High levels of γ-glutamyl hydrolase (GGH) are associated with poor prognosis and unfavorable clinical outcomes in invasive breast cancer.

BACKGROUND: Previously, we performed analysis of gene expression in 46 axillary lymph node negative tumors and identified molecular gene signatures that resulted in different clinical outcomes. The aim of this study was to determine the correlation of γ-glutamyl hydrolase (GGH), fatty acid amide hydrolase (FAAH), Pirin (PIR) and TAF5-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa (TAF5L), selected from identified gene signatures, with clinical outcomes as well as classical clinicopathological characteristics in primary invasive breast cancer patients. METHODS: The protein levels of GGH, FAAH, PIR and TAF5L were assessed by immunohistochemistry (IHC) on a panel of 80 primary invasive breast tumors. Quantitative real-time PCR (qRT-PCR) and western blot analysis were performed to verify the expression levels of the candidate biomarkers. Patient disease-specific survival (DSS) and recurrence-free survival (RFS) were evaluated using the Kaplan-Meier method. The prognostic biomarkers were identified by univariate analysis with a log-rank test and by multivariate analysis with Cox proportional hazards regression models. RESULTS: The GGH and FAAH protein levels were significantly up-regulated in invasive breast cancer tumors compared with adjacent non-cancerous tissues. Furthermore, the protein levels of GGH and FAAH were significantly correlated in tumor tissues. Tumoral GGH protein expression was significantly correlated with shorter DSS and RFS. Furthermore, the protein expression of GGH was positively correlated with undifferentiated tumors (BRE grade III) and ER/PR expressing tumors. Multivariate regression analysis showed that only GGH protein expression independently predicts DSS. No such correlations were found for FAAH, PIR and TAF5L protein expression. However, elevated protein levels of FAAH were positively associated with high number of lymph node involvement and upregulated levels of PIR were positively related with lymph node metastasis. The TAF5L was pronouncedly down-regulated in primary invasive breast cancer tissues compared to matched adjacent non-cancerous tissues. CONCLUSION: These data show for the first time that cytoplasmic GGH might play a relevant role in the development and progression of invasive breast cancer, warranting further investigations. Our findings suggest that GGH serve as a potential biomarker of unfavorable clinical outcomes over short-term follow-up in breast cancer. The GGH may be a very attractive targeted therapy for selected patients.

Suplementación con ácido fólico y prevención de recurrencia de adenomas colorrectales; revisión sistemática / Folic acid supplementation and colorrectal adenoma recurrence; systematic review

Antecedentes: Estudios observacionales muestran que los niveles de folatos podrían estar asociados con el desarrollo de adenomas y cáncer colorrectal, sugiriendo que la suplementación de ácido fólico podría tener un efecto preventivo. Objetivo: Revisar sistemáticamente la evidencia científica proveniente de estudios clínicos randomizados con placebo y controlados que permitan conocer los efectos de la suplementación del ácido fólico sobre la recurrencia de adenomas colorrectales. Material y método: Revisión sistemática en Medline, vía Pubmed de estudios clínicos randomizados, con placebo y controlados a doble ciego y sus referencias, que evalúen específicamente el efecto de la suplementación de ácido sobre la recurrencia de adenomas colorrectales. Resultados: Siete estudios clínicos randomizados que cumplían los criterios de inclusión fueron seleccionados y evaluados. Conclusión: Los estudios seleccionados no permiten concluir que la suplementación de ácido fólico tenga un efecto beneficioso sobre la recurrencia de adenomas colorrectales. Se observa en algunos estudios diferencias de riesgo según tipo de folatos que sugieren revisar los criterios y niveles de suplementación en algunos subgrupos de población con mayores riesgos (AU)

