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1.
Int J Mol Sci ; 19(7)2018 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-29932110

RESUMO

Interleukin-1β (IL-1β) is a prominent pro-inflammatory cytokine that is implicated in a variety of autoimmune diseases and plays an important role in host defense against infections. IL-1β activity increases with its increasing binding capacity to IL-1 receptors (IL-1Rs). Thus, numerous studies have targeted the discovery of molecules modulating the interactions between IL-1β and IL-1R1. We have conducted an IL-1R1 structure-based virtual screening to identify small molecules that could alter IL-1β activity, using in silico computational analysis. Sixty compounds from commercial libraries were predicted to bind to IL-1R1, and their influence on cytokine production in IL-1β-stimulated gingival fibroblasts (GFs) was determined. Of these, only (2-(1,2-diphenyl-1H-indol-3-yl)ethanamine (DPIE) showed a synergistic increase in inflammatory molecules and cytokine production (IL-6, IL-8, and COX-2) at both mRNA and protein levels in IL-1β-stimulated GFs. The enhancing activity of DPIE in IL-1β-induced cytokine production increased in a dose-dependent manner without cytotoxicity. This pattern was also observed in IL-1β-stimulated primary human periodontal ligament cells (PDLs). Furthermore, we measured the impact of DPIE on the IL-1β⁻IL-1R1 system using surface plasmon resonance and demonstrated that DPIE increased the binding affinity of IL-1β to IL-1R1. These data indicate that DPIE boosts IL-1β signaling by enhancing the binding of IL-1β to IL-1R1 in oral primary cells.


Assuntos
Aminas/farmacologia , Citocinas/metabolismo , Fibroblastos/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Interleucina-1beta/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Aminas/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Gengiva/citologia , Humanos , Estrutura Molecular , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptores Tipo I de Interleucina-1/metabolismo , Bibliotecas de Moléculas Pequenas/química , Ressonância de Plasmônio de Superfície
2.
Int J Mol Sci ; 18(5)2017 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-28489026

RESUMO

Organic peroxides and hydroperoxides are skin tumor promoters. Free radical derivatives from these compounds are presumed to be the prominent mediators of tumor promotion. However, the molecular targets of these species are unknown. Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) are tumor suppressors that play important roles in cell growth, proliferation, and cell survival by negative regulation of phosphoinositol-3-kinase/protein kinase B signaling. PTEN is reversibly oxidized in various cells by exogenous and endogenous hydrogen peroxide. Oxidized PTEN is converted back to the reduced form by cellular reducing agents, predominantly by the thioredoxin (Trx) system. Here, the role of tert-butyl hydroperoxide (t-BHP) in redox regulation of PTEN was analyzed by using cell-based and in vitro assays. Exposure to t-BHP led to oxidation of recombinant PTEN. In contrast to H2O2, PTEN oxidation by t-BHP was irreversible in HeLa cells. However, oxidized PTEN was reduced by exogenous Trx system. Taken together, these results indicate that t-BHP induces PTEN oxidation and inhibits Trx system, which results in irreversible PTEN oxidation in HeLa cells. Collectively, these results suggest a novel mechanism of t-BHP in the promotion of tumorigenesis.


Assuntos
Peróxido de Hidrogênio/farmacologia , PTEN Fosfo-Hidrolase/química , terc-Butil Hidroperóxido/farmacologia , Células HeLa , Humanos , Oxirredução , PTEN Fosfo-Hidrolase/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Tiorredoxinas/metabolismo
3.
Methods ; 77-78: 58-62, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25637034

RESUMO

PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS-PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys(124) or nearby Cys(71) was replaced with Ser suggesting that Cys(124) and Cys(71) are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells.


