RESUMEN
Astaxanthin (AST) is a product made from marine organisms that has been used as an anti-cancer supplement. It reduces pontin expression and induces apoptosis in SKBR3, a breast cancer cell line. Using Western blotting and qRT-PCR analyses, this study revealed that in the T47D and BT20 breast cancer cell lines, AST inhibits expression of pontin and mutp53, as well as the Oct4 and Nanog cancer stem cell (CSC) stemness genes. In addition, we explored the mechanism by which AST eradicates breast cancer cells using pontin siRNAs. Pontin knockdown by pontin siRNA reduced proliferation, Oct4 and Nanog expression, colony and spheroid formation, and migration and invasion abilities in breast cancer cells. In addition, reductions in Oct4, Nanog, and mutp53 expression following rottlerin treatment confirmed the role of pontin in these cells. Therefore, pontin may play a central role in the regulation of CSC properties and in cell proliferation following AST treatment. Taken together, these findings demonstrate that AST can repress CSC stemness genes in breast cancer cells, which implies that AST therapy could be used to improve the efficacy of other anti-cancer therapies against breast cancer cells.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Portadoras/genética , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN Helicasas/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Invasividad Neoplásica , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteína p53 Supresora de Tumor/genética , Xantófilas/farmacologíaRESUMEN
Astaxanthin (AST) is related to apoptosis but the details of the mechanism of how AST makes apoptosis is not clear. The present study investigated apoptotic effects of AST to SKBR3, a breast cancer cell line in detail. Cell viability assay showed cellular proliferation and morphological changes of the cells were observed under AST treatment. FACS analysis indicated that AST blocked cell cycle progression at G0/G1, suppressed proliferation dose-dependently, and induced apoptosis of the cells. The apoptosis of the cells by AST was further demonstrated through the decreased expression level of mutp53 and cleaved a PARP-1 fragment, respectively. In addition, AST induced the intrinsic apoptosis of the cells by activation of Bax/Bcl2, cleaved caspase-3, and cleaved caspase-9 as well as the phosphorylation of ERK1/2, JNK, and p38. Furthermore, AST decreased production of intracellular reactive oxygen species as well as modulated expressions of superoxide dismutases and Pontin, an anti-apoptotic factor. Co-immunoprecipitation assay revealed AST reduced interaction between Pontin and mutant p53. Taken together, these studies proved that AST regulates the expression of apoptotic molecules to induce intrinsic apoptosis of the cells, suggesting AST therapy might provide an alternative for improving the efficacies of other anti-cancer therapies for breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Línea Celular Tumoral/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Xantófilas/farmacología , Xantófilas/uso terapéutico , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Rhus verniciflua Stokes has long been used as a food supplement and traditional herbal medicine for various ailments in East Asia. We evaluated the anticancer effects of Rhus verniciflua Stokes extract (RVSE) on MCF-7 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, annexin V/7-AAD staining, and western blotting. In addition, the gallic acid content of RVSE was assayed using high-performance liquid chromatography. RVSE inhibited the growth of MCF-7 cells in a dose-dependent manner by inducing apoptosis in the sub-G1 phase. RVSE also significantly increased the number of apoptotic cells and increased the expression of p53 and p21 in a dose-dependent manner. Furthermore, RVSE treatment increased the Bax:Bcl-2 ratio and the levels of apoptosis-related factors, such as cleaved caspase-3 and -9 and PARP, in MCF-7 cells. Our findings suggest that the proapoptotic effect of RVSE on MCF-7 cells is mediated by p53, p21, and the intrinsic mitochondrial cascade. Thus, RVSE shows promise for the prevention and treatment of breast cancer.
