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1.
Ecotoxicol Environ Saf ; 208: 111693, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396024

RESUMO

Arsenic is a natural chemical element that is strongly associated with bladder cancer. Understanding the underlying mechanisms behind the association between arsenic and bladder cancer as well as identifying effective preventive interventions will help reduce the incidence and mortality of this disease. The epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties play key roles in cancer development and progression. Here, we reported that chronic exposure to arsenic resulted in EMT and increased levels of the CSC marker CD44 in human uroepithelial cells. Furthermore, IL-8 promoted a mesenchymal phenotype and upregulated CD44 by activating the ERK, AKT and STAT3 signaling. Phosphorylation of the human epidermal growth factor receptor 2 (HER2) was key for arsenic-induced IL-8 overexpression and depended on the simultaneous activation of the MAPK, JNK, PI3K/AKT and GSK3ß signaling pathways. We also found that genistein inhibited arsenic-induced HER2 phosphorylation and downregulated its downstream signaling pathways, thereby inhibiting progression of EMT, and reducing CD44 expression levels. These results demonstrate that the HER2/IL-8 axis is related to the acquisition of an EMT phenotype and CSCs in arsenic-treated cells. The inhibitory effects of genistein on EMT and CSCs provide a new perspective for the intervention and potential chemotherapy against arsenic-induced bladder cancer.


Assuntos
Arsênico/toxicidade , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Interleucina-8/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptor ErbB-2/genética , Bexiga Urinária/metabolismo , Arsênico/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/imunologia , Humanos , Receptores de Hialuronatos/genética , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bexiga Urinária/citologia
2.
Nat Commun ; 11(1): 5485, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127883

RESUMO

Cancer patient classification using predictive biomarkers for anti-cancer drug responses is essential for improving therapeutic outcomes. However, current machine-learning-based predictions of drug response often fail to identify robust translational biomarkers from preclinical models. Here, we present a machine-learning framework to identify robust drug biomarkers by taking advantage of network-based analyses using pharmacogenomic data derived from three-dimensional organoid culture models. The biomarkers identified by our approach accurately predict the drug responses of 114 colorectal cancer patients treated with 5-fluorouracil and 77 bladder cancer patients treated with cisplatin. We further confirm our biomarkers using external transcriptomic datasets of drug-sensitive and -resistant isogenic cancer cell lines. Finally, concordance analysis between the transcriptomic biomarkers and independent somatic mutation-based biomarkers further validate our method. This work presents a method to predict cancer patient drug responses using pharmacogenomic data derived from organoid models by combining the application of gene modules and network-based approaches.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Aprendizado de Máquina , Organoides/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Bexiga Urinária/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Desenvolvimento de Medicamentos/métodos , Fluoruracila/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Organoides/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Transcriptoma , Bexiga Urinária/efeitos dos fármacos , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo
3.
Life Sci ; 261: 118468, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32961232

RESUMO

AIMS: RacGTPase-mediated proliferation and smooth muscle contraction in the lower urinary tract has been recently suggested and may offer putative targets for treamtment of lower urinary tract symptoms. However, RacGTPase function for proliferation of detrusor smooth muscle cells is unknown and the specificity of Rac inhibitors has been questioned. Here, we examined effects of Rac1 knockdown and of the Rac inhibitors NSC23766 and EHT1864 in human bladder smooth muscle cells (hBSMCs). MAIN METHODS: Rac1 expression was silenced by shRNA expression. Effects of silencing and Rac inhibitors were assessed by CCK-8 assay, EdU staining, RT-PCR, colony formation assay, flow cytometry, and phalloidin staining. KEY FINDINGS: Silencing of Rac1 expression reduced the viability (up to 83% compared to scramble shRNA) and proliferation (virtually completely in proliferation assay), increased apoptosis (124%) and the number of dead cells (51%), and caused breakdown of actin organization (56% reduction of polymerized actin compared to scramble shRNA). Effects on proliferation, viability, and actin organization were mimicked by NSC23766 and EHT1864, while both compounds showed divergent effects on cell death (32-fold increase of dead cells by EHT1864, but not NSC23766). Effects of NSC23766 and EHT1864 on viability of hBSMCs were not altered by Rac1 knockdown. SIGNIFICANCE: Rac1 promotes proliferation, viability, and cytoskeletal organization, and suppresses apoptosis in bladder smooth muscle cells, which may be relevant in overactive bladder or diabetes-related bladder dysfunction. NSC23766 and EHT1864 mimick these effects, but may act Rac1-independently, by shared and divergent effects.


