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Novel role of sirtuin related mechanisms in overload-induced hypertrophy of skeletal muscle in rat
C. Nyakas / Free Radical Biology and Medicine 96 (2016) S21–S31
O-04
The antileishmanial activity of xanthohumol but not of resveratrol is mediated by mitochondrial inhibition Lars Gille 1, Alexandra Lackova 1, Katrin Staniek 1, Silvia Steinbauer 1, Gerald Pichler 1, Walter Jäger 2, Lianet Monzote 3 1
Institute of Pharmacology and Toxicology, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria 2 Division of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria 3 Parasitology Department, Institute of Tropical Medicine “Pedro Kouri”, Havana, Cuba
Resveratrol (Res) and xanthohumol (Xan) are naturally occurring antioxidants and constituents of human nutrition. Besides effects on mammalian cells also their effect on mammalian pathogens, such as protozoa, is of interest. Leishmania are eukaryotic protozoal organisms which infect humans and other mammals, threatening millions of people in (sub-) tropical regions. The treatment of leishmaniasis is difficult due to toxicity and resistance development for current drugs. For Res and Xan little is known about their actions on leishmanial parasites and their mitochondria as putative drug target. Therefore, we compared the antileishmanial activity of Res and Xan and their modes of action with respect to mitochondria. Antileishmanial activities of xenobiotics were assessed in Leishmania amazonensis amastigotes (LaA) and compared to peritoneal macrophages from mouse (PMM) using viability assays. Furthermore, mechanistic studies regarding mitochondrial functions were conducted in L. tarentolae promastigotes (LtP) and mitochondrial fractions isolated from LtP. Assays of individual mitochondrial complex activities, membrane potential and superoxide radical formation were performed by photometry, fluorimetry and electron spin resonance spectroscopy, respectively. Xan and Res demonstrated antileishmanial activity in LaA (IC50: Xan 6.9 7 0.3 mM, Res 13.9 7 1.7 mM) while they had less influence on the viability of PMM (IC50: Xan 69.5 7 9.8 mM, Res 4 438 mM). In contrast to Res, Xan strongly inhibited oxygen consumption in LtP. Further studies demonstrated that this was based on the inhibition of the mitochondrial electron transfer complex II/III by Xan. This effect was not observed with Res. However, both Xan and Res resulted in mitochondrial uncoupling. Neither Xan nor Res increased mitochondrial superoxide release in LtP. These data show that the antioxidants Res and Xan may have antileishmanial activity, however, only in case of Xan mitochondrial inhibition is involved in the mechanism. http://dx.doi.org/10.1016/j.freeradbiomed.2016.04.171 O-05
Generation of oxidative stress as an anticancer therapeutic strategy Tiantian He 1,2, Elie Hatem 1,2, Laurence Vernis 1,2, Meng-Er Huang 1,2 1
Centre National de la Recherche Scientifique, UMR3348 "Genotoxic Stress and Cancer", Orsay, France 2 Institut Curie, Centre de Recherche, Orsay, France
Aim: Intracellular reactive oxygen species (ROS) and reductive/oxidative (redox) homeostasis play a key role in regulating cell survival/
S23
death. Among the key players of redox system, peroxiredoxin 1 (PRX1) is a major antioxidant and signaling regulator. PRX1 is frequently over-expressed in various cancer cells, which is thought to be associated with carcinogenesis, metastasis and drug resistance. We have investigated whether and how modulations of intracellular redox system through PRX1 knockdown affect cancer cell sensitivity to a set of chemotherapy drugs or ROS-generating molecules. Materials and Methods: Cancer cell lines with PRX1 gene transiently or stably knocked-down and corresponding controls were exposed to a set of anticancer molecules, including vinblastine, taxol, doxorubicin, daunorubicin, actinomycin D, 5-fluorouracil, βlapachone (β-lap) and vitamin K3 (vitK3). Cytotoxic effects were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay, cell clonogenic assay, annexin V/propidium iodide double staining. Global ROS accumulation and compartment-specific H2O2 generation were determined respectively by redox-sensitive chemical probes and H2O2-sensitive sensor HyPer. Oxidation of endogenous antioxidant proteins including TRX1, TRX2 and PRX3 was monitored by redox western blot. Results: PRX1 knockdown significantly