Gabriele Hakim

Low levels of selenium compounds are selectively toxic for a human neuron cell line through ROS/RNS increase and apoptotic process activation.

Organic and inorganic selenium compounds were used to examine whether low selenium concentration is able to trigger apoptotic degeneration in a human neuron cell line in vitro and to explore changes in reactive oxygen and nitrogen species and antioxidant protein content during the apoptotic processes. The results indicated that: (1) SKNBE neuroblastoma cells treated with sodium selenite, sodium selenate and seleno-methionine (0.1, 0.5 and 0.5 μM, respectively) for 24h exhibited a viability decrease, unlike kidney or prostatic cells; (2) the PARP (poly-ADP-ribose-polymerase) degradation and caspase activation detected by Western blot and flow cytometry fluorimetric examination showed induction of apoptosis; (3) during selenium treatment, a ROS/RNS increase occurred despite the GSH increment, as revealed by fluorimetric analysis; (4) the RNS production could be blocked by a peroxynitrite scavenger; (5) after exposure to selenium compounds, the concentration of nitric oxide synthase, manganese superoxide dismutase (SOD2), P-NF-kB (phospho nuclear factor kB), glutathione reductase and glutathione peroxidase increased, whereas that of P-ERK (phospho extracellular signal-regulated kinase) decreased; (6) selenium presence induced copper/zinc superoxide dismutase (SOD1) translocation into mitochondria, in a way similar to what is observed in amyotrophic lateral sclerosis (ALS). This study supports epidemiologic studies showing the possibility that excess environmental exposure to Se represents a risk factor for a devastating human neurodegenerative disease.

Doxorubicin induces early lipid peroxidation associated with changes in glucose transport in cultured cardiomyocytes

Doxorubicin (DOX) has not only chronic, but also acute toxic effects in the heart, ascribed to the generation of reactive oxygen species (ROS). Focusing on the DOX-induced early biochemical changes in rat cardiomyocytes, we demonstrated that lipid peroxidation is an early event, in fact conjugated diene production increased after 1-h DOX exposure, while cell damage, evaluated as lactate dehydrogenase (LDH) release, was observed only later, when at least one third of the cell antioxidant defences were consumed. Cell pre-treatment with alpha-tocopherol (TC) inhibited both conjugated diene production and LDH release. In cardiomyocytes, DOX treatment caused a maximal increase in glucose uptake at 1 h, demonstrating that glucose transport may represent an early target for DOX. At longer times, as the cell damage become significant, the glucose uptake stimulation diminished. Immunoblotting of glucose transporter isoform GLUT1 in membranes after 1-h DOX exposure revealed an increase in GLUT1 amount similar to the increase in transport activity; both effects were inhibited by alpha TC. Early lipid peroxidation evokes an adaptive response resulting in an increased glucose uptake, presumably to restore cellular energy. The regulation of nutrient transport mechanisms in cardiomyocytes may be considered an early event in the development of the cardiotoxic effects of the anthracycline.

The Mechanism of Iron (III) Stimulation of Lipid Peroxidation

A study conducted on Fe2+ autoxidation showed that its rate was extremely slow at acidic pH values and increased by increasing the pH; it was stimulated by Fe3+ addition but the stimulation did not present a maximum at a Fe2+/Fe3+ ratio approaching 1:1. The species generated during Fe(3+)-catalyzed Fe2+ autoxidation was able to oxidize deoxyribose; the increased Fe2+ oxidation observed at higher pHs was paralleled by increased deoxyribose degradation. The species generated during Fe(3+)-catalyzed Fe2+ autoxidation could not initiate lipid peroxidation in phosphatidylcholine liposomes from which lipid hydroperoxides (LOOH) had been removed by treatment with triphenylphosphine. Neither Fe2+ oxidation nor changes in the oxidation index of the liposomes due to lipid peroxidation were observed at pHs where the Fe3+ effect on Fe2+ autoxidation and on deoxyribose degradation was evident. In our experimental system, a Fe2+/Fe3+ ratio ranging from 1:3 to 2:1 was unable to initiate lipid peroxidation in LOOH-free phosphatidylcholine liposomes. By contrast Fe3+ stimulated the peroxidation of liposomes where increasing amounts of cumene hydroperoxide were incorporated. These results argue against the participation of Fe3+ in the initiation of LOOH-independent lipid peroxidation and suggest its possible involvement in LOOH-dependent lipid peroxidation.

Iron (III) Stimulation of Lipid Hydroperoxide-Dependent Lipid Peroxidation

In an experimental system where both Fe2+ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl2 and FeCl3 on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe2+ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl2/FeCl3 ratio when PC liposomes deprived of LOOH by triphenylphosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl2. The FeCl2 concentration at which Fe2+ oxidation was maximal, defined as critical Fe2+ concentration [Fe2+]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl3 addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe2+]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl3 addition. The experimental data obtained indicate the kinetics of LOOH-dependent lipid peroxidation depends on the Fe2+/Fe3+ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl3 does not affect the LOOH-independent but the LOOH-dependent lipid peroxidation; and suggest that the Fe3+ endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.

NAD(P)H oxidase isoform Nox2 plays a prosurvival role in human leukaemia cells.

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.

