RESUMO
The receptor-binding domain (RBD) is crucial for understanding how severe acute respiratory syndrome coronavirus (SARS-CoV-2) recognizes and infects host cells. Chitooligosaccharide (CS) exhibits diverse antiviral activities, with its derivatives showing remarkable efficacy in blocking SARS-CoV-2 infection. Thus, this study employed spectroscopy, virus-infected cell experiments, and molecular simulation to investigate the molecular interactions between CS and SARS-CoV-2 RBD, as well as their mechanisms. In spectroscopic experiments, all four CS variants with different molecular weights formed interactions with the RBD. These variants increased the resistance of HEK293ACE2 cells to SARS-CoV-2 invasion. Molecular docking revealed that the four CS variants could bind to the RBD through hydrogen bonding or salt-bridge interactions, forming stable complexes. Chitotetraose provided stronger protection to HEK293ACE2 cells compared to other CS variants and displayed higher molecular docking scores. Further investigation into the optimal docking conformation of chitotetraose was conducted through molecular dynamics simulation methods. This study lays a solid theoretical foundation and provides a scientific basis for the development of targeted RBD inhibitors, as well as drug screening and application against novel coronaviruses.
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Anoctamin 1 (ANO1), a drug target for various cancers, including prostate and oral cancers, is an intracellular calcium-activated chloride ion channel that plays various physiopathological roles, especially in the induction of cancer growth and metastasis. In this study, we tested a novel compound isolated from Schisandra sphenanthera, known as schisandrathera D, for its inhibitory effect on ANO1. Schisandrathera D dose-dependently suppressed the ANO1 activation-mediated decrease in fluorescence of yellow fluorescent protein; however, it did not affect the adenosine triphosphate-induced increase in the intracellular calcium concentration or forskolin-induced cystic fibrosis transmembrane conductance regulator activity. Specifically, schisandrathera D gradually decreased the levels of ANO1 protein and significantly reduced the cell viability in ANO1-expressing cells when compared to those in ANO1-knockout cells. These effects could be attributed to the fact that schisandrathera D displayed better binding capacity to ANO1 protein than the previously known ANO1 inhibitor, Ani9. Finally, schisandrathera D increased the levels of caspase-3 and cleaved poly (ADP-ribose) polymerase 1, thereby indicating that its anticancer effect is mediated through apoptosis. Thus, this study highlights that schisandrathera D, which reduces ANO1 protein levels, has apoptosis-mediated anticancer effects in prostate and oral cancers, and thus, can be further developed into an anticancer agent.
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Anoctamin 1 (ANO1) is a calcium-activated chloride channel found in various cell types and is overexpressed in non-small cell lung cancer (NSCLC), a major cause of cancer-related mortality. With the rising interest in development of druggable compounds for NSCLC, there has been a corresponding rise in interest in ANO1, a novel drug target for NSCLC. However, as ANO1 inhibitors that have been discovered simultaneously exhibit both the functions of an inhibition of ANO1 channel as well as a reduction of ANO1 protein levels, it is unclear which of the two functions directly causes the anticancer effect. In this study, verteporfin, a chemical compound that reduces ANO1 protein levels was identified through high-throughput screening. Verteporfin did not inhibit ANO1-induced chloride secretion but reduced ANO1 protein levels in a dose-dependent manner with an IC50 value of ~300 nM. Moreover, verteporfin inhibited neither P2Y receptor-induced intracellular Ca2+ mobilization nor cystic fibrosis transmembrane conductance regulator (CFTR) channel activity, and molecular docking studies revealed that verteporfin bound to specific sites of ANO1 protein. Confirming that verteporfin reduces ANO1 protein levels, we then investigated the molecular mechanisms involved in its effect on NSCLC cells. Interestingly, verteporfin decreased ANO1 protein levels, the EGFR-STAT3 pathway as well as ANO1 mRNA expression. Verteporfin reduced the viability of ANO1-expressing cells (PC9, and gefitinib-resistant PC9) and induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage. However, it did not affect hERG channel activity. These results show that the anticancer mechanism of verteporfin is caused via the down-regulation of ANO1.
