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Biomedical subjects

Alfonso Pompella

Publications and source records attributed to Alfonso Pompella.

At least 19 recordsLinked to original sources
PubMed2006

Modulation of cell growth and cisplatin sensitivity by membrane gamma-glutamyltransferase in melanoma cells.

The plasma membrane enzyme gamma-glutamyltransferase (GGT) is regarded as critical for the maintenance of intracellular levels of glutathione (GSH). GGT expression has been implicated in drug resistance through elevation of intracellular GSH. The dependence of intracellular GSH on GGT expression was not conclusively ascertained. The present study was designed to investigate the role of GGT and of intracellular GSH levels in modulating proliferation and sensitivity to cisplatin of melanoma cells. GGT transfection resulted in increased growth, both in vitro and in tumour xenografts. In addition, GGT-transfected cells exhibited reduced sensitivity to cisplatin associated with lower DNA platination. A decrease in intracellular GSH levels, rather than an increase, was observed in GGT-transfected cells; moreover, in cysteine-deficient conditions, the expression of GGT did not provide transfected cells with the ability of utilising extracellular GSH. In conclusion, these results indicate that GGT activity confers a growth advantage unrelated with intracellular glutathione supply, and are consistent with the interpretation that cisplatin resistance is the consequence of modifications of cellular pharmacokinetics as a result of extracellular drug inactivation by thiol metabolites originated by GGT-mediated GSH cleavage.

Antineoplastic Agents↗
PubMed2006

[The prognostic role of gamma-glutamyltransferase for cardiovascular events].

Serum gamma-glutamyltransferasis activity is a low-cost, highly sensitive laboratory test. Though it is currently considered as an index of hepato-biliary dysfunction and alcohol abuse, recent epidemiological and pathologic studies have proposed its independent role in the pathogenesis and clinical evolution of cardiovascular diseases brought by atherosclerosis, associated with a gamma-glutamyltransferase-linked prooxidant effect of glutathione catabolism within the plaque.

Atherosclerosis↗
PubMed2005

Beta-lipoprotein- and LDL-associated serum gamma-glutamyltransferase in patients with coronary atherosclerosis.

Elevation of serum gamma-glutamyltransferase (GGT) activity is a risk factor for myocardial infarction and stroke. GGT activity can catalyze the oxidation of low-density lipoprotein (LDL), a process involved in the pathogenesis of atherosclerosis. Serum GGT is partially adsorbed onto circulating LDL, and catalytically active GGT has been found within atherosclerotic plaques, colocalizing with oxidized LDL and foam cells. We investigated the the nature of the LDL-associated GGT, the degree of correlation between total serum GGT levels and beta-lipoprotein (beta-LP)-associated GGT, and whether this association is altered in subjects with coronary artery disease (CAD). LDL-bound GGT showed an entire, amphiphilic heavy chain, but the association was easily lost during LDL purification by affinity chromatography. When the activity of GGT associated with polycation-precipitated beta-lipoproteins was assayed, an identical immunoreactive GGT was found in Western blot, and a statistically significant linear correlation was found between total serum GGT levels and the corresponding beta-LP-bound activities (p<0.0001) in controls and patients with CAD. Nevertheless, subjects with CAD presented a lower ratio of beta-LP-bound GGT to total serum GGT respect to controls (p<0.05) and healthy subjects with elevated serum GGT (p<0.01). In addition, a relative decrease of total serum GGT was observed in CAD subjects of older age as compared to younger ones (p<0.005).

Biomarkers↗
PubMed2005

Prooxidant reactions promoted by soluble and cell-bound gamma-glutamyltransferase activity.

Recent studies have provided evidence for the prooxidant roles played by molecular species originating during the catabolism of glutathione (GSH) effected by gamma-glutamyltransferase (GGT), an enzyme normally present in serum and on the outer surface of numerous cell types. The reduction of metal ions by GSH catabolites is capable of inducing the redox cycling processes, leading to the production of reactive oxygen species and other free radicals. Through the action of these reactive compounds, cell membrane GGT activity can ultimately produce oxidative modifications on a variety of molecular targets, involving oxidation and/or S-thiolation of protein thiol groups in the first place. This chapter is a survey of the procedures most suitable to reveal GGT-dependent prooxidant reactions and their effects at the cellular and extracellular level, including methods in histochemistry, cytochemistry, and biochemistry, with special reference to methods for the evaluation of protein thiol redox status.

