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1.
Anticancer Res ; 40(1): 109-119, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31892559

RESUMO

BACKGROUND/AIM: Although molecular targeting therapy is an attractive treatment for cancer, resistance eventually develops in most cases. Here, we evaluated chemotherapeutic efficacy on non-small cell lung cancer (NSCLC) with acquired resistance to epidermal growth factor receptor inhibitors mechanistically. MATERIALS AND METHODS: Antitumor effects of taxotere were evaluated using multiple models, including xenograft, and patient-derived models developed from adenocarcinoma cancer patients. Protein expressions were analyzed after drug treatment. RESULTS: Taxotere inhibited tumor growth of NSCLC cells harboring drug resistance, and reduced the expression of phosphorylated MET proto-oncogene, receptor tyrosine kinase (MET). A tumor-inhibitory effect of taxotere was also demonstrated in vivo in xenografts in mice, patient-derived primary lung tumor cells and patient-derived xenograft with concomitant repression of phosphorylated MET expression. Chemotherapeutic and MET-targeting drug exhibited a synergistic cell growth-inhibitory effect. CONCLUSION: These results suggest that the anticancer drug taxane may be an adjuvant for lung tumors exhibiting enhanced signaling of MET networks.


Assuntos
Antineoplásicos/farmacologia , Docetaxel/farmacologia , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Respir Physiol Neurobiol ; 271: 103292, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31542455

RESUMO

The interaction between chronic inflammation and neural dysfunction points to a link between the nervous and immune systems in the airways. In particular, environmental exposure to nanoparticles (NPs), defined as particulate matter having one dimension <100 nm, is associated with an enhanced risk of childhood and adult asthma. However, the impact of NPs on the neural response in asthma remains to be determined. This study determined the impact of NPs on neuroinflammation in a mouse model of allergic asthma. Ovalbumin (OVA) sensitized mice were treated with saline (Sham), OVA challenged and exposed to 200 µg/m3 NPs 1 h a day for 3 days on days 21-23 in a closed-system chamber attached to a ultrasonic nebulizer. The effect of NPs on the levels of neuropeptides, transient receptor potential vanilloid 1 (TRPV1), TRPV4, P2 × 4, and P2 × 7 was assessed by enzyme-linked immunosorbent assays, immunoblotting, and immunohistochemistry. NP exposure increased airway inflammation and responsiveness in OVA mice, and these increases were augmented in OVA plus NP-exposed mice. The lung tissue levels of TRPV1, TRPV4, P2 × 4, and P2 × 7 were increased in OVA mice, and these increases were augmented in OVA plus NP-exposed mice. The substance P, adenosine triphosphate (ATP), and calcitonin gene-related peptide (CGRP) levels in bronchoalveolar lavage fluid were increased in OVA mice, and these increases were augmented in OVA plus NP-exposed mice. Bradykinin, ATP, and CGRP were dose dependently increased in NP-exposed normal human bronchial epithelial (NHBE) cells. The calcium concentration was increased in NHBE cells exposed to NPs for 8 h. These results indicate that neuroinflammation can be involved in the pathogenesis of bronchial asthma and that NPs can exacerbate asthma via neuromediator release.

3.
Chem Res Toxicol ; 32(1): 139-145, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30608172

RESUMO

Acrolein, an α/ß-unsaturated aldehyde, is volatile at room temperature. It is a respiratory irritant found in environmental tobacco smoke, which can be generated during cooking or endogenously at sites of injury. An acute high concentration of uncontrolled irritant exposure can lead to an asthma-like syndrome known as reactive airways dysfunction syndrome (RADS). However, whether acrolein can induce RADS remains poorly understood. The aim of study is to develop a RADS model of acrolein inhalation in mice and to clarify the mechanism of RADS. Mice were treated with ovalbumin (OVA) and exposed to acrolein (5 ppm/10 min). Airway hyper-responsiveness (AHR) was measured on days 24 and 56, and samples were collected on days 25 and 57. Tight junction protein, antioxidant-associated protein, and vascular endothelial growth factor (VEGF) levels were estimated by Western blotting and immunohistochemical staining. Reactive oxygen species (ROS) was calculated using enzyme linked immunosorbent assays. Acrolein or OVA groups exhibited an increase in airway inflammatory cells and AHR compared to a sham group. These effects were further increased in mice in the OVA + acrolein exposure group than in the OVA exposure group and persisted in the acrolein exposure group for 8 weeks. CLDNs, carbonyls, VEGF, Nrf2, and Keap1 were observed in the lungs. Our data demonstrate that acrolein induces RADS and that ROS, angiogenesis, and tight junction proteins are involved in RADS in a mouse model.


