RESUMEN
Measures of fitness are valuable tools to predict species' responses to environmental changes, like increased water temperature. Aerobic scope (AS) is a measure of an individual's capacity for aerobic processes, and frequently used as a proxy for fitness. However, AS is complicated by individual variation found not only within a species, but within similar body sizes as well. Maximum metabolic rate (MMR), one of the factors determining AS, is constrained by an individual's ability to deliver and extract oxygen (O2) at the tissues. Recently, data has shown that red blood cell carbonic anhydrase (RBC CA) is rate-limiting for O2 delivery in red drum (Sciaenops ocellatus). We hypothesized increased temperature impacts MMR and RBC CA activity in a similar manner, and that an individual's RBC CA activity drives individual variation in AS. Red drum were acutely exposed to increased temperature (+6 °C; 22 °C to 28 °C) for 24 h prior to exhaustive exercise and intermittent-flow respirometry at 28 °C. RBC CA activity was measured before temperature exposure and after aerobic performance. Due to enzymatic thermal sensitivity, acute warming increased individual RBC CA activity by 36%, while there was no significant change in the control (22 °C) treatment. Interestingly, average MMR of the acute warming treatment was 36% greater than that of control drum. However, we found no relationships between individual RBC CA activity and their respective MMR and AS at either temperature. While warming similarly affects RBC CA activity and MMR, RBC CA activity is not a predictor of individual MMR.
Asunto(s)
Anhidrasas Carbónicas/biosíntesis , Metabolismo Energético , Eritrocitos/citología , Consumo de Oxígeno , Oxígeno/metabolismo , Aerobiosis , Animales , Cambio Climático , Peces , Calor , Perciformes/fisiología , Frecuencia Respiratoria , TemperaturaRESUMEN
Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes with a pivotal potential role in the biomimetic CO2 capture process (CCP) because these biocatalysts catalyse the simple but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons in all life kingdoms. The CAs are among the fastest known enzymes, with kcat values of up to 106 s-1 for some members of the superfamily, providing thus advantages when compared with other CCP methods, as they are specific for CO2. Thermostable CAs might be used in CCP technology because of their ability to perform catalysis in operatively hard conditions, typical of the industrial processes. Moreover, the improvement of the enzyme stability and its reuse are important for lowering the costs. These aspects can be overcome by immobilising the enzyme on a specific support. We report in this article that the recombinant thermostable SspCA (α-CA) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense can been heterologously produced by a high-density fermentation of Escherichia coli cultures, and covalently immobilised onto the surface of magnetic Fe3O4 nanoparticles (MNP) via carbodiimide activation reactions. Our results demonstrate that using a benchtop bioprocess station and strategies for optimising the bacterial growth, it is possible to produce at low cost a large amount SspCA. Furthermore, the enzyme stability and storage greatly increased through the immobilisation, as SspCA bound to MNP could be recovered from the reaction mixture by simply using a magnet or an electromagnetic field, due to the strong ferromagnetic properties of Fe3O4.
Asunto(s)
Anhidrasas Carbónicas/biosíntesis , Bacterias Gramnegativas Quimiolitotróficas/enzimología , Nanopartículas de Magnetita/química , Anhidrasas Carbónicas/metabolismo , Bacterias Gramnegativas Quimiolitotróficas/crecimiento & desarrollo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismoRESUMEN
Tissue hypoxia contributes to solid tumor pathogenesis by activating a series of adaptive programs. We previously showed that hypoxia promotes the preferential expansion and maintenance of CD133 positive human glioma stem cells (GSC) in a hypoxia inducible factor 1 alpha (HIF-1α)-dependent mechanism. Here, we examined the activity of digitoxin (DT), a cardiac glycoside and a putative inhibitor of HIF-1α, on human GSC in vitro and in vivo. During hypoxic conditions (1% O2), we observed the effect of DT on the intracellular level of HIF-1α and the extracellular level of vascular endothelial growth factor (VEGF) in human GSC. We found that DT at clinically achievable concentrations, suppressed HIF-1α accumulation during hypoxic conditions in human GSC and established glioma cell lines. DT treatment also significantly attenuated hypoxia-induced expression of VEGF, a downstream target of HIF-1α. Exposure to DT also reduced hypoxia-induced activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Furthermore, DT potently inhibited neurosphere formation, and decreased CD133 expression even at concentrations that were not overtly cytotoxic. Lastly, treatment with DT reduced GSC engraftment in an in vivo xenograft model of glioblastoma. Intraperitoneal injections of DT significantly inhibited the growth of established glioblastoma xenografts, and suppressed expression of HIF-1α and carbonic anhydrase (CA9), a surrogate marker of hypoxia. Taken together, these results suggest that DT at clinically achievable concentration functions as an inhibitor of HIF-1α, worthy of further investigations in the therapy of glioblastoma.
