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The Effect of Nitric Oxide on Mitochondrial Metabolism and Proliferation in Breast Cancer Cells
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Juan Du , Malvika Rawal1, Justin C Moser1, Kristen E Olney1, Garry R Buettner1, and Joseph J Cullen1,2 1 2 The University of Iowa, VA Hospital, Iowa Pharmacological ascorbate has been shown to induce toxicity in a various cancer cell lines both in vitro and in vivo. Pharmacological concentrations of ascorbate produce H2O2 via the formation of ascorbate radical (Asc•−). The rate of ascorbate oxidation is principally a function of the level of catalytically active iron and copper in solution. For example, catalytic iron in cell culture media contributes significantly to the rate of H2O2 generation. Deferoxamine (DFO) is an iron-chelator that renders iron catalytically inactive with respect to ascorbate oxidation. The addition of the iron-chelators DFO and dipyridyl (DPD) to DMEM high glucose media in the presence of ascorbate (2 mM) reversed the ascorbate-induced flux of H2O2 and reversed ascorbateinduced toxicity. In addition, increasing intracellular iron concentration by pre-incubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate-induced cytotoxicity as demonstrated by decreased clonogenic survival in pancreatic cancer cells. Furthermore, ascorbate (10 mM) treatment also increased the amount of transition metal irons in cells and tumor tissues. This study suggests that iron plays an important role in ascorbateinduced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the efficacy of pharmacological ascorbate. (Supported by VA Merit grant, NIH grants CA166800 CA137230)
doi:10.1016/j.freeradbiomed.2012.10.107
79 5HGXFWLYH&DUER[\ODWLRQ0D\$IIHFW5HGR[6WDWHRI &DQFHU&HOOV 1
Ales Dvorak , Katarina Smolkova1, Jaroslav Zelenka1, Libor Vitek2, and Petr Jezek1 1 2 ASCR, Prague, Czech Republic, Charles University of Prague, Prague, Czech Republic Reductive carboxylation (RC, reverse Krebs cycle reaction of 2oxoglutarate) is indispensable metabolic pathway which plays an important role in viability of cancer cells. RC is supplemented by anaplerotic pathways - largely due to glutaminolysis. Glutamine is transformed by transaminases to 2-oxoglutarate which can be further converted by mitochondrial isocitrate dehydrogenase 2 (IDH2) to isocitrate or to oncometabolite 2-hydroxyglutarate (2HG) with concomitant NADPH consumption. Isocitrate can be transformed to citrate by the reversed aconitase and citrate is exported from the matrix to the cytosol. This RC glutaminolysis may help highly malignant tumors to survive. 2-HG is formed only by IDH2 with a R172K point mutation (1) and cannot be further metabolized. Both reactions may affect reactive oxygen species (ROS) production and antioxidant protection of glutathione because of NADPH consumption. Thus we have studied redox balance, ROS production and reduced/oxidized glutathione ration (GSH/GSSG) in cancer cell lines HTB-126, HepG2 and SHSY5Y and in cells where RC was inhibited by IDH2 silencing. We have measured incorporation of 13C from glutamine to metabolites of 13 RC by GC-MS, namely C citrate, malate and 2-HG, for all also upon hypoxia and presence of respiratory inhibitors. In hypoxia, this incorporation was much higher as compared to normoxia. 13 Oligomycin increased incorporation of C into citrate, malate and 2-HG, however, FCCP decreased it. It supports hypothesis that RC is reciprocally dependent on OXPHOS activity. RC
S42
semiquantification was taken as differences to measurements in cells with IDH2 silencing or respiratory inhibitors. Also mitochondrial superoxide production and GSH/GSSG were quantified in relation to IDH2 expression and activity. Amount of GSSG was decreased in cell lines with IDH2 silencing. We have investigated activity of IDH2 in matrix which affects redox changes that subsequently influence redox balance, and activities of redox-sensitive mitochondrial enzymes. 1. Ward P.S., et.al., Cancer Cell 17, 225-234, 2010 Supported by Grant Agency of Charles University No. 426411
doi:10.1016/j.freeradbiomed.2012.10.108
80 7KH(IIHFWRI1LWULF2[LGHRQ0LWRFKRQGULDO 0HWDEROLVPDQG3UROLIHUDWLRQLQ%UHDVW&DQFHU&HOOV 1,2
