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1.
Biochem Biophys Res Commun ; 503(2): 950-955, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-29932914

RESUMO

G protein-coupled receptor (GPCR) activation-mediated PKA and PKC pathways have been recognized to be important in ovarian physiology. Expression of regulator of G-protein signaling 2 (RGS2) has been reported in ovarian granulosa cells. The detailed mechanisms in PKA- and PKC-regulated RGS2 expression and cellular translocation in granulosa cells remain mostly unclear. PKA activator 8-bromo-cAMP and PKC activator phorbol-12, 13-didecanoate appeared to rapidly elevate both protein and mRNA levels and promoter activation of RGS2 gene. Two consensus Sp1 elements within the shortest 78 bp fragment of RGS2 promoter sequence were essential for the full responsiveness to PKA and PKC. PKC activation appeared to increase the RGS2 translocation from nucleus to cytosol. PKA- and PKC-mediated RGS2 transcription in a Sp-1-dependent manner and a PKC-mediated RGS2 intracellular translocation were noted in granulosa cells.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células da Granulosa/metabolismo , Proteína Quinase C/metabolismo , Proteínas RGS/genética , Transdução de Sinais , Ativação Transcricional , Animais , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Camundongos , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas RGS/análise , Proteínas RGS/metabolismo
2.
Innate Immun ; 23(1): 54-66, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27821648

RESUMO

Cyclooxygenase-2 (COX-2) and IL-8 are two inflammatory mediators induced by protein kinase C (PKC) via various stimuli. Both contribute significantly to cancer progression. Bufalin, a major active component of the traditional Chinese medicine Chan Su, is known to induce apoptosis in various cancer cells. This study clarifies the role and mechanism of bufalin action during PKC regulation of COX-2/IL-8 expression and investigates the associated impact on breast cancer. Using MB-231 breast cancer cells, bufalin augments PKC induction of COX-2/IL-8 at both the protein and mRNA levels, and the production of prostaglandin E2 (PGE2) and IL-8. The MAPK and NF-κB pathways are involved in both the PKC-mediated and bufalin-promoted PKC regulation of COX-2/IL-8 production. Bufalin increases PKC-induced MAPKs phosphorylation and NF-κB nuclear translocation. PGE2 stimulates the proliferation/migration of breast cancer cells. Furthermore, PKC-induced matrix metalloproteinase 3 expression is enhanced by bufalin. Bufalin significantly enhances breast cancer xenograft growth, which is accompanied by an elevation in COX-2/IL-8 expression. In conclusion, bufalin seems to promote the inflammatory response in vitro and in vivo, and this occurs, at least in part, by targeting the MAPK and NF-κB pathways, which then enhances the growth of breast cancer cells.


Assuntos
Neoplasias da Mama/metabolismo , Bufanolídeos/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Interleucina-8/metabolismo , Glândulas Mamárias Humanas/efeitos dos fármacos , Animais , Neoplasias da Mama/patologia , Ciclo-Oxigenase 2/genética , Dinoprostona/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Interleucina-8/genética , Células MCF-7 , Glândulas Mamárias Humanas/patologia , Medicina Tradicional Chinesa , Camundongos , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Int J Mol Sci ; 17(8)2016 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-27548147

RESUMO

Cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) are two important inflammatory mediators in ovulation. Ghrelin may modulate inflammatory signaling via growth hormone secretagogue receptors. We investigated the role of ghrelin in KGN human ovarian granulosa cells using protein kinase C (PKC) activator phorbol 12, 13-didecanoate (PDD) and synthetic ghrelin analog growth hormone releasing peptide-2 (GHRP-2). GHRP-2 attenuated PDD-induced expression of protein and mRNA, the promoter activity of COX-2 and IL-8 genes, and the secretion of prostaglandin E2 (PGE2) and IL-8. GHRP-2 promoted the degradation of PDD-induced COX-2 and IL-8 proteins with the involvement of proteasomal and lysosomal pathways. PDD-mediated COX-2 production acts via the p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways; PDD-mediated IL-8 production acts via the p38, JNK and ERK pathways. GHRP-2 reduced the PDD-induced phosphorylation of p38 and JNK and activator protein 1 (AP-1) reporter activation and PDD-induced NF-κB nuclear translocation and reporter activation. The inhibitors of mitogen-activated protein kinase phosphatase-1 (MKP-1) and protein phosphatase 2 (PP2A) reduced the inhibitory effect of GHRP-2 on PDD-induced COX-2 and IL-8 expression. Our findings demonstrate an anti-inflammatory role for ghrelin (GHRP-2) in PKC-mediated inflammation of granulosa cells, at least in part, due to its inhibitory effect on PKC-induced activation of p38, JNK and NF-κB, possibly by targeting to MKP-1 and PP2A.


