RESUMO
OBJECTIVES: The impact of genetic variants (single nucleotide polymorphisms [SNPs]) in the clinical heterogeneity of ulcerative colitis (UC) remains unclear. We showed that patients with UC exhibit a deficiency in MGAT5 glycogene transcription in intestinal T cells associated with a hyperimmune response. Herein, we evaluated whether MGAT5 SNPs might functionally impact on T cells glycosylation and plasma IgG glycome in patients with UC, as well as in UC clinical outcomes. METHODS: Three selected MGAT5 SNPs (rs3814022, rs4953911, and rs1257220), previously associated with severity of autoimmune disease or with plasma glycome composition in healthy individuals, were functionally evaluated in patients with UC through analysis of MGAT5 mRNA levels in colonic (n = 14) and circulating (n = 24) T cells and through profiling the plasma IgG Fc glycosylation (n = 152). MGAT5 SNPs were genotyped in 931 patients with UC from 2 European cohorts and further associated with patients' prognosis. Targeted next-generation sequencing for MGAT5 coding and regulatory regions was also performed. RESULTS: MGAT5 SNPs were shown to be functionally associated with low transcription levels of MGAT5 in colonic and circulating T cells from patients with UC and with agalactosylation of IgGs, often associated with a proinflammatory phenotype. The SNPs rs3814022 and rs4953911 were further associated with the need of biologics. Next-generation sequencing data further revealed a combination of MGAT5 SNPs that stratify patients with UC according to their severity. DISCUSSION: Our results revealed that MGAT5 SNPs have a phenotypic impact on T cells glycosylation and in plasma IgG glycome composition associated with UC pathogenesis. MGAT5 SNPs display a tendency in the association with a worse disease course in patients with UC.
Assuntos
Colite Ulcerativa/genética , Imunoglobulina G/sangue , N-Acetilglucosaminiltransferases/genética , Linfócitos T/imunologia , Adulto , Estudos de Coortes , Colite Ulcerativa/sangue , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/imunologia , Feminino , Glicosilação , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Índice de Gravidade de Doença , Linfócitos T/metabolismo , Adulto JovemRESUMO
Mucosal T lymphocytes from patients with ulcerative colitis (UC) were previously shown to display a deficiency in branched N-glycosylation associated with disease severity. However, whether this glycosylation pathway shapes the course of the T cell response constituting a targeted-specific mechanism in UC remains largely unknown. In this study, we demonstrated that metabolic supplementation of ex vivo mucosal T cells from patients with active UC with N-acetylglucosamine (GlcNAc) resulted in enhancement of branched N-glycosylation in the T cell receptor (TCR), leading to suppression of T cell growth, inhibition of the T helper 1 (Th1)/Th17 immune response, and controlled T cell activity. We further demonstrated that mouse models displaying a deficiency in the branched N-glycosylation pathway (MGAT5-/-, MGAT5+/-) exhibited increased susceptibility to severe forms of colitis and early-onset disease. Importantly, the treatment of these mice with GlcNAc reduced disease severity and suppressed disease progression due to a controlled T cell-mediated immune response at the intestinal mucosa. In conclusion, our human ex vivo and preclinical results demonstrate the targeted-specific immunomodulatory properties of this simple glycan, proposing a therapeutic approach for patients with UC.
