibroblasts, B cells, and T cells. We found that urothelial cells in males demonstrated a higher growth rate. Moreover, female fibroblasts produced more extracellular matrix, including seven collagen genes that may mediate BCa progression. Furthermore, the results showed that B cells in female bladders exhibited more B-cell activated signals and a higher expression of immunoglobulin genes. We also found that T cells in female bladders exhibited more T-cell activated signals. These different biological functions and properties of these cell populations may correlate with sex differences in UTI and BCa, and result in different disease processes and outcomes.ConclusionsOur study provides reasonable insights for further studies of sex-based physiological and pathological disparities in the human bladder, which will contribute to the understanding of epidemiological differences in UTI and BCa.

Targeting the PRC2-dependent epigenetic program alleviates urinary tract infections

Urinary tract infection (UTI) is a pervasive health problem worldwide. Patients with a history of UTIs suffer increased risk of recurrent infections, a major risk of antibiotic resistance. Here, we show that bladder infections induce expression of Ezh2 in bladder urothelial cells. Ezh2 is the methyltransferase of polycomb repressor complex 2 (PRC2)-a potent epigenetic regulator. Urothelium-specific inactivation of PRC2 results in reduced urine bacterial burden, muted inflammatory response, and decreased activity of the NF-κB signaling pathway. PRC2 inactivation also facilitates proper regeneration after urothelial damage from UTIs, by attenuating basal cell hyperplasia and increasing urothelial differentiation. In addition, treatment with Ezh2-specific small-molecule inhibitors improves outcomes of the chronic and severe bladder infections in mice. These findings collectively suggest that the PRC2-dependent epigenetic reprograming controls the amplitude of inflammation and severity of UTIs and that Ezh2 inhibitors may be a viable non-antibiotic strategy to manage chronic and severe UTIs.

Abstract 2884: Cancer-associated fibroblasts potentiate subcutaneous tumor growth in a novel bladder cancer mouse model with a genotype dependency

Abstract Introduction Interrogation of the relationship of genotype (mutation in relevant gene) and phenotype (sensitivity to therapy) in targeted therapy would be facilitated by the use of isogenic in vitro and in vivo models. The PI3K/AKT/mTOR pathway is widely activated but heretofore untargeted in bladder cancer. We sought to develop an immunocompetent CRISPR-edited bladder cancer mouse model that recapitulates common PI3K/AKT/mTOR mutations to study a genotype-phenotype relationship. Our in vitro CRISPR-edited organoids are immunogenic in vivo, as such, we explored utilizing Cancer-Associated Fibroblasts (CAFs) to mediate immunosuppression and promote a pro-tumorigenic environment. Methods and Results We isolated bladder urothelial cells from Rosa26 LSL-Cas9/GFP transgenic mice and grew them as organoids. Trp53 and Rb1 were co-edited to establish ‘base organoids’. As proof of concept, Pten was edited in base organoids in anticipation of developing a series of organoids with different mTOR pathway genotypes. We found that Pten-KO base organoids were 9x more sensitive to ipatasertib than Pten WT organoids. The edited urothelial cells were engrafted into mouse bladder to further test kinase inhibitor susceptibility as preclinical evidence to support human clinical trials. However, the cells successfully engrafted but disappeared after 3-4 weeks before tumors formed. Subcutaneous allografts were also rejected after 3-4 weeks and showed CD45+ immune infiltration during rejection. When cells were injected subcutaneously with CAFs, tumors persisted for 12 weeks, but only in organoids with both Trp53 loss and loss of murine chromosome 4qC4 (which is homologous to 9p21 loss in humans). 9p21 contains 3 genes which are co-deleted in ~40% of bladder cancers: MTAP, CDKN2A, and CDKN2B. Histopathological analysis of these masses confirmed their malignant nature and revealed an immune-excluded architecture. IHC staining of 9p21/Trp53 KO tumors for CD206 and CD11c revealed greater recruitment of M2 macrophages than M1 macrophages. Conclusions CAFs remodel the tumor-microenvironment in bladder cancer via the recruitment of pro-tumorigenic M2 macrophages. This may occur in a genotype-specific manner and 9p21 loss may be a requirement for some genotypes to be tumorigenic. Citation Format: Henkel Lee Valentine, Uttam Satyal, Alexander Metz, Laura Bukavina, Philip H. Abbosh. Cancer-associated fibroblasts potentiate subcutaneous tumor growth in a novel bladder cancer mouse model with a genotype dependency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2884.

