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Correlation between Mitochondrial Morphology and Functionality after Oxidative Stress
470a
Tuesday, March 1, 2016
polymers confer distinct mechanical and biological properties to single bacteria and to mature biofilms. When individual bacteria attach to a surface, intracellular levels of cyclic-diGMP increase. Cyclic-di-GMP is required to change gene expression to initiate the transition to the biofilm state. What specific cues control cyclic-di-GMP production were previously unknown - we show that this is controlled by mechanical shear stress, which is primarily impacted by bacterial motility and the EPS coating on bacteria. This opens up the possibility of making a new class of anti-biofilm surface, by using a 2D fluid that cannot sustain a lateral shear stress and thereby preventing activation of the cyclic-di-GMP signal. P. aeruginosa biofilm infections in the cystic fibrosis (CF) lung often last for decades, ample time for the infecting strain(s) to evolve. Production of alginate is well-known to tend to increase during CF infections. More recently, it is becoming recognized that CF infections also evolve to increase PSL production. Alginate chemically protects biofilms, but also makes them softer and weaker. Here, we show that PSL stiffens and strengthens biofilms, and that increased PSL production in biofilms grown from CF clinical isolates completely rescues the mechanical weakening caused by alginate.
Mitochondrial Cell Life and Death 2315-Pos Board B459 Dark Hypericin Affects Several Sub-Cellular Levels Katarina Stroffekova1, Veronika Huntosova2, Marta Novotova3, Zuzana Nichtova3, Tibor Kozar2, Pavol Miskovsky4. 1 Biophysics, PJ Safarik University, Kosice, Slovakia, 2Center of Interdisciplinary Biosciences, PJ Safarik University, Kosice, Slovakia, 3 Muscle Cell Research, Institute of Molecular Physiology and Genetics SAV, Bratislava, Slovakia, 4Biophysics and Center of Interdisciplinary Biosciences, PJ Safarik University, Kosice, Slovakia. Photosensitizers (PSs) in photodynamic therapy (PDT) are administered systemically with preferential accumulation in malignant cells; however, exposure of non-malignant cells to PS may be clinically relevant if PS molecules affect the pro-apoptotic cascade without illumination. The important PS characteristic is low dark cytotoxicity. Originally, Hypericin (Hyp) as PS displayed minimal dark cytotoxicity and preferential accumulation in tumor cells, however recently, evidence to contrary appeared. The molecular mechanisms underlying Hyp dark toxicity may be due to its interaction with different molecules at the Hyp accumulation sites including mitochondria, and they are not understood in detail. Our previous study demonstrated that in human glioma U87 MG cells, Hyp affected localization of pro-apoptotic Bax and Bak, from Bcl2 family of proteins that are key regulators of apoptosis and mitogenesis. To understand the mechanisms underlying Hyp dark toxicity better, we investigated the Hyp effect on mitochondrial function and cell metabolism in U87 MG and human coronary artery endothelial (HCAEC) cells. We have found that Hyp in HCAEC displayed significant dark cytotoxicity in contrast to U87 MG cells. Further, Hyp significantly affected mitochondria function in U87 MG by turning them towards glycolysis, but it did not have this effect in HCAEC. In addition, in both cell types Hyp in the dark triggered morphological changes in mitochondria and the ER and GA ultrastructure indicative of altered protein synthesis. Lastly, in U87 MG and HCAEC Hyp affected the distribution of Bcl2 and Bax proteins via hydrophobic interaction at their BH1-BH3 domain. Overall, our results indicate that Hyp in the dark had effects at several sub-cellular levels similar to small mitochondria targeting molecules (mitocans), and thus Hyp should be explored as mitocan. Support: EU 7FP PIRG06-GA-2009256580, CELIM 316310; EU SF ITMS 26240120040; VEGA -1-0111-12, APVV-0134-11 and APVV-0242-11 2316-Pos Board B460 Correlation between Mitochondrial Morphology and Functionality after Oxidative Stress Zuzana Nadova1, Lenka Lenkavska2, Alexandra Fragola3, Stephanie Bonneau4, Franck Sureau4, Pavol Miskovsky1. 