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1.
Medicine (Baltimore) ; 100(4): e24525, 2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33530281

RESUMO

BACKGROUND: Anoctamin-1 (ANO1) plays a pivotal role in cancer progression. A meta-analysis was conducted to assess the potential prognostic role of ANO1 in cancers. METHODS: A total of 1760 patients from 7 eligible studies were included into the analysis. Pooled hazard ratios or odds ratios were extracted and calculated with a random-effects model, and analyses of heterogeneity bias were conducted. RESULTS: Our results showed that over expression of ANO1 was significantly correlated with poor overall survival in all cancers (HR = 1.52; 95% CI: 1.19-1.92; P = .0006). Subgroup analysis indicated that there was a significant association between over expression of ANO1 and poor prognosis breast cancer (HR = 3.24; 95% CI: 1.74-6.04), head and neck squamous cell carcinoma (HR = 1.14; 95% CI: 1.00-1.30), esophageal squamous cell carcinoma (HR = 1.93; 95% CI: 1.07-3.50), gastric cancer (HR = 1.62; 95% CI: 1.12-2.34) and colorectal cancer (HR = 1.38; 95% CI: 1.03-1.85). In addition, over expression of ANO1 was not associated with TNM stage, histological grade, lymph node metastasis, tumor size, age and gender. However, ANO1 was significantly associated with human epidermal growth factor receptor 2, but not associated with progesterone receptor or estrogen receptor in breast cancer. CONCLUSIONS: Our results indicate that ANO1 can be a predictive factor for prognosis of cancer.


Assuntos
Anoctamina-1/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Biomarcadores/sangue , Feminino , Expressão Gênica , Humanos , Masculino , Neoplasias/mortalidade , Modelos de Riscos Proporcionais
2.
Nat Commun ; 12(1): 785, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542223

RESUMO

The binding of cytoplasmic Ca2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca2+.


Assuntos
Anoctamina-1/metabolismo , Cálcio/metabolismo , Ativação do Canal Iônico , Proteínas de Neoplasias/metabolismo , Anoctamina-1/genética , Anoctamina-1/ultraestrutura , Sítios de Ligação/genética , Cátions Bivalentes/metabolismo , Cloretos/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutagênese , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/ultraestrutura , Ligação Proteica , Conformação Proteica em alfa-Hélice
3.
Nat Commun ; 12(1): 786, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542228

RESUMO

The anion channel TMEM16A is activated by intracellular Ca2+ in a highly cooperative process. By combining electrophysiology and autocorrelation analysis, we investigated the mechanism of channel activation and the concurrent rearrangement of the gate in the narrow part of the pore. Features in the fluctuation characteristics of steady-state current indicate the sampling of intermediate conformations that are successively occupied during gating. The initial step is related to conformational changes induced by Ca2+ binding, which is ensued by rearrangements that open the pore. Mutations in the gate shift the equilibrium of transitions in a manner consistent with a progressive destabilization of this region during pore opening. We come up with a mechanism of channel activation where the binding of Ca2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation.


Assuntos
Anoctamina-1/metabolismo , Cálcio/metabolismo , Ativação do Canal Iônico , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Regulação Alostérica , Anoctamina-1/genética , Anoctamina-1/ultraestrutura , Sítios de Ligação/genética , Cátions Bivalentes/metabolismo , Cloretos/metabolismo , Células HEK293 , Humanos , Cinética , Método de Monte Carlo , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/ultraestrutura , Técnicas de Patch-Clamp , Distribuição de Poisson , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice
4.
Nat Commun ; 11(1): 4320, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859916

RESUMO

In autosomal dominant polycystic kidney disease (ADPKD) multiple bilateral renal cysts gradually enlarge, leading to a decline in renal function. Transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1) are known to drive cyst enlargement. Here we demonstrate that loss of Pkd1 increased expression of TMEM16A and CFTR and Cl- secretion in murine kidneys, with TMEM16A essentially contributing to cyst growth. Upregulated TMEM16A enhanced intracellular Ca2+ signaling and proliferation of Pkd1-deficient renal epithelial cells. In contrast, increase in Ca2+ signaling, cell proliferation and CFTR expression was not observed in Pkd1/Tmem16a double knockout mice. Knockout of Tmem16a or inhibition of TMEM16A in vivo by the FDA-approved drugs niclosamide and benzbromarone, as well as the TMEM16A-specific inhibitor Ani9 largely reduced cyst enlargement and abnormal cyst cell proliferation. The present data establish a therapeutic concept for the treatment of ADPKD.