MP03-08 INCREASED EXPRESSION OF PRO-FIBROTIC AMPHIREGULIN IN RADIATION CYSTITIS

You have accessJournal of UrologyCME1 Apr 2023MP03-08 INCREASED EXPRESSION OF PRO-FIBROTIC AMPHIREGULIN IN RADIATION CYSTITIS Bernadette Zwaans, Sabrina Mota, Elijah Ward, Sarah Bartolone, and Michael Chancellor Bernadette ZwaansBernadette Zwaans More articles by this author , Sabrina MotaSabrina Mota More articles by this author , Elijah WardElijah Ward More articles by this author , Sarah BartoloneSarah Bartolone More articles by this author , and Michael ChancellorMichael Chancellor More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Radiation cystitis (RC) is a painful chronic bladder condition that results from radiation therapy for pelvic cancers and for which no safe and effective therapy exists. RC has a long latency; it can take years before We previously developed a pre-clinical model that closely mimics the human condition. We sought to use this model to identify and validate proteins that are altered after RT and that may drive RC. METHODS: Female C57Bl/6 mice received a single dose of 40Gy irradiation. Bladders were harvested at 1 week, 4 weeks, 3 months and 6 months after irradiation, RNA was harvested, mRNA sequencing was performed at paired-end 150bp on the Illumina NovaSeq6000 with a target of 30 million reads per sample, and bioinformatics analysis was performed. Findings of the RNA sequencing were validated using qPCR analysis in bladder tissues and human urothelial cells (SV-HUC-1). We subsequently assessed levels of our protein of interest in urine samples from prostate cancer survivors with history of external beam radiation therapy using ELISA. RESULTS: RNAseq analysis identified amphiregulin (AREG) to be significantly increased in mouse bladder tissues in response to irradiation (Figure 1). AREG expression in the bladder significantly increased over time starting at 1-week post-RT and continuing up to 6 months post-RT. AREG is a member of the Epidermal Growth Factor (EGF) family and AREG has been implicated in fibrosis in various organ systems including kidney, skin, heart, lung, and liver. These findings were validated with qPCR. Exposure of human urothelial cells to radiation also resulted in increased AREG RNA and protein expression in a dose dependent manner. In addition, AREG was detectable in the urine of prostate cancer survivors with history of pelvic radiation therapy and was significantly higher in the subset of patients with diagnosis of RC (Figure 1). CONCLUSIONS: We have identified increased AREG in bladder tissue and urothelial cells after radiation exposure. AREG has been implicated in pathological tissue fibrosis in various organ systems. As such, AREG may play an important role in mediating radiation cystitis in cancer survivors. AREG may be Further research is needed to determine if AREG could be a novel target to treat and detect bladder fibrosis. Source of Funding: Research reported in this abstract was supported by NIDDK of NIH by award K01DK114334 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e23 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bernadette Zwaans More articles by this author Sabrina Mota More articles by this author Elijah Ward More articles by this author Sarah Bartolone More articles by this author Michael Chancellor More articles by this author Expand All Advertisement PDF downloadLoading ...

Artesunate Exhibits Synergy With Cisplatin and Cytotoxicity for Upper Tract and Bladder Urothelial Carcinoma Cells.

Urothelial carcinoma (UC) may arise from the urothelium of the upper tract and the bladder. Cisplatin-based therapy remains the gold standard for UC treatment. The poor 5-year survival rate of UC patients creates an urgent need to develop new drugs for advanced UC therapy. Artesunate (ART), a traditional Chinese medicine for treating malaria, is a potential anticancer agent, but its antigrowth effects on upper tract and bladder UC have not been investigated. The antigrowth effect of ART in HT 1376 (bladder UC cells) and BFTC 909 [upper tract urothelial carcinoma (UTUC) cells] was determined by the CCK-8 assay. Flow cytometric analysis was used to evaluate the cell cycle distribution and apoptosis. The cell cycle, apoptosis, and autophagy-related protein expression were analyzed by western blotting. The efficacy of combination treatment with cisplatin was determined by the Calcusyn software. ART induced HT 1376 and BFTC 909 cell death in a concentration- and time-dependent manner, inducing G2/M cell-cycle arrest. ART induced apoptosis and redox imbalance in HT 1376 and BFTC 909 cells. Application of the reactive oxygen species (ROS) scavenger, N-acetyl-L-cysteine (NAC), attenuated cell death in ART-treated UC cells. BFTC 909 cells show a better response after ART treatment. ART may be a candidate drug for treating UTUC and bladder UC while increasing the therapeutic effect of cisplatin.