Assuntos
Anoctamina-1 , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteínas de Neoplasias , Verteporfina , Anoctamina-1/genética , Anoctamina-1/metabolismo , Cálcio/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Canais de Cloreto/metabolismo , Regulação para Baixo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Verteporfina/farmacologiaRESUMO
Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.
Assuntos
Anoctamina-1/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Dietilestilbestrol/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Anoctamina-1/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dietilestilbestrol/metabolismo , Humanos , Neoplasias Pulmonares , Proteínas de Neoplasias/metabolismo , Transdução de SinaisRESUMO
mTORopathies are a heterogeneous group of neurological disorders characterized by malformations of cortical development (MCD), enhanced cellular mechanistic target of rapamycin (mTOR) signaling, and epilepsy that results from mutations in mTOR pathway regulatory genes. Homozygous mutations (del exon 9-13) in the pseudokinase STE20-related kinase adaptor alpha (STRAD-α; STRADA), an mTOR modulator, are associated with Pretzel Syndrome (PS), a neurodevelopmental disorder within the Old Order Mennonite Community characterized by megalencephaly, intellectual disability, and intractable epilepsy. To study the cellular mechanisms of STRADA loss, we generated CRISPR-edited Strada mouse N2a cells, a germline mouse Strada knockout (KO-/-) strain, and induced pluripotent stem cell (iPSC)-derived neurons from PS individuals harboring the STRADA founder mutation. Strada KO in vitro leads to enhanced mTOR signaling and iPSC-derived neurons from PS individuals exhibit enhanced cell size and mTOR signaling activation, as well as subtle alterations in electrical firing properties e.g., increased input resistance, a more depolarized resting membrane potential, and decreased threshold for action potential (AP) generation. Strada-/- mice exhibit high rates of perinatal mortality and out of more than 100 litters yielding both WT and heterozygous pups, only eight Strada-/- animals survived past P5. Strada-/- mice are hypotonic and tremulous. Histopathological examination (n = 5 mice) revealed normal gross brain organization and lamination but all had ventriculomegaly. Ectopic neurons were seen in all five Strada-/- brains within the subcortical white matter mirroring what is observed in human PS brain tissue. These distinct experimental platforms demonstrate that STRADA modulates mTOR signaling and is a key regulator of cell size, neuronal excitability, and cortical lamination.
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BACKGROUND: The prognostic role of neutrophil-to-lymphocyte ratio (NLR) has been proposed in head and neck squamous cell carcinoma (HNSCC). However, it is currently unclear which cutoff values of NLR could consistently and independently differentiate HNSCC patients to better and worse prognosis groups. METHODS: We performed a meta-analysis of prognostic significance of pretreatment NLR values, using data extracted from 24 relevant articles. Main outcomes were overall survival (OS) and disease-free survival (DFS) in HNSCC patients. Pooled hazard ratio (HR) and 95% confidence intervals (95%CI) were calculated using the random effect model for outcomes. Impacts of NLR cutoff values across the studies were assessed with a meta-regression analysis. Results were validated using an independent data set of patients (n = 540). RESULTS: Pretreatment high NLR values above the cutoff were significantly associated with shorter OS (HR = 1.96, 95%CI = 1.66-2.31) and DFS (HR = 1.90, 95%CI = 1.41-2.54). Of note, NLR cutoffs ranging from 1.9 to 6.0 did not affect HR of OS or DFS in meta-regression analyses. In an independent cohort, any NLR cutoff between 2 and 6 produced significant HR of OS, similarly. Instead of binary cutoffs, three subgroups of NLR (< 2, 2 to 6, and ≥ 6) showed significant differences of OS in survival analyses. CONCLUSIONS: Meta-analyses confirmed that pretreatment NLR values above the cutoff were associated with shorter survival in HNSCC patients. However, the binary cutoffs of NLR values were variable across studies. Rather, pretreatment NLR values below 2 and above 6 using a three-tier classification (< 2, 2 to 6, and ≥ 6) could consistently imply better and worse prognosis in HNSCC patients, which could be readily translated to clinics.
