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1.
J Cancer Res Clin Oncol ; 146(1): 75-86, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31754833

RESUMO

PURPOSE: The enzymes gamma-glutamyl hydrolase (GGH) and folylpolyglutamate synthetase (FPGS) regulate intracellular folate concentrations needed for cell proliferation, DNA synthesis, and repair. High GGH expression affects 5-FU thymidylate synthase (TS) inhibition and is a risk factor for various malignancies. Here, the clinical significance of GGH and FPGS expression was investigated in Stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1. METHODS: Surgical specimens of cancer tissue and adjacent normal mucosa, obtained from 253 patients with previously untreated gastric cancer, were examined. GGH and FPGS mRNA expression was measured by qPCR to evaluate their clinicopathological significance in gastric cancer patients after curative resection. RESULTS: While FPGS expression showed no significant differences between the cancerous and normal samples, GGH expression was higher in cancer tissue than in adjacent normal mucosa. High GGH expression was correlated with age, histological type, and vascular invasion. Overall survival (OS) of patients with high GGH mRNA expression was significantly poorer than of patients with low GGH expression. Multivariate analysis showed that high GGH expression was an independent prognostic factor of OS (HR: 2.58, 95% CI 1.29-5.16). Patients who received S-1 adjuvant treatment showed a significantly poor OS between high GGH/low FPGS and low GGH/high FPGS. Patients without adjuvant treatment showed no significant difference. CONCLUSION: GGH expression was significantly higher in gastric cancer tissue than in adjacent normal mucosa. High GGH and low FPGS expression is a useful independent predictor of poor outcomes in stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/biossíntese , Peptídeo Sintases/biossíntese , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/enzimologia , gama-Glutamil Hidrolase/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Quimioterapia Adjuvante , Combinação de Medicamentos , Feminino , Mucosa Gástrica/enzimologia , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Ácido Oxônico/administração & dosagem , Peptídeo Sintases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia , Tegafur/administração & dosagem , gama-Glutamil Hidrolase/genética
2.
Biomed Pharmacother ; 112: 108725, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30970523

RESUMO

Glucarpidase, also known as carboxypeptidase G2, is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels "biobetter" variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. Km, Vmax and Kcat measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase. Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase.


Assuntos
Desenho de Drogas , Pró-Fármacos , Pseudomonas putida/genética , gama-Glutamil Hidrolase/genética , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/farmacocinética , Clonagem Molecular , Estabilidade Enzimática , Terapia Enzimática/métodos , Metotrexato/efeitos adversos , Metotrexato/farmacocinética , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Mutação Puntual , Pró-Fármacos/administração & dosagem , Pró-Fármacos/uso terapêutico , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , gama-Glutamil Hidrolase/imunologia , gama-Glutamil Hidrolase/uso terapêutico
3.
Clin Rheumatol ; 37(12): 3221-3228, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30022368

RESUMO

This study investigated the impact of seven polymorphisms in genes of folate transport and (de)glutamation pathway on methotrexate polyglutamate levels and response in patients with rheumatoid arthritis. This prospective study included patients with rheumatoid arthritis. They were treated with methotrexate (up to 25 mg per week) for 24 weeks and categorized by EULAR response criteria into responders (good and moderate) and non-responders. Using real-time Taqman discrimination assay, SNPs were genotyped-rs1045642 (ABCB1 3435C>T), rs1128503 (ABCB1 1236C>T), rs10106 (FPGS 1994A>G), rs1544105 (FPGS G>A), rs11545078 (GGH 452C>T), rs3758149 (GGH -401C>T), and rs1051266 (RFC1 80G>A). RBC methotrexate polyglutamate1-5(MTX-glu1-5) levels were determined at 4, 8, 16, and 24 weeks using by reverse phase HPLC using C-18 column followed by post column photo-oxidation. This study included 117 patients with rheumatoid arthritis (M:F = 14:103). The mean dose of methotrexate at 24 weeks was 22.0 ± 4.0 mg, with data on DAS28(3) at 24 weeks available in 96 patients-61 responders and 35 non-responders. Minor allele of GGH 452C>T had an association with non-response (odds ratio 2.9, 95% CI 1.4-5.6) and assuming the dominance of C, the recessive genetic model found GGH 452C>T CC genotype (odds ratio 9.5, 95% CI 1.2 to 76.0) was significantly associated with response. However, there was no difference in MTX-glu1-5 levels among the various genotypes of this SNP (p = 0.9). Other SNPs were neither associated with response nor with alteration in methotrexate polyglutamate levels. On logistic regression, GGH 452C>T CC genotype and DAS28(3) at baseline were independent predictors of response. GGH 452C>T CC genotype was associated with response to methotrexate. None of the SNPs affected MTX-glu1-5levels.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Ácido Fólico/metabolismo , Metotrexato/análogos & derivados , Ácido Poliglutâmico/análogos & derivados , Polimorfismo de Nucleotídeo Único , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Adulto , Alelos , Artrite Reumatoide/metabolismo , Feminino , Genótipo , Humanos , Índia , Modelos Logísticos , Masculino , Metotrexato/administração & dosagem , Metotrexato/farmacocinética , Pessoa de Meia-Idade , Razão de Chances , Peptídeo Sintases/genética , Ácido Poliglutâmico/administração & dosagem , Ácido Poliglutâmico/farmacocinética , Estudos Prospectivos , Proteína de Replicação C/genética , gama-Glutamil Hidrolase/genética
4.
Med Sci (Paris) ; 34(12): 1111-1114, 2018 12.
Artigo em Francês | MEDLINE | ID: mdl-30623769
5.
Cancer Chemother Pharmacol ; 79(6): 1077-1085, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28417167