Assuntos
Actinas/metabolismo , Aminoquinolinas/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Pirimidinas/farmacologia , Pironas/farmacologia , Quinolinas/farmacologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Inativação Gênica , Células HEK293 , Humanos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Bexiga Urinária/citologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
4.
PLoS One ; 15(9): e0235384, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32925915

RESUMO

Interstitial cystitis/bladder pain syndrome (IC) is a debilitating condition of chronic pelvic pain with unknown etiology. Recently, we used a genetic approach in a murine model of IC to identify the lipase acyloxyacyl hydrolase (AOAH) as a modulator of pelvic pain. We found that AOAH-deficient mice have elevated pelvic pain responses, and AOAH immunoreactivity was detected along the bladder-brain axis. Lipidomic analyses identified arachidonic acid (AA) and its metabolite PGE2 as significantly elevated in the sacral spinal cord of AOAH-deficient mice, suggesting AA is a substrate for AOAH. Here, we quantified the effects of AOAH on phospholipids containing AA. Spinal cord lipidomics revealed increased AA-containing phosphatidylcholine in AOAH-deficient mice and concomitantly decreased AA-phosphatidylethanolamine, consistent with decreased CoA-independent transferase activity (CoIT). Overexpression of AOAH in cell cultures similarly altered distribution of AA in phospholipid pools, promoted AA incorporation, and resulted in decreased membrane fluidity. Finally, administration of a PGE2 receptor antagonist reduced pelvic pain in AOAH-deficient mice. Together, these findings suggest that AOAH represents a potential CoA-independent AA transferase that modulates CNS pain pathways at the level of phospholipid metabolism.


Assuntos
Ácido Araquidônico/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Cistite Intersticial/metabolismo , Dor Pélvica/metabolismo , Fosfolipídeos/metabolismo , Animais , Cistite Intersticial/complicações , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Dor Pélvica/complicações , Bexiga Urinária/metabolismo
5.
Am J Physiol Renal Physiol ; 319(2): F257-F283, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32628539

RESUMO

Urinary bladder smooth muscle (UBSM), also known as detrusor smooth muscle, forms the bladder wall and ultimately determines the two main attributes of the organ: urine storage and voiding. The two functions are facilitated by UBSM relaxation and contraction, respectively, which depend on UBSM excitability shaped by multiple ion channels. In this review, we summarize the current understanding of key ion channels establishing and regulating UBSM excitability and contractility. They include excitation-enhancing voltage-gated Ca2+ (Cav) and transient receptor potential channels, excitation-reducing K+ channels, and still poorly understood Cl- channels. Dynamic interplay among UBSM ion channels determines the overall level of Cav channel activity. The net Ca2+ influx via Cav channels increases global intracellular Ca2+ concentration, which subsequently triggers UBSM contractility. Here, for each ion channel type, we describe UBSM tissue/cell expression (mRNA and protein) profiles and their role in regulating excitability and contractility of UBSM in various animal species, including the mouse, rat, and guinea pig, and, most importantly, humans. The currently available data reveal certain interspecies differences, which complicate the translational value of published animal research results to humans. This review highlights recent developments, findings on genetic knockout models, pharmacological data, reports on UBSM ion channel dysfunction in animal bladder disease models, and the very limited human studies currently available. Among all gaps in present-day knowledge, the unknowns on expression and functional roles for ion channels determined directly in human UBSM tissues and cells under both normal and disease conditions remain key hurdles in the field.


Assuntos
Canais Iônicos/metabolismo , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Bexiga Urinária/metabolismo , Animais , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Contração Muscular/fisiologia
6.
Am J Pathol ; 190(8): 1752-1762, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32339497

