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2.
Braz. j. med. biol. res ; 47(12): 1021-1028, 12/2014. tab, graf
Artículo en Inglés | LILACS | ID: lil-727663

RESUMEN

DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.


Asunto(s)
Animales , Humanos , Masculino , Antineoplásicos/farmacología , Metilación de ADN/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , S-Adenosilmetionina/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Factor C de Crecimiento Endotelial Vascular/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Carcinogénesis/efectos de los fármacos , Metilación de ADN/genética , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/fisiología , Xenoinjertos/efectos de los fármacos , Inmunohistoquímica , Ratones Desnudos , Oncogenes/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , ARN Mensajero/análisis , Neoplasias Gástricas/metabolismo , Factor C de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor C de Crecimiento Endotelial Vascular/genética
3.
J Biosci ; 2008 Jun; 33(2): 269-77
Artículo en Inglés | IMSEAR | ID: sea-111352

RESUMEN

Most drugs and xenobiotics induce the expression of cytochrome P450 (CYP) enzymes, which reduce the bioavailability of the inducer and/or co-administered drugs. Therefore, evaluation of new drug candidates for their effect on CYP expression is an essential step in drug development. The available methods for this purpose are expensive and not amenable to high-throughput screening. We developed a fluorescence-based in vivo assay using transgenic Caenorhabditis elegans worms that express the green fluorescent protein (GFP) under the control of various CYP promoters. Using this assay, we found striking similarities between the worm CYPs and their human orthologs in their response to treatment with various drugs. For example,the antibiotic rifampicin, one of the strongest inducers of the human gene CYP3A4, was the strongest inducer of the worm ortholog CYP13A7. Since worms can be easily grown in liquid medium in microtitre plates, the assay described in this paper is suitable for the screening of a large number of potential lead compounds in the drug discovery process.


Asunto(s)
Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente/genética , Secuencia de Bases , Caenorhabditis elegans/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/química , ADN de Helmintos , Evaluación Preclínica de Medicamentos/métodos , Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/efectos de los fármacos , Homología de Secuencia de Aminoácido
4.
Experimental & Molecular Medicine ; : 195-204, 2007.
Artículo en Inglés | WPRIM | ID: wpr-90613

RESUMEN

The BubR1 mitotic-checkpoint protein monitors proper attachment of microtubules to kinetochores, and links regulation of chromosome-spindle attachment to mitotic-checkpoint signaling. Thus, disruption of BubR1 activity results in a loss of checkpoint control, chromosomal instability caused by a premature anaphase, and/or the early onset of tumorigenesis. The mechanisms by which deregulation and/or abnormalities of BubR1 expression operate, however, remain to be elucidated. In this study, we demonstrate that levels of BubR1 expression are significantly increased by demethylation. Bisulfite sequencing analysis revealed that the methylation status of two CpG sites in the essential BubR1 promoter appear to be associated with BubR1 expression levels. Associations of MBD2 and HDAC1 with the BubR1 promoter were significantly relieved by addition of 5-aza-2'-deoxycytidine, an irreversible DNA methyltransferase inhibitor. However, genomic DNA isolated from 31 patients with colorectal carcinomas exhibited a +84A/G polymorphic change in approximately 60% of patients, but this polymorphism had no effect on promoter activity. Our findings indicate that differential regulation of BubR1 expression is associated with changes in BubR1 promoter hypermethylation patterns, but not with promoter polymorphisms, thus providing a novel insight into the molecular regulation of BubR1 expression in human cancer cells.


Asunto(s)
Humanos , Azacitidina/farmacología , Secuencia de Bases , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Histona Desacetilasas/metabolismo , Células Jurkat , Datos de Secuencia Molecular , Neoplasias/genética , Polimorfismo Genético/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Transcripción Genética/efectos de los fármacos
5.
Experimental & Molecular Medicine ; : 445-452, 2006.
Artículo en Inglés | WPRIM | ID: wpr-200504

RESUMEN

We investigated the effect of tilianin upon inducible nitric oxide synthesis in the plasma of low-density lipoprotein receptor knock-out (Ldlr-/-) mice fed with high cholesterol diet and in primary peritoneal macrophages of Ldlr-/- mice. High cholesterol diet induced nitric oxide production in the plasma of Ldlr-/- mice. Tilianin reduced the level of nitric oxide (NO) in plasma from Ldlr-/- mice induced by the high cholesterol diet. Tilianin also inhibited the NO production from the primary culture of peritoneal macrophages treated with lipopolysaccharide. The inhibition of NO production was caused by the suppression of inducible nitric oxide synthase (iNOS) gene expression in peritoneal macrophages isolated from Ldlr-/- mice. Moreover, tilianin inhibited the transcriptional activation of iNOS promoter that has NF-kappa B binding element. Thus, these results provide the first evidence that tilianin inhibit iNOS expression and production of NO and may act as a potential anti-inflammatory agent.