RESUMO

PURPOSE: Preoperative chemoradiotherapy (CRT) using 5-fluorouracil (5-FU)-based chemotherapy is the standard of care for rectal cancer. The effect of additional chemotherapy during the period between the completion of radiotherapy and surgery remains unclear. Predictive factors for CRT may differ between combination chemotherapy with S-1 and with tegafur-uracil/leucovorin (UFT/LV). METHODS: The subjects were 54 patients with locally advanced rectal cancer who received preoperative CRT with S-1 or UFT/LV. The pathological tumor response was assessed according to the tumor regression grade (TRG). The expression levels of 18 CRT-related genes were determined using RT-PCR assay. RESULTS: A pathological response (TRG 1-2) was observed in 23 patients (42.6%). In a multivariate logistic regression analysis for pathological response, the overall expression levels of four genes, HIF1A, MTHFD1, GGH and TYMS, were significant, and the accuracy rate of the predictive model was 83.3%. The effects of the gene expression levels of GGH on the response differed significantly according to the treatment regimen. The total pathological response rate of both high-GGH patients in the S-1 group and low-GGH patients in the UFT/LV group was 58.3%. CONCLUSION: Additional treatment with 5-FU-based chemotherapy during the interval between radiotherapy and surgery is not beneficial in patients who have received 5-FU-based CRT. The expression levels of four genes, HIF1A, MTHFD1, GGH and TYMS, in tumor tissues can predict the response to preoperative CRT including either S-1 or UFT/LV. In particular, the gene expression level of GGH in tumor tissues may be a useful biomarker for the appropriate use of S-1 and UFT/LV in CRT.


Assuntos
Adenocarcinoma/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimiorradioterapia/métodos , Neoplasias Retais/terapia , gama-Glutamil Hidrolase/biossíntese , gama-Glutamil Hidrolase/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/cirurgia , Adulto , Idoso , Antídotos/administração & dosagem , Antimetabólitos Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais/sangue , Terapia Combinada , Combinação de Medicamentos , Feminino , Fluoruracila/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucovorina/administração & dosagem , Masculino , Pessoa de Meia-Idade , Ácido Oxônico/administração & dosagem , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/cirurgia , Reprodutibilidade dos Testes , Tegafur/administração & dosagem , Resultado do Tratamento
6.
PLoS One ; 12(3): e0173472, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28278270

RESUMO

BACKGROUND: γ-Glutamyl hydrolase (GGH) regulates intracellular folates and antifolates such as methotrexate (MTX) for proper nucleotide biosynthesis and antifolate-induced cytotoxicity, respectively. In addition to genetic polymorphism and karyotypic abnormalities, methylation of CpG island 1 (CpG1) in the promoter region is found to modulate GGH activity by reducing GGH mRNA expression in acute lymphoblastic leukemia (ALL) cells. We aim to investigate methylation status of two CpG islands (CpG1 and CpG2) in the GGH promoter region in pediatric patients with ALL and acute myelogenous leukemia (AML). METHODS: 70B-ALL, 29 AML, 10 ITP (idiopathic thrombocytopenic purpura) and 40 healthy children are recruited in the present study. MS-HRM (methylation-sensitive high-resolution melting) and bisulfite sequencing PCR (BSP) are used to detect methylation change and its level in CpG1 and CpG2 in the GGH promoter region. GGH mRNA expression is quantified by real-time PCR. Correlation between CpG island methylation and GGH mRNA expression is assessed by statistical software. RESULTS: Methylations of CpG1 are detected in leukemia cells samples obtained from 30.9% (21/68) of patients with ALL and 20.7% (6/29) of patients with AML. These methylations are not detected in the controls. Methylations of CpG2 are detected in leukemia cell samples obtained from 44.1% (30/68) of the ALL patients and 37.9% (11/29) of the AML patients. These percentages are significantly higher than that observed in the control cell samples: 6.0% (3/50) (Fisher's exact test, P = 0.000). The abundance of CpG1 methylation in all leukemia cell samples is classified as Grade I (methylation level is less than 10%) and the abundance of CpG2 methylation in leukemia cell samples is classified in separate grades. Our results indicate that methylation of CpG1 or hypermethylation (the methylation level is greater than 10%) of CpG2 could significantly reduce GGH mRNA expression in leukemia cells from the ALL and AML patients (ALL-CpG1: t = 4.632, P = 0.000; ALL-CpG2: t = 3.250, P = 0.006; AML-CpG1: t = -2.254, P = 0.037; AML-CpG2: t = 1.328, P = 0.202). CONCLUSION: Either methylation of CpG1 or hypermethylation of CpG2 in GGH promoter region can significantly reduce GGH mRNA expression in pediatric patients with acute leukemia, which can improve the response to treatment.


