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1.
Acta Med Okayama ; 74(4): 307-317, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32843762

RESUMO

We examined the effect of D-Tagatose on the growth of oral bacteria including Streptococcus mutans (S. mutans). Saliva collected from 10 healthy volunteers was plated on BHI medium (to culture total oral bacteria) and MBS medium (to culture S. mutans, specifically). Agar plates of BHI or MBS containing xylitol or D-Tagatose were cultured under aerobic or anaerobic conditions. We then counted the number of colonies. In BHI plates containing D-Tagatose, a complete and significant reduction of bacteria occurred under both aerobic and anaerobic conditions. In MSB medium, significant reduction of S. mutans was also observed. We then performed a doubleblind parallel randomized trial with 19 healthy volunteers. They chewed gum containing xylitol, D-Tagatose, or both for 4 weeks, and their saliva was collected weekly and plated on BHI and MSB media. These plates were cultured under anaerobic conditions. Total bacteria and S. mutans were not effectively reduced in either the D-Tagatose or xylitol gum group. However, S. mutans was significantly reduced in volunteers chewing gum containing both D-Tagatose and xylitol. Thus, D-Tagatose inhibited the growth of S. mutans and many types of oral bacteria, indicating that D-Tagatose intake may help prevent dental caries, periodontitis, and many oral diseases.


Assuntos
Cárie Dentária/prevenção & controle , Hexoses/administração & dosagem , Streptococcus mutans/efeitos dos fármacos , Edulcorantes/administração & dosagem , Adulto , Goma de Mascar , Método Duplo-Cego , Feminino , Humanos , Masculino , Projetos Piloto , Saliva/microbiologia , Streptococcus mutans/crescimento & desenvolvimento , Xilitol/administração & dosagem
2.
J Cell Biochem ; 119(6): 4627-4635, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29266408

RESUMO

Proper balance between lipolysis and lipogenesis in adipocytes determines the release of free fatty acids (FFA) and glycerol, which is crucial for whole body lipid homeostasis. Although, dysregulation of lipid homeostasis contributes to various metabolic complications such as insulin resistance, the regulatory mechanism remains elusive. This study clarified the individual and combined roles for glucocorticoid receptor (GCR) and peroxisome proliferator-activated receptor (PPAR)γ pathways in lipid metabolism of adipocytes. In mature 3T3-L1 adipocytes, GCR activation using dexamethasone upregulated adipose triglyceride lipase (ATGL) and downregulated phosphoenolpyruvate carboxykinase (PEPCK), resulting in enhanced glycerol release into the medium. In contrast, PPARγ ligand pioglitazone modestly upregulated ATGL and hormone sensitive lipase (HSL), but markedly enhanced PEPCK and glycerol kinase (GK), thereby suppressed glycerol release. Dexamethasone showed permissive like effect on PPARγ target genes including perilipin A and aP2, therefore co-administration of dexamethasone and pioglitazone demonstrated synergistic upregulation of these enzymes excepting PEPCK, of which downregulation by dexamethasone was abolished by pioglitazone to the level above control. Thus, the excessive glycerol release was prevented as the net outcome of the co-administration. Consistently, the bodipy stain demonstrated that dexamethasone reduced the amount of cytosolic lipid, which was preserved in co-treated adipocytes. Moreover, silencing of PPARγ suppressed the synergistic effects of co-treatment on the lipolytic and lipogenic genes, and therefore the GCR pathway indeed involves PPARγ. In conclusion, crosstalk between GCR and PPARγ is largely synergistic but counter-regulatory in lipogenic genes, of which enhancement prevents excessive glycerol and possibly FFA release by glucocorticoids into the circulation.