Asunto(s)
Anhidrasas Carbónicas/biosíntesis , Digitoxina/farmacología , Glioblastoma/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Células Madre Neoplásicas/patología , Antígeno AC133/metabolismo , Animales , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Ratones , Ratones SCID , Interferencia de ARN , ARN Interferente Pequeño/genética , Esferoides Celulares , Células Tumorales Cultivadas , Hipoxia Tumoral/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We used isobaric mass tagging (iTRAQ) and lectin affinity capture mass spectrometry (MS)-based workflows for global analyses of parotid saliva (PS) and whole saliva (WS) samples obtained from patients diagnosed with primary Sjögren's Syndrome (pSS) who were enrolled in the Sjögren's International Collaborative Clinical Alliance (SICCA) as compared with two control groups. The iTRAQ analyses revealed up- and down-regulation of numerous proteins that could be involved in the disease process (e.g., histones) or attempts to mitigate the ensuing damage (e.g., bactericidal/permeability increasing fold containing family (BPIF) members). An immunoblot approach applied to independent sample sets confirmed the pSS associated up-regulation of ß2-microglobulin (in PS) and down-regulation of carbonic anhydrase VI (in WS) and BPIFB2 (in PS). Beyond the proteome, we profiled the N-glycosites of pSS and control samples. They were enriched for glycopeptides using lectins Aleuria aurantia and wheat germ agglutinin, which recognize fucose and sialic acid/N-acetyl glucosamine, respectively. MS analyses showed that pSS is associated with increased N-glycosylation of numerous salivary glycoproteins in PS and WS. The observed alterations of the salivary proteome and N-glycome could be used as pSS biomarkers enabling easier and earlier detection of this syndrome while lending potential new insights into the disease process.
Asunto(s)
Glicoproteínas/metabolismo , Proteoma/genética , Saliva/metabolismo , Síndrome de Sjögren/metabolismo , Anhidrasas Carbónicas/biosíntesis , Femenino , Glicoproteínas/química , Glicosilación , Humanos , Lectinas/química , Masculino , Ácido N-Acetilneuramínico/metabolismo , Glándula Parótida/química , Glándula Parótida/metabolismo , Saliva/química , Síndrome de Sjögren/genética , Síndrome de Sjögren/patologíaRESUMEN
Human carbonic anhydrase XIV (CA XIV), a transmembrane protein, highly expressed in the central nervous system, is difficult to recombinantly express and purify in large scale for the measurements of inhibitor binding and drug design. CA XIV belongs to the family of twelve catalytically active CA isoforms in the human body. Disorders in the expression of CA XIV cause serious diseases and CA XIV has been described as a possible drug target for the treatment of epilepsy, some retinopathies, and skin tumors. In this study, the effect of different promoters, E. coli strains, and the length of recombinant CA XIV protein construct were analyzed for the production CA XIV in large scale by using affinity purification. Active site titration by inhibitors and the isothermal titration calorimery revealed over 96% purity of the protein. Enzymatic activity of the purified CA XIV was determined by following the CO2 hydration using the stopped-flow technique. Several inhibitors were discovered that exhibited selectivity towards CA XIV over other CA isoforms and could be developed as drugs.
