RESUMEN
AIM: Platelet-activating factor acetyl hydrolase 1B1 (PAFAH1B1, also known as Lis1) is a protein essentially involved in neurogenesis and mostly studied in the nervous system. As we observed a significant expression of PAFAH1B1 in the vascular system, we hypothesized that PAFAH1B1 is important during angiogenesis of endothelial cells as well as in human vascular diseases. METHOD: The functional relevance of the protein in endothelial cell angiogenic function, its downstream targets and the influence of NONHSAT073641, a long non-coding RNA (lncRNA) with 92% similarity to PAFAH1B1, were studied by knockdown and overexpression in human umbilical vein endothelial cells (HUVEC). RESULTS: Knockdown of PAFAH1B1 led to impaired tube formation of HUVEC and decreased sprouting in the spheroid assay. Accordingly, the overexpression of PAFAH1B1 increased tube number, sprout length and sprout number. LncRNA NONHSAT073641 behaved similarly. Microarray analysis after PAFAH1B1 knockdown and its overexpression indicated that the protein maintains Matrix Gla Protein (MGP) expression. Chromatin immunoprecipitation experiments revealed that PAFAH1B1 is required for active histone marks and proper binding of RNA Polymerase II to the transcriptional start site of MGP. MGP itself was required for endothelial angiogenic capacity and knockdown of both, PAFAH1B1 and MGP, reduced migration. In vascular samples of patients with chronic thromboembolic pulmonary hypertension (CTEPH), PAFAH1B1 and MGP were upregulated. The function of PAFAH1B1 required the presence of the intact protein as overexpression of NONHSAT073641, which was highly upregulated during CTEPH, did not affect PAFAH1B1 target genes. CONCLUSION: PAFAH1B1 and NONHSAT073641 are important for endothelial angiogenic function.
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1-Alquil-2-acetilglicerofosfocolina Esterasa/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , 1-Alquil-2-acetilglicerofosfocolina Esterasa/genética , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Células Cultivadas , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Femenino , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/fisiología , Tromboembolia/complicaciones , Tromboembolia/metabolismo , Cicatrización de Heridas , Proteína Gla de la MatrizRESUMEN
The ALOX5 gene encodes 5-lipoxygenase (5-LO), a key enzyme of inflammatory reactions, which is transcriptionally activated by trichostatin A (TSA). Physiologically, 5-LO expression is induced by calcitriol and/or transforming growth factor-ß. Regulation of 5-LO mRNA involves promoter activation and elongation control within the 3'-portion of the ALOX5 gene. Here we focused on the ALOX5 promoter region. Transcriptional initiation was associated with an increase in histone H3 lysine 4 trimethylation in a TSA-inducible manner. Therefore, we investigated the effects of the MLL (mixed lineage leukemia) protein and its derivatives, MLL-AF4 and AF4-MLL, respectively. MLL-AF4 was able to enhance ALOX5 promoter activity by 47-fold, which was further stimulated when either vitamin D receptor and retinoid X receptor or SMAD3/SMAD4 were co-transfected. In addition, we investigated several histone deacetylase inhibitors (HDACi) in combination with gene knockdown experiments (HDAC1-3, MLL). We were able to demonstrate that a combined inhibition of HDAC1-3 induces ALOX5 promoter activity in an MLL-dependent manner. Surprisingly, a constitutive activation of ALOX5 by MLL-AF4 was inhibited by class I HDAC inhibitors, by relieving inhibitory functions deriving from MLL.Conversely, a knockdown of MLL increased the effects mediated by MLL-AF4. Thus, HDACi treatment seems to switch 'inactive MLL' into 'active MLL' and overwrites the dominant functions deriving from MLL-AF4.
RESUMEN
5-Lipoxygenase (5-LO) is the key enzyme in leukotriene biosynthesis. Leukotrienes are key mediators of inflammation, allergic and innate immune reactions. 5-LO expression is mainly restricted to a variety of immune competent cells including B-lymphocytes, granulocytes and monocytes/macrophages. Here, we studied the effects of the growth or differentiation factors TGFbeta, 1,25(OH)2D3, GM-CSF and TNFalpha on 5-LO mRNA and protein expression and on 5-LO activity in human monocytes. We found that cultivation of monocytes under standard culture conditions downregulates 5-LO mRNA expression which could be prevented by addition of the four factors. Monocyte 5-LO activity was serum-dependent and cultivation of the cells in serum-free medium strongly downregulated cellular 5-LO activity which could be prevented by TGFbeta, 1,25(OH)2D3, GM-CSF and TNFalpha to different extents. The protein kinase A activator dibutyryl-cAMP blocked the effects of the four factors. The data suggest that 5-LO expression and activity in monocytes is strongly regulated by pro- and anti-inflammatory growth or differentiation factors.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Factores de Diferenciación de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Monocitos/enzimología , Transducción de Señal/efectos de los fármacos , Western Blotting , Células Cultivadas , Citocinas/farmacología , Regulación hacia Abajo , Humanos , Monocitos/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Suero , Regulación hacia ArribaRESUMEN
This review addresses current and future perspectives of nanogel technology for nanomedicine. The synthetic methodologies and material properties of nanogels prepared by chemical meanings are discussed in detail, and examples that illustrate the different methodologies are presented. Applications in the fields of drug and gene delivery, smart imaging modalities, responsive materials, and multivalency as a therapeutic approach highlight the enormous potential of the functional nanogels as novel polymeric platforms for biomedicine.