1 Dept. of Biophysics, Center for Interdisciplinary Biosciences, University P.J.Safarik, Faculty of Science, Kosice, Slovakia, 2Dept. of Biophysics, University P.J.Safarik, Faculty of Science, Kosice, Slovakia, 3aboratoire Physique et Etude de Mate´riaux, Universite´ Pierre et Marie Curie, Paris, France, 4Laboratoire Jean Perrin, Universite Pierre et Marie Curie, Paris, France. Mitochondria play a key role in the energy metabolism of cells, in the process of oxidative stress development as well as in the program of cell death progression. Redox imbalances lead to oxidative stress, changes in mitochondrial membrane potential, affects permeability of the membranes and leads to release of proapoptotic proteins from mitochondrial intermembrane space. Inner organization of mitochondria appears as a key of the accurate operating of the
organelle IMM alterations are often associated with structure disorganization and mitochondrial dysfunction, and consequently in numerous pathologies. We investigated the effect of reactive species on shape and structure of mitochondria in Hela cells. ROS production was induced by photoactivated hypericin. Inner structure of mitochondria was investigated by 3D structuredillumination microscopy. By using confocal microscopy and flow cytometry we shown, that increased light dose leads to significant increase of ROS, affects mitochondrial organization and shape a finally leads to cell death. Acknowledgement: This work is supported by 7FP EU CELIM 316310, EU Structural Fund ITMS 26240120040 and APVV-0242-11. 2317-Pos Board B461 Oxidative Stress and JNK Activation cause Mitochondrial Dysfunction and Cell Death in Hepatocarcinoma after VDAC-Tubulin Antagonists Eduardo Maldonado1, David N. DeHart1, Diana Fang1, Kareem Heslop1, Monika Beck Gooz1, John Lemasters1,2. 1 Pharmaceutical & Biomedical Sciences, Medical University of South Carolina, Charleston, SC, USA, 2IInstitute of Theoretical & Experimental Biophysics, Pushchino, Russian Federation. Background: Flux of metabolites into mitochondria sustains membrane potential (DJ) and reactive oxygen species (ROS) formation. Free a,b-tubulin dimers close VDAC in vitro and high free tubulin in cancer cells closes VDAC. The small molecule erastin antagonizes the inhibition of tubulin on VDAC. Here, we hypothesized that VDAC-tubulin antagonists (VTA) open VDAC, increase mitochondrial metabolism, decrease glycolysis and activate c-jun N-terminal kinase (JNK), culminating in mitochondrial dysfunction and death of hepatocarcinoma (HCC) cells. Our AIM was to evaluate the effects of VTAs on mitochondrial DJ, NAD(P)Hþ and ROS, lactate generation, JNK activation and cell killing in HepG2 and Huh7 HCC cells. Methods: Confocal/multiphoton fluorescence microscopy assessed DJ (tetramethylrhodamine methylester), ROS (chloromethyldichlorofluorescein [cmDCF]; MitoSOX Red) and NAD(P)H (autofluorescence). JNK was assessed by Western blotting and cell killing by propidium iodide fluorometry. Results: Erastin and small molecules X1-2 identified in a high-throughput screen increased DJ, NAD(P)H and cmDCF and MitoSOX fluorescence. The mitochondrial antioxidant MitoQ blocked ROS increases. Mitochondrial hyperpolarization was followed by depolarization. Additionally, erastin and X1 activated JNK. JNK inhibitors (JNK VIII and SP600125, 30 mM) blocked X1-induced