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , PTEN Fosfo-Hidrolase/análise , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Oxirredução , PTEN Fosfo-Hidrolase/genética , Coelhos , Proteínas Supressoras de Tumor/genética
4.
Mol Cell Biochem ; 398(1-2): 147-56, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25234193

RESUMO

Piperine, a kind of natural alkaloid found in peppers, has been reported to exhibit anti-oxidative and anti-tumor activities, both in vitro and in vivo. Interleukin-6 (IL-6) is an important cytokine that activates the signal transduction, promotes tumor cell metastasis, and induces malignancy, including in gastric cancer. However, the effects of piperine on IL-6 expression in gastric cancer cells have not yet been well defined. In this study, we investigated the effects of piperine on the IL-6 expression, and examined the underlying signaling pathways via RT-PCR, promoter studies and Western blotting in human gastric cancer TMK-1 cells. Our results showed that piperine inhibited interleukin-1ß (IL-1ß)-induced IL-6 expression in a dose-dependent manner. In addition, piperine also inhibited IL-6 promoter activity. Experiments with mitogen-activated protein kinase (MAPK) inhibitors and dominant negative mutant p38 MAPK indicated that p38 MAPK was essential for IL-6 expression in the TMK-1 cells. Additionally, signal transducer and activator of transcription 3 (STAT3) was also involved in the IL-1ß-induced IL-6 expression in gastric cancer cells. Piperine inhibited IL-1ß-induced p38 MAPK and STAT3 activation and, in turn, blocked the IL-1ß-induced IL-6 expression. Furthermore, gastric cancer cells pretreated with IL-1ß showed markedly enhanced invasiveness, which was partially abrogated by treatment with IL-6 siRNA, piperine, and inhibitors of p38 MAPK and STAT3. These results suggest that piperine may exert at least part of its anti-cancer effect by controlling IL-6 expression through the suppression of p38 MAPK and STAT3.


Assuntos
Alcaloides/farmacologia , Benzodioxóis/farmacologia , Interleucina-1beta/farmacologia , Interleucina-6/metabolismo , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Fator de Transcrição STAT3/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Interleucina-6/genética , Mutação , Invasividade Neoplásica , Piridinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , UDPglucose 4-Epimerase/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética
5.
Biochem Biophys Res Commun ; 407(1): 175-80, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21371429

RESUMO

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H(2)O(2)-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.


Assuntos
Flavodoxina/metabolismo , Glutarredoxinas/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Glutarredoxinas/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Dados de Sequência Molecular , Oxirredução , PTEN Fosfo-Hidrolase/genética , Saccharomyces cerevisiae/genética
6.
Anim Cells Syst (Seoul) ; 23(1): 1-9, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30834153

RESUMO

Paxillin is a focal adhesion adaptor protein, heavily phosphorylated at multiple tyrosine residues, as well as at serine 273 (S273), and is known to be critical for cytoskeleton rearrangement and cell migration. We previously found that paxillin plays a regulatory role in IL-3-dependent survival of Ba/F3 cells, a mouse pro-B cell line. In this study, by using overexpressed His6 tagged-paxillin as a bait, we found that DDX42, a DEAD-box RNA helicase, interacted with paxillin, inhibited apoptosis, and promoted polarization of Ba/F3 cells. His6 tagged-paxillin was stably overexpressed in Ba/F3 cells, pulled-down from cell lysates with Ni+-NTA beads, and analyzed by one-dimensional SDS-PAGE followed by LC-MS. We found that DDX42 co-precipitated with paxillin, as demonstrated by western blotting analysis of His6 tagged-paxillin precipitates with anti-DDX42 antibodies and His6 tagged-DDX42 precipitates with anti-paxillin antibodies. In addition, we observed a preferential interaction of DDX42 with the paxillin mutant, S273A, compared to the S273D mutant. Furthermore, DDX42 overexpression in Ba/F3 cells delayed the apoptosis induced by IL-3 deprivation and promoted restoration of the elongated shape in Ba/F3 cells induced by IL-3 re-supply after a 6 h-deprivation. These results suggested that DDX42 interacts with paxillin and participates in IL-3-dependent cell survival, as well as in the cytoskeletal rearrangements underlying polarization of Ba/F3 cells.