Asunto(s)
Anhidrasas Carbónicas/biosíntesis , Anhidrasas Carbónicas/aislamiento & purificación , Escherichia coli , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Escherichia coli/genética , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Carbonic anhydrase plays a key role in CO2 transport, acid-base and ion regulation and metabolic processes in vertebrates. While several carbonic anhydrase isoforms have been identified in numerous vertebrate species, basal lineages such as the cyclostomes have remained largely unexamined. Here we investigate the repertoire of cytoplasmic carbonic anhydrases in the sea lamprey (Petromyzon marinus), that has a complex life history marked by a dramatic metamorphosis from a benthic filter-feeding ammocoete larvae into a parasitic juvenile which migrates from freshwater to seawater. We have identified a novel carbonic anhydrase gene (ca19) beyond the single carbonic anhydrase gene (ca18) that was known previously. Phylogenetic analysis and synteny studies suggest that both carbonic anhydrase genes form one or two independent gene lineages and are most likely duplicates retained uniquely in cyclostomes. Quantitative PCR of ca19 and ca18 and protein expression in gill across metamorphosis show that the ca19 levels are highest in ammocoetes and decrease during metamorphosis while ca18 shows the opposite pattern with the highest levels in post-metamorphic juveniles. We propose that a unique molecular switch occurs during lamprey metamorphosis resulting in distinct gill carbonic anhydrases reflecting the contrasting life modes and habitats of these life-history stages.
Asunto(s)
Anhidrasas Carbónicas , Citoplasma , Proteínas de Peces , Regulación Enzimológica de la Expresión Génica/fisiología , Petromyzon , Animales , Dióxido de Carbono/metabolismo , Anhidrasas Carbónicas/biosíntesis , Anhidrasas Carbónicas/genética , Citoplasma/enzimología , Citoplasma/genética , Proteínas de Peces/biosíntesis , Proteínas de Peces/genética , Branquias/enzimología , Petromyzon/genética , Petromyzon/metabolismoRESUMEN
The aim of this work was to study the co-production of the carbonic anhydrase, C-phycocyanin and allophycocyanin during cyanobacteria growth. Spirulina sp. LEB 18 demonstrated a high potential for simultaneously obtaining the three products, achieving a carbonic anhydrase (CA) productivity of 0.97U/L/d and the highest C-phycocyanin (PC, 5.9µg/mL/d) and allophycocyanin (APC, 4.3µg/mL/d) productivities. In the extraction study, high extraction yields were obtained from Spirulina using an ultrasonic homogenizer (CA: 25.5U/g; PC: 90mg/g; APC: 70mg/g). From the same biomass, it was possible to obtain three biomolecules that present high industrial value.
Asunto(s)
Anhidrasas Carbónicas/biosíntesis , Ficobiliproteínas/biosíntesis , Spirulina/metabolismo , Synechococcus/metabolismo , BiomasaRESUMEN
Elevated sweat chloride levels, failure to thrive (FTT), and lung disease are characteristic features of cystic fibrosis (CF, OMIM #219700). Here we describe variants in CA12 encoding carbonic anhydrase XII in two pedigrees exhibiting CF-like phenotypes. Exome sequencing of a white American adult diagnosed with CF due to elevated sweat chloride, recurrent hyponatremia, infantile FTT and lung disease identified deleterious variants in each CA12 gene: c.908-1 G>A in a splice acceptor and a novel frameshift insertion c.859_860insACCT. In an unrelated consanguineous Omani family, two children with elevated sweat chloride, infantile FTT, and recurrent hyponatremia were homozygous for a novel missense variant (p.His121Gln). Deleterious CFTR variants were absent in both pedigrees. CA XII protein was localized apically in human bronchiolar epithelia and basolaterally in the reabsorptive duct of human sweat glands. Respiratory epithelial cell RNA from the adult proband revealed only aberrant CA12 transcripts and in vitro analysis showed greatly reduced CA XII protein. Studies of ion transport across respiratory epithelial cells in vivo and in culture revealed intact CFTR-mediated chloride transport in the adult proband. CA XII protein bearing either p.His121Gln or a previously identified p.Glu143Lys missense variant localized to the basolateral membranes of polarized Madin-Darby canine kidney (MDCK) cells, but enzyme activity was severely diminished when assayed at physiologic concentrations of extracellular chloride. Our findings indicate that loss of CA XII function should be considered in individuals without CFTR mutations who exhibit CF-like features in the sweat gland and lung.