Assuntos
Ilhas de CpG/genética , Metilação de DNA , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiões Promotoras Genéticas/genética , gama-Glutamil Hidrolase/genética , Sequência de Bases , Criança , Pré-Escolar , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino
7.
Protein Eng Des Sel ; 30(4): 321-331, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28160000

RESUMO

Carboxypeptidase G2 (CPG2) is an Food and Drug Administration (FDA)-approved enzyme drug used to treat methotrexate (MTX) toxicity in cancer patients receiving MTX treatment. It has also been used in directed enzyme-prodrug chemotherapy, but this strategy has been hampered by off-site activation of the prodrug by the circulating enzyme. The development of a tumor protease activatable CPG2, which could be achieved using a circular permutation of CPG2 fused to an inactivating 'prodomain', would aid in these applications. We report the development of a protease accessibility-based screen to identify candidate sites for circular permutation in proximity of the CPG2 active site. The resulting six circular permutants showed similar expression, structure, thermal stability, and, in four cases, activity levels compared to the wild-type enzyme. We rationalize these results based on structural models of the permutants obtained using the Rosetta software. We developed a cell growth-based selection system, and demonstrated that when fused to periplasm-directing signal peptides, one of our circular permutants confers MTX resistance in Escherichia coli with equal efficiency as the wild-type enzyme. As the permutants have similar properties to wild-type CPG2, these enzymes are promising starting points for the development of autoinhibited, protease-activatable zymogen forms of CPG2 for use in therapeutic contexts.


Assuntos
Mutação , gama-Glutamil Hidrolase , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Estabilidade Enzimática , gama-Glutamil Hidrolase/biossíntese , gama-Glutamil Hidrolase/química , gama-Glutamil Hidrolase/genética
8.
Int J Mol Sci ; 18(2)2017 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-28146062

RESUMO

γ-glutamyl-hydrolase (GGH) is a ubiquitously-expressed enzyme that regulates intracellular folate metabolism for cell proliferation, DNA synthesis, and repair. Employing GGH immunohistochemistry on a tissue microarray with 12,427 prostate cancers, we found that GGH expression was negative to low in normal prostate epithelium, whereas 88.3% of our 10,562 interpretable cancers showed GGH expression. GGH staining was considered as low intensity in 49.6% and as high intensity in 38.6% of cancers. High GGH expression was linked to the TMPRSS2:ERG-fusion positive subset of cancers (p < 0.0001), advanced pathological tumor stage, and high Gleason grade (p < 0.0001 each). Further analysis revealed that these associations were merely driven by the subset of ERG-negative cancers, High GGH expression was weakly linked to early biochemical recurrence in ERG negative cancers (p < 0.0001) and independent from established histo-pathological parameters. Moreover, GGH expression was linked to features of genetic instability, including presence of recurrent deletions at 3p, 5q, 6q, and 10q (PTEN, p ≤ 0.01 each), as well as to accelerated cell proliferation as measured by Ki67 immunohistochemistry (p < 0.0001). In conclusion, the results of our study identify GGH as an ERG subtype specific molecular marker with modest prognostic relevance, which may have clinical relevance if analyzed in combination with other molecular markers.


Assuntos
Biomarcadores Tumorais , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/mortalidade , Regulador Transcricional ERG/deficiência , gama-Glutamil Hidrolase/metabolismo , Proliferação de Células , Expressão Gênica , Humanos , Imuno-Histoquímica , Metástase Linfática , Masculino , Margens de Excisão , Gradação de Tumores , Estadiamento de Neoplasias , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Prognóstico , Antígeno Prostático Específico , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/cirurgia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Recidiva , Deleção de Sequência , Regulador Transcricional ERG/genética , gama-Glutamil Hidrolase/genética
9.
Oncotarget ; 8(6): 9388-9398, 2017 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-27566582