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Nanomedicina/métodos , Polietilenglicoles/química , Polietileneimina/química , Tecnología Biomédica/métodos , Sistemas de Liberación de Medicamentos/métodos , Humanos , NanogelesRESUMEN
BACKGROUND AND PURPOSE: The potency of many 5-lipoxygenase (5-LOX) inhibitors depends on the cellular peroxide tone and the mechanism of 5-LOX enzyme activation. Therefore, new inhibitors that act regardless of the mode of enzyme activation need to be developed. Recently, we identified a novel class of thiazolinone-based compounds as potent 5-LOX inhibitors. Here, we present the molecular pharmacological profile of (Z)-5-(4-methoxybenzylidene)-2-(p-tolyl)-5H-thiazol-4-one, compound C06. EXPERIMENTAL APPROACH: Inhibition of 5-LOX product formation was determined in intact cells [polymorphonuclear leukocytes (PMNL), rat basophilic leukaemia-1, RAW264.7] and in cell-free assays [homogenates, 100, 000×g supernatant (S100), partially purified 5-LOX] applying different stimuli for 5-LOX activation. Inhibition of peroxisome proliferator-activated receptor (PPAR), cytosolic phospholipase A(2) (cPLA(2) ), 12-LOX, 15-LOX-1 and 15-LOX-2 as well as cyclooxygenase-2 (COX-2) were measured in vitro. KEY RESULTS: C06 induced non-cytotoxic, direct 5-LOX inhibition with IC(50) values about 0.66 µM (intact PMNL, PMNL homogenates) and approximately 0.3 µM (cell-free PMNL S100, partially purified 5-LOX). Action of C06 was independent of the stimulus used for 5-LOX activation and cellular redox tone and was selective for 5-LOX compared with other arachidonic acid binding proteins (PPAR, cPLA(2) , 12-LOX, 15-LOX-1, 15-LOX-2, COX-2). Experimental results suggest an allosteric binding distinct from the active site and the C2-like domain of 5-LOX. CONCLUSIONS AND IMPLICATIONS: C06 was identified as a potent selective direct 5-LOX inhibitor exhibiting a novel and unique mode of action, different from other established 5-LOX inhibitors. This thiazolinone may possess potential for intervention with inflammatory and allergic diseases and certain types of cancer.
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Inhibidores de la Lipooxigenasa/farmacología , Tiazoles/farmacología , Sitio Alostérico , Animales , Araquidonato 5-Lipooxigenasa/química , Araquidonato 5-Lipooxigenasa/metabolismo , Sistema Libre de Células , Humanos , Técnicas In Vitro , Inhibidores de la Lipooxigenasa/química , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Oxidación-Reducción , Ratas , Tiazoles/química , Células U937RESUMEN
BACKGROUND AND PURPOSE: 5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) representing a potential target for pharmacological intervention with inflammation and allergic disorders. Although many LT synthesis inhibitors are effective in simple in vitro test systems, they frequently fail in vivo due to lack of efficacy. Here, we attempted to assess the pharmacological potential of the previously identified 5-LO inhibitor 2-(4-(biphenyl-4-ylamino)-6-chloropyrimidin-2-ylthio)octanoic acid (HZ52). EXPERIMENTAL APPROACH: We evaluated the efficacy of HZ52 in vivo using carrageenan-induced pleurisy in rats and platelet-activating factor (PAF)-induced lethal shock in mice. We also characterized 5-LO inhibition by HZ52 at the cellular and molecular level in comparison with other types of 5-LO inhibitor, that is, BWA4C, ZM230487 and hyperforin. KEY RESULTS: HZ52, 1.5 mg·kg⻹ i.p., prevented carrageenan-induced pleurisy accompanied by reduced LTB(4) levels and protected mice (10 mg·kg⻹, i.p.) against PAF-induced shock. Detailed analysis in cell-based and cell-free assays revealed that inhibition of 5-LO by HZ52 (i) does not depend on radical scavenging properties and is reversible; (ii) is not impaired by an increased peroxide tone or by elevated substrate concentrations; and (iii) is little affected by the cell stimulus or by phospholipids, glycerides, membranes or Ca²âº. CONCLUSIONS AND IMPLICATIONS: HZ52 is a promising new type of 5-LO inhibitor with efficacy in vivo and with a favourable pharmacological profile. It possesses a unique 5-LO inhibitory mechanism different from classical 5-LO inhibitors and seemingly lacks the typical disadvantages of former classes of LT synthesis blockers.