Asunto(s)
Ratones , Masculino , Animales , Tirosina/análogos & derivados , Distribución Tisular , Seno Aórtico/metabolismo , Receptores de LDL/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico/biosíntesis , FN-kappa B/metabolismo , Ratones Noqueados , Inflamación/metabolismo , Glicósidos/farmacología , Flavonoides/farmacología , Regulación hacia Abajo/efectos de los fármacos , Aterosclerosis/metabolismo
6.
Experimental & Molecular Medicine ; : 204-210, 2004.
Artículo en Inglés | WPRIM | ID: wpr-217501

RESUMEN

Mammalian epithelia produce the various antimicrobial peptides against the bacterial or viral infection, thereby acting as the active immune modulators in the innate immunity. In this study, we examined the effects of the various proinflammatory cytokines or LPS on cell viability and antimicrobial beta-defensin gene expressions in human corneal epithelial cells. Results showed that the cytokines or LPS did not exert severe cytotoxic effects on the cells, and that beta-defensin 1 was constitutively expressed, while beta-defensin 2 was specifically induced by IL-1beta, supporting the idea that these cytokines or LPS involve the defense mechanism in the cornea. Furthermore, the reporter and gel shift assay to define the induction mechanism of beta-defensin 2 by IL-1beta demonstrated that the most proximal NF-kB site on the promoter region of beta-defensin 2 was not critical for the process. Data obtained from the normal or patients with the varying ocular diseases showed that our in vitro results were relevant in the clinical settings. Our results clearly demonstrated that beta-defensin 1 and 2 are important antimicrobial peptides in the corneal tissues, and that the mechanistic induction process of beta-defensin 2 by IL-1beta is not solely dependent on proximal NF-kB site activation, thus suggesting that the long distal portion of the promoter is needed for the full responsiveness toward IL-1beta.


Asunto(s)
Humanos , Unión Competitiva , Supervivencia Celular , Células Cultivadas , Enfermedades de la Córnea/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Epitelio Corneal/efectos de los fármacos , Expresión Génica , Interferón gamma/metabolismo , Interleucina-1/farmacología , Lipopolisacáridos/metabolismo , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , beta-Defensinas/biosíntesis
7.
Experimental & Molecular Medicine ; : 380-386, 2004.
Artículo en Inglés | WPRIM | ID: wpr-119638

RESUMEN

The early growth response gene-1 (Egr-1) is a tumor suppressor which plays an important role in cell growth, differentiation and apoptosis. Egr-1 has been shown to be down-regulated in many types of tumor tissues. Trifluoperazine (TFP), a phenothiazine class of antipsychotics, restored serum-induced Egr-1 expression in several cancer cell lines. We investigated the effect of Egr-1 expression on the TFP-induced inhibition of cell growth. Ectopic expression of Egr-1 enhanced the TFP-induced antiproliferative activity and downregulated cyclin D1 level in U87MG glioma cells. Our results suggest that antipsychotics TFP exhibits antiproliferative activity through up-regulation of Egr-1.


Asunto(s)
Humanos , Ciclo Celular , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Proteínas de Unión al ADN/genética , Expresión Génica , Glioma/metabolismo , Proteínas Inmediatas-Precoces/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Factores de Transcripción/genética , Trifluoperazina/farmacología , Células Tumorales Cultivadas
8.
Experimental & Molecular Medicine ; : 23-29, 2003.
Artículo en Inglés | WPRIM | ID: wpr-77001

RESUMEN

Mammals have two major isoforms of acetyl-CoA carboxyase (ACC). The 275 kDa beta-form (ACC beta) is predominantly in heart and skeletal muscle while the 265 kDa alpha-form (ACC alpha) is the major isoform in lipogenic tissues such as liver and adipose tissue. ACC alpha is thought to control fatty acid oxidation by means of the ability of malonyl-CoA to inhibit carnitine palmitoyl-CoA transferase-1 (CPT-1), which is a rate-limiting enzyme of fatty acid oxidation in mitochondria. Previously, it was reported that MyoD and other muscle regulating factors (MRFs) up-regulate the expression of ACC beta by interactions between these factors and several cis-elements of ACC beta promoter. We described here that ACC beta expression mediated by MRFs is regulated by retinoic acids. Endogenous expression of ACCb in differentiated H9C2 myotube was significantly increased by retinoic acid treatment. However, on transient transfection assay in H9C2 myoblast, ACC beta promoter activity was suppressed by RXRa and more severely by RAR alpha. These effects on ACCb expression in myoblasts and myotubes by RXR alpha and RAR alpha seem to be mediated by their interactions with MRFs because no consensus sequence for RXR alpha and RAR alpha has been found in ACC beta promoter and retinoic acid receptors did not affect this promoter activities by itself. In transient transfection in NIH3T3 fibroblast, the activation of ACC beta promoter by MyoD, main MRF in myoblast, was significantly suppressed by RAR alpha and to a less extent by RXR alpha while the RXR alpha drastically augmented the activation by MRF4, major MRF in myotube. These results explained that retinoic acids differentially affected the action of MRFs according to their types and RXR alpha specially elevates the expression of muscle specific genes by stimulating the action of MRF4.


Asunto(s)
Animales , Ratones , Células 3T3 , Acetil-CoA Carboxilasa/genética , Diferenciación Celular , Células Cultivadas , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteína MioD/metabolismo , Mioblastos/efectos de los fármacos , Factores Reguladores Miogénicos/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Receptores de Ácido Retinoico/genética , Activación Transcripcional , Factores de Transcripción/genética , Tretinoina/farmacología
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