Assuntos
Células da Granulosa/metabolismo , Inflamação/tratamento farmacológico , Oligopeptídeos/farmacologia , Proteína Quinase C/metabolismo , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Grelina/metabolismo , Células da Granulosa/efeitos dos fármacos , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Interleucina-8/metabolismo , Ésteres de Forbol/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/metabolismo
4.
Innate Immun ; 22(6): 452-65, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27312705

RESUMO

Ovulation is a critical inflammation-like event that is central to ovarian physiology. IL-1ß is an immediate early pro-inflammatory cytokine that regulates production of several other inflammatory mediators, such as cyclooxygenase 2 (COX)-2 and IL-8. NS-398 is a selective inhibitor of COX-2 bioactivity and thus this drug is able to mitigate the COX-2-mediated production of downstream prostaglandins and the subsequent inflammatory response. Here we have investigated the action of NS-398 using a human ovarian granulosa cell line, KGN, by exploring IL-1ß-regulated COX-2 and IL-8 expression. First, NS-398, instead of reducing inflammation, appeared to further enhance IL-1ß-mediated COX-2 and IL-8 production. Using selective inhibitors targeting various signaling molecules, MAPK and NF-κB pathways both seemed to be involved in the impact of NS-398 on IL-1ß-induced COX-2 and IL-8 expression. NS-398 also promoted IL-1ß-mediated NF-κB p65 nuclear translocation but had no effect on IL-1ß-activated MAPK phosphorylation. Flow cytometry analysis demonstrated that NS-398, in combination with IL-1ß, significantly enhanced cell cycle progression involving IL-8. Our findings demonstrate a clear pro-inflammatory function for NS-398 in the IL-1ß-mediated inflammatory response of granulosa cells, at least in part, owing to its augmenting effect on the IL-1ß-induced activation of NF-κB.


Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Células da Granulosa/efeitos dos fármacos , Inflamação/imunologia , Interleucina-8/metabolismo , Nitrobenzenos/farmacologia , Ovulação/imunologia , Sulfonamidas/farmacologia , Linhagem Celular , Ciclo-Oxigenase 2/genética , Dinoprostona/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/imunologia , Humanos , Interleucina-1beta/imunologia , Interleucina-8/genética , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
Br J Clin Pharmacol ; 80(4): 755-61, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25808421

RESUMO

AIMS: Cholangiocarcinoma (CCA) is the second most common primary liver cancer in the world. Due to the lack of effective treatments, the survival rate of CCA is low and it is usually considered difficult to diagnose early. To date, no effective strategies for the prevention of CCA have been developed. Statins are cholesterol-lowering agents which possess pleiotropic properties and the use of statins may reduce cancer risk. The aim of the study was to investigate the effect of statin use on the risk of CCA. METHODS: We used nationwide insurance data to perform a case-control study including 3174 CCA patients diagnosed in 2002-2011 and 3174 propensity score matched controls. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated to assess the association between CCA risk and statin use by type of statin and dose. RESULTS: Patients with CCA were slightly younger than controls with mean ages of 67.4 (SD 12.3) and 68.5 (SD 13.2) years (P = 0.001), respectively, and had less users of statins (22.7 vs. 26.5%, P < 0.001). The overall adjusted OR of statin use associated CCA was 0.80 (95% CI 0.71, 0.90) and lowered for those with longer medications. The OR ranged from 0.65 to 0.77. Stronger dose-response association was seen when using lovastatin. CONCLUSIONS: Statin use is associated with reduced risk of CCA and there is a dose-response relationship between the use of statins and risk of CCA.


Assuntos
Neoplasias dos Ductos Biliares/epidemiologia , Colangiocarcinoma/epidemiologia , Uso de Medicamentos/estatística & dados numéricos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Idoso , Estudos de Casos e Controles , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição de Risco , Taiwan/epidemiologia
6.
Innate Immun ; 21(6): 635-46, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25783839