Assuntos
Adipócitos/metabolismo , Lipólise , PPAR gama/metabolismo , Receptores de Glucocorticoides/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Dexametasona/farmacologia , Camundongos , PPAR gama/genética , Pioglitazona/farmacologia , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/genética
3.
Exp Cell Res ; 358(2): 343-351, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28689812

RESUMO

Hypoxia predisposes renal fibrosis. This study was conducted to identify novel approaches to ameliorate the pathogenic effect of hypoxia. Using human proximal tubular epithelial cells we showed that a pan-phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) dose and time dependently downregulated hypoxia-inducible factor 1α (HIF-1α) mRNA expression, which was further augmented by addition of a transcriptional inhibitor, actinomycin D. IBMX also increased the cellular cyclic adenosine monophosphate (cAMP) level. Luciferase assay showed that blocking of protein kinase A (PKA) using H89 reduced, while 8-Br-cAMP agonized the repression of HIF-1α promoter activity in hypoxic condition. Deletion of cAMP response element binding sites from the HIF-1α promoter abrogated the effect of IBMX. Western blot and immunofluorescent study confirmed that the CoCl2 induced increased HIF-1α protein in whole cell lysate and in nucleus was reduced by the IBMX. Through this process, IBMX attenuated both CoCl2 and hypoxia induced mRNA expressions of two pro-fibrogenic factors, platelet-derived growth factor B and lysyl oxidase. Moreover, IBMX reduced production of a mesenchymal transformation factor, ß-catenin; as well as protected against hypoxia induced cell-death. Taken together, our study showed novel evidence that the PDE inhibitor IBMX can downregulate the transcription of HIF-1α, and thus may attenuate hypoxia induced renal fibrosis.


Assuntos
1-Metil-3-Isobutilxantina/farmacologia , Células Epiteliais/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-sis/metabolismo , Transdução de Sinais/efeitos dos fármacos , Xantinas/farmacologia , beta Catenina/metabolismo
4.
Biochem Biophys Res Commun ; 486(1): 76-82, 2017 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-28263744

RESUMO

How nutritional excess leads to inflammatory responses in metabolic syndrome is not well characterized. Here, we evaluated the effects of ω-3 polyunsaturated fatty acid specific G-protein coupled receptor 120 (GPR120) activation on inflammatory pathways in adipocytes, and the influence of this process on macrophage migration. Using 3T3-L1 adipocytes, we found that agonizing GPR120 using its synthetic ligand, GSK137647, attenuated both basal and lipopolysaccharide-induced production of interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2). Moreover, the intervention reduced the phosphorylation of nuclear factor kappa B inhibitor alpha (IκBα) and nuclear translocation of nuclear factor kappa-B p65 subunit (p65). Furthermore, the silencing of GPR120 itself reduced IL-6 and CCL2 mRNA expression. Inhibition of protein kinase C (PKC) augmented the down-regulatory effect of GSK137647 on IL-6 and CCL2 mRNA. Using a luciferase assay to measure promoter activity of the IL-6 gene in mouse embryonic fibroblasts, we demonstrated that exogenous transfection of GPR120 alone reduced the promoter activity, which was augmented by GSK137647. Inhibition of PKC further reduced the promoter activity. Nevertheless, RAW 264.7 macrophages grown in conditioned medium collected from GSK137647-treated adipocytes attenuated the expressions of matrix metalloproteinases-9 and -3, and tissue inhibitor of metalloproteinase-1. Conditioned medium also inhibited the lipopolysaccharide-induced migration of these macrophages. Taken together, these findings provide critical evidence that although GPR120 is associated with a PKC-mediated pro-inflammatory pathway, the direct inhibitory effects of GPR120 on the nuclear factor kappa B pathway are anti-inflammatory. Moreover, GPR120 activity can attenuate the adipocyte-mediated enhanced production of extracellular matrix-modulating factors in macrophages and can reduce their migration by a paracrine mechanism.


Assuntos
Adipócitos/metabolismo , Adipocinas/metabolismo , Mediadores da Inflamação/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipocinas/genética , Animais , Western Blotting , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Inibidor de NF-kappaB alfa/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Interferência de RNA , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Transcrição RelA/metabolismo
6.
Biogerontology ; 18(1): 55-68, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27734200