7.
Genes Genomics ; 41(2): 241-248, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30604146

RESUMO

BACKGROUND: Ba/F3, a mouse pro-B cell line, is dependent on IL-3 for its survival and proliferation. IL-3 withdrawal causes cells to round, stop in G1 phase, then undergo apoptosis. Additionally, IL-3 is known to induce tyrosine phosphorylation of paxillin, a scaffold and signaling protein. We previously determined that overexpression of paxillin prohibited Ba/F3 cell apoptosis induced by IL-3 withdrawal. OBJECTIVE: Address whether phosphorylation is essential for the anti-apoptotic effect of overexpressed paxillin. METHODS: Mutations were introduced into paxillin cDNA at five phosphorylation sites-Y31F, Y40F, Y118F, Y181F, S273A, or S273D. After overexpression of paxillin mutants in Ba/F3 cells, the apoptotic proportions of cell populations were measured by an annexin V conjugation assay while cells were undergoing IL-3 withdrawal. RESULTS: The anti-apoptotic effect of paxillin overexpression was abolished by site-directed mutagenesis replacing Y31, Y40, Y118, and Y181 with phenylalanine, and S273 with aspartic acid. In contrast, the mutation replacing S273 with alanine had no effect on the anti-apoptotic effect. CONCLUSION: The above results suggest that paxillin-mediated phosphorylation at Y31, Y40, Y118, and Y181 is essential for the anti-apoptotic effect of paxillin overexpression in Ba/F3 cells and contributes to the cell survival signaling pathway triggered by IL-3. Conversely, phosphorylation at S273 is involved in the negative regulation of the anti-apoptotic action of overexpressed paxillin.


Assuntos
Apoptose , Paxilina/metabolismo , Motivos de Aminoácidos , Animais , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Interleucina-3/metabolismo , Camundongos , Paxilina/química , Paxilina/genética , Fosforilação
8.
Genes Genomics ; 41(3): 373-379, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30610621

RESUMO

BACKGROUND: Nitric oxide synthases (NOSs) are a unique family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. Atherogenic action of oxidized low-density lipoproteins (oxLDL) may be mediated partly by the formation of NO in endothelial cells. OBJECTIVE: The objective of this study was to identify sources of reactive oxygen species (ROS) causing native LDL (nLDL)-induced senescence of cultured human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were treated with nLDL and NO production was assessed using Griess reagent as substrate and spectrophotometry in the absence or presence of specific inhibitors of endothelial NOS (eNOS) and inducible NOS (iNOS). In addition, expression levels of eNOS and iNOS were measured with ELISA and western blotting, and ROS was evaluated using 2',7'-dichlorofluorescin diacetate (DCF-DA) and a fluorescence microplate reader. RESULTS: NO formation in nLDL-treated HUVECs was significantly increased. Long-term treatment with nLDL up-regulated both eNOS and iNOS proteins. Such increase of NO production in HUVECs induced by nLDL was significantly suppressed by treatment with iNOS-selective inhibitor 1400 W, but not by the eNOS-selective inhibitor L-NIO. Native LDL treatment uncoupled Hsp90, the regulatory binding protein of eNOS, from the enzyme in HUVECs. Native LDL also significantly increased ROS production in HUVECs. CONCLUSION: These findings suggest that oxidative stress originated from induction of iNOS and eNOS could be a causative factor for nLDL-induced senescence of HUVECs.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Lipoproteínas LDL/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Humanos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo
9.
Neurosci Lett ; 443(1): 17-22, 2008 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-18672029

