Search

The modulation of H2O2 production by NADPH oxidase (Nox), on vascular endothelial growth factor (VEGF) stimulation, affects the redox signaling linked to cancer cell proliferation. H2O2 signal transduction involves reversible oxidation of thiol proteins, leading to the formation of cysteine sulfenic acids, responsible for the temporary inactivation of many phosphatases. These events imply that H2O2 reaches its intracellular targets. As Aquaporin-8 (AQP8) has been demonstrated to funnel Nox-produced H2O2 across the plasma membrane, this study aims to elucidate the role of AQP8 in the redox signaling occurring in human leukaemia B1647 cells that constitutively produce VEGF. AQP8 overexpression or silencing resulted in the modulation of VEGF ability of increasing or decreasing respectively, H2O2 intracellular level. Moreover, data obtained by a dimedone-based immunochemical method for sulfenic acid detection demonstrate that the expression of AQP8 can modulate the amplitude of downstream events, altering the activity of redox-sensitive targets. In particular, AQP8 affected VEGF-induced redox signaling by increasing the sulfenation of the tumor suppressor PTEN, which resulted in its inactivation and, in turn, caused Akt activation. Therefore, the dimedone based method for easily monitoring cellular protein sulfenation allowed to demonstrate, for the first time, the role of AQP8 on the fine tune of cysteine oxidation in target proteins involved in leukaemia cell proliferation pathways.

Caveolae/lipid rafts are membrane-rich cholesterol domains endowed with several functions in signal transduction and caveolin-1 (Cav-1) has been reported to be implicated in regulating multiple cancer-associated processes, ranging from tumor growth to multidrug resistance and angiogenesis. Vascular endothelial growth factor receptor-2 (VEGFR-2) and Cav-1 are frequently colocalized, suggesting an important role played by this interaction on cancer cell survival and proliferation. Thus, our attention was directed to a leukemia cell line (B1647) that constitutively produces VEGF and expresses the tyrosine-kinase receptor VEGFR-2. We investigated the presence of VEGFR-2 in caveolae/lipid rafts, focusing on the correlation between reactive oxygen species (ROS) production and glucose transport modulation induced by VEGF, peculiar features of tumor proliferation. In order to better understand the involvement of VEGF/VEGFR-2 in the redox signal transduction, we evaluated the effect of different compounds able to inhibit VEGF interaction with its receptor by different mechanisms, corroborating the obtained results by immunoprecipitation and fluorescence techniques. Results here reported showed that, in B1647 leukemia cells, VEGFR-2 is present in caveolae through association with Cav-1, demonstrating that caveolae/lipid rafts act as platforms for negative modulation of VEGF redox signal transduction cascades leading to glucose uptake and cell proliferation, suggesting therefore novel potential targets.

Caffeic, syringic, and protocatechuic acids are phenolic acids derived directly from food intake or come from the gut metabolism of polyphenols. In this study, the antioxidant activity of these compounds was at first evaluated in membrane models, where caffeic acid behaved as a very effective chain-breaking antioxidant, whereas syringic and protocatechuic acids were only retardants of lipid peroxidation. However, all three compounds acted as good scavengers of reactive species in cultured cells subjected to exogenous oxidative stress produced by low level of H2O2. Many tumour cells are characterised by increased ROS levels compared with their noncancerous counterparts. Therefore, we investigated whether phenolic acids, at low concentrations, comparable to those present in human plasma, were able to decrease basal reactive species. Results show that phenolic acids reduced ROS in a leukaemia cell line (HEL), whereas no effect was observed in normal cells, such as HUVEC. The compounds exhibited no toxicity to normal cells while they decreased proliferation in leukaemia cells, inducing apoptosis. In the debate on optimal ROS-manipulating strategies in cancer therapy, our work in leukaemia cells supports the antioxidant ROS-depleting approach.

The mechanism involved in the prosurvival effect of interleukin-3 on the human acute myeloid leukaemia cell line M07e is investigated. A decrease in intracellular reactive oxygen species (ROS) content, glucose transport activity and cell survival was observed in the presence of inhibitors of plasma membrane ROS sources, such as diphenylene iodonium and apocynin, and by small interference RNA for Nox2. Moreover, IL-3 incubation stimulated the synthesis of Nox2 cytosolic sub-unit p47phox and glucose transporter Glut1. Thus, the inhibition of ROS generation by Nox inhibitors stimulated apoptosis showing that ROS production, induced by IL-3 via Nox2, protects leukaemic cells from cell death. Also incubation with receptor tyrosine kinase inhibitors, such as anti-leukaemic drugs blocking the stem cell factor receptor (c-kit), showed similar effects, hinting that IL-3 transmodulates c-kit phosphorylation. These mechanisms may play an important role in acute myeloid leukaemia treatment, representing a novel therapeutic target.

The discovery of superoxide-generating enzymes homologues of phagocytic NAD(P)H oxidase, the Nox family, has led to the concept that reactive oxygen species (ROS) are “intentionally” generated with biological functions in various cell types. In this study, by treating an acute leukaemic cell line with different antioxidants, ROS generation was shown to be crucially involved in the modulation of glucose transport (mediated by Glut1), which is frequently up-regulated in cancer cells. Then, this study tried to elucidate ROS source(s) and mechanisms by which ROS are involved in Glut1 activity regulation. Results prove that Nox2 and Nox4 are the candidates and that phosphorylation processes are important in the regulation of glucose uptake on which cancer cells rely. On the whole, data suggest that both Glut1 and Nox homologues may be considered new potential targets in the treatment of leukaemia.