RESUMO

Deterioration of adipocyte function due to increased oxidative stress predisposes patients to metabolic disorders in advanced age. However, the roles of tumor suppressors in such conditions remain largely unknown. Therefore, we aimed to address their dynamics in aged adipocytes using a long-term culture model. We compared 3T3-L1 adipocytes at 17-19 days (long-term) with those at 8-10 days (short-term) after initiation of adipogenic induction for mimicking 'aged' and 'young' adipocytes, respectively. H2O2 release and dihydroethidium (DHE) staining was increased, while superoxide dismutase (SOD) activity was reduced in long-term cultured adipocytes, which is suggestive of enhanced oxidative stress in this group. Moreover, qRT-PCR revealed increased mRNAs of Nox4 (a subunit of NADPH oxidase complex), Ccl2 (a proinflammatory chemokine) and Il6 [a marker of senescence-associated secretory phenotype (SASP)] along with decreased levels of Pparγ, Adipoq and Slc2a4 (genes related to glucose metabolism). These alterations were associated with increased expression of the tumor suppressors alternate-reading-frame protein p19Arf (Arf) and p16Ink4a. However, silencing of Arf reduced mRNAs of Adipoq and Slc2a4 and enhanced release of Il6. The effect was opposite in Arf overexpressing adipocytes, which showed reduced superoxide production as assessed with DHE staining and SOD activity. Western blots showed that Arf negatively regulates the phosphorylation of Akt. Luciferase assay in Hela cells additionally suggested that Arf negatively regulates Il6 transcriptional activity through a PI3 K/Akt mediated pathway. These findings strongly suggest that the enhanced Arf expression in oxidative stress plays compensatory protective roles against aging-related dysregulation of gene expression in adipocytes.


Assuntos
Adipócitos/metabolismo , Envelhecimento/metabolismo , Senescência Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Células 3T3-L1 , Animais , Células HeLa , Humanos , Camundongos , Regulação para Cima/fisiologia
7.
Tohoku J Exp Med ; 238(2): 131-41, 2016 02.
Artigo em Inglês | MEDLINE | ID: mdl-26829886

RESUMO

Glucose is a major energy source for mammalian cells and is transported into cells via cell-specific expression of various glucose transporters (GLUTs). Especially, cancer cells require massive amounts of glucose as an energy source for their dysregulated growth and thus over-express GLUTs. d-allose, a C-3 epimer of d-glucose, is one of rare sugars that exist in small quantities in nature. We have shown that d-allose induces the tumor suppressor gene coding for thioredoxin interacting protein (TXNIP) and inhibits cancer cell growth by G1 cell cycle arrest. It has also been reported that GLUTs including GLUT1 are over-expressed in many cancer cell lines, which may contribute to larger glucose utilization. Since d-allose suppresses the growth of cancer cells through the upregulation of TXNIP expression, our present study focused on whether d-allose down-regulates GLUT1 expression via TXNIP expression and thus suppresses cancer cell growth. Western blot and real-time PCR analyses revealed that d-allose significantly induced TXNIP expression and inhibited GLUT1 expression in a dose-dependent manner in three human cancer cell lines: hepatocellular carcinoma (HuH-7), Caucasian breast adenocarcinoma (MDA-MB-231), and neuroblastoma (SH-SY5Y). In these cell lines, d-allose treatment inhibited cell growth. Importantly, d-allose treatment decreased glucose uptake, as measured by the uptake of 2-deoxy d-glucose. Moreover, the reporter assays showed that d-allose decreased the expression of luciferase through the hypoxia response element present in the tested promoter region. These results suggest that d-allose may cause the inhibition of cancer growth by reducing both GLUT1 expression and glucose uptake.


Assuntos
Transportador de Glucose Tipo 1/genética , Glucose/farmacologia , Neoplasias/genética , Neoplasias/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta , Regulação para Cima/efeitos dos fármacos
8.
Tohoku J Exp Med ; 240(1): 67-78, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27600583

RESUMO

Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium-binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis.


Assuntos
Proteínas Nucleares/metabolismo , Estresse Oxidativo , Fosfoproteínas Fosfatases/metabolismo , Proteínas S100/metabolismo , Caspases/metabolismo , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Peróxido de Hidrogênio/toxicidade , MAP Quinase Quinase Quinase 5/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos
9.
Biochem J ; 458(1): 141-52, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24295050

