RESUMEN
BACKGROUND AND PURPOSE: Azithromycin has been reported to modify activation of macrophages towards the M2 phenotype. Here, we have sought to identify the mechanisms underlying this modulatory effect of azithromycin on human monocytes, classically activated in vitro. EXPERIMENTAL APPROACH: Human blood monocytes were primed with IFN-γ for 24 h and activated with LPS for 24 h. Azithromycin, anti-inflammatory and lysosome-affecting agents were added 2 h before IFN-γ. Cytokine and chemokine expression was determined by quantitative PCR and protein release by ELISA. Signalling molecules were determined by Western blotting and transcription factor activation quantified with a DNA-binding ELISA kit. KEY RESULTS: Azithromycin (1.5-50 µM) dose-dependently inhibited gene expression and/or release of M1 macrophage markers (CCR7, CXCL 11 and IL-12p70), but enhanced CCL2, without altering TNF-α or IL-6. Azithromycin also enhanced the gene expression and/or release of M2 macrophage markers (IL-10 and CCL18), and the pan-monocyte marker CD163, but inhibited that of CCL22. The Toll-like receptor (TLR) 4 signalling pathway was modulated, down-regulating NF-κB and STAT1 transcription factors. The inhibitory profile of azithromycin differed from that of dexamethasone, the phosphodiesterase-4 inhibitor roflumilast and the p38 kinase inhibitor SB203580 but was similar to that of the lysosomotropic drug chloroquine. Effects of concanamycin and NH4Cl, which also act on lysosomes, differed significantly. CONCLUSIONS AND IMPLICATIONS: Azithromycin modulated classical activation of human monocytes by inhibition of TLR4-mediated signalling and possible effects on lysosomal function, and generated a mediator expression profile that differs from that of monocyte/macrophage phenotypes so far described.
Asunto(s)
Azitromicina/farmacología , Monocitos/efectos de los fármacos , Antiinflamatorios/farmacología , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL22/genética , Quimiocina CCL22/metabolismo , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/genética , Lisosomas/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Nm23-H1/NDPKA and Nm23-H2/NDPKB belong to a large family of NDP kinases, group of structurally and functionally closely related enzymes. The Nm23/NDPs are known to catalyse the transfer of terminal phosphates from ATP to other NTPs and dNTPs. Besides their role in the maintenance of the cells NTP pool the nm23 genes/proteins are known to have additional different biological functions, the most important being its metastasis suppressor activity. The complete picture of roles, actions and targets of nm23 genes/proteins is yet to be discovered. Our goal was to identify the downstream targets of Nm23-H2 by subjecting Nm23-H2 overexpressing CAL 27 cells (oral squamous cell carcinoma of the tongue) to microarray analysis. Using this powerful technology we identified genes, groups of genes and signalling pathways that could be clustered into several groups: apoptosis related genes, cell cycle and DNA damage, TGFbeta (transforming growth factor beta) signalling pathway and related molecules, WNT signalling pathway, differentiation and epithelial structural and related molecules, cell adhesion, metalloproteinases and their inhibitors, vesicular transport related molecules, proteasome associated, ubiquitin mediated proteolysis and several metabolic pathways. Based on these results we suggest that nm23-H2 might have an important role in oral squamous cell carcinoma which is to be confirmed by future studies.
Asunto(s)
Carcinoma de Células Escamosas/genética , Perfilación de la Expresión Génica , Neoplasias de la Boca/genética , Nucleósido Difosfato Quinasas NM23/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Línea Celular Tumoral , Progresión de la Enfermedad , Humanos , Transducción de Señal , TransfecciónRESUMEN
This paper describes the use of potentiometric titration to determine the relevant acid-base properties of 5-hydroxypyrazine-2-carboxylic acid (5OH-PYCA), an important intermediate in the production of tuberculostatics. The data obtained were used for calculation of the dissociation constants of 5OH-PYCA. It was found that 5OH-PYCA dissociates in two steps, with the corresponding dissociation constants pK (a1)=3.42 and pK (a2)=7.96, designating 5OH-PYCA as a medium weak acid (1st step). The distribution diagram of dissociated species and the buffer-strength diagram of 5OH-PYCA provide useful information about its behaviour at different pH. The ionic equilibria data obtained can be used for selection of the optimum pH for biotransformation of pyrazine-2-carboxylic acid (PYCA) and for prediction of pH changes during the biotransformation. These data can also be used for selection of the optimum pH for precipitating 5OH-PYCA in downstream processing. All computations have been optimized by mathematical modelling using Solver.