Asunto(s)
Anhidrasas Carbónicas/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Enfermedades Pulmonares/genética , Sudor/metabolismo , Adolescente , Adulto , Animales , Anhidrasas Carbónicas/biosíntesis , Anhidrasas Carbónicas/metabolismo , Niño , Preescolar , Cloruros/metabolismo , Fibrosis Quística/metabolismo , Fibrosis Quística/fisiopatología , Perros , Femenino , Regulación Enzimológica de la Expresión Génica , Homocigoto , Humanos , Pulmón/enzimología , Pulmón/patología , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/fisiopatología , Células de Riñón Canino Madin Darby , Masculino , Mutación , Linaje , FenotipoRESUMEN
For various types of tumor therapy, it is suggested that co-targeting of tumor microenvironment, mainly tumor vasculature, mediates tumor response mechanisms. Immunohistochemistry for glucose transporter-1 (GLUT-1), carbonic anhydrase-IX (CAIX), Ki-67, and von Willebrand factor VIII for microvessel density (MVD) were performed on formalin-fixed paraffin-embedded samples of canine oral malignant neoplasms. Polarographic oxygen measurements (median pO2) and perfusion data via contrast-enhanced power Doppler ultrasound (median vascularity, median blood volume) provided additional information. Ninety-two samples were analyzed: sarcomas (n = 32), carcinomas (n = 30), and malignant melanomas (n = 30). Polarographic oxygen and perfusion data was available in 22.8% (sarcomas n = 9, carcinomas n = 7, melanomas n = 5), and 27.1% (sarcomas n = 10, carcinomas n = 8, melanomas n = 7) of cases, respectively. GLUT-1 expression was detected in 46.7% of all samples, and was generally weak. CAIX expression was found in 34.8% of all samples. Median Ki-67 score and MVD count was 19% and 17, respectively. The evaluation of the GLUT-1 score and continuous data showed significantly lower GLUT-1 levels in sarcomas (mean 5.1%, SD 6.2) versus carcinomas and melanomas (mean 16.5%/ 19.0%, SD 17.3/ 20.9, p = 0.001). The expression of CAIX correlated mildly positively with GLUT-1 (p = 0.018, rho = 0.250) as well as with Ki-67 (p = 0.014, rho = 0.295). MVD showed a significantly lower level in melanomas (mean 12.6, SD 7.7) versus sarcomas and carcinomas (mean 21.8/ 26.9, SD 13.0/20.4, p = 0.001). Median vascularity and blood volume were significantly lower in sarcomas (mean 10.4%, SD 11.0, and mean 6.3%, SD 6.5, respectively) versus carcinomas (mean 39.2%, SD 16.4 and mean 33.0%, SD 25.6, respectively) and melanomas (mean 36.0%, SD 18.3, and 31.5%, SD 24.5). Between the 3 histological groups, there was neither a significant difference in the GLUT-1 and CAIX score and continuous data, nor the Ki67 score, or polarographic oxygen measurements. GLUT-1 continuous data and Ki-67 (p<0.001, rho = 0.403), as well as Ki-67 and MVD (p = 0.029, rho = 0.228) correlated positively and a mild correlation was found between vascularity and GLUT-1 (p = 0.043, rho = 0.408). GLUT-1, CAIX, proliferative index and MVD levels were established as microenvironmental descriptors with the purpose of creating a baseline in order to follow changes seen in the tumor microenvironment after hypofractionated radiation with high doses.
Asunto(s)
Anhidrasas Carbónicas/biosíntesis , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Transportador de Glucosa de Tipo 1/biosíntesis , Microcirculación , Neoplasias de la Boca , Proteínas de Neoplasias/biosíntesis , Animales , Hipoxia de la Célula , Perros , Neoplasias de la Boca/irrigación sanguínea , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patologíaRESUMEN
PURPOSE: The aim of study was to test by immunohistochemical (IHC) staining whether carbonic anhydrase (CA) 9 and 12 have an effect on sentinel lymph node (SLN) metastasis in early breast cancer and to find clinicopathologic factors associated with SLN metastasis. MATERIALS AND METHODS: Between June 2003 and June 2011, medical records of 470 patients diagnosed with breast cancer with pT1-2, pN0-2, and M0 were reviewed. Of these 470, 314 patients who underwent SLN biopsy±axillary dissection were subjects of this study. Using tissue microarray, IHC staining for CA9 and CA12 was performed. Clinicopathologic factors such as patient age, tumour size, lymphatic invasion, hormone receptor status, and the Ki-67 labeling index were analysed together. RESULTS: The mean age of all patients was 51.7 years. The mean number of harvested SLN was 3.62, and 212 patients (67.5%) had negative SLN. Lymphatic invasion, the Ki-67 labelling index of primary tumours, and CA9 staining of stromal cells, were independent risk factors for SLN metastasis in the multivariate analysis. In 33 patients (10.5%) without the three risk factors, no patient had SLN metastasis. In 80 patients without lymphatic invasion of primary tumours or CA9 staining of stromal cells, only four patients (5%) had positive SLN. CONCLUSION: CA9 staining of stromal cells is an independent risk factor for SLN metastasis as well as lymphatic invasion and a low Ki-67 labelling index of primary tumours in patients with early breast cancer. IHC staining of primary tumours for CA12was not associated with SLN metastasis.