RESUMO

Inter-individual differences in toxic symptoms and pharmacokinetics of high-dose methotrexate (MTX) treatment may be caused by genetic variants in the MTX pathway. Correlations between polymorphisms and pharmacokinetic parameters and the occurrence of hepato- and myelotoxicity were studied. Single nucleotide polymorphisms (SNPs) of the ABCB1, ABCC1, ABCC2, ABCC3, ABCC10, ABCG2, GGH, SLC19A1 and NR1I2 genes were analyzed in 59 patients with osteosarcoma. Univariate association analysis and Bayesian network-based Bayesian univariate and multilevel analysis of relevance (BN-BMLA) were applied. Rare alleles of 10 SNPs of ABCB1, ABCC2, ABCC3, ABCG2 and NR1I2 genes showed a correlation with the pharmacokinetic values and univariate association analysis. The risk of toxicity was associated with five SNPs in the ABCC2 and NR1I2 genes. Pharmacokinetic parameters were associated with four SNPs of the ABCB1, ABCC3, NR1I2, and GGH genes, and toxicity was shown to be associated with ABCC1 rs246219 and ABCC2 rs717620 using the univariate and BN-BMLA method. BN-BMLA analysis detected relevant effects on the AUC0-48 in the following SNPs: ABCB1 rs928256, ABCC3 rs4793665, and GGH rs3758149. In both univariate and multivariate analyses the SNPs ABCB1 rs928256, ABCC3 rs4793665, GGH rs3758149, and NR1I2 rs3814058 SNPs were relevant. These SNPs should be considered in future dose individualization during treatment.


Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Neoplasias Ósseas/tratamento farmacológico , Metotrexato/administração & dosagem , Osteossarcoma/tratamento farmacológico , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adolescente , Fatores Etários , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/farmacocinética , Área Sob a Curva , Teorema de Bayes , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Distribuição de Qui-Quadrado , Criança , Feminino , Genótipo , Meia-Vida , Humanos , Masculino , Taxa de Depuração Metabólica , Metotrexato/efeitos adversos , Metotrexato/farmacocinética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Análise Multivariada , Razão de Chances , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Farmacogenética , Fenótipo , Receptor de Pregnano X , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Fatores de Risco , Resultado do Tratamento , gama-Glutamil Hidrolase/genética , gama-Glutamil Hidrolase/metabolismo
10.
Sci Rep ; 6: 35615, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27752107

RESUMO

We investigated major determinants of the intracellular concentrations of methotrexate polyglutamates (MTXPGs) in patients with rheumatoid arthritis (RA). In 271 RA patients on stable oral low dose weekly pulse MTX therapy, the concentrations of MTXPGs in red blood cells (RBCs) were measured by liquid chromatography-electrospray ionization-tandem mass spectrometry. Polymerase chain reaction-restriction fragment length polymorphism analysis was performed to determine the genotypes of solute carrier family 19 member 1 (SLC19A1), folylpolyglutamate synthase (FPGS), and gamma-glutamyl hydrolase (GGH). The mean total MTXPG concentration and the concentrations of individual MTXPGs increased dose-dependently, but reached a plateau at MTX doses >10 mg weekly. The MTXPG3-5/1-2 ratio was lower in patients with adverse events related to MTX than in patients without adverse events. Three polymorphisms of FPGS significantly influenced the MTXPG3-5/1-2 ratio in RBCs, while polymorphisms of SLC19A1 and GGH had no impact. The minor allele frequencies of 2 FPGS genotypes were significantly increased in our patients compared with a Caucasian population. FPGS may have a major role in regulating intracellular polyglutamation of MTX in RA patients receiving low-dose weekly MTX therapy.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Metotrexato/análogos & derivados , Peptídeo Sintases/metabolismo , Ácido Poliglutâmico/análogos & derivados , Proteína Carregadora de Folato Reduzido/genética , gama-Glutamil Hidrolase/genética , Idoso , Artrite Reumatoide/genética , Eritrócitos/metabolismo , Feminino , Frequência do Gene , Genótipo , Humanos , Japão , Masculino , Metotrexato/metabolismo , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Peptídeo Sintases/genética , Ácido Poliglutâmico/metabolismo , Ácido Poliglutâmico/uso terapêutico , Polimorfismo Genético
11.
Oncotarget ; 7(38): 61970-61987, 2016 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-27566557

RESUMO

This study aimed to identify associations between germline polymorphisms and risk of high-grade osteosarcoma (HGOS) development, event-free survival (EFS) and toxicity in HGOS patients treated with neo-adjuvant chemotherapy and surgery.Germline polymorphisms of 31 genes known to be relevant for transport or metabolism of all four drugs used in HGOS chemotherapy (methotrexate, doxorubicin, cisplatin and ifosfamide) were genotyped in 196 patients with HGOS and in 470 healthy age and gender-matched controls. Of these 196 HGOS patients, a homogeneously treated group of 126 patients was considered for survival analyses (survival cohort). For 57 of these, treatment-related toxicity data were available (toxicity cohort).Eleven polymorphisms were associated with increased risk of developing HGOS (p < 0.05). The distribution of polymorphisms in patients was characterized by a higher Shannon entropy. In the survival cohort (n = 126, median follow-up = 126 months), genotypes of ABCC2_1249A/G, GGH_452T/C, TP53_IVS2+38G/C and CYP2B6*6 were associated with EFS (p < 0.05). In the toxicity cohort (n = 57), genotypes of ABCB1_1236T/C, ABCC2_1249A/G, ABCC2_3972A/G, ERCC1_8092T/G, XPD_23591A/G, XRCC3_18067T/C, MTHFR_1298A/C and GGH_16T/C were associated with elevated risk for toxicity development (p < 0.05).The results obtained in this retrospective study indicate that the aforementioned germline polymorphisms significantly impact on the risk of HGOS development, EFS and the occurrence of chemotherapy-related toxicity. These findings should be prospectively validated with the aim of optimizing and tailoring HGOS treatment in the near future.