Background: Observational studies show that folate levels may be associated with the development of adenomas and colorectal cancer, suggesting that folic acid supplementation may have a preventive effect. Aim: Systematic review of scientific evidence from randomized placebo-controlled clinical studies to identify the effects of folic acid supplementation on the recurrence of colorectal adenomas. Material and methods: Medline via Pubmed systematic review of randomized clinical trials, double-blind and placebo-controlled and references, specifically to evaluate the effect of acid supplementation on the recurrence of colorectal adenomas Results: Seven randomized clinical trials that met the inclusion criteria were selected and evaluated for analysis based on pre established criteria. Conclusions: The selected studies do not support that folic acid supplementation is beneficial in recurrence of colorectal adenomas. We observed in some studies differences in risk by type of folate suggesting to review the criteria and levels of supplementation in some population subgroups with higher risks (AU)

Hyperpolarized (13)C magnetic resonance detection of carboxypeptidase G2 activity.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene-directed enzyme prodrug therapy (GDEPT). Employing dynamic nuclear polarization (DNP) and natural abundance (13)C magnetic resonance spectroscopy (MRS), we observed the CPG2-mediated conversion of a novel hyperpolarized reporter probe 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu) to 3,5-difluorobenzoic acid (3,5-DFBA) and L-glutamic acid (L-Glu) in vitro. Isotopic labeling of the relevant nuclei with (13)C in 3,5-DFBGlu or related substrates will yield a further factor of 100 increase in the signal-to-noise. We discuss the feasibility of translating these experiments to generate metabolic images of CPG2 activity in vivo.

Concentraciones elevadas de homocisteína en el síndrome de ovario poliquístico / Increased homocysteine levels in polycystic ovary syndrome

Fundamento y objetivo: En las mujeres con síndrome de ovario poliquístico (SOP) es frecuente que haya factores de riesgo que predispongan a tener enfermedad cardiovascular. Se sabe que la hiperhomocisteinemia es un factor de riesgo independiente para esta enfermedad. El objetivo del presente estudio ha sido conocer si las mujeres jóvenes con SOP presentan concentraciones elevadas de homocisteína, y su posible relación con las de folato y vitamina B12. Pacientes y método: Se seleccionó a 39 mujeres con SOP, con una edad media (desviación estándar [DE]) de 28,9 (5,8) años, y 39 mujeres sanas de edad similar, y en todas ellas se evaluaron: tabaquismo, ciclos menstruales, grado de hirsutismo, índice de masa corporal, presencia de síndrome metabólico y concentraciones de homocisteína, lípidos, glucosa, creatinina, folato, vitamina B12, folitropina (FSH), lutropina (LH) y androstendiona. Resultados: Los ciclos menstruales, el grado de hirsutismo, los valores de androstendiona y LH y la relación LH/FSH eran más elevados, como se esperaba, en las pacientes con SOP. Además, las pacientes presentaban valores más elevados de homocisteína (media [DE] de 9,1 [2,1] frente a 6,4 [1,8] mmol/l; p 110 mg/dl) (el 23 frente al 2,5%; p = 0,01) y unos valores más bajos de folato (media [DE] de 7,6 [3,7] frente a 10,2 [3,6] ng/ml; p = 0,02). En una regresión lineal múltiple, se comprobó una asociación negativa entre las concentraciones de homocisteína y las de folato (r2 = 0,05; p = 0,02). Conclusiones: La homocisteinemia es más elevada en las mujeres con SOP y se asocia negativamente a las concentraciones de folato

Background and objective: Women with polycystic ovary syndrome (PCOS) exhibit frequently risk factors that predispose to cardiovascular disease. Hyperhomocysteinemia is an independent risk factor for this disease. The aim of this study was to know whether young women with PCOS have increased homocysteine levels. We also analyzed their possible relation with folate and vitamin B12 levels. Patients and method: Thirty nine patients with PCOS were studied; (age: mean [standard deviation] 28.9 [5.8] years), and 39 healthy women similar in age. We evaluated in all of them: smoking, menstrual cycles, hirsutism, body mass index, metabolic syndrome and levels of homocysteine, lipids, glucose, creatinine, folate, vitamin B12, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and androstendione. Results: Menstrual cycles, hirsutism, androstendione, LH levels and LH/FSH were higher, as we expected, in patients with PCOS. Moreover, patients had increased homocysteine (9.1 [2.1] vs 6.4 [1.8] mmol/L; p 110 mg/dl) (23% vs 2.5%; p =.01) and lower folate levels (7.6 [3.7] vs 10.2 [3.6] ng/ml; p = 0.02). A multiple linear regression showed a negative association between homocysteine and folate levels (r2 = 0.05; p =.02). Conclusions: Homocysteinemia is increased in women with PCOS, and it is negatively associated with folate levels