Assuntos
Acroleína/efeitos adversos , Alérgenos/efeitos adversos , Asma Ocupacional/induzido quimicamente , Exposição Ambiental/efeitos adversos , Ovalbumina/efeitos adversos , Hipersensibilidade Respiratória/induzido quimicamente , Acroleína/administração & dosagem , Administração por Inalação , Alérgenos/administração & dosagem , Animais , Asma Ocupacional/diagnóstico , Claudinas/análise , Claudinas/metabolismo , Feminino , Proteína 1 Associada a ECH Semelhante a Kelch/análise , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/análise , Fator 2 Relacionado a NF-E2/metabolismo , Ovalbumina/administração & dosagem , Hipersensibilidade Respiratória/diagnóstico , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
Lung ; 196(6): 681-689, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30182154

RESUMO

PURPOSE: Annexin A5 (ANXA5) has a potential role in cellular signal transduction, inflammation, and fibrosis. However, the exact role of ANXA5 in asthma remains to be clarified. The aims of the present study were to investigate ANXA5 protein expression in a mouse model of asthma and pollutant exposure and to elucidate the relationships between clinical variables and plasma ANXA5 levels in patients with asthma. METHODS: A murine model of asthma induced by ovalbumin (OVA) and titanium dioxide (TiO2) nanoparticles has been established using BALB/c mice, and we examined ANXA5 expression and lung fibrosis using this model. Moreover, we also compared ANXA5 plasma levels in patients with controlled vs. exacerbated asthma. RESULTS: ANXA5 protein levels were lower in lung tissue from OVA + OVA mice than in control mice. Lung ANXA5, connective tissue growth factor (CTGF), and transforming growth factor ß1 (TGF-ß1) protein levels were higher in OVA + TiO2-exposed mice than in control or OVA + OVA mice. Although Dermatophagoides pteronyssinus (Derp1) treatment increased lung ANXA5 protein levels in MRC-5 cells and A549 epithelial cells, it decreased lung ANXA5 levels in NHBE cells. Treatment with TiO2 nanoparticles increased lung ANXA5, CTGF, and TGF-ß1 protein levels in MRC-5 cells, A549 epithelial cells, and NHBE cells. Plasma ANXA5 levels were lower in asthmatic patients than in healthy controls, and they were significantly enriched in patients with exacerbated asthma compared with those with controlled asthma (P < 0.05). ANXA5 levels were correlated with pulmonary function as assessed by spirometry. CONCLUSION: Our results imply that ANXA5 plays a potential role in asthma pathogenesis and may be a promising marker for exacerbated bronchial asthma and exposure to air pollutants.


Assuntos
Anexina A5/metabolismo , Antígenos de Dermatophagoides/farmacologia , Asma/diagnóstico , Asma/fisiopatologia , Células A549/metabolismo , Idoso , Poluentes Atmosféricos/efeitos adversos , Animais , Asma/etiologia , Asma/patologia , Biomarcadores/sangue , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Dermatophagoides pteronyssinus , Modelos Animais de Doenças , Progressão da Doença , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Nanopartículas/efeitos adversos , Ovalbumina , Fibrose Pulmonar/patologia , Titânio/efeitos adversos , Fator de Crescimento Transformador beta1/metabolismo , Capacidade Vital
5.
Allergy Asthma Immunol Res ; 10(5): 533-542, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30088372

RESUMO

PURPOSE: The tight junction protein claudin-5 (CLDN5) is critical to the control of endothelial cellular polarity and pericellular permeability. The role of CLDN5 in chronic obstructive pulmonary disease (COPD) remains unclear. The aim of this study was to investigate the association between CLDN5 levels and clinical variables in patients with COPD. METHODS: In total, 30 patients with COPD and 30 healthy controls were enrolled in the study. The plasma CLDN5 level was checked in patients with stable or exacerbated COPD and in healthy controls. RESULTS: The mean plasma CLDN5 level of patients with COPD was 0.63 ± 0.05 ng/mL and that of healthy controls was 6.9 ± 0.78 ng/mL (P = 0.001). The mean plasma CLDN5 level was 0.71 ± 0.05 ng/mL in exacerbated COPD patients and 0.63 ± 0.04 ng/mL in patients with stable COPD (P < 0.05). The plasma CLDN5 level among COPD subjects was correlated with the smoking amount (r = -0.530, P = 0.001). The plasma CLDN5 level in stable COPD patients was correlated with forced expiratory volume in one second (FEV1, %pred.) (r = -0.481, P = 0.037). CONCLUSIONS: The plasma CLDN5 level was not correlated with age. CLDN5 may be involved in the pathogenesis of COPD. Further studies having a larger sample size will be needed to clarify CLDN5 in COPD.