Animals↗
PubMed2005

Advanced oxidation protein products in serum of patients with myotonic disease type I: association with serum gamma-glutamyltransferase and disease severity.

Steinert's disease (myotonic dystrophy type 1; MD) is caused by a CTG trinucleotide expansion on 19q13.3. Although the pathogenic mechanism underlying multisystem involvement in MD is still unclear, a role of oxidative stress in this disease has been suggested. We investigated 39 MD patients to assess the plasma concentration of advanced oxidation protein products (AOPPs) and gamma-glutamyltransferase (GGT) and related them to clinical severity scores. Plasma AOPP levels (p=0.021), total serum GGT activity (p=0.0005) and GGT activity associated with low-density lipoprotein (p=0.0021) were significantly higher in patients than in controls. There was significant correlation between serum GGT levels and AOPPs (r=0.5831; p=0.0022). A statistically significant increase in serum GGT with age was found in MD patients (p=0.0193). Receiver operating characteristic curve analysis showed that higher AOPP levels were significantly associated with extra-muscular signs of the disease, i.e., cataracts and heart involvement (area under the curve+/-SE=0.908+/-0.083), but not with muscular involvement. The concomitant increment in GGT and AOPPs indicates a possible role of oxidative stress in the pathogenesis of MD type 1, while the association of increased AOPP levels with extra-muscular signs of the disease suggests that individual susceptibility to oxidative stress can modulate the extra-muscular phenotype of the disease.

Adolescent↗
PubMed2004

Plasma membrane gamma-glutamyltransferase activity facilitates the uptake of vitamin C in melanoma cells.

Adequate cellular transport of ascorbic acid (AA) and its oxidation product dehydroascorbate (DHA) is assured through specific carriers. It was shown that vitamin C is taken up as DHA by most cell types, including cancer cells, via the facilitative GLUT transporters. Thus, AA oxidation to DHA can be considered a mechanism favoring vitamin C uptake and intracellular accumulation. We have investigated whether such an AA-oxidizing action might be provided by plasma membrane gamma-glutamyltransferase (GGT), previously shown to function as an autocrine source of prooxidants. The process was studied using two distinct human metastatic melanoma clones. It was observed that the Me665/2/60 clone, expressing high levels of membrane GGT activity, was capable of effecting the oxidation of extracellular AA, accompanied by a marked increase of intracellular AA levels. The phenomenon was not observed with Me665/2/21 cells, possessing only traces of membrane GGT. On the other hand, AA oxidation and stimulation of cellular uptake were indeed observed after transfection of 2/21 cells with cDNA coding for GGT. The mechanism of GGT-mediated AA oxidation was investigated in acellular systems, including GGT and its substrate glutathione. The process was observed in the presence of redox-active chelated iron(II) and of transferrin or ferritin, i.e., two physiological iron sources. Thus, membrane GGT activity-often expressed at high levels in human malignancies-can oxidize extracellular AA and promote its uptake efficiently.

Ascorbic Acid↗
PubMed2004

The significance of serum gamma-glutamyltransferase in cardiovascular diseases.

Since early after the introduction of serum gamma-glutamyltransferase (GGT) in clinical practice as a reliable and widely employed laboratory test, epidemiological and prospective studies have repeatedly shown that this activity possesses a prognostic value for morbidity and mortality. The association is independent of possibly concomitant conditions of liver disease, and notably, a significant independent correlation of serum GGT exists with the occurrence of cardiovascular diseases (myocardial infarction, stroke). Experimental work has documented that active GGT is present in atherosclerotic plaques of coronary as well as in cerebral arteries. These findings, and the recently recognized functions of GGT in the generation of reactive oxygen species, indicate that serum GGT represents a true marker of cardiovascular diseases and underlying atherosclerosis. Further insights into potential therapeutic interest will probably be derived from studies investigating the origin of GGT activity in plaque tissue.

