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Allen J Bard - One of the best experts on this subject based on the ideXlab platform.
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inhibition of the mrp1 mediated transport of the Menadione glutathione conjugate thiodione in hela cells as studied by secm
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Dipankar Koley, Allen J BardAbstract:Oxidative stress induced in live HeLa cells by Menadione (2-methyl-1,4-napthaquinone) was studied in real time by scanning electrochemical microscopy (SECM). The hydrophobic molecule Menadione diffuses through a living cell membrane where it is toxic to the cell. However, in the cell it is conjugated with glutathione to form thiodione. Thiodione is then recognized and transported across the cell membrane via the ATP-driven MRP1 pump. In the extracellular environment, thiodione was detected by the SECM tip at levels of 140, 70, and 35 µM upon exposure of the cells to Menadione concentrations of 500, 250, and 125 µM, respectively. With the aid of finite element modeling, the kinetics of thiodione transport was determined to be 1.6 × 10-7 m/s, about 10 times faster than Menadione uptake. Selective inhibition of these MRP1 pumps inside live HeLa cells by MK571 produced a lower thiodione concentration of 50 µM in presence of 500 µM Menadione and 50 µM MK571. A similar reduced (50% drop) thiodione efflux was observed in the presence of monoclonal antibody QCRL-4, a selective blocking agent of the MRP1 pumps. The reduced thiodione flux confirmed that thiodione was transported by MRP1, and that glutathione is an essential substrate for MRP1-mediated transport. This finding demonstrates the usefulness of SECM in quantitative studies of MRP1 inhibitors and suggests that monoclonal antibodies can be a useful tool in inhibiting the transport of these MDR pumps, and thereby aiding in overcoming multidrug resistance.
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scanning electrochemical microscopy of Menadione glutathione conjugate export from yeast cells
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Janine Mauzeroll, Allen J BardAbstract:The uptake of Menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between Menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV–visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a Menadione-containing solution, the export of the conjugate from the cells with time could be measured. Similar experiments were performed on immobilized yeast cell aggregates stressed by a Menadione solution. From the export of the Menadione-glutathione conjugate detected at a 1-μm-diameter electrode situated 10 μm from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted. Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of Menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster.
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cyclic voltammetric and scanning electrochemical microscopic study of Menadione permeability through a self assembled monolayer on a gold electrode
Langmuir, 2002Co-Authors: Celine Cannes, Frederic Kanoufi, Allen J BardAbstract:Menadione (2-methyl-1,4-naphthoquinone) reduction and menadiol oxidation at octadecanethiol (C18SH) monolayer modified gold electrodes were investigated by cyclic voltammetry (CV) and scanning electrochemical microscopy (SECM). The modified electrode acts as a better barrier toward the ferrocyanide transport than toward the Menadione species. This difference is attributed to permeation of the organic substrates into the hydrophobic monolayer. A simple model is proposed and applied to extract the rate constant of the kinetically limiting permeation step from the cyclic voltammograms. However, a better estimate of the transport properties is obtained by SECM. The same trends are observed with CV and SECM, and a similar pH dependence shows the loss of an intermediate formed during Menadione reduction from the monolayer with increasing pH. This loss can probably be assigned to the rapid expulsion of the more hydrophilic reduced species from the monolayer.
Julien Verrax - One of the best experts on this subject based on the ideXlab platform.
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role of glycolysis inhibition and poly adp ribose polymerase activation in necrotic like cell death caused by ascorbate Menadione induced oxidative stress in k562 human chronic myelogenous leukemic cells
International Journal of Cancer, 2007Co-Authors: Julien Verrax, Julie Stockis, Henryk Taper, Stephanie Vanbever, Pedro Buc CalderonAbstract:Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of Menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the Menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/Menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/Menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/Menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells.
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oxidative stress by ascorbate Menadione association kills k562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice
Biochemical Pharmacology, 2006Co-Authors: Julien Verrax, Julie Stockis, Aurelie Tison, Henryk Taper, Pedro Buc CalderonAbstract:The effect of oxidative stress induced by the ascorbate/Menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances Menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/Menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H(2)O(2) as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/Menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/Menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and Menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/Menadione association in cancer therapy.