6.
Environ Toxicol ; 33(7): 798-806, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29673049

RESUMO

Claudins (CLDNs) are a major transmembrane protein component of tight junctions (TJs) in endothelia and epithelia. CLDNs are not only essential for sustaining the role of TJs in cell permeability but are also vital for cell signaling through protein-protein interactions. Ozone induces oxidative stress and lung inflammation in humans and experimental models, but the impact of ozone on claudins remains poorly understood. This study was to determine the expression of TJ proteins, such as claudin 3, 4, 5, and 14 following ozone exposure. Mice were exposed to 0.1, 1, or 2 ppm of ozone or ambient air for 6 h for 3 days. The impact of ozone on CLDNs, Nrf2, Keap1, and reactive oxygen species (ROS) were estimated using immunoblotting, immunohistochemical staining, confocal imaging, and ELISA analysis in mice and bronchial epithelial cells. Mice exposed to ozone experienced increased airway inflammatory cell infiltration and bronchial hyper-responsiveness compared to control mice. Additionally, CLDN3, CLDN4, ROS, Nrf2, and Keap1 protein expression increased, and lung CLDN14 protein expression decreased, in mice exposed to ozone compared with control mice. These results indicate that CLDNs are involved in airway inflammation following ozone exposure, suggesting that ozone affects TJ proteins through oxidative mechanisms.


Assuntos
Claudinas/metabolismo , Pulmão/efeitos dos fármacos , Ozônio/toxicidade , Junções Íntimas/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Citocinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Korean J Intern Med ; 33(4): 807-814, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29334723

RESUMO

Background/Aims: The methacholine bronchial provocation test (MBPT) is used to detect and quantify airway hyper-responsiveness (AHR). Since improvements in the severity of asthma are associated with improvements in AHR, clinical studies of asthma therapies routinely use the change of airway responsiveness as an objective outcome. The aim of this study was to assess the relationship between serial MBPT and clinical profiles in patients with asthma. METHODS: A total of 323 asthma patients were included in this study. The MBPT was performed on all patients beginning at their initial diagnosis until asthma was considered controlled based on the Global Initiative for Asthma guidelines. A responder was defined by a decrease in AHR while all other patients were considered non-responders. RESULTS: A total of 213 patients (66%) were responders, while 110 patients (34%) were non-responders. The responder group had a lower initial PC20 (provocative concentration of methacholine required to decrease the forced expiratory volume in 1 second by 20%) and longer duration compared to the non-responder group. Members of the responder group also had superior qualities of life, compared to members of the non-responder group. Whole blood cell counts were not related to differences in PC20; however, eosinophil concentration was. No differences in sex, age, body mass index, smoking history, serum immunoglobulin E, or frequency of acute exacerbation were observed between responders and non-responders. Conclusions: The initial PC20, the duration of asthma, eosinophil concentrations, and quality-of-life may be useful variables to identify improvements in AHR in asthma patients.


Assuntos
Asma , Testes de Provocação Brônquica , Asma/diagnóstico , Hiper-Reatividade Brônquica , Broncoconstritores , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Cloreto de Metacolina , Pessoa de Meia-Idade , Valores de Referência , Estudos Retrospectivos
8.
BMC Pulm Med ; 18(1): 1, 2018 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-29301525

RESUMO

BACKGROUND: Annexin-A1 (ANXA1) is a glucocorticoid-induced protein with multiple actions in the regulation of inflammatory cell activation. The anti-inflammatory protein ANXA1 and its N-formyl peptide receptor 2 (FPR2) have protective effects on organ fibrosis. However, the exact role of ANXA1 in asthma remains to be determined. The aim of this study was to identify the role of ANXA1 in bronchial asthma. METHODS: In mice sensitized and challenged with ovalbumin (OVA-OVA mice) and mice sensitized with saline and challenged with air (control mice), we investigated the potential links between ANXA1 levels and bronchial asthma using ELISA, immunoblotting, and immunohistochemical staining. Moreover, we also determined ANXA1 levels in blood from 50 asthmatic patients (stable and exacerbated states). RESULTS: ANXA1 protein levels in lung tissue and bronchoalveolar lavage fluid were significantly higher in OVA-OVA mice compared with control mice. FPR2 protein levels in lung tissue were significantly higher in OVA-OVA mice compared with control mice. Plasma ANXA1 levels were increased in asthmatic patients compared with healthy controls. Plasma ANXA1 levels were significantly lower in exacerbated patients compared with stable patients with bronchial asthma (p < 0.05). The plasma ANXA1 levels in controlled asthmatic patients were correlated with forced expiratory volume in 1 s (FEV1) (r = - 0.191, p = 0.033) and FEV1/forced vital capacity (FVC) (r = -0.202, p = 0.024). CONCLUSION: These results suggest that ANXA1 may be a potential marker and therapeutic target for asthma.