Assuntos
Anoctamina-1/genética , Anoctamina-1/metabolismo , Cistos/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Animais , Anoctamina-1/efeitos dos fármacos , Benzobromarona/farmacologia , Canais de Cálcio , Proliferação de Células , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , Cistos/tratamento farmacológico , Cistos/genética , Modelos Animais de Doenças , Cães , Células Epiteliais/metabolismo , Humanos , Rim/metabolismo , Rim/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Néfrons/metabolismo , Niclosamida/farmacologia , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/genética
5.
Am J Physiol Renal Physiol ; 319(3): F394-F402, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32686521

RESUMO

Stress urinary incontinence (SUI) is more common in women than in men, and sex differences in anatomic structure and physiology have been suggested as causes; however, the underlying cellular and molecular mechanisms remain unclear. The spontaneous tone (STT) of the urethra has been shown to have a fundamental effect on preventing the occurrence of SUI. Here, we investigated whether the urethral STT exhibited sex differences. First, we isolated urethral smooth muscle (USM) and detected STT in female mice and women. No STT was found in male mice or men. Furthermore, caffeine induced increased contractility and intracellular Ca2+ concentration in urethrae from female mice compared with male mice. EACT [an N-aroylaminothiazole, anoctamin-1 (ANO1) activator] elicited increased intracellular Ca2+ concentration and stronger currents in female mice than in male mice. Moreover, ANO1 expression in single USM cells from women and female mice was almost twofold higher than that found in cells from men and male mice. In summary, ANO1 in USM contributes to sex differences in urethral spontaneous tone. This finding may provide new guidance for the treatment of SUI in women and men.


Assuntos
Anoctamina-1/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Uretra/citologia , Incontinência Urinária por Estresse/metabolismo , Adulto , Animais , Anoctamina-1/genética , Cálcio/farmacologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Uretra/fisiologia , Adulto Jovem
6.
Tunis Med ; 98(2): 168-171, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32395809

RESUMO

BACKGROUND: Mammary analogue secretory carcinoma is a rare new entity of low-grade malignant tumor of salivary glands. It shared the same histologic features and the chromosomal translocation t(12;15)(p13;q25) as secretory carcinoma of the breast. AIM: To highlight the diagnosis approaches and the attitude of management in a case of MASC which is the first case reported in Tunisia. Reported case: A case of MASC of the lower left jugal mucosa was reviewed for its microscopic and immunohistochemical features. Fluorescence in situ hybridization (FISH) for the ETV6-NTRK3 translocation was performed. Surgery was the only treatment required in this case. No signs of local or regional recurrence during the one-year follow-up were noticed. COMMENTARIES: Secretory carcinoma was confused with other salivary gland tumors especially acinic cell carcinoma due to their morphological similarities, making diagnosis dilemma. Fluorescence in-situ hybridization (FISH) is the one definitive finding to confirm the diagnosis of MASC and to differentiate it from the other types of salivary gland tumor. At the present time, no specific therapy is available for patients with MASC.


Assuntos
Carcinoma Secretor Análogo ao Mamário/diagnóstico , Anoctamina-1/análise , Anoctamina-1/metabolismo , Bochecha/patologia , Análise Citogenética , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Mamoglobina A/análise , Mamoglobina A/metabolismo , Carcinoma Secretor Análogo ao Mamário/genética , Carcinoma Secretor Análogo ao Mamário/cirurgia , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/genética , Proteínas S100/análise , Proteínas S100/metabolismo , Tunísia
7.
Mol Pharmacol ; 98(1): 61-71, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32358165