RESUMEN
Astaxanthin (AST) is known to exhibit antioxidative and antitumor properties, therefore, the present study investigated its other potential medical applications. AST was observed to exhibit antiallergic and antiinflammatory effects in a dinitrofluorobenzene (DNFB)induced contact dermatitis (CD) mouse model and RBL2H3 cell lines. The topical application of AST effectively inhibited the enlargement of ear thickness and increase in weight, which occurred following repeated application of DNFB. Furthermore, topical application of different concentrations of AST inhibited inflammatory hyperplasia, edema, spongiosis, and the infiltration of mononuclear cells and mast cells in the ear tissue. In addition, the levels of TNFα and IFNγ produced were decreased by application of AST in vivo, and treatment of RBL2H3 cells with AST inhibited the release of histamine and ßhexosaminidase in vitro. Taken together, these data suggested that AST may be used to treat patients with allergic skin diseases through a mechanism, which may be associated with that involved in antiinflammatory or anti-allergic activities.
Asunto(s)
Antiinflamatorios/uso terapéutico , Dermatitis por Contacto/tratamiento farmacológico , Dermatitis por Contacto/patología , Dinitrofluorobenceno , Animales , Degranulación de la Célula/efectos de los fármacos , Línea Celular , Dermatitis por Contacto/inmunología , Oído/patología , Humanos , Interferón gamma/análisis , Interferón gamma/inmunología , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/patología , Ratones Endogámicos BALB C , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/inmunología , Xantófilas/uso terapéuticoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The root bark of Dictamnus dasycarpus Turcz., family Rutaceae is a well known anti-inflammatory agent for skin diseases such as eczema, pruritus and urticaria in Eastern countries. MATERIALS AND METHODS: We investigated the effects of methanol extract of Dictamnus dasycarpus root bark (MEDD) on Intercellular Adhesion Molecule-1 (ICAM-1) expression, epidermal hyperplasia and immune cell infiltration in 1-fluoro-2,4-dinitrofluorobenzene (DNFB)-induced contact dermatitis (CD) mice. We also investigated its effects on the expression of ICAM-1, binding capacity to THP-1 cells, cytokine and chemokine production, and phosphorylation of NF-κB in human keratinocytes (HaCaT cells). RESULTS: Topical application of MEDD effectively inhibited ICAM-1 expression and epidermal hyperplasia in inflamed tissues. MEDD treatment also inhibited immune cell infiltration induced by DNFB. In addition, treatment with MEDD reduced surface expression and total amount of ICAM-1in HaCaT cells and effectively lowered the capacity to bind to THP-1 cells. MEDD also lowered the levels of IL-6, IL-8, monokine induced by gamma interferon (MIG), monocyte chemotactic protein-1 (MCP-1) and regulated on activation, normal T cell expressed and secreted (RANTES). Finally, MEDD treatment prevented activation of the NF-κB pathway induced by TNF-α in HaCaT cells. CONCLUSIONS: These data indicate that root bark of Dictamnus dasycarpus has the potential for treatment of inflammatory skin diseases as a complementary or alternative medicine to corticosteroids. In addition, they suggest that the anti-inflammatory effects of Dictamnus dasycarpus on CD are involved in the regulation of ICAM-1 expression and cytokine and chemokine secretion through down-regulation of the NF-κB signaling pathway in keratinocytes.