Assuntos
Anexina A1/sangue , Asma/sangue , Pulmão/fisiopatologia , Adulto , Idoso , Animais , Anexina A1/análise , Asma/induzido quimicamente , Asma/fisiopatologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Estudos de Casos e Controles , Feminino , Volume Expiratório Forçado , Humanos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Ovalbumina , Exacerbação dos Sintomas , Capacidade Vital
9.
Proteome Sci ; 16: 2, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29375273

RESUMO

Background: Acrolein (allyl Aldehyde) as one of smoke irritant exacerbates chronic airway diseases and increased in sputum of patients with asthma and chronic obstructive lung disease. But underlying mechanism remains unresolved. The aim of study was to identify protein expression in human lung microvascular endothelial cells (HMVEC-L) exposed to acrolein. Methods: A proteomic approach was used to determine the different expression of proteins at 8 h and 24 h after treatment of acrolein 30 nM and 300 nM to HMVEC-L. Treatment of HMVEC-L with acrolein 30 nM and 300 nM altered 21 protein spots on the two-dimensional gel, and these were then analyzed by MALDI-TOF MS. Results: These proteins included antioxidant, signal transduction, cytoskeleton, protein transduction, catalytic reduction. The proteins were classified into four groups according to the time course of their expression patterns such as continually increasing, transient increasing, transient decreasing, and continually decreasing. For validation immunohistochemical staining and Western blotting was performed on lung tissues from acrolein exposed mice. Moesin was expressed in endothelium, epithelium, and inflammatory cells and increased in lung tissues of acrolein exposed mice compared with sham treated mice. Conclusions: These results indicate that some of proteins may be an important role for airway disease exacerbation caused by acrolein exposure.

10.
Allergy Asthma Immunol Res ; 10(1): 25-33, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29178675

RESUMO

PURPOSE: Claudin-4 has been reported to function as a paracellular sodium barrier and is one of the 3 major claudins expressed in lung alveolar epithelial cells. However, the possible role of claudin-4 in bronchial asthma has not yet been fully studied. In this study, we aimed to elucidate the role of claudin-4 in the pathogenesis of bronchial asthma. METHODS: We determined claudin-4 levels in blood from asthmatic patients. Moreover, using mice sensitized and challenged with OVA, as well as sensitized and challenged with saline, we investigated whether claudin-4 is involved in the pathogenesis of bronchial asthma. Der p1 induced the inflammatory cytokines in NHBE cells. RESULTS: We found that claudin-4 in blood from asthmatic patients was increased compared with that from healthy control subjects. Plasma claudin-4 levels were significantly higher in exacerbated patients than in control patients with bronchial asthma. The plasma claudin-4 level was correlated with eosinophils, total IgE, FEV1% pred, and FEV1/FVC. Moreover, lung tissues from the OVA-OVA mice showed significant increases in transcripts and proteins of claudin-4 as well as in TJ breaks and the densities of claudin-4 staining. When claudin-4 was knocked down by transfecting its siRNA, inflammatory cytokine expressions, which were induced by Der p1 treatment, were significantly increased. CONCLUSIONS: These findings thus raise the possibility that regulation of lung epithelial barrier proteins may constitute a therapeutic approach for asthma.