Asunto(s)
Neoplasias de la Mama , Anhidrasa Carbónica IX/biosíntesis , Metástasis Linfática , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Anhidrasas Carbónicas/biosíntesis , Humanos , Persona de Mediana Edad , Biopsia del Ganglio Linfático CentinelaRESUMEN
Though recent studies have revealed that stem cells of many tissues are harbored in hypoxic microenvironment, little is known about the relationship between hypoxia and intestinal crypt base, where intestinal stem cells are supposed to exist. In this study, we focused on carbonic anhydrase IX (CA9), a hypoxia-inducible membrane-tethered protein, in normal intestinal crypt base, adenoma and early colorectal cancer. Using surgically resected human colorectal cancer specimen, we searched for the expression pattern and functional association of CA9 in human adult normal intestinal epithelia, adenoma and early colorectal cancer by immunofluorescent and immunohistochemical staining, flow cytometry, and quantitative real-time-polymerase chain reaction. We demonstrated that almost all crypt base slender cells in ileum and crypt base cells with eosinophilic structure in their basal cytoplasm in right and left colon were CA9+ with the ratio of 25 to 40%, and that adenoma and T1 colorectal cancer showed broad expression of CA9. Flow cytometrically sorted CA9+ population showed increased mRNA level of a Wnt signaling factor AXIN2. In conclusion, these observations indicate that CA9 expression in normal crypt base cells has association with intestinal epithelial stemness and CA9 may be involved in the carcinogenesis of colorectal cancer.
Asunto(s)
Adenoma/genética , Antígenos de Neoplasias/biosíntesis , Proteína Axina/biosíntesis , Anhidrasas Carbónicas/biosíntesis , Neoplasias Colorrectales/genética , Adenoma/patología , Adenoma/cirugía , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/genética , Proteína Axina/genética , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/cirugía , Femenino , Citometría de Flujo , Humanos , Mucosa Intestinal/metabolismo , Intestinos/patología , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
Hypoxia, a common phenomenon during the development of malignant solid tumors including breast cancer, serves to propagate a cascade of molecular pathways triggered by hypoxia-inducible factor-1α (HIF-1α). Carbonic anhydrase IX (CA IX), one of the target genes of HIF-1α, has been reported to be involved in progression of malignant tumors. The objective of this study was to investigate the expression of HIF-1α and CA IX in hypoxia, involvement of CA IX in the regulation of migration and invasion/metastasis and its prognostic significance in breast cancer. We used cobalt chloride (CoCl2) as a hypoxia-mimetic agent and found that the expression of HIF-1α protein, CA IX mRNA and protein, is effectively upregulated, except for HIF-1α mRNA. Data showed that the elevated CA IX expression is closely related to in vitro cell migration and invasion, and the underlying mechanism of this process may be associated with epithelial-mesenchymal transition (EMT). The study of clinical tissue samples also demonstrated that CA IX is an independent prognostic marker that may serve as a useful clinical biomarker for predicting tumor progression and the invasion/metastasis of breast cancer. These results provide further insight into the role of CA IX in tumor progression and put forward further strong evidence as well as new consideration for CA IX target therapy.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Biomarcadores de Tumor/biosíntesis , Neoplasias de la Mama/genética , Anhidrasas Carbónicas/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/genética , Hipoxia de la Célula/efectos de los fármacos , Proliferación Celular/genética , Cobalto/toxicidad , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Células MCF-7 , Persona de Mediana Edad , Pronóstico , Activación Transcripcional/efectos de los fármacosRESUMEN