Asunto(s)
Antiinflamatorios/farmacología , Citocinas/metabolismo , Dictamnus , Molécula 1 de Adhesión Intercelular/metabolismo , Extractos Vegetales/farmacología , Animales , Antiinflamatorios/uso terapéutico , Adhesión Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dermatitis por Contacto/tratamiento farmacológico , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/metabolismo , Dermatitis por Contacto/patología , Dinitrofluorobenceno , Oído/patología , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/fisiología , Masculino , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Fitoterapia , Corteza de la Planta , Extractos Vegetales/uso terapéutico , Raíces de Plantas , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Schizandrae fructus (SF), the fruit of Schisandra chinensis, has been used for the treatment of cough, wheezing, dry mouth, hepatitis, cardiovascular disease, and as a tonic and astringent in China, Japan, and Korea. OBJECTIVE: Investigation of the antiasthmatic effects of SF. MATERIALS AND METHODS: We investigated the effects of SF on airway hyperresponsiveness (AHR) to methacholine, production levels of antigen-specific antibodies, and histopathological changes in the lung tissue in a mouse model (Balb/c) of asthma induced by repeated intranasal instillation of an antigen. RESULTS: SF lowered AHR to methacholine (P < 0.05), antigen-specific immunoglobulin E (IgE) level (P < 0.01), and immune cell infiltration in mice with asthma. Prednisolone (PD) effectively decreased AHR (P < 0.01), total antibody (P < 0.01) and IgE (P < 0.01) levels, and immune cell infiltration. SF and PD did not affect the levels of antigen-specific IgG1 and IgG2a antibodies. CONCLUSION: Our data suggest that SF has possible application as an antiasthmatic drug. We also suggest that SF could be used as a complementary or alternative medicine to glucocorticoids.
RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The root bark of Dictamnus dasycarpus Turcz. is widely used as a medicinal herb for treatment of skin diseases such as eczema, pruritus and urticaria in China, Japan and Korea. MATERIALS AND METHODS: We investigated the effects of methanol extract of Dictamnus dasycarpus Turcz., root bark (MEDD) on ear thickness, ear weights, histopathological changes such as hyperplasia, edema, spongiosis and immune cell infiltration and cytokine productions in 1-fluoro-2,4-dinitrofluorobenzene (DNFB)-induced contact dermatitis (CD) mice. We also investigated its effects on degranulation of histamine and ß-hexosaminidase and related mechanisms using RBL-2H3 cells. RESULTS: Topical application of MEDD effectively inhibited enlargement of ear thickness and weight (P<0.05). MEDD treatment also inhibited hyperplasia, edema and spongiosis induced by DNFB. Treatment with 300 µg/ear of MEDD suppressed the increase in IFN-γ and TNF-α levels (P<0.05). In addition, treatment with >50 µg/mL MEDD reduced the level of ß-hexosaminidase release, while >100 µg/mL MEDD lowered the level of histamine release in a dose-dependent manner (P<0.05). Finally, MEDD treatment prevented phosphorylation of p38 MAPK induced by phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 in RBL-2H3 cells. CONCLUSIONS: These data indicate that root bark of Dictamnus dasycarpus Turcz. has the potential for use in the treatment of allergic skin diseases. Furthermore, they suggest that root bark of Dictamnus dasycarpus Turcz. is involved in decreasing degranulation of MCs via inhibition of the p38 MAPK pathway as well as in the inhibition of Th1 skewing reactions.
Asunto(s)
Antialérgicos/uso terapéutico , Antiinflamatorios/uso terapéutico , Dermatitis Alérgica por Contacto/tratamiento farmacológico , Dictamnus , Extractos Vegetales/uso terapéutico , Animales , Antialérgicos/farmacología , Antiinflamatorios/farmacología , Proliferación Celular/efectos de los fármacos , Dermatitis Alérgica por Contacto/etiología , Dermatitis Alérgica por Contacto/patología , Dinitrofluorobenceno , Oído/patología , Edema/tratamiento farmacológico , Edema/patología , Histamina/metabolismo , Interferón gamma/metabolismo , Masculino , Metanol/química , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Corteza de la Planta , Extractos Vegetales/farmacología , Raíces de Plantas , Solventes/química , Factor de Necrosis Tumoral alfa/metabolismo , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The dried root of Sophora flavescens Aiton (Sophorae radix, SR) has long been used in traditional medicine for the treatment of fever and swelling in eastern countries. MATERIALS AND METHODS: The present study investigated the anti-allergic and anti-inflammatory effects of SR using 1-fluoro-2,4-dinitrofluorobenzene (DNFB)-induced contact dermatitis mouse model and in vitro using RBL-2H3 cells. RESULTS: In mice, the topical application of 10 mg/mL of SR effectively inhibited enlargement of ear thickness and weight induced by repeated painting with DNFB. Topical application of SR also inhibited hyperplasia, edema, spongiosis and infiltration of mononuclear cells in ear tissue. In addition, production levels of interferon-gamma and tumor necrosis factor-alpha were decreased by SR in vivo. Finally, the release of histamine and ß-hexosaminidase, and migration were inhibited by treatment with SR. CONCLUSIONS: These data indicate the potential of SR in treating patients with allergic skin diseases and also suggest that related mechanisms are involved in anti-inflammatory action on the Th 1 skewing reaction and inhibition against recruitment and degranulation of mast cells.