RESUMO

LPS can activate the inflammatory cascades by inducing various inflammatory mediators, such as prostaglandin E(2) (PGE(2)) resulting from cyclooxygenase-2 (COX-2), and NO produced by inducible NO synthase (iNOS). Lysophosphatidic acid (LPA) has been demonstrated to participate in inflammation. This study aimed to clarify the impact and the involving mechanisms of LPA on LPS-incurred inflammation in macrophages. First, LPA appeared to attenuate LPS-induced protein and mRNA expression of COX-2 and iNOS genes, as well as production of PGE(2) and NO. By using selective inhibitors targeting various signaling players, the inhibitory G protein alpha subunit (Gα(i)) seemed to be involved in the effect of LPA; p38, ERK and NF-κB were involved in the LPS-mediated COX-2/PGE(2) pathway; and p38, JNK, phosphoinositide-3-kinase and NF-κB were involved in the LPS-mediated iNOS/NO pathway. LPA was able to diminish LPS-induced phosphorylation of p38 and Akt, as well as NF-κB p65 nuclear translocation. By utilization of inhibitors of COX-2 and iNOS, there appeared to be no modulation between the COX-2/PGE(2) and the iNOS/NO signaling pathways. Our findings demonstrate a clear anti-inflammatory role of LPA acting via Gα(i) in LPS-mediated inflammatory response in macrophages, owing, at least in part, to its suppressive effect on LPS-induced activation of p38, Akt and NF-κB.


Assuntos
Anti-Inflamatórios/farmacologia , Ciclo-Oxigenase 2/metabolismo , Lisofosfolipídeos/farmacologia , Macrófagos/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Linhagem Celular , Ciclo-Oxigenase 2/genética , Dinoprostona/metabolismo , Regulação para Baixo , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética
7.
J Cell Physiol ; 230(9): 2240-51, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25754990

RESUMO

Breast cancer is a common cancer leading to many deaths among females. Cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) are two highly expressed inflammatory mediators to be induced by the protein kinase C (PKC) signaling via various inflammatory stimuli and both contribute significantly to cancer metastasis/progression. Glucosamine has been shown to act as an anti-inflammation molecule. The aim of this study was to clarify the role and acting mechanism of glucosamine during the PKC-regulation of COX-2/IL-8 expression and the associated impact on breast cancer. In MCF-7 breast cancer cells, glucosamine effectively suppresses the PKC induction of COX-2 and IL-8 promoter activity, mRNA and protein levels, as well as the production of prostaglandin E(2) (PGE(2)) and IL-8. Glucosamine is able to promote COX-2 protein degradation in a calpain-dependent manner and IL-8 protein degradation in calpain-dependent and proteasome-dependent manners. The MAPK and NF-κB pathways are involved in PKC-induced COX-2 expression, but only the NF-κB pathway is involved in PKC-induced IL-8 expression. Glucosamine attenuates PKC-mediated IκBα phosphorylation, nuclear NF-κB translocation, and NF-κB reporter activation. Both PGE(2) and IL-8 promote cell proliferation and IL-8 induces cell migration; thus, glucosamine appears to suppress PKC-induced cell proliferation and migration. Furthermore, glucosamine significantly inhibits the growth of breast cancer xenografts and this is accompanied by a reduction in COX-2 and IL-8 expression. In conclusion, glucosamine seems to attenuate the inflammatory response in vitro and in vivo and this occurs, at least in part by targeting to the NF-κB signaling pathway, resulting in an inhibition of breast cancer cell growth.


Assuntos
Neoplasias da Mama/genética , Ciclo-Oxigenase 2/biossíntese , Interleucina-8/biossíntese , Proteína Quinase C/metabolismo , Animais , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Glucosamina/administração & dosagem , Glucosamina/genética , Humanos , Inflamação/genética , Inflamação/patologia , Células MCF-7 , Camundongos , RNA Mensageiro/biossíntese , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Free Radic Biol Med ; 69: 208-18, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24486342

RESUMO

Cigarette smoking causes persistent lung inflammation that is mainly regulated by redox-sensitive pathways. We have reported that cigarette smoke (CS) activates a NADPH oxidase-dependent reactive oxygen species (ROS)-sensitive AMP-activated protein kinase (AMPK) signaling pathway leading to induction of lung inflammation. Glucosamine, a dietary supplement used to treat osteoarthritis, has antioxidant and anti-inflammatory properties. However, whether glucosamine has similar beneficial effects against CS-induced lung inflammation remains unclear. Using a murine model we show that chronic CS exposure for 4 weeks increased lung levels of 4-hydroxynonenal (an oxidative stress biomarker), phospho-AMPK, and macrophage inflammatory protein 2 and induced lung inflammation; all of these CS-induced events were suppressed by chronic treatment with glucosamine. Using human bronchial epithelial cells, we demonstrate that cigarette smoke extract (CSE) sequentially activated NADPH oxidase; increased intracellular levels of ROS; activated AMPK, mitogen-activated protein kinases (MAPKs), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription proteins 3 (STAT3); and induced interleukin-8 (IL-8). Additionally, using a ROS scavenger, a siRNA that targets AMPK, and various pharmacological inhibitors, we identified the signaling cascade that leads to induction of IL-8 by CSE. All these CSE-induced events were inhibited by glucosamine pretreatment. Our findings suggest a novel role for glucosamine in alleviating the oxidative stress and lung inflammation induced by chronic CS exposure in vivo and in suppressing the CSE-induced IL-8 in vitro by inhibiting both the ROS-sensitive NADPH oxidase/AMPK/MAPK signaling pathway and the downstream transcriptional factors NF-κB and STAT3.


