RESUMEN
The complex enzyme network responsible for glycan synthesis suffers significant changes during the first steps of tumor development, leading to the early formation of tumor-associated glycan signatures. Among the glycosylation pathways, changes in fucosylation emerged as one of most important features in cancer. Αlpha-1,3/4-fucosyltransferase (FUT3) has been linked to pro-tumor and anti-tumor pathways depending on the cancer type. The present study aimed to understand the gene and protein expression profiles of FUT3 in three different and independent cohorts composed by invasive breast cancer patients: Local Brazilian population, METABRIC and TCGA. FUT3 transcripts and protein were measured in the Brazilian population by real-time PCR and Western blotting, respectively. Clinical records and FUT3 levels from public METABRIC and TCGA cohorts were accessed through CBioPortal database. FUT3 expression was analyzed in each cohort using the appropriated statistic tools. Survival meta-analysis in triple negative patients was performed using five independent cohorts including GSE41119, GSE47994 and GSE86945, data obtained from GEO repository available at NCBI database, and METABRIC and TCGA. Our analysis showed that high FUT3 levels were consistently associated to reduced invasive breast cancer patients overall survival. This finding is particularly significant in triple negative patients. These results together with the previously knowledge regarding the involvement of FUT3 in pro-tumor and anti-tumor mechanisms led us to purpose a model for FUT3 expression regulation throughout breast cancer establishment and progression.
Asunto(s)
Biomarcadores de Tumor/genética , Fucosiltransferasas/genética , Neoplasias de la Mama Triple Negativas/genética , Biomarcadores de Tumor/metabolismo , Femenino , Fucosiltransferasas/metabolismo , Humanos , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Supervivencia , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
FUT3 gene is responsible for encode an homonymous α1,3/4-fucosyltransferase involved in the synthesis of sialyl-Lewis antigens. FUT3-fucosylated glycoconjugates play key roles in pathways involved in tumor biology and metastasis, such as cellular ligation to E-selectins, TGF-ß-induced epithelial-mesenchymal transition, NK cell-mediated tumor cytotoxicity and apoptosis. Tumor-associated FUT3 promoter polymorphism rs2306969 (-6951 C> T, position related to the gene's translation start site) has been linked to breast, ovarian and intestinal gastric cancer. Although non-coding polymorphisms accounts for the majority of variations founded in breast cancer, their functional roles are still poorly understood. This study aimed to investigate the impact of different alleles for this variation in FUT3 expression of invasive breast tumors. A luciferase reporter assay was performed using two breast tumor cell lines to evaluate respectively the impact of FUT3 rs2306969 (-6951 CC) and (-6951 TT) on protein expression. Gene and protein expressions were also measured in twenty-nine fresh biopsies of invasive breast tumors. Rs2306969 did not significantly influence FUT3 expression in both used systems. However, this study is defiant since the biological role of this polymorphism in breast cancer and other tumor types could be linked to cis/trans modulation of other genes, respond to different environmental stimuli or impact gene expression only in association with other variations. Rs2306969 did not modulate FUT3 expression in breast tumors under non-stimulated conditions. Nevertheless, our study contributes to the notably challenging task that is to understand how non-coding polymorphisms can drive the overall risk in cancer development.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Fucosiltransferasas/genética , Alelos , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Femenino , Fucosiltransferasas/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad/genética , Humanos , Polimorfismo de Nucleótido Simple/genética , Factores de Riesgo , Factor de Crecimiento Transformador beta/genéticaAsunto(s)