and specifically enhances several cancer cell lines’ sensitivity to β-lap and vitK3, two structurally related quinone molecules. Interestingly, β-lap triggers NADPH:quinone oxidoreductase (NQO1)-dependent ROS generation and activation of mitogen-activated protein kinase pathway leading to cell death. On the other hand, PRX1 knockdown up-regulates NRH:quinone oxidoreductase 2 (NQO2) activity which is largely responsible for vitK3-induced ROS accumulation and cell death. Conclusion: Our data provide evidence that PRX1 could be an interesting anticancer target for modulating intracellular redox environment. More generally, these data support the view that redox modulation and enhanced generation of oxidative stress could be an alternative anticancer strategy to enhance cancer cell sensitivity to ROS-generating drugs or to overcome some types of drug resistance. http://dx.doi.org/10.1016/j.freeradbiomed.2016.04.172 O-06
Novel role of sirtuin related mechanisms in overloadinduced hypertrophy of skeletal muscle in rat Erika Koltai 1, Zoltán Bori 1, Clovis Chaber 2, Hervé Dubouchaud 2, Tomoko Funakoshi 3, Shuichi Machida 3, Zsolt Radák 1 1
University of Physical Education, Research Institute of Sport Sciences, Budapest, Hungary 2 Université Joseph Fourier, Laboratoire de Bioénergétique Fondamentale et Appliquée, Grenoble, France 3 Department of Exercise Physiology, Graduate School of Health and Sports Science & Medicine, Juntendo University, Tokyo, Japan
Introduction: Skeletal muscle is a dynamic organ, which can change its metabolic rate by manyfold during intensive contractions. The multi-nucleated myocytes as a result of resistance training can readily increase their size, indeed, the role of satellite cells in this process is still contradictory. The major member of sirtuin protein family, SIRT1 has been implicated as one of the key regulator in cell metabolism, but its involvement in muscle hypertrophy has not been investigated. SIRT1 has been shown to deacetylate eNOS, FOXO1 and activate Akt (Lee and Goldberg, 2013). Therefore, we suggested that SIRT1 can be a potential regulator of prosynthetic
S24
C. Nyakas / Free Radical Biology and Medicine 96 (2016) S21–S31
and catabolic processes during compensatory hypertrophy of skeletal muscle. Methods: Middle aged (15 months) male Wistar rats were used in the study and assigned to control and hypertrophy (N ¼7) groups. Overload of the plantaris muscle was performed bilaterally via removal of their major synergistic muscles, the gastrocnemius and soleus, as described previously. The surgical procedure was performed under deep anesthesia. The overload period lasted for 2 weeks. Plantaris muscle samples frozen in liquid nitrogen were analyzed by immunohistochemistry, ELISA, western blot and fluorescence technic, and the differences between groups were calculated by t-test (p o0.05). Results: Fourteen days of overloading resulted more than 30% increase in the mass of plantaris muscle (po0.01) compared to control group. Histochemical evaluation of satellite cell marker, Pax7 proteins showed significantly elevated Pax7 levels in hypertrophy group. The markers of muscle differentiation, MyoD and PCNA levels were significantly higher in hypertrophy group compared to control. The level of NAD dependent SIRT1 increased by hypertrophy and the NAMPT levels showed similar changing pattern. p53 is deacetylated by SIRT1 and the level of acp53 decreased in hypertrophy group. Contrary, the eNOS and Akt levels elevated significantly by muscle hypertrophy, while the FOXO1 protein concentration decreased by this treatment. The changes in the levels of ubiquitin ligase and protein ubiquitination suggest, that during hypertrophy ubiquitin mediated signaling pathway also activated. Discussion: Our data suggest that novel regulatory role of SIRT1 in overload-induce hypertrophy of skeletal muscle. The rate of NAD þ biosynthesis and the SIRT1 activity - judged by acp53 level - further support the activation of SIRT1-related pathways during hypertrophy. SIRT1 modulates prosynthetic pathways through eNOS, Akt activation and down-regulation of catabolic processes by inhibiting FOXO1 and proteasome’s protein degradation. References: Lee, D., Goldberg, A.L., 2013. SIRT1 protein, by blocking the activities of transcription factors FoxO1 and FoxO3, inhibits muscle atrophy and promotes muscle growth. J Biol Chem 288, 3051530526.