hyperpolarization/depolarization and decreased ROS formation. Lactate generation after X1 decreased by 60%. Both X1 and X2 killed cancer cells (~93% and ~76% respectively), which the antioxidant N-acetylcysteine (100 mM) blocked and JNKVIII and SP600125 decreased. By contrast, X1-2 caused <25% cell death in primary rat hepatocytes. X1 (5 mg/kg, i.p. daily) decreased tumor growth in a Huh7 xenograft model. Conclusion: AVTs are anti-Warburg compounds that promote mitochondrial metabolism, inhibit glycolysis and cause mitochondrial generation of ROS, which in turn leads to JNK activation, mitochondrial dysfunction and selective death of cancer cells that is prevented by antioxidants and JNK inhibitors. 2318-Pos Board B462 Study of the NIR Light Induced Effects on Neuroblastoma N2A Cells with Parkinson’s-Like Features Lenka Koptasikova1, Veronika Huntosova2, Emmanuel Gerelli3, Pavol Miskovsky2, Georges Wagnieres3, Katarina Stroffekova1. 1 Department of Biophysics, PJ Safarik University in Kosice, Kosice, Slovakia, 2Center for Interdisciplinary Biosciences, PJ Safarik University in Kosice, Kosice, Slovakia, 3Laboratory of Organometallic and Medicinal Chemistry, Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology in Lausanne, Lausanne, Switzerland. Parkinson’s disease is an incurable neurodegenerative disorder associated with losses of dopaminergic neurones. The underlying causes of this loss are unknown, but photobiomodulation (PBM) with near-infrared (NIR) light may be a promising approach to slow down or even stop the neurodegenerative process. NIR has optimal penetration in biological tissues and can thus provide a minimally-invasive treatment. However, the mechanisms underlying the PBM effects, including an increase in ATP synthesis, modulation of reactive oxygen species, and induction of transcription factors are not fully understood. The aim of our study was to examine PBM effects on neuroblastoma N2A cells. We focused our study on the cell proliferation, mitochondria function and cell metabolism. We induced Parkinson’s-like features in N2A cells by rotenone treatment. We then compared the PBM effects, induced by an illumination at 808 nm, in intact N2A cells, treated or not with rotenone. Overall, we observed a significant stimulation of cell proliferation in control and rotenone-treated cells after PBM. This effect was accompanied by an increased oxygen consumption rate by mitochondria and increased mitochondrial membrane
Tuesday, March 1, 2016
polymers confer distinct mechanical and biological properties to single bacteria and to mature biofilms. When individual bacteria attach to a surface, intracellular levels of cyclic-diGMP increase. Cyclic-di-GMP is required to change gene expression to initiate the transition to the biofilm state. What specific cues control cyclic-di-GMP production were previously unknown - we show that this is controlled by mechanical shear stress, which is primarily impacted by bacterial motility and the EPS coating on bacteria. This opens up the possibility of making a new class of anti-biofilm surface, by using a 2D fluid that cannot sustain a lateral shear stress and thereby preventing activation of the cyclic-di-GMP signal. P. aeruginosa biofilm infections in the cystic fibrosis (CF) lung often last for decades, ample time for the infecting strain(s) to evolve. Production of alginate is well-known to tend to increase during CF infections. More recently, it is becoming recognized that CF infections also evolve to increase PSL production. Alginate chemically protects biofilms, but also makes them softer and weaker. Here, we show that PSL stiffens and strengthens biofilms, and that increased PSL production in biofilms grown from CF clinical isolates completely rescues the mechanical weakening caused by alginate.