The hypoxic environment is a crucial component of the cancer stem cell niche and it is capable of eliciting stem cell features in cancer cells. We previously reported that SNAI2 up-regulates the expression of Carbonic Anhydrase iso-enzyme 9 (CA9) in hypoxic MCF7 cells. Here we show that SNAI2 down-regulates miR34a expression in hypoxic MCF7 cell-derived mammospheres. Next, we report on the capability of miR34a to decrease CA9 mRNA stability and CA9 protein expression. We also convey that the over-expression of cloned CA9-mRNA-3'UTR increases the mRNA half-life and protein levels of two miR34a targets JAGGED1 and NOTCH3. The data here reported shows that the SNAI2-dependent down-regulation of miR34a substantially contributes to the post-transcriptional up-regulation of CA9, and that CA9-mRNA-3'UTR acts as an endogenous microRNA sponge. We conclude that CA9/miR34 interplay shares in the hypoxic regulation of mammospheres and therefore, may play a relevant role in the hypoxic breast cancer stem cell niche.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Neoplasias de la Mama/genética , Anhidrasas Carbónicas/biosíntesis , Hipoxia de la Célula/genética , MicroARNs/genética , Antígenos de Neoplasias/genética , Neoplasias de la Mama/patología , Proteínas de Unión al Calcio/biosíntesis , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoxia/genética , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Proteína Jagged-1 , Células MCF-7 , Proteínas de la Membrana/biosíntesis , MicroARNs/biosíntesis , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptor Notch3 , Receptores Notch/biosíntesis , Proteínas Serrate-Jagged , Factores de Transcripción de la Familia Snail , Factores de Transcripción/biosíntesisRESUMEN
Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.
Asunto(s)
Proteínas Sanguíneas/biosíntesis , Proteínas Musculares/biosíntesis , Músculo Esquelético/metabolismo , Proteoma , Animales , Biopsia , Anhidrasas Carbónicas/biosíntesis , Forma MM de la Creatina-Quinasa/biosíntesis , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Human carbonic anhydrase IX (CA IX) is overexpressed in a number of solid tumors and is considered to be a marker for cellular hypoxia that it is not produced in most normal tissues. CA IX contributes to the acidification of the extracellular matrix, which, in turn, favors tumor growth and metastasis. Therefore, CA IX is considered to be a promising anti-cancer drug target. However, the ability to specifically target CA IX is challenging due to the fact that the human genome encodes 15 different carbonic anhydrase isoforms that have a high degree of homology. Furthermore, structure-based drug design of CA IX inhibitors so far has been largely unsuccessful due to technical difficulties regarding the expression and crystallization of the enzyme. Currently, only one baculovirus-produced CA IX structure in complex with a nonspecific CA inhibitor, acetazolamide, is available in Protein Data Bank. We have developed an efficient system for the production of the catalytic domain of CA IX in methylotrophic yeast Pichia pastoris. The produced protein can be easily crystallized in the presence of inhibitors, as we have demonstrated for several 2-thiophene-sulfonamide compounds. We have also observed significant differences in the binding mode of chemically identical compounds to CA IX and CA II, which can be further exploited in the design of CA IX-specific inhibitors.