Assuntos
Carcinoma de Células Escamosas/sangue , Neoplasias de Cabeça e Pescoço/sangue , Intervalo Livre de Doença , Humanos , Contagem de Leucócitos , Contagem de Linfócitos , Prognóstico , Análise de SobrevidaRESUMO
PURPOSE: The prognostic impact of circulating tumor cells (CTC) on disease recurrence, progression and survivals in patients with head and neck squamous cell carcinoma (HNSCC) has not been adequately described. The objective of this study was to determine the impacts of the presence of CTC on loco-regional recurrence and survival of HNSCC patients by conducting a systematic review and meta-analysis. METHODS: A comprehensive search for articles published between 1990 and 2016 was conducted and data from these studies were extracted, using the MEDLINE, Cochrane Library, and EMBASE databases. The main outcomes were overall survival (OS) and recurrence-free survival (RFS) of HNSCC patients. Pooled hazard ratio (HR) and 95% confidence intervals (95%CI) were calculated using the random effect model for outcomes. The quality of the studies, heterogeneity and publication bias were assessed with the appropriate statistical methods. RESULTS: Six eligible studies with 429 patients were identified. The presence of CTC was significantly associated shorter RFS (HR = 4.88 [95%CI: 1.93-12.35], P < 0.001). However, it could not predict patients' OS (HR = 1.92 [95%CI: 0.93-3.96], P = 0.078). The following analyses using univariable values of each study also made the similar results (HR = 1.70 [95%CI: 0.83-3.45] for OS, HR = 3.79 [95%CI: 2.02-7.13] for RFS). Heterogeneity and publication bias were not significant, except one enrolled study. CONCLUSIONS: The presence of CTC is not a significant prognostic indicator for OS of patients with HNSCC, although it could reflect the outcomes of loco-regional disease.
Assuntos
Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/patologia , Células Neoplásicas Circulantes/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Humanos , Prognóstico , Recidiva , Análise de SobrevidaRESUMO
BACKGROUND: Postoperative radiation and chemotherapy are routinely applied for microscopic residual diseases; however, treatment outcomes are not optimal, and patients frequently suffer from treatment-related toxicities. To search for an effective and less-toxic adjuvant treatment for patients with high risk of recurrence, the preventive effect of anti-programmed cell death protein 1 (PD-1) treatment was evaluated in an in vivo animal model of post-surgical tumor recurrence. MATERIALS AND METHODS: An animal model of postsurgical tumor recurrence (SCCVII tumors in C3H mice) was established by reinoculating tumor cells (105 cells) into surgical wound of primary tumor resection. Initial and recurrent tumors were compared by an immunohistochemistry and complementary DNA microarray. Using this in vivo model, tumor recurrence rates were evaluated in the animals receiving anti-PD-1 treatments. Animals were rechallenged with tumor cells, and interferon gamma secretion from spleen cells was analyzed to determine tumor-specific antitumor immunity. RESULTS: FoxP3high cell population was significantly elevated in recurrent tumors compared with that in primary tumors. Some immune response-related factors (granzyme F, neuronal leucine-rich repeat protein 1, myosin heavy chain 3, and transmembrane protein 8C) showed significant differences between primary and recurrent tumors. In this animal model, anti-PD-1 treatments significantly suppressed tumor recurrence. Importantly, tumor induction was significantly reduced when anti-PD-1-treated mice were rechallenged with tumor cells. Tumor cell-specific interferon gamma production was increased in these animals. CONCLUSIONS: Postoperative anti-PD-1 treatment significantly reduced recurrence in a cancer ablation surgical wound in an in vivo model of tumor recurrence. Our data lay the preclinical groundwork for the future clinical validation of adjuvant anti-PD-1 treatments in patients.