Protective Effect of Purinergic P2X7 Receptor Inhibition on Acrolein-Induced Urothelial Cell Damage.

Patients undergoing chemotherapy with cyclophosphamide experience cystitis due to excretion of a toxic metabolite, acrolein. Cystitis, an inflammation of the bladder, is associated with damage to the integrity of the urothelial barrier. The purinergic P2X7 receptor (P2X7R) is increasingly recognized for its role in inflammation and cell death. P2X7R is expressed abundantly on the bladder urothelium. The aim of this study was to investigate the role of P2X7R in acrolein-induced inflammatory damage in primary cultured porcine bladder urothelial cells. Confluent urothelial cells in culture were treated with acrolein to induce damage; also, with the P2X7R selective antagonist, A804598. Cell viability assay, immunocytochemistry, and trans-epithelial electrical resistance (TEER) studies were carried out to investigate the effect of treatments on urothelial cell function. Acrolein induced a significant reduction in urothelial cell viability, which was protected by the presence of A804598 (10 µM). The urothelial barrier function, indicated by TEER values, was also significantly reduced by acrolein, whereas pre-incubation with P2X7R antagonist significantly protected the urothelial cell barrier from acrolein-induced TEER reduction. The structure of urothelial cell tight junctions was similarly impacted by acrolein treatment, showing the fragmentation of zona occludens-1 (ZO-1) immunoreactivity. Pre-treatment of cells with A804598 countered against the actions of acrolein and maintained ZO-1 expression level and cell structure. The damaging effect of acrolein on urothelial cells integrity could be impaired by inhibition of P2X7R, therefore P2X7R blockade may be a possible therapy in patients with bladder cystitis caused by cyclophosphamide treatment.

Targeting HNRNPU to overcome cisplatin resistance in bladder cancer

PurposeThe overall response of cisplatin-based chemotherapy in bladder urothelial carcinoma (BUC) remains unsatisfactory due to the complex pathological subtypes, genomic difference, and drug resistance. The genes that associated with cisplatin resistance remain unclear. Herein, we aimed to identify the cisplatin resistance associated genes in BUC.Experimental designThe cytotoxicity of cisplatin was evaluated in six bladder cancer cell lines to compare their responses to cisplatin. The T24 cancer cells exhibited the lowest sensitivity to cisplatin and was therefore selected to explore the mechanisms of drug resistance. We performed genome-wide CRISPR screening in T24 cancer cells in vitro, and identified that the gene heterogeneous nuclear ribonucleoprotein U (HNRNPU) was the top candidate gene related to cisplatin resistance. Epigenetic and transcriptional profiles of HNRNPU-depleted cells after cisplatin treatment were analyzed to investigate the relationship between HNRNPU and cisplatin resistance. In vivo experiments were also performed to demonstrate the function of HNRNPU depletion in cisplatin sensitivity.ResultsSignificant correlation was found between HNRNPU expression level and sensitivity to cisplatin in bladder cancer cell lines. In the high HNRNPU expressing T24 cancer cells, knockout of HNRNPU inhibited cell proliferation, invasion, and migration. In addition, loss of HNRNPU promoted apoptosis and S-phase arrest in the T24 cells treated with cisplatin. Data from The Cancer Genome Atlas (TCGA) demonstrated that HNRNPU expression was significantly higher in tumor tissues than in normal tissues. High HNRNPU level was negatively correlated with patient survival. Transcriptomic profiling analysis showed that knockout of HNRNPU enhanced cisplatin sensitivity by regulating DNA damage repair genes. Furthermore, it was found that HNRNPU regulates chemosensitivity by affecting the expression of neurofibromin 1 (NF1).ConclusionsOur study demonstrated that HNRNPU expression is associated with cisplatin sensitivity in bladder urothelial carcinoma cells. Inhibition of HNRNPU could be a potential therapy for cisplatin-resistant bladder cancer.

Surface Shave: Revealing the Apical-Restricted Uroglycome.