Assuntos
Neoplasias Ósseas/genética , Osteossarcoma/genética , Polimorfismo Genético , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Criança , Cisplatino/administração & dosagem , Estudos de Coortes , Intervalo Livre de Doença , Doxorrubicina/administração & dosagem , Feminino , Genótipo , Humanos , Ifosfamida/administração & dosagem , Itália , Masculino , Metotrexato/administração & dosagem , Pessoa de Meia-Idade , Terapia Neoadjuvante , Metástase Neoplásica , Resultado do Tratamento , Adulto Jovem , gama-Glutamil Hidrolase/genética
12.
Protein Expr Purif ; 127: 44-52, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27374188

RESUMO

Due to its applications in the treatment of cancer and autoimmune diseases, the 42 kDa zinc-dependent metalloenzyme carboxypeptidase G2 (CPG2) is of great therapeutic interest. An X-ray crystal structure of unliganded CPG2 reported in 1997 revealed the domain architecture and informed early rational drug design efforts, however further efforts at co-crystallization of CPG2 with ligands, substrates or inhibitors have not been reported. Thus key features of CPG2 such as the location of the active site, the presence of additional ligand-binding sites, stability, oligomeric state, and the molecular basis of activity remain largely unknown, with the current working understanding of CPG2 activity based primarily on computational modelling. To facilitate renewed efforts in CPG2 structural biology, we report the first high-yield (250 mg L(-1)) recombinant expression (and purification) of soluble and active CPG2 using the Escherichia coli expression system. We used this protocol to produce full-length enzyme, as well as protein fragments corresponding to the individual catalytic and dimerization domains, and the activity and stability of each construct was characterised. We adapted our protocol to allow for uniform incorporation of NMR labels ((13)C, (15)N and (2)H) and present preliminary solution-state NMR spectra of high quality. Taken together, our results offer a route for production and solution-state characterization that supports renewed effort in CPG2 structural biology as well as design of significantly truncated CPG2 proteins, which retain activity while yielding (potentially) improved immunogenicity.


Assuntos
Proteínas de Bactérias , Escherichia coli/metabolismo , Expressão Gênica , Pseudomonas/genética , gama-Glutamil Hidrolase , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/genética , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Pseudomonas/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , gama-Glutamil Hidrolase/biossíntese , gama-Glutamil Hidrolase/química , gama-Glutamil Hidrolase/genética , gama-Glutamil Hidrolase/isolamento & purificação
13.
J Cardiothorac Surg ; 11(1): 85, 2016 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-27387303

RESUMO

BACKGROUND: Thymic carcinoma (TC) is a rare type of malignant neoplasm that develops in the anterior mediastinum and associated with poor prognosis. Type B3 thymoma (B3) occasionally demonstrates malignant tumor characteristics, especially in the advanced stage. We investigated the expressions of tumor-related genes in resected TC and B3 specimens. METHODS: TC and B3 specimens resected from 1999 through 2012 were investigated. Tumor segments were collected from the specimens by micro-dissection to extract mRNA, then RT-PCR was performed according to Dannenberg's tumor profile method for semi-quantitation of tumor-related gene mRNA. To compare with other types of cancer, data from lung cancer (LC) cases in our database were also examined. RESULTS: The gene expression levels of thymidylate synthase were significantly higher in TC and B3 as compared to LC specimens (p < 0.02), while no difference were observed between TC and B3 tumors. The ratio of folypolyglutamyl synthase (FPGS) to gamma-glutamyl hydrolase (GGH) mRNA was significantly lower in TC than in B3 (p < 0.05), with lower FPGS/GGH in those tumors related to overall survival. Also, the gene expression of vascular endothelial growth factor (VEGF) was significantly higher in TC as compared to B3 (p = 0.04), with higher VEGF gene expression in TC and B3 specimens related to overall survival of affected patients. Epidermal growth factor receptor (EGFR) expression was significantly higher in B3 as compared to both TC and LC specimens (p < 0.01). However, there were no EGFR gene mutations detected in any of the specimens. CONCLUSIONS: These results indicate that elevated expressions of the tumor-related genes FPGS/GGH and VEGF are correlated with malignancy of TC and B3 tumors. Additional examinations will be necessary to investigate their chemosensitivity.