RESUMO

The biological processes of urothelial carcinogenesis are not fully understood, particularly regarding the relationship between specific genetic events, cell of origin, and molecular subtypes of subsequent tumors. N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced mouse bladder cancer is widely accepted as a useful model that recapitulates the pathway of human bladder tumorigenesis from dysplasia to invasive cancer via carcinoma in situ. However, the long and variable time of tumorigenesis often hinders efficient preclinical or translational research. We hypothesized that Trp53 mutation in specific types of urothelial cells facilitates efficient development of clinically relevant bladder cancer. Using lineage tracing, we showed that Trp53 mutation in Krt5-expressing cells resulted in more efficient tumorigenesis of mouse muscle-invasive bladder cancer (MIBC) with squamous differentiation compared with Trp53 mutation in Upk2-expressing cells, or wild-type or hemizygous Trp53 in the entire urothelium. Mouse MIBC that developed at 24 weeks of BBN treatment showed morphologic and genetic similarities to the basal squamous subtypes of human MIBC, irrespective of pre-induction of Trp53 mutation or whether the cell of origin was Krt5- or Upk2-expressing cells. Our findings suggest that intermediate cells as well as basal cells also can give rise to basal-like MIBC, with pre-induction of Trp53 mutation accelerating MIBC. Thus, in BBN chemical carcinogenesis, pre-induction of Trp53 mutation in basal cells facilitates efficient modeling of the basal squamous subtype of human MIBC.


Assuntos
Carcinogênese/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células de Transição/genética , Queratina-5/genética , Proteína Supressora de Tumor p53/genética , Neoplasias da Bexiga Urinária/genética , Bexiga Urinária/patologia , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Queratina-5/metabolismo , Camundongos , Mutação , Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
7.
Am J Physiol Renal Physiol ; 318(4): F901-F910, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32116016

RESUMO

The signaling pathways and effectors that drive the response of the bladder to nonmalignant insults or injury are incompletely defined. Interrogation of biological systems has been revolutionized by the ability to generate high-content data sets that capture information on a variety of biomolecules in cells and tissues, from DNA to RNA to proteins. In oncology, such an approach has led to the identification of cancer subtypes, improved prognostic capability, and has provided a basis for precision treatment of patients. In contrast, systematic molecular characterization of benign bladder disorders has lagged behind, such that our ability to uncover novel therapeutic interventions or increase our mechanistic understanding of such conditions is limited. Here, we discuss existing literature on the application of omics approaches, including transcriptomics and proteomics, to urinary tract conditions characterized by pathological tissue remodeling. We discuss molecular pathways implicated in remodeling, challenges in the field, and aspirations for omics-based research in the future.


Assuntos
Genômica , Análise de Célula Única , Biologia de Sistemas , Doenças da Bexiga Urinária/genética , Doenças da Bexiga Urinária/metabolismo , Bexiga Urinária/metabolismo , Animais , Epigênese Genética , Epigenômica , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença , Humanos , Fenótipo , Proteômica , Transcriptoma , Bexiga Urinária/patologia , Bexiga Urinária/fisiopatologia , Doenças da Bexiga Urinária/patologia , Doenças da Bexiga Urinária/fisiopatologia
8.
Eur J Histochem ; 64(2)2020 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-32214283

RESUMO

Although the incidence varies with age and gender, urothelial bladder cancer is a relatively frequently occurring malignancy with variable clinical behavior that often has high recurrence rates. In this study, we analyzed the tumor tissues of 224 patients with pTa, pT1, and pT2 urinary bladder cancer. We performed a histomorphologic analysis and immunohistochemistry for p53, Ki-67, and E-cadherin, which were selected as markers of the malignant process. For pTa and pT1, univariate analyses of cancer-specific survival (CSS), progression-free survival (PFS), and recurrence-free survival (RFS) were calculated using the Kaplan-Meier method, the log-rank test and Cox regression. Multivariate analysis was performed by a Cox regression analysis. Ki-67 (P<0.001) was significantly associated with CSS, but the highest association was shown for E-cadherin (P<0.001). For pT1 and pTa, the Kaplan-Meier analysis and the log-rank test revealed significantly worse PFS for patients with higher levels of Ki-67 (P<0.001) and lower levels of E-cadherin (P<0.001). Based on these obtained results, it can be clearly stated that Ki-67 and E-cadherin expression levels are associated with CSS, PFS and RFS. The clinical utility of these markers is valuable for pTa and pT1 urinary bladder cancer and should be further verified with prospective multi-center trials.


