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1.
ACS Appl Mater Interfaces ; 14(28): 31715-31726, 2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35798541

RESUMO

The gated state of anion channels is involved in the regulation of proliferation and migration of tumors. Specific regulators are urgently needed for efficacious cancer ablation. For this purpose, it is essential to understand the molecular mechanisms of interaction between the regulators and anion channels and apply this knowledge to regulate anion channels. Transmembrane 16A (TMEM16A) is the molecular basis of the calcium-activated chloride channels. It is an anion channel activated by Ca2+, and the inhibition of TMEM16A is associated with a decrease in tumorigenesis. Herein, we characterized a natural compound procyanidin (PC) as an efficacious and selective inhibitor of TMEM16A with an IC50 of 10.6 ± 0.6 µM. Our research revealed the precise sites (D383, R535, and E624) of electrostatic interactions between PC and TMEM16A. Near-infrared (NIR)-light-responsive photothermal conjugated polymer nanoparticles encapsulating PC (CPNs-PC) were established to remotely target and regulate the TMEM16A anion channel. Upon NIR irradiation, CPNs-PC downregulated the signaling pathway downstream of TMEM16A and arrested the cell cycle progression of cancer cells and improved the bioavailability of PC. The tumor inhibition ratio of CPNs-PC was superior to PC by 13.4%. Our findings enabled the development of a strategy to accurately and remotely regulate anion channels to promote tumor regression using NIR-light-responsive conjugated polymer nanoparticles containing specific inhibitors of TMEM16A.


Assuntos
Canais de Cloreto , Transdução de Sinais , Ânions , Anoctamina-1/metabolismo , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Polímeros/metabolismo
2.
Front Biosci (Landmark Ed) ; 27(7): 216, 2022 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-35866397

RESUMO

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy characterized by abnormal tubular epithelial proliferation and fluid secretion. Anoctamin 1 (ANO1) is a calcium-dependent chloride channel. However, how ANO1 contributes to ADPKD is largely unexplored. METHODS: Kidney tissues from ADPKD patients, Pkd1RC/RC mice model, WT9-7 human PKD1+⁣/- cells, and 3D culture models in vitro were used. Localization of ANO1 and cilium length were investigated by confocal immunofluorescence. RESULTS: We found that ANO1 was consistently upregulated in human and mouse PKD kidneys. Intriguingly, ANO1 located in a vesicle-like pattern at the ciliary base but not on the ciliary surface. ANO1 deficiency enhanced ciliogenesis and the ciliary dosage of polycystin-2 in human PKD cells, and reduced cyst formation in 3D culture models. Moreover, inhibition of ANO1 abolished the activation of STAT3 and ERK pathways in PKD cells. CONCLUSIONS: Our data indicate ANO1 is a negative regulator for both cilia length and cilia trafficking of polycystin-2 and provide mechanistic insights regarding the therapeutic potential of ANO1 pathway in ADPKD treatment.


Assuntos
Anoctamina-1 , Rim Policístico Autossômico Dominante , Canais de Cátion TRPP , Animais , Anoctamina-1/genética , Anoctamina-1/metabolismo , Cílios/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Proteínas de Neoplasias , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo
3.
Int J Mol Sci ; 23(9)2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35563558