RESUMO

We examined the involvement of oxidative stress in neuronal cell death induced by taxol, a microtubule-stabilizing anti-cancer drug and investigated whether NADPH oxidase plays a role in taxol-induced neuronal cell death in mouse cortical cultures. Cell death was assessed by measuring lactate dehydrogenase in the bathing media after 24-h exposure to taxol. Taxol (30-1000 nM) induced the concentration-dependent neuronal death with apoptotic features. The neuronal death induced by taxol was significantly attenuated not only by anti-apoptotic drugs such as z-VAD-fmk and cycloheximide but also by antioxidants such as trolox, ascorbic acid and tempol. Vinblastine, a microtubule-depolymerizing anti-cancer drug, also induced neuronal death. The neuronal cell death induced by vinblastine was also attenuated by z-VAD-fmk, but not by antioxidants and NADPH oxidase inhibitors. Exposure the cortical cultures to taxol for 80 min formed neurite beadings visualized by fluorescence immunocytochemistry for tubulin. Treatment with either trolox or apocynin, an NADPH oxidase inhibitor, did not affect formation of the neurite beadings. RT-PCR and Western blot analysis revealed that exposure to taxol increased the expression of p47(phox) and gp91(phox) and induced translocation of the p47(phox) to the membrane in cortical cultures. Exposure to taxol markedly increased cellular 2,7-dichlorofluorescin diacetate fluorescence, an indicator for reactive oxygen species. Apocynin and trolox markedly inhibited the taxol-induced increase of the fluorescence. Moreover, treatment with NADPH oxidase inhibitors or suppression of gp91(phox) by siRNA significantly attenuated the taxol-induced neuronal death. These results indicate that taxol induces oxidative neuronal apoptosis by enhancing the activity of NADPH oxidase.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Córtex Cerebral/citologia , NADPH Oxidases/metabolismo , Neurônios/efeitos dos fármacos , Paclitaxel/farmacologia , ATPases Associadas a Diversas Atividades Celulares , Animais , Animais Recém-Nascidos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Cromanos/farmacologia , DNA Helicases/genética , DNA Helicases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Fármacos Neuroprotetores/farmacologia , Compostos Orgânicos , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Fatores de Tempo , Vimblastina/farmacologia
10.
Chonnam Med J ; 53(3): 196-202, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29026707

RESUMO

ß-Amyloid peptide (Aß) is the main component of senile plaques in patients with Alzheimer's disease, and is known to be a main pathogenic factor of the disease. Recent evidence indicates that activation of NADPH oxidase (NOX) in microglia or astrocytes may be a source of Aß-induced reactive oxygen species (ROS). We investigated the role of neuronal NOX in Aß-induced neuronal death in mouse mixed cortical cultures. Cell death was assessed by measuring lactate dehydrogenase efflux to bathing media 24 or 48 hr after exposure to Aß25-35, a fragment of Aß with an equivalent neurotoxic effect. Aß25-35 induced neuronal death in concentration- and time- dependent manners with apoptotic features. Neuronal death was significantly attenuated, not only by anti-apoptotic drugs, such as z-VAD-fmk and cycloheximide, but also by antioxidants, such as trolox, ascorbic acid, and epigallocatethin gallate. We also demonstrated that treatment with 20 µM Aß25-35 increased fluorescent signals in mixed cortical cultures, but produced only weak signals in pure astrocyte cultures in the presence of 2',7'-dichlorofluorescin diacetate (DCF-DA), an indicator for intracellular ROS. Increased DCF-DA fluorescence was markedly inhibited, not only by trolox, but also by selective NOX inhibitors, such as apocynin and AEBSF. Western blot analyses revealed that Aß25-35 increased the expression of gp91phox, a main subunit of NOX in cells. The above antioxidants, apocynin, and AEBSF significantly attenuated neuronal death induced by Aß25-35. Furthermore, the gp91phox-specific siRNA-based knockdown of NOX significantly inhibited neuronal death. These results suggest that activation of neuronal NOX is involved in Aß25-35-induced neuronal death.