Acute regulation of glucose transport in a human megakaryocytic cell line: Difference between growth factors and H2O2

The present study was undertaken to: (i) compare the effect of some hematopoietic growth factors, like interleukine-3, thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, stem cell factor, and reactive oxygen species such as H(2)O(2) on glucose uptake in a human leukemic megakaryocytic cell line, M07; (ii) investigate the changes in kinetic parameters of the transport activity induced by these stimuli; and (iii) evaluate the effect of genistein, a tyrosine kinase inhibitor, on the glucose uptake activation by the cited agents. The results are as follows: (i) exposure of M07 cells to thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, and stem cell factor resulted in a rapid stimulation of glucose transport; interleukine-3-treated cells exhibited no increase in the rate of glucose uptake, although M07 proliferation is interleukine-3 dependent; a rapid glucose transport enhancement was also observed when M07 cells were exposed to low doses of H(2)O(2); (ii) the transport kinetic parameters point out that an important difference exists between the effect of cytokines and that of H(2)O(2): cytokines increased predominantly the affinity for glucose, while H(2)O(2) raised both the V(max) and K(m) values; (iii) the isoflavone genistein, at a very low concentration, inhibited the stem cell factor- or H(2)O(2)-induced stimulation of hexose transport, reversing the variations of K(m) and V(max), but it did not affect the transport activity of granulocyte-megakaryocyte colony-stimulating factor-treated cells; and (iv) catalase completely abolished the stimulatory action of H(2)O(2) on glucose transport and slightly prevented the effect of stem cell factor, while caffeic acid phenethyl ester was only able to affect the activation due to stem cell factor.

Nox-generated ROS modulate glucose uptake in a leukaemic cell line.

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.

Effect of Plasma Membrane Cholesterol Depletion on Glucose Transport Regulation in Leukemia Cells

GLUT1 is the predominant glucose transporter in leukemia cells, and the modulation of glucose transport activity by cytokines, oncogenes or metabolic stresses is essential for their survival and proliferation. However, the molecular mechanisms allowing to control GLUT1 trafficking and degradation are still under debate. In this study we investigated whether plasma membrane cholesterol depletion plays a role in glucose transport activity in M07e cells, a human megakaryocytic leukemia line. To this purpose, the effect of cholesterol depletion by methyl-β-cyclodextrin (MBCD) on both GLUT1 activity and trafficking was compared to that of the cytokine Stem Cell Factor (SCF). Results show that, like SCF, MBCD led to an increased glucose transport rate and caused a subcellular redistribution of GLUT1, recruiting intracellular transporter molecules to the plasma membrane. Due to the role of caveolae/lipid rafts in GLUT1 stimulation in response to many stimuli, we have also investigated the GLUT1 distribution along the fractions obtained after non ionic detergent treatment and density gradient centrifugation, which was only slightly changed upon MBCD treatment. The data suggest that MBCD exerts its action via a cholesterol-dependent mechanism that ultimately results in augmented GLUT1 translocation. Moreover, cholesterol depletion triggers GLUT1 translocation without the involvement of c-kit signalling pathway, in fact MBCD effect does not involve Akt and PLCγ phosphorylation. These data, together with the observation that the combined MBCD/SCF cell treatment caused an additive effect on glucose uptake, suggest that the action of SCF and MBCD may proceed through two distinct mechanisms, the former following a signalling pathway, and the latter possibly involving a novel cholesterol dependent mechanism.

Induction of apoptosis in a human leukemic cell line via reactive oxygen species modulation by antioxidants.

In the human acute myeloid leukemia cell line M07e, the growth factor interleukin-3 (IL-3) induces ROS formation, positively affecting Glut1-mediated glucose uptake and cell survival. The effect of IL-3 and exogenous hydrogen peroxide on cell viability seems to be mediated through inhibition of the cell death commitment, as shown by apoptotic markers such as caspase activities, apoptotic nuclei, and changes in the amount of proteins belonging to the Bcl-2 family. The pivotal role of ROS is confirmed using various antioxidants, such as EUK-134, ebselen, TEMPO, and hydroxylamine probe. In fact, these antioxidants, acting through different mechanisms, decrease glucose transport activity and cell proliferation activated by IL-3 or by low concentrations of hydrogen peroxide. Moreover, antioxidants foster programmed cell death commitment, as shown by the cited apoptotic parameters. EUK-134, a combined superoxide dismutase/catalase mimetic, opposes the effects of IL-3 and H(2)O(2), decreasing phosphorylation levels of signaling enzymes such as Akt, Src tyrosine kinase, and ERK. Results show that ROS production induced by IL-3 can protect leukemic cells from apoptosis, the effect being counteracted by antioxidants. This mechanism may play an important role in supporting acute myeloid leukemia treatment, thus representing a novel therapeutic strategy.

Effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylmethoxy coumarin) on cyclic nucleotide phosphodiesterases in human platelets

The effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylmethoxy coumarin), an inhibitor of platelet aggregation, on cyclic nucleotide metabolism was investigated. AD6 inhibited selectively human platelet cyclic GMP phosphodiesterase, which was separated from cyclic AMP phosphodiesterase by DEAE-cellulose chromatography. Addition of AD6 to washed platelets increased cyclic GMP levels significantly in two minutes. These results could be useful in elucidating the action of AD6 on intact platelets.