RESUMO

The blood-brain barrier (BBB) is essential for the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) comprise the BBB, and thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying molecular mechanisms remain unclear. In the present study, the molecular components of Ca2+-activated Cl- (ClCa) channels and their physiological roles were examined using mouse BCECs (mBCECs) and a cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was strongly expressed in mBCECs and t-BBEC117 cells. In t-BBEC117 cells, whole-cell Cl- currents were sensitive to the ClCa channel blockers, 100 µM niflumic acid and 10 µM T16Ainh-A01, and were also reduced markedly by small-interfering RNA (siRNA) knockdown of TMEM16A. Importantly, block of ClCa currents with ClCa channel blockers or TMEM16A siRNA induced membrane hyperpolarization. Moreover, treatment with TMEM16A siRNA caused an increase in resting cytosolic Ca2+ concentration ([Ca2+]cyt). T16Ainh-A01 reduced cell viability in a concentration-dependent manner. Either ClCa channel blockers or TMEM16A siRNA also curtailed cell proliferation and migration. Furthermore, ClCa channel blockers attenuated the trans-endothelial permeability. In combination, these results strongly suggest that TMEM16A contributes to ClCa channel conductance and can regulate both the resting membrane potential and [Ca2+]cyt in BCECs. Our data also reveal how these BCECs may be involved in the maintenance of BBB functions, as both the proliferation and migration are altered following changes in channel activity. SIGNIFICANCE STATEMENT: In brain capillary endothelial cells (BCECs) of the blood-brain barrier (BBB), TMEM16A is responsible for Ca2+-activated Cl- channels and can regulate both the resting membrane potential and cytosolic Ca2+ concentration, contributing to the proliferation and migration of BCECs. The present study provides novel information on the molecular mechanisms underlying the physiological functions of BCECs in the BBB and a novel target for therapeutic drugs for disorders associated with dysfunctions in the BBB.


Assuntos
Anoctamina-1/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Animais , Anoctamina-1/antagonistas & inibidores , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Bovinos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Ácido Niflúmico/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia
8.
Int J Clin Oncol ; 25(6): 1145-1154, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32240440

RESUMO

BACKGROUND: Increase of the Ca2+-activated chloride channel TMEM16A is contribute to tumorigenesis. However, the expression level of TMEM16A and its underlying molecular mechanism for TMEM16Apromotingliver carcinogenesis is remains unknown. METHODS: In the present study, the expression of TMEM16A in hepatocellular carcinoma (HCC) tissues were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), Western blot and immunohistochemical. Cell proliferation was detected using CCK-8, EdU staining and colony formation methods. Flow cytometry was carried out for detecting cell cycle distribution and apoptosis rate. Migration and invasion abilities were analyzed using transwell and wound healing assay. Western blot method was performed to analyze protein expression. RESULTS: Here, we found TMEM16A was significantly increased in HCC tissues, and a higher TMEM16A expression levels were detected in larger tumor size, higher tumor grade, with distant metastasis and poor differentiation. Moreover, overexpression of TMEM16A promoted HCC growth, migration and invasion, and suppressed apoptosis in vitro and in vivo. Knockdown of TMEM16A inhibited HCC growth, migration and invasion, and induced apoptosis in vitro and in vivo. Furthermore, TMEM16A regulated PI3K/AKT-MAKP signaling pathway. CONCLUSION: Our data indicate that TMEM16A may represent a novel biomarker of HCC and may be a potential therapeutic target for diagnosis and therapy.


Assuntos
Anoctamina-1/metabolismo , Apoptose , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/metabolismo , Animais , Anoctamina-1/genética , Carcinoma Hepatocelular/genética , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Sci Rep ; 10(1): 6644, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313203

RESUMO

Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca2+-activated Cl- channel responsible for essential physiological processes. Each monomer harbours a pore and a Ca2+-binding pocket; the voltage-dependent binding of two intracellular Ca2+ ions to the pocket gates the pore. However, in the absence of intracellular Ca2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca2+.


Assuntos
Anoctamina-1/metabolismo , Cálcio/metabolismo , Cloretos/metabolismo , Proteínas Recombinantes de Fusão/genética , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Anoctamina-1/antagonistas & inibidores , Anoctamina-1/genética , Antracenos/farmacologia , Cátions Bivalentes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Camundongos , Mutação , Técnicas de Patch-Clamp , Plasmídeos/química , Plasmídeos/metabolismo , Prótons , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Taninos/farmacologia , Transfecção
10.
Diagn Pathol ; 15(1): 23, 2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32164724

