Redox Mechanisms Underlying the Cytostatic Effects of Boric Acid on Cancer Cells-An Issue Still Open.

Boric acid (BA) is the dominant form of boron in plasma, playing a role in different physiological mechanisms such as cell replication. Toxic effects have been reported, both for high doses of boron and its deficiency. Contrasting results were, however, reported about the cytotoxicity of pharmacological BA concentrations on cancer cells. The aim of this review is to briefly summarize the main findings in the field ranging from the proposed mechanisms of BA uptake and actions to its effects on cancer cells.

Biocompatibility of Subperiosteal Dental Implants: Effects of Differently Treated Titanium Surfaces on the Expression of ECM-Related Genes in Gingival Fibroblasts.

INTRODUCTION: Titanium alloys currently are the most used material for the manufacture of dental endosseous implants. However, in partially or totally edentulous patients, varying degrees of maxillary bone resorption usually occur, making the application of these devices difficult or even impossible. In these cases, a suitable alternative is offered by subperiosteal implants, whose use is undergoing a revival of interest following the introduction of novel, computer-assisted manufacturing techniques. Several procedures have been developed for the modification of titanium surfaces so to improve their biocompatibility and integration with bone. Information is, however, still incomplete as far as the most convenient surface modifications to apply with subperiosteal implants, in which an integration with soft mucosal tissues is just as important. OBJECTIVES: The present study aimed at evaluating whether different treatments of titanium surfaces can produce different effects on the viability, attachment, and differentiation of gingival fibroblasts, i.e., the cell type mainly involved in osteointegration as well as the healing of soft tissues injured by surgical procedures, in order to verify whether any of the treatments are preferable under these respects. METHODOLOGY: The human immortalized gingival fibroblast (CRL-4061 line) were cultured in the presence of titanium specimens previously treated with five different procedures for surface modification: (i) raw machined (Ti-1); (ii) electropolished (Ti-2); (iii) sand-blasted acid-etched (Ti-3); (iv) Al Ti Color™ proprietary procedure (Ti-4); and (v) anodized (Ti-5). At different times of incubation, viability and proliferation of cells, was determined along with the changes in the expression patterns of ECM-related genes involved in fibroblast attachment and differentiation: vinculin, fibronectin, collagen type I-alpha 1 chain, focal adhesion kinase, integrin ß-1, and N-cadherin. Three different experiments were carried out for each experimental point. The release from fibroblasts of endothelin-1 was also analyzed as a marker of inflammatory response. The proliferation and migration of fibroblasts were evaluated by scratch tests. RESULTS: None of the five types of titanium surface tested significantly affected the fibroblasts' viability and proliferation. The release of endothelin-1 was also not significantly affected by any of the specimens. On the other hand, all titanium specimens significantly stimulated the expression of ECM-related genes at varying degrees. The proliferation and migration abilities of fibroblasts were also significantly stimulated by all types of titanium surface, with a higher-to-lower efficiency in the order: Ti-3 > Ti-4 > Ti-5 > Ti-2 > Ti-1, thus identifying sandblasting acid-etching as the most convenient treatment. CONCLUSIONS: Our observations suggest that the titanium alloys used for manufacturing subperiosteal dental implants do not produce cytotoxic or proinflammatory effects on gingival fibroblasts, and that sandblasting acid-etching may be the surface treatment of choice as to stimulate the differentiation of gingival fibroblasts in the direction of attachment and migration, i.e., the features allegedly associated with a more efficient implant osteointegration, wound healing, and connective tissue seal formation.

Enhancement of ferroptosis by boric acid and its potential use as chemosensitizer in anticancer chemotherapy.

Ferroptosis is a form of regulated cell death (RCD) characterized by intracellular iron ion accumulation and reactive oxygen species (ROS)-induced lipid peroxidation. Ferroptosis in cancer and ferroptosis-related anticancer drugs have recently gained interest in the field of cancer treatment. Boron is an essential trace element playing an important role in several biological processes. Recent studies have described contrasting effects of boric acid (BA) in cancer cells, ranging from protective/mitogenic to damaging/antiproliferative. Interestingly, boron has been shown to interfere with critical factors involved in ferroptosis-intracellular glutathione and lipid peroxidation in the first place. Thus, the present study was aimed to verify the ability of boron to modulate the ferroptotic process in HepG2 cells, a model of hepatocellular carcinoma. Our results indicate that-when used at high, pharmacological concentrations-BA can increase intracellular ROS, glutathione, and TBARS levels, and enhance ferroptosis induced by RSL3 and erastin. Also, high BA concentrations can directly induce ferroptosis, and such BA-induced ferroptosis can add to the cytotoxic effects of anticancer drugs sorafenib, doxorubicin and cisplatin. These observations suggest that BA could be exploited as a chemo-sensitizer agent in order to overcome cancer drug resistance in selected conditions. However, the possibility of reaching suitably high concentrations of BA in the tumor microenvironment will need to be further investigated.