Assuntos
Neoplasias Pulmonares/genética , Timoma/genética , Timoma/patologia , Neoplasias do Timo/genética , Neoplasias do Timo/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Receptores ErbB/genética , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Peptídeo Sintases/genética , RNA Mensageiro/análise , RNA Neoplásico/análise , Taxa de Sobrevida , Timidilato Sintase/genética , Fator A de Crescimento do Endotélio Vascular/genética , gama-Glutamil Hidrolase/genética
14.
J Clin Pharmacol ; 56(12): 1563-1569, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27146084

RESUMO

Sex differences in the prevalence of autoimmune diseases such as rheumatoid arthritis (RA) are well known, but little is known about those differences in relation to therapeutic response. Reduced folate carrier-1 (RFC-1), folypolyformyl glutamate synthase (FPGS), and γ-glutamyl hydrolase (GGH) are important transporters and enzymes that convert methotrexate (MTX) in the body. This study investigated the sex differences in mRNA expression of RFC-1, FPGS, and GGH in 190 unrelated healthy Japanese people. The genotypes and mRNA expression were determined using the real-time PCR method. Significant differences between men and women were observed in RFC-1, FPGS, and GGH mRNA expression. The mRNA expression of FPGS and GGH was greater in women than that in men, but the expression of RFC-1 was less in the former than the latter. In stratified analysis by genotype, significant differences in sex-specific mRNA expression were observed in G/G of FPGS, C/C of GGH 452, and C/C of GGH -401. All showed greater mRNA expression in women than in men. In the 5 single-nucleotide polymorphisms RFC-1 80G>A, RFC-1 -43T>C, FPGS 1994G>A, GGH 452C>T, and GGH -401C>T examined, the FPGS 1994 G/G (1.46-fold), GGH 452 C/C (2.16-fold), and GGH -401 C/C (2.68-fold) genotypes showed significantly higher mRNA expression in women than in men. Healthy Japanese adults in this study showed sex-specific differences in mRNA expression that differed among RFC-1, FPGS, and GGH. Therefore, the relationship between genetic polymorphisms and mRNA expression including sex differences might contribute to the variation in the efficacy/toxicity of MTX in patients with RA.


Assuntos
Grupo com Ancestrais do Continente Asiático , Proteínas de Membrana Transportadoras/biossíntese , Peptídeo Sintases/biossíntese , RNA Mensageiro/biossíntese , Caracteres Sexuais , gama-Glutamil Hidrolase/biossíntese , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Peptídeo Sintases/genética , Vigilância da População , RNA Mensageiro/genética , Adulto Jovem , gama-Glutamil Hidrolase/genética
15.
Int J Mol Med ; 37(2): 319-28, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26676887

RESUMO

To establish the individualized treatment of patients with colorectal cancer, factors associated with chemotherapeutic effects should be identified. However, to the best of our knowledge, few studies are available on this topic, although it is known that the prognosis of patients and sensitivity to chemotherapy depend on the location of the tumor and that the tumor location is important for individualized treatment. In this study, primary tumors obtained from 1,129 patients with colorectal cancer were used to measure the mRNA expression levels of the following genes associated with the effects of standard chemotherapy for colorectal cancer: 5-fluorouracil (5-FU)-related thymidylate synthase (TYMS), dihydropyrimidine dehydrogenase (DPYD) and thymidine phosphorylase (TYMP); folate-related dihydrofolate reductase (DHFR), folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH); irinotecan-related topoisomerase I (TOP1); oxaliplatin-related excision repair cross-complementing 1 (ERCC1); biologic agent-related vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Large-scale population analysis was performed to determine the association of gene expression with the clinicopathological features, in particular, the location of the colorectal cancer. From the results of our analysis of the mRNA expression of these 10 genes, we noted the strongest correlation between DPYD and TYMP, followed by TYMS and DHFR. The location of the colorectal cancer was classified into 4 regions (the right­ and left-sided colon, rectosigmoid and rectum) and was compared with gene expression. A significant difference in all genes, apart from VEGF, was noted. Of the remaining 9 genes, the highest expression of TYMS and DPYD was observed in the right­sided colon; the highest expression of GGH and EGFR was noted in the left-sided colon; the highest expression of DHFR, FPGS, TOP1 and ERCC1 was noted in the rectosigmoid, whereas TYMP expression was approximately equivalent in the right-sided colon and rectum, and higher than that in other locations. The data generated from this study may prove to be useful for the development of individualized chemotherapeutic treatments for patients with colorectal cancer, and will mean that the tumor location is taken into account.


Assuntos
Neoplasias Colorretais/genética , DNA Topoisomerases Tipo I/biossíntese , Proteínas de Ligação a DNA/biossíntese , Endonucleases/biossíntese , Receptores ErbB/biossíntese , Peptídeo Sintases/biossíntese , Tetra-Hidrofolato Desidrogenase/biossíntese , Timidina Fosforilase/biossíntese , gama-Glutamil Hidrolase/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , DNA Topoisomerases Tipo I/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Receptores ErbB/genética , Feminino , Fluoruracila/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Irinotecano , Masculino , Pessoa de Meia-Idade , Compostos Organoplatínicos/administração & dosagem , Oxaliplatina , Peptídeo Sintases/genética , Prognóstico , RNA Mensageiro/biossíntese , Tetra-Hidrofolato Desidrogenase/genética , Timidina Fosforilase/genética , gama-Glutamil Hidrolase/genética
16.
PLoS One ; 10(7): e0130810, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26158264

RESUMO

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.