11.
Acta Neurobiol Exp (Wars) ; 77(1): 18-30, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28379213

RESUMO

Obesity is associated with consumption of energy-dense diets and development of systemic inflammation. Gut microbiota play a role in energy harvest and inflammation and can influence the change from lean to obese phenotypes. The nucleus of the solitary tract (NTS) is a brain target for gastrointestinal signals modulating satiety and alterations in gut-brain vagal pathway may promote overeating and obesity. Therefore, we tested the hypothesis that high-fat diet­induced changes in gut microbiota alter vagal gut-brain communication associated with increased body fat accumulation. Sprague-Dawley rats consumed a low energy­dense rodent diet (LFD; 3.1 kcal/g) or high energy­dense diet (HFD, 5.24 kcal/g). Minocycline was used to manipulate gut microbiota composition. 16S Sequencing was used to determine microbiota composition. Immunofluorescence against IB4 and Iba1 was used to determine NTS reorganization and microglia activation. Nodose ganglia from LFD rats were isolated and co-cultured with different bacteria strains to determine neurotoxicity. HFD altered gut microbiota with increases in Firmicutes/Bacteriodetes ratio and in pro-inflammatory Proteobacteria proliferation. HFD triggered reorganization of vagal afferents and microglia activation in the NTS, associated with weight gain. Minocycline-treated HFD rats exhibited microbiota profile comparable to LFD animals. Minocycline suppressed HFD­induced reorganization of vagal afferents and microglia activation in the NTS, and reduced body fat accumulation. Proteobacteria isolated from cecum of HFD rats were toxic to vagal afferent neurons in culture. Our findings show that diet­induced shift in gut microbiome may disrupt vagal gut­brain communication resulting in microglia activation and increased body fat accumulation.


Assuntos
Tecido Adiposo/metabolismo , Dieta Hiperlipídica , Microbioma Gastrointestinal/fisiologia , Núcleo Solitário/fisiologia , Nervo Vago/fisiologia , Vias Aferentes/fisiologia , Animais , Antibacterianos/farmacologia , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Fezes/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/genética , Bactérias Gram-Negativas/isolamento & purificação , Lectinas/metabolismo , Lipopolissacarídeos/sangue , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Minociclina/farmacologia , Gânglio Nodoso/metabolismo , Gânglio Nodoso/microbiologia , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Ratos , Ratos Sprague-Dawley , Núcleo Solitário/efeitos dos fármacos , Núcleo Solitário/metabolismo , Fatores de Tempo , Nervo Vago/efeitos dos fármacos
12.
Allergy Asthma Immunol Res ; 9(3): 257-264, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28293932

RESUMO

PURPOSE: Nanoparticles (NPs) may cause cell and tissue damage, leading to local and systemic inflammatory responses and adverse effects on health due to the inhalation of particulate matter. The inflammasome is a major regulator of inflammation through its activation of pro-caspase-1, which cleaves pro-interleukin-1ß (pro-IL-1ß) into its mature form and may induce acute and chronic immune responses to NPs. However, little is known about the response of the inflammasome to NP exposure via the airways in asthma. The aim of this study was to identify the impact of titanium dioxide (TiO2) NPs on inflammasome in a mouse model of allergic asthma. METHODS: Mice were treated with ovalbumin (OVA) or TiO2 NPs. IL-1ß, IL-18, NAIP, CIITA, HET-E, TP-2 (NACHT), leucine-rich repeat (LRR), pyrin domain-containing protein 3 (NLRP3), and caspase-1 were assessed by Western blotting. Caspase-1 was assessed by immunohistochemistry (IHC). Levels of reactive oxygen species (ROS)-as markers of oxidative damage-and the mediators 8-isoprostane and carbonyl were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Airway hyperresponsiveness (AHR) and inflammation were increased in OVA-sensitized/challenged mice, and these responses were exacerbated by exposure to TiO2 NPs. NP treatment increased IL-1ß and IL-18 expression in OVA-sensitized/challenged mice. NPs augmented the expression of NLRP3 and caspase-1, leading to production of active caspase-1 in the lung. Caspase-1 expression was increased and exacerbated by TiO2 NP exposure in OVA-sensitized/challenged mice. ROS levels tended to be increased in OVA-sensitized/challenged and OVA-sensitized/challenged-plus-TiO2 NP-exposed mice. CONCLUSIONS: Our data demonstrated that inflammasome activation occured in asthmatic lungs following NP exposure, suggesting that targeting the inflammasome may assist in controling NP-induced airway inflammation and hyperresponsiveness.