In M07e cells, a human megakaryocytic leukaemia line, reactive oxygen species (ROS) are generated in response to cytokines acting as intracellular messengers to modulate glucose transport. The aim of this work was to study the signal cascade involved in the acute glucose transport activation in cells exposed to growth factors, such as granulocyte macrophage-colony stimulation factor (GM-CSF) and thrombopoietin (TPO), to better understand some aspects of the aberrant proliferation in leukaemia. Results confirm ROS involvement in modulation of glucose transport in this cell line. Furthermore, GM-CSF and TPO produced changes in Glut1 phosphorylation and specific inhibitors employed to identify protein kinases involved in Glut activation by these cytokines proved that Akt, PLC gamma, Syk and the Src family take part in signal transduction leading to Glut1 activation.

Alzheimer’s disease is the most common and lethal neurodegenerative disorder. The major hallmarks of Alzheimer’s disease are extracellular aggregation of amyloidβpeptides and, the presence of intracellular neurofibrillary tangles formed by precipitation/aggregation of hyperphosphorylated tau protein. The etiology of Alzheimer’s disease is multifactorial and a full understanding of its pathogenesis remains elusive. Some years ago, it has been suggested that glycation may contribute to both extensive protein cross-linking and oxidative stress in Alzheimer’s disease. Glycation is an endogenous process that leads to the production of a class of compounds known as advanced glycation end products (AGEs). Interestingly, increased levels of AGEs have been observed in brains of Alzheimer’s disease patients. Methylglyoxal, a reactive intermediate of cellular metabolism, is the most potent precursor of AGEs and is strictly correlated with an increase of oxidative stress in Alzheimer’s disease. Many studies are showing that methylglyoxal and methylglyoxal-derived AGEs play a key role in the etiopathogenesis of Alzheimer's disease.

In the last decade, the generation and the role of reactive oxygen species (ROS), particularly hydrogen peroxide, in cell signalling transduction pathways have been intensively studied, and it is now clear that an increase of ROS level affects cellular growth and proliferation pathways related to cancer development. Hydrogen peroxide (H2O2) has been long thought to permeate biological membranes by simple diffusion since recent evidence challenged this notion disclosing the role of aquaporin water channels (AQP) in mediating H2O2 transport across plasma membranes. We previously demonstrated that NAD(P)H oxidase (Nox)-generated ROS sustain glucose uptake and cellular proliferation in leukaemia cells. The aim of this study was to assess whether specific AQP isoforms can channel Nox-produced H2O2 across the plasma membrane of leukaemia cells affecting downstream pathways linked to cell proliferation. In this work, we demonstrate that AQP inhibition caused a decrease in intracellular ROS accumulation in leukaemia cells both when H2O2 was produced by Nox enzymes and when it was exogenously added. Furthermore, AQP8 overexpression or silencing resulted to modulate VEGF capacity of triggering an H2O2 intracellular level increase or decrease, respectively. Finally, we report that AQP8 is capable of increasing H2O2-induced phosphorylation of both PI3K and p38 MAPK and that AQP8 expression affected positively cell proliferation. Taken together, the results here reported indicate that AQP8 is able to modulate H2O2 transport through the plasma membrane affecting redox signalling linked to leukaemia cell proliferation.

Extracts fromStevia rebaudianaBertoni, a plant native to Central and South America, have been used as a sweetener since ancient times. Currently,Steviaextracts are largely used as a noncaloric high-potency biosweetener alternative to sugar, due to the growing incidence of type 2 diabetes mellitus, obesity, and metabolic disorders worldwide. Despite the large number of studies onSteviaand steviol glycosidesin vivo, little is reported concerning the cellular and molecular mechanisms underpinning the beneficial effects on human health. The effect of four commercialSteviaextracts on glucose transport activity was evaluated in HL-60 human leukaemia and in SH-SY5Y human neuroblastoma cells. The extracts were able to enhance glucose uptake in both cellular lines, as efficiently as insulin. Our data suggest that steviol glycosides could act by modulating GLUT translocation through the PI3K/Akt pathway since treatments with both insulin andSteviaextracts increased the phosphorylation of PI3K and Akt. Furthermore,Steviaextracts were able to revert the effect of the reduction of glucose uptake caused by methylglyoxal, an inhibitor of the insulin receptor/PI3K/Akt pathway. These results corroborate the hypothesis thatSteviaextracts could mimic insulin effects modulating PI3K/Akt pathway.

Reactive oxygen species (ROS) has been increasingly recognised as intracellular messengers in signal transduction following receptor activation by a variety of bioactive peptides including growth factors, cytokines and hormones. In this study ROS production and glucose transport activity were evaluated in the growth factor dependent M07e cells and in B1647 cells, not requiring additional hematopoietic cytokines for growth: the aim was to investigate whether ROS could be involved in the regulation of Glut1-mediated glucose uptake in both cell lines. The effect of the synthetic superoxide and hydrogen peroxide scavenger EUK-134 on DOG uptake activity and intracellular ROS formation supports the concept of reactive oxygen species as signalling molecules. In order to investigate ROS generation sources, diphenyleneiodonium, an inhibitor of flavoprotein centres and apocynin, an inhibitor of NAD(P)H oxidase, were used: they inhibit both ROS production and glucose uptake activation. All these data support the hypothesis that ROS can contribute to the regulation of glucose transport, not only in M07e cells but also in B1647 cells; we could speculate that one possible source of ROS, linked somehow with Glut1 activity, can be a NAD(P)H oxidase similar to that one present in phagocytic cells.