- Email: [email protected]
Insight the mechanism of ferroptosis inhibition by ferrostatin-1
S120
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
2
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
Medicinal plants have been a prolific source of new bioactive compounds with anticancer activity. Parvifloron D (ParvD) is an abietane diterpenoid isolated from Plectranthus ecklonii acetonic maceration extract. ParvD was previously shown to have a strong reducing capacity (DPPH) and to protect DNA from oxidative breakage (plasmid strand break analysis). In addition, ParvD has shown cytotoxic and pro-apoptotic effects in leukemia and melanoma models. Herein, the anticancer effect of ParvD was evaluated in MDA-MB-231 human breast cancer cells. ParvD (0.1–10 μM) decreased cell viability in a concentration-dependent manner. Treatment with 5 μM ParvD significantly increased the percentage of apoptotic nuclei. Cell exposure to 3 μM ParvD increased the sub-G1 population. Treatment with ParvD (1 μM) had no effect on cell-substrate attachment when cell detachment was induced with EDTA. The effect of ParvD on cell chemotaxis and invasion was evaluated by a transwell assay. ParvD (1 μM) significantly reduced cell migration and invasion, which are determinant processes for the formation of metastases. In summary, the redox-active natural compound ParvD showed interesting anticancer properties and should be further studied towards a potential therapeutic application.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.394
P-248
Coenzyme Q10 or creatine counteract pravastatininduced liver redox changes in hypercholesterolemic mice Ana Carolina Marques, Estela Natasha Brandt Busanello, Helena Coutinho Franco Oliveira, Anibal Eugenio Vercesi Universidade Estadual de Campinas - UNCAMP, São Paulo, Brasil
Statins are the preferred therapy to treat hypercholesterolemia and decrease coronary heart disease. However, previous studies have reported mitochondrial dysfunctions of several experimental models submitted to diverse statins treatments. The aim of the present study was to investigate whether treatment with doses of pravastatin during 3 months induces hepatotoxicity in LDL receptor knockout mice (LDLr-/-), a model for human familial hypercholesterolemia. We evaluated liver mitochondrial function parameters, antioxidant enzymes activities and protein and lipid oxidation markers. We observed a higher mitochondrial H2O2 production rate, decreased activity of aconitase and increased MPT. Among several antioxidant enzymes, only G6PD activity was increased in treated mice. The presence of several oxidized lipid species was detected in pravastatin group but protein oxidation markers were not altered. Diet supplementation with the antioxidants CoQ10 or creatine fully reversed all pravastatin effects. Taken together, our results show that pravastatin chronic treatment induced liver mitochondrial redox imbalance that may explain the hepatic side effects reported in a small number of patients, and the co-treatment with safe antioxidants neutralize these side effects.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.395
P-249
Protein sulphenic acid formation during CD4 þ T cell activation Ali Hussein Remtulla 1, Irundika H.K. Dias 1, Ivana Milic 1, Andrew Devitt 1, Helen R. Griffiths 1,2 1 2
Aston University, Life & Health Sciences, Birmingham, UK University of Surrey, Health and Medical Sciences, Guildford, UK
T cell receptor signalling requires a reduced cell surface protein environment and increased reactive oxygen species (ROS) inhibit activation. The main objective of this study was to identify sulphenated proteins on the membrane of T cells at rest, during activation and under oxidising conditions. Naïve CD4 þ T cells were activated using anti-CD3/CD28 antibodies in the presence or absence H2O2 (20 mM) for 24 h. Activated CD4 þ T cells in the presence of H2O2 show a significant decrease in CD25 expression by twofold and IL-2 secretion (0.6770.14ng/ml versus 1.16 70.09ng/ml). NAC (10 mM) did not reverse the effect of H2O2 on IL-2 or CD25 after 24 h. CD4 þ T cell-membrane proteins were isolated using MEM-PR kit after T cell activation with or without H2O2 and sulphenated proteins were captured by a dimedone based technique. Captured proteins were identified by LC-MS/MS and the table below shows the number of sulphenated proteins; Activation alone did not affect the number of sulphenated membrane proteins. Activation in the presence of H2O2, which associated with lower intracellular calcium flux, show a reduction in number of sulphenated proteins possibly due to disulphide bond formation. Time
Unactivated
Activated
Unactivatedþ H2O2
Activatedþ H2O2
30 min 24 hr
58 45
59 41
63 58
43 48