This study aimed to investigate the highly differentiated urothelial apical surface glycome. The functions of the mammalian urothelium, lining the majority of the urinary tract and providing a barrier against toxins in urine, are dependent on the correct differentiation of urothelial cells, relying on protein expression, modification, and complex assembly to regulate the formation of multiple differentiated cell layers. Protein glycosylation, a poorly studied aspect of urothelial differentiation, contributes to the apical glycome and is implicated in the development of urothelial diseases. To enable surface glycome characterization, we developed a method to collect tissue apical surface N- and O-glycans. A simple, novel device using basic laboratory supplies was developed for enzymatic shaving of the luminal bladder urothelial surface, with subsequent release and mass spectrometric analysis of apical surface O- and N-glycans, the first normal mammalian urothelial N-glycome to be defined. Trypsinization of superficial glycoproteins was tracked using immunolabeling of the apically expressed uroplakin 3a protein to optimize enzymatic release, without compromising the integrity of the superficial urothelial layer. The approach developed for releasing apical tissue surface glycans allowed for comparison with the N-glycome of the total porcine bladder urothelial cells and thus identification of apical surface glycans as candidates implicated in the urothelial barrier function. Data are available in MassIve: MSV000087851.

Induction of apoptosis through inactivation of ROS-dependent PI3K/Akt signaling pathway by platycodin D in human bladder urothelial carcinoma cells.

Platycodin D (PD) is a triterpenoid saponin, a major bioactive constituent of the roots of Platycodon grandiflorum, which is well known for possessing various pharmacological properties. However, the anti-cancer mechanism of PD in bladder cancer cells remains poorly understood. In the current study, we investigated the effect of PD on the growth of human bladder urothelial carcinoma cells. PD treatment significantly reduced the cell survival of bladder cancer cells associated with induction of apoptosis and DNA damage. PD inhibited the expression of inhibitor of apoptosis family members, activated caspases, and induced cleavage of poly (ADP-ribose) polymerase. PD also increased the release of cytochrome c into the cytoplasm by disrupting the mitochondrial membrane potential while upregulating the expression ratio of Bax to Bcl-2. The PD-mediated anti-proliferative effect was significantly inhibited by pre-treatment with a pancaspase inhibitor, but not by an inhibitor of necroptosis. Moreover, PD suppressed the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, and the apoptosis-inducing effect of PD was further enhanced by a PI3K inhibitor. In addition, PD increased the accumulation of reactive oxygen species (ROS), whereas N-acetyl cysteine (NAC), an ROS inhibitor, significantly attenuated the growth inhibition and inactivation of the PI3K/Akt/mTOR signaling caused by PD. Furthermore, NAC significantly suppressed apoptosis, DNA damage, and decreased cell viability induced by PD treatment. Collectively, our findings indicated that PD blocked the growth of bladder urothelial carcinoma cells by inducing ROS-mediated inactivation of the PI3K/Akt/mTOR signaling.

Spatiotemporal Expression of SHH/GLI Signaling in Human Fetal Bladder Development.

Objectives: Sonic hedgehog (SHH) signaling is important in bladder development. Mice with defective hedgehog signaling develop bladder anomalies. Clinically, urinary tract malformations are reported in human fetuses and infants with mutations of SHH and related signaling pathway genes. Information on the expression of SHH and associated signaling genes in normal human bladder development is fragmentary. This study determined the temporal and spatial expression patterns of SHH signaling pathway components in human fetal bladders by immunohistochemistry (IHC).Material and Methods: Twenty-four bladder specimens from 16 male and 8 female human fetuses aged 12- to 36-week (wk) were obtained from the First Affiliated Hospital of Xi'an Jiaotong University. The tissue slides were processed for IHC staining with SHH, Patched1 (PTC-1), Patched2 (PTC-2), Smoothened (SMO), GLI1 and proliferating cell nuclear antigen (PCNA). The expression levels of each gene were analyzed by semi-quantitative histological scoring system.Results: High intensity of SHH and SMO expression was detected in developing bladder urothelial cells, with no staining in lamina propria (LP), but with minimal expression of SMO in differentiating smooth muscle (SM) layers. The spatial distribution pattern of PTC1 and GLI1 was more complex with minimal expression in the LP layer, moderate expression in the SM layer, and high expression in the urothelium. PTC2 expression was mainly localized in the urothelium and LP, but no expression in the SM layer. All of the SHH signaling components were detected in fetal bladder tissues throughout the development, with expression peaks at 12- and 23-wk, coinciding with high cell proliferation as indicated by PCNA staining in the cell nuclei of urothelium and SM.Conclusions: The autocrine SHH signaling in the developing urothelium, and paracrine SHH signaling in the developing smooth muscle layer, mediated by SMO, PTC-1 and GLI1 were demonstrated during human bladder development. Expression of SHH signaling components peaked at 12-and 23-wk. The first expression peak at 12-wk may relate to urothelium growth, SM induction, and dilation of the bladder cavity. The second expression peaked at 23-wk may relate to urothelium and SM layer differentiation.