RESUMEN
Natural silicate materials, including zeolite clinoptilolite, have been shown to exhibit diverse biological activities and have been used successfully as a vaccine adjuvant and for the treatment of diarrhea. We report a novel use of finely ground clinoptilolite as a potential adjuvant in anticancer therapy. Clinoptilolite treatment of mice and dogs suffering from a variety of tumor types led to improvement in the overall health status, prolongation of life-span, and decrease in tumors size. Local application of clinoptilolite to skin cancers of dogs effectively reduced tumor formation and growth. In addition, toxicology studies on mice and rats demonstrated that the treatment does not have negative effects. In vitro tissue culture studies showed that finely ground clinoptilolite inhibits protein kinase B (c-Akt), induces expression of p21WAF1/CIP1 and p27KIP1 tumor suppressor proteins, and blocks cell growth in several cancer cell lines. These data indicate that clinoptilolite treatment might affect cancer growth by attenuating survival signals and inducing tumor suppressor genes in treated cells.
Asunto(s)
Adyuvantes Farmacéuticos/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas , Zeolitas/uso terapéutico , Adyuvantes Farmacéuticos/efectos adversos , Adyuvantes Farmacéuticos/farmacología , Envejecimiento/fisiología , Animales , Apoptosis/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Proteínas de Ciclo Celular/análisis , División Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclinas/análisis , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/patología , Perros , Femenino , Células HeLa , Humanos , Masculino , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Neoplasias/patología , Neoplasias/veterinaria , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/análisis , Zeolitas/efectos adversos , Zeolitas/farmacologíaRESUMEN
In order to assess mean diameters and blood flow velocities (BFV), Color Doppler Flow Imaging (CDFI) of vertebral arteries (VA) was performed. Five hundred and ninety six persons without carotid disease or symptoms related to vertebrobasilar system were analyzed by CDFI of VA. Mean right VA diameter was 3.37 +/- 0.6 mm and left 3.55 +/- 0.61 mm. Women had thinner VA (p < 0.05). Left VA was wider (p < 0.05). Mean right BFV was 48.31 +/- 14.09 cm/s and left 48.93 +/- 13.94 cm/s. Females had higher BFV (p < 0.05). BFV didn't very with age (p > 0.05). The VA hypoplasia was present in 2.34%, asymmetry in 15% (left VA dominant in 64%). Visualisation of V1 and V2 segment was possible in 100% and of the origin in 81.7% on the right, and 80.7% on the left side. CDFI is a reliable method for evaluation of VA. Left VA was wider. Women had thinner VA. Hypoplasia was present in 2.34% and asymmetry in 15%.
Asunto(s)
Velocidad del Flujo Sanguíneo , Ultrasonografía Doppler en Color , Arteria Vertebral/diagnóstico por imagen , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valores de Referencia , Arteria Vertebral/fisiologíaRESUMEN
Relatively little is known about molecular genetic events that participate in the genesis and progression of hemangiopericytoma. In this study, we describe two cases of hemangiopericytoma accompanied by severe hypoglycemia. Tumor cells from patient 1 exhibited insulin-growth factor I (IGF I) and insulin-like growth factor I receptor (IGF IR) mRNA transcripts. Tumor cells from patient 2 exhibited IGF II, IGF IR and IGF binding proteins 1-3 mRNA. Serum from patient 2 contained IGF II, mostly in a large molecular form ("big" IGF II); the IGF II level did not change after the tumor removal. The presence of IGF IR in tumor cells was confirmed by immunoprecipitation with antibodies that recognize human IGF IR subunit (visualized as a 460-kDa band). The hemangiopericytoma cells derived from patient 1 expressed 210000 IGF I receptors/cell. Specific binding of IGF I to the tumor cell membrane fraction was higher in tissue from patient 1, while the tissue of patient 2 showed relatively low IGF I binding. In contrast, IGF II binding was much higher in tissue from patient 2. Both tumor tissues showed positive immunostaining for c-Jun; one tumor showed strong immunostaining for c-Myc, H-Ras and p53, while the other exhibited strong reaction with H-Ras antibodies only. No loss of the heterozygosity at the genes APC, NFI and nm23-H1 loci in tumor tissue obtained from patient 1 was found. In effect, our results suggest multiple molecular genetic changes in hemangiopericytoma -- activation of some oncogenes and the IGF growth factor family. IGF ligands together with IGF IR could be responsible for hypoglycemia and perhaps the transformed phenotype.