11.
Free Radic Biol Med ; 112: 277-286, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28774816

RESUMO

Intracellular redox status influences the oxidation and enzyme activity of the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). Cumene hydroperoxide (CuHP), an organic hydroperoxide, is a known tumor promoter. However, molecular targets and action mechanism of CuHP in tumor promotion have not been well characterized. In this study, we investigated the effect of CuHP on the redox state of PTEN in HeLa cells. In addition, the intracellular reducing system of oxidized PTEN was analyzed using a biochemical approach and the effect of CuHP on this reducing system was also analyzed. While PTEN oxidized by hydrogen peroxide is progressively converted to its reduced form, PTEN was irreversibly oxidized by exposure to CuHP in HeLa cells. A combination of protein fractionation and mass analysis showed that the reducing system of PTEN was comprised of NADPH, thioredoxin reductase (TrxR), and thioredoxin (Trx). Although CuHP-mediated PTEN oxidation was not reversible in cells, CuHP-oxidized PTEN was reactivated by the exogenous Trx system, indicating that the cellular Trx redox system for PTEN is inactivated by CuHP. We present evidence that PTEN oxidation and the concomitant inhibition of thioredoxin by CuHP results in irreversible oxidation of PTEN in HeLa cells. In addition, ablation of peroxiredoxin (Prdx) enhanced CuHP-induced PTEN oxidation in cells. These results provide a new line of evidence that PTEN might be a crucial determinant of cell fate in response to cellular oxidative stress induced by organic hydroperoxides.


Assuntos
Derivados de Benzeno/farmacologia , Carcinógenos/farmacologia , Fibroblastos/efeitos dos fármacos , PTEN Fosfo-Hidrolase/química , Tiorredoxina Redutase 1/metabolismo , Tiorredoxinas/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , NADP/metabolismo , Oxirredução , Estresse Oxidativo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxina Redutase 1/genética , Tiorredoxinas/genética
12.
Sci Rep ; 7(1): 10222, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28860541

RESUMO

COACH syndrome is an autosomal recessive developmental disorder, a subtype of Joubert syndrome and related disorders, characterized by cerebellar vermis hypoplasia, oligophrenia, ataxia, coloboma, and hepatic fibrosis. Although mutations in TMEM67 (transmembrane protein 67)/MKS3 (Meckel-Gruber syndrome, type 3) were reported to cause COACH syndrome, this causality has not verified by functional studies. In a 20-year-old Korean man, we found cerebellar ataxia, isolated elevation in serum γ-glutamyl transpeptidase (γ-GTP) activity, oligophrenia, the molar tooth sign (MTS) in the brain MR images and congenital hepatic fibrosis (CHF). Two novel compound heterozygous mutations were found in TMEM67 in the patient: i) missense mutation (c.395 G > C and p.Gly132Ala) in exon 3, and ii) deletion in exon 26 (c.2758delT and p.Tyr920ThrfsX40). Western blotting showed that the p.Tyr920ThrfsX40 mutation accelerates turnover of the TMEM67 protein. Although wild-type human TMEM67 RNA rescued phenotypes of zebrafish embryos injected with anti-sense oligonucleotide morpholinos against tmem67, the two human TMEM67 RNAs individually harboring the two mutations did not. Finally, Wnt signaling, but not Hedgehog signaling, was suppressed in tmem67 morphants. To the best of our knowledge, this is the first report verifying the causality between COACH syndrome and TMEM67, which will further our understanding of molecular pathogenesis of the syndrome.


Assuntos
Anormalidades Múltiplas/genética , Ataxia/genética , Encéfalo/anormalidades , Colestase/genética , Coloboma/genética , Hepatopatias/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Anormalidades Múltiplas/tratamento farmacológico , Anormalidades Múltiplas/metabolismo , Animais , Ataxia/tratamento farmacológico , Ataxia/metabolismo , Encéfalo/metabolismo , Colestase/tratamento farmacológico , Colestase/metabolismo , Coloboma/tratamento farmacológico , Coloboma/metabolismo , Modelos Animais de Doenças , Células HEK293 , Humanos , Hepatopatias/tratamento farmacológico , Hepatopatias/metabolismo , Masculino , Morfolinos/administração & dosagem , Morfolinos/farmacologia , Mutação de Sentido Incorreto , Deleção de Sequência , Via de Sinalização Wnt , Adulto Jovem , Peixe-Zebra
13.
Cancer Res ; 64(12): 4235-43, 2004 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-15205336