Assuntos
Anti-Inflamatórios/administração & dosagem , Glucosamina/administração & dosagem , Pneumonia/tratamento farmacológico , Fumar/efeitos adversos , Proteínas Quinases Ativadas por AMP/biossíntese , Animais , Células Cultivadas , Humanos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pneumonia/patologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Fator de Transcrição STAT3/biossíntese , Transdução de Sinais/efeitos dos fármacos
9.
Am J Respir Cell Mol Biol ; 49(6): 1110-9, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23898954

RESUMO

Acute inflammation is often observed during acute lung injury (ALI) and acute respiratory distress syndrome. Glucosamine is known to act as an anti-inflammatory molecule. The effects of glucosamine on acute lung inflammation and its associated mechanisms remain unclear. The present study sought to address how glucosamine plays an anti-inflammatory role in acute lung inflammation in vivo and in vitro. Using the LPS intratracheal instillation-elicited rat lung inflammation model, we found that glucosamine attenuated pulmonary edema and polymorphonuclear leukocyte infiltration, as well as the production of TNF-α, IL-1ß, cytokine-induced neutrophil chemoattractant (CINC)-1, macrophage inflammatory protein (MIP)-2, and nitric oxide (NO) in the bronchoalveolar lavage fluid (BALF) and in the cultured medium of BALF cells. The expression of TNF-α, IL-1ß, IFN-γ, CINC-1, MIP-2, monocyte chemotactic protein-1, and inducible NO synthase (iNOS) in LPS-inflamed lung tissue was also suppressed by glucosamine. Using the rat alveolar epithelial cell line L2, we noted that the cytokine mixture (cytomix)-regulated production and mRNA expression of CINC-1 and MIP-2, NO production, the protein and mRNA expression of iNOS, iNOS mRNA stability, and iNOS promoter activity were all inhibited by glucosamine. Furthermore, glucosamine reduced LPS-mediated NF-κB signaling by decreasing IκB phosphorylation, p65 nuclear translocation, and NF-κB reporter activity. Overexpression of the p65 subunit restored the inhibitory action of glucosamine on cytomix-regulated NO production and iNOS expression. In conclusion, glucosamine appears to act as an anti-inflammatory molecule in LPS-induced lung inflammation, at least in part by targeting the NF-κB signaling pathway.


Assuntos
Glucosamina/administração & dosagem , Lipopolissacarídeos/toxicidade , Pneumonia/etiologia , Pneumonia/prevenção & controle , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Linhagem Celular , Citocinas/farmacologia , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Pneumonia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
10.
Pulm Pharmacol Ther ; 26(2): 195-204, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23128353

RESUMO

Airway epithelial cells play an important role against intruding pathogens. Glucosamine, a commonly used supplemental compound, has recently begun to be regarded as a potential anti-inflammatory molecule. This study aimed to uncover how glucosamine impacts on cellular proliferation in human alveolar epithelial cells (A549) and bronchial epithelial cells (HBECs). With trypan blue-exclusion assay, we observed that glucosamine (10, 20, 50 mM) caused a decrease in cell number at 24 and 48 h; with a flow cytometric analysis, we also noted an enhanced cell accumulation within the G(0)/G(1) phase at 24 h and induction of late apoptosis at 24 and 48 h by glucosamine (10, 20, 50 mM) in A549 cells and HBECs. Examination of phosphorylation in retinoblastoma (Rb) protein, we found an inhibitory effect by glucosamine at 20 and 50 mM. Glucosamine at 50 mM was demonstrated to elevate both the mRNA and protein expression of p53 and heme oxygenase-1 (HO-1), but also caused a reduction in p21 protein expression. In addition, glucosamine attenuated p21 protein stability via the proteasomal proteolytic pathway, as well as inducing p21 nuclear accumulation. Altogether, our results suggest that a high dose of glucosamine may inhibit cell proliferation through apoptosis and disturb cell cycle progression with a halt at G(0)/G(1) phase, and that this occurs, at least in part, by a reduction in Rb phosphorylation together with modulation of p21, p53 and HO-1 expression, and nuclear p21 accumulation.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Glucosamina/farmacologia , Pulmão/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/análise , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos , Pulmão/citologia , Fosforilação , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/análise
11.
FEBS Lett ; 585(9): 1375-81, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21510939

RESUMO

Regulator of G-protein signaling protein (RGS)-2 is a modulator of anxiety and dysregulation of oxidative stress is implicated in anxiety. Also, RGS2 expression is reported to be induced by oxidative stress. Thus, if oxidative stress induces RGS2 expression and lack of RGS2 causes anxiety, then mechanisms that link RGS2 and oxidative stress potentially critical to anxiety must be revealed. Our study is the first to suggest role of RGS2 in regulation of enzymes involved in antioxidant defense namely glyoxalase-1 and glutathione reductase-1 via activation of p38 MAPK and PKC pathways in an Sp-1 dependent manner.