Assuntos
Anticorpos/uso terapêutico , Recidiva Local de Neoplasia/prevenção & controle , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos/imunologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Camundongos Endogâmicos C3H , Recidiva Local de Neoplasia/imunologia , Cuidados Pós-Operatórios , Receptor de Morte Celular Programada 1/imunologiaRESUMO
Timely generation and normal maturation of ependymal cells along the aqueduct are critical for preventing physical blockage between the third and fourth ventricles and the development of fetal non-communicating hydrocephalus. Our study identifies Yap, the downstream effector of the evolutionarily conserved Hippo pathway, as a central regulator for generating developmentally controlled ependymal cells along the ventricular lining of the aqueduct. Yap function is necessary for proper proliferation of progenitors and apical attachment of ependymal precursor cells. Importantly, an injury signal initiated by lysophosphatidic acid (LPA), an upstream regulator of Yap that can cause fetal haemorrhagic hydrocephalus, deregulates Yap in the developing aqueduct. LPA exposure leads to the loss of N-cadherin concentrations at the apical endfeet, which can be partially restored by forced Yap expression and more efficiently by phosphomimetic Yap. These results reveal a novel function of Yap in retaining tissue junctions during normal development and after fetal brain injury.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Aqueduto do Mesencéfalo/metabolismo , Epêndima/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hidrocefalia/metabolismo , Fosfoproteínas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Caderinas/metabolismo , Proteínas de Ciclo Celular , Aqueduto do Mesencéfalo/patologia , Epêndima/patologia , Doenças Fetais , Hidrocefalia/induzido quimicamente , Hidrocefalia/patologia , Imuno-Histoquímica , Lisofosfolipídeos/toxicidade , Camundongos , Fosfoproteínas/metabolismo , Proteínas de Sinalização YAPRESUMO
Through their biased localization and function within the cell, polarity complex proteins are necessary to establish the cellular asymmetry required for tissue organization. Well-characterized germinal zones, mitogenic signals and cell types make the cerebellum an excellent model for addressing the crucial function of polarity complex proteins in the generation and organization of neural tissues. Deletion of the apical polarity complex protein Pals1 in the developing cerebellum results in a remarkably undersized cerebellum with disrupted layers in poorly formed folia and strikingly reduced granule cell production. We demonstrate that Pals1 is not only essential for cerebellum organogenesis, but also for preventing premature differentiation and thus maintaining progenitor pools in cerebellar germinal zones, including cerebellar granule neuron precursors in the external granule layer. In the Pals1 mouse mutants, the expression of genes that regulate the cell cycle was diminished, correlating with the loss of the proliferating cell population of germinal zones. Furthermore, enhanced Shh signaling through activated Smo cannot overcome impaired cerebellar cell generation, arguing for an epistatic role of Pals1 in proliferation capacity. Our study identifies Pals1 as a novel intrinsic factor that regulates the generation of cerebellar cells and Pals1 deficiency as a potential inhibitor of overactive mitogenic signaling.