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Antiinflamatorios/farmacología , Araquidonato 5-Lipooxigenasa/metabolismo , Caprilatos/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Pirimidinas/farmacología , Animales , Araquidonato 5-Lipooxigenasa/biosíntesis , Carragenina , Leucotrieno B4/antagonistas & inhibidores , Leucotrieno B4/metabolismo , Masculino , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/metabolismo , Factor de Activación Plaquetaria/efectos adversos , Factor de Activación Plaquetaria/farmacología , Pleuresia/inducido químicamente , Pleuresia/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Choque/inducido químicamente , Choque/metabolismoRESUMEN
BACKGROUND AND PURPOSE Arachidonic acid derivatives play a central role in inflammation processes. Arachidonic acid is metabolized by several enzymes, particularly cyclooxygenases (COX), 5-lipoxygenase (5-LOX) and microsomal prostaglandin E-synthase-1 (mPGES-1) to pro-inflammatory mediators. EXPERIMENTAL APPROACH We determined the effect of LP105, a pirinixic acid derivative which acts as inhibitor of 5-LOX, COX and mPGES-1, on aortic aneurysm development in mice and on 5-LOX activity in murine monocytes. KEY RESULTS In a monocyte cell line (RAW264.7), LP105 inhibited 5-LOX in whole cells (IC(50) : 1-3 µM) and in supernatants (IC(50) : â¼10 µM). Oral administration of LP105 to mice resulted in therapeutic tissue and plasma levels. Aortic aneurysms were induced in ApoE(-/-) mice by angiotensin II (AngII) and LP105 (5 mg·day(-1) per animal) was co-administered to a subgroup. Compared with animals receiving AngII alone, the LP105+AngII group showed a lower heart rate, a trend towards reduced heart to body weight ratio but similar hypertensive responses. AngII alone significantly increased aortic weight and diameter but co-treatment with LP105+AngII prevented these changes. LC/MS-MS studies revealed increased 15-hydroxytetraenoic acid (15-HETE) and 14,15-epoxyeicosatrienoic acid (14,15-EET) plasma levels in LP105-treated animals. In the murine kidney, mRNAs of EET-generating or metabolizing enzymes and of 5-LOX and 15-LOX were unaffected by LP105. LP105 also did not inhibit the EET-metabolizing soluble epoxide hydrolase. CONCLUSIONS AND IMPLICATIONS LP105 was a potent inhibitor of monocyte 5-LOX and reduced AngII-induced vascular remodelling in mice. A shift of arachidonic acid metabolism to the protective EET pathway may contribute to the beneficial effects of LP105.
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Aneurisma de la Aorta/patología , Araquidonato 5-Lipooxigenasa/metabolismo , Cardiotónicos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Inhibidores de la Lipooxigenasa/farmacología , Pirimidinas/farmacología , Administración Oral , Angiotensina II/administración & dosificación , Angiotensina II/toxicidad , Animales , Aorta/patología , Aneurisma de la Aorta/metabolismo , Araquidonato 5-Lipooxigenasa/sangre , Cardiotónicos/farmacocinética , Cardiotónicos/uso terapéutico , Sistema Cardiovascular/efectos de los fármacos , Línea Celular , Inhibidores de la Ciclooxigenasa/uso terapéutico , Modelos Animales de Enfermedad , Epóxido Hidrolasas/sangre , Epóxido Hidrolasas/metabolismo , Inyecciones Subcutáneas , Inhibidores de la Lipooxigenasa/uso terapéutico , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Prostaglandina-E Sintasas , Pirimidinas/metabolismo , Pirimidinas/uso terapéuticoRESUMEN
BACKGROUND AND PURPOSE: Certain 5-lipoxygenase (5-LO) inhibitors exhibit anti-carcinogenic activities against 5-LO overexpressing tumour types and cultured tumour cells. It has been proposed therefore that 5-LO products significantly contribute to tumour cell proliferation. To date, the relationship between the inhibitory mechanisms of 5-LO inhibitors, which vary widely, and tumour cell viability has not been evaluated. This study addresses the anti-proliferative and cytotoxic potency of a number of 5-LO inhibitors with different inhibitory mechanisms in 5-LO-positive and 5-LO-negative tumour cells. EXPERIMENTAL APPROACH: Cell viability was measured by the WST-1 assay; cell proliferation was assessed using the bromodeoxyuridine (BrdU) incorporation assay. Cell death was analysed by annexin V staining, Western blot analysis of PARP (poly ADP-ribose polymerase) cleavage and a cytotoxicity assay. 5-LO product formation was quantified by a 5-LO activity assay. KEY RESULTS: The common 5-LO inhibitors AA-861, Rev-5901 and MK-886 induced cytotoxic and anti-proliferative effects in 5-LO-positive Capan-2 pancreatic cancer cells; BWA4C and CJ-13,610 only caused anti-proliferative effects, while zileuton failed to impair cell viability. Moreover, the concentrations of the 5-LO inhibitors required to induce anti-proliferation and cytotoxicity highly exceeded those for suppression of 5-LO. Supplementation with mitogenic 5-LO products failed to protect Capan-2 cells from the effects of 5-LO inhibitors. Finally, the cytotoxic and anti-proliferative 5-LO inhibitors also potently reduced the viability of 5-LO-deficient tumour cell lines (HeLa, Panc-1 and U937). CONCLUSIONS AND IMPLICATIONS: Certain 5-LO inhibitors cause cytotoxic and anti-proliferative effects independently of suppression of 5-LO activity. Thus, the role of 5-LO overexpression in tumour cell viability remains unclear and requires further elucidation.