John M Egner , Anne R Dier2, Shiqui Li2, Ching-Fang Chang2, and Neil Hogg2 1 2 Carthage College, WI, Medical College of Wisconsin The mitochondrion is emerging as a key regulator of cancer cell growth because of their critical role in producing energy and biosynthetic intermediates required for rapid replication and proliferation. Nitric oxide (NO) is known to control mitochondrial oxidative phosphorylation through its direct chemical interactions with mitochondrial proteins (e.g. cytochrome c oxidase) and activation of multiple signaling pathways. Despite the wellcharacterized anti-proliferative effects of NO in several cancer types, the direct link between NO-dependent control of mitochondrial function and subsequent changes in cell growth has not been established. We hypothesize that NO controls proliferative responses of breast cancer cells through downregulation of mitochondrial function. Growth curve analysis of four breast cancer cell lines (MCF7, T-47D, MB231, and MB468) demonstrated a decrease in cell proliferation with exposure to increasing concentration of the NO donor DetaNONOate (Deta/NO). Further studies in MCF7 cells investigated the effects of Deta/NO on cellular bioenergetics using the Seahorse Bioscience XF24 Analyzer. Deta/NO caused a time- and concentration-dependent decrease in multiple mitochondrial function parameters and a concomitant stimulation of the extracellular acidification rate, an indicator of glycolysis. Changes in mitochondrial function were accompanied by decreased expression of cytochrome c oxidase subunits I and Vb with no change in ATP synthase Į subunit levels. Loss of cytochrome c oxidase subunit expression was not reversed 24 h after Deta/NO exposure. Taken together, these data indicate that NO decreases mitochondrial function, in part through regulating levels of cytochrome c oxidase, which may be linked to its anti-proliferative effects in breast cancer cells. These findings also provide important insight into the biological effects of NO and its potential role in therapeutic targeting of metabolism in cancer.
doi:10.1016/j.freeradbiomed.2012.10.109
81 1LWUDWHG+VS 5HJXODWHV0LWRFKRQGULDO0HPEUDQH 3RWHQWLDODQG2[\JHQ&RQVXPSWLRQLQ&DQFHU&HOOV 1
Sarah B. Gitto , Alvaro G. Estévez1, and María C. Franco1 1 University of Central Florida Peroxynitrite production and nitrotyrosine formation are associated with several pathologies, including cancer. Tyrosine nitration is found in malignant gliomas and invasive breast
SFRBM 2012
Juan Du , Malvika Rawal1, Justin C Moser1, Kristen E Olney1, Garry R Buettner1, and Joseph J Cullen1,2 1 2 The University of Iowa, VA Hospital, Iowa Pharmacological ascorbate has been shown to induce toxicity in a various cancer cell lines both in vitro and in vivo. Pharmacological concentrations of ascorbate produce H2O2 via the formation of ascorbate radical (Asc•−). The rate of ascorbate oxidation is principally a function of the level of catalytically active iron and copper in solution. For example, catalytic iron in cell culture media contributes significantly to the rate of H2O2 generation. Deferoxamine (DFO) is an iron-chelator that renders iron catalytically inactive with respect to ascorbate oxidation. The addition of the iron-chelators DFO and dipyridyl (DPD) to DMEM high glucose media in the presence of ascorbate (2 mM) reversed the ascorbate-induced flux of H2O2 and reversed ascorbateinduced toxicity. In addition, increasing intracellular iron concentration by pre-incubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate-induced cytotoxicity as demonstrated by decreased clonogenic survival in pancreatic cancer cells. Furthermore, ascorbate (10 mM) treatment also increased the amount of transition metal irons in cells and tumor tissues. This study suggests that iron plays an important role in ascorbateinduced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the efficacy of pharmacological ascorbate. (Supported by VA Merit grant, NIH grants CA166800 CA137230)
doi:10.1016/j.freeradbiomed.2012.10.107
79 5HGXFWLYH&DUER[\ODWLRQ0D\$IIHFW5HGR[6WDWHRI &DQFHU&HOOV 1