Mitochondrial Cell Life and Death 2315-Pos Board B459 Dark Hypericin Affects Several Sub-Cellular Levels Katarina Stroffekova1, Veronika Huntosova2, Marta Novotova3, Zuzana Nichtova3, Tibor Kozar2, Pavol Miskovsky4. 1 Biophysics, PJ Safarik University, Kosice, Slovakia, 2Center of Interdisciplinary Biosciences, PJ Safarik University, Kosice, Slovakia, 3 Muscle Cell Research, Institute of Molecular Physiology and Genetics SAV, Bratislava, Slovakia, 4Biophysics and Center of Interdisciplinary Biosciences, PJ Safarik University, Kosice, Slovakia. Photosensitizers (PSs) in photodynamic therapy (PDT) are administered systemically with preferential accumulation in malignant cells; however, exposure of non-malignant cells to PS may be clinically relevant if PS molecules affect the pro-apoptotic cascade without illumination. The important PS characteristic is low dark cytotoxicity. Originally, Hypericin (Hyp) as PS displayed minimal dark cytotoxicity and preferential accumulation in tumor cells, however recently, evidence to contrary appeared. The molecular mechanisms underlying Hyp dark toxicity may be due to its interaction with different molecules at the Hyp accumulation sites including mitochondria, and they are not understood in detail. Our previous study demonstrated that in human glioma U87 MG cells, Hyp affected localization of pro-apoptotic Bax and Bak, from Bcl2 family of proteins that are key regulators of apoptosis and mitogenesis. To understand the mechanisms underlying Hyp dark toxicity better, we investigated the Hyp effect on mitochondrial function and cell metabolism in U87 MG and human coronary artery endothelial (HCAEC) cells. We have found that Hyp in HCAEC displayed significant dark cytotoxicity in contrast to U87 MG cells. Further, Hyp significantly affected mitochondria function in U87 MG by turning them towards glycolysis, but it did not have this effect in HCAEC. In addition, in both cell types Hyp in the dark triggered morphological changes in mitochondria and the ER and GA ultrastructure indicative of altered protein synthesis. Lastly, in U87 MG and HCAEC Hyp affected the distribution of Bcl2 and Bax proteins via hydrophobic interaction at their BH1-BH3 domain. Overall, our results indicate that Hyp in the dark had effects at several sub-cellular levels similar to small mitochondria targeting molecules (mitocans), and thus Hyp should be explored as mitocan. Support: EU 7FP PIRG06-GA-2009256580, CELIM 316310; EU SF ITMS 26240120040; VEGA -1-0111-12, APVV-0134-11 and APVV-0242-11 2316-Pos Board B460 Correlation between Mitochondrial Morphology and Functionality after Oxidative Stress Zuzana Nadova1, Lenka Lenkavska2, Alexandra Fragola3, Stephanie Bonneau4, Franck Sureau4, Pavol Miskovsky1. 1 Dept. of Biophysics, Center for Interdisciplinary Biosciences, University P.J.Safarik, Faculty of Science, Kosice, Slovakia, 2Dept. of Biophysics, University P.J.Safarik, Faculty of Science, Kosice, Slovakia, 3aboratoire Physique et Etude de Mate´riaux, Universite´ Pierre et Marie Curie, Paris, France, 4Laboratoire Jean Perrin, Universite Pierre et Marie Curie, Paris, France. Mitochondria play a key role in the energy metabolism of cells, in the process of oxidative stress development as well as in the program of cell death progression. Redox imbalances lead to oxidative stress, changes in mitochondrial membrane potential, affects permeability of the membranes and leads to release of proapoptotic proteins from mitochondrial intermembrane space. Inner organization of mitochondria appears as a key of the accurate operating of the
organelle IMM alterations are often associated with structure disorganization and mitochondrial dysfunction, and consequently in numerous pathologies. We investigated the effect of reactive species on shape and structure of mitochondria in Hela cells. ROS production was induced by photoactivated hypericin. Inner structure of mitochondria was investigated by 3D structuredillumination microscopy. By using confocal microscopy and flow cytometry we shown, that increased light dose leads to significant increase of ROS, affects mitochondrial organization and shape a finally leads to cell death. Acknowledgement: This work is supported by 7FP EU CELIM 316310, EU Structural Fund ITMS 26240120040 and APVV-0242-11. 