RESUMO

BACKGROUND: Low-grade fibromyxoid sarcoma (LGFMS) is a rare fibroblastic tumor often involving deep tissue of trunk and lower extremities in young to middle-aged patients. Rarely, LGFMS can occur in other sites including head and neck, chest, abdomen and female reproductive system. Three cases of LGFMS in mesentery of small intestine have been reported and all have conventional histologic features. Herein we reported a unique case of LGFMS in mesentery of small intestine. CASE PRESENTATION: A 43 year-old male with chief complaint of lower back pain for 4 years presented to our hospital. Physical exam reveal a firm, non-tender, non-distended, mobile large abdominal mass, which was shown on abdominal CT as a 10 cm retroperitoneal tumor. Biopsy revealed a spindle cell neoplasm in a myxoid background with a delicate vascular network. Tumor resection was performed. Gross examination of the resected specimen showed a 10.8 cm, tan-white, smooth, firm, lobulated mesenteric mass with bulging and gelatinous cut surface and confined within small bowel serosa. Microscopic examination demonstrated foci epithelioid cords and whorls with prominent atypia, in additional of regular, bland-appearing spindle cells in a fibrous and myxoid stroma and osseous metaplasia. The tumor cells stained diffusely positive for DOG1 with moderate staining density, and diffusely and strongly positive for MUC4. Rearrangement involving FUS (16p11.2) gene was identified with break-apart probe and confirmed by Anchored Multiplex PCR. A final diagnosis of low-grade fibromyxoid sarcoma was rendered. CONCLUSION: Our case highlights the importance of including LGFMS in the differential diagnosis of mesenteric tumors and the DOG1 positivity which could represent a potential diagnostic pitfall.


Assuntos
Anoctamina-1/metabolismo , Fibroma/patologia , Fibrossarcoma/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias Retroperitoneais/patologia , Adulto , Biomarcadores Tumorais/análise , Fibroma/metabolismo , Fibrossarcoma/metabolismo , Rearranjo Gênico , Humanos , Intestino Delgado/patologia , Masculino , Mesentério/patologia , Proteína FUS de Ligação a RNA/genética , Neoplasias Retroperitoneais/metabolismo
11.
Am J Physiol Gastrointest Liver Physiol ; 318(4): G763-G771, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32090602

RESUMO

TMEM16A is a Ca2+-activated Cl- channel in the apical membrane of biliary epithelial cells, known as cholangiocytes, which contributes importantly to ductular bile formation. Whereas cholangiocyte TMEM16A activity is regulated by extracellular ATP-binding membrane purinergic receptors, channel expression is regulated by interleukin-4 (IL-4) through an unknown mechanism. Therefore, the aim of the present study was to identify the signaling pathways involved in TMEM16A expression and cholangiocyte secretion. Studies were performed in polarized normal rat cholangiocyte monolayers, human Mz-Cha-1 biliary cells, and cholangiocytes isolated from murine liver tissue. The results demonstrate that all the biliary models expressed the IL-4Rα/IL-13Rα1 receptor complex. Incubation of cholangiocytes with either IL-13 or IL-4 increased the expression of TMEM16A protein, which was associated with an increase in the magnitude of Ca2+-activated Cl- currents in response to ATP in single cells and the short-circuit current response in polarized monolayers. The IL-4- and IL-13-mediated increase in TMEM16A expression was also associated with an increase in STAT6 phosphorylation. Specific inhibition of JAK-3 inhibited the increase in TMEM16A expression and the IL-4-mediated increase in ATP-stimulated currents, whereas inhibition of STAT6 inhibited both IL-4- and IL-13-mediated increases in TMEM16A expression and ATP-stimulated secretion. These studies demonstrate that the cytokines IL-13 and IL-4 regulate the expression and function of biliary TMEM16A channels through a signaling pathway involving STAT6. Identification of this regulatory pathway provides new insight into biliary secretion and suggests new targets to enhance bile formation in the treatment of cholestatic liver disorders.NEW & NOTEWORTHY The Ca2+-activated Cl- channel transmembrane member 16A (TMEM16A) has emerged as an important regulator of biliary secretion and hence, ductular bile formation. The present studies represent the initial description of the regulation of TMEM16A expression in biliary epithelium. Identification of this regulatory pathway involving the IL-4 and IL-13 receptor complex and JAK-3 and STAT-6 signaling provides new insight into biliary secretion and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.