RESUMO

FKBP38 (FK506-binding protein 38), a membrane-anchored TPR (tetratricopeptide repeat)-containing immunophilin, regulates signalling pathways such as cell survival, apoptosis, proliferation and metastasis. However, the mechanisms that regulate the activity of FKBP38 are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR proteins, such as Hop [Hsp70 (heat-shock protein of 70 kDa)/Hsp90-organizing protein], kinesin-light chain, Tom70 (translocase of outer mitochondrial membrane 70), FKBP52, CyP40 (cyclophilin 40), CHIP (C-terminus of Hsc70-interacting protein) and PP5 (protein phosphatase 5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore we have hypothesized that Ca2+/S100 proteins can interact with FKBP38 and regulate its function. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, S100B and S100P specifically interact with FKBP38 and inhibit the interaction of FKBP38 with Bcl-2 and Hsp90. Overexpression of permanently active S100P in Huh-7 cells inhibited the interaction of FKBP38 with Bcl-2, resulting in the suppression of Bcl-2 stability. The association of the S100 proteins with FKBP38 provides a Ca2+-dependent regulatory mechanism of the FKBP38-mediated signalling pathways.


Assuntos
Cálcio/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas S100/fisiologia , Proteínas de Ligação a Tacrolimo/metabolismo , Sítios de Ligação , Linhagem Celular , Humanos , Ligação Proteica , Proteínas S100/metabolismo
10.
Acta Med Okayama ; 69(2): 105-11, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25899632

RESUMO

We examined and compared the inhibitory effects of D-tagatose on the growth, acid production, and water-insoluble glucan synthesis of GS5, a bacterial strain of Streptococcus mutans, with those of xylitol, D-psicose, L-psicose and L-tagatose. GS5 was cultured for 12h in a medium containing 10% (w/v) of xylitol, D-psicose, L-psicose, D-tagatose or L-tagatose, and the inhibitory effect of GS5 growth was assessed. Each sugar showed different inhibitory effects on GS5. Both D-tagatose and xylitol significantly inhibited the acid production and water-insoluble glucan synthesis of GS5 in the presence of 1% (w/v) sucrose. However, the inhibitory effect of acid production by D-tagatose was significantly stronger than that of xylitol in presence of sucrose.


Assuntos
Ácidos/metabolismo , Glucanos/metabolismo , Hexoses/farmacologia , Streptococcus mutans/classificação , Streptococcus mutans/metabolismo , Sacarose/farmacologia , Frutose/farmacologia , Concentração de Íons de Hidrogênio , Quelantes de Ferro/farmacologia , Técnicas Microbiológicas , Streptococcus mutans/crescimento & desenvolvimento , Xilitol/farmacologia
11.
J Biol Chem ; 288(10): 7158-68, 2013 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-23344957

RESUMO

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca(2+)/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca(2+)/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca(2+)/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca(2+)/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca(2+)-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Fatores Quimiotáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas S100/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação/genética , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Fatores Quimiotáticos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Fatores de Transcrição de Choque Térmico , Humanos , Lisina/genética , Lisina/metabolismo , Mutação , Proteínas de Neoplasias/genética , Prolina/genética , Prolina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas S100/genética , Transdução de Sinais , Proteína Smad1/genética , Proteína Smad1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
12.
J Biol Chem ; 287(17): 13787-98, 2012 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-22399290

RESUMO

PP5 is a unique member of serine/threonine phosphatases comprising a regulatory tetratricopeptide repeat (TPR) domain and functions in signaling pathways that control many cellular responses. We reported previously that Ca(2+)/S100 proteins directly associate with several TPR-containing proteins and lead to dissociate the interactions of TPR proteins with their client proteins. Here, we identified protein phosphatase 5 (PP5) as a novel target of S100 proteins. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, and S100B proteins specifically interact with PP5-TPR and inhibited the PP5-Hsp90 interaction. In addition, the S100 proteins activate PP5 by using a synthetic phosphopeptide and a physiological protein substrate, Tau. Overexpression of S100A1 in COS-7 cells induced dephosphorylation of Tau. However, S100A1 and permanently active S100P inhibited the apoptosis signal-regulating kinase 1 (ASK1) and PP5 interaction, resulting the inhibition of dephosphorylation of phospho-ASK1 by PP5. The association of the S100 proteins with PP5 provides a Ca(2+)-dependent regulatory mechanism for the phosphorylation status of intracellular proteins through the regulation of PP5 enzymatic activity or PP5-client protein interaction.