Asunto(s)
Antígenos de Neoplasias/química , Anhidrasas Carbónicas/química , Neoplasias/enzimología , Acetazolamida/farmacología , Antígenos de Neoplasias/biosíntesis , Antineoplásicos/farmacología , Baculoviridae/metabolismo , Anhidrasa Carbónica IX , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/biosíntesis , Clonación Molecular , Cristalización , Bases de Datos de Proteínas , Humanos , Isoenzimas , Modelos Moleculares , Pichia/enzimología , Relación Estructura-Actividad , Especificidad por Sustrato , Difracción de Rayos XRESUMEN
BACKGROUND: There is significant evidence that severe tumor hypoxia may cause resistance to chemoradiotherapy and promote metastatic spread in locally advanced carcinoma of the uterine cervix. Some clinical investigations have suggested that high expression of hypoxia-inducible factor-1α (HIF-1α) and/or its target gene carbonic anhydrase IX (CAIX) may be useful biomarkers of tumor hypoxia and poor outcome in cervical cancer. Here, we challenged this view by investigating possible associations between HIF-1α expression, CAIX expression, fraction of hypoxic tissue, and lymph node metastasis in experimental human tumors. METHODS: Tumors of two cervical carcinoma xenograft lines (CK-160 and TS-415) were included in the study. Pimonidazole was used as a hypoxia marker, and tumor hypoxia, HIF-1α expression, and CAIX expression were detected by immunohistochemistry. Metastatic status was assessed by examining external lymph nodes in the inguinal, axillary, interscapular, and submandibular regions and internal lymph nodes in the abdomen and mediastinum. RESULTS: Tissue regions staining positive for pimonidazole, HIF-1α, or CAIX were poorly colocalized, both in CK-160 and TS-415 tumors. The expression of HIF-1α or CAIX did not correlate with the fraction of hypoxic tissue in any of the two tumor lines. Furthermore, clinically relevant associations between HIF-1α or CAIX expression and lymph node metastasis were not found. CONCLUSION: Because significant associations between HIF-1α expression, CAIX expression, fraction of hypoxic tissue, and incidence of lymph node metastases could not be detected in any of two preclinical models of human cervical cancer, it is not realistic to believe that high expression of HIF-1α or CAIX can be useful biomarkers of tumor hypoxia and poor outcome in a highly heterogeneous disease like cervical carcinoma.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Biomarcadores de Tumor/biosíntesis , Anhidrasas Carbónicas/biosíntesis , Carcinoma de Células Escamosas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Neoplasias del Cuello Uterino/metabolismo , Animales , Anhidrasa Carbónica IX , Carcinoma de Células Escamosas/patología , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias del Cuello Uterino/patología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Carbonic anhydrase IX (CAIX) is strongly induced by hypoxia and its overexpression is associated with poor therapeutic outcome in cancer. Here, we report that hypoxia promotes tumour heterogeneity through the epigenetic regulation of CAIX. Based on hypoxic CAIX expression we identify and characterize two distinct populations of tumour cells, one that has inducible expression of CAIX and one that does not. The CAIX+ve population is enriched with cells expressing cancer stem cell markers and which have high self-renewal capacity. We show that differential CAIX expression is due to differences in chromatin structure. To further investigate the relationship between chromatin organization and hypoxic induction of CAIX expression we investigated the effect of JQ1 an inhibitor of BET bromodomain proteins and A366 a selective inhibitor of the H3K9 methyltransferase G9a/GLP. We identified that these drugs were able to modulate hypoxic CAIX expression induction. This further highlights the role of epigenetic modification in adaption to hypoxia and also in regulation of heterogeneity of cells within tumours. Interestingly, we identified that the two subpopulations show a differential sensitivity to HDAC inhibitors, NaBu or SAHA, with the CAIX positive showing greater sensitivity to treatment. We propose that drugs modulating chromatin regulation of expression may be used to reduce heterogeneity induced by hypoxia and could in combination have significant clinical consequences.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Anhidrasas Carbónicas/biosíntesis , Inhibidores de Histona Desacetilasas/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Animales , Antígenos de Neoplasias/genética , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/genética , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Inducción Enzimática , Femenino , Células HCT116 , Xenoinjertos , Humanos , Isoenzimas/biosíntesis , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Madre Neoplásicas/patologíaRESUMEN
While normal kidneys are relatively sensitive to ionizing radiation (IR), renal cell carcinoma (RCC) is considered radioresistant. Carbonic anhydrase IX (CA9), an enzyme that maintains intracellular pH by carbon dioxide dissolution, is upregulated in the majority of RCC, but not in normal kidneys. Since regulation of intracellular pH may enhance radiation effects, we hypothesized that inhibition of CA9 may radiosensitize RCC. Clonogenic survival assay of human clear cell RCC 786-O and murine RCC RAG cells in the presence of a pharmacological CA9 inhibitor or with shRNA-mediated knockdown of CA9 was performed to investigate the response to IR in vitro (single dose or fractionated) and in vivo. Extracellular pH changes were measured in vitro. Treatment with AEBS [4-(2-aminoethyl)benzene sulfonamide], a sulfonamide, was used as a pharmacological inhibitor of the enzymatic activity of CA9. Nude mice bearing subcutaneous xenografts of 786-O cells stably expressing CA9 shRNA or scrambled control were irradiated (6 Gy). Tumor growth was followed longitudinally in the 786-O-bearing mice receiving AEBS (50-200 µg/ml drinking water) or control (vehicle only) which were irradiated (6 Gy) and compared with mice receiving either IR or AEBS alone. In vitro inhibition of CA9 activity or expression significantly sensitized RCC cells to the effects of IR (p<0.05), an effect even more significant when hypofractionated IR was applied. In vivo irradiated xenografts from RCC cells transfected with CA9 shRNA were significantly smaller compared to irradiated xenografts from the scrambled shRNA controls (p<0.05). RCC xenografts from mice treated with AEBS in combination with IR grew significantly slower than all controls (p<0.05). Inhibition of CA9 expression or activity resulted in radiation sensitization of RCC in a preclinical mouse model.