Assuntos
Fagos Bacilares/enzimologia , Bacillus subtilis/virologia , Genoma Microbiano , Proteínas Virais/metabolismo , gama-Glutamil Hidrolase/metabolismo , Sequência de Aminoácidos , Fagos Bacilares/classificação , Fagos Bacilares/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Eletroforese em Gel de Ágar , Genoma Bacteriano/genética , Dados de Sequência Molecular , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/metabolismo , Prófagos/enzimologia , Prófagos/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas Virais/genética , gama-Glutamil Hidrolase/genética
17.
Pharmacogenomics ; 16(4): 383-91, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25823786

RESUMO

AIM: Pemetrexed is a commonly used chemotherapeutic agent for lung adenocarcinoma patients. We investigated the impact of the genetic polymorphisms on the therapeutic efficacy of pemetrexed in lung adenocarcinoma patients. MATERIALS & METHODS: We performed genotying of 51 polymorphisms of 13 genes in 243 lung adenocarcinoma patients treated with pemetrexed as a single agent for second or more line of therapy. RESULTS: Total 12 polymorphisms in six genes were showed statistical significances in univariate analysis. After a false-discovery rate correction, the associations between GGH rs16930092 (p = 0.034) and rs10464903 (p = 0.034), and progression-free survival (PFS) were still conserved. Two polymorphisms in ATIC and GGH genes were associated with therapeutic efficacy in multivariate analysis: ATIC rs12995526 for tumor response (p = 0.014) and for overall survival (p = 0.006), and GGH rs16930092 (p = 0.009) for PFS. CONCLUSION: This study shows that polymorphisms on genes related to the metabolic pathway of pemetrexed, especially, ATIC and GGH genes, would have a therapeutic implication in pemetrexed-treated patients with lung adenocarcinoma. Original submitted 10 May 2013; Revision submitted 27 June 2014.


Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Hidroximetil e Formil Transferases/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Complexos Multienzimáticos/genética , Nucleotídeo Desaminases/genética , gama-Glutamil Hidrolase/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adulto , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Estudos de Associação Genética , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Pemetrexede/administração & dosagem , Pemetrexede/efeitos adversos , Prognóstico , Resultado do Tratamento
18.
Planta ; 240(3): 575-83, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25000918

RESUMO

MAIN CONCLUSION: Arabidopsis was engineered to produce 21.2 % punicic acid in the seed oil. Possible molecular factors limiting further accumulation of the conjugated fatty acid were investigated. Punicic acid (18:3Δ(9cis,11trans,13cis) ) is a conjugated linolenic acid isomer and is a main component of Punica granatum (pomegranate) seed oil. Medical studies have shown that punicic acid is a nutraceutical with anti-cancer and anti-obesity properties. It has been previously demonstrated that the conjugated double bonds in punicic acid are produced via the catalytic action of fatty acid conjugase (FADX), which is a homolog of the oleate desaturase. This enzyme catalyzes the conversion of the Δ(12)-double bond of linoleic acid (18:2Δ(9cis,12cis) ) into conjugated Δ(11trans) and Δ(13cis) -double bonds. Previous attempts to produce punicic acid in transgenic Arabidopsis thaliana seeds overexpressing P. granatum FADX resulted in a limited accumulation of punicic acid of up to 4.4 %, accompanied by increased accumulation of oleic acid (18:1∆(9cis) ), suggesting that production of punicic acid in some way inhibits the activity of oleate desaturase (Iwabuchi et al. 2003). In the current study, we applied a new strategy to enhance the production of punicic acid in a high linoleic acid A. thaliana fad3/fae1 mutant background using the combined expression of P. granatum FADX and FAD2. This approach led to the accumulation of punicic acid at the level of 21 % of total fatty acids and restored the natural proportion of oleic acid observed in the A. thaliana fad3/fae1 mutant. In addition, we provide new insights into the high oleate phenotype and describe factors limiting the production of punicic acid in genetically engineered plants.


Assuntos
Ácidos Graxos Dessaturases/metabolismo , Ácidos Linolênicos/biossíntese , Lythraceae/enzimologia , Sementes/metabolismo , gama-Glutamil Hidrolase/metabolismo , Arabidopsis/metabolismo , Ácidos Graxos Dessaturases/genética , Lythraceae/genética , Fosfatidilcolinas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Triglicerídeos/metabolismo , gama-Glutamil Hidrolase/genética
19.
Cancer Chemother Pharmacol ; 74(2): 283-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24908438