Dioclea/química , Lectinas de Unión a Manosa/química , Lectinas de Plantas/química , Semillas/química , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalización , Glioma , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Unión a Manosa/farmacología , Simulación del Acoplamiento Molecular , Lectinas de Plantas/aislamiento & purificación , Lectinas de Plantas/farmacología , Unión Proteica , Conformación Proteica , RatasRESUMEN
Lectins are multidomain proteins that specifically recognize various carbohydrates. The structural characterization of these molecules is crucial in understanding their function and activity in systems and organisms. Most cancer cells exhibit changes in glycosylation patterns, and lectins may be able to recognize these changes. In this work, Dioclea lasiocarpa seed lectin (DLL) was structurally characterized. The lectin presented a high degree of similarity with other lectins isolated from legumes, presenting a jelly roll motif and a metal-binding site stabilizing the carbohydrate-recognition domain. DLL demonstrated differential interactions with carbohydrates, depending on type of glycosidic linkage present in ligands. As observed by the reduction of cell viability in C6 cells, DLL showed strong antiglioma activity by mechanisms involving activation of caspase 3.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Dioclea/química , Glioma/patología , Lectinas de Plantas/química , Lectinas de Plantas/farmacología , Animales , Antineoplásicos/metabolismo , Metabolismo de los Hidratos de Carbono , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Simulación del Acoplamiento Molecular , Lectinas de Plantas/metabolismo , Conformación Proteica , Ratas , Semillas/químicaRESUMEN
Lectins are proteins, or glycoproteins, capable of reversibly binding to specific mono- or oligosaccharides via a noncatalytic domain. The Diocleinae subtribe presents lectins with high structural similarity, but different effects based on biological activity assays. This variability results from small structural differences. Therefore, in this context, the present study aimed to perform a structural analysis of the lectin from Dioclea lasiophylla Mart. ex Benth seeds (DlyL) and evaluate its inflammatory effect. To accomplish this, DlyL was purified in a single step by affinity chromatography on Sephadex® G-50 matrix. DlyL primary structure was determined through a combination of tandem mass spectrometry and DNA sequencing. DlyL showed high similarity with other species from the same genus. Its theoretical three-dimensional structure was predicted by homology modelling, and the protein was subjected to ligand screening with monosaccharides, oligosaccharides and complex N-glycans by molecular docking. Stability and binding of the lectin with α-methyl-d-mannoside were assessed by molecular dynamics. DlyL showed acute inflammatory response with hypernociceptive effect in the paw edema model, possibly by interaction with glycans present at the cell surface.
Asunto(s)
Lectinas/química , Simulación del Acoplamiento Molecular , Dioclea/química , Simulación de Dinámica Molecular , Monosacáridos/química , Oligosacáridos/química , Espectrometría de Masas en TándemRESUMEN
The three-dimensional structure of Dioclea reflexa seed lectin (DrfL) was studied in detail by a combination of X-ray crystallography, molecular docking and molecular dynamics. DrfL was purified by affinity chromatography using Sephadex G-50 matrix. Its primary structure was obtained by mass spectrometry, and crystals belonging to orthorhombic space group P212121 were grown by the vapor diffusion method at 293K. The crystal structure was solved at 1.765Å and was very similar to that of other lectins from the same subtribe. The structure presented Rfactor and Rfree of 21.69% and 24.89%, respectively, with no residues in nonallowed regions of Ramachandran plot. Similar to other Diocleinae lectins, DrfL was capable of relaxing aortic rings via NO induction, with CRD participation, albeit with low intensity (32%). In silico analysis results demonstrated that DrfL could strongly interact with complex N-glycans, components of blood vessel glycoconjugates. Despite the high similarity among Diocleinae lectins, it was also reported that each lectin has unique CRD properties that influence carbohydrate binding, resulting in different biological effects presented by these molecules.