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Antineoplásicos/farmacología , Araquidonato 5-Lipooxigenasa/efectos de los fármacos , Inhibidores de la Lipooxigenasa/farmacología , Neoplasias/tratamiento farmacológico , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/metabolismo , Western Blotting , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/patología , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
The speed of immune recovery after allo-SCT is of central importance to overcome infectious complications and relapse. To evaluate the immune reconstitution of pediatric patients concerning overall survival, we developed a three-component multivariate model and generated a reference domain of ellipsoidal shape on the basis of normal leukocyte subtype values of 100 healthy children and adolescents. The leukocyte subtypes include absolute nos. of leukocytes, CD14(+) monocytes, lymphocytes, CD3(+) T cells, CD3(+)CD4(+) helper T cells, CD3(+)CD8(+) cytotoxic T cells, CD3(-)CD56(+) natural killer-cells and CD19(+) B cells, all of which are correlated, thus, requiring the application of multivariate as opposed to multiple univariate modeling. According to their immune reconstitution, 32 pediatric patients post allo-SCT were classified into low-risk and high-risk groups on the basis of our new model. Therefore, we evaluated if the patients reached the ellipsoid of normal leukocyte sub-population values post SCT. We detected a significantly higher number of long-time survivors among the low-risk group compared with the high-risk group at days 200 (P=0.001) and 300 (P<0.0001). This is superior to our previously published univariate analysis. Combined with the clinical observation, a classification into risk groups based on an extended patient cohort may represent a predictor for complications.
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Trasplante de Células Madre Hematopoyéticas , Recuento de Linfocitos , Subgrupos de Linfocitos T , Adolescente , Niño , Preescolar , Estudios de Cohortes , Femenino , Supervivencia de Injerto , Humanos , Inmunidad Celular , Células Asesinas Naturales , Masculino , Monocitos , Análisis Multivariante , Valores de Referencia , Medición de Riesgo , Análisis de Supervivencia , Trasplante Homólogo , Adulto JovenRESUMEN
BACKGROUND AND PURPOSE: Licofelone is a dual inhibitor of the cyclooxygenase and 5-lipoxygenase (5-LO) pathway, and has been developed for the treatment of inflammatory diseases. Here, we investigated the molecular mechanisms underlying the inhibition by licofelone of the formation of 5-LO products. EXPERIMENTAL APPROACH: The efficacy of licofelone to inhibit the formation of 5-LO products was analysed in human isolated polymorphonuclear leukocytes (PMNL) or transfected HeLa cells, as well as in cell-free assays using respective cell homogenates or purified recombinant 5-LO. Moreover, the effects of licofelone on the subcellular redistribution of 5-LO were studied. KEY RESULTS: Licofelone potently blocked synthesis of 5-LO products in Ca(2+)-ionophore-activated PMNL (IC(50)=1.7 microM) but was a weak inhibitor of 5-LO activity in cell-free assays (IC(50)>>10 microM). The structures of licofelone and MK-886, an inhibitor of the 5-LO-activating protein (FLAP), were superimposable. The potencies of both licofelone and MK-886 in ionophore-activated PMNL were impaired upon increasing the concentration of arachidonic acid, or under conditions where 5-LO product formation was evoked by genotoxic, oxidative or hyperosmotic stress. Furthermore, licofelone prevented nuclear redistribution of 5-LO in ionophore-activated PMNL, as had been observed for FLAP inhibitors. Finally, licofelone as well as MK-886 caused only moderate inhibition of the synthesis of 5-LO products in HeLa cells, unless FLAP was co-transfected. CONCLUSIONS AND IMPLICATIONS: Our data suggest that the potent inhibition of the biosynthesis of 5-LO products by licofelone requires an intact cellular environment and appears to be due to interference with FLAP.