Assuntos
Proliferação de Células/genética , Cerebelo/embriologia , Proteínas de Membrana/metabolismo , Neurogênese/fisiologia , Núcleosídeo-Fosfato Quinase/metabolismo , Células-Tronco/citologia , Animais , Ciclo Celular/genética , Cerebelo/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neuroglia/citologia , Núcleosídeo-Fosfato Quinase/genética , Organogênese/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Receptor SmoothenedRESUMO
Tuberous sclerosis complex (TSC) is associated with neurodevelopmental abnormalities, including defects in neuronal migration. However, the alterations in cell signaling mechanisms critical for migration and final positioning of neurons in TSC remain unclear. Our detailed cellular analyses reveal that reduced Tsc2 in newborn neurons causes abnormalities in leading processes of migrating neurons, accompanied by significantly delayed migration. Importantly, we demonstrate that Reelin-Dab1 signaling is aberrantly regulated in TSC mouse models and in cortical tubers from TSC patients owing to enhanced expression of the E3 ubiquitin ligase Cul5, a known mediator of pDab1 ubiquitination. Likewise, mTORC1 activation by Rheb overexpression generates similar neuronal and Reelin-Dab1 signaling defects, and directly upregulates Cul5 expression. Inhibition of mTORC1 by rapamycin treatment or by reducing Cul5 largely restores normal leading processes and positioning of migrating neurons. Thus, disrupted Reelin-Dab1 signaling is critically involved in the neuronal migration defects of TSC.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Serina Endopeptidases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Moléculas de Adesão Celular Neuronais/genética , Movimento Celular/genética , Movimento Celular/fisiologia , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas da Matriz Extracelular/genética , Feminino , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Modelos Biológicos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Neurogênese/fisiologia , Proteína Reelina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Mucins are high molecular weight proteins that make up the major components of mucus. Hypersecretion of mucus is a feature of several chronic inflammatory airway diseases. MUC8 is an important component of airway mucus, and its gene expression is upregulated in nasal polyp epithelium. Little is known about the molecular mechanisms of MUC8 gene expression. We first observed overexpression of activator protein-2 alpha (AP2 alpha) in human nasal polyp epithelium. We hypothesized that AP2 alpha overexpression in nasal polyp epithelium correlates closely with MUC8 gene expression. We demonstrated that phorbol 12-myristate 13-acetate (PMA) treatment of the airway epithelial cell line NCI-H292 increases MUC8 gene and AP2 alpha expression. In this study, we sought to determine which signal pathway is involved in PMA-induced MUC8 gene expression. The results show that the protein kinase C and mitogen-activating protein/ERK kinase (MAPK) pathways modulate MUC8 gene expression. PD98059 or ERK1/2 siRNA and RO-31-8220 or PKC siRNA significantly suppress AP2 alpha as well as MUC8 gene expression in PMA-treated cells. To verify the role of AP2 alpha, we specifically knocked down AP2 alpha expression with siRNA. A significant AP2 alpha knock-down inhibited PMA-induced MUC8 gene expression. While dominant negative AP2 alpha decreased PMA-induced MUC8 gene expression, overexpressing wildtype AP2 alpha increased MUC8 gene expression. Furthermore, using lentiviral vectors for RNA interference in human nasal polyp epithelial cells, we confirmed an essential role for AP2 alpha in MUC8 gene expression. From these results, we concluded that PMA induces MUC8 gene expression through a mechanism involving PKC, ERK1/2, and AP2 alpha activation in human airway epithelial cells.
Assuntos
Células Epiteliais/metabolismo , Expressão Gênica , Mucinas/genética , Fator de Transcrição AP-2/genética , Adolescente , Adulto , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mucinas/metabolismo , Mucosa Nasal/metabolismo , Pólipos Nasais/genética , Pólipos Nasais/metabolismo , Interferência de RNA , Mucosa Respiratória/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-2/metabolismo , Regulação para Cima/efeitos dos fármacos , Adulto JovemRESUMO
Lysozyme is a major component of airway epithelial secretions, acts as cationic anti-microbial protein for innate immunity. Although lysozyme plays an important role in airway defense and is a key component of airway secretions under inflammatory conditions, little is understood about the regulation of its expression and the associated signaling pathway. We wanted to examine whether Phorbol 12-myristate 13-acetate (PMA), one of PKC activators, treatment of the airway epithelial cell line NCI-H292 increases lysozyme gene expression. In this study, we sought to determine which signal molecules are involved in PMA-induced lysozyme gene expression. We found that PKC and mitogen-activating protein/ERK2 kinase are essential for PMA-induced lysozyme expression and also mediate the PMA-induced activation of c-Myb protein. We identified a proximal region of the lysozyme promoter essential for promoter activity containing c-Myb transcription factor binding site. Additionally, by site-directed promoter mutagenesis, we identified that c-Myb preferred the CAA motif of the -85/-73 region of the lysozyme promoter. Finally, we showed that overexpression of c-Myb without PMA treatment increased the lysozyme promoter activity and protein expression. From these results, we conclude that PMA induces overexpression of lysozyme via ERK1/2 MAP kinase-c-Myb signaling pathways in NCI-H292 cells.