RESUMO

We cloned recently an alternatively spliced variant of KAI1 mRNA that lacked exon 7 at the COOH-terminal region and showed differences in metastasis suppression when compared with the wild-type KAI1. These findings indicated that the COOH-terminal region of KAI1 is critical for its metastasis suppressor function. In this study, we isolated a cDNA clone of VANGL1, a member of the tetraspanin protein family, which interacted specifically with the COOH-terminal cytoplasmic domain of KAI1 in the yeast two-hybrid system. We renamed it KAI1 COOH-terminal interacting tetraspanin (KITENIN). We found that KITENIN-overexpressing CT-26 mouse colon cancer cells showed increased tumorigenicity and early hepatic metastasis in vivo, as well as increased invasiveness and adhesion to fibronectin in vitro compared with parental cells. Moreover, increased levels of KITENIN were observed in a human gastric tumor and its metastatic tissues, compared with the normal adjacent mucosa. Our results indicate that KITENIN promotes adhesion and invasion of cancer cells in vitro and in vivo, and suggest that KITENIN participates in the regulation of the tumor formation and metastasis by interacting with KAI1, a metastasis suppressor and antisense KITENIN strategy that can be used to inhibit metastasis in various cancers.


Assuntos
Antígenos CD , Proteínas de Transporte/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas , Neoplasias Gástricas/metabolismo , Processamento Alternativo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Matriz Extracelular/metabolismo , Genes Supressores de Tumor , Humanos , Proteína Kangai-1 , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/secundário , Glicoproteínas de Membrana/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Tetraspaninas
14.
PLoS One ; 10(4): e0124007, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25875631

RESUMO

Cell invasion is a crucial mechanism of cancer metastasis and malignancy. Matrix metalloproteinase-9 (MMP-9) is an important proteolytic enzyme involved in the cancer cell invasion process. High expression levels of MMP-9 in gastric cancer positively correlate with tumor aggressiveness and have a significant negative correlation with patients' survival times. Recently, mechanisms suppressing MMP-9 by phytochemicals have become increasingly investigated. Chrysin, a naturally occurring chemical in plants, has been reported to suppress tumor metastasis. However, the effects of chrysin on MMP-9 expression in gastric cancer have not been well studied. In the present study, we tested the effects of chrysin on MMP-9 expression in gastric cancer cells, and determined its underlying mechanism. We examined the effects of chrysin on MMP-9 expression and activity via RT-PCR, zymography, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin inhibited phorbol-12-myristate 13-acetate (PMA)-induced MMP-9 expression in a dose-dependent manner. Using AP-1 decoy oligodeoxynucleotides, we confirmed that AP-1 was the crucial transcriptional factor for MMP-9 expression. Chrysin blocked AP-1 via suppression of the phosphorylation of c-Jun and c-Fos through blocking the JNK1/2 and ERK1/2 pathways. Furthermore, AGS cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by chrysin and MMP-9 antibody. Our results suggest that chrysin may exert at least part of its anticancer effect by controlling MMP-9 expression through suppression of AP-1 activity via a block of the JNK1/2 and ERK1/2 signaling pathways in gastric cancer AGS cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 9 da Matriz/genética , Fator de Transcrição AP-1/genética , Anticorpos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Mucosa Gástrica/metabolismo , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais , Estômago/efeitos dos fármacos , Estômago/patologia , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/metabolismo
15.
Int J Oncol ; 46(4): 1835-43, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25625479

RESUMO

Cell invasion is one of crucial reasons for cancer metastasis and malignancy. Recepteur d'origine Nantais (RON) has been reported to play an important role in the cancer cell invasion process. High accumulation and activation of RON has been implicated in gastric adenocarcinoma AGS cells. Chrysin is a naturally occurring phytochemical, a type of flavonoid, which has been reported to suppress tumor metastasis. However, the effects of chrysin on RON expression in gastric cancer are not well studied. In the present study, we examined whether chrysin affects RON expression in gastric cancer, and if so, its underlying mechanism. We examined the effect of chrysin on RON expression and activity, via RT-PCR, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin significantly inhibited endogenous and inducible RON expression in a dose-dependent manner. After demonstrating that Egr-1 and NF-κB are the critically required transcription factors for RON expression, we discovered that chrysin suppressed Egr-1 and NF-κB transcription factor activities. Additionally, the phorbol-12-myristate-13-acetate- (PMA) induced cell invasion was partially abrogated by chrysin and an RON antibody. Our results suggest that chrysin has anticancer effects at least by suppressing RON expression through blocking Egr-1 and NF-κB in gastric cancer AGS cells.