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.396
P-250
Insight the mechanism of ferroptosis inhibition by ferrostatin-1 Giovanni Miotto, Monica Rossetto, Antonella Roveri, Rina Venerando, Ana-Marija Vučković, Maria Luisa Di Paolo, Valentina Bosello-Travain, Mattia Zaccarin, Matilde Maiorino, Stefano Toppo, Fulvio Ursini, Giorgio Cozza Department of Molecular Medicine, University of Padova, Padova, Italy
Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and hence prevented by GPx4. Ferrostatin-1 (Fer-1) inhibits ferroptosis more efficiently than phenolic antioxidants. Previous studies on the reaction of Fer-1 adopted the kinetic test where a diazo-compound generates the hydroperoxyl radical to be reduced. However, this “chain breaking” effect is not satisfying for ferrous iron dependent peroxidation. New chain reactions, indeed, are primed from hydroperoxides produced by the antioxidant. On liposomes containing traces of lipid hydroperoxides and exposed to ferrous iron we disclosed by oxi-lipidomics the species produced and deduced the pattern of radical reactions. Although
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
Fer-1 inhibits peroxidation, the pattern of oxidized species produced from pre-existing hydroperoxides was practically identical to that observed following exhaustive lipid peroxidation. This supported the notion that the anti-ferroptotic activity of Fer-1 descends from the scavenging of alkoxyl radicals generated by ferrous iron from lipid hydroperoxides. Thereof, Fer-1, in the presence if iron, eliminates lipid hydroperoxides and this produces the same anti-ferroptotic effect as GPx4, although generating a series of oxidized species but not just hydroxy fatty acids derivatives.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.397
P-251
S121
Lipids are targets of reactive nitrogen species (RNS). Nitro-fatty acids (NO2-FA) are well-known products of RNS and have been associated with anti-inflammatory and cytoprotective effects. NO2-FAs play their biological actions mainly by covalent adduction to key peptides/proteins, modulating their roles. Nitroxidation of phospholipids (PL) has been recently detected in biological samples, but their biological effects were not addressed. We hypothesize that nitrated PL may react with peptide/proteins. We performed in vitro biomimetic assays to synthetize adducts of nitrated POPC (NO2-POPC), already detected in biological samples, and GSH. The formation of NO2-POPC-GSH adducts was studied by ESI-MS and MS2, and low and high energy CID using different MS platforms. Typical product ions observed under MS2 conditions are b, y and immonium ions bearing NO2POPC covalently-linked that unequivocally confirmed the presence of the lipid-peptide adduct. In summary, the characterization of nitro PL-peptide adducts by MS and MS2 allowed the identification of specific reporter ions to be used to pinpoint these adductions in biological systems.
E-mail address: [email protected]
Smad4 loss is associated with decreased oxidative metabolism in pancreatic cancer and affects sensitivity to mitochondrial therapy
Acknowledgements Funds from H2020 (Project MASSTRPLAN, Grant number 675132), QOPNA (FCT UID/QUI/00062/2013) & RNEM (LISBOA-01–0145-FEDER402–022125)
Zuzana Ezrova, Lukas Werner, Jan Stursa, Jiri Neuzil, Stepana Boukalova
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.399
Czech Academy of Sciences, Prague, Czech Republic P-253 Pancreatic cancer is one of the most challenging types of tumors to treat. This is likely caused by a population of resistant cancer cells (CS), which are reported to rely on functional mitochondria. We have shown that mitochondrial targeting is an efficient way to eliminate CS. At the same time, we believe that loss of Smad4, a mediator of transforming growth factor 3 (TGF3) pathway, might be the cause of metabolic reprogramming resulting in mitochondrial dysfunction of pancreatic cancer cells (PCC). We demonstrated that respiration of Smad4þ PCC was substantially decreased when TGF3 was applied. By contrast, Smad4-/- PCC as well as Smad4 knocked out (KO) cells showed lower basal respiration level and they were insensitive to TGF3 treatment. Furthermore, we observed significant changes in mitochondrial fragmentation of Smad4 þ PCC after TGF3 treatment, while Smad4 KO PCC remained unaffected. Finally, the Smad4 expression profile correlated with the vulnerability of PCC to mitochondrial-targeted inhibitor of complex I, MitoMet. Our results suggest that loss of Smad4 leads to metabolic reprogramming of PCC, which consequently impacts the sensitivity to mitochondrial-targeted therapy. Based on these findings, we propose a new treatment approach aimed at efficient elimination of PCC.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.398