Asunto(s)
Genes Supresores de Tumor , Hemangiopericitoma/metabolismo , Hemangiopericitoma/patología , Hipoglucemia/complicaciones , Hipoglucemia/metabolismo , Oncogenes , Somatomedinas/biosíntesis , Neoplasias Abdominales/genética , Neoplasias Abdominales/metabolismo , Neoplasias Abdominales/patología , Hemangiopericitoma/genética , Humanos , Hipoglucemia/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Masculino , Persona de Mediana Edad , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/metabolismo , Neoplasias Peritoneales/patología , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/biosíntesis , Receptor IGF Tipo 1/metabolismo , Somatomedinas/metabolismoRESUMEN
This analysis of 32 pairs of human squamous cell lung carcinomas and normal matched control DNA demonstrates that loss of heterozygosity (LOH) is infrequent at the nm23-H1 locus, affecting only 2 of the 18 informative cases. Both LOH cases were in the tumor stage IIIA. One tumor was of poor and the other of moderate histological grade. These and an additional 34 tumor samples were also analyzed immunohistochemically for the presence of nm23-H1 protein. Of the 66 cases tested for the presence of nm23-H1 protein 54 were negative. Eight samples exhibited up to 35% positive cells (with weak immunostaining intensity) and four between 35% and 70% (moderate immunostaining intensity); no sample showed more than 70% positive cells. Noncancerous lung parts contained no nm23-H1 protein. nm23-H1 expression was independent of TNM stage, grade, tumor size, and patient's survival. Two samples with LOH were negative for nm23-H1 protein. We therefore conclude that neither loss of heterozygosity of the nm23-H1 gene nor the intensity of specific protein expression are related to squamous cell lung carcinoma development and progression.
Asunto(s)
Carcinoma de Células Escamosas/genética , Genes Relacionados con las Neoplasias , Pérdida de Heterocigocidad , Neoplasias Pulmonares/genética , Proteínas de Unión al GTP Monoméricas , Nucleósido-Difosfato Quinasa , Factores de Transcripción/genética , Anciano , Carcinoma de Células Escamosas/metabolismo , ADN de Neoplasias/análisis , Repeticiones de Dinucleótido , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/metabolismo , Masculino , Persona de Mediana Edad , Nucleósido Difosfato Quinasas NM23 , Polimorfismo Genético , Factores de Transcripción/metabolismoRESUMEN
This study evaluates the potential contribution of the nm23-H1 gene to malignant transformation in patients with renal cell carcinoma. Using specific oligonucleotide primers for the nm23-H1 microsatellite repetitive sequence, gene instability was followed by polymerase chain reaction/loss of heterozygosity assay on 54 tumor specimens and the corresponding normal tissue samples. We also determined, immunohistochemically, the relative concentration and localization of the nm23-H1 protein product. From 77.7% informative cases, DNA from 6 tumors exhibited loss of heterozygosity, regardless of the tumor stage (TNM). Out of 39 samples analyzed, 30 were negative for Nm23-H1 protein, while the others were only slightly positive. No correlation with tumor stage was found. Normal renal tissue was also negative for this protein. Our results provide the evidence for loss of heterozygosity, followed by means of microsatellite tandem-repeat polymorphism, at the nm23-H1 locus in renal cell carcinoma. However, since no correlation was found between the tumor stage or metastatic potential on the one hand, and allelic loss and specific protein expression on the other, it seems that nm23-H1 does not play a key role in the invasiveness of this tumor type.
Asunto(s)
Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Proteínas de Unión al GTP Monoméricas , Nucleósido-Difosfato Quinasa , Factores de Transcripción/genética , Cromosomas Humanos Par 17 , ADN de Neoplasias/genética , Humanos , Riñón/química , Pérdida de Heterocigocidad , Repeticiones de Microsatélite , Nucleósido Difosfato Quinasas NM23 , Metástasis de la NeoplasiaRESUMEN
The experiments were designed to assess whether DNA could be recovered from archival, fixed, paraffin embedded specimens, 1-39 years old, for use in PCR and Southern blot analyses. Specimen fixed in either 10% formalin, Carnoy's or AMeX fixative produced almost equally abundant 318 bp long beta-actin fragment, after 2 x 120 min deparaffinization time, proteinase K DNA extraction and 40, or more, amplification cycles. Prolonged deparaffinization time and phenol/chloroform extraction did not influence DNA quality for PCR. Formalin fixed tissues can be successfully used for DNA/PCR of shorter fragments (318 bp) even if they are up to 39 years old. Longer fragment (720 bp) was successfully amplified from 1 and 10 years old specimens, also investigated whether DNA suitable for hybridization studies could be prepared from fixed tissue. Formalin caused irreversible DNA damages which were greater with prolonged fixation time making it unsuitable for hybridization studies, but still suitable for PCR amplification. Carnoy's and AMeX fixation resulted in consistently high yield of high molecular size DNA suitable for use in hybridization studies and PCR respectively. DNA from Papanicolaou stained smears was successfully amplified by PCR as well. Of all stains used in the preparation of smears, only eosin was detectable as a greenish band in agarose gels under ultraviolet illumination.