Assuntos
Acetilglucosamina/farmacologia , Linfócitos T CD4-Positivos/imunologia , Colite Ulcerativa/imunologia , N-Acetilglucosaminiltransferases/fisiologia , Polissacarídeos/metabolismo , Imunidade Adaptativa , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Citocinas/metabolismo , Glicosilação , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
OBJECTIVE: Colorectal cancer (CRC) is the second leading cause of cancer death in occidental countries. Chronic inflammatory bowel disease (crohn's disease and ulcerative colitis) is associated with an increased risk for CRC development. The aim of this work was to investigate the relationship between inflammatory status and absorption of nutrients with a role in CRC pathogenesis. MATERIALS AND METHODS: In this experimental study, we evaluated the in vitro effect of tumour necrosis factor-alpha (TNF-α), interferon-γ (IF-γ), and acetylsalicylic acid on 14C-butyrate (14C- BT), 3H-folic acid (3H-FA) uptake, and on proliferation, viability and differentiation of Caco-2 and IEC-6 cells in culture. RESULTS: The proinflammatory cytokines TNF-α and INF-γ were found to decrease uptake of a low concentration of 14C-BT (10 µM) by Caco-2 (tumoral) and IEC-6 (normal) intestinal epithelial cell lines. However, the effect of TNF-α and INF-γ in IEC-6 cells is most probably related to a cytotoxic and antiproliferative impact. In contrast, INF-γ increases uptake of a high concentration (10 mM) of 14C-BT in Caco-2 cells. The anticarcinogenic effect of BT (10 mM) in these cells is not affected by the presence of this cytokine. On the other hand, acetylsalicylic acid stimulates 14C-BT uptake by Caco-2 cells and potentiates its antiproliferative effect. Finally, both TNF-α and INF-γ cause a significant decrease in 3H-FA uptake by Caco-2 cells. CONCLUSION: The inflammatory status has an impact upon cellular uptake of BT and FA, two nutrients with a role in CRC pathogenesis. Moreover, the anti-inflammatory acetylsalicylic acid potentiates the anticarcinogenic effect of BT in Caco-2 cells by increasing its cellular uptake.
RESUMO
Inflammation is the driving force in inflammatory bowel disease (IBD) and its link to oxidative stress and carcinogenesis has long been accepted. The antioxidant system of the intestinal mucosa in IBD is compromised resulting in increased oxidative injury. This defective antioxidant system may be the result of genetic variants in antioxidant genes, which can represent susceptibility factors for IBD, namely Crohn's disease (CD) and ulcerative colitis (UC). Single nucleotide polymorphisms (SNPs) in the antioxidant genes SOD2 (rs4880) and GPX1 (rs1050450) were genotyped in a Portuguese population comprising 436 Crohn's disease and 367 ulcerative colitis patients, and 434 healthy controls. We found that the AA genotype in GPX1 is associated with ulcerative colitis (OR = 1.93, adjusted P-value = 0.037). Moreover, we found nominal significant associations between SOD2 and Crohn's disease susceptibility and disease subphenotypes but these did not withstand the correction for multiple testing. These findings indicate a possible link between disease phenotypes and antioxidant genes. These results suggest a potential role for antioxidant genes in IBD pathogenesis and should be considered in future association studies.
Assuntos
Doenças Inflamatórias Intestinais/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Colite Ulcerativa/genética , Doença de Crohn/genética , Feminino , Predisposição Genética para Doença/genética , Genótipo , Glutationa Peroxidase/genética , Humanos , Masculino , Pessoa de Meia-Idade , Superóxido Dismutase/genética , Glutationa Peroxidase GPX1RESUMO
Human hotspot TERT promoter (TERTp) mutations have been reported in a wide range of tumours. Several studies have shown that TERTp mutations are associated with clinicopathological features; in some instances, TERTp mutations were considered as biomarkers of poor prognosis. The rs2853669 SNP, located in the TERT promoter region, was reported to modulate the increased TERT expression levels induced by the recurrent somatic mutations. In this study we aimed to determine the frequency and prognostic value of TERTp mutations and TERT rs2853669 SNP in 504 gliomas from Portuguese and Brazilian patients. TERTp mutations were detected in 47.8% of gliomas (216/452). Glioblastomas (GBM) exhibited the highest frequency of TERTp mutations (66.9%); in this glioma subtype, we found a significant association between TERTp mutations and poor prognosis, regardless of the population. Moreover, in a multivariate analysis, TERTp mutations were the only independent prognostic factor. Our data also showed that the poor prognosis conferred by TERTp mutations was restricted to GBM patients carrying the rs2853669 A allele and not in those carrying the G allele. In conclusion, the presence of TERTp mutations was associated with worse prognosis in GBM patients, although such association depended on the status of the rs2853669 SNP. The status of the rs2853669 SNP should be taken in consideration when assessing the prognostic value of TERTp mutations in GBM patients. TERTp mutations and the rs2853669 SNP can be used in the future as biomarkers of glioma prognosis.