Asunto(s)
Antígenos de Neoplasias/genética , Anhidrasas Carbónicas/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/radioterapia , Tolerancia a Radiación/genética , Animales , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/efectos de los fármacos , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/biosíntesis , Anhidrasas Carbónicas/efectos de los fármacos , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , ARN Interferente Pequeño/genética , Radiación Ionizante , Sulfonamidas/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sleep disordered breathing (SDB) represents a spectrum of disorders, including habitual snoring and obstructive sleep apnea (OSA). Sleep disordered breathing is characterized by chronic intermittent hypoxia, airflow limitation, and recurrent arousals, which may lead to tissue hypoperfusion, hypoxia, and inflammation. In this study, we aimed to examine whether SDB during pregnancy was associated with histopathologic evidence of chronic placental hypoxia and/or uteroplacental underperfusion. The placentas of women with OSA (n â=â 23) and habitual snoring (n â=â 78) as well as nonsnorer controls (n â=â 47) were assessed for histopathologic and immunohistochemical markers of chronic hypoxia and uteroplacental underperfusion. Fetal normoblastemia was significantly more prevalent in SDB placentas than in those of nonsnorer controls (34.6% and 56.5% in snorers and OSA, respectively, versus 6.4% in controls). Expression of the tissue hypoxia marker carbonic anhydrase IX (CAIX) was more common in OSA placentas than controls (81.5% and 91.3% in snorers and OSA, respectively, versus 57.5% in controls). Adjusting for confounders such as body mass index, diabetes mellitus, or chronic hypertension did not alter the results. The uteroplacental underperfusion scores were similar among the 3 groups. Our findings suggest that SDB during pregnancy is associated with fetoplacental hypoxia, as manifested by fetal normoblastemia and increased placental carbonic anhydrase IX immunoreactivity. The clinical implications and underlying mechanisms remain to be determined.
Asunto(s)
Hipoxia/etiología , Placenta/patología , Síndromes de la Apnea del Sueño/complicaciones , Adolescente , Adulto , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/biosíntesis , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/análisis , Anhidrasas Carbónicas/biosíntesis , Femenino , Humanos , Hipoxia/patología , Inmunohistoquímica , Embarazo , Adulto JovenRESUMEN
Carbonic anhydrase IX (CAIX) is reportedly overexpressed in several types of carcinomas and is generally considered a marker of malignancy. The current study investigated the association between membrane expression of CAIX and the clinicopathological characteristics in oral squamous cell carcinoma (OSCC) patients. The study used immunohistochemistry to examine CAIX expression in 271 OSCC specimens by tissue microarray (TMA) and assessed the effect of CAIX overexpression and knockdown on migration of oral cancer cells in vitro. We found that CAIX expression was associated with more advanced clinical stages (p = 0.030) and positive lymph node metastasis (p = 0.026). Importantly, CAIX expression was correlated with a poorer patient prognosis in a univariate survival analysis (p = 0.025). Moreover, CAIX suppression by small interfering RNA (siRNA) significantly reduced cellular migration in OECM-1 oral cancer cell. In conclusion, our study showed that the expression of CAIX in OSCC samples can predict the progression of OSCC and survival of OSCC patients in Taiwan.