Folatos y su disponibilidad en alimentos evaluados por cromatografía líquida (HPLC). Validación y aplicaciones en calida nutricional de alimentos / Folates and their Availability in Foods Evaluated by Liquid Chromatography (HPLC). Validation and Application in Food Nutritional Quality

En este artículo se presenta el método cromatográfico de mayor precisión para la determinación de las formas químicas (folatos) de la vitamina B9 (ácido fólico o folacina) en alimentos, así como su disponibilidad (mono o poliglutamatos). Se trata de un método basado en la cromatografía líquida de alta resolución (HPLC), cuyo protocolo permite la extracción de la vitamina, su purificación y posterior separación de las distintas formas de los folatos. Este método ha sido validado sobre estándares y alimentos de referencia certificados y comparado con otros grupos de investigación frente a métodos microbiológicos (AU)

In this article it is presented the most precise chromatographic method to determine the different forms of B9 vitamin (folic acid or folacin) in foods, besides their availability (mono or polyglutamates). It is based in high performance liquid chromatographic method (HPLC) which protocol permits the extraction, purification and separation of folic acid and its different derivatives forms. This method has been validated with the standards and certified reference materials and compared with others investigation groups versus microbiological assays (AU)

Gene-directed enzyme prodrug therapy with a mustard prodrug/carboxypeptidase G2 combination.

The gene for the bacterial enzyme carboxypeptidase G2 (CPG2) was expressed internally in mammalian cells. Mammalian-expressed CPG2 had kinetic properties indistinguishable from bacterially expressed CPG2. Human tumor cell lines A2780, SK-OV-3 (ovarian adenocarcinomas), LS174T, and WiDr (colon carcinomas) were engineered to express constitutively either CPG2 or bacterial beta-galactosidase. These cell lines were subjected to a gene-directed enzyme prodrug therapy regime, using the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA). The lines which expressed CPG2 had enhanced sensitivity to CMDA. Comparing IC50S, WiDr-CPG2 and SK-OV-3-CPG2 were 11-16-fold more sensitive, whereas A2780-CPG2 and LS174T-CPG2 were approximately 95-fold more sensitive than the corresponding control lines. CPG2-expressing cells and control cells were mixed in differing proportions and then treated with prodrug. Total kill occurred when only approximately 12% of cells expressed CPG2 with the WiDr and SK-OV-3 lines and when only 4-5% of cells expressed CPG2 with the LS174T and A2780 lines, indicating a substantial bystander effect. These results establish this CPG2 enzyme/CMDA prodrug system as an effective combination for the gene-directed enzyme prodrug therapy approach.

A rapid colorimetric assay for gamma-glutamyl hydrolase (conjugase).

A simple procedure for the measurement of gamma-glutamyl hydrolase (conjugase) activity is described. Glutamic acid released from pteroylpenta-gamma-glutamate by hog kidney and chicken pancreas conjugases was quantitated using the dye 4,4'-bis(dimethylamino)benzophenone hydrazone. The procedure involves hydrolysis of the folylpoly-gamma-glutamate substrate by conjugase, conversion of glutamate to alpha-ketoglutarate by L-glutamate dehydrogenase and colorimetric measurement of the BDBH derivative of alpha-ketoglutarate. The release of as little as one nmol of glutamic acid from the substrate can be measured by this procedure, which is well suited for the assay of a variety of conjugase preparations. In addition, the method should provide a general assay for the enzymatic hydrolysis of various folate and antifolate polyglutamates.