Assuntos
Antioxidantes/metabolismo , Homeostase , Neurônios/metabolismo , Proteínas RGS/metabolismo , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA Antissenso/genética , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Peróxido de Hidrogênio/farmacologia , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Carbonilação Proteica/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteínas RGS/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
Free Radic Biol Med ; 50(11): 1492-502, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21376115

RESUMO

Cigarette smoke (CS) increases chemokine production in lung epithelial cells (LECs), but the pathways involved are not completely understood. AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, may modulate inflammation. Here, we show that cigarette smoke extract sequentially activated NADPH oxidase; increased intracellular reactive oxygen species (ROS) level; activated AMPK, NF-κB, and STAT3; and induced interleukin 8 (IL-8) in human LECs. Inhibition of NADPH oxidase activation by apocynin or siRNA targeting p47(phox) (a subunit of NADPH oxidase) attenuated the increased intracellular ROS level, AMPK activation, and IL-8 induction. Removal of intracellular ROS by N-acetylcysteine reduced the AMPK activation and IL-8 induction. Prevention of AMPK activation by Compound C or AMPK siRNA lessened the activation of both NF-κB and STAT3 and the induction of IL-8. Abrogation of the activation of NF-κB and STAT3 by BAY11-7085 and AG490, respectively, attenuated the IL-8 induction. We additionally show that chronic CS exposure in mice promoted AMPK phosphorylation and expression of MIP-2α (an IL-8 homolog) in LECs and lungs, as well as lung inflammation, all of which were reduced by Compound C treatment. Thus, a novel NADPH oxidase-dependent, ROS-sensitive AMPK signaling is important for CS-induced IL-8 production in LECs and possibly lung inflammation.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Interleucina-8/biossíntese , Pneumonia/metabolismo , Mucosa Respiratória/metabolismo , Fumar/efeitos adversos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Acetofenonas/farmacologia , Animais , Linhagem Celular , Humanos , Interleucina-8/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Extratos Vegetais/administração & dosagem , Pneumonia/tratamento farmacológico , Pneumonia/etiologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Transdução de Sinais/efeitos dos fármacos
13.
Free Radic Biol Med ; 50(1): 47-54, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21034810

RESUMO

α-Lipoic acid (α-LA), a key cofactor in cellular energy metabolism, has protective activities in atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined whether α-LA affects foam cell formation and its underlying molecular mechanisms in murine macrophages. Treatment with α-LA markedly attenuated oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, which was due to increased cholesterol efflux. Additionally, α-LA treatment dose-dependently increased protein levels of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 but had no effect on the protein expression of SR-A, CD36, or SR-BI involved in cholesterol homeostasis. Furthermore, α-LA increased the mRNA expression of ABCA1 and ABCG1. The upregulation of ABCA1 and ABCG1 by α-LA depended on liver X receptor α (LXRα), as evidenced by an increase in the nuclear levels of LXRα and LXRE-mediated luciferase activity and its prevention of the expression of ABCA1 and ABCG1 after inhibition of LXRα activity by the pharmacological inhibitor geranylgeranyl pyrophosphate (GGPP) or knockdown of LXRα expression with small interfering RNA (siRNA). Consistently, α-LA-mediated suppression of oxLDL-induced lipid accumulation was abolished by GGPP or LXRα siRNA treatment. In conclusion, LXRα-dependent upregulation of ABCA1 and ABCG1 may mediate the beneficial effect of α-LA on foam cell formation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Células Espumosas/efeitos dos fármacos , Lipoproteínas/genética , Receptores Nucleares Órfãos/fisiologia , Ácido Tióctico/farmacologia , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células Cultivadas , Células Espumosas/metabolismo , Células Espumosas/patologia , Humanos , Lipoproteínas/metabolismo , Lipoproteínas LDL/metabolismo , Receptores X do Fígado , Camundongos , Receptores Nucleares Órfãos/antagonistas & inibidores , Receptores Nucleares Órfãos/metabolismo , Fosfatos de Poli-Isoprenil/farmacologia , RNA Interferente Pequeno/farmacologia , Elementos de Resposta/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Transfecção , Regulação para Cima/efeitos dos fármacos
14.
Cardiovasc Res ; 88(3): 415-23, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-20615914