Asunto(s)
Antialérgicos/uso terapéutico , Antiinflamatorios/uso terapéutico , Dermatitis Alérgica por Contacto/tratamiento farmacológico , Edema/tratamiento farmacológico , Extractos Vegetales/uso terapéutico , Sophora , Animales , Antialérgicos/farmacología , Antiinflamatorios/farmacología , Degranulación de la Célula/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Dermatitis Alérgica por Contacto/inmunología , Dinitrofluorobenceno , Edema/inducido químicamente , Edema/inmunología , Edema/patología , Liberación de Histamina , Interferón gamma/inmunología , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/fisiología , Ratones , Ratones Endogámicos BALB C , Fitoterapia , Extractos Vegetales/farmacología , Raíces de Plantas , Ratas , Factor de Necrosis Tumoral alfa/inmunología , beta-N-Acetilhexosaminidasas/inmunologíaRESUMEN
Plasminogen activator inhibitor-1 (PAI-1) is an important regulator of numerous pathophysiological processes such as inflammation, thrombosis, angiogenesis and tumor metastasis. Its expression is induced by hypoxia at the transcriptional level, via the hypoxia inducible factor-1 (HIF-1) or -2 (HIF-2). In this study, we elucidated the mechanism of transcriptional regulation of mouse PAI-1 gene by hypoxia in mouse hepatoma cells. We searched for hypoxia response elements (HREs) of murine PAI-1 promoter using several molecular biological assays. DNAse I hypersensitivity assay first suggested that PAI-1 gene expression is up-regulated by protein-DNA interactions at the -3.6- and -3-kb upstream regions of the PAI-1 gene transcription start site. An approximately 6.4-kb region of DNA containing the 5'-flanking promoter region of the PAI-1 gene was isolated, mapped, and cloned into reporter gene assay vectors and sequenced. Luciferase reporter gene assay subsequently identified two functional HREs, located around -3.6 kb of the 5'-flanking promoter region of PAI-1 gene that were responsible for the enhancement of luciferase reporter gene activity. Mutation of the HREs in this fragment abolished luciferase reporter gene activity. Finally, in vitro and in vivo protein-DNA interaction assays confirmed binding of the two HREs to HIF-1 or HIF-2 protein. Our results show that two HREs located around -3.6 kb of the 5'-flanking promoter region of the mouse PAI-1 gene function as hypoxia enhancers, which, alongside other regulatory regions, control PAI-1 gene transcription by HIF-1 or HIF-2 under hypoxic environments in mouse hepatoma cells.