Assuntos
Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Antígeno Ki-67/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Intervalo Livre de Progressão , Recidiva , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia
9.
Sci Rep ; 10(1): 4116, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139747

RESUMO

Inflammatory mediators may have a role in various lower urinary tract disorders. Histamine is known to induce significant increases in both the tension and frequency of spontaneous phasic contractions in both urothelium with lamina propria (U&LP) and detrusor muscle via the activation of H1 receptor in juvenile animal models. However, it is unclear whether age affects these contractile responses to histamine. This study assessed the histamine receptor subtypes mediating contraction in juvenile and adult porcine bladders and compared the urothelium with lamina propria and detrusor responses to histamine. Isolated tissue bath studies were conducted using strips of porcine U&LP and detrusor obtained from juvenile (6 months) and adult (3 years) animals exposed to histamine receptor agonists and antagonists. Treatment with histamine (100 µM) in U&LP of juvenile animals caused increases in baseline tension by 47.84 ± 6.52 mN/g (p < 0.001, n = 51) and by 50.76 ± 4.10 mN/g (p < 0.001, n = 55) in adult animals. Furthermore, the frequency of spontaneous phasic contractions was significantly enhanced in response to histamine in U&LP of both juvenile and adult tissues (p < 0.001 for both age groups). Treatment with an H2 agonist in U&LP of juvenile animals decreased baseline tension by 13.97 ± 3.45 mN/g (n = 12, p < 0.05), but had no effect in adult animals. Inhibition of H1 receptors resulted in significantly reduced contractile responses of U&LP and detrusor to histamine in both juvenile and adult animals (p < 0.05). Treatment with an H2 receptor antagonist significantly enhanced contractions in juvenile preparations (n = 10, p < 0.05) but had no effect in adult preparations (n = 8). In detrusor, treatment with histamine (100 µM) in juvenile tissues showed a significantly higher increase in baseline tension of 19.10 ± 4.92 mN/g (n = 51) when compared to adult tissues exhibiting increases of 8.21 ± 0.89 mN/g (n = 56, p < 0.05). The increases in the baseline tension were significantly inhibited by the presence of H1 receptor antagonists in both juvenile and adult detrusor preparations. Treatment with either the H2 receptor antagonist or agonist in detrusor had no effect on both juvenile and adult tissues. Therefore, the histamine receptor system may play an essential role in the maintenance of bladder function or in bladder dysfunction observed in some lower urinary tract disorders.


Assuntos
Histamina/metabolismo , Membrana Mucosa/metabolismo , Receptores Histamínicos/metabolismo , Bexiga Urinária/metabolismo , Urotélio/metabolismo , Envelhecimento/metabolismo , Animais , Feminino , Histamina/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos , Técnicas In Vitro , Contração Muscular/efeitos dos fármacos , Suínos , Bexiga Urinária/efeitos dos fármacos , Urotélio/efeitos dos fármacos
10.
Neurourol Urodyn ; 39(3): 926-934, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32049380

RESUMO

AIM: To characterize purinergic signaling in overactive bladder (OAB). METHODS: Mucosal biopsies were taken by flexible cystoscopy from patients with storage symptoms referred to Urology Departments of collaborating hospitals. Immunohistochemistry (n = 12) and Western blot analysis (n = 28) were used to establish the qualitative and quantitative expression profile of P2Y6 in human mucosa. Participants from the general population provided a mid-stream urine sample. Bioluminescent assays were used to quantify adenosine triphosphate (ATP; n = 66) and adenosine diphosphate (ADP; n = 60) concentrations, which were normalized to creatinine (Cr) concentration. All participants completed a questionnaire (International Consultation on Incontinence Questionnaire - Overactive Bladder) to score urinary symptoms of OAB. RESULTS: P2Y6 immunoreactivity, more prominent in the urothelium (colocalized with the uroepithelial marker pan-cytokeratin), was more greatly expressed in OAB compared to age- and sex-matched controls (benign prostatic hyperplasia) without OAB symptoms. Mucosal P2Y6 was positively correlated only with incontinence (P = .009). Both urinary ATP and its hydrolysis product, ADP, an agonist to P2Y6, were positively correlated with total OAB symptom score (P = .010 and P = .042, respectively). CONCLUSIONS: The positive correlation of P2Y6 only with incontinence may indicate a different phenotype in OAB wet and warrants further investigation. Positive correlations of ATP and ADP with total OAB symptom score demonstrate upregulation in purinergic signaling in OAB; shown previously only in animal models. Further research is required to validate whether purinoceptors are indeed new therapeutic targets for this highly prevalent symptom complex.