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Acetatos/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Inhibidores de la Lipooxigenasa , Proteínas de la Membrana/antagonistas & inhibidores , Pirroles/farmacología , Proteínas Activadoras de la 5-Lipooxigenasa , Acetatos/química , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/metabolismo , Ácido Araquidónico/química , Ácido Araquidónico/farmacología , Arsenitos/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Calcimicina/farmacología , Calcio/farmacología , Proteínas Portadoras/biosíntesis , Sistema Libre de Células , Células Cultivadas , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Indoles/química , Indoles/farmacología , Antagonistas de Leucotrieno/farmacología , Leucotrienos/biosíntesis , Leucotrienos/química , Inhibidores de la Lipooxigenasa/farmacología , Proteínas de la Membrana/biosíntesis , Estructura Molecular , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/metabolismo , Pirroles/química , Quinolinas/farmacología , Compuestos de Sodio/farmacología , TransfecciónRESUMEN
BACKGROUND: The biologic role of 1,25-dihydroxyvitamin D(3), such as anti-inflammatory functions, reduction of cytokine production by T cells and immunoglobulin production by B cells, is well established. However, its clinical use as an immunosuppressive agent is limited because of the hypercalcemic toxicity occurring after systemic application. The purpose of this study was to investigate the immunmodulatory effects of 22-ene-25-oxa-vitamin D (ZK156979), a novel low calcemic vitamin D analogue. MATERIALS AND METHODS: Human peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using the Ficoll Hypaque technique, cultured for 24 h and treated with different concentrations of ZK156979 ranging from 10(-5) to 10(-10) mol L(-1) compared with 1,25-dihydroxyvitamin D(3)[10(-5)-10(-10) mol L(-1)] following phytohaemagglutinin (PHA) stimulation. Interferon gamma (IFNgamma), tumour necrosis factor alpha (TNFalpha), interleukin 1 beta (IL-1beta), interleukin 10 (IL-10) and interleukin 4 (IL-4) secretion in supernatants were measured by ELISA. RESULTS: ZK156979 inhibited the PHA-induced Th1-response (IFNgamma and TNFalpha levels) and the macrophage-product IL-1beta in a concentration-dependent manner (10(-10)-10(-5) mol L(-1)) with the efficiency on cytokine expression compared with 1,25-dihydroxyvitamin D(3) being slightly reduced. In contrast, ZK156979 and 1,25-dihydroxyvitamin D(3) both affected the Th2 response, leading to significantly increased IL-10- and IL-4 secretion. CONCLUSIONS: ZK156979 is a member of novel vitamin D analogues revealing prominent immunomodulatory and suppressive characteristics with distinctive inhibition of Th1-cytokines whereas the Th2 compartment is augmented, thus providing a considerable therapeutic potential in T-cell -mediated diseases.
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Citocinas/inmunología , Inmunosupresores/farmacología , Linfocitos T Colaboradores-Inductores/inmunología , Vitamina D/análogos & derivados , Calcitriol/inmunología , Calcitriol/farmacología , Células Cultivadas , Citocinas/efectos de los fármacos , Relación Dosis-Respuesta Inmunológica , Humanos , Interferón gamma/análisis , Interleucina-1/inmunología , Interleucina-10/análisis , Interleucina-4/análisis , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Factor de Necrosis Tumoral alfa/análisis , Vitamina D/farmacologíaRESUMEN
Butyrate in combination with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] produces a synergistic effect on cell differentiation of human colon cancer cells (Caco-2). The objective of this study was to confirm the role of the vitamin D receptor (VDR) in butyrate-induced cell differentiation of Caco-2. We studied the effects of the novel VDR antagonist ZK 191732 on butyrate-induced cell differentiation and on p21Waf1/Cip1 expression. Butyrate induced cell differentiation which was further enhanced after addition of 1,25-(OH)2D3. Experiments using ZK 191732 indicate that the synergistic effect of butyrate and 1,25-(OH)2D3 was due to butyrate-induced upregulation of VDR. While butyrate alone increased expression of p21Waf1/Cip1 and combined exposure of butyrate and 1,25-(OH)2D3 resulted in a synergistic amplification, p21Waf1/Cip1 expression did not change from the control level after treatment with butyrate plus ZK 191732. These data further imply that butyrate-induced differentiation and p21Waf1/Cip1 expression of Caco-2 cells occur via upregulation of VDR.