Asunto(s)
Carcinoma Hepatocelular/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Neoplasias Hepáticas/genética , Elementos de Respuesta/fisiología , Roedores/genética , Serpina E2/genética , Factores de Transcripción/fisiología , Animales , Carcinoma Hepatocelular/metabolismo , Hipoxia de la Célula/genética , Células Cultivadas , Femenino , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Especificidad de la Especie , Factores de Transcripción/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
In order to reduce of the manufacturing cost of bacterial cellulose (BC), BC production by Acetobacter sp. V6 was investigated in shaking culture using molasses and corn steep liquor (CSL) as the sole carbon and nitrogen sources, respectively. The highest BC production was obtained with Ca3(PO4)2-treated molasses. Maximum BC yield (2.21+/-0.04 g/l) was obtained at 5% (w/v) total sugar in molasses. In improved medium containing molasses and CSL, BC production was observed in the medium after 1 day of incubation and increased rapidly thereafter with maximum yield (3.12+/-0.03 g/l) at 8 days. This value was approximately twofold higher than the yield in the complex medium. Physical properties of BC from the complex and molasses media were studied using Fourier-transform infrared (FT-IR) spectroscopy and X-ray diffractometer. By FT-IR, all the BC were found to be of cellulose type I, the same as typical native cellulose. The relative crystallinity of BC produced in the complex and molasses media were 83.02 and 67.27%, respectively. These results suggest that molasses and CSL can be useful low-cost substrates for BC production by Acetobacter sp. V6 without supplementation with expensive nitrogen complexes such as yeast extract and polypeptone, leading to the reduction in the production costs.
Asunto(s)
Acetobacter/enzimología , Celulosa/metabolismo , Medios de Cultivo/metabolismo , Melaza , Zea mays/metabolismo , Celulosa/química , Análisis Costo-Beneficio , Medios de Cultivo/economía , Concentración de Iones de Hidrógeno , Melaza/economía , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos XRESUMEN
Membrane proteins possess significant hydrophobic domains and are likely to deplete their native activity immobilized on the solid surface relative to those occurring in a membrane environment. To investigate an efficient immobilization method, calix[4]crown-ether monolayer as an artificial protein linker system was constructed on the gold surface and characterized by Fourier transform infrared reflection absorption spectroscopy (FTIR-RAS), atomic force microscopy (AFM) and cyclic voltammetry (CV). Integrin alpha(v)beta3 was functionally immobilized onto the monolayer and the integrin-vitronectin interaction was investigated by surface plasmon resonance (SPR). It was found that calix[4]crown-ether was assembled as a monolayer on the gold surface. Orientation and accessibility of integrin alpha(v)beta3 was assessed by sensitive binding of its natural ligand, vitronectin at pg mL(-1) level. Moreover, surface coverage of integrin layer and thickness calculated through SPR curve simulation verified that integrin layer was a monolayer in activated form. In combination with the SPR method, this calix[4]crown monolayer provided a reliable and simple experimental platform for the investigation of isolated membrane proteins under experimental conditions resembling those of their native properties.
Asunto(s)
Calixarenos/química , Integrina alfaVbeta3/química , Vitronectina/química , Electroquímica , Microscopía de Fuerza Atómica , Estructura Molecular , Espectroscopía Infrarroja por Transformada de Fourier , Resonancia por Plasmón de Superficie/métodosRESUMEN
Redox sensitive, pro-inflammatory nuclear transcription factor NF-kappaB plays a key role in both inflammation and aging processes. In a redox state disrupted by oxidative stress, pro-inflammatory genes are upregulated by the activation of NF-kappaB through diverse kinases. Thus, the search and characterization of new substances that modulate NF-kappaB are of recent research interest. Cinnamaldehyde (CNA) is the major component of cinnamon bark oil, which has been widely used as a flavoring agent in foodstuffs such as beverages and ice cream. In the present study, CNA was examined for its molecular modulation of inflammatory NF-kappaB activation via the redox-related NIK/IKK and MAPK pathways through the reduction of oxidative stress. Results show that age-related NF-kappaB activation upregulated NF-kappaB targeting genes, inflammatory iNOS, and COX-2, all of which were inhibited effectively by CNA. Our study further shows that CNA inhibited the activation of NF-kappaB via three signal transduction pathways, NIK/IKK, ERK, and p38 MAPK. Our results indicate that CNA's antioxidative effect and the restoration of redox balance were responsible for its anti-inflammatory action. Thus, the significance of the current study is the new information revealing the anti-inflammatory properties of CNA and the role it plays in the regulation of age-related alterations in signal transduction pathways.