Supplementation of Probiotic Butyricicoccus pullicaecorum Mediates Anticancer Effect on Bladder Urothelial Cells by Regulating Butyrate-Responsive Molecular Signatures.

In bladder cancer, urothelial carcinoma is the most common histologic subtype, accounting for more than 90% of cases. Pathogenic effects due to the dysbiosis of gut microbiota are localized not only in the colon, but also in regulating bladder cancer distally. Butyrate, a short-chain fatty acid produced by gut microbial metabolism, is mainly studied in colon diseases. Therefore, the resolution of the anti-cancer effects of butyrate-producing microbes on bladder urothelial cells and knowledge of the butyrate-responsive molecules must have clinical significance. Here, we demonstrate a correlation between urothelial cancer of the bladder and Butyricicoccus pullicaecorum. This butyrate-producing microbe or their metabolite, butyrate, mediated anti-cancer effects on bladder urothelial cells by regulating cell cycle, cell growth, apoptosis, and gene expression. For example, a tumor suppressor against urothelial cancer of the bladder, bladder cancer-associated protein, was induced in butyrate-treated HT1376 cells, a human urinary bladder cancer cell line. In conclusion, urothelial cancer of the bladder is a significant health problem. To improve the health of bladder urothelial cells, supplementation of B. pullicaecorum may be necessary and can further regulate butyrate-responsive molecular signatures.

An experimental model of the epithelial to mesenchymal transition and pro-fibrogenesis in urothelial cells related to bladder pain syndrome/interstitial cystitis.

BackgroundSuitable in vitro models are needed to investigate urothelial epithelial to mesenchymal transition (EMT) and pro-fibrogenesis phenotype in bladder pain syndrome/interstitial cystitis (BPS/IC). This study is to establish a novel experimental BPS/IC cell model and explore how different concentrations of tumor necrosis factor (TNF)-α influence the EMT and pro-fibrogenesis phenotype of urothelial cells.MethodsSV-HUC-1 urothelial cells were cultured with 2, 10, or 50 ng/mL TNF-α to mimic chronic inflammatory stimulation. The EMT and pro-fibrogenesis phenotype, including production of collagen I and pro-fibrosis cytokines, were estimated after 72 h of culture.ResultsThe bladder urothelial cells of BPS/IC exhibited upregulated vimentin, TNF-α and TNF receptor, downregulated E-cadherin, and increased collagen I. Higher concentrations of TNF-α (10 and 50 ng/mL) produced an obvious mesenchymal morphology, enhanced invasion and migratory capacity, increased expression of vimentin, and decreased expression of E-cadherin. Collagen I was increased in cells treated with 2 and 10 ng/mL TNF-α after 72 h. Secretion of interleukin (IL)-6 and IL-8 was promoted with 10 and 50 ng/mL TNF-α, while that of IL-1β or transforming growth factor-β was unaffected. Slug and Smad2 were upregulated by TNF-α after 72 h. The Smad pathway was activated most strongly with 10 ng/mL TNF-α and Slug pathway activation was positively correlated with the concentration of TNF-α.ConclusionsSustained 10 ng/mL TNF-α stimulation induced the EMT and pro-fibrogenesis phenotype resembling BPS/IC in SV-HUC-1 cells. Minor inflammatory stimulation induced the pro-fibrogenesis phenotype while severe inflammatory stimulation was more likely to produce significant EMT changes. Different degrees of activation of the Slug and Smad pathways may underlie this phenomenon.