Assuntos
Anoctamina-1/metabolismo , Regulação da Expressão Gênica/fisiologia , Fígado/metabolismo , Receptores de Interleucina-13/metabolismo , Receptores de Interleucina-4/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Anoctamina-1/genética , Ácidos e Sais Biliares , Ductos Biliares/metabolismo , Linhagem Celular , Cloretos , Fenômenos Eletrofisiológicos , Humanos , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Masculino , Camundongos , Técnicas de Patch-Clamp , Ratos , Receptores de Interleucina-13/genética , Receptores de Interleucina-4/genética , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo
12.
Am J Respir Crit Care Med ; 201(8): 946-954, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31898911

RESUMO

Rationale: Enhancing non-CFTR (cystic fibrosis transmembrane conductance regulator)-mediated anion secretion is an attractive therapeutic approach for the treatment of cystic fibrosis (CF) and other mucoobstructive diseases.Objectives: To determine the effects of TMEM16A potentiation on epithelial fluid secretion and mucociliary clearance.Methods: The effects of a novel low-molecular-weight TMEM16A potentiator (ETX001) were evaluated in human cell and animal models of airway epithelial function and mucus transport.Measurements and Main Results: Potentiating the activity of TMEM16A with ETX001 increased the Ca2+-activated Cl- channel activity and anion secretion in human bronchial epithelial (HBE) cells from patients with CF without impacting calcium signaling. ETX001 rapidly increased fluid secretion and airway surface liquid height in CF-HBE cells under both static conditions and conditions designed to mimic the shear stress associated with tidal breathing. In ovine models of mucus clearance (tracheal mucus velocity and mucociliary clearance), inhaled ETX001 was able to accelerate clearance both when CFTR function was reduced by administration of a pharmacological blocker and when CFTR was fully functional.Conclusions: Enhancing the activity of TMEM16A increases epithelial fluid secretion and enhances mucus clearance independent of CFTR function. TMEM16A potentiation is a novel approach for the treatment of patients with CF and non-CF mucoobstructive diseases.


Assuntos
Anoctamina-1/efeitos dos fármacos , Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Depuração Mucociliar/efeitos dos fármacos , Muco/efeitos dos fármacos , Administração por Inalação , Animais , Anoctamina-1/metabolismo , Brônquios/citologia , Sinalização do Cálcio/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , Técnicas de Patch-Clamp , Respiração , Mucosa Respiratória/citologia , Ovinos , Traqueia/efeitos dos fármacos , Traqueia/metabolismo
13.
Gastric Cancer ; 23(1): 118-125, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31041650

RESUMO

BACKGROUND: A multidisciplinary approach based on guidelines and pathological diagnosis by specialized pathologists are important for improving the prognosis and QoL of GIST patients. This study examined the adherence to the guidelines and the concordance of the pathological diagnosis of high-risk GISTs. PATIENTS AND METHODS: Among 541 patients with high-risk GISTs recruited to the prospective registry between Dec. 2012 and Dec. 2015, 534 patients were analyzed after central pathology with KIT and DOG1 IHC and genotyping of KIT and PDGFRA. RESULTS: Of the 534 patients, 432 (81%) received imatinib adjuvant therapy at a starting dose of 400 or 300 mg/day. Multivariate analysis indicated that age (HR 0.71; 95% CI 0.58-0.88), tumor size (HR for > 10 cm vs < 5 cm, 3.87; 95% CI 1.72-8.74), mitosis (HR for > 10 vs < 5, 3.54; 95% CI 1.84-6.79), tumor rupture (HR 3.69; 95% CI 1.43-9.52) and performance status (HR 0.55; 95% CI 0.31-0.99) were independently related to adjuvant therapy. Among the 534 high-risk GISTs diagnosed locally, 19 tumors (3.6%) were diagnosed as non-GISTs, and the other 93 (18.1%) GISTs were reclassified into lower risk categories by central pathology. Among 10 patients with non-GISTs and 8 patients with PDGFRA D842V mutations, 4 (40%) and 3 (38%) patients, respectively, continued the therapy after receiving the central pathology results. CONCLUSIONS: The adherence to guidelines and the concordance of pathological diagnoses were comparatively good for high-risk GISTs. Central pathology may contribute to improved diagnosis, but further refinements may be required.