RESUMO

Volume-regulated anion channel (VRAC) is ubiquitously expressed and plays a pivotal role in vertebrate cell volume regulation. A heterologous complex of leucine-rich repeat containing 8A (LRRC8A) and LRRC8B-E constitutes the VRAC, which is involved in various processes such as cell proliferation, migration, differentiation, intercellular communication, and apoptosis. However, the lack of a potent and selective inhibitor of VRAC limits VRAC-related physiological and pathophysiological studies, and most previous VRAC inhibitors strongly blocked the calcium-activated chloride channel, anoctamin 1 (ANO1). In the present study, we performed a cell-based screening for the identification of potent and selective VRAC inhibitors. Screening of 55,000 drug-like small-molecules and subsequent chemical modification revealed 3,3'-((2-hydroxy-3-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (VI-116), a novel potent inhibitor of VRAC. VI-116 fully inhibited VRAC-mediated I- quenching with an IC50 of 1.27 ± 0.18 µM in LN215 cells and potently blocked endogenous VRAC activity in PC3, HT29 and HeLa cells in a dose-dependent manner. Notably, VI-116 had no effect on intracellular calcium signaling up to 10 µM, which completely inhibited VRAC, and showed high selectivity for VRAC compared to ANO1 and ANO2. However, DCPIB, a VRAC inhibitor, significantly affected ATP-induced increases in intracellular calcium levels and Eact-induced ANO1 activation. In addition, VI-116 showed minimal effect on hERG K+ channel activity up to 10 µM. These results indicate that VI-116 is a potent and selective VRAC inhibitor and a useful research tool for pharmacological dissection of VRAC.


Assuntos
Sinalização do Cálcio , Proteínas de Membrana , Ânions , Anoctamina-1/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias
4.
Nat Commun ; 13(1): 2798, 2022 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-35589730

RESUMO

TMEM16A, a calcium-activated chloride channel involved in multiple cellular processes, is a proposed target for diseases such as hypertension, asthma, and cystic fibrosis. Despite these therapeutic promises, its pharmacology remains poorly understood. Here, we present a cryo-EM structure of TMEM16A in complex with the channel blocker 1PBC and a detailed functional analysis of its inhibition mechanism. A pocket located external to the neck region of the hourglass-shaped pore is responsible for open-channel block by 1PBC and presumably also by its structural analogs. The binding of the blocker stabilizes an open-like conformation of the channel that involves a rearrangement of several pore helices. The expansion of the outer pore enhances blocker sensitivity and enables 1PBC to bind at a site within the transmembrane electric field. Our results define the mechanism of inhibition and gating and will facilitate the design of new, potent TMEM16A modulators.


Assuntos
Cálcio , Canais de Cloreto , Anoctamina-1/genética , Anoctamina-1/metabolismo , Cálcio/metabolismo , Canais de Cloreto/metabolismo
5.
Nat Commun ; 13(1): 2899, 2022 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-35610255

RESUMO

Osteoclast over-activation leads to bone loss and chloride homeostasis is fundamental importance for osteoclast function. The calcium-activated chloride channel Anoctamin 1 (also known as TMEM16A) is an important chloride channel involved in many physiological processes. However, its role in osteoclast remains unresolved. Here, we identified the existence of Anoctamin 1 in osteoclast and show that its expression positively correlates with osteoclast activity. Osteoclast-specific Anoctamin 1 knockout mice exhibit increased bone mass and decreased bone resorption. Mechanistically, Anoctamin 1 deletion increases intracellular Cl- concentration, decreases H+ secretion and reduces bone resorption. Notably, Anoctamin 1 physically interacts with RANK and this interaction is dependent upon Anoctamin 1 channel activity, jointly promoting RANKL-induced downstream signaling pathways. Anoctamin 1 protein levels are substantially increased in osteoporosis patients and this closely correlates with osteoclast activity. Finally, Anoctamin 1 deletion significantly alleviates ovariectomy induced osteoporosis. These results collectively establish Anoctamin 1 as an essential regulator in osteoclast function and suggest a potential therapeutic target for osteoporosis.