Arteriosclerosis↗
PubMed2003

The changing faces of glutathione, a cellular protagonist.

Glutathione (GSH) has been described for a long time just as a defensive reagent against the action of toxic xenobiotics (drugs, pollutants, carcinogens). As a prototype antioxidant, it has been involved in cell protection from the noxious effect of excess oxidant stress, both directly and as a cofactor of glutathione peroxidases. In addition, it has long been known that GSH is capable of forming disulfide bonds with cysteine residues of proteins, and the relevance of this mechanism ("S-glutathionylation") in regulation of protein function is currently receiving confirmation in a series of research lines. Rather paradoxically, however, recent studies have also highlighted the ability of GSH-and notably of its catabolites-to promote oxidative processes, by participating in metal ion-mediated reactions eventually leading to formation of reactive oxygen species and free radicals. A crucial role in these phenomena is played by membrane bound gamma-glutamyltransferase activity. The significance of GSH as a major factor in regulation of cell life, proliferation, and death, should be regarded as the integrated result of all these roles it can play.

Animals↗
PubMed2003

Possible role of membrane gamma-glutamyltransferase activity in the facilitation of transferrin-dependent and -independent iron uptake by cancer cells.

BACKGROUND: The molecular mechanisms by which iron is physiologically transported trough the cellular membranes are still only partially understood. Several studies indicate that a reduction step of ferric iron to ferrous is necessary, both in the case of transferrin-mediated and transferrin-independent iron uptake. Recent studies from our laboratory described gamma-glutamyltransferase activity (GGT) as a factor capable to effect iron reduction in the cell microenvironment. GGT is located on the outer aspect of plasma membrane of most cell types, and is often expressed at high levels in malignant tumors and their metastases. The present study was aimed at verifying the possibility that GGT-mediated iron reduction may participate in the process of cellular iron uptake. RESULTS: Four distinct human tumor cell lines, exhibiting different levels of GGT activity, were studied. The uptake of transferrin-bound iron was investigated by using 55Fe-loaded transferrin, as well as by monitoring fluorimetrically the intracellular iron levels in calcein-preloaded cells. Transferrin-independent iron uptake was investigated using 55Fe complexed by nitrilotriacetic acid (55Fe-NTA complex).The stimulation of GGT activity, by administration to cells of the substrates glutathione and glycyl-glycine, was generally reflected in a facilitation of transferrin-bound iron uptake. The extent of such facilitation was correlated with the intrinsic levels of the enzyme present in each cell line. Accordingly, inhibition of GGT activity by means of two independent inhibitors, acivicin and serine/boric acid complex, resulted in a decreased uptake of transferrin-bound iron. With Fe-NTA complex, the inhibitory effect - but not the stimulatory one - was also observed. CONCLUSION: It is concluded that membrane GGT can represent a facilitating factor in iron uptake by GGT-expressing cancer cells, thus providing them with a selective growth advantage over clones that do not possess the enzyme.

Journal Article↗
PubMed2003

Redox modulation of protein kinase/phosphatase balance in melanoma cells: the role of endogenous and gamma-glutamyltransferase-dependent H2O2 production.

Alterations of protein kinase and protein phosphatase activities have been described in a number of tumors. Redox changes, such as in conditions of oxidant stress, have been reported to affect the cellular protein kinase/phosphatase balance. A basal production of reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)), exists in tumor cells, and the membrane-bound ecto-enzyme gamma-glutamyltransferase (GGT)-overexpressed in a variety of malignant tumors-is one of the mechanisms capable of promoting such a production. The present study was aimed to verify the interactions of GGT activity with protein phosphatase and kinase activities in Me665/2/60 melanoma cells, expressing high levels of this enzyme and exhibiting both basal and GGT-dependent production of hydrogen peroxide. An increase of total phosphatase as well as tyrosine phosphatase activities was observed after treatment of cells with both micromolar H(2)O(2) and GGT stimulation. Accordingly, stimulation of GGT resulted in decreased levels of phosphotyrosine. On the other hand, when serine/threonine phosphatase activities were selectively analyzed, both H(2)O(2) treatment and GGT stimulation caused their down-regulation.The data reported suggest that basal conditions of oxidant stress in melanoma may represent a factor contributing to the redox regulation of protein phosphorylation, and that GGT-mediated prooxidant reactions may participate in the process. As basal oxidant stress and expression of GGT activity are present in a variety of malignant tumors besides melanoma, these phenomena likely represent general mechanisms participating in the alteration of intracellular transduction during carcinogenesis.