Assuntos
Antineoplásicos/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/genética , Flavonoides/farmacologia , NF-kappa B/metabolismo , Receptores Proteína Tirosina Quinases/genética , Neoplasias Gástricas/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Humanos , NF-kappa B/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo
16.
Int J Oncol ; 45(4): 1760-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25069788

RESUMO

Cadmium exposure has been linked to human cancers, including stomach cancer. In this study, the effects of cadmium on urokinase-type plasminogen activator receptor (uPAR) expression in human gastric cancer cells and the underlying signal transduction pathways were investigated. Cadmium induced uPAR expression in a time- and concentration-dependent manner. Cadmium also induced uPAR promoter activity. Additionally, cadmium induced the activation of extracellular signal regulated kinase-1/2 (ERK-1/2), p38 mitogen-activated protein kinase (MAPK), and the activation of c-Jun amino terminal kinase (JNK). A specific inhibitor of MEK-1 (PD98059) inhibited cadmium-induced uPAR expression, while JNK and p38 MAPK inhibitors did not. Expression vectors encoding dominant-negative MEK-1 (pMCL-K97M) also prevented cadmium-induced uPAR promoter activity. Site-directed mutagenesis and electrophoretic mobility shift studies showed that sites for the transcription factors nuclear factor (NF)-κB and activator protein-1 (AP-1) were involved in cadmium-induced uPAR transcription. Suppression of the cadmium-induced uPAR promoter activity by a mutated-type NF-κB-inducing kinase and I-κB and an AP-1 decoy oligonucleotide confirmed that the activation of NF-κB and AP-1 are essential for cadmium-induced uPAR upregulation. Cells pretreated with cadmium showed markedly enhanced invasiveness and this effect was partially abrogated by uPAR-neutralizing antibodies and by inhibitors of ERK-1/2, NF-κB, and AP-1. These results suggest that cadmium induces uPAR expression via ERK-1/2, NF-κB, and AP-1 signaling pathways and, in turn, stimulates cell invasiveness in human gastric cancer AGS cells.


Assuntos
Cádmio/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Neoplasias Gástricas/metabolismo , Fator de Transcrição AP-1/metabolismo , Quinase Induzida por NF-kappaB
17.
Chonnam Med J ; 50(1): 6-14, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24855601

RESUMO

This study was designed to evaluate the efficacy of an orally administered aqueous extract of glutinous rice (GRE) to protect against acute gastric mucosal lesions induced by ethanol, indomethacin, and water immersion restraint stress in rats and to characterize the active substances responsible for the protection. GRE was shown to dose-dependently prevent the gastric lesions induced by the above ulcerogenic treatments at doses of 30 to 300 mg/kg. GRE treatment increased the gastric mucin content and partially blocked the ethanol-induced depletion of the gastric mucus layer. Also, it increased the nonprotein sulfhydryl concentration in the gastric mucosa. The gastroprotective action of GRE was markedly enhanced by co-treatment with 4-8 mg/kg tea extracts. The activity of GRE was completely lost by heat treatment at 80℃ for 3 min or treatment with 0.01% pepsin at 37℃ for 1 h. Protein extraction studies indicated that prolamins are involved in the gastroprotective activity of GRE. Our results suggest that glutinous rice proteins are useful for the prevention and treatment of gastritis and peptic ulcer.