Asunto(s)
Acroleína/análogos & derivados , Envejecimiento/metabolismo , Antioxidantes/farmacología , FN-kappa B/metabolismo , Acroleína/farmacología , Envejecimiento/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Aromatizantes/farmacología , Quinasa I-kappa B/efectos de los fármacos , Quinasa I-kappa B/fisiología , Masculino , FN-kappa B/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas Endogámicas F344 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
HIF-1alpha is a transcription factor that acts as a master regulator of gene expression induced by hypoxia. Recent studies have demonstrated that the potent inflammatory factor, lipopolysaccharide (LPS), can also activate HIF-1alpha in myeloid cells. However, the molecular mechanisms at the transcriptional level of HIF-1alpha induction by LPS remained undefined. Here, we investigated the regulatory mechanism of HIF-1alpha expression by LPS in hepatocytes and identified that LPS-induced HIF-1alpha mediate gene transcription of a typical inflammatory mediator, tumor-necrosis factor alpha (TNFalpha). Increased HIF-1alpha gene expression by LPS was defined in a series of hepatic cell lines by RT-PCR, Western blotting and promoter transactivation assay. The JNK signaling and c-Jun activation were required to induce the HIF-1alpha gene transcription by LPS. The finding that a cascade transcriptional activation of distinct set of transcription factors, c-Jun and HIF-1alpha, in response to LPS stimulation associates with induction of TNFalpha gene transcription lends new insights into the functional mechanisms by which complex patterns of gene regulation on LPS-derived HIF activation are achieved.
Asunto(s)
Regulación de la Expresión Génica/genética , Hepatocitos/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/fisiopatología , Hipoxia de la Célula/fisiología , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Hepatocitos/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inflamación/inducido químicamente , Inflamación/fisiopatología , Mediadores de Inflamación/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif (10)PQENDE(15) in STP-A11 reveals that Glu (E)(12) residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.
Asunto(s)
Herpesvirus Saimiriino 2/metabolismo , FN-kappa B/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Línea Celular , Detergentes , Humanos , Iones , FN-kappa B/agonistas , Unión Proteica , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Factor de Transcripción STAT3/metabolismo , Solubilidad , Factor de Transcripción AP-1/agonistasRESUMEN
Until now, it is known that hypoxia increases the glycolytic enzyme expression at the transcriptional level. Here, we show evidence that hypoxia increases hepatic glucose output and HIF-1 and ATF-2-mediated transactivation of phosphoenolpyruvate carboxykinase (PEPCK), which plays a critical role as a rate-limiting enzyme in gluconeogenesis, gene in liver. HIF-1 directly bound to the specific PEPCK promoter region through its cognate binding element and found as an active complex with coactivator CBP. Additionally, ATF-2 was also involved to regulate hypoxia-dependent PEPCK transcription in the transcriptional complex with HIF-1 and CBP. Interestingly, retinoic acid (RA) signaling induced the recruitment of HIF-1 on the PEPCK promoter, resulting from the functional interaction of HIF-1 and ATF-2 with coactivator CBP. Taken together, these results suggest that hypoxia signaling leads the hepatic glucose production and release via the increased gene expression of gluconeogenic enzymes, possibly playing a role in providing glucose to other tissues, such as endothelial, brain and muscle cells.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Gluconeogénesis , Hipoxia/metabolismo , Hígado/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Activación Transcripcional , Animales , Secuencia de Bases , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Hipoxia/enzimología , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Hígado/enzimología , Proteínas Nucleares/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Regiones Promotoras Genéticas , Ratas , Factores de Transcripción/metabolismoRESUMEN