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Butiratos/farmacología , Diferenciación Celular/efectos de los fármacos , Receptores de Calcitriol/fisiología , Tiazoles/farmacología , Células CACO-2 , Diferenciación Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Interacciones Farmacológicas , Expresión Génica/efectos de los fármacos , Humanos , Receptores de Calcitriol/biosíntesis , Receptores de Calcitriol/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Tributyrin, a prodrug of natural butyrate, has been evaluated with an aim to overcome pharmacokinetic drawbacks of natural butyrate as a drug, i.e., its rapid metabolization and inability to achieve pharmacologic concentrations in neoplastic cells. We studied the effects of tributyrin on growth, differentiation and vitamin D receptor expression in Caco-2 cells, a human colon cancer cell line. Tributyrin was more potent in inhibiting growth and inducing cell differentiation than natural butyrate. The effect was further enhanced after addition of physiologic concentrations of dihydroxycholecalciferol [(OH)2D3]. The synergistic effect of tributyrin and (OH)2D3 in Caco-2 cells was due to tributyrin-induced overexpression of the vitamin D receptor, as measured by reverse transcriptase-polymerase chain reaction. Treatment with tributyrin increased binding of (OH)2D3 to its receptor 1.5-fold, without any change in receptor affinity. We conclude that tributyrin may, at least in part, exert its growth-reducing and differentiation-inducing effect in Caco-2 cells by an upregulation of the vitamin D receptor; this may provide a useful therapeutic approach in chemoprevention and treatment of colorectal cancer by the two nutrients occurring naturally in human diet.
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Células CACO-2/efectos de los fármacos , Dihidroxicolecalciferoles/farmacología , Profármacos/farmacología , Triglicéridos/farmacología , Fosfatasa Alcalina/análisis , Butiratos/farmacología , Células CACO-2/patología , Recuento de Células , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , ADN/biosíntesis , Regulación hacia Abajo , Combinación de Medicamentos , Sinergismo Farmacológico , HumanosRESUMEN
Butyrate, a short-chain fatty acid produced in the colon, as well as its prodrug tributyrin, reduce proliferation and increase differentiation of colon cancer cells. p21(Waf1/Cip1) and p27(Kip1) are negative regulators of cell cycle and are thought to have a key function in the differentiation of various cell lines. We studied the effects of butyrate on differentiation, VDR expression, as well as on p21(Waf1/Cip1) and p27(Kip1) expression in human colon cancer cells (Caco-2). Butyrate induced cell differentiation, which was further enhanced after addition of 1,25-dihydroxycholecalciferol. Synergistic effect of butyrate and dihydroxycholecalciferol in Caco-2 cells was due to butyrate-induced overexpression of VDR. While butyrate as well as dihydroxycholecalciferol increased p21(Waf1/Cip1) and p27(Kip1) expression, in contrast combined exposure of butyrate and dihydroxycholecalciferol resulted in a synergistic amplification of p21(Waf1/Cip1), but not of p27(Kip1) expression. These data imply that butyrate selectively increases p21(Waf1/Cip1) expression via upregulation of VDR in Caco-2 cells.
Asunto(s)
Butiratos/farmacología , Células CACO-2/efectos de los fármacos , Calcitriol/farmacología , Proteínas de Ciclo Celular , Ciclinas/metabolismo , Proteínas Supresoras de Tumor , Fosfatasa Alcalina/metabolismo , Western Blotting , Células CACO-2/citología , Células CACO-2/metabolismo , Diferenciación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , ARN Mensajero/metabolismo , Receptores de Calcitriol/biosíntesis , Receptores de Calcitriol/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Triglicéridos/farmacología , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The 5-lipoxygenase (5-LO) pathway in human CD34(+) hematopoietic progenitor cells, which were induced to differentiate into dendritic cells (DCs) by cytokines in vitro and in DCs of lymphoid tissues in situ, was examined. Extracts prepared from HPCs contained low levels of 5-LO or 5-LO-activating protein. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plus tumor necrosis factor-alpha (TNF-alpha) promoted DC differentiation and induced a strong rise in 5-LO and FLAP expression. Fluorescence-activated cell sorter (FACS) analyses identified a major DC population coexpressing human leukocyte antigen (HLA)-DR/CD80 and monocytic or Langerhans cell markers. Transforming growth factor-beta1 (TGF-beta-1), added to support DC maturation, strongly promoted the appearance of CD1a(+)/Lag(+) Langerhans-type cells as well as mature CD83(+) DCs. TGF-beta-1 further increased 5-LO and FLAP expression, recruited additional cells into the 5-LO(+) DC population, and promoted production of 5-hydroxyeicosatetraenoic acid and leukotriene B(4) in response to calcium (Ca(++)) ionophore A23187. These in vitro findings were corroborated by 5-LO expression in distinct DC phenotypes in vivo. Scattered 5-LO and FLAP in situ hybridization signals were recorded in cells of paracortical T-lymphocyte-rich areas and germinal centers (GCs) of lymph nodes (LNs) and tonsil and in cells of mucosae overlying the Waldeyer tonsillar ring. 5-LO protein localized to both CD1a(+) immature DCs and to CD83(+) mature interdigitating DCs of T-lymphocyte-rich areas of LNs and tonsil. As DCs have the unique ability to initiate naive lymphocyte activation, our data support the hypothesis that leukotrienes act at proximal steps of adaptive immune responses. (Blood. 2000;96:3857-3865)
Asunto(s)
Antígenos CD34/análisis , Araquidonato 5-Lipooxigenasa/metabolismo , Células Dendríticas/enzimología , Araquidonato 5-Lipooxigenasa/biosíntesis , Araquidonato 5-Lipooxigenasa/genética , Ácido Araquidónico/farmacología , Calcimicina/farmacología , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Factores Quimiotácticos/biosíntesis , Citocinas/farmacología , Células Dendríticas/citología , Sangre Fetal/citología , Citometría de Flujo , Antígenos HLA-DR , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Inmunohistoquímica , Inmunofenotipificación , Ionóforos/farmacología , Leucotrieno B4/biosíntesis , Ganglios Linfáticos/enzimología , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Macrófagos/enzimología , Tonsila Palatina/enzimologíaRESUMEN
Changes in the osmolarity of the airway surface fluid have been described to be involved in the pathogenesis of exercise induced asthma, and are suggested as the major cause of the lung disease in cystic fibrosis. In this study, we examined the signaling pathway of hyperosmotic challenge to interleukin-8 (IL-8). Hyperosmolarity (NaCl) caused a time- and concentration-dependent increase in IL-8 expression and secretion in bronchial epithelial cells. These effects could be blocked by antioxidants, such as DMSO, DMTU, DTT, and beta-mercaptoethanol, suggesting an involvement of reactive oxygen intermediates (ROI) in the signal transduction of hyperosmolarity-induced IL-8 synthesis. Since IL-8 is regulated by MAP kinases, we examined the influence of MAP kinase inhibitors on hyperosmolarity-induced IL-8 expression. The results show that this induction is regulated by p38 MAPK and not by ERK1/2. Furthermore, antioxidants blocked the activation of p38 MAPK induced by hyperosmolarity. These results suggest that ROIs are critical for p38 MAPK mediated IL-8 expression by hyperosmolarity.
Asunto(s)
Bronquios/metabolismo , Interleucina-8/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Antioxidantes/farmacología , Bronquios/citología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Concentración Osmolar , Presión Osmótica , Fosforilación , Factores de Tiempo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
In contrast to neutrophils or B-lymphocytes, cells of the monocytic lineage like rat macrophages, human peripheral blood monocytes and Mono Mac 6 cells contain a strong inhibitor of 5-lipoxygenase (5-LO) activity, which scavenges hydroperoxides and inhibits 5-LO activity in broken-cell preparations in the absence of exogenously added thiols. Chromatographic purification of the inhibitor from the human monocytic cell line Mono Mac 6 and amino acid sequence analysis revealed that the inhibitory factor is glutathione peroxidase-1 (GPx-1). In contrast to the peroxidase activity of GPx-1, 5-LO inhibition by GPx-1 was supported by beta-mercaptoethanol and there was no absolute requirement for millimolar concentrations of glutathione or dithiothreitol. These cofactor characteristics suggest that both activities address distinct catalytic properties of GPx-1. 5-LO inhibition by GPx-1 was not due to direct GPx-5-LO protein-protein interactions, since GPx-1 did not bind to immobilized 5-LO. Interestingly, 5-LO derived from granulocytes was significantly more resistant against GPx-1 inhibition than B-lymphocytic 5-LO, which correlates with the respective cellular 5-LO activities. In summary, the data suggest that, in addition to previously reported phospholipid hydroperoxide glutathione peroxidase (GPx-4), GPx-1 is an efficient inhibitor of 5-LO even at low thiol concentrations, and is involved in the regulation of cellular 5-LO activity in various cell types.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Glutatión Peroxidasa/metabolismo , Monocitos/enzimología , Animales , Catálisis , Células Cultivadas , Inhibidores Enzimáticos/metabolismo , Estabilidad de Enzimas , Humanos , Inhibidores de la Lipooxigenasa , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Unión Proteica , Ratas , Selenio/farmacología , Compuestos de Sulfhidrilo/metabolismo , Glutatión Peroxidasa GPX1RESUMEN