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Glioblastoma/genética , Glioblastoma/mortalidade , Mutação , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Telomerase/genética , Adolescente , Adulto , Idoso , Alelos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Criança , Pré-Escolar , Feminino , Genótipo , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Lactente , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Adulto JovemRESUMO
Cell immortalization has been considered for a long time as a classic hallmark of cancer cells. Besides telomerase reactivation, such immortalization could be due to telomere maintenance through the "alternative mechanism of telomere lengthening" (ALT) but the mechanisms underlying both forms of reactivation remained elusive. Mutations in the coding region of telomerase gene are very rare in the cancer setting, despite being associated with some degenerative diseases. Recently, mutations in telomerase (TERT) gene promoter were found in sporadic and familial melanoma and subsequently in several cancer models, notably in gliomas, thyroid cancer and bladder cancer. The importance of these findings has been reinforced by the association of TERT mutations in some cancer types with tumour aggressiveness and patient survival. In the first part of this review, we summarize the data on the biology of telomeres and telomerase, available methodological approaches and non-neoplastic diseases associated with telomere dysfunction. In the second part, we review the information on telomerase expression and genetic alterations in the most relevant types of cancer (skin, thyroid, bladder and central nervous system) on record, and discuss the value of telomerase as a new biomarker with impact on the prognosis and survival of the patients and as a putative therapeutic target.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias/genética , Regiões Promotoras Genéticas , Telomerase/genética , Humanos , Biologia Molecular/métodos , Mutação , Neoplasias/diagnóstico , Neoplasias/mortalidade , Prognóstico , Taxa de Sobrevida , Telômero/fisiologiaAssuntos
Neoplasias das Glândulas Suprarrenais/genética , Efeito Fundador , Mutação em Linhagem Germinativa/genética , Paraganglioma/genética , Feocromocitoma/genética , Succinato Desidrogenase/genética , Adulto , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , PortugalRESUMO
Our aim was to investigate the effect of oxidative stress upon butyrate uptake at the intestinal epithelial level. For this, IEC-6 cells were treated with tert-butylhydroperoxide 3000µM (tBOOH), which increased levels of oxidative stress biomarkers, while maintaining cellular viability. The effect of tBOOH upon uptake of [(14)C]butyrate ([(14)C]BT) (10µM) can be summarized as follows: (a) it caused a reduction in the intracellular accumulation of [(14)C]BT over time, (b) it strongly reduced total [(14)C]BT uptake but did not affect Na(+)-independent uptake of [(14)C]BT, and (c) it did not affect the kinetics of [(14)C]BT uptake at 37°C, but increased uptake at 4°C. Moreover, tBOOH increased the efflux of [(14)C]BT not mediated by breast cancer resistance protein. We thus conclude that tBOOH strongly inhibits Na(+)-coupled monocarboxylate cotransporter 1 (SMCT1)-mediated, but not H(+)-coupled monocarboxylate transporter (MCT1)-mediated butyrate uptake; moreover, it increases uptake and efflux of butyrate by passive diffusion. tBOOH did not affect the mRNA expression levels of MCT1 and SMCT1 nor their cell membrane insertion. Rather, its effect was dependent on extracellular signal regulated kinase 1/2 and protein tyrosine kinase activation and on the generation of reactive oxygen species by NADPH and xanthine oxidases and was partially prevented by the polyphenols quercetin and resveratrol. In conclusion, tBOOH is an effective inhibitor of SMCT1-mediated butyrate transport in non-tumoral intestinal epithelial cells. Given the important role played by butyrate in the intestine, this mechanism may contribute to the procarcinogenic and proinflammatory effect of oxidative stress at this level.