RESUMO

AIMS: Accumulation of foam cells in the intima is a hallmark of early-stage atherosclerotic lesions. Ginkgo biloba extract (EGb761) has been reported to exert anti-oxidative and anti-inflammatory properties in atherosclerosis, yet the significance and the molecular mechanisms of action of EGb761 in the formation of macrophage foam cells are not fully understood. METHODS AND RESULTS: Treatment with EGb761 resulted in a dose-dependent decrease in oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, a consequence that was due to a decrease in cholesterol uptake and an increase in cholesterol efflux. Additionally, EGb761 significantly down-regulated the mRNA and protein expression of class A scavenger receptor (SR-A) by decreasing expression of activator protein 1 (AP-1); however, EGb761 increased the protein stability of ATP-binding cassette transporter A1 (ABCA1) by reducing calpain activity without affecting ABCA1 mRNA expression. Small interfering RNA (siRNA) targeting haem oxygenase-1 (HO-1) abolished the EGb761-induced protective effects on the expression of AP-1, SR-A, ABCA1, and calpain activity. Accordingly, EGb761-mediated suppression of lipid accumulation in foam cells was also abrogated by HO-1 siRNA. Moreover, the lesion size of atherosclerosis was smaller in EGb761-treated, apolipoprotein E-deficient mice compared with the vehicle-treated mice, and the expression of HO-1, SR-A, and ABCA1 in aortas was modulated similar to that observed in macrophages. CONCLUSION: These findings suggest that EGb761 confers a protection from the formation of foam cells by a novel HO-1-dependent regulation of cholesterol homeostasis in macrophages.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Heme Oxigenase-1/metabolismo , Extratos Vegetais/farmacologia , Receptores Depuradores Classe A/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Calpaína/metabolismo , Colesterol/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Espumosas/patologia , Homeostase/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout
15.
Eur J Pharmacol ; 635(1-3): 219-26, 2010 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-20307528

RESUMO

Inflammation is a complex process involving cytokine production to regulate host defense cascades in order to clear pathogenic agents. Upregulation of inflammatory cytokines, such as IL-6 and IL-8 by bacteria infection, occurs in pulmonary tissues and has been demonstrated to be critical to the lung inflammatory response. Glucosamine, primarily identified as an anti-arthritis supplement, has been also regarded as a potential anti-inflammatory agent. Thus we hypothesized that lipopolysaccharide (LPS) would activate IL-6 and IL-8 expressions in human primary bronchial epithelial cells and glucosamine could attenuate such an effect. The RT-PCR, real-time PCR, and ELISA analyses demonstrated that LPS-induced mRNAs encoding IL-6 and IL-8 and the subsequent secretion of IL-6 and IL-8 were inhibited by glucosamine treatment. MTT, alamarBlue, and annexin V apoptosis assays all suggested that this inhibition effect was not due to a cytotoxic effect mediated by glucosamine. Using the inhibitors of the MAP kinases and NFkappaB, it was revealed that p38, JNK and ERK, as well as NFkappaB, are all involved in LPS-induced IL-8 secretion; however only p38 is involved in LPS-induced IL-6 secretion. Immunoblot analysis further demonstrated that LPS-mediated phosphorylation of JNK and ERK, but not the LPS-induced NFkappaB translocation, was inhibited by glucosamine. Altogether, our results indicate that glucosamine can potently suppress LPS-induced inflammatory cytokine expression, at least in part via attenuation of MAPK activation.


Assuntos
Brônquios/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Glucosamina/farmacologia , Inflamação/tratamento farmacológico , Lipopolissacarídeos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosamina/uso terapêutico , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
J Cell Biochem ; 108(2): 489-98, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19626664

RESUMO

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL-1beta and IL-8, has been noted in prostate cancer patients and IL-8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL-1beta regulates IL-8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti-inflammatory agent and thus we hypothesized that if IL-1beta activated IL-8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU-145, PC-3, and LNCaP, were used to evaluate the effects of IL-1beta and glucosamine on IL-8 expression using ELISA and RT-PCR analyses. IL-1beta elevated IL-8 mRNA expression and subsequent IL-8 secretion. Glucosamine significantly inhibited IL-1beta-induced IL-8 secretion. IL-8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL-8 in IL-1beta-dependent PC-3 cell migration was demonstrated by wound-healing and transwell migration assays. Inhibitors of MAPKs and NFkappaB were used to pinpoint MAPKs but not NFkappaB being involved in IL-1beta-mediated IL-8 production. IL-1beta-provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL-1beta can activate the MAPK pathways resulting in an induction of IL-8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL-1beta-mediated activation of MAPKs and therefore reduces IL-8 production; this, in turn, attenuates cell proliferation/migration.