Assuntos
Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Muramidase/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Sistema Respiratório/citologia , Acetato de Tetradecanoilforbol/farmacologia , Anti-Infecciosos , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Muramidase/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de TranscriçãoRESUMO
The goal of this study was to investigate the effect of epicatechin gallate (ECG), a component of green tea polyphenols, on the signal pathway for oxidative stress-induced intracellular reactive oxygen species (ROS) generation and MUC5AC overexpression in normal human nasal epithelial (NHNE) cells. Passage-2 NHNE cells were used, and ECG was administered before stimulation with exogenous hydrogen peroxide (H(2)O(2)). MUC5AC gene and protein levels were measured by real-time PCR and dot blot analysis. Western blot analysis and immunocytofluorescence study were performed for detecting the activity of epidermal growth factor receptor (EGFR). Exogenous H(2)O(2) increases intracellular ROS generation, leading to the overexpression of MUC5AC. The phosphorylation and internalization of EGFR were associated with this ROS generation. ECG decreased the phosphorylation and internalization of EGFR at the cell surface of NHNE cells, resulting in the attenuation of exogenous H(2)O(2)-induced intracellular ROS generation and MUC5AC overexpression. ECG may be a therapeutic material against oxidative stress-induced ROS generation and mucus hypersecretion in airways.
Assuntos
Antioxidantes/farmacologia , Catequina/análogos & derivados , Receptores ErbB/metabolismo , Mucina-5AC/metabolismo , Mucosa Nasal/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Western Blotting , Catequina/farmacologia , Proliferação de Células , Células Cultivadas , Células Epiteliais/metabolismo , Receptores ErbB/genética , Imunofluorescência , Humanos , Peróxido de Hidrogênio/farmacologia , Mucina-5AC/genética , Mucosa Nasal/citologia , Mucosa Nasal/metabolismo , Oxidantes/farmacologia , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
MUC5B is a major mucin of the human respiratory tract, and it is not clear how MUC5B expression is regulated in various airway diseases. The goal of this study was to determine the mechanisms by which 17beta-estradiol induces MUC5B gene expression in airway epithelial cells. It was found that E2, a sex hormone, stimulates MUC5B gene overexpression by interaction with estrogen receptor alpha (ERalpha) and by acting through extracellular signal-regulated kinase 1/2 (ERK1/2)-mitogen-activated protein kinase (MAPK). Pretreatment with ER antagonist ICI 182,780 blocked both E2-induced ERK1/2-MAPK activation and MUC5B gene expression. It was also found that the activation of p90 ribosomal S 6 protein kinase 1 (RSK1), cAMP-response element-binding protein (CREB), and cAMP-response element (CRE) (-956 region of the MUC5B promoter)-responsive signaling cascades via ERK1/2 MAPK are crucial aspects of the intracellular mechanisms that mediate MUC5B gene expression. Taken together, these studies give additional insights into the molecular mechanism of hormone-induced MUC5B gene expression and enhance our understanding of abnormal mucin secretion in response to hormonal imbalances.