Assuntos
Difosfato de Adenosina/urina , Trifosfato de Adenosina/urina , Membrana Mucosa/metabolismo , Receptores Purinérgicos P2/metabolismo , Bexiga Urinária Hiperativa/metabolismo , Bexiga Urinária/metabolismo , Incontinência Urinária/metabolismo , Urotélio/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Creatinina/urina , Cistoscopia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Hiperplasia Prostática/fisiopatologia , Inquéritos e Questionários , Bexiga Urinária/patologia , Bexiga Urinária/fisiopatologia , Bexiga Urinária Hiperativa/patologia , Bexiga Urinária Hiperativa/fisiopatologia , Incontinência Urinária/patologia , Incontinência Urinária/fisiopatologia
11.
Virchows Arch ; 477(2): 269-277, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32034486

RESUMO

Carcinoma in situ (CIS) is believed to be a precursor of muscle-invasive carcinomas that may arise from these flat high-grade, superficial urothelial lesions. CIS accounts for approximately 10% of all bladder tumors. Therapeutic options for urothelial CIS are limited, and in order to inhibit disease progression and recurrence, current guidelines recommend transurethral resection (TURBT) followed by intravesical administration of Bacillus of Calmette-Guerin (BCG). Approximately 30-40% of patients fail the BCG therapy with recurrence and progression of disease. In the present study, we examined the expression of PD-L1 both in neoplastic epithelial cells and in stromal inflammatory cells in patients with diagnosis of CIS primary responders and not responders to BCG therapy, in order to verify if the PD-L1 expression could identify patients resistant to BCG treatment. Moreover, we analyzed on the same cases the immunoreactivities of anti-PD-L1 MoAbs such as SP263, C23, and SP142. Our results have showed that PD-L1 expression in tumor cells and in immune cell compartment is higher in BCG-unresponsive group than in BCG responders, but only the PD-L1 22C3 expression in tumor cells seems to be associated with recurrence of disease (p = 0.035; OR 0.1204; CI 95% from 0.0147 to 1.023). Hence, our data suggest that the PD-L1 22C3 expression could help to identify CIS that fail the BCG therapy, supporting the hypothesis that enhanced levels of intratumoral PD-L1 22C3 expressed by the tumor cells may explain the failure of BCG immunotherapy.


Assuntos
Recidiva Local de Neoplasia/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Bexiga Urinária/metabolismo , Neoplasias Urológicas/metabolismo , Idoso , Anticorpos Monoclonais/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/patologia , Neoplasias Urológicas/patologia
12.
Gene ; 738: 144475, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32081697

RESUMO

INTRODUCTION: In this article, we utilized Ingenuity® Pathway Analysis (IPA®) bioinformatics analysis software and Metascape® bioinformatics analysis website tools to analyse the possible mechanism of ERH affecting tumourigenesis (proliferation and apoptosis) in bladder cancer (BC) T24 cells. METHODS: The ERH gene was knocked down, and BC T24 cells were divided into ERH normal and knockdown groups. Affymetrix® gene expression microarrays were performed to obtain a differentially expressed gene list (DEGL) between the 2 groups. IPA® data analyses contain five modules: disease and function analysis, upstream analysis, regulator effects analysis, canonical pathway analysis and molecular network analysis. The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were analysed by Metascape®. RESULTS: The results of the gene expression profiling chip and the DEGL showed that 344 genes were upregulated and 254 genes were downregulated. The IPA® and Metascape® pathway analyses showed that the ERH gene may affect proliferation and apoptosis by affecting the apoptosis, cell cycle, Toll-like receptor (TLR), NF-κB or TGF-beta signalling pathways. Upstream analysis determined that the ERH gene may regulate TNF and NK-κB in the BC T24 cell lines. The ERH gene may be involved in the "cell death and survival" molecular network in BC T24 cells. ERH may be a regulator of KITLG through TNF. CONCLUSIONS: The ERH gene may affect apoptosis through the TLR, NF-κB, TNF or TGF-beta signalling pathways in BC T24 cells, and may be a regulator of KITLG to ultimately activate the growth of malignant tumours.


Assuntos
Proteínas de Ciclo Celular/genética , Fatores de Transcrição/genética , Neoplasias da Bexiga Urinária/genética , Apoptose/genética , Carcinogênese/genética , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Biologia Computacional/métodos , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes/métodos , Ontologia Genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Software , Fatores de Transcrição/metabolismo , Bexiga Urinária/metabolismo
13.
J Pharmacol Sci ; 142(3): 127-130, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31889618

RESUMO

In vitro and in vivo binding sites of [3H]-labeled 5-hydroxymethyltolterodine (5-HMT), a new radioligand for labeling muscarinic receptors in rat tissues were characterized. Specific [3H]5-HMT binding in rat tissues was saturable and of high affinity in each tissue. The dissociation constant (Kd) was significantly lower in bladder and heart than in submaxillary gland. Significant levels of in vivo specific [3H]5-HMT binding by intravenous injection of the radioligand were detected in tissues, except for cerebral cortex. Thus, [3H]5-HMT was shown to specifically label muscarinic receptors in rat tissues, suggesting a useful radioligand for labeling muscarinic receptors with high affinity.