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ascorbate potentiates the cytotoxicity of Menadione leading to an oxidative stress that kills cancer cells by a non apoptotic caspase 3 independent form of cell death
Apoptosis, 2004Co-Authors: Julien Verrax, Henryk Taper, Julie Cadrobbi, Carole Marques, Yvette Habraken, Jacques Piette, Pedro Buck CalderonAbstract:Hepatocarcinoma cells (TLT) were incubated in the presence of ascorbate and Menadione, either alone or in combination. Cell death was only observed when such compounds were added simultaneously, most probably due to hydrogen peroxide (H2O2) generated by ascorbate-driven Menadione redox cycling. TLT cells were particularly sensitive to such an oxidative stress due to its poor antioxidant status. DNA strand breaks were induced by this association but this process did not correspond to oligosomal DNA fragmentation (a hallmark of cell death by apoptosis). Neither caspase-3-like DEVDase activity, nor processing of procaspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) were observed in the presence of ascorbate and Menadione. Cell death induced by such an association was actively dependent on protein phosphorylation since it was totally prevented by preincubating cells with sodium orthovanadate, a tyrosine phosphatase inhibitor. Finally, while H2O2, when administered as a bolus, strongly enhances a constitutive basal NF-kappaB activity in TLT cells, their incubation in the presence of ascorbate and Menadione results in a total abolition of such a constitutive activity.
Pedro Buc Calderon - One of the best experts on this subject based on the ideXlab platform.
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role of glycolysis inhibition and poly adp ribose polymerase activation in necrotic like cell death caused by ascorbate Menadione induced oxidative stress in k562 human chronic myelogenous leukemic cells
International Journal of Cancer, 2007Co-Authors: Julien Verrax, Julie Stockis, Henryk Taper, Stephanie Vanbever, Pedro Buc CalderonAbstract:Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of Menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the Menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/Menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/Menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/Menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells.
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oxidative stress by ascorbate Menadione association kills k562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice
Biochemical Pharmacology, 2006Co-Authors: Julien Verrax, Julie Stockis, Aurelie Tison, Henryk Taper, Pedro Buc CalderonAbstract:The effect of oxidative stress induced by the ascorbate/Menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances Menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/Menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H(2)O(2) as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/Menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/Menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and Menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/Menadione association in cancer therapy.
Oleg Vsevolodovich Gerasimenko - One of the best experts on this subject based on the ideXlab platform.
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calcium elevation in mitochondria is the main ca2 requirement for mitochondrial permeability transition pore mptp opening
Journal of Biological Chemistry, 2009Co-Authors: Heidi K Baumgartner, Julia Vladimirovna Gerasimenko, Christopher Thorne, Pawel E Ferdek, Tullio Pozzan, Alexei V Tepikin, O H Petersen, Robert Sutton, Alastair J M Watson, Oleg Vsevolodovich GerasimenkoAbstract:We have investigated in detail the role of intra-organelle Ca2+ content during induction of apoptosis by the oxidant Menadione while changing and monitoring the Ca2+ load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, Menadione was found to induce repetitive cytosolic Ca2+ responses because of the release of Ca2+ from both ER and acidic stores. Ca2+ responses to Menadione were accompanied by elevation of Ca2+ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca2+ stores did not necessarily prevent Menadione-induced apoptosis. High mitochondrial Ca2+ at the time of Menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca2+ with 10 μm RU360, then caspase-9 activation did not occur irrespective of the content of other Ca2+ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca2+ with pericam. We conclude that elevated Ca2+ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.
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Menadione induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells
Journal of Biological Chemistry, 2006Co-Authors: Dabid N Criddle, Stuart Gillies, Heidi K Baumgartnerwilson, Mohammed Jaffar, E Chinje, Sarah Passmore, Michael Chvanov, Stephanie L Barrow, Oleg Vsevolodovich GerasimenkoAbstract:Oxidative stress may be an important determinant of the severity of acute pancreatitis. One-electron reduction of oxidants generates reactive oxygen species (ROS) via redox cycling, whereas two-electron detoxification, e.g. by NAD(P)H:quinone oxidoreductase, does not. The actions of Menadione on ROS production and cell fate were compared with those of a non-cycling analogue (2,4-dimethoxy-2-methylnaphthalene (DMN)) using real-time confocal microscopy of isolated perfused murine pancreatic acinar cells. Menadione generated ROS with a concomitant decrease of NAD(P)H, consistent with redox cycling. The elevation of ROS was prevented by the antioxidant N-acetyl-l-cysteine but not by the NADPH oxidase inhibitor diphenyliodonium. DMN produced no change in reactive oxygen species per se but significantly potentiated Menadione-induced effects, probably via enhancement of one-electron reduction, since DMN was found to inhibit NAD(P)H:quinone oxidoreductase detoxification. Menadione caused apoptosis of pancreatic acinar cells that was significantly potentiated by DMN, whereas DMN alone had no effect. Furthermore, bile acid (taurolithocholic acid 3-sulfate)-induced caspase activation was also greatly increased by DMN, whereas DMN had no effect per se. These results suggest that acute generation of ROS by Menadione occurs via redox cycling, the net effect of which is induction of apoptotic pancreatic acinar cell death. Two-electron detoxifying enzymes such as NAD(P)H:quinone oxidoreductase, which are elevated in pancreatitis, may provide protection against excessive ROS and exert an important role in determining acinar cell fate.