MP25-04 IDENTIFYING METABOLIC AND GENOMIC SIGNATURES AS BIOMARKERS FOR UROPATHOGENIC E COLI AND ASYMPTOMATIC BACTERIURIA

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder I (MP25)1 Sep 2021MP25-04 IDENTIFYING METABOLIC AND GENOMIC SIGNATURES AS BIOMARKERS FOR UROPATHOGENIC E COLI AND ASYMPTOMATIC BACTERIURIA Benjamin Abelson, Grace Morales, Gerald Van Horn, Allison R Eberly, Douglass B Clayton, Jonathan E Schmitz, and Maria Hadjifrangiskou Benjamin AbelsonBenjamin Abelson More articles by this author , Grace MoralesGrace Morales More articles by this author , Gerald Van HornGerald Van Horn More articles by this author , Allison R EberlyAllison R Eberly More articles by this author , Douglass B ClaytonDouglass B Clayton More articles by this author , Jonathan E SchmitzJonathan E Schmitz More articles by this author , and Maria HadjifrangiskouMaria Hadjifrangiskou More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002022.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Uropathogenic Escherichia coli (UPEC) is the most common cause of cystitis, though the presence of E. coli in urine does not universally cause symptoms. We aim to identify the metabolic and genomic signatures of E. coli isolates that distinguish cystitis-causing strains from E. coli strains present in asymptomatic bacteriuria (ASB). METHODS: We used a biobank to identify E. coli isolates from adult patients. The cohort was divided into cystitis strains and asymptomatic bacteriuria strains based on medical record review. A subset of these isolates were grown in pooled human urine under hypoxic conditions and then subjected to global metabolomics. For genomic sequencing, isolates were grown overnight in lysogeny broth, samples pelleted then subjected to genomic DNA isolation, whole-genome sequencing and analysis. RESULTS: Pooled metabolomic analysis of a subset of 26 ASB and 77 cystitis isolates demonstrates lower levels of guanosine but higher levels of adenosine in the supernatant of asymptomatic bacteriuria E. coli isolates. Given that both metabolites are implicated in purine metabolism, this supplements previously published work from our group demonstrating that purine biosynthesis is necessary for uropathogenic E. coli survival with bladder urothelial cells. 138 isolates were processed for genomic analysis. This included isolates from 126 women (91.6%) and 68 (49.3%) cystitis strains. Our development of a reproducible pipeline for urinary bacterial isolate whole genome sequencing and gene assembly demonstrates the feasibility for developing continued translational analysis to examine clinical specimens for genomic signatures. Genomic analysis of known purine synthesis-related genes did demonstrate single nucleotide polymorphisms (SNPs) that may alter single amino acids, but these results did not cluster based on the clinical presentation of the isolate source. CONCLUSIONS: UPEC differ from strains isolated from patients with asymptomatic bacteria in terms of the levels of purine synthesis metabolites, though examination of known purine synthesis gene SNPs does not reveal clustering based on clinical data. Further work using genome wide association studies to identify SNPs in non-coding regions along with additional metagenomics studies will be needed to identify further metabolic and genomic signatures that differentiate uropathogenic E. coli from asymptomatic strains. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e454-e455 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Benjamin Abelson More articles by this author Grace Morales More articles by this author Gerald Van Horn More articles by this author Allison R Eberly More articles by this author Douglass B Clayton More articles by this author Jonathan E Schmitz More articles by this author Maria Hadjifrangiskou More articles by this author Expand All Advertisement Loading ...

Heat injury-induced tRNA fragment tRF-Ile-AAT-1 inhibits proliferation and invasion of bladder urothelial cancer cells

目的探讨热损伤诱导高度恶性与低度恶性膀胱尿路上皮癌(bladder urothelial cancer, BUC)细胞之间差异表达的tRNA碎片对BUC细胞增殖与侵袭的影响。 方法对高度恶性BUC细胞株T24和低度恶性BUC细胞株5637行热损伤处理。对热损伤前后细胞进行tRNA碎片(transfer RNA stress-induced fragments, tRFs)测序，筛选出热损伤后在T24中表达下降且在5637细胞中表达上升的tRFs，并对其行细胞株及临床标本RT-PCR检测及验证。构建目的tRF的单链模拟物(mimic)和反义链(inhibitor)，并转染至热损伤后的T24及5637细胞中，分组为: 相对应的Heat组、载体对照组、Mimic组、Inhibitor组。通过CCK-8、流式细胞术、Transwell实验检测tRF对BUC细胞增殖、凋亡、迁移及侵袭的调控作用。 结果通过测序及细胞株RT-PCR检测，所筛选出的tRFs分别为tRF-chrM.Ser-GCT、tRF-Ile-AAT-1，而只有tRF-Ile-AAT-1在低级别BUC患者行第2次经尿道膀胱肿瘤电切(transurethral resection of bladder tumor, TURBT)所取标本中表达量较第1次TURBT的标本显著升高[(1.61±0.16)倍，P 结论热损伤诱导的tRNA碎片tRF-Ile-AAT-1，可抑制BUC细胞的增殖与侵袭能力。