Assuntos
Cálcio/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas S100/metabolismo , Animais , Células COS , Proteínas de Ligação ao Cálcio/metabolismo , Chlorocebus aethiops , Regulação Enzimológica da Expressão Gênica , Glutationa Transferase/metabolismo , Glicoproteínas/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , MAP Quinase Quinase Quinase 5/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Transdução de Sinais , Ressonância de Plasmônio de Superfície/métodos , Proteínas tau/metabolismo
13.
BMC Cancer ; 13: 468, 2013 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-24112473

RESUMO

BACKGROUND: Accumulating evidence has suggested the importance of glutamate signaling in cancer growth, yet the signaling pathway has not been fully elucidated. N-methyl-D-aspartic acid (NMDA) receptor activates intracellular signaling pathways such as the extracellular-signal-regulated kinase (ERK) and forkhead box, class O (FOXO). Suppression of lung carcinoma growth by NMDA receptor antagonists via the ERK pathway has been reported. However, series of evidences suggested the importance of FOXO pathways for the regulation of normal and cancer cell growth. In the liver, FOXO1 play important roles for the cell proliferation such as hepatic stellate cells as well as liver metabolism. Our aim was to investigate the involvement of the FOXO pathway and the target genes in the growth inhibitory effects of NMDA receptor antagonist MK-801 in human hepatocellular carcinoma. METHODS: Expression of NMDAR1 in cancer cell lines from different tissues was examined by Western blot. NMDA receptor subunits in HepG2, HuH-7, and HLF were examined by reverse transcriptase polymerase chain reaction (RT-PCR), and growth inhibition by MK-801 and NBQX was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of MK-801 on the cell cycle were examined by flow cytometry and Western blot analysis. Expression of thioredoxin-interacting protein (TXNIP) and p27 was determined by real-time PCR and Western blotting. Activation of the FOXO pathway and TXNIP induction were examined by Western blotting, fluorescence microscopy, Chromatin immunoprecipitation (ChIP) assay, and reporter gene assay. The effects of TXNIP on growth inhibition were examined using the gene silencing technique. RESULTS: NMDA receptor subunits were expressed in all cell lines examined, and MK-801, but not NBQX, inhibited cell growth of hepatocellular carcinomas. Cell cycle analysis showed that MK-801 induced G1 cell cycle arrest by down-regulating cyclin D1 and up-regulating p27. MK-801 dephosphorylated Thr24 in FOXO1 and induced its nuclear translocation, thus increasing transcription of TXNIP, a tumor suppressor gene. Knock-down of TXNIP ameliorated the growth inhibitory effects of MK-801. CONCLUSIONS: Our results indicate that functional NMDA receptors are expressed in hepatocellular carcinomas and that the FOXO pathway is involved in the growth inhibitory effects of MK-801. This mechanism could be common in hepatocellular carcinomas examined, but other mechanisms such as ERK pathway could exist in other cancer cells as reported in lung carcinoma cells. Altered expression levels of FOXO target genes including cyclin D1 and p27 may contribute to the inhibition of G1/S cell cycle transition. Induction of the tumor suppressor gene TXNIP plays an important role in the growth inhibition by MK-801. Our report provides new evidence that FOXO-TXNIP pathway play a role in the inhibition of the hepatocellular carcinoma growth by MK-801.


Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/metabolismo , Maleato de Dizocilpina/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Transdução de Sinais , Carcinoma Hepatocelular/genética , Proteínas de Transporte/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo
14.
J Surg Res ; 183(2): 976-81, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23541813