Activating signal cointegrator-2 (ASC-2), a novel coactivator, is amplified in several cancer cells and known to interact with mitogenic transcription factors, including serum response factor, activating protein-1, and nuclear factor-kappaB, suggesting the physiological role of ASC-2 in the promotion of cell proliferation. Here, we show that the expression pattern of ASC-2 was correlated with that of E2F-1 for protein increases at G(1) and S phase. Furthermore, cells stably overexpressing ASC-2 had an increased cell proliferation profile. These results prompted us to examine the functional interaction of ASC-2 and E2F-1. Biochemical evidence of protein interaction indicated that the transactivation domain of E2F-1 interacted with the COOH-terminal region of ASC-2. The importance of the E2F-1-ASC-2 interaction was supported by the demonstration that the coexpression of ASC-2 and E2F-1 synergistically transactivated E2F-1-driven gene transcription and the acetylation of E2F-1 protein was necessary for ASC-2-mediated transcriptional coactivation. Interestingly, overexpression of ASC-2 increased the endogenous protein level of E2F-1 in cells, resulting from the prolonged protein stability of E2F-1. Taken together, these results suggest that the cancer-amplified transcriptional coactivator ASC-2 may promote cell proliferation through enhancement of E2F-1-dependent transactivation of the expression of genes associated with cell cycle progression that may be available to favor tumor growth in vivo.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Animales , Sitios de Unión/genética , Proteínas Portadoras/genética , Ciclo Celular/genética , Ciclo Celular/fisiología , División Celular/genética , División Celular/fisiología , Proteínas de Unión al ADN/genética , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Expresión Génica , Células HeLa , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Células 3T3 NIH , Coactivadores de Receptor Nuclear , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transactivadores/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Activación Transcripcional , Técnicas del Sistema de Dos HíbridosRESUMEN
We previously presented that calmodulin-dependent kinase IV (CaMKIV) mutually interacts with NF-kappa B and phosphorylates it directly, inducing the increased transcriptional regulation dependent on NF-kappa B target genes [J. Biol. Chem. 276 (2001) 20005]. Here, we show that Ser(535) residue is phosphorylated by CaMKIV. S535A mutant of p65 was specifically defective in transactivation of NF-kappa B target gene expression induced by CaMKIV. While coexpression of active CaMKIV with wild-type p65 led to a recovery from etoposide-induced apoptosis and an increase of Bcl-2 protein in cells, cells expressing S535A mutant did not. Taken together these results suggest that phosphorylated NF-kappa B p65 on Ser(535) by CaMKIV increases NF-kappa B target gene expression, including anti-apoptotic gene, hence leading to inhibition of apoptosis.
Asunto(s)
Apoptosis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , FN-kappa B/metabolismo , Activación Transcripcional , Proteína de Unión a CREB , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Mutación , FN-kappa B/química , FN-kappa B/genética , Proteínas Nucleares/metabolismo , Co-Represor 2 de Receptor Nuclear , Fosforilación , Proteínas Represoras/metabolismo , Serina/metabolismo , Transactivadores/metabolismo , Factor de Transcripción ReIARESUMEN
Disruption of the actin cytoskeleton in subconfluent mesenchymal cells induces chondrogenic differentiation via protein kinase C (PKC) alpha signaling. In this study, we investigated the role of p38 mitogen-activated protein (MAP) kinase in the chondrogenic differentiation of mesenchymal cells that is induced by depolymerization of the actin cytoskeleton. Treatment of mesenchymal cells derived from chick embryonic limb buds with cytochalasin D (CD) disrupted the actin cytoskeleton with concomitant chondrogenic differentiation. The chondrogenesis was accompanied by an increase in p38 MAP kinase activity and inhibition of p38 MAP kinase with SB203580 blocked chondrogenesis. Together these results suggest an essential role for p38 MAP kinase in chondrogenesis. In addition, inhibition of p38 MAP kinase did not alter CD-induced increased expression and activity of PKC alpha, whereas down-regulation of PKC by prolonged exposure of cells to phorbol ester inhibited CD-induced p38 MAP kinase activation. Our results therefore suggest that PKC is involved in the regulation of chondrogenesis induced by disruption of the actin cytoskeleton via a p38 MAP kinase signaling pathway.