progression of aging, disease or decomposition of bio matter. In this study we investigate the mechanisms of protein modification of lysozyme, glucose-6-phosphate dehydrogenase and ribonuclease A, subjected to damage by either peroxyl radicals from 2,2'-azobis(2amidinopropane) (AAPH) in the presence of O2, or singlet oxygen generated by Rose Bengal in presence of O2 and light. Aims: To investigate the role of aromatic amino acids as target for protein oxidation; To identify key intermediates in protein crosslinking and scission; To explore factors directing the ongoing reactions and outcomes. Results: The chosen proteins undergo different reaction pathways on oxidation resulting in intermolecular cross-linking (RNAse A), intramolecular cross-linking (glucose-6-phosphate dehydrogenase) or fragmentation (lysozyme). In each case, main oxidation targets were methionine and the aromatic amino acids histidine (His), tyrosine (Tyr) and tryptophan (Trp). Oxidized amino acids including di-tyrosine and N-formylkynurenine (NFK) were detected by Western blotting and UHPLC. In addition, the proteins were treated with trypsin in either 16O- or 18 O-water and were analyzed by mass spectrometry. Protein-derived peptides cross-linked by Tyr-Tyr, TyrTrp, and a novel Tyr-Lys linkage have been identified. Sequence and structure appear to be important factors in determining the major reaction pathways. Two tyrosine residues located on different chains are found in touching distance in the X-ray structure of RNAse A. Glucose-6-phosphate dehydrogenase has several spatially close Tyr and Trp residues. We hypothesize that fragmentation of lysozyme is favored by a unique spatial arrangement of Tyr, Trp and the scission site on the backbone between an asparagine and glycine residue. While oxidation of small peptides and amino acids is well-described, less is known how different mechanisms rank during the oxidation of intact proteins. Our findings indicate that reaction at Tyr and Trp residues appear to be of particular relevance, as these are among the most readily oxidized amino acids and have been shown to engage in fundamental processes such as cross-linking and scission. This provides clues for understanding formation and accumulation of oxidation products in biological samples.
http://dx.doi.org/10.1016/j.freeradbiomed.2016.04.173 O-07
Cross-linking and scission initiated by protein oxidation – evidence for involvement of tyrosine and tryptophan residues Fabian Leinisch 1, Michele Mariotti 2, Per Hägglund 2, Michael J. Davies 1 1
Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark 2 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark Background: Protein oxidation can occur at varied reaction sites by numerous mechanisms, each causing specific modifications to the protein. This alters the properties of the protein and can manifest in
Figure 1: Glucose-6-phosphate dehydrogenase oxidation has been shown, by mass spectroscopy, to result in cross-linking via Tyr450Tyr460 and Trp202- Tyr206. http://dx.doi.org/10.1016/j.freeradbiomed.2016.04.174
O-04
The antileishmanial activity of xanthohumol but not of resveratrol is mediated by mitochondrial inhibition Lars Gille 1, Alexandra Lackova 1, Katrin Staniek 1, Silvia Steinbauer 1, Gerald Pichler 1, Walter Jäger 2, Lianet Monzote 3 1
Institute of Pharmacology and Toxicology, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria 2 Division of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria 3 Parasitology Department, Institute of Tropical Medicine “Pedro Kouri”, Havana, Cuba
Resveratrol (Res) and xanthohumol (Xan) are naturally occurring antioxidants and constituents of human nutrition. Besides effects on mammalian cells also their effect on mammalian pathogens, such as protozoa, is of interest. Leishmania are eukaryotic protozoal organisms which infect humans and other mammals, threatening millions of people in (sub-) tropical regions. The treatment of leishmaniasis is difficult due to toxicity and resistance development for current drugs. For Res and Xan little is known about their actions on leishmanial parasites and their mitochondria as putative drug target. Therefore, we compared the antileishmanial activity of Res and Xan and their modes of action with respect to mitochondria. Antileishmanial activities of xenobiotics were assessed in Leishmania amazonensis amastigotes (LaA) and compared to peritoneal macrophages from mouse (PMM) using viability assays. Furthermore, mechanistic studies regarding mitochondrial functions were conducted in L. tarentolae promastigotes (LtP) and mitochondrial fractions isolated from LtP. Assays of individual mitochondrial complex activities, membrane potential and superoxide radical formation were performed by photometry, fluorimetry and electron spin resonance spectroscopy, respectively. Xan and Res demonstrated antileishmanial activity in LaA (IC50: Xan 6.9 7 0.3 mM, Res 13.9 7 1.7 mM) while they had less influence on the viability of PMM (IC50: Xan 69.5 7 9.8 mM, Res 4 438 mM). In contrast to Res, Xan strongly inhibited oxygen consumption in LtP. Further studies demonstrated that this was based on the inhibition of the mitochondrial electron transfer complex II/III by Xan. This effect was not observed with Res. However, both Xan and Res resulted in mitochondrial uncoupling. Neither Xan nor Res increased mitochondrial superoxide release in LtP. These data show that the antioxidants Res and Xan may have antileishmanial activity, however, only in case of Xan mitochondrial inhibition is involved in the mechanism. http://dx.doi.org/10.1016/j.freeradbiomed.2016.04.171 O-05