The dark side of gamma-glutamyltransferase (GGT): Pathogenic effects of an 'antioxidant' enzyme.

Having long been regarded as just a member in the cellular antioxidant systems, as well as a clinical biomarker of hepatobiliary diseases and alcohol abuse, gamma-glutamyltransferase (GGT) enzyme activity has been highlighted by more recent research as a critical factor in modulation of redox equilibria within the cell and in its surroundings. Moreover, due to the prooxidant reactions which can originate during its metabolic function in selected conditions, experimental and clinical studies are increasingly involving GGT in the pathogenesis of several important disease conditions, such as atherosclerosis, cardiovascular diseases, cancer, lung inflammation, neuroinflammation and bone disorders. The present article is an overview of the laboratory findings that have prompted an evolution in interpretation of the significance of GGT in human pathophysiology.

Induction of Gamma-Glutamyltransferase Activity and Consequent Pro-oxidant Reactions in Human Macrophages Exposed to Crocidolite Asbestos.

Asbestos is the main causative agent of malignant pleural mesothelioma. The variety known as crocidolite (blue asbestos) owns the highest pathogenic potential, due to the dimensions of its fibers as well as to its content of iron. The latter can in fact react with macrophage-derived hydrogen peroxide in the so called Fenton reaction, giving rise to highly reactive and mutagenic hydroxyl radical. On the other hand, hydroxyl radical can as well originate after thiol-dependent reduction of iron, a process capable of starting its redox cycling. Previous studies showed that glutathione (GSH) is one such thiol, and that cellular gamma-glutamyltransferase (GGT) can efficiently potentiate GSH-dependent iron redox cycling and consequent oxidative stress. As GGT is expressed in macrophages and is released upon their activation, the present study was aimed at verifying the hypothesis that GSH/GGT-dependent redox reactions may participate in the oxidative stress following the activation of macrophages induced by crocidolite asbestos. Experiments in acellular systems confirmed that GGT-mediated metabolism of GSH can potentiate crocidolite-dependent production of superoxide anion, through the production of highly reactive dipeptide thiol cysteinyl-glycine. Cultured THP-1 macrophagic cells, as well as isolated monocytes obtained from healthy donors and differentiated to macrophages in vitro, were investigated as to their expression of GGT and the effects of exposure to crocidolite. The results show that crocidolite asbestos at subtoxic concentrations (50-250 ng/1000 cells) can upregulate GGT expression, which raises the possibility that macrophage-initiated, GSH/GGT-dependent pro-oxidant reactions may participate in the pathogenesis of tissue damage and inflammation consequent to crocidolite intoxication.

γ-Glutamyltransferase enzyme activity of cancer cells modulates L-γ-glutamyl-p-nitroanilide (GPNA) cytotoxicity.

L-γ-Glutamyl-p-nitroanilide (GPNA) is widely used to inhibit the glutamine (Gln) transporter ASCT2, but recent studies have demonstrated that it is also able to inhibit other sodium-dependent and independent amino acid transporters. Moreover, GPNA is a well known substrate of the enzyme γ-glutamyltransferase (GGT). Our aim was to evaluate the effect of GGT-mediated GPNA catabolism on cell viability and Gln transport. The GGT-catalyzed hydrolysis of GPNA produced cytotoxic effects in lung cancer A549 cells, resulting from the release of metabolite p-nitroaniline (PNA) rather than from the inhibition of Gln uptake. Interestingly, compounds like valproic acid, verapamil and reversan were able to increase the cytotoxicity of GPNA and PNA, suggesting a key role of intracellular detoxification mechanisms. Our data indicate that the mechanism of action of GPNA is more complex than believed, and further confirm the poor specificity of GPNA as an inhibitor of Gln transport. Different factors may modulate the final effects of GPNA, ranging from GGT and ASCT2 expression to intracellular defenses against xenobiotics. Thus, other strategies - such as a genetic suppression of ASCT2 or the identification of new specific inhibitors - should be preferred when inhibition of ASCT2 function is required.

The S-thiolating activity of membrane gamma-glutamyltransferase: formation of cysteinyl-glycine mixed disulfides with cellular proteins and in the cell microenvironment.