Assuntos
Compostos Radiofarmacêuticos/metabolismo , Receptores Muscarínicos/metabolismo , Bexiga Urinária/metabolismo , Animais , Glicina Hidroximetiltransferase , Técnicas In Vitro , Masculino , Ratos Sprague-Dawley
14.
FASEB J ; 34(2): 2126-2146, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31909533

RESUMO

Bladder dysfunction is associated with the overexpression of the intermediate filament (IF) proteins desmin and vimentin in obstructed bladder smooth muscle (BSM). However, the mechanisms by which these proteins contribute to BSM dysfunction are not known. Previous studies have shown that desmin and vimentin directly participate in signal transduction. In this study, we hypothesized that BSM dysfunction associated with overexpression of desmin or vimentin is mediated via c-Jun N-terminal kinase (JNK). We employed a model of murine BSM tissue in which increased expression of desmin or vimentin was induced by adenoviral transduction to examine the sufficiency of increased IF protein expression to reduce BSM contraction. Murine BSM strips overexpressing desmin or vimentin generated less force in response to KCl and carbachol relative to the levels in control murine BSM strips, an effect associated with increased JNK2 phosphorylation and reduced myosin light chain (MLC20 ) phosphorylation. Furthermore, desmin and vimentin overexpressions did not alter BSM contractility and MLC20 phosphorylation in strips isolated from JNK2 knockout mice. Pharmacological JNK2 inhibition produced results qualitatively similar to those caused by JNK2 knockout. These findings suggest that inhibition of JNK2 may improve diminished BSM contractility associated with obstructive bladder disease.


Assuntos
Desmina/biossíntese , Sistema de Sinalização das MAP Quinases , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Contração Muscular , Músculo Liso/metabolismo , Bexiga Urinária/metabolismo , Vimentina/biossíntese , Animais , Desmina/genética , Camundongos , Camundongos Knockout , Proteína Quinase 9 Ativada por Mitógeno/genética , Músculo Liso/citologia , Bexiga Urinária/citologia , Vimentina/genética
15.
J Pharmacol Exp Ther ; 372(1): 63-72, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31636173

RESUMO

The objective of this study was to investigate the role of ATP in cholinergic neurotransmission in the urinary bladder of control men and of patients obstructed as a result of benign prostatic hyperplasia (BPH). Human detrusor samples were collected from 41 patients who submitted to transvesical prostatectomy resulting from BPH and 26 male organ donors. The release of [3H]acetylcholine ([3H]ACh) was evoked by electrical field stimulation (10 Hz, 200 pulses) in urothelium-denuded detrusor strips. Myographic recordings were performed to test detrusor strip sensitivity to ACh and ATP. Nerve-evoked [3H]ACh release was 1.5-fold higher in detrusor strips from BPH patients compared with controls. This difference was abolished after desensitization of ionotropic P2X1-3 receptors with an ATP analog, α,ß-methylene ATP (30 µM, applied for 15 minutes). TNP-ATP (10 nM, a preferential P2X2/3 antagonist) and A317491 (100 nM, a selective P2X3 antagonist) were about equipotent in decreasing nerve-evoked [3H]ACh release in control detrusor strips, but the selective P2X1 receptor antagonist NF023 (3 µM) was devoid of effect. The inhibitory effect of TNP-ATP (10 nM) increased from 27% ± 9% to 43% ± 6% in detrusor strips of BPH patients, but the effect of A317491 (100 nM) [3H]ACh release unaltered (20% ± 2% vs. 24% ± 4%). The amplitude of ACh (0.1-100 µM)-induced myographic recordings decreased, whereas sensitivity to ATP (0.01-3 mM) increased in detrusor strips from BPH patients. Besides the well characterized P2X1 receptor-mediated contractile activity of ATP in pathologic human bladders, we show here for the first time that cholinergic hyperactivity in the detrusor of BPH patients is facilitated by activation of ATP-sensitive P2X2/3 heterotrimers. SIGNIFICANCE STATEMENT: Bladder outlet obstruction often leads to detrusor overactivity and reduced bladder compliance in parallel to atropine-resistant increased purinergic tone. Our data show that P2X1 purinoceptors are overexpressed in the detrusor of patients with benign prostatic hyperplasia. Besides the P2X1 receptor-mediated detrusor contractions, ATP favors nerve-evoked acetylcholine release via the activation of prejunctional P2X2/3 excitatory receptors in these patients Thus, our hypothesis is that manipulation of the purinergic tone may be therapeutically useful to counteract cholinergic overstimulation in obstructed patients.