RESUMO

BACKGROUND: Recently, one of the rare sugars, D-allose, has received attention from many researchers because of its availability for mass production and its various physiological functions. Among these, an antioxidative effect has been strongly suggested. In this study, we investigated whether this effect is also applicable to the field of skin surgery. METHODS: In ischemia-reperfusion injury model using the rat abdominal skin island flap (male Wistar rats, n = 110), D-allose was injected intravenously 15 min before 8-h ischemia. The survival area (%) was measured by digital photographic assessment 1 wk after surgery, and multiple comparisons (Fisher's protected least significant difference) were carried out. Histopathological examination (neutrophilic infiltration into dermis in hematoxylin and eosin stain) and immunostaining (of ectodermal dysplasia-1 (ED1)-positive cells/flap) were assessed. Myeloperoxidase (MPO) activity in the skin flap (sampling at the time of 8 h after reperfusion) was measured spectrophotometrically, and Student t-test was performed. RESULTS: D-allose extended the survival of the remaining flaps, and a dose greater than 30 mg (0.1 mg/g) was necessary to be effective. The flap survival rates in the 30, 60, and 150 mg groups were significantly higher than that in the control (saline) group: 75.87 ± 5.90, 79.27 ± 7.81, and 77.87 ± 6.20 versus 50.53 ± 9.66, respectively (P < 0.05). ED1-positive cells/flap in 60 mg of D-allose and control (saline) were 78 ± 25.7 versus 124 ± 15.8, respectively (P = 0.08). The MPO activity in the D-allose 60 mg group was 0.40 ± 0.04, and that in the control (saline) was 0.72 ± 0.12. D-allose significantly reduced the skin tissue MPO activity (P < 0.05) compared with that in the control (saline) group. CONCLUSIONS: We proved that D-allose has a reducing effect against ischemia-reperfusion injury on the skin island flap model, and the mechanism is related to inhibiting the activity of neutrophils in the skin tissues. Compared with chemo-synthetic materials, rare sugars are safer for our bodies as well as the environment; therefore, this rare sugar project is expected to lead to the development of a safer antioxidant for skin flap surgery.


Assuntos
Antioxidantes/uso terapêutico , Glucose/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Pele/irrigação sanguínea , Retalhos Cirúrgicos/irrigação sanguínea , Abdome/irrigação sanguínea , Abdome/patologia , Abdome/cirurgia , Animais , Relação Dose-Resposta a Droga , Masculino , Modelos Animais , Peroxidase/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Pele/metabolismo , Pele/patologia , Retalhos Cirúrgicos/patologia , Retalhos Cirúrgicos/cirurgia
15.
Biochem Biophys Res Commun ; 425(4): 717-23, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22877751

RESUMO

Rare sugar D-psicose has cropped up as a non-toxic and effective compound to protect and preserve pancreatic ß-islets in the growing type 2 diabetes mellitus (T2DM) rats through the regulation of glucose and fat metabolism. The present study was undertaken to examine the effect of rare sugar D-psicose on the protection of pancreatic ß-islets using Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a T2DM model. Treated rats were fed with 5% D-psicose or 5% D-glucose supplemented drinking water, and only water in the control for 13 weeks. A non-diabetic Long-Evans Tokushima Otsuka (LETO), fed with water served as a counter control of OLETF. D-Psicose significantly attenuated progressive ß-islet fibrosis and preserved islets, evaluated by hematoxylin-eosin staining, Masson's trichrome staining and immunostainings of insulin and α-smooth muscle actin (SMA). D-Psicose significantly reduced increase in body weight and abdominal fat deposition. Oral glucose tolerance test (OGTT) showed reduced blood glucose levels suggesting the improvement of insulin resistance. All these data suggests that D-psicose protected and preserved pancreatic ß-islets through the maintenance of hyperglycemia and by the prevention of fat accumulation in OLETF rats.


Assuntos
Citoproteção , Diabetes Mellitus Tipo 2/tratamento farmacológico , Frutose/administração & dosagem , Resistência à Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Obesidade/tratamento farmacológico , Gordura Abdominal/efeitos dos fármacos , Gordura Abdominal/patologia , Actinas/metabolismo , Adipocinas/sangue , Animais , Índice de Massa Corporal , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Fibrose , Glucose/metabolismo , Teste de Tolerância a Glucose , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Obesidade/sangue , Ratos , Ratos Endogâmicos OLETF
16.
Biochem Biophys Res Commun ; 405(1): 7-12, 2011 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-21187061