13.
Am J Respir Cell Mol Biol ; 55(2): 170-5, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27163839

RESUMO

Animal model systems are invaluable for examining human diseases. Our laboratory recently established a mouse model of nasal polyps (NPs) and investigated similarities and differences between this mouse model and human NPs. We especially focus on the hypothesis that B cell activation occurs during NP generation in the murine model. After induction of ovalbumin-induced allergic rhinosinusitis, 6% ovalbumin and Staphylococcus aureus enterotoxin B (10 ng) were instilled into the nasal cavity of mice three times per week for 8 weeks. The development of structures that somewhat resemble NPs (which we will refer to as NPs) was confirmed by hematoxylin and eosin staining. The mRNA and protein levels of various inflammatory cell markers and mediators were measured by real-time PCR in nasal tissue and by ELISA in nasal lavage fluid (NLF), respectively. Total Ig isotype levels in NLF were also quantitated using the Mouse Ig Isotyping Multiplex kit (EMD Millipore, Billerica, MA) on a Luminex 200 instrument (Life Technologies, Grand Island, NY). Similar to human NPs, there were significant increases in gene expression of inflammatory cell markers, such as CD19, CD138, CD11c, and mast cell protease-6 in nasal tissue samples of the NP group compared with those of the control group. In further investigations of B cell activation, mRNA expressions of B cell activating factor and a proliferation-inducing ligand were found to be significantly increased in mouse NP tissue. B cell-activating factor protein concentration and IgA and IgG1 levels in NLF were significantly higher in the NP group compared with the control group. In this study, the NP mouse model demonstrated enhanced B cell responses, which are reminiscent of B cell responses in human NPs.


Assuntos
Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Pólipos Nasais/imunologia , Pólipos Nasais/patologia , Animais , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Biomarcadores/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Inflamação/patologia , Camundongos Endogâmicos BALB C , Líquido da Lavagem Nasal , Pólipos Nasais/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Oncotarget ; 6(35): 37257-68, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26484565

RESUMO

Wnt2 is implicated in various human cancers. However, it remains unknown how Wnt2 is upregulated in human cancer and contributes to tumorigenesis. Here we found that Wnt2 is highly expressed in colorectal cancer (CRC) cells. In addition to co-expression of Wnt2 with Wnt/ß-catenin target genes in CRC, knockdown or knockout of Wnt2 significantly downregulates Wnt/ß-catenin target gene expression in CRC cells. Importantly, depletion or ablation of endogenous Wnt2 inhibits CRC cell proliferation. Similarly, neutralizing secreted Wnt2 reduces Wnt target gene expression and suppresses CRC cell proliferation. Conversely, Wnt2 increases cell proliferation of intestinal epithelial cells. Intriguingly, WNT2 expression is transcriptionally silenced by EZH2-mediated H3K27me3 histone modification in non-CRC cells, However, WNT2 expression is de-repressed by the loss of PRC2's promoter occupancy in CRC cells. Our results reveal the unexpected roles of Wnt2 in complementing Wnt/ß-catenin signaling for CRC cell proliferation.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias Colorretais/metabolismo , Mucosa Intestinal/metabolismo , Via de Sinalização Wnt , Proteína Wnt2/metabolismo , beta Catenina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Comunicação Autócrina , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Histonas/metabolismo , Humanos , Mucosa Intestinal/patologia , Metilação , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , Fatores de Tempo , Transcrição Genética , Transfecção , Proteína Wnt2/genética , beta Catenina/genética
15.
Mol Cancer Ther ; 14(11): 2613-22, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26351320

RESUMO

The MET receptor tyrosine kinase, the receptor for hepatocyte growth factor (HGF), has been implicated in cancer growth, invasion, migration, angiogenesis, and metastasis in a broad variety of human cancers, including human hepatocellular carcinoma (HCC). Recently, MET was suggested to be a potential target for the personalized treatment of HCC with an active HGF-MET signaling pathway. However, the mechanisms of resistance to MET inhibitors need to be elucidated to provide effective treatment. Here, we show that HCC cells exhibit different sensitivities to the MET inhibitor PHA665752, depending on the phosphorylation status of FGFR. Treatment of cells expressing both phospho-FGFR and phospho-MET with the inhibitor PHA665752 did not cause growth inhibition and cell death, whereas treatment with AZD4547, a pan-FGFR inhibitor, resulted in decreased colony formation and cleavage of caspase-3. Moreover, silencing of endogenous FGFR1 and FGFR2 by RNAi of HCC cells expressing phospho-FGFR, phospho-FGFR2, and phospho-MET overcame the resistance to PHA665752 treatment. Treatment of primary cancer cells from patients with HCC expressing both phospho-FGFR and phospho-MET with PHA665752 did not induce cell death, whereas AZD4547 treatment induced cell death through the cleavage of caspase-3. In addition, treatment of cells resistant to PHA665752 with AZD4547 abrogated the activation of downstream effectors of cell growth, proliferation, and survival. On the basis of these results, we conclude that the FGFR pathway is critical for HCC survival, and that targeting this pathway with AZD4547 may be beneficial for the treatment of patients with HCC-expressing phospho-FGFR and phospho-MET.