Generation of oxidative stress as an anticancer therapeutic strategy Tiantian He 1,2, Elie Hatem 1,2, Laurence Vernis 1,2, Meng-Er Huang 1,2 1
Centre National de la Recherche Scientifique, UMR3348 "Genotoxic Stress and Cancer", Orsay, France 2 Institut Curie, Centre de Recherche, Orsay, France
Aim: Intracellular reactive oxygen species (ROS) and reductive/oxidative (redox) homeostasis play a key role in regulating cell survival/
S23
death. Among the key players of redox system, peroxiredoxin 1 (PRX1) is a major antioxidant and signaling regulator. PRX1 is frequently over-expressed in various cancer cells, which is thought to be associated with carcinogenesis, metastasis and drug resistance. We have investigated whether and how modulations of intracellular redox system through PRX1 knockdown affect cancer cell sensitivity to a set of chemotherapy drugs or ROS-generating molecules. Materials and Methods: Cancer cell lines with PRX1 gene transiently or stably knocked-down and corresponding controls were exposed to a set of anticancer molecules, including vinblastine, taxol, doxorubicin, daunorubicin, actinomycin D, 5-fluorouracil, βlapachone (β-lap) and vitamin K3 (vitK3). Cytotoxic effects were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay, cell clonogenic assay, annexin V/propidium iodide double staining. Global ROS accumulation and compartment-specific H2O2 generation were determined respectively by redox-sensitive chemical probes and H2O2-sensitive sensor HyPer. Oxidation of endogenous antioxidant proteins including TRX1, TRX2 and PRX3 was monitored by redox western blot. Results: PRX1 knockdown significantly and specifically enhances several cancer cell lines’ sensitivity to β-lap and vitK3, two structurally related quinone molecules. Interestingly, β-lap triggers NADPH:quinone oxidoreductase (NQO1)-dependent ROS generation and activation of mitogen-activated protein kinase pathway leading to cell death. On the other hand, PRX1 knockdown up-regulates NRH:quinone oxidoreductase 2 (NQO2) activity which is largely responsible for vitK3-induced ROS accumulation and cell death. Conclusion: Our data provide evidence that PRX1 could be an interesting anticancer target for modulating intracellular redox environment. More generally, these data support the view that redox modulation and enhanced generation of oxidative stress could be an alternative anticancer strategy to enhance cancer cell sensitivity to ROS-generating drugs or to overcome some types of drug resistance. http://dx.doi.org/10.1016/j.freeradbiomed.2016.04.172 O-06
Novel role of sirtuin related mechanisms in overloadinduced hypertrophy of skeletal muscle in rat Erika Koltai 1, Zoltán Bori 1, Clovis Chaber 2, Hervé Dubouchaud 2, Tomoko Funakoshi 3, Shuichi Machida 3, Zsolt Radák 1 1
University of Physical Education, Research Institute of Sport Sciences, Budapest, Hungary 2 Université Joseph Fourier, Laboratoire de Bioénergétique Fondamentale et Appliquée, Grenoble, France 3 Department of Exercise Physiology, Graduate School of Health and Sports Science & Medicine, Juntendo University, Tokyo, Japan
Introduction: Skeletal muscle is a dynamic organ, which can change its metabolic rate by manyfold during intensive contractions. The multi-nucleated myocytes as a result of resistance training can readily increase their size, indeed, the role of satellite cells in this process is still contradictory. The major member of sirtuin protein family, SIRT1 has been implicated as one of the key regulator in cell metabolism, but its involvement in muscle hypertrophy has not been investigated. SIRT1 has been shown to deacetylate eNOS, FOXO1 and activate Akt (Lee and Goldberg, 2013). Therefore, we suggested that SIRT1 can be a potential regulator of prosynthetic
S24
C. Nyakas / Free Radical Biology and Medicine 96 (2016) S21–S31