Assuntos
Células Epiteliais/metabolismo , Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mucina-5B/biossíntese , Mucosa Respiratória/metabolismo , Adulto , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Células Epiteliais/citologia , Estradiol/análogos & derivados , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Fulvestranto , Regulação da Expressão Gênica/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mucosa Respiratória/citologia , Elementos de Resposta/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases, and MUC5AC is a major airway mucin. It is well known that reactive oxygen species (ROS) may be involved in the pathogenesis of various inflammatory airway diseases. The purpose of this study was to identify which secreted mucin genes are induced by exogenous hydrogen peroxide and the mechanism by which these genes are up-regulated in normal human nasal epithelial (NHNE) cells. Exogenous H(2)O(2) induced the ligand-independent activation of epidermal growth factor receptors (EGFR) and the subsequent activation of ERK1 mitogen-activated protein kinase, resulting in the induction of intracellular ROS generation. Through this signal pathway, exogenous H(2)O(2) markedly induced overexpression of the MUC5AC gene alone. In addition, Nox4, a subtype of nonphagocytic NADPH oxidase, was found to play a key role in intracellular ROS generation and exogenous H(2)O(2)-induced MUC5AC gene expression in NHNE cells.
Assuntos
Células Epiteliais/metabolismo , Mucinas/metabolismo , NADPH Oxidases/metabolismo , Mucosa Nasal/metabolismo , Estresse Oxidativo , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Receptores ErbB/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Caliciformes/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Ligantes , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mucina-5AC , Mucinas/genética , NADPH Oxidase 4 , NADPH Oxidases/genética , Nariz/efeitos dos fármacos , Fosforilação , Espécies Reativas de Oxigênio/metabolismoRESUMO
It has been reported that the proinflammatory cytokine interleukin-1beta (IL-1beta) induces mucus hypersecretion in normal human nasal epithelial (NHNE) cells and that the MAP kinase pathway may be an important signal pathway in IL-1beta-induced MUC5AC gene expression. Green tea (Camellia sinensis) polyphenols are potent anti-inflammatory agents and have been shown to inhibit inflammation in tumor cell lines and cultured respiratory epithelial cells. In this study, we examined the effect of (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, on IL-1beta-induced MUC5AC gene expression and secretion in NHNE cells. After cells had been treated with IL-1beta (10 ng/ml) and pretreated with EGCG (10, 50 and 100 microM), mRNA expression of MUC5AC was determined by real-time polymerase chain reaction. The suppression of each signal pathway protein was determined by Western blot analysis after treatment with IL-1beta and EGCG, respectively. IL-1beta increased MUC5AC gene expression and MUC5AC secretion. EGCG markedly suppressed IL-1beta-induced MUC5AC gene expression and MUC5AC secretion via suppression of the phosphorylation of ERK MAP kinase, MSK1, and transcription factor, cAMP response element-binding protein. IL-1beta increased the number of cells staining positive with MUC5AC antibodies, and EGCG treatment decreased this number. Our data suggest that EGCG may be an effective inhibitor of IL-1beta-induced mucus hypersecretion.
Assuntos
Catequina/análogos & derivados , Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/antagonistas & inibidores , Mucinas/genética , Mucinas/metabolismo , Mucosa Nasal/efeitos dos fármacos , Catequina/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , Neoplasias Pulmonares , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mucina-5AC , Mucosa Nasal/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The proper homeostasis of the liquid lining the surface of the middle ear cavity is vitally important for maintaining a fluid-free middle ear cavity. Disruption of this homeostasis leads to fluid collection in the middle ear cavity and results in otitis media with effusion. We demonstrated the molecular and functional expression of the Na+/H+ exchanger (NHE)s in normal human middle ear epithelial (NHMEE) cells. We also evaluated the role of NHEs in fluid absorption and the effect of dexamethasone on NHE function and NHE-dependent fluid absorption in NHMEE cells. Western blot analysis was performed for NHE1, -2, and -3 in NHMEE cells. The fluid absorption rate was measured after liquid application on the luminal surface of the cells. Intracellular pH (pHi) was measured using the pH-sensitive fluorescent probe bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM. NHE activity was determined as Na+-induced pHi recovery from an acid load achieved by luminal exposure to 40 mmol/l NH4Cl. NHE1, -2 and -3 were all expressed in the NHMEE cells. The pHi recovery rate was suppressed by inhibition of NHE2 and -3 with HOE694 at concentrations greater than 50 microM. Inhibition of NHE3 with 650 microM of HOE694 or S3226 significantly decreased the fluid absorption rate. Dexamethasone increased the Na+-induced pHi recovery rate which was reversed by the inhibition of NHE3 with 650 microM of HOE694. Dexamethasone treatment up-regulated NHE3 expression in a dose-dependent manner. The fluid absorption rate was increased by treatment with dexamethasone (10(-7) M) and reversed by the inhibition of NHE3. In summary, we have shown that NHE3 are involved in the regulation of both pHi and fluid absorption on the luminal surface of NHMEE cells. Dexamethasone stimulates NHE3 expression and NHE3-dependent fluid absorption in NHMEE cells. These findings provide a new insight into mechanisms that regulate periciliary fluid and the therapeutic mechanisms behind steroid treatment of otitis media with effusion.