The aim of the study was the development of vitamin D receptor agonists with decreased metabolic stability for the topical treatment of psoriasis and related hyperproliferative skin diseases. Calcitriol analogues 1, 2, 3, all of which contain modifications in the side chain, were synthesized. The obtained analogues were full agonists when the induction of CD14 expression in HL-60 cells, the induction of 5-lipoxygenase activity in Mono Mac 6 cells, and the inhibition of phytohemagglutinin (PHA)-stimulated lymphocyte proliferation were studied. The EC(50) value of the most active compound 1 was 1.2 nM in the CD14 assay and 1 nM in the 5-lipoxygenase assay, whereas calcitriol gave EC(50) values in these assays of 3.7 and 9 nM, respectively. In the lymphocyte proliferation assay, compound 1 and calcitriol had IC(50) values of 0.3 and 2.8 nM, respectively. All three compounds had receptor binding affinities similar to that of calcitriol. The compounds showed a decreased metabolic stability in rat liver homogenates and had a 50-fold lower affinity for the vitamin D-binding protein than calcitriol, which suggests that calcitriol analogues are metabolized more rapidly after systemic uptake or application. When injected into rats, the analogues displayed an approximately 100-fold lower hypercalcemic effect than calcitriol. In summary, our study presents three new and potent vitamin D receptor agonists with interesting profiles for development as antipsoriatic drugs.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Receptores de Calcitriol/agonistas , Vitamina D/farmacología , Animales , Calcitriol/efectos adversos , Calcitriol/farmacología , Agonistas de los Canales de Calcio/farmacología , Estabilidad de Medicamentos , Femenino , Células HL-60 , Humanos , Hipercalcemia/inducido químicamente , Linfocitos/efectos de los fármacos , Ratas , Porcinos , Vitamina D/análogos & derivadosRESUMEN
B-lymphocytes express 5-lipoxygenase (5-LO) protein but cellular leukotriene production is suppressed by selenium-dependent peroxidases. Thus it was of interest to check whether reactive oxygen species (ROS) which are released under inflammatory conditions can stimulate B-lymphocyte 5-LO and counteract peroxidase-mediated suppression of cellular 5-LO activity. It was found that 5-LO in the Epstein-Barr virus-transformed B-lymphocytic cell line BL41-E95-A is activated by addition of hydrogen peroxide or xanthine/xanthine oxidase and after increasing the oxidative state of the cell by azodicarboxylic acid bis(dimethylamide). Generation of endogenous ROS from mitochondria by antimycin A also lead to a threefold upregulation of 5-LO activity in B-cells. There was almost no detectable endogenous superoxide formation in BL41-E95-A cells after stimulation with 4beta-phorbol 12-myristate 13-acetate. Co-incubation experiments with BL41-E95-A cells and granulocytes demonstrated that granulocyte-derived ROS can activate B-lymphocyte 5-LO. Addition of superoxide dismutase and/or catalase to the B-lymphocyte/granulocyte co-incubations and to B-lymphocyte homogenates revealed that the 5-LO activation is due to the superoxide-derived release of hydroperoxides or hydrogen peroxide from granulocytes. The data suggest that ROS formation plays an important role in the regulation of cellular 5-LO activity in B-lymphocytes. As leukotrienes affect B-cell functions like cell proliferation, activation and maturation, this finding provides a new link between the formation of ROS and the regulation of immune responses.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Linfocitos B/enzimología , Granulocitos/metabolismo , Especies Reactivas de Oxígeno , Línea Celular , Activación Enzimática , HumanosRESUMEN
Butyrate, a short-chain fatty acid, modulates proliferation and differentiation of normal and neoplastic colonocytes. We examined the expression of 5-lipoxygenase (5-LO) and its metabolites in human colorectal carcinoma (Caco-2) cells, exposed to differentiation-inducing doses of butyrate. Treatment with butyrate significantly increased 5-lipoxygenase mRNA and protein in comparison to nontreated cells. Cyclooxygenases (COX)-1 and COX-2 mRNA were not significantly influenced by the treatment. However, 5-LO activity, low in nontreated cells, increased only minimally after butyrate, and its metabolic product (5-HETE) was detectable neither in control nor in treated cells. In contrast, 15-HETE (a product of 15-LO, which is also upregulated by butyrate) rose significantly. We conclude that, although being overexpressed by butyrate on mRNA and protein level, 5-LO remains inactive in differentiating Caco-2 cells. This is likely to be due either to some associated actions of butyrate, or to 5-LO-inhibition by 15-HETE, concomitantly induced by butyrate treatment.