RESUMO

PURPOSE: To investigate the correlation between common genetic polymorphisms of folylpolyglutamate synthase (FPGS), gamma-glutamyl hydrolase (GGH), and methylenetetrahydrofolate reductase (MTHFR) and serum levels of methotrexate (MTX) in Chinese children with acute lymphoblastic leukemia (ALL). METHODS: Ninety-one children with ALL who received high-dose MTX were recruited. The polymorphisms FPGS (rs1544105 G>A), GGH (rs3758149 C>T), and MTHFR (rs1801133 C>T) were genotyped through polymerase chain reaction-restriction fragment length polymorphism analysis. Serum MTX was measured by fluorescence polarization immunoassay. The association between targeted polymorphisms and MTX concentration-to-dose (C/D) ratios was assessed, and between targeted polymorphisms and the percent of MTX above the therapeutic threshold (40 µmol/L). RESULTS: The minor allele frequencies of rs1544105 G (34.1%), rs3758149 T (19.2%), and rs1801133 C (48.4%) observed in our population were significantly lower than those reported for European populations (64.2, 30.8, and 69.0%, respectively). The association between the GGH rs3758149 polymorphism and MTX C/D was gender-specific; in girls, the MTX C/D at 24 h of GGH rs3758149 CC carriers (12.09 µmol/L per g/m(2)) was significantly lower than that of CT or TT carriers (16.80 µmol/L per g/m(2)). The percent of serum MTX above the therapeutic threshold in GGH rs3758149 CC carriers (18.3%) was significantly lower than that of CT and TT carriers (38.7%). The MTX C/D ratios at 24 h and the percent of MTX >40 µmol/L for the A-T-T (three variant alleles) haplotype were significantly higher than those for other haplotypes combined (P < 0.05). CONCLUSIONS: These data indicate that FPGS rs1544105, GGH rs3758149, and MTHFR rs1801133 polymorphisms contribute to the variability of MTX pharmacokinetics, and their genotyping may be useful to reduce toxicities associated with MTX therapy.


Assuntos
Metotrexato/sangue , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Peptídeo Sintases/genética , Polimorfismo de Nucleotídeo Único/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , gama-Glutamil Hidrolase/genética , Adolescente , Grupo com Ancestrais do Continente Asiático/genética , Criança , Pré-Escolar , DNA/análise , DNA/genética , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Lactente , Masculino , Metotrexato/administração & dosagem , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Prognóstico
20.
Clin Exp Rheumatol ; 32(3): 324-32, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24447348

RESUMO

OBJECTIVES: Methotrexate (MTX) is the cornerstone medication in the treatment of rheumatoid arthritis (RA). We examined whether single nucleotide polymorphisms (SNPs) in enzymes of the folic acid pathway (folylpoly-gamma-glutamate synthetase [FPGS], gamma-glutamyl hydrolase [GGH], and methylenetetrahydrofolate reductase [MTHFR]) associate with significant adverse events (SigAE). METHODS: Patients (n=319) enrolled in the Veterans Affairs RA (VARA) registry taking MTX were genotyped for HLA-DRB1-SE and the following SNPs: FPGS (rs7033913, rs10760503, rs10106), GGH (12548933, rs7010484, rs4617146, rs719235, rs11988534), MTHFR (rs1801131, rs1801133). AE were abstracted from the medical record using a structured instrument. SigAE were defined as an AE leading to MTX discontinuation. Covariates included: age, gender, race, RA antibody status, tobacco, RA disease duration between diagnosis and MTX course, Charlson-Deyo comorbidity index, glucocorticoids, use of prior RA medications, and mean 4-variable disease activity score. Cox regression was performed to determine factors associated with time-to-SigAE. A p-value ≤ 0.005 established significance in the final model. RESULTS: The presence of ≥ 1 copy of the minor allele in MTHFR rs1801131 was associated with an increased hazard ratio (HR) of SigAE (HR 3.05, 95% CI 1.48-6.29, p-value 0.003 and HR 3.88, 95% CI 1.62-9.28, p-value 0.002 for heterozygotes and homozygotes for the minor allele, respectively). An interaction term, between FPGS rs7033913 heterozygotes and GGH rs11988534 homozygotes for the minor allele, had a p-value <0.0001. CONCLUSIONS: RA subjects taking MTX may have decreased time-to-SigAE with ≥ 1 copy of the minor allele in MTHFR rs1801131. Further investigation is warranted, as these SNPs may indicate susceptibility to MTX toxicity.


Assuntos
Artrite Reumatoide , Ácido Fólico/metabolismo , Metotrexato/toxicidade , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Peptídeo Sintases/genética , gama-Glutamil Hidrolase/genética , Idoso , Idoso de 80 Anos ou mais , Antirreumáticos/administração & dosagem , Antirreumáticos/toxicidade , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Feminino , Ácido Fólico/genética , Genótipo , Humanos , Masculino , Metotrexato/administração & dosagem , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Pessoa de Meia-Idade , Peptídeo Sintases/metabolismo , Polimorfismo de Nucleotídeo Único , Sistema de Registros , Estados Unidos , Veteranos , gama-Glutamil Hidrolase/metabolismo
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