Assuntos
Anoctamina-1/metabolismo , Reabsorção Óssea , Osteoporose , Animais , Reabsorção Óssea/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Osteoporose/metabolismo , Ovariectomia , Ligante RANK/genética , Ligante RANK/metabolismo
6.
J Cancer Res Clin Oncol ; 148(8): 2045-2068, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35471604

RESUMO

Dysregulation of gene amplification, cell-signaling-pathway transduction, epigenetic and transcriptional regulation, and protein interactions drives tumor-cell proliferation and invasion, while ion channels also play an important role in the generation and development of tumor cells. Overexpression of Ca2+-activated Cl- channel anoctamin 1 (ANO1) is shown in numerous cancer types and correlates with poor prognosis. However, the mechanisms involved in ANO1-mediated malignant cellular transformation and the role of ANO1 in tumor immunity remain unknown. In this review, we discuss recent studies to determine the role of ANO1 in tumorigenesis and provide novel insights into the role of ANO1 in the context of tumor immunity. Furthermore, we analyze the roles and potential mechanisms of ANO1 in different types of cancers, and provide novel notions for the role of ANO1 in the tumor microenvironment and for potential use of ANO1 in clinical applications. Our review shows that ANO1 is involved in tumor immunity and microenvironment, and may, therefore, be an effective biomarker and therapeutic drug target.


Assuntos
Canais de Cloreto , Neoplasias , Anoctamina-1/genética , Anoctamina-1/metabolismo , Carcinogênese , Proliferação de Células , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Canais de Cloreto/farmacologia , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Microambiente Tumoral
7.
Proc Natl Acad Sci U S A ; 119(12): e2100670119, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35286200

RESUMO

Squamous cell carcinoma of the head and neck (SCCHN) is a devastating disease that continues to have low cure rates despite the recent advances in therapies. Cisplatin is the most used chemotherapy agent, and treatment failure is largely driven by resistance to this drug. Amplification of chromosomal band 11q13 occurs in ∼30% of SCCHN tumors. This region harbors the ANO1 gene that encodes the TMEM16A ion channel, which is responsible for calcium-activated chloride transport in epithelial tissues. TMEM16A overexpression is associated with cisplatin resistance, and high TMEM16A levels correlate with decreased survival. However, the mechanistic underpinning of this effect remains unknown. Lysosomal biogenesis and exocytosis have been implicated in cancer because of their roles in the clearance of damaged organelles and exocytosis of chemotherapeutic drugs and toxins. Here, we show that TMEM16A overexpression promotes lysosomal biogenesis and exocytosis, which is consistent with the expulsion of intracellular cisplatin. Using a combination of genetic and pharmacologic approaches, we find that TMEM16A promotes lysosomal flux in a manner that requires reactive oxygen species, TRPML1, and the activation of the ß-catenin­melanocyte-inducing transcription factor pathway. The lysosomal inhibitor hydroxychloroquine (HCQ) synergizes with cisplatin in killing SCCHN cells in vitro. Using a murine model of SCCHN, we show that HCQ and cisplatin retard the growth of cisplatin-resistant patient-derived xenografts in vivo. We propose that TMEM16A enables cell survival by the up-regulation of lysosomal sequestration and exocytosis of the cytotoxic drugs. These results uncover a model of treatment for resistance in cancer, its reversal, and a role for TMEM16A.


Assuntos
Anoctamina-1 , Antineoplásicos , Cisplatino , Neoplasias de Cabeça e Pescoço , Proteínas de Neoplasias , Anoctamina-1/genética , Anoctamina-1/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Canais de Cloreto , Cisplatino/farmacologia , Humanos , Lisossomos/metabolismo , Proteínas de Neoplasias/metabolismo
8.
Biochim Biophys Acta Mol Basis Dis ; 1868(6): 166370, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35231545

RESUMO

The Ca2+-activated Cl- channel ANO1 is widely expressed in epithelial cells, and ANO1 upregulation is implicated in the oncogenesis of many epithelium-originated cancers. However, whether ANO1 plays a causal role in the tumorigenesis of colorectal cancer remains largely unknown. Here, we show that ANO1 channel protein is upregulated in human colorectal cancer tissue samples and its upregulation is correlated with the TNM staging, histological type, pathological differentiation and poor prognosis. Knockdown or pharmacological inhibition of ANO1 suppresses colorectal cancer cell proliferation and induces cell apoptosis. Furthermore, ANO1 knockdown inhibits the growth of subcutaneous xenograft tumors implanted with colorectal cancer HT-29 cells in nude mice. Mechanically, knockdown of endogenous ANO1 inactivates the Wnt/ß-catenin signaling through downregulating critical components, such as Frizzled protein 1, ß-catenin and upregulating GSK3ß. Taken together, our results demonstrate that ANO1 upregulation is involved in the tumorigenesis of colorectal cancer, and inhibition of ANO1 upregulation or inactivating downstream Wnt/ß-catenin signaling may have therapeutic potential for colorectal cancer.