Properties of pteroylpolyglutamate hydrolase in pancreatic juice of the pig.

The function of pteroylpolyglutamate hydrolase (PPH) of pancreatic secretion in the hydrolysis of dietary polyglutamyl folates (PteGlun) in humans is unclear. In this study, PPH was detected in pancreatic juice collected from pigs during both fasting and postprandial conditions. The secretion of PPH was markedly increased following feeding. Pancreatic PPH showed the following characteristics: 1) endo/random hydrolysis of gamma-glutamyl peptide bonds of Pte-Glun substrates, yielding folic acid as the terminal product; 2) maximum activity at pH 4.0-4.5 and maximum stability at pH 7.0; 3) stimulation of activity by Zn2+ and 2-mercaptoethanol; 4) Km values for pteroyltriglutamate (PteGlu3) of 28.7 microM at pH 4.0 and 9.1 microM at pH 5.0; 5) apparent molecular weight of 29,000; and 6) isoelectric point within the range of 8.5-9.0. On the basis of PPH activity, volume of the postprandial secretion and pH profile of enzyme activity, it is suggested that pancreatic PPH may act in vivo in folate digestion and absorption to initiate the deconjugation of dietary PteGlun prior to the action of jejunal brush border PPH.

Role of folylpolyglutamate synthetase in the regulation of methotrexate polyglutamate formation in H35 hepatoma cells.

The effect of culture conditions on the glutamylation of methotrexate by intact H35 hepatoma cells and folylpolyglutamate synthetase (FPGS) activity in the corresponding crude extracts has been examined. The rate of cellular glutamylation of methotrexate observed in rapidly dividing cultures was 4-fold higher than confluent cultures, and was accompanied by an increase in extract FPGS activity (2.2-fold). The depletion of cellular folates produced comparable increases in both cellular methotrexate glutamylation and extract FPGS activity (approximately 1.8-fold). Near-quantitative reductions in cellular methotrexate glutamylation were caused by media additions of reduced folates and methotrexate to confluent cultures of wild-type and folate-depleted H35 cells. However, these produced relatively modest reductions in FPGS activity in the corresponding crude extracts (approximately 50%). Methionine exclusion resulted in a greater than 50% decrease in FPGS activity in crude extracts of these cells compared to extracts of control cultures. The combination of methionine exclusion and folinic acid addition lowered the FPGS activity to less than 25% that of control. The data suggest that the changes in the glutamylation rate of methotrexate in whole cells due to culture conditions such as folate restriction, reduced folate addition, methionine exclusion, and growth state are at least in part a consequence of alterations in FPGS activity. This conclusion is consistent with the proposition that the metabolism of slow-acting substrates for FPGS (such as 4-amino antifolates and their corresponding polyglutamates) may be sensitive to changes in enzyme levels or activity (Cook et al., Biochemistry, 26: 530-539, 1987). Analysis of the products formed by FPGS from extracts using methotrexate as the substrate revealed no significant amounts of polyglutamate species higher than 4-NH2-10-CH3-PteGlu3. In contrast, when using the thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid as the starting substrate under identical assay conditions, FPGS from extracts catalyzed the formation of predominantly long chain polyglutamate derivatives (Glu4 and higher). These results reflect the relative efficacy of methotrexate and N10-propargyl-5,8-dideazafolic acid, as well as their polyglutamate derivatives, as substrates for FPGS.

High-performance liquid chromatographic assay for pteroylpolyglutamate hydrolase.

A highly sensitive assay for pteroylpolyglutamate hydrolase is described employing high-performance liquid chromatography (HPLC) with ultraviolet detection at 280 nm. The method is based on the separation of pteroylpolyglutamates containing various glutamyl residues on a C18 muBondapak reversed-phase column. Individual pteroylpolyglutamates are eluted by a gradient of 2.5-8.5% acetonitrile in 0.1 M potassium phosphate buffer (pH 6.0) within 20 min. The polyglutamates with higher glutamyl residues were less well retained in the reversed-phase column. The relationship between the peak area and the amount of pteroylpolyglutamate was observed to be linear over the range 10 pmol to 2.5 nmol. Human serum pteroylpolyglutamate hydrolase was studied using pteroylpentaglutamate as substrate in 0.1 M sodium acetate buffer (pH 4.5). The enzyme appeared to function as an exopeptidase based on the detection of intermediates, pteroyltetra-, tri-, and -diglutamate, and the product, pteroylmonoglutamate. Using the HPLC assay, extracts of Plasmodium falciparum were found not to contain detectable enzyme activity.