Asunto(s)
Dioclea/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Lectinas de Plantas/química , Lectinas de Plantas/farmacología , Semillas/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Manósidos/química , Manósidos/metabolismo , Lectinas de Plantas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Dominios Proteicos , Ratas , Vasodilatadores/química , Vasodilatadores/metabolismo , Vasodilatadores/farmacologíaRESUMEN
Fucosylated glycans synthesized by α1,3/4-fucosyltransferase (FUT3) enzyme play an important role in breast cancer prognosis and metastasis, being involved in the binding of circulating tumor cells to the endothelium and being related to tumor stage, metastatic potential and chemoresistance. Despite the pro-tumor action of this enzyme, studies have demonstrated its role in natural killer-induced cytotoxicity through the recognition of sialyl Lewis X by C-type lectin receptors and through extrinsic apoptosis pathway triggered by Apo2L-TRAIL. This study aimed to investigate the expression pattern of FUT3 in invasive breast carcinoma (IDC) from patients of Pernambuco state, Northeast of Brazil, and genotype FUT3 promoter region to identify possible SNPs that could be associated with variations in FUT3 expression. Immunohistochemistry assay was used to access the FUT3 expression in normal (n=11) and tumor tissues (n=85). DNA sequencing was performed to genotype the FUT3 promoter region in patients with IDC (n=109) and healthy controls (n=110). Our results demonstrated that the absence of FUT3 enzyme is related to breast's IDC. The non-expression of FUT3 was more frequent in larger lesions and also in HER2 negative IDC tumors. Genomic analysis showed that two variations localized in FUT3 promoter region are possibly associated with IDC. Our results suggest that minor allele T of SNP rs73920070 (-6933 C>T) confers protection whereas minor allele T of SNP rs2306969 (-6951 C>T) triggers to susceptibility to IDC in the population of Pernambuco state, Northeast of Brazil.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patología , Fucosiltransferasas/genética , Regulación Neoplásica de la Expresión Génica/genética , Regiones Promotoras Genéticas , Brasil , Femenino , Fucosiltransferasas/metabolismo , Humanos , Inmunohistoquímica/métodos , Antígeno Lewis X/metabolismoRESUMEN
This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens . Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results.
Asunto(s)
Queso/microbiología , Enterococcus faecium/aislamiento & purificación , Enterococcus faecium/fisiología , Manipulación de Alimentos/métodos , Inocuidad de los Alimentos , Adhesión Bacteriana , Brasil , Fenómenos Químicos , Ácidos Cólicos/metabolismo , Ácidos Cólicos/toxicidad , Clostridium perfringens/química , Clostridium perfringens/fisiología , Enterococcus faecium/química , Escherichia coli/química , Escherichia coli/fisiología , Tracto Gastrointestinal/química , Interacciones Hidrofóbicas e Hidrofílicas , Inactivación Metabólica , Viabilidad Microbiana/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Factores de Virulencia/análisis , Factores de Virulencia/genéticaRESUMEN
This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens. Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results.
Asunto(s)
Queso/microbiología , Enterococcus faecium/aislamiento & purificación , Enterococcus faecium/fisiología , Inocuidad de los Alimentos , Manipulación de Alimentos/métodos , Adhesión Bacteriana , Brasil , Fenómenos Químicos , Ácidos Cólicos/metabolismo , Ácidos Cólicos/toxicidad , Clostridium perfringens/química , Clostridium perfringens/fisiología , Enterococcus faecium/química , Escherichia coli/química , Escherichia coli/fisiología , Tracto Gastrointestinal/química , Interacciones Hidrofóbicas e Hidrofílicas , Inactivación Metabólica , Viabilidad Microbiana/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Factores de Virulencia/análisis , Factores de Virulencia/genéticaRESUMEN
This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens. Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results.(AU)
Asunto(s)
Queso/microbiología , Enterococcus faecium/aislamiento & purificación , Enterococcus faecium/fisiología , Manipulación de Alimentos/métodos , Adhesión Bacteriana , Brasil , Ácidos Cólicos/metabolismo , Ácidos Cólicos/toxicidad , Clostridium perfringens/químicaRESUMEN
The aim of this study was to determine the prevalence of the bla (SHV) gene in Klebsiella pneumoniae isolates from hospital and community infections and from the normal microbiota of healthy individuals in Recife, PE, Brazil. Fifty-two K. pneumoniae isolates were analyzed regarding the presence of the bla (SHV) gene, using PCR, and eight isolates were analyzed by DNA sequencing. This gene was detected in 16 isolates from hospital infections, four from community infections, and nine from the normal microbiota. This was the first study to find the bla (SHV) gene in K. pneumoniae isolates from the normal microbiota. Through DNA sequencing of eight K. pneumoniae isolates from hospital and community infections, with a resistance phenotype indicative of extended-spectrum ß-lactamase production, a new SHV variant named SHV-122 was found. We also detected the presence of bla (SHV-1), bla (SHV-11), bla (SHV-28), and bla (SHV-108). The results show that in Recife, Brazil, K. pneumoniae isolates that presented resistance to oxyimino-ß-lactams had high prevalence and diversity of the bla (SHV) gene. We also conclude that there was a high presence of the bla (SHV) gene among isolates from the normal microbiota of healthy individuals.