18.
Mol Cells ; 35(6): 533-42, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23677378

RESUMO

Oxidative stress promotes damage to cellular proteins, lipids, membranes and DNA, and plays a key role in the development of cancer. Reactive oxygen species disrupt redox homeostasis and promote tumor formation by initiating aberrant activation of signaling pathways that lead to tumorigenesis. We used shotgun proteomics to identify proteins containing oxidation-sensitive cysteines in tissue specimens from colorectal cancer patients. We then compared the patterns of cysteine oxidation in the membrane fractions between the tumor and non-tumor tissues. Using nano-UPLC-MS(E) proteomics, we identified 31 proteins containing 37 oxidation-sensitive cysteines. These proteins were observed with IAM-binding cysteines in non-tumoral region more than tumoral region of CRC patients. Then using the Ingenuity pathway program, we evaluated the cellular canonical networks connecting those proteins. Within the networks, proteins with multiple connections were related with organ morphology, cellular metabolism, and various disorders. We have thus identified networks of proteins whose redox status is altered by oxidative stress, perhaps leading to changes in cellular functionality that promotes tumorigenesis.


Assuntos
Neoplasias Colorretais/metabolismo , Cisteína/metabolismo , Redes e Vias Metabólicas , Estresse Oxidativo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Biologia Computacional , Simulação por Computador , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Oxirredução , Proteômica/métodos
19.
Korean J Med Educ ; 24(3): 233-40, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25813132

RESUMO

PURPOSE: A considerable number of medical students drop out due to low academic achievement, and these students have a high probability of repeated failure experiences. This study investigated the personal and academic problems of these students to help develop student support systems. METHODS: First-year (n=146) and second-year (n=119) medical students were asked to complete questionnaires. The questionnaires consisted of personality traits and the students' management of/satisfaction with school life. RESULTS: Students who had already dropped out accounted for 17.4% of the study subjects. The most common reason for dropping out was low academic achievement, and the most difficult part of taking a leave of absence from school was psychological anxiety. The group who dropped out had significantly lower levels of emotional stability, sociability, responsibility, dominance, masculinity, and superiority and more vulnerable mental states compared with those who did not drop out. They also expressed less motivation with regard to medical science and less satisfaction with school life than did the group that did not drop out. Those who dropped out tended not to prepare for exams, and they managed their time ineffectively. They also tried to resolve their difficulties alone and rarely sought help from teachers. CONCLUSION: More intimate student-teacher relationships should be established, and teachers should be encouraged to meet and interact with their students on a regular basis. Additionally, personality inventories should be used to assist in efforts to understand students, especially to identify hidden social and emotional problems.

20.
Redox Rep ; 16(4): 181-6, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21888769

RESUMO

Exposure of cells to hydrogen peroxide or platelet-derived growth factor (PDGF) induced Akt phosphorylation and oxidation of phosphatase and tensin homolog (PTEN). The Cys124 and Cys71 residues of PTEN were critical for the formation of a disulfide bond and the intermediate glutathionylation in the process of reduction of the disulfide bond. To determine which specific tyrosine residues of the PDGF beta receptor (PDGFßR) is involved in PDGF-induced PTEN oxidation and Akt phosphorylation, we investigated a kinase activity-deficient mutant and PDGFßR mutants where the tyrosine residues in the binding site for phosphoinositide 3-kinase (PI3K), GTPase-activating protein of Ras, Src homology 2 domain containing protein-tyrosine phosphatase-2, and phospholipase C-1 were replaced by Phe. Both PTEN oxidation and Akt phosphorylation did not occur in response to PDGF in the kinase-deficient mutant and in the PDGFßR mutant with a mutation in the PI3K binding site (Tyr740 and Tyr751). Thus, the kinase activity and the constituent Tyr740 and Tyr751 residues of PDGFßR in the cells stimulated with PDGF are responsible for the oxidation of PTEN and the Akt phosphorylation.


Assuntos
Peróxido de Hidrogênio/farmacologia , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/genética , Sítios de Ligação/genética , Técnicas de Cultura de Células , Ativação Enzimática , Glutationa/metabolismo , Humanos , Mutação , Oxirredução/efeitos dos fármacos , PTEN Fosfo-Hidrolase/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos
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