Assuntos
Anti-Inflamatórios/farmacologia , Glucosamina/farmacologia , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Neoplasias da Próstata/metabolismo , Análise de Variância , Anti-Inflamatórios/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Técnicas de Cocultura , Regulação Neoplásica da Expressão Gênica , Glucosamina/metabolismo , Humanos , Interleucina-1beta/farmacologia , Interleucina-8/genética , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , Fosforilação , Neoplasias da Próstata/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Fatores de Tempo
17.
Pulm Pharmacol Ther ; 22(4): 286-96, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19254777

RESUMO

Cigarette smoking is the major cause of chronic obstructive pulmonary disease, which is associated with increased oxidative stress and numbers of apoptotic endothelial cells in the lungs. Ginkgo biloba extract (EGb) is a therapeutic agent for disorders such as vascular insufficiency and Alzheimer's disease. Although EGb is known to possess antioxidant functions, its ability to alleviate cigarette smoke-induced pathophysiological consequences has not been elucidated. We investigated the cytoprotective effects and therapeutic mechanisms of EGb against oxidative stress and apoptosis induced by cigarette smoke extract (CSE) in human pulmonary artery endothelial cells (HPAECs). Challenge with CSE (160 microg/ml) caused a reduction in cell viability, an increase in intracellular reactive oxygen species and an acceleration of caspase-dependent apoptosis in HPAECs, all of which were alleviated by pretreatment with EGb (100 microg/ml). N-acetylcysteine (an antioxidant) also reduced both the CSE-induced oxidative stress and apoptosis, indicating that the former response triggered the latter. Additionally, EGb produced activation of ERK, JNK and p38 [three major mitogen-activated protein kinases (MAPKs)], an increase in the nuclear level of nuclear factor erythroid-2-related factor 2 (Nrf2) and upregulation of heme oxygenase-1 (HO-1, a stress-responsive protein with antioxidant function). Pretreatment with inhibitors of MAPKs abolished both EGb-induced Nrf2 nuclear translocation and HO-1 upregulation. Small interfering RNAs targeting HO-1 prevented EGb-induced HO-1 upregulation and also abolished the antioxidant, anti-apoptotic and cytoprotective effects of EGb in HPAECs insulted with CSE. We conclude that EGb confers protection from oxidative stress-related apoptosis induced by CSE in HPAECs and its therapeutic effects depend on transcriptional upregulation of HO-1 by EGb via the MAPKs/Nrf2 pathway.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Heme Oxigenase-1/fisiologia , Pulmão/efeitos dos fármacos , Extratos Vegetais/farmacologia , Fumaça/efeitos adversos , Fumar/patologia , Western Blotting , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Células Endoteliais/enzimologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Humanos , Imuno-Histoquímica , Pulmão/citologia , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , RNA/biossíntese , RNA/genética , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fumaça/análise
18.
Cardiovasc Res ; 82(3): 468-75, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19307231

RESUMO

AIMS: Valsartan, a selective angiotensin II type 1 receptor (AT1R) blocker, has beneficial effects in the cardiovascular system in part by its increase of nitric oxide (NO) bioavailability, yet the mechanisms are unclear. We investigated the molecular mechanisms underlying this effect in endothelial cells (ECs). METHODS AND RESULTS: NO production was examined by Griess reagent assay, DAF-2 DA fluorescence staining and cGMP ELISA kits. Protein interaction was determined by western blotting and immunoprecipitation. Treating bovine or human aortic ECs with valsartan increased NO production, as evidenced by elevated level of stable NO metabolites and intracellular cGMP. Valsartan increased the phosphorylation but not the protein level of endothelial NO synthase (eNOS). Inhibition of phosphoinositide-3 kinase (PI3K)/Akt and Src pathways by specific inhibitors suppressed valsartan-induced NO release. In addition, valsartan increased the tyrosine residue phosphorylation of AT1R, which was attenuated by inhibition of Src but not PI3K activities. Valsartan also suppressed the interaction of eNOS and AT1R, which was blocked by Src or PI3K inhibition. CONCLUSION: Valsartan-induced NO production in ECs is mediated through Src/PI3K/Akt-dependent phosphorylation of eNOS. Valsartan-induced AT1R phosphorylation depends on Src but not PI3K, whereas valsartan-induced suppression of AT1R-eNOS interaction depends on Src/PI3K/Akt signalling. These results indicate a novel vasoprotective mechanism of valsartan in upregulating NO production in ECs.