Assuntos
Trifosfato de Adenosina/metabolismo , Tono Muscular , Receptores Purinérgicos P2X1/metabolismo , Obstrução do Colo da Bexiga Urinária/metabolismo , Acetilcolina/metabolismo , Adulto , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Contração Muscular , Fenóis/farmacologia , Compostos Policíclicos/farmacologia , Multimerização Proteica , Antagonistas do Receptor Purinérgico P2X/farmacologia , Suramina/análogos & derivados , Suramina/farmacologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Bexiga Urinária/fisiopatologia , Obstrução do Colo da Bexiga Urinária/fisiopatologia
16.
Pharmacology ; 105(3-4): 135-144, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31614352

RESUMO

Distigmine bromide (distigmine) is a reversible carbamate cholinesterase (ChE) inhibitor that is used to treat myasthenia gravis. In Japan, it is also used as a remedy for urination disorder (underactive bladder). The most distinctive pharmacological feature of distigmine is its long-lasting action compared to that of other ChE inhibitors. In animals and humans, distigmine was reported to inhibit acetylcholinesterase (AChE) and improve myasthenia gravis for an extended period. Few studies have examined the sustainability of this enhancing effect on the contractile function of urinary bladder smooth muscle. In addition, the cause of this long-lasting feature remains unclear. In this review, we present our findings for the long-lasting feature of distigmine on isolated urinary bladder contraction and in vivo urinary function of guinea pig. We also present our results on the mechanism of its long-lasting sustainability using recombinant human AChE.


Assuntos
Contração Muscular/efeitos dos fármacos , Compostos de Piridínio/farmacologia , Bexiga Urinária/efeitos dos fármacos , Animais , Inibidores da Colinesterase/farmacologia , Cobaias , Humanos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Fatores de Tempo , Bexiga Urinária/metabolismo
17.
Chemistry ; 26(8): 1789-1799, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-31605633

RESUMO

The synthesis of ruthenium(II) phthalocyanines (RuPcs) endowed with one carbohydrate unit-that is, glucose, galactose and mannose-and a dimethylsulfoxide (DMSO) ligand at the two axial coordination sites, respectively, is described. Two series of compounds, one unsubstituted at the periphery, and the other one bearing eight PEG chains at the isoindole meta-positions, have been prepared. The presence of the axial DMSO unit significantly increases the phthalocyanine singlet oxygen quantum yields, related to other comparable RuPcs. The compounds have been evaluated for PDT treatment in bladder cancer cells. In vitro studies have revealed high phototoxicity for RuPcs unsubstituted at their periphery. The phototoxicity of PEG-substituted RuPcs has been considerably improved by repeated light irradiation. The choice of the axial carbohydrate introduced little differences in the cellular uptake for both series of photosensitizers, but the phototoxic effects were considerably higher for compounds bearing mannose units.


Assuntos
Compostos Organometálicos/química , Fármacos Fotossensibilizantes/síntese química , Oxigênio Singlete/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Luz , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Espectrofotometria , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo
18.
Neurourol Urodyn ; 39(1): 125-132, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31609506

RESUMO

INTRODUCTION: Past research has demonstrated that the urethral tonus is mainly under sympathetic control. Since 5 years, a beta 3-adrenoceptor (ADRB3) agonist is available in the treatment of overactive bladder syndrome. The presence of ADRB3 within the human urethra has not been demonstrated to date. Presence of ADRB3 in the urethra could influence urethral tonus. The aim of this study is to investigate the presence of ADRB3 in the human female urethra. MATERIAL AND METHODS: We performed anatomical studies in five female specimens. Three specimens were obtained from the body donation program, two from female patients with muscle-invasive bladder cancer, where radical resection of bladder and urethra was performed. The urethra up till the bladder neck was separated from the rest of the bladder and freshly obtained for this study. For demonstrating ADRB3 expression, we used rabbit polyclonal anti-human ADRB3 LS-A4198. RESULTS: Expression of ADBR3 was demonstrated in the epithelial layer of all urethral parts, except at the level of the meatus. The level of ADRB3 expression was highest in the mid urethra. There was no direct contact between ADRB3 and nerve tissue. ADRB3 expression was also demonstrated in the stratified muscle layer at the level of the external urethral sphincter. CONCLUSIONS: This is the first study to demonstrate the expression of ADRB3 in the human female urethra. There is an absence of a direct connection between ADRB3 and nerve tissue.