P-252
Study of covalent adduction of nitrated phospholipids to peptides using tandem mass spectrometry in different instruments
Effects of methanol extract of Moringa oleifera leaves on indomethacin-induced free radical systemic toxicity in rats Ubachukwu Chidiebere Chiemerie, Taiwo Victor Olusegun Department of Veterinary Pathology, University of Ibadan, Ibadan, Nigeria
We investigated the effects of methanol extract of Moringa oleifera leaves (MEMO) against indomethacin (Indo) induced systemic toxicity. Indo is known to cause free radicals-induced blood and tissue pathology. Sixty male Wistar rats, aged 14–16 weeks were randomly divided into 8 groups. Group 1, distilled water; Group 2, MEMO 250 mg/kg; Group 3, MEMO 500 mg/kg; Group 4, Indo 125 mg/kg; Group 5, MEMO 250 mg/ kgþ Indo 125 mg/kg; Group 6, MEMO 500 mg/kgþIndo 125 mg/kg; Group 7, Indo 125 mg/kgþMEMO 250 mg/kg; Group 8, Indo 125 mg/kgþ MEMO 500 mg/kg. After 24 hours, Indo-treated rats showed moderately severe anaemia, neutrophilia, elevated serum levels of ALT, AST, ALP, malondialdehyde, nitric oxide, myeloperoxidase, and decreased serum levels of superoxide dismutase and total glutathione. Histologic lesions in various organs of all Indo-treated rats were those of congestion, haemorrhage, thrombosis, degeneration and necrosis of parenchymal cells. The results of this study show that MEMO is protective against free radical-induced cytotoxic actions of Indo. Follow-up dosing of MEMO however seems to be necessary to sustain the anti-oxidative stress activity.
E-mail address: [email protected] Keywords: Oxidative stress; Free Radicals; Indomethacin; Moringa oleifera; Toxicology
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.400 Javier-Fernando Montero-Bullón, Tânia Melo, M Rosário M Domingues, Pedro Domingues Mass Spectrometry Centre, Department of Chemistry & QOPNA, University of Aveiro, Aveiro, Portugal
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
2
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
Medicinal plants have been a prolific source of new bioactive compounds with anticancer activity. Parvifloron D (ParvD) is an abietane diterpenoid isolated from Plectranthus ecklonii acetonic maceration extract. ParvD was previously shown to have a strong reducing capacity (DPPH) and to protect DNA from oxidative breakage (plasmid strand break analysis). In addition, ParvD has shown cytotoxic and pro-apoptotic effects in leukemia and melanoma models. Herein, the anticancer effect of ParvD was evaluated in MDA-MB-231 human breast cancer cells. ParvD (0.1–10 μM) decreased cell viability in a concentration-dependent manner. Treatment with 5 μM ParvD significantly increased the percentage of apoptotic nuclei. Cell exposure to 3 μM ParvD increased the sub-G1 population. Treatment with ParvD (1 μM) had no effect on cell-substrate attachment when cell detachment was induced with EDTA. The effect of ParvD on cell chemotaxis and invasion was evaluated by a transwell assay. ParvD (1 μM) significantly reduced cell migration and invasion, which are determinant processes for the formation of metastases. In summary, the redox-active natural compound ParvD showed interesting anticancer properties and should be further studied towards a potential therapeutic application.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.394
P-248
Coenzyme Q10 or creatine counteract pravastatininduced liver redox changes in hypercholesterolemic mice Ana Carolina Marques, Estela Natasha Brandt Busanello, Helena Coutinho Franco Oliveira, Anibal Eugenio Vercesi Universidade Estadual de Campinas - UNCAMP, São Paulo, Brasil
Statins are the preferred therapy to treat hypercholesterolemia and decrease coronary heart disease. However, previous studies have reported mitochondrial dysfunctions of several experimental models submitted to diverse statins treatments. The aim of the present study was to investigate whether treatment with doses of pravastatin during 3 months induces hepatotoxicity in LDL receptor knockout mice (LDLr-/-), a model for human familial hypercholesterolemia. We evaluated liver mitochondrial function parameters, antioxidant enzymes activities and protein and lipid oxidation markers. We observed a higher mitochondrial H2O2 production rate, decreased activity of aconitase and increased MPT. Among several antioxidant enzymes, only G6PD activity was increased in treated mice. The presence of several oxidized lipid species was detected in pravastatin group but protein oxidation markers were not altered. Diet supplementation with the antioxidants CoQ10 or creatine fully reversed all pravastatin effects. Taken together, our results show that pravastatin chronic treatment induced liver mitochondrial redox imbalance that may explain the hepatic side effects reported in a small number of patients, and the co-treatment with safe antioxidants neutralize these side effects.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.395