RESUMO

A rare sugar, D-psicose has progressively been evaluated as a unique metabolic regulator of glucose and lipid metabolism, and thus represents a promising compound for the treatment of type 2 diabetes mellitus (T2DM). The present study was undertaken to examine the underlying effector organs of D-psicose in lowering blood glucose and abdominal fat by exploiting a T2DM rat model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Rats were fed 5% D-psicose or 5% D-glucose supplemented in drinking water, and only water in the control for 13 weeks and the protective effects were compared. A non-diabetic Long-Evans Tokushima Otsuka (LETO), fed with water served as a counter control of OLETF. After 13 weeks feeding, D-psicose treatment significantly reduced the increase in body weight and abdominal fat mass. Oral glucose tolerance test (OGTT) showed the reduced blood glucose and insulin levels suggesting the improvement of insulin resistance in OLETF rats. Oil-red-O staining elucidated that D-psicose significantly reduced lipid accumulation in the liver. Immunohistochemical analysis showed D-psicose induced glucokinase translocation from nucleus to cytoplasm of the liver which enhances glucokinase activity and subsequent synthesis of glycogen in the liver. D-psicose also protected the pathological change of the ß-cells of pancreatic islets. These data demonstrate that D-psicose controls blood glucose levels by reducing lipotoxicity in liver and by preserving pancreatic ß-cell function.


Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Frutose/uso terapêutico , Resistência à Insulina , Gordura Abdominal/efeitos dos fármacos , Gordura Abdominal/patologia , Animais , Peso Corporal/efeitos dos fármacos , Núcleo Celular/enzimologia , Citoplasma/enzimologia , Citoproteção , Ingestão de Alimentos/efeitos dos fármacos , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/patologia , Glucoquinase/metabolismo , Teste de Tolerância a Glucose , Homeostase/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Endogâmicos OLETF
17.
Biochem J ; 412(2): 223-31, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18271754

RESUMO

The mechanisms of NO inhibition of CaMK [Ca(2+)/CaM (calmodulin)-dependent protein kinase] II activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser(847) was sensitive to an inhibitor of CaMKs, KN-93, and was enhanced by inhibition of nNOS with 7NI (7-nitroindazole). Enzyme activity of CaMKII following in situ treatment with 7NI was also increased. The in vitro activity of CaMKII was inhibited by co-incubation either with nNOS and L-arginine or with NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) and DEA-NONOate [diethylamine-NONOate (diazeniumdiolate)]. Once inhibited by these treatments, CaMKII was observed to undergo full reactivation on the addition of a reducing reagent, DTT (dithiothreitol). In transfected cells expressing CaMKII and nNOS, treatment with the calcium ionophore A23187 further revealed nNOS phosphorylation at Ser(847), which was enhanced by 7NI and CaMKII S-nitrosylation. Mutated CaMKII (C6A), in which Cys(6) was substituted with an alanine residue, was refractory to 7NI-induced enhancement of nNOS phosphorylation or to CaMKII S-nitrosylation. Furthermore, we could identify Cys(6) as a direct target for S-nitrosylation of CaMKII using MS. In addition, treatment with glutamate caused an increase in CaMKII S-nitrosylation in rat hippocampal slices. This glutamate-induced S-nitrosylation was blocked by 7NI. These results suggest that inactivation of CaMKII mediated by S-nitrosylation at Cys(6) may contribute to NO-induced neurotoxicity in the brain.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Animais , Benzilaminas/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular Tumoral , Cisteína/metabolismo , Ativação Enzimática , Hipocampo/citologia , Hipocampo/metabolismo , Hidrazinas/metabolismo , Indazóis/metabolismo , Doadores de Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/genética , Inibidores de Proteínas Quinases/metabolismo , Ratos , S-Nitroso-N-Acetilpenicilamina/metabolismo , Serina/metabolismo , Sulfonamidas/metabolismo , Hormônio Liberador de Tireotropina/metabolismo
18.
Methods Mol Biol ; 1929: 367-377, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710285