Assuntos
Butiratos/farmacocinética , Absorção Intestinal , Mucosa Intestinal/metabolismo , Estresse Oxidativo , Animais , Antioxidantes/farmacologia , Transporte Biológico , Células CACO-2 , Linhagem Celular , Humanos , Mucosa Intestinal/citologia , Transportadores de Ácidos Monocarboxílicos/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Simportadores/metabolismo , Temperatura , Fatores de Tempo , terc-Butil Hidroperóxido/toxicidadeRESUMO
Folic acid (FA) is a vitamin essential for normal cellular functions, growth, and development. Because humans cannot synthesize this micronutrient, it must be obtained from dietary sources through intestinal absorption. The intestinal tract is a major target for oxidative stress. Our aim was to investigate the effect of oxidative stress upon the uptake of FA by Caco-2 cells. Oxidative stress was induced by exposure of the cells to tert-butyl hydroperoxide (TBH) for 1 h. TBH (3,000 µM) induced an increase in biomarkers of oxidative stress, while maintaining cell viability and proliferation. In relation to the apical uptake of (3)H-FA, TBH (3,000 µM) reduced the cellular accumulation of (3)H-FA (10 nM), although the characteristics (kinetics, pH dependence, and inhibitory profile) of (3)H-FA uptake were not changed. This effect was associated with a decrease in the mRNA steady-state levels of proton-coupled folate transporter and folate receptor alpha and of the efflux transporter multidrug resistance protein 2. Moreover, TBH (3,000 µM) did not affect the noncarrier-mediated apical uptake of (3)H-FA. Finally, the effect of TBH upon (3)H-FA apical uptake was not dependent on protein kinase A, protein kinase C, mitogen-activated protein kinases, phosphoinositide 3-kinase, nuclear factor kappa B, and protein tyrosine kinases, but was completely prevented by dietary polyphenols (resveratrol, quercetin, and EGCG). These results suggest that oxidative stress at the intestinal level may result in a reduction in the intestinal absorption of dietary FA and that polyphenolic dietary components may offer protection against oxidative stress-induced inhibition of intestinal FA absorption.
Assuntos
Ácido Fólico/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Estresse Oxidativo , 1-Fosfatidilinositol 4-Quinase/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Proliferação de Células , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor 1 de Folato/genética , Humanos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Polifenóis/farmacologia , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transportador de Folato Acoplado a Próton/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , terc-Butil Hidroperóxido , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological and experimental studies suggest that bile acids may play a role in CRC etiology. Our aim was to characterize the effect of the primary bile acid chenodeoxycholic acid (CDCA) upon(14) C-BT uptake in tumoral (Caco-2) and non-tumoral (IEC-6) intestinal epithelial cell lines. A 2-day exposure to CDCA markedly and concentration-dependently inhibited (14) C-BT uptake by IEC-6 cells (IC(50) = 120 µM), and, less potently, by Caco-2 cells (IC(50) = 402 µM). The inhibitory effect of CDCA upon (14) C-BT uptake did not result from a decrease in cell proliferation or viability. In IEC-6 cells: (1) uptake of (14) C-BT involves both a high-affinity and a low-affinity transporter, and CDCA acted as a competitive inhibitor of the high-affinity transporter; (2) CDCA inhibited both Na(+)-coupled monocarboxylate cotransporter 1 (SMCT1)- and H(+)-coupled monocarboxylate transporter 1 (MCT1)-mediated uptake of (14) C-BT; (3) CDCA significantly increased the mRNA expression level of SMCT1; (4) inhibition of (14) C-BT uptake by CDCA was dependent on CaM, MAP kinase (ERK1/2 and p38 pathways), and PKC activation, and reduced by a reactive oxygen species scavenger. Finally, BT (5 mM) decreased IEC-6 cell viability and increased IEC-6 cell differentiation, and CDCA (100 µM) reduced this effect. In conclusion, CDCA is an effective inhibitor of (14) C-BT uptake in tumoral and non-tumoral intestinal epithelial cells, through inhibition of both H(+) -coupled MCT1- and SMCT1-mediated transport. Given the role played by BT in the intestine, this mechanism may contribute to the procarcinogenic effect of CDCA at this level.