Assuntos
Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Idoso , Anoctamina-1/metabolismo , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Quimioterapia Adjuvante , Feminino , Tumores do Estroma Gastrointestinal/mortalidade , Fidelidade a Diretrizes , Humanos , Mesilato de Imatinib/efeitos adversos , Mesilato de Imatinib/uso terapêutico , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Estudos Prospectivos , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Sistema de Registros
14.
Ann Diagn Pathol ; 44: 151440, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31862519

RESUMO

Chondroblastoma is a relatively uncommon, primary benign bone tumor, frequently identified in young individuals. Despite its classical radiologic and histopathological features, at times, it is fraught with a diagnostic challenge, especially differentiating it from a giant cell tumor of bone (GCTB); an osteosarcoma and a chondrosarcoma. Lately, few studies have shown the diagnostic utility of immunohistochemical (IHC) expression DOG1 antibody in chondroblastomas. The present study was undertaken to evaluate IHC expression of S100 protein, DOG1 and p63 in 36 chondroblastomas. From January 2013 to July 2019 (6-year duration), 106 chondroblastomas were diagnosed, with IHC staining performed in 36 cases. Conventional Hematoxylin and Eosin stained microsections and IHC stained sections were reviewed in 36 cases. IHC staining of p63 (intranuclear), S100 protein (nuclear and cytoplasmic) and DOG1 (cytoplasmic membranous) was recorded in various cases. Seventy-four tumors occurred in males and 32 in females, within age-range of 7-55 years (average = 18.6), frequently in tibia (33/106; 31.1%), followed by femur (26, 24.5%) humerus (22, 20.7%), calcaneum (5) and scapula (4). IHC staining for S100P was positive in 33/36cases (91.7%); DOG1 in 16/19 (84.2%) cases and p63 in 10/15cases (66.6%). DOG1 immunostaining was negative in 25 various other tumors. Sensitivity and specificity for S100P, DOG1and p63 in chondroblastomas was (91.6%, 59.3%); (84.2%, 100%) and (66.6%, 46.6%), respectively. P63 was positively expressed in 15/27 (55.5%) GCTBs. S100 protein and DOG1 can be utilized for a confirmatory diagnosis of a chondroblastoma, especially for differentiating it from its other differentials, such as GCTB, in view of certain associated therapeutic implications. P63 is not useful in that scenario.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/patologia , Condroblastoma/patologia , Tumor de Células Gigantes do Osso/patologia , Osteossarcoma/patologia , Adolescente , Adulto , Anoctamina-1/metabolismo , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/metabolismo , Criança , Condroblastoma/diagnóstico por imagem , Condroblastoma/metabolismo , Feminino , Tumor de Células Gigantes do Osso/diagnóstico por imagem , Tumor de Células Gigantes do Osso/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Osteossarcoma/diagnóstico por imagem , Osteossarcoma/metabolismo , Proteínas S100/metabolismo , Sensibilidade e Especificidade , Adulto Jovem
15.
Am J Physiol Lung Cell Mol Physiol ; 318(2): L287-L295, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31747299

RESUMO

TMEM16A (anoctamin 1) is an important calcium-activated chloride channel in airway smooth muscle (ASM). We have previously shown that TMEM16A antagonists such as benzbromarone relax ASM and have proposed TMEM16A antagonists as novel therapies for asthma treatment. However, TMEM16A is also expressed on airway epithelium, and TMEM16A agonists are being investigated as novel therapies for cystic fibrosis. There are theoretical concerns that agonism of TMEM16A on ASM could lead to bronchospasm, making them detrimental as airway therapeutics. The TMEM16A agonist Eact induced a significant contraction of human ASM and guinea pig tracheal rings in an ex vivo organ bath model. Pretreatment with two different TMEM16A antagonists, benzbromarone or T16Ainh-A01, completely attenuated these Eact-induced contractions. Pretreatment with Eact alone augmented the maximum acetylcholine contraction. Pretreatment of A/J mice in vivo with nebulized Eact caused an augmentation of methacholine-induced increases in airway resistance measured by the forced oscillatory technique (flexiVent). Pretreatment with the TMEM16A antagonist benzbromarone significantly attenuated methacholine-induced increases in airway resistance. In in vitro cellular studies, TMEM16A was found to be expressed more abundantly in ASM compared with epithelial cells in culture (8-fold higher in ASM). Eact caused an increase in intracellular calcium in human ASM cells that was completely attenuated by pretreatment with benzbromarone. Eact acutely depolarized the plasma membrane potential of ASM cells, which was attenuated by benzbromarone or nifedipine. The TMEM16A agonist Eact modulates ASM contraction in both ex vivo and in vivo models, suggesting that agonism of TMEM16A may lead to clinically relevant bronchospasm.