P-249
Protein sulphenic acid formation during CD4 þ T cell activation Ali Hussein Remtulla 1, Irundika H.K. Dias 1, Ivana Milic 1, Andrew Devitt 1, Helen R. Griffiths 1,2 1 2
Aston University, Life & Health Sciences, Birmingham, UK University of Surrey, Health and Medical Sciences, Guildford, UK
T cell receptor signalling requires a reduced cell surface protein environment and increased reactive oxygen species (ROS) inhibit activation. The main objective of this study was to identify sulphenated proteins on the membrane of T cells at rest, during activation and under oxidising conditions. Naïve CD4 þ T cells were activated using anti-CD3/CD28 antibodies in the presence or absence H2O2 (20 mM) for 24 h. Activated CD4 þ T cells in the presence of H2O2 show a significant decrease in CD25 expression by twofold and IL-2 secretion (0.6770.14ng/ml versus 1.16 70.09ng/ml). NAC (10 mM) did not reverse the effect of H2O2 on IL-2 or CD25 after 24 h. CD4 þ T cell-membrane proteins were isolated using MEM-PR kit after T cell activation with or without H2O2 and sulphenated proteins were captured by a dimedone based technique. Captured proteins were identified by LC-MS/MS and the table below shows the number of sulphenated proteins; Activation alone did not affect the number of sulphenated membrane proteins. Activation in the presence of H2O2, which associated with lower intracellular calcium flux, show a reduction in number of sulphenated proteins possibly due to disulphide bond formation. Time
Unactivated
Activated
Unactivatedþ H2O2
Activatedþ H2O2
30 min 24 hr
58 45
59 41
63 58
43 48
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.396
P-250
Insight the mechanism of ferroptosis inhibition by ferrostatin-1 Giovanni Miotto, Monica Rossetto, Antonella Roveri, Rina Venerando, Ana-Marija Vučković, Maria Luisa Di Paolo, Valentina Bosello-Travain, Mattia Zaccarin, Matilde Maiorino, Stefano Toppo, Fulvio Ursini, Giorgio Cozza Department of Molecular Medicine, University of Padova, Padova, Italy
Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and hence prevented by GPx4. Ferrostatin-1 (Fer-1) inhibits ferroptosis more efficiently than phenolic antioxidants. Previous studies on the reaction of Fer-1 adopted the kinetic test where a diazo-compound generates the hydroperoxyl radical to be reduced. However, this “chain breaking” effect is not satisfying for ferrous iron dependent peroxidation. New chain reactions, indeed, are primed from hydroperoxides produced by the antioxidant. On liposomes containing traces of lipid hydroperoxides and exposed to ferrous iron we disclosed by oxi-lipidomics the species produced and deduced the pattern of radical reactions. Although
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
Fer-1 inhibits peroxidation, the pattern of oxidized species produced from pre-existing hydroperoxides was practically identical to that observed following exhaustive lipid peroxidation. This supported the notion that the anti-ferroptotic activity of Fer-1 descends from the scavenging of alkoxyl radicals generated by ferrous iron from lipid hydroperoxides. Thereof, Fer-1, in the presence if iron, eliminates lipid hydroperoxides and this produces the same anti-ferroptotic effect as GPx4, although generating a series of oxidized species but not just hydroxy fatty acids derivatives.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.397
P-251
S121
Lipids are targets of reactive nitrogen species (RNS). Nitro-fatty acids (NO2-FA) are well-known products of RNS and have been associated with anti-inflammatory and cytoprotective effects. NO2-FAs play their biological actions mainly by covalent adduction to key peptides/proteins, modulating their roles. Nitroxidation of phospholipids (PL) has been recently detected in biological samples, but their biological effects were not addressed. We hypothesize that nitrated PL may react with peptide/proteins. We performed in vitro biomimetic assays to synthetize adducts of nitrated POPC (NO2-POPC), already detected in biological samples, and GSH. The formation of NO2-POPC-GSH adducts was studied by ESI-MS and MS2, and low and high energy CID using different MS platforms. Typical product ions observed under MS2 conditions are b, y and immonium ions bearing NO2POPC covalently-linked that unequivocally confirmed the presence of the lipid-peptide adduct. In summary, the characterization of nitro PL-peptide adducts by MS and MS2 allowed the identification of specific reporter ions to be used to pinpoint these adductions in biological systems.