Assuntos
Dexametasona/farmacologia , Células Epiteliais/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/fisiologia , Absorção/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Western Blotting , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Orelha Média/citologia , Células Epiteliais/metabolismo , Deslocamentos de Líquidos Corporais/efeitos dos fármacos , Guanidinas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Metacrilatos/farmacologia , Sódio/farmacocinética , Trocador 1 de Sódio-Hidrogênio , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonas/farmacologiaRESUMO
MUC8 gene expression is overexpressed in nasal polyp epithelium and is also increased by treatment with inflammatory mediators in nasal epithelial cells. These data suggest that MUC8 may be one of important mucin genes expressed in human airway. However, the mechanisms of various inflammatory mediator-induced MUC8 gene expression in normal nasal epithelial cells remain unclear. We examined the mechanism by which prostaglandin E(2) (PGE2), an arachidonic acid metabolite, increases MUC8 gene expression levels. Here, we show that ERK mitogen-activated protein kinase is essential for PGE2-induced MUC8 gene expression in normal human nasal epithelial cells and that p90 ribosomal S 6 protein kinase 1 (RSK1) mediates the PGE2-induced phosphorylation of cAMP-response element binding protein. Our results also indicate that cAMP-response element at the -803 region of the MUC8 promoter is an important site of PGE2-induced MUC8 gene expression. In conclusion, this study gives insights into the molecular mechanism of PGE2-induced MUC8 gene expression in human airway epithelial cells.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mucinas/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases , Nariz/citologia , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologiaRESUMO
Various genetic and environmental factors appear to be involved in systemic lupus erythematosus (SLE). Epstein-Barr virus (EBV) is among the environmental factors that are suspected of predisposing to SLE, based on the characteristics of EBV itself and on sequence homologies between autoantigens and EBV antigens. In addition, higher titers of anti-EBV antibodies and increased EBV seroconversion rates have been observed in SLE patients as compared with healthy control individuals. Serologic responses do not directly reflect EBV status within the body. Clarification of the precise status of EBV infection in SLE patients would help to improve our understanding of the role played by EBV in this disease. In the present study we determined EBV types in SLE patients (n = 66) and normal control individual (n = 63) by direct PCR analysis of mouthwash samples. We also compared EBV load in blood between SLE patients (n = 24) and healthy control individuals (n = 29) using semiquantitative PCR assay. The number of infections and EBV type distribution were similar between adult SLE patients and healthy control individuals (98.5% versus 94%). Interestingly, the EBV burden in peripheral blood mononuclear cells (PBMCs) was over 15-fold greater in SLE patients than in healthy control individuals (mean +/- standard deviation: 463 +/- 570 EBV genome copies/3 microg PBMC DNA versus 30 +/- 29 EBV genome copies/3 microg PBMC DNA; P = 0.001), suggesting that EBV infection is abnormally regulated in SLE. The abnormally increased proportion of EBV-infected B cells in the SLE patients may contribute to enhanced autoantibody production in this disease.