RESUMO
UDP-glucose-dehydrogenase (UGDH) synthesizes UDP-glucuronic acid. It is involved in epirubicin detoxification and hyaluronan synthesis. This work aimed to evaluate the effect of UGDH knockdown on epirubicin response and hyaluronan metabolism in MDA-MB-231 breast cancer cells. Additionally, the aim was to determine UGDH as a possible prognosis marker in breast cancer. We studied UGDH expression in tumors and adjacent tissue from breast cancer patients. The prognostic value of UGDH was studied using a public Kaplan-Meier plotter. MDA-MB-231 cells were knocked-down for UGDH and treated with epirubicin. Epirubicin-accumulation and apoptosis were analyzed by flow cytometry. Hyaluronan-coated matrix and metabolism were determined. Autophagic-LC3-II was studied by Western blot and confocal microscopy. Epirubicin accumulation increased and apoptosis decreased during UGDH knockdown. Hyaluronan-coated matrix increased and a positive modulation of autophagy was detected. Higher levels of UGDH were correlated with worse prognosis in triple-negative breast cancer patients that received chemotherapy. High expression of UGDH was found in tumoral tissue from HER2--patients. However, UGDH knockdown contributes to epirubicin resistance, which might be associated with increases in the expression, deposition and catabolism of hyaluronan. The results obtained allowed us to propose UGDH as a new prognostic marker in breast cancer, positively associated with development of epirubicin resistance and modulation of extracellular matrix.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Ácido Hialurônico/biossíntese , Neoplasias de Mama Triplo Negativas/enzimologia , Uridina Difosfato Glucose Desidrogenase/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Epirubicina/farmacologia , Feminino , Humanos , Prognóstico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The Salmonella PmrA-PmrB system controls the expression of genes necessary for polymyxin B resistance. Four loci were previously identified as part of the regulon, and interaction of PmrA with the promoter region of three of them was observed. Here we characterized the interaction of PmrA with the promoter region of ugd, previously suggested to be regulated indirectly by PmrA. Our results indicate that PmrA controls the expression of ugd by interacting with a specific sequence in the promoter region of this gene.
Assuntos
Proteínas de Bactérias/metabolismo , Regiões Promotoras Genéticas , Salmonella enterica/enzimologia , Uridina Difosfato Glucose Desidrogenase/genética , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , DNA Bacteriano , Dados de Sequência Molecular , Fosforilação , Salmonella enterica/genéticaRESUMO
Uridine 5'-diphosphate galacturonic acid (UDP-GalA) is a substrate for the galacturonosyltransferases that synthesize the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II. Pectin synthesis occurs in the Golgi and it is hypothesized that UDP-GalA is transported into the lumen of the Golgi by membrane-localized transporters. To study the transport and metabolism of UDP-GalA in the Golgi, UDP-GalA labeled in the uridine moiety is required. Here we present a high-yield method for the synthesis of [(3)H]UDP-GalA from [(3)H]UTP and Glc-1-P by sequential reactions catalyzed by UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and UDP-GlcA-4-epimerase and the separation of the reaction products over a Dionex CarboPac PA1 anion-exchange column using high-performance anion-exchange chromatography (HPAEC). Approximately half of the [(3)H]UTP was converted into [(3)H]UDP-GalA and the remaining 50% was recovered as [(3)H]UDP-GlcA. Both products were purified and the identity of the [(3)H]UDP-GalA was confirmed by its conversion into [(3)H]UDP-GlcA by UDP-GlcA-4-epimerase. The enzymatic synthesis of diverse nucleotide sugars radiolabeled in the nucleotide by the use of nucleotide-converting enzymes, combined with the high-resolution separation of the nucleotide sugars and their purification by HPAEC, can provide unique substrates required for the study of diverse nucleotide sugar transporters.
Assuntos
Complexo de Golgi/metabolismo , Açúcares de Uridina Difosfato/síntese química , Açúcares de Uridina Difosfato/metabolismo , Proteínas de Transporte/metabolismo , Cromatografia por Troca Iônica/métodos , Indicadores e Reagentes , Pirofosfatase Inorgânica , Marcação por Isótopo/métodos , Pirofosfatases , Trítio , UDPglucose 4-Epimerase , UTP-Glucose-1-Fosfato Uridililtransferase , Uridina Difosfato Glucose Desidrogenase , Açúcares de Uridina Difosfato/isolamento & purificaçãoAssuntos
Galactosemias/sangue , Galactosemias/terapia , Uridina Difosfato Galactose/deficiência , Criança , Cromatografia Líquida de Alta Pressão , Eritrócitos/química , Galactosemias/complicações , Humanos , Espectroscopia de Ressonância Magnética , Uridina/uso terapêutico , Uridina Difosfato Galactose/sangue , Uridina Difosfato Galactose/isolamento & purificação , Uridina Difosfato Glucose/sangue , Uridina Difosfato Glucose/isolamento & purificação , Uridina Difosfato Glucose DesidrogenaseRESUMO
To settle the ongoing controversy regarding differential uridine diphosphoglucose (UDPG) and uridine diphosphogalactose (UDPGal) content of erythrocytes, which may be important in evaluating the metabolic abnormality in patients with galactosemia, we derived a combined enzymatic-high-performance liquid chromatography (HPLC) assay. Uridine diphosphoglucuronate (UDPGA), the unique product of UDPG dehydrogenase activity, was separated and quantified by HPLC in extracts of human erythrocytes. The quantity of UDPGA produced in cell filtrates incubated with the enzyme corresponds to the amount of UDPG directly determined by HPLC. The amount of UDPGA produced was independent of the enzyme purity or activity used. On the other hand, the amounts of UDPG estimated by fluorometric measurement of the production of reduced nicotinamide adenine dinucleotide varied with the enzyme purity and activity. The combined enzymatic-HPLC method confirms the direct determinations of UDPG content of normal erythrocytes. The results indicate that, under appropriate conditions, the fluorometric-based assay will give accurate estimates of UDPG, but the direct HPLC method yields consistent and correct UDPG and UDPGal determinations.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Eritrócitos/química , Uridina Difosfato Glucose/sangue , Humanos , Valores de Referência , Uridina Difosfato Galactose/sangue , Uridina Difosfato Glucose/metabolismo , Uridina Difosfato Glucose DesidrogenaseRESUMO
Impurities in a reagent dehydrogenase caused overestimates of erythrocytic uridine diphosphate glucose and accounted for clinically important differences in results between those of one group of investigators using enzymatic methods and those of two other groups using enzymatic methods, high-performance liquid chromatography, and nuclear magnetic resonance. These data have relevance for the current debate regarding the pathophysiologic changes in galactosemia.