Assuntos
Anoctamina-1 , Neoplasias Colorretais , Proteínas de Neoplasias , Animais , Anoctamina-1/genética , Anoctamina-1/metabolismo , Carcinogênese/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células HT29 , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regulação para Cima
9.
Int J Mol Sci ; 23(3)2022 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-35163502

RESUMO

The TMEM16A/anoctamin-1 calcium-activated chloride channel (CaCC) contributes to a range of vital functions, such as the control of vascular tone and epithelial ion transport. The channel is a founding member of a family of 10 proteins (TMEM16x) with varied functions; some members (i.e., TMEM16A and TMEM16B) serve as CaCCs, while others are lipid scramblases, combine channel and scramblase function, or perform additional cellular roles. TMEM16x proteins are typically activated by agonist-induced Ca2+ release evoked by Gq-protein-coupled receptor (GqPCR) activation; thus, TMEM16x proteins link Ca2+-signalling with cell electrical activity and/or lipid transport. Recent studies demonstrate that a range of other cellular factors-including plasmalemmal lipids, pH, hypoxia, ATP and auxiliary proteins-also control the activity of the TMEM16A channel and its paralogues, suggesting that the TMEM16x proteins are effectively polymodal sensors of cellular homeostasis. Here, we review the molecular pathophysiology, structural biology, and mechanisms of regulation of TMEM16x proteins by multiple cellular factors.


Assuntos
Anoctamina-1/metabolismo , Anoctaminas/metabolismo , Canais de Cloreto/metabolismo , Animais , Anoctaminas/fisiologia , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Humanos , Transporte de Íons/fisiologia , Proteínas de Transferência de Fosfolipídeos/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-35202806

RESUMO

The calcium-activated chloride channel TMEM16A (ANO1) supports the passive movement of chloride ions across membranes and controls critical cell functions. Here we study the block of wild-type and mutant TMEM16A channels expressed in HEK293 cells by oleic acid, a monounsaturated omega-9 fatty acid beneficial for cardiovascular health. We found that oleic acid irreversibly blocks TMEM16A in a dose- and voltage-dependent manner at low intracellular Ca2+. We tested whether oleic acid interacted with the TMEM16A pore, varying the permeant anion concentration and mutating pore residues. Lowering the permeating anion concentration in the intracellular side did nothing but the blockade was intensified by increasing the anion concentration in the extracellular side. However, the blockade of the pore mutants E633A and I641A was voltage-independent, and the I641A IC50, a mutant with the inner hydrophobic gate in disarray, increased 16-fold. Furthermore, the uncharged methyl-oleate blocked 20-24% of the wild-type and I641A channels regardless of voltage. Our findings suggest that oleic acid inhibits TMEM16A by an allosteric mechanism after the electric field drives oleic acid's charged moiety inside the pore. Block of TMEM16A might be why oleic acid has a beneficial impact on the cardiovascular system.