Assuntos
Anoctamina-1/agonistas , Anoctamina-1/metabolismo , Pulmão/metabolismo , Tono Muscular , Músculo Liso/metabolismo , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Acetilcolina/farmacologia , Animais , Anoctamina-1/genética , Hiper-Reatividade Brônquica/fisiopatologia , Broncoconstrição/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Cobaias , Humanos , Fosfatos de Inositol/biossíntese , Cloreto de Metacolina/farmacologia , Contração Muscular/efeitos dos fármacos , Tono Muscular/efeitos dos fármacos , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Acta Pharmacol Sin ; 41(2): 208-217, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31484993

RESUMO

TMEM16A Ca2+-activated chloride channel (CaCC) plays an essential role in vascular homeostasis. In this study we investigated the molecular mechanisms underlying downregulation of TMEM16A CaCC activity during hypertension. In cultured basilar artery smooth muscle cells (BASMCs) isolated from 2k2c renohypertesive rats, treatment with angiotensin II (0.125-1 µM) dose-dependently increased endophilin A2 levels and decreased TMEM16A expression. Similar phenomenon was observed in basilar artery isolated from 2k2c rats. We then used whole-cell recording to examine whether endophilin A2 could regulate TMEM16A CaCC activity in BASMCs and found that knockdown of endophilin A2 significantly enhanced CaCC activity, whereas overexpression of endophilin A2 produced the opposite effect. Overexpression of endophilin A2 did not affect the TMEM16A mRNA level, but markedly decreased TMEM16A protein level in BASMCs by inducing ubiquitination and autophagy of TMEM16A. Ubiquitin-binding receptor p62 (SQSTM1) could bind to ubiquitinated TMEM16A and resulted in a process of TMEM16A proteolysis in autophagosome/lysosome. These data provide new insights into the regulation of TMEM16A CaCC activity by endophilin A2 in BASMCs, which partly explains the mechanism of angiotensin-II-induced TMEM16A inhibition during hypertension-induced vascular remodeling.


Assuntos
Aciltransferases/metabolismo , Anoctamina-1/metabolismo , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Aciltransferases/genética , Angiotensina II/metabolismo , Animais , Autofagia/fisiologia , Células Cultivadas , Regulação para Baixo , Técnicas de Silenciamento de Genes , Hipertensão/fisiopatologia , Masculino , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Remodelação Vascular/fisiologia
17.
Biochem J ; 476(24): 3705-3719, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31790150

RESUMO

Platinum-containing drugs such as cisplatin and carboplatin are routinely used for the treatment of many solid tumors including squamous cell carcinoma of the head and neck (SCCHN). However, SCCHN resistance to platinum compounds is well documented. The resistance to platinum has been linked to the activity of divalent transporter ATP7B, which pumps platinum from the cytoplasm into lysosomes, decreasing its concentration in the cytoplasm. Several cancer models show increased expression of ATP7B; however, the reason for such an increase is not known. Here we show a strong positive correlation between mRNA levels of TMEM16A and ATP7B in human SCCHN tumors. TMEM16A overexpression and depletion in SCCHN cell lines caused parallel changes in the ATP7B mRNA levels. The ATP7B increase in TMEM16A-overexpressing cells was reversed by suppression of NADPH oxidase 2 (NOX2), by the antioxidant N-Acetyl-Cysteine (NAC) and by copper chelation using cuprizone and bathocuproine sulphonate (BCS). Pretreatment with either chelator significantly increased cisplatin's sensitivity, particularly in the context of TMEM16A overexpression. We propose that increased oxidative stress in TMEM16A-overexpressing cells liberates the chelated copper in the cytoplasm, leading to the transcriptional activation of ATP7B expression. This, in turn, decreases the efficacy of platinum compounds by promoting their vesicular sequestration. We think that such a new explanation of the mechanism of SCCHN tumors' platinum resistance identifies novel approach to treating these tumors.


Assuntos
Anoctamina-1/metabolismo , Cisplatino/farmacologia , ATPases Transportadoras de Cobre/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteínas de Neoplasias/metabolismo , Anoctamina-1/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , ATPases Transportadoras de Cobre/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Neoplasias/genética , Regulação para Cima
18.
Cell Calcium ; 84: 102103, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31683182

RESUMO

Recently there has been a flurry of interest in the regulation of the homo-dimeric calcium-activated chloride channel ANO1 (also known as TMEM16A) by phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). These recent studies show that upon Ca2+ binding, PI(4,5)P2 cooperates to maintain the conductive state of ANO1. PI(4,5)P2 does so by binding to sites or modules on the protein's cytosolic side. These findings add a new function to the PI(4,5)P2 repertoire and a new dimension to ANO1 gating.