Studies on the nature of transition-metal-ion-mediated binding of triazine dyes to enzymes. The interaction of procion red MX-8B with carboxypeptidase G-2.

Several reactive azoic dichlorotriazinyl dyes specifically and irreversibly inactivate the folate-degrading enzyme carboxypeptidase G-2 at a site competitive with the enzyme substrates methotrexate (4-amino-N10-methylfolic acid) and p-aminobenzoyl-L-glutamate. Although the less reactive monochlorotriazinyl dye, Procion red H-8BN, is unable to inactivate the enzyme, it is capable of marked inhibition of inactivation by dichlorotriazinyl dyes in the presence of Zn2+. Zinc ions and, to a lesser extent other first row transition metal ions, significantly enhance the affinity of Procion red H-8BN and its analogues Procion red MX-8B and Procion red MX-2B, for carboxypeptidase G-2. It is proposed that this effect is mediated through the formation of a specific tetracoordinate Zn2+ complex between the azo linkage and adjacent sulphonate and hydroxyl functions of the dye and an appropriate ligand on the protein. Carboxypeptidase G-2 quantitatively inactivated with the dichlorotriazinyl dye, Procion red MX-8B, contains approximately 1 mol dye/mol subunit of Mr 42000. Proteolytic cleavage of the labelled enzyme and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a principal red peptide which on amino acid sequence analysis results in the identification of the dye binding domain. The affinity label, Procion red MX-8B, is believed to be attached to the hydroxylic side chain of Thr-279.

Intestinal folate conjugase activity and folate absorption in the ethanol-fed pregnant rat.

The effect of chronic alcohol consumption on intestinal folate conjugase activity and on the absorption of both mono- and polyglutamyl folates was evaluated in pregnant rats. Female rats were given either 20% ethanol in drinking water and rat chow ad libitum (alcohol group) or were fed rat chow and water ad libitum (controls). After four weeks on this regimen all animals were bred and the alcohol group was changed to 30% ethanol in water. Conjugase activity, in vivo folate absorption, and fetal and placental weights were measured on day 20 of gestation. The controls consumed more food than the alcohol group, which was reflected in significantly higher body weights of the control group. Fetal weights of the alcohol group were significantly lower than those of the controls, but litter size and placental weights were not significantly different. No significant differences were found in intestinal conjugase activity between the two groups of animals, but the intestinal absorption of both folate mono- and pentaglutamate were significantly higher in the alcohol than in the control group. The absorption of folate pentaglutamate was higher than that of folate monoglutamate in both groups of animals but the differences were not significant. These findings indicate that ethanol ingestion during pregnancy does not seem to interfere with either folate polyglutamate hydrolysis or folate absorption.

Radioassay of the folate-hydrolyzing enzyme activity, and the distribution of the enzyme in biological cells and tissues.

A sensitive radioassay method has been developed to quantitate the activity of the folate-hydrolyzing enzyme which catalyzes the hydrolysis of folic acid to pteroic acid and glutamic acid. The method is based on analyzing [2-14C]pteroic acid separated by a thin-layer chromatography on an Avicel SF cellulose plate using 0.1 M potassium phosphate buffer, pH 7.0, as a solvent. This method was found to be more sensitive than a conventional photometric method to determine the activity of the folate-hydrolyzing enzyme. High activities of the enzyme were found in Crithidia fasciculata ATCC 12857, Neurospora crassa IFO 6979 and rat liver. Smaller activities of the enzyme were widely distributed in other microbial cells and mammalian tissues.