Assuntos
Canais de Cloreto , Ácido Oleico , Ânions/metabolismo , Anoctamina-1/genética , Anoctamina-1/metabolismo , Cálcio/metabolismo , Canais de Cloreto/química , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ácido Oleico/farmacologia
11.
J Biol Chem ; 298(3): 101731, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35176281

RESUMO

Lung cancer has the highest mortality among cancers worldwide due to its high incidence and lack of the effective cures. We have previously demonstrated that the membrane ion channel TMEM16A is a potential drug target for the treatment of lung adenocarcinoma and have identified a pocket of inhibitor binding that provides the basis for screening promising new inhibitors. However, conventional drug discovery strategies are lengthy and costly, and the unpredictable side effects lead to a high failure rate in drug development. Therefore, finding new therapeutic directions for already marketed drugs may be a feasible strategy to obtain safe and effective therapeutic drugs. Here, we screened a library of over 1400 Food and Drug Administration-approved drugs through virtual screening and activity testing. We identified a drug candidate, Zafirlukast (ZAF), clinically approved for the treatment of asthma, that could inhibit the TMEM16A channel in a concentration-dependent manner. Molecular dynamics simulations and site-directed mutagenesis experiments showed that ZAF can bind to S387/N533/R535 in the nonselective inhibitor binding pocket, thereby blocking the channel pore. Furthermore, we demonstrate ZAF can target TMEM16A channel to inhibit the proliferation and migration of lung adenocarcinoma LA795 cells. In vivo experiments showed that ZAF can significantly inhibit lung adenocarcinoma tumor growth in mice. Taken together, we identified ZAF as a novel TMEM16A channel inhibitor with excellent anticancer activity, and as such, it represents a promising candidate for future preclinical and clinical studies.


Assuntos
Adenocarcinoma de Pulmão , Anoctamina-1 , Indóis , Neoplasias Pulmonares , Fenilcarbamatos , Sulfonamidas , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Anoctamina-1/antagonistas & inibidores , Anoctamina-1/metabolismo , Canais de Cloreto , Indóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Fenilcarbamatos/farmacologia , Sulfonamidas/farmacologia
12.
Urology ; 165: 157-163, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35167881

RESUMO

OBJECTIVE: To investigate the effect of hypertriglyceridemia (HTG) on the Anoctamin 1 (ANO1)-centered signal on urinary incontinence. METHODS: Female mice were fed with high fat diet to set up HTG mice model. ANO1 expression in urethrae were detected by Flow Cytometry and Western Blotting. The habit of micturition was recorded through urine spot test. Urethral Smooth Muscle Myography was used to record the urethral spontaneous tone (STT) and contraction. Patch-clamp Recording confirmed the currents of ANO1 in urethral smooth muscle cells. RESULTS: HTG mice showed almost abolished STT and significantly decreased contraction induced by EACT. Meanwhile, a change in urination habits was detected in HTG mice which suggested that mice could not hold the urethral pressure. Furthermore, ANO1 expression in single urethral smooth muscle  cells from HTG mice decreased almost 30% than that found in cells from normal group. Moreover, EACT (an N-aroylaminothiazole, ANO1 activator) induced decreased intracellular calcium ([Ca2+]i) and currents in single urethral smooth muscle cells from HTG mice. CONCLUSION: These results suggested that hypertriglyceridemia decreased the STT on urethral motility by down-regulating the expression of ANO1 in urethral smooth muscles. These findings provide a new perspective on the mechanism of urinary incontinence which is common in HTG patients.


Assuntos
Anoctamina-1 , Hipertrigliceridemia , Incontinência Urinária , Animais , Anoctamina-1/metabolismo , Cálcio/metabolismo , Regulação para Baixo , Feminino , Hipertrigliceridemia/complicações , Hipertrigliceridemia/metabolismo , Camundongos , Miócitos de Músculo Liso/metabolismo
13.
Elife ; 112022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35129434

RESUMO

Before hearing onset (postnatal day 12 in mice), inner hair cells (IHCs) spontaneously fire action potentials, thereby driving pre-sensory activity in the ascending auditory pathway. The rate of IHC action potential bursts is modulated by inner supporting cells (ISCs) of Kölliker's organ through the activity of the Ca2+-activated Cl--channel TMEM16A (ANO1). Here, we show that conditional deletion of Ano1 (Tmem16a) in mice disrupts Ca2+ waves within Kölliker's organ, reduces the burst-firing activity and the frequency selectivity of auditory brainstem neurons in the medial nucleus of the trapezoid body (MNTB), and also impairs the functional refinement of MNTB projections to the lateral superior olive. These results reveal the importance of the activity of Kölliker's organ for the refinement of central auditory connectivity. In addition, our study suggests the involvement of TMEM16A in the propagation of Ca2+ waves, which may also apply to other tissues expressing TMEM16A.