Assuntos
Anti-Hipertensivos/farmacologia , Células Endoteliais/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Tetrazóis/farmacologia , Valina/análogos & derivados , Animais , Aorta/citologia , Bovinos , Células Cultivadas , Humanos , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Valina/farmacologia , Valsartana , Quinases da Família src/metabolismo
19.
Life Sci ; 84(3-4): 97-104, 2009 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-19041881

RESUMO

AIMS: Resistin promotes macrophage-foam cell formation, but the mechanisms are unclear. In macrophages, lipid uptake is regulated by scavenger receptors (SR-A and CD36), while the cholesterol efflux is regulated by SR-BI, ATP-binding cassette transporter-A1 (ABCA1) and ABCG1. We investigated the mechanisms underlying the dysregulation by resistin of these regulators leading to promotion of lipid accumulation in bone marrow-derived macrophages. MAIN METHODS: Western blotting, real-time PCR and oil red O staining were performed. KEY FINDINGS: Resistin exacerbated lipid accumulation in oxLDL-treated macrophages. Resistin treatment of oxLDL-untreated macrophages showed increased SR-A and CD36 mRNA and protein levels, and decreased ABCA1 protein level, while having no effect on SR-BI or ABCG1 expression. Up-regulation of SR-A and CD36 by resistin resulted from activation of AP-1 and PPARgamma, respectively, and this was confirmed by the lack of activation of either after AP-1 inhibition using curcumin or SP600125, or PPARgamma inhibition using GW9662, respectively. The down-regulation of ABCA1 by resistin was not accompanied by a reduced mRNA level or an activation of LXRalpha/RXR, but resulted from enhanced protein degradation as revealed by the abolition of the down-regulation after inhibition of the proteasome pathway using ALLN or MG-132. A combined inhibition by SP600125, GW9662 and ALLN prevented resistin-induced exacerbation of lipid accumulation in oxLDL-treated macrophages. SIGNIFICANCE: Resistin promotes foam cell formation via dysregulation of SR-A, CD36 and ABCA1. SR-A and CD36 are transcriptionally up-regulated by resistin through AP-1 and PPARgamma, respectively, whereas ABCA1 is down-regulated by resistin through proteasome-mediated enhancement of protein degradation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Antígenos CD36/fisiologia , Metabolismo dos Lipídeos , Macrófagos/metabolismo , Resistina/fisiologia , Receptores Depuradores Classe A/fisiologia , Transportador 1 de Cassete de Ligação de ATP , Animais , Colesterol/metabolismo , Lipoproteínas LDL/fisiologia , PPAR gama/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Ratos , Ratos Sprague-Dawley , Fator de Transcrição AP-1/fisiologia
20.
J Cell Biochem ; 105(3): 922-30, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18726905

RESUMO

Adipocyte differentiation is a complex process involving several signaling pathways. Molecular mechanisms regulating the very early stage of adipocyte differentiation is not fully appreciated yet. Several inducible genes at the early stage of preadipocyte differentiation have been identified, including the regulator of G protein signaling 2 (RGS2), a member of the RGS protein superfamily. This study aimed to clarify the precise induction profile of RGS2 and to determine the essential transcription element(s) regulating RGS2 expression in differentiating 3T3-L1 preadipocytes. RGS2 mRNA expression was elevated immediately at 1 h after differentiation initiation and it remained high until the late stage of differentiation. The putative promoter sequence (approximately 3,000 bp) of the mouse RGS2 gene was isolated and the RGS2 promoter activity was significantly upregulated 3 h after inducing differentiation. The primary signaling pathway leading to RGS2 transcriptional activation appeared to be cAMP-dependent. Sequential deletion and site-directed mutagenesis strategies demonstrate that the RGS2 promoter sequence truncated down to 78 bp in size retained full inducibility by the differentiation stimuli. Mutation of a Sp1 site within the 78 bp region significantly blocked promoter activity. In addition, high expression of Sp1 transcription factor was noted prior to and paralleling the differentiation process. Taken together, our data suggest that RGS2 transcription is immediately induced via a cAMP-dependent pathway after initiation of 3T3-L1 differentiation and the RGS2 mRNA level remains consistently high throughout the differentiation progression. A Sp1 site within RGS2 promoter appeared to be a crucial response element to regulate RGS2 transcription.


Assuntos
Adipócitos/metabolismo , Diferenciação Celular/genética , Proteínas RGS/genética , Transdução de Sinais , Células-Tronco/metabolismo , Transcrição Genética , Células 3T3-L1 , Adipócitos/citologia , Animais , Clonagem Molecular , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Camundongos , Regiões Promotoras Genéticas , Proteínas RGS/metabolismo , RNA Mensageiro/metabolismo , Células-Tronco/citologia
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