Cell Membrane↗
PubMed2002

Enhanced resistance of HeLa cells to cisplatin by overexpression of gamma-glutamyltransferase.

Gamma-glutamyltransferase (GGT), which is a key enzyme for the cellular glutathione (GSH) homeostasis, was shown to be overexpressed in human tumor cells selected for resistance to cisplatin and to influence the resistance of experimental tumors in vivo. We first established that cisplatin treatment of HeLa cells was accompanied by an early 3-fold induction of GGT synthesis, enhancing the possibility that this enzyme plays an important role in the cell defenses against this anticancer drug. This role was then studied using a GGT-transfected HeLa cell line (HeLa-GGT) exhibiting 10 times the activity of the parental HeLa cells (120-150 and 10-14 mU/mg protein, respectively). Both cell lines showed comparable intracellular GSH levels and cisplatin resistance when cultured in high (250 microM) or low (50 microM) cysteine-containing medium. When 50 microM of GSH were included in the low-cysteine culture medium only HeLa-GGT cells partially recovered their intracellular GSH and exhibited an increased resistance to cisplatin. Cisplatin treatment also inhibited GGT-dependent production of reactive oxygen species, a process depending on the availability of cysteinylglycine produced during GSH catabolism. Furthermore, we showed that cisplatin forms adducts with cysteinylglycine 10 times more rapidly than with GSH, and that these adducts were formed only in the extracellular medium of HeLa GGT cells. This extracellular mechanism could at least partially account for the increased resistance of GGT-rich cells to cisplatin.

Antineoplastic Agents↗
PubMed2002

Extra-cellular thiol metabolism in clones of human metastatic melanoma with different gamma-glutamyl transpeptidase expression: implications for cell response to platinum-based drugs.

Thiol redox status can affect important functions both intracellularly and extracellularly. The plasma membrane enzyme gamma-glutamyl transpeptidase (GGT), which plays a crucial role in cellular handling of thiols, is often expressed in malignant tumors, including melanoma, although its expression levels may vary widely among different tumors or cells of the same tumor. In an attempt to better understand the functional significance of GGT overexpression, we have examined the relationships between intra- and extra-cellular thiol metabolism and GGT expression. Intra- and extra-cellular distribution of glutathione and other low mol. wt. thiols and disulfides was investigated in two different Me665/2 human melanoma clones that originated from the same metastasis, but exhibiting high (2/60 clone) and low (2/21 clone) GGT activity. Intracellular content of glutathione was lower in GGT-rich 2/60 cells, in spite of high GGT expression. A lower utilization of extracellular cystine was also observed in these cells. In both clones, a direct secretion of cysteine in the extracellular medium was detected, which was independent of GGT-mediated catabolism of extracellular glutathione. Substantial amounts of glutathione, GSSG and glutathione-cysteine disulfide were accumulated extracellularly only in the case of GGT-poor 2/21 cells, while the same event was apparent in 2/60 cells only after the following inhibition of GGT activity. When exposed to the trinuclear platinum compound BBR 3464 or hydrogen peroxide, which are very reactive for sulfur-containing nucleophiles, the 2/60 clone showed higher sensitivity than the 2/21 clone to both agents. These results suggest that the clone-specific balance between transport of sulfur aminoacids and GGT activity results in profound differences in the capability of each clone to modify the thiol redox status of the extracellular milieu. The finding may have important implications in tumor cell behavior with particular reference to chemosensitivity, since thiols are recognized factors in modulation of cell sensitivity to platinum-based anticancer drugs.

Antineoplastic Agents↗