Assuntos
Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Indóis/farmacologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Microscopia Confocal , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Pirazóis/farmacologia , Interferência de RNA , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sulfonas/farmacologia
16.
Oncotarget ; 4(12): 2523-31, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24344114

RESUMO

K-Ras mutation is detected in over 30% of human malignancies. In particular, 90% of human pancreatic cancers are initiated by K-Ras mutation. Thus, selective elimination of K-Ras mutated cells would be a plausible strategy to prevent or cure the malignancies. In our previous reports, it has been revealed that oncogenic K-Ras promotes the exocytosis of p53 with Snail. In this study, we have followed the final destination of extracellular p53, which is secreted by the Snail complex. Here we provide evidences that p53, exported from K-Ras-mutated cells, is specifically re-endocytosed by oncogenic K-Ras-containing cancer cells. The p53 DNA-binding domain directly associates with caveolin-1 and enters K-Ras mutated cells through early endosome-mediated endocytosis. Using a serial deletion approach, we revealed that a fragment of human p53 extending from 93-143 amino acids (AA) is responsible for binding with caveolin-1 and for endocytosis. In contrast, p53-Snail binding occurs at the 143-193 aa region. Finally, through in vivo study, we confirmed that injected recombinant p53 could be up-taken by tumor tissues, constructed by oncogenic K-Ras transformed MEF cells. In contrast, the tumors formed by H-Ras mutated MEF cells did not accumulate the injected p53 protein. These results indicate that the p53 fragment might be useful as a specific delivery tool into K- Ras mutated cells as well as a diagnostic method.


Assuntos
Caveolina 1/metabolismo , Genes ras , Mutação , Neoplasias Pancreáticas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Caveolina 1/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Endossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/genética , Proteína Supressora de Tumor p53/genética
17.
Cell Cycle ; 12(14): 2277-90, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24067370

RESUMO

Renal cell carcinomas (RCCs) are frequently occurring genitourinary malignancies in the aged population. A morphological characteristic of RCCs is an irregular nuclear shape, which is used to index cancer grades. Other features of RCCs include the genetic inactivation of the von Hippel-Lindau gene, VHL, and p53 genetic-independent inactivation. An aberrant nuclear shape or p53 suppression has not yet been demonstrated. We examined the effect of progerin (an altered splicing product of the LMNA gene linked to Hutchinson Gilford progeria syndrome; HGPS) on the nuclear deformation of RCCs in comparison to that of HGPS cells. In this study, we showed that progerin was suppressed by pVHL and was responsible for nuclear irregularities as well as p53 inactivation. Thus, progerin suppression can ameliorate nuclear abnormalities and reactivate p53 in response to genotoxic addition. Furthermore, we found that progerin was a target of pVHL E3 ligase and suppressed p53 activity by p14/ARF inhibition. Our findings indicate that the elevated expression of progerin in RCCs results from the loss of pVHL and leads to p53 inactivation through p14/ARF suppression. Interestingly, we showed that progerin was expressed in human leukemia and primary cell lines, raising the possibility that the expression of this LMNA variant may be a common event in age-related cancer progression.


Assuntos
Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Leucemia/genética , Proteínas Nucleares/genética , Precursores de Proteínas/genética , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Deleção de Genes , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Lamina Tipo A , Leucemia/metabolismo , Leucemia/patologia , Proteínas Nucleares/metabolismo , Forma das Organelas/genética , Cultura Primária de Células , Precursores de Proteínas/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
18.
BMC Syst Biol ; 7: 85, 2013 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-24004452