Assuntos
Anoctamina-1/metabolismo , Tronco Encefálico/metabolismo , Cálcio/metabolismo , Cóclea/metabolismo , Audição , Neurônios/metabolismo , Potenciais de Ação , Animais , Vias Auditivas , Ondas Encefálicas , Canais de Cloreto/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Camundongos , Camundongos Knockout
14.
Cells ; 10(11)2021 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-34831090

RESUMO

Cystic fibrosis (CF) is the most common of rare hereditary diseases in Caucasians, and it is estimated to affect 75,000 patients globally. CF is a complex disease due to the multiplicity of mutations found in the CF transmembrane conductance regulator (CFTR) gene causing the CFTR protein to become dysfunctional. Correctors and potentiators have demonstrated good clinical outcomes for patients with specific gene mutations; however, there are still patients for whom those treatments are not suitable and require alternative CFTR-independent strategies. Although CFTR is the main chloride channel in the lungs, others could, e.g., anoctamin-1 (ANO1 or TMEM16A), compensate for the deficiency of CFTR. This review summarizes the current knowledge on calcium-activated chloride channel (CaCC) ANO1 and presents ANO1 as an exciting target in CF.


Assuntos
Anoctamina-1/metabolismo , Fibrose Cística/tratamento farmacológico , Animais , Anoctamina-1/química , Fibrose Cística/patologia , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Modelos Biológicos
15.
Protein Pept Lett ; 28(12): 1338-1348, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34749600

RESUMO

TMEM16A mediates the calcium-activated transmembrane flow of chloride ions and a variety of physiological functions. The binding of cytoplasmic calcium ions of TMEM16A and the consequent conformational changes of it are the key issues to explore the structure-function relationship. In recent years, researchers have explored this issue through electrophysiological experiments, structure resolving, molecular dynamic simulation, and other methods. The structures of TMEM16 family members determined by cryo-Electron microscopy (cryo-EM) and X-ray crystallization provide the primary basis for the investigation of the molecular mechanism of TMEM16A. However, the binding and activation mechanism of calcium ions in TMEM16A are still unclear and controversial. This mini-review discusses four Ca2+ sensing sites of TMEM16A and analyzes activation properties of TMEM16A by them, which will help understand the structure-function relationship of TMEM16A and throw light on the molecular design targeting the TMEM16A channel.


Assuntos
Anoctamina-1/química , Cálcio/química , Proteínas de Neoplasias/química , Animais , Anoctamina-1/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Proteínas de Neoplasias/metabolismo , Relação Estrutura-Atividade
16.
Int J Mol Sci ; 22(21)2021 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-34769467

RESUMO

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including oral squamous cell carcinoma (OSCC). OSCC is a highly aggressive cancer and the most common oral malignancy. ANO1 has been proposed as a potential candidate for targeted anticancer therapy. In this study, we performed a cell-based screening to identify novel regulators leading to the downregulation of ANO1, and discovered cinobufagin, which downregulated ANO1 expression in oral squamous cell carcinoma CAL-27 cells. ANO1 protein levels were significantly reduced by cinobufagin in a dose-dependent manner with an IC50 value of ~26 nM. Unlike previous ANO1 inhibitors, short-term (≤10 min) exposure to cinobufagin did not alter ANO1 chloride channel activity and ANO1-dependent intestinal smooth muscle contraction, whereas long-term (24 h) exposure to cinobufagin significantly reduced phosphorylation of STAT3 and mRNA expression of ANO1 in CAL-27 cells. Notably, cinobufagin inhibited cell proliferation of CAL-27 cells expressing high levels of ANO1 more potently than that of ANO1 knockout CAL-27 cells. In addition, cinobufagin significantly reduced cell migration and induced caspase-3 activation and PARP cleavage in CAL-27 cells. These results suggest that downregulation of ANO1 by cinobufagin is a potential mechanism for the anticancer effect of cinobufagin in OSCC.