Assuntos
Anoctamina-1/metabolismo , Cálcio/metabolismo , Citoesqueleto/metabolismo , Citosol/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Anoctamina-1/genética , Sinalização do Cálcio , Humanos , Ativação do Canal Iônico
19.
Life Sci Alliance ; 2(6)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31732694

RESUMO

Airway mucus obstruction is the main cause of morbidity in cystic fibrosis, a disease caused by mutations in the CFTR Cl- channel. Activation of non-CFTR Cl- channels such as TMEM16A can likely compensate for defective CFTR. However, TMEM16A was recently described as a key driver in mucus production/secretion. Here, we have examined whether indeed there is a causal relationship between TMEM16A and MUC5AC production, the main component of respiratory mucus. Our data show that TMEM16A and MUC5AC are inversely correlated during differentiation of human airway cells. Furthermore, we show for the first time that the IL-4-induced TMEM16A up-regulation is proliferation-dependent, which is supported by the correlation found between TMEM16A and Ki-67 proliferation marker during wound healing. Consistently, the notch signaling activator DLL4 increases MUC5AC levels without inducing changes neither in TMEM16A nor in Ki-67 expression. Moreover, TMEM16A inhibition decreased airway surface liquid height. Altogether, our findings demonstrate that up-regulation of TMEM16A and MUC5AC is only circumstantial under cell proliferation, but with no causal relationship between them. Thus, although essential for airway hydration, TMEM16A is not required for MUC5AC production.


Assuntos
Anoctamina-1/metabolismo , Mucina-5AC/biossíntese , Muco/metabolismo , Proteínas de Neoplasias/metabolismo , Mucosa Respiratória/metabolismo , Transporte Biológico , Brônquios/citologia , Brônquios/metabolismo , Linhagem Celular , Células Cultivadas , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Humanos , Mucina-5AC/metabolismo , RNA Interferente Pequeno/metabolismo , Mucosa Respiratória/citologia , Transdução de Sinais
20.
Ann Diagn Pathol ; 43: 151408, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31629156

RESUMO

Epithelial membrane antigen (EMA) and DOG1 are used as marker of epithelial cells, particularly the luminal cells, of salivary gland tumours. The aim of this study was to compare the EMA and DOG1 expression in tumours of minor salivary glands. Cases of pleomorphic adenoma (PA), basal cell adenoma (BCA), canalicular adenoma (CA), adenoid cystic carcinoma (ACC), polymorphous adenocarcinoma (PAC), mucoepidermoid carcinoma (MEC) and epithelial-myoepithelial carcinoma (EMC) were submitted to immunohistochemistry for EMA and DOG1. In PA and BCA, EMA and DOG1 were observed in luminal cells, while in CA the tumour cells were negative for both proteins. The EMA and DOG1 pattern expression detected in EMC was similar to that one observed in benign tumours. In ACC, both myoepithelial e epithelial expressed EMA and DOG-1. PAC tumour cells were only positive for DOG1, whereas MEC were only positive for EMA. In conclusion, EMA and DOG1 expression in benign salivary gland tumours was similar to normal salivary gland tissue and can be used as good marker of tumoral cells derived from intercalated ducts or its progenitor cells, while in malignant salivary gland tumours EMA expression is, however, better used as an indicator of aggressive behavior than a marker of luminal cells.


Assuntos
Anoctamina-1/metabolismo , Mucina-1/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias das Glândulas Salivares/patologia , Glândulas Salivares Menores/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenoma/metabolismo , Adenoma/patologia , Adenoma Pleomorfo/metabolismo , Adenoma Pleomorfo/patologia , Biomarcadores Tumorais/metabolismo , Carcinoma/metabolismo , Carcinoma/patologia , Carcinoma Adenoide Cístico/metabolismo , Carcinoma Adenoide Cístico/patologia , Carcinoma Mucoepidermoide/metabolismo , Carcinoma Mucoepidermoide/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Imuno-Histoquímica/métodos , Neoplasias das Glândulas Salivares/ultraestrutura , Glândulas Salivares Menores/patologia , Glândulas Salivares Menores/ultraestrutura
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