Assuntos
Receptores Adrenérgicos beta 3/metabolismo , Uretra/metabolismo , Bexiga Urinária/metabolismo , Feminino , Humanos , Músculo Liso/metabolismo
19.
Biochem Biophys Res Commun ; 521(3): 555-561, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31677792

RESUMO

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infection (UTI), a common bacterial infectious disease. This bacterium invades the urinary tract cells, where it aggregates, and subsequently forms multicellular colonies termed intracellular bacterial communities (IBCs). The motility of the bacteria plays a key role in the mechanism of virulence in the host bladder. Here, we show that CytR is a modulator of bacterial internalization and aggregation within the bladder epithelial cells sustained by CRP in UPEC. Mutational analyses and gel-shift assays indicated that CytR represses the expression of flhD, thereby encoding a master regulator for flagellar expression that is responsible for bacterial motility when CRP is present, whereas CRP is an activator of flhD expression. Thus, elevated flagellar expression was involved in promoted virulence in the cytR mutant. These combined observations suggest another regulatory layer of flagellar expression and the role of CytR in UPEC virulence.


Assuntos
Proteína Receptora de AMP Cíclico/metabolismo , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Infecções Urinárias/metabolismo , Escherichia coli Uropatogênica/fisiologia , Linhagem Celular , Proteína Receptora de AMP Cíclico/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Proteínas Repressoras/genética , Transativadores/genética , Transativadores/metabolismo , Bexiga Urinária/citologia , Bexiga Urinária/metabolismo , Bexiga Urinária/microbiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/patogenicidade
20.
Am J Pathol ; 190(1): 108-124, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31654636

RESUMO

Keratinocyte growth factor (KGF) improves cyclophosphamide-induced bladder injury. To understand the mechanisms, we subcutaneously administered KGF to mice 24 hours before i.p. cyclophosphamide administration, followed by histologic assays and immunostaining. In vehicle (phosphate-buffered saline)-pretreated mice, nonapoptotic superficial cell death from 2 to 6 hours and apoptosis in intermediate and basal cells from 4 to 24 hours was observed after cyclophosphamide. Despite superficial cell loss, KGF suppressed intermediate and basal cell apoptosis, likely via AKT signaling. At 6 and 24 hours after cyclophosphamide, KGF-pretreated mice also had apparent extracellular signal-regulated kinase (ERK)-driven proliferation of mostly keratin 5 (KRT5)+/KRT14- intermediate cells. At 1 to 28 days after cyclophosphamide treatment, mostly KRT14+ basal progenitor cells proliferated in response to injury, peaking at 3 days in both treatment groups; however, proliferation rates were lower in the KGF group at 3 days, consistent with less injury. Three days after injury, unlike controls, KGF-pretreated mice had regenerated superficial cells. At 10 and 28 days after cyclophosphamide treatment, KGF-pretreated mice had little proliferation and marked restoration of urothelial layers, whereas the phosphate-buffered saline group had ongoing regeneration. Administration of KGF to uninjured mice reproduced ERK-driven KRT5+/KRT14- proliferation seen in injured mice; KRT14+ cells were unaffected. KGF pretreatment blocks cyclophosphamide-induced intermediate and basal cell apoptosis, likely by phosphorylated AKT, and drives phosphorylated ERK-mediated KRT5+/KRT14- cell proliferation, leading to early urothelial regeneration.


Assuntos
Antineoplásicos Alquilantes/toxicidade , Ciclofosfamida/toxicidade , Cistite/prevenção & controle , Citoproteção , Fator 7 de Crescimento de Fibroblastos/metabolismo , Bexiga Urinária/lesões , Animais , Proliferação de Células , Cistite/induzido quimicamente , Cistite/metabolismo , Cistite/patologia , Feminino , Fator 7 de Crescimento de Fibroblastos/genética , Camundongos , Regeneração , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia
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