E-mail address: [email protected]
Smad4 loss is associated with decreased oxidative metabolism in pancreatic cancer and affects sensitivity to mitochondrial therapy
Acknowledgements Funds from H2020 (Project MASSTRPLAN, Grant number 675132), QOPNA (FCT UID/QUI/00062/2013) & RNEM (LISBOA-01–0145-FEDER402–022125)
Zuzana Ezrova, Lukas Werner, Jan Stursa, Jiri Neuzil, Stepana Boukalova
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.399
Czech Academy of Sciences, Prague, Czech Republic P-253 Pancreatic cancer is one of the most challenging types of tumors to treat. This is likely caused by a population of resistant cancer cells (CS), which are reported to rely on functional mitochondria. We have shown that mitochondrial targeting is an efficient way to eliminate CS. At the same time, we believe that loss of Smad4, a mediator of transforming growth factor 3 (TGF3) pathway, might be the cause of metabolic reprogramming resulting in mitochondrial dysfunction of pancreatic cancer cells (PCC). We demonstrated that respiration of Smad4þ PCC was substantially decreased when TGF3 was applied. By contrast, Smad4-/- PCC as well as Smad4 knocked out (KO) cells showed lower basal respiration level and they were insensitive to TGF3 treatment. Furthermore, we observed significant changes in mitochondrial fragmentation of Smad4 þ PCC after TGF3 treatment, while Smad4 KO PCC remained unaffected. Finally, the Smad4 expression profile correlated with the vulnerability of PCC to mitochondrial-targeted inhibitor of complex I, MitoMet. Our results suggest that loss of Smad4 leads to metabolic reprogramming of PCC, which consequently impacts the sensitivity to mitochondrial-targeted therapy. Based on these findings, we propose a new treatment approach aimed at efficient elimination of PCC.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.398
P-252
Study of covalent adduction of nitrated phospholipids to peptides using tandem mass spectrometry in different instruments
Effects of methanol extract of Moringa oleifera leaves on indomethacin-induced free radical systemic toxicity in rats Ubachukwu Chidiebere Chiemerie, Taiwo Victor Olusegun Department of Veterinary Pathology, University of Ibadan, Ibadan, Nigeria
We investigated the effects of methanol extract of Moringa oleifera leaves (MEMO) against indomethacin (Indo) induced systemic toxicity. Indo is known to cause free radicals-induced blood and tissue pathology. Sixty male Wistar rats, aged 14–16 weeks were randomly divided into 8 groups. Group 1, distilled water; Group 2, MEMO 250 mg/kg; Group 3, MEMO 500 mg/kg; Group 4, Indo 125 mg/kg; Group 5, MEMO 250 mg/ kgþ Indo 125 mg/kg; Group 6, MEMO 500 mg/kgþIndo 125 mg/kg; Group 7, Indo 125 mg/kgþMEMO 250 mg/kg; Group 8, Indo 125 mg/kgþ MEMO 500 mg/kg. After 24 hours, Indo-treated rats showed moderately severe anaemia, neutrophilia, elevated serum levels of ALT, AST, ALP, malondialdehyde, nitric oxide, myeloperoxidase, and decreased serum levels of superoxide dismutase and total glutathione. Histologic lesions in various organs of all Indo-treated rats were those of congestion, haemorrhage, thrombosis, degeneration and necrosis of parenchymal cells. The results of this study show that MEMO is protective against free radical-induced cytotoxic actions of Indo. Follow-up dosing of MEMO however seems to be necessary to sustain the anti-oxidative stress activity.
E-mail address: [email protected] Keywords: Oxidative stress; Free Radicals; Indomethacin; Moringa oleifera; Toxicology
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.400 Javier-Fernando Montero-Bullón, Tânia Melo, M Rosário M Domingues, Pedro Domingues Mass Spectrometry Centre, Department of Chemistry & QOPNA, University of Aveiro, Aveiro, Portugal