and catabolic processes during compensatory hypertrophy of skeletal muscle. Methods: Middle aged (15 months) male Wistar rats were used in the study and assigned to control and hypertrophy (N ¼7) groups. Overload of the plantaris muscle was performed bilaterally via removal of their major synergistic muscles, the gastrocnemius and soleus, as described previously. The surgical procedure was performed under deep anesthesia. The overload period lasted for 2 weeks. Plantaris muscle samples frozen in liquid nitrogen were analyzed by immunohistochemistry, ELISA, western blot and fluorescence technic, and the differences between groups were calculated by t-test (p o0.05). Results: Fourteen days of overloading resulted more than 30% increase in the mass of plantaris muscle (po0.01) compared to control group. Histochemical evaluation of satellite cell marker, Pax7 proteins showed significantly elevated Pax7 levels in hypertrophy group. The markers of muscle differentiation, MyoD and PCNA levels were significantly higher in hypertrophy group compared to control. The level of NAD dependent SIRT1 increased by hypertrophy and the NAMPT levels showed similar changing pattern. p53 is deacetylated by SIRT1 and the level of acp53 decreased in hypertrophy group. Contrary, the eNOS and Akt levels elevated significantly by muscle hypertrophy, while the FOXO1 protein concentration decreased by this treatment. The changes in the levels of ubiquitin ligase and protein ubiquitination suggest, that during hypertrophy ubiquitin mediated signaling pathway also activated. Discussion: Our data suggest that novel regulatory role of SIRT1 in overload-induce hypertrophy of skeletal muscle. The rate of NAD þ biosynthesis and the SIRT1 activity - judged by acp53 level - further support the activation of SIRT1-related pathways during hypertrophy. SIRT1 modulates prosynthetic pathways through eNOS, Akt activation and down-regulation of catabolic processes by inhibiting FOXO1 and proteasome’s protein degradation. References: Lee, D., Goldberg, A.L., 2013. SIRT1 protein, by blocking the activities of transcription factors FoxO1 and FoxO3, inhibits muscle atrophy and promotes muscle growth. J Biol Chem 288, 3051530526.
progression of aging, disease or decomposition of bio matter. In this study we investigate the mechanisms of protein modification of lysozyme, glucose-6-phosphate dehydrogenase and ribonuclease A, subjected to damage by either peroxyl radicals from 2,2'-azobis(2amidinopropane) (AAPH) in the presence of O2, or singlet oxygen generated by Rose Bengal in presence of O2 and light. Aims: To investigate the role of aromatic amino acids as target for protein oxidation; To identify key intermediates in protein crosslinking and scission; To explore factors directing the ongoing reactions and outcomes. Results: The chosen proteins undergo different reaction pathways on oxidation resulting in intermolecular cross-linking (RNAse A), intramolecular cross-linking (glucose-6-phosphate dehydrogenase) or fragmentation (lysozyme). In each case, main oxidation targets were methionine and the aromatic amino acids histidine (His), tyrosine (Tyr) and tryptophan (Trp). Oxidized amino acids including di-tyrosine and N-formylkynurenine (NFK) were detected by Western blotting and UHPLC. In addition, the proteins were treated with trypsin in either 16O- or 18 O-water and were analyzed by mass spectrometry. Protein-derived peptides cross-linked by Tyr-Tyr, TyrTrp, and a novel Tyr-Lys linkage have been identified. Sequence and structure appear to be important factors in determining the major reaction pathways. Two tyrosine residues located on different chains are found in touching distance in the X-ray structure of RNAse A. Glucose-6-phosphate dehydrogenase has several spatially close Tyr and Trp residues. We hypothesize that fragmentation of lysozyme is favored by a unique spatial arrangement of Tyr, Trp and the scission site on the backbone between an asparagine and glycine residue. While oxidation of small peptides and amino acids is well-described, less is known how different mechanisms rank during the oxidation of intact proteins. Our findings indicate that reaction at Tyr and Trp residues appear to be of particular relevance, as these are among the most readily oxidized amino acids and have been shown to engage in fundamental processes such as cross-linking and scission. This provides clues for understanding formation and accumulation of oxidation products in biological samples.
http://dx.doi.org/10.1016/j.freeradbiomed.2016.04.173 O-07
Cross-linking and scission initiated by protein oxidation – evidence for involvement of tyrosine and tryptophan residues Fabian Leinisch 1, Michele Mariotti 2, Per Hägglund 2, Michael J. Davies 1 1
Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark 2 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark Background: Protein oxidation can occur at varied reaction sites by numerous mechanisms, each causing specific modifications to the protein. This alters the properties of the protein and can manifest in
Figure 1: Glucose-6-phosphate dehydrogenase oxidation has been shown, by mass spectroscopy, to result in cross-linking via Tyr450Tyr460 and Trp202- Tyr206. http://dx.doi.org/10.1016/j.freeradbiomed.2016.04.174