2317-Pos Board B461 Oxidative Stress and JNK Activation cause Mitochondrial Dysfunction and Cell Death in Hepatocarcinoma after VDAC-Tubulin Antagonists Eduardo Maldonado1, David N. DeHart1, Diana Fang1, Kareem Heslop1, Monika Beck Gooz1, John Lemasters1,2. 1 Pharmaceutical & Biomedical Sciences, Medical University of South Carolina, Charleston, SC, USA, 2IInstitute of Theoretical & Experimental Biophysics, Pushchino, Russian Federation. Background: Flux of metabolites into mitochondria sustains membrane potential (DJ) and reactive oxygen species (ROS) formation. Free a,b-tubulin dimers close VDAC in vitro and high free tubulin in cancer cells closes VDAC. The small molecule erastin antagonizes the inhibition of tubulin on VDAC. Here, we hypothesized that VDAC-tubulin antagonists (VTA) open VDAC, increase mitochondrial metabolism, decrease glycolysis and activate c-jun N-terminal kinase (JNK), culminating in mitochondrial dysfunction and death of hepatocarcinoma (HCC) cells. Our AIM was to evaluate the effects of VTAs on mitochondrial DJ, NAD(P)Hþ and ROS, lactate generation, JNK activation and cell killing in HepG2 and Huh7 HCC cells. Methods: Confocal/multiphoton fluorescence microscopy assessed DJ (tetramethylrhodamine methylester), ROS (chloromethyldichlorofluorescein [cmDCF]; MitoSOX Red) and NAD(P)H (autofluorescence). JNK was assessed by Western blotting and cell killing by propidium iodide fluorometry. Results: Erastin and small molecules X1-2 identified in a high-throughput screen increased DJ, NAD(P)H and cmDCF and MitoSOX fluorescence. The mitochondrial antioxidant MitoQ blocked ROS increases. Mitochondrial hyperpolarization was followed by depolarization. Additionally, erastin and X1 activated JNK. JNK inhibitors (JNK VIII and SP600125, 30 mM) blocked X1-induced hyperpolarization/depolarization and decreased ROS formation. Lactate generation after X1 decreased by 60%. Both X1 and X2 killed cancer cells (~93% and ~76% respectively), which the antioxidant N-acetylcysteine (100 mM) blocked and JNKVIII and SP600125 decreased. By contrast, X1-2 caused <25% cell death in primary rat hepatocytes. X1 (5 mg/kg, i.p. daily) decreased tumor growth in a Huh7 xenograft model. Conclusion: AVTs are anti-Warburg compounds that promote mitochondrial metabolism, inhibit glycolysis and cause mitochondrial generation of ROS, which in turn leads to JNK activation, mitochondrial dysfunction and selective death of cancer cells that is prevented by antioxidants and JNK inhibitors. 2318-Pos Board B462 Study of the NIR Light Induced Effects on Neuroblastoma N2A Cells with Parkinson’s-Like Features Lenka Koptasikova1, Veronika Huntosova2, Emmanuel Gerelli3, Pavol Miskovsky2, Georges Wagnieres3, Katarina Stroffekova1. 1 Department of Biophysics, PJ Safarik University in Kosice, Kosice, Slovakia, 2Center for Interdisciplinary Biosciences, PJ Safarik University in Kosice, Kosice, Slovakia, 3Laboratory of Organometallic and Medicinal Chemistry, Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology in Lausanne, Lausanne, Switzerland. Parkinson’s disease is an incurable neurodegenerative disorder associated with losses of dopaminergic neurones. The underlying causes of this loss are unknown, but photobiomodulation (PBM) with near-infrared (NIR) light may be a promising approach to slow down or even stop the neurodegenerative process. NIR has optimal penetration in biological tissues and can thus provide a minimally-invasive treatment. However, the mechanisms underlying the PBM effects, including an increase in ATP synthesis, modulation of reactive oxygen species, and induction of transcription factors are not fully understood. The aim of our study was to examine PBM effects on neuroblastoma N2A cells. We focused our study on the cell proliferation, mitochondria function and cell metabolism. We induced Parkinson’s-like features in N2A cells by rotenone treatment. We then compared the PBM effects, induced by an illumination at 808 nm, in intact N2A cells, treated or not with rotenone. Overall, we observed a significant stimulation of cell proliferation in control and rotenone-treated cells after PBM. This effect was accompanied by an increased oxygen consumption rate by mitochondria and increased mitochondrial membrane