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Menadione induced apoptosis roles of cytosolic ca2 elevations and the mitochondrial permeability transition pore
Journal of Cell Science, 2002Co-Authors: Julia Vladimirovna Gerasimenko, Alexei V Tepikin, O H Petersen, Oleg Vsevolodovich Gerasimenko, Altaf Palejwala, Alistair J M WatsonAbstract:In normal pancreatic acinar cells, the oxidant Menadione evokes repetitive cytosolic Ca(2+) spikes, partial mitochondrial depolarisation, cytochrome c release and apoptosis. The physiological agonists acetylcholine and cholecystokinin also evoke cytosolic Ca(2+) spikes but do not depolarise mitochondria and fail to induce apoptosis. Ca(2+) spikes induced by low agonist concentrations are confined to the apical secretory pole of the cell by the buffering action of perigranular mitochondria. Menadione prevents mitochondrial Ca(2+) uptake, which permits rapid spread of Ca(2+) throughout the cell. Menadione-induced mitochondrial depolarisation is due to induction of the permeability transition pore. Blockade of the permeability transition pore with bongkrekic acid prevents activation of caspase 9 and 3. In contrast, the combination of antimycin A and acetylcholine does not cause apoptosis but elicits a global cytosolic Ca(2+) rise and mitochondrial depolarisation without induction of the permeability transition pore. Increasing the cytosolic Ca(2+) buffering power by BAPTA prevents cytosolic Ca(2+) spiking, blocks the Menadione-elicited mitochondrial depolarisation and blocks Menadione-induced apoptosis. These results suggest a twin-track model in which both intracellular release of Ca(2+) and induction of the permeability transition pore are required for initiation of apoptosis.
Henryk Taper - One of the best experts on this subject based on the ideXlab platform.
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role of glycolysis inhibition and poly adp ribose polymerase activation in necrotic like cell death caused by ascorbate Menadione induced oxidative stress in k562 human chronic myelogenous leukemic cells
International Journal of Cancer, 2007Co-Authors: Julien Verrax, Julie Stockis, Henryk Taper, Stephanie Vanbever, Pedro Buc CalderonAbstract:Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of Menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the Menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/Menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/Menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/Menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells.
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oxidative stress by ascorbate Menadione association kills k562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice
Biochemical Pharmacology, 2006Co-Authors: Julien Verrax, Julie Stockis, Aurelie Tison, Henryk Taper, Pedro Buc CalderonAbstract:The effect of oxidative stress induced by the ascorbate/Menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances Menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/Menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H(2)O(2) as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/Menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/Menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and Menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/Menadione association in cancer therapy.
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ascorbate potentiates the cytotoxicity of Menadione leading to an oxidative stress that kills cancer cells by a non apoptotic caspase 3 independent form of cell death
Apoptosis, 2004Co-Authors: Julien Verrax, Henryk Taper, Julie Cadrobbi, Carole Marques, Yvette Habraken, Jacques Piette, Pedro Buck CalderonAbstract:Hepatocarcinoma cells (TLT) were incubated in the presence of ascorbate and Menadione, either alone or in combination. Cell death was only observed when such compounds were added simultaneously, most probably due to hydrogen peroxide (H2O2) generated by ascorbate-driven Menadione redox cycling. TLT cells were particularly sensitive to such an oxidative stress due to its poor antioxidant status. DNA strand breaks were induced by this association but this process did not correspond to oligosomal DNA fragmentation (a hallmark of cell death by apoptosis). Neither caspase-3-like DEVDase activity, nor processing of procaspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) were observed in the presence of ascorbate and Menadione. Cell death induced by such an association was actively dependent on protein phosphorylation since it was totally prevented by preincubating cells with sodium orthovanadate, a tyrosine phosphatase inhibitor. Finally, while H2O2, when administered as a bolus, strongly enhances a constitutive basal NF-kappaB activity in TLT cells, their incubation in the presence of ascorbate and Menadione results in a total abolition of such a constitutive activity.