RESUMO

S100A6 is a member of the EF-hand Ca2+-binding protein family, which plays important roles in a wide variety of Ca2+ signaling in the cells, as well as in pathophysiological conditions. Herein, we describe analytical protocols for evaluating the interaction of S100A6 with multiple target proteins in vitro, including biotinylated S100A6 overlay, glutathione-S-transferase (GST)-precipitation, surface plasmon resonance, and a GST-precipitation assay in living cells. These methods will elucidate the detailed molecular mechanisms of S100A6/target interactions and further improve our understanding of the physiological significance of S100A6-mediated Ca2+ signaling. Moreover, they may be used to evaluate other physical S100/target interactions.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteína A6 Ligante de Cálcio S100/química , Proteína A6 Ligante de Cálcio S100/metabolismo , Animais , Biotinilação , Células COS , Sinalização do Cálcio , Precipitação Química , Chlorocebus aethiops , Humanos , Immunoblotting , Cinética , Ligação Proteica , Ressonância de Plasmônio de Superfície
19.
Int J Oncol ; 32(2): 377-85, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18202760

RESUMO

'Rare sugars' are defined as monosaccharides that exist in nature but are only present in limited quantities. The development of mass production method of rare sugars revealed some interesting physiological effects of these on animal cells, but the mechanisms have not been well studied. We examined the effect of D-allose on the proliferation of cancer cells and the underlying molecular mechanism of the action. The HuH-7 hepatocellular carcinoma cells were treated with various monosaccharides for 48 h and D-allose was shown to inhibit cell growth by 40% in a dose-dependent manner. D-allose induced G1 cell cycle arrest but not apoptosis. The microarray analysis revealed that D-allose significantly up-regulated thioredoxin interacting protein (TXNIP) gene expression, which is often suppressed in tumor cells and western blot analysis confirmed its increase at protein level. The overexpression of TXNIP also induced G1 cell cycle arrest. Analysis of cell cycle regulatory genes showed p27kip1, a key regulator of G1/S cell cycle transition, to be increased at the protein but not the transcriptional level. Protein interaction between TXNIP and jab1, and p27kip1 and jab1, was observed, suggesting stabilization of p27kip1 protein by the competitive inhibition of jab1-mediated nuclear export of p27kip1 by TXNIP. In addition, increased interaction and nuclear localization of TXNIP and p27kip1 were apparent after D-allose treatment. Our findings surprisingly suggest that D-allose, a simple monosaccharide, may act as a novel anticancer agent via unique TXNIP induction and p27kip1 protein stabilization.


Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/biossíntese , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Fase G1 , Regulação Neoplásica da Expressão Gênica , Glucose/farmacologia , Neoplasias Hepáticas/metabolismo , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Modelos Biológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Biochem J ; 401(2): 391-8, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-16984226

RESUMO

Evidence is presented that RSK1 (ribosomal S6 kinase 1), a downstream target of MAPK (mitogen-activated protein kinase), directly phosphorylates nNOS (neuronal nitric oxide synthase) on Ser847 in response to mitogens. The phosphorylation thus increases greatly following EGF (epidermal growth factor) treatment of rat pituitary tumour GH3 cells and is reduced by exposure to the MEK (MAPK/extracellular-signal-regulated kinase kinase) inhibitor PD98059. Furthermore, it is significantly enhanced by expression of wild-type RSK1 and antagonized by kinase-inactive RSK1 or specific reduction of endogenous RSK1. EGF treatment of HEK-293 (human embryonic kidney) cells, expressing RSK1 and nNOS, led to inhibition of NOS enzyme activity, associated with an increase in phosphorylation of nNOS at Ser847, as is also the case in an in vitro assay. In addition, these phenomena were significantly blocked by treatment with the RSK inhibitor Ro31-8220. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and decrease of NOS activity. Within minutes of adding EGF to transfected cells, RSK1 associated with nNOS and subsequently dissociated following more prolonged agonist stimulation. EGF-induced formation of the nNOS-RSK1 complex was significantly decreased by PD98059 treatment. Treatment with EGF further revealed phosphorylation of nNOS on Ser847 in rat hippocampal neurons and cerebellar granule cells. This EGF-induced phosphorylation was partially blocked by PD98059 and Ro31-8220. Together, these data provide substantial evidence that RSK1 associates with and phosphorylates nNOS on Ser847 following mitogen stimulation and suggest a novel role for RSK1 in the regulation of nitric oxide function in brain.


Assuntos
Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sequência de Aminoácidos , Animais , Cerebelo/citologia , Cerebelo/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/fisiologia , Hipocampo/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Fosforilação , Ratos , Serina/metabolismo
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