Assuntos
Anoctamina-1/genética , Bufanolídeos/farmacologia , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Proteínas de Neoplasias/genética , Animais , Anoctamina-1/metabolismo , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Bucais/genética , Proteínas de Neoplasias/metabolismo , Células Tumorais Cultivadas
17.
Int J Mol Sci ; 22(20)2021 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-34681590

RESUMO

Lung cancer has the highest rate of incidence and mortality among all cancers. Most chemotherapeutic drugs used to treat lung cancer cause serious side effects and are susceptible to drug resistance. Therefore, exploring novel therapeutic targets for lung cancer is important. In this study, we evaluated the potential of TMEM16A as a drug target for lung cancer. Homoharringtonine (HHT) was identified as a novel natural product inhibitor of TMEM16A. Patch-clamp experiments showed that HHT inhibited TMEM16A activity in a concentration-dependent manner. HHT significantly inhibited the proliferation and migration of lung cancer cells with high TMEM16A expression but did not affect the growth of normal lung cells in the absence of TMEM16A expression. In vivo experiments showed that HHT inhibited the growth of lung tumors in mice and did not reduce their body weight. Finally, the molecular mechanism through which HHT inhibits lung cancer was explored by western blotting. The findings showed that HHT has the potential to regulate TMEM16A activity both in vitro and in vivo and could be a new lead compound for the development of anti-lung-cancer drugs.


Assuntos
Anoctamina-1/antagonistas & inibidores , Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Mepesuccinato de Omacetaxina/farmacologia , Animais , Anoctamina-1/metabolismo , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/metabolismo , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Mepesuccinato de Omacetaxina/química , Mepesuccinato de Omacetaxina/metabolismo , Mepesuccinato de Omacetaxina/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Transplante Heterólogo
18.
FASEB J ; 35(10): e21897, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34473378

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of bilateral renal cysts which enlarge continuously, leading to compression of adjacent intact nephrons. The growing cysts lead to a progressive decline in renal function. Cyst growth is driven by enhanced cell proliferation and chloride secretion into the cyst lumen. Chloride secretion is believed to occur mainly by the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR), with some contribution by the calcium-activated chloride channel TMEM16A. However, our previous work suggested TMEM16A as a major factor for renal cyst formation. The contribution of CFTR to cyst formation has never been demonstrated in an adult ADPKD mouse model. We used mice with an inducible tubule-specific Pkd1 knockout, which consistently develop polycystic kidneys upon deletion of Pkd1. Cellular properties, ion currents, and cyst development in these mice were compared with that of mice carrying a co-deletion of Pkd1 and Cftr. Knockout of Cftr did not reveal any significant impact on cyst formation in the ADPKD mouse model. Furthermore, knockout of Cftr did not attenuate the largely augmented cell proliferation observed in Pkd1 knockout kidneys. Patch clamp analysis on primary renal epithelial cells lacking expression of Pkd1 indicated an only marginal contribution of CFTR to whole cell Cl- currents, which were clearly dominated by calcium-activated TMEM16A currents. In conclusion, CFTR does not essentially contribute to renal cyst formation in mice caused by deletion of Pkd1. Enhanced cell proliferation and chloride secretion is caused primarily by upregulation of the calcium-activated chloride channel TMEM16A.