Previous studies have documented that activity of the plasma membrane enzyme gamma-glutamyltransferase (GGT) is accompanied by prooxidant processes, with production of reactive oxygen species and oxidation of cellular protein thiols. The present work was aimed to verify the occurrence and extent of S-thiolation mediated by GGT and characterize the molecular species involved in mixed disulfide formation. Experiments show that the cysteinyl-glycine (CG) originating from cellular GGT-mediated glutathione (GSH) metabolism can efficiently thiolate cellular proteins, as well as proteins present in the extracellular environment. With cells presenting high levels of GGT expression, basal levels of CG-containing protein mixed disulfides are detectable, in cellular proteins, as well as in proteins of the culture medium. Stimulation of GGT activity in these cells by administration of substrates results in an increase of CG mixed disulfide formation and a concomitant decrease of GSH-containing disulfides, likely due to GGT-dependent removal of GSH from the system. The findings reported suggest that binding of CG ("protein S-cysteylglycylation") may represent an as yet unrecognized function of membrane GGT, likely playing a regulatory role(s) in the cell and its surroundings.

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.

Redox modulation of NF-kappaB nuclear translocation and DNA binding in metastatic melanoma. The role of endogenous and gamma-glutamyl transferase-dependent oxidative stress.

AIMS AND BACKGROUND: The transcription factor NF-kappaB is implicated in the expression of genes involved in cell proliferation, apoptosis and metastasis. In melanoma, high constitutive levels of NF-kappaB activation are usually observed. NF-kappaB is regulated by oxidation/reduction (redox) processes, and the occurrence of constitutive oxidative stress in melanoma cells has been documented. Recent studies of our laboratories showed that the membrane-bound gamma-glutamyl transferase (GGT) enzyme activity--expressed by a number of malignancies, including melanoma--can act as a basal source of superoxide, hydrogen peroxide and other prooxidants. METHODS: In the present study we utilized the 2/60 clone of Me665/2 human metastatic melanoma, which displays high levels of GGT activity, in order to verify if the presence of this enzyme--through the promotion of redox processes--may influence the activation status of NF-kappaB. The latter was evaluated by determining the nuclear translocation of the p65 subunit (by immunoblot), the DNA binding of NF-kappaB (by electrophoretic mobility shift assay) and its transcriptional activity (by gene transactivation studies). RESULTS: Me665/2/60 cells displayed a basal production of hydrogen peroxide. Stimulation of GGT activity by its substrates glutathione and glycyl-glycine caused additional production of hydrogen peroxide, up to levels approx. double the basal levels. Nuclear translocation of the NF-kappaB p65 subunit, DNA-binding and gene transactivation were thus investigated in Me665/2/60 cells whose GGT activity was modulated by means of substrates or inhibitors. Stimulation of GGT activity resulted in increased nuclear translocation of p65, while on the other hand NF-kappaB DNA binding and gene transactivation were paradoxically decreased. NF-kappaB DNA binding could be restored by treating cell lysates with the thiol-reducing agent dithiothreitol (DTT). Treatment of cells with exogenous hydrogen peroxide did not affect NF-kappaB activation status. CONCLUSIONS: Altogether, the data obtained indicate that GGT activity may impair the redox status of thiols that is critical for NF-kappaB DNA binding and gene transactivation, through the production of prooxidant species allegedly distinct from hydrogen peroxide. GGT activity therefore appears to be an additional factor in modulation of NF-kappaB transcriptional activity in melanoma, capable of hindering NF-kappaB DNA binding even in conditions where continuous oxidative stress would favor NF-kappaB nuclear translocation.

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.

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.

Glutathione catabolism as a signaling mechanism.

Glutathione (GSH) is the main intracellular thiol antioxidant, and as such participates in a number of cellular antitoxic and defensive functions. Nevertheless, non-antioxidant functions of GSH have also been described, e.g. in modulation of cell proliferation and immune response. Recent studies from our and other laboratories have provided evidence for a third functional aspect of GSH, i.e. the prooxidant roles played by molecular species originating during its catabolism by the membrane ectoenzyme gamma-glutamyl transpeptidase (GGT). The reduction of metal ions effected by GSH catabolites is capable to induce redox cycling processes leading to the production of reactive oxygen species (superoxide, hydrogen peroxide), as well as of other free radicals. Through the action of these reactive compounds, GSH catabolism can ultimately lead to oxidative modifications on a variety of molecular targets, involving oxidation and/or S-thiolation of protein thiol groups in the first place. Modulating effects of this kind have been observed on several important, redox-sensitive components of the signal transduction chains, such as cell surface receptors, protein phosphatase activities and transcription factors. Against this background, the prooxidant reactions induced by GSH catabolism appear to represent a novel, as yet unrecognized mechanism for modulation of cellular signal transduction.