Ales Dvorak , Katarina Smolkova1, Jaroslav Zelenka1, Libor Vitek2, and Petr Jezek1 1 2 ASCR, Prague, Czech Republic, Charles University of Prague, Prague, Czech Republic Reductive carboxylation (RC, reverse Krebs cycle reaction of 2oxoglutarate) is indispensable metabolic pathway which plays an important role in viability of cancer cells. RC is supplemented by anaplerotic pathways - largely due to glutaminolysis. Glutamine is transformed by transaminases to 2-oxoglutarate which can be further converted by mitochondrial isocitrate dehydrogenase 2 (IDH2) to isocitrate or to oncometabolite 2-hydroxyglutarate (2HG) with concomitant NADPH consumption. Isocitrate can be transformed to citrate by the reversed aconitase and citrate is exported from the matrix to the cytosol. This RC glutaminolysis may help highly malignant tumors to survive. 2-HG is formed only by IDH2 with a R172K point mutation (1) and cannot be further metabolized. Both reactions may affect reactive oxygen species (ROS) production and antioxidant protection of glutathione because of NADPH consumption. Thus we have studied redox balance, ROS production and reduced/oxidized glutathione ration (GSH/GSSG) in cancer cell lines HTB-126, HepG2 and SHSY5Y and in cells where RC was inhibited by IDH2 silencing. We have measured incorporation of 13C from glutamine to metabolites of 13 RC by GC-MS, namely C citrate, malate and 2-HG, for all also upon hypoxia and presence of respiratory inhibitors. In hypoxia, this incorporation was much higher as compared to normoxia. 13 Oligomycin increased incorporation of C into citrate, malate and 2-HG, however, FCCP decreased it. It supports hypothesis that RC is reciprocally dependent on OXPHOS activity. RC
S42
semiquantification was taken as differences to measurements in cells with IDH2 silencing or respiratory inhibitors. Also mitochondrial superoxide production and GSH/GSSG were quantified in relation to IDH2 expression and activity. Amount of GSSG was decreased in cell lines with IDH2 silencing. We have investigated activity of IDH2 in matrix which affects redox changes that subsequently influence redox balance, and activities of redox-sensitive mitochondrial enzymes. 1. Ward P.S., et.al., Cancer Cell 17, 225-234, 2010 Supported by Grant Agency of Charles University No. 426411
doi:10.1016/j.freeradbiomed.2012.10.108
80 7KH(IIHFWRI1LWULF2[LGHRQ0LWRFKRQGULDO 0HWDEROLVPDQG3UROLIHUDWLRQLQ%UHDVW&DQFHU&HOOV 1,2
John M Egner , Anne R Dier2, Shiqui Li2, Ching-Fang Chang2, and Neil Hogg2 1 2 Carthage College, WI, Medical College of Wisconsin The mitochondrion is emerging as a key regulator of cancer cell growth because of their critical role in producing energy and biosynthetic intermediates required for rapid replication and proliferation. Nitric oxide (NO) is known to control mitochondrial oxidative phosphorylation through its direct chemical interactions with mitochondrial proteins (e.g. cytochrome c oxidase) and activation of multiple signaling pathways. Despite the wellcharacterized anti-proliferative effects of NO in several cancer types, the direct link between NO-dependent control of mitochondrial function and subsequent changes in cell growth has not been established. We hypothesize that NO controls proliferative responses of breast cancer cells through downregulation of mitochondrial function. Growth curve analysis of four breast cancer cell lines (MCF7, T-47D, MB231, and MB468) demonstrated a decrease in cell proliferation with exposure to increasing concentration of the NO donor DetaNONOate (Deta/NO). Further studies in MCF7 cells investigated the effects of Deta/NO on cellular bioenergetics using the Seahorse Bioscience XF24 Analyzer. Deta/NO caused a time- and concentration-dependent decrease in multiple mitochondrial function parameters and a concomitant stimulation of the extracellular acidification rate, an indicator of glycolysis. Changes in mitochondrial function were accompanied by decreased expression of cytochrome c oxidase subunits I and Vb with no change in ATP synthase Į subunit levels. Loss of cytochrome c oxidase subunit expression was not reversed 24 h after Deta/NO exposure. Taken together, these data indicate that NO decreases mitochondrial function, in part through regulating levels of cytochrome c oxidase, which may be linked to its anti-proliferative effects in breast cancer cells. These findings also provide important insight into the biological effects of NO and its potential role in therapeutic targeting of metabolism in cancer.
doi:10.1016/j.freeradbiomed.2012.10.109
81 1LWUDWHG+VS 5HJXODWHV0LWRFKRQGULDO0HPEUDQH 3RWHQWLDODQG2[\JHQ&RQVXPSWLRQLQ&DQFHU&HOOV 1
Sarah B. Gitto , Alvaro G. Estévez1, and María C. Franco1 1 University of Central Florida Peroxynitrite production and nitrotyrosine formation are associated with several pathologies, including cancer. Tyrosine nitration is found in malignant gliomas and invasive breast
SFRBM 2012