Assuntos
Anoctamina-1/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Cistos/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Anoctamina-1/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Cistos/genética , Cistos/patologia , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Knockout , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/genética
19.
Eur J Pharmacol ; 910: 174491, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34506779

RESUMO

The interstitial cells of Cajal (ICCs) play an important role in coordinated gastrointestinal motility. The present study aimed to elucidate whether or how ICCs are involved in the lower esophageal sphincter (LES) relaxation induced by stimulation of the nicotinic acetylcholine receptor. The application of 1,1-dimethyl-4-phenyl-piperazinium (DMPP; a nicotinic acetylcholine receptor agonist) induced a transient relaxation in the circular smooth muscle of the porcine LES. DMPP-induced relaxation was abolished by not only 1 µM tetrodotoxin but also the inhibition of ICC activity by pretreatment with 100 µM carbenoxolone (a gap junction inhibitor), pretreatment with 100 µM CaCCinh-A01 (an anoctamin-1 blocker acting as a calcium-activated chloride channel inhibitor), and pretreatment with Cl--free solution. However, pretreatment with 100 µM Nω-nitro-L-arginine methyl ester had little effect on DMPP-induced relaxation. Furthermore, DMPP-induced relaxation was inhibited by pretreatment with 1 mM suramin, a purinergic P2 receptor antagonist, but not by 1 µM VIP (6-28), a vasoactive intestinal peptide (VIP) receptor antagonist. Stimulation of the purinergic P2 receptor with adenosine triphosphate (ATP) induced relaxation, which was abolished by the inhibition of ICC activity by pretreatment with CaCCinh-A01. In conclusion, membrane hyperpolarization of the ICCs via the activation of anoctamin-1 plays a central role in DMPP-induced relaxation. ATP may be a neurotransmitter for inhibitory enteric neurons, which stimulate the ICCs. The ICCs act as the interface of neurotransmission of nicotinic acetylcholine receptor in order to induce LES relaxation.


Assuntos
Esfíncter Esofágico Inferior/fisiologia , Células Intersticiais de Cajal/metabolismo , Relaxamento Muscular/fisiologia , Receptores Nicotínicos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Anoctamina-1/metabolismo , Esfíncter Esofágico Inferior/efeitos dos fármacos , Motilidade Gastrointestinal/efeitos dos fármacos , Células Intersticiais de Cajal/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Neurônios/metabolismo , Neurotransmissores/metabolismo , Agonistas Nicotínicos/farmacologia , Suínos
20.
Am J Physiol Cell Physiol ; 321(5): C798-C811, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34524930

RESUMO

Altered esophageal ion transport mechanisms play a key role in inflammatory and cancerous diseases of the esophagus, but epithelial ion processes have been less studied in the esophagus because of the lack of a suitable experimental model. In this study, we generated three-dimensional (3D) esophageal organoids (EOs) from two different mouse strains and characterized the ion transport processes of the EOs. EOs form a cell-filled structure with a diameter of 250-300 µm and were generated from epithelial stem cells as shown by FACS analysis. Using conventional PCR and immunostaining, the presence of Slc26a6 Cl-/HCO3- anion exchanger (AE), Na+/H+ exchanger (NHE), Na+/HCO3- cotransporter (NBC), cystic fibrosis transmembrane conductance regulator (CFTR), and anoctamin 1 Cl- channels was detected in EOs. Microfluorimetric techniques revealed high NHE, AE, and NBC activities, whereas that of CFTR was relatively low. In addition, inhibition of CFTR led to functional interactions between the major acid-base transporters and CFTR. We conclude that EOs provide a relevant and suitable model system for studying the ion transport mechanisms of esophageal epithelial cells, and they can be also used as preclinical tools to assess the effectiveness of novel therapeutic compounds in esophageal diseases associated with altered ion transport processes.


Assuntos
Células Epiteliais/metabolismo , Esôfago/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Organoides/metabolismo , Células-Tronco/metabolismo , Animais , Anoctamina-1/genética , Anoctamina-1/metabolismo , Antiporters/genética , Antiporters/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Esôfago/citologia , Feminino , Transporte de Íons , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos Endogâmicos C57BL , Organoides/citologia , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo
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