RESUMO

BACKGROUND: HMLEs (HMLE-SNAIL and Kras-HMLE, Kras-HMLE-SNAIL pairs) serve as excellent model system to interrogate the effect of SNAIL targeted agents that reverse epithelial-to-mesenchymal transition (EMT). We had earlier developed a SNAIL-p53 interaction inhibitor (GN-25) that was shown to suppress SNAIL function. In this report, using systems biology and pathway network analysis, we show that GN-25 could cause reversal of EMT leading to mesenchymal-to-epithelial transition (MET) in a well-recognized HMLE-SNAIL and Kras-HMLE-SNAIL models. RESULTS: GN-25 induced MET was found to be consistent with growth inhibition, suppression of spheroid forming capacity and induction of apoptosis. Pathway network analysis of mRNA expression using microarrays from GN-25 treated Kras-HMLE-SNAIL cells showed an orchestrated global re-organization of EMT network genes. The expression signatures were validated at the protein level (down-regulation of mesenchymal markers such as TWIST1 and TWIST2 that was concurrent with up-regulation of epithelial marker E-Cadherin), and RNAi studies validated SNAIL dependent mechanism of action of the drug. Most importantly, GN-25 modulated many major transcription factors (TFs) such as inhibition of oncogenic TFs Myc, TBX2, NR3C1 and led to enhancement in the expression of tumor suppressor TFs such as SMAD7, DD1T3, CEBPA, HOXA5, TFEB, IRF1, IRF7 and XBP1, resulting in MET as well as cell death. CONCLUSIONS: Our systems and network investigations provide convincing pre-clinical evidence in support of the clinical application of GN-25 for the reversal of EMT and thereby reducing cancer cell aggressiveness.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Mesoderma/efeitos dos fármacos , Naftoquinonas/farmacologia , Fenótipo , Biologia de Sistemas , Fatores de Transcrição/antagonistas & inibidores , Transcrição Genética/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Inativação Gênica , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , RNA Interferente Pequeno/genética , Fatores de Transcrição da Família Snail , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção
19.
Neoplasia ; 15(7): 727-37, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23814485

RESUMO

p53, a strong tumor suppressor protein, is known to be involved in cellular senescence, particularly premature cellular senescence. Oncogenic stresses, such as Ras activation, can initiate p53-mediated senescence, whereas activation of the Ras-mitogen-activated protein kinase (MAPK) pathway can promote cell proliferation. These conflicting facts imply that there is a regulatory mechanism for balancing p53 and Ras-MAPK signaling. To address this, we evaluated the effects of p53 on the extracellular signal-regulated kinase (ERK) activation and found that p53 could suppress ERK activation through de novo synthesis. Through several molecular biologic analyses, we found that RKIP, an inhibitor of Raf kinase, is responsible for p53-mediated ERK suppression and senescence. Overexpression of RKIP can induce cellular senescence in several types of cell lines, including p53-deficient cells, whereas the elimination of RKIP by siRNA or forced expression of ERK blocks p53-mediated cellular senescence. These results suggested that RKIP is an essential protein for cellular senescence. Moreover, modification of the p53 serine 46 residue was critical for RKIP induction and ERK suppression as well as cellular senescence. These results indicated that RKIP is a novel p53 target gene that is responsible for p53-mediated cellular senescence and tumor suppressor protein expression.


Assuntos
Senescência Celular/genética , Dano ao DNA , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Ativação Enzimática , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Transdução de Sinais , Inibidores da Topoisomerase II/farmacologia
20.
Lab Invest ; 93(6): 663-76, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23608757

RESUMO

Parkinson's disease (PD) is the second leading neurodegenerative disease, and is known to be induced by environmental factors or genetic mutations. Among the verified genetic mutations of PD, Parkin, isolated from the PARK2 locus, shows an autosomal recessive inheritance pattern and is known to be an E3 ligase. However, the physiological target of Parkin and the molecular mechanism of Parkin-deficiency-induced PD have not been clearly demonstrated until now. It has recently been proposed that inflammation, suggesting as a causal factor for PD, is enhanced by Parkin deficiency. Thus, we examined the relationship between inflammation-related factors and Parkin. Here, we provide the evidence that Parkin suppresses inflammation and cytokine-induced cell death by promoting the proteasomal degradation of TRAF2/6 (TNF-α receptor-associated factor 2/6). Overexpression of Parkin can reduce the half-lives of TRAF2 and TRAF6, whereas si-Parkin can extend them. However, mutant Parkins did not alter the expression of TRAF2/6. Thus, loss of Parkin enhances sensitivity to TNF-α- or IL-1ß-induced JNK activation and NF-κB activation. Indeed, si-Parkin-induced apoptosis is suppressed by the knockdown of TRAF6 or TRAF2. We also observed elevated expression levels of TRAF6 and a reduction of IκB in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse model. Moreover, elevated expression levels or aggregation of TRAF6 were detected in approximately half of the human PD tissues (7/15 cases) and 2 cases, respectively. In addition, TRAF6 and Parkin expression levels show a reverse relationship in human PD tissues. Our results strongly suggest that the reduction of Parkin or overexpression of TRAF2/6 by chronic inflammation would be the reason for occurrence of PD.


Assuntos
Doença de Parkinson/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Morte Celular , Citocinas/metabolismo , Citosol/metabolismo , Feminino , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética
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