外源性氧化应激诱导的线粒体超氧阴离子决定肿瘤细胞命运:一项基于单个细胞的研究（英文）
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摘要
目的:通过细胞外过氧化氢(H_2O_2)的刺激建立单个人肺癌SPC-A-1细胞的氧化压力模型。创新点:氧自由基(ROS)涉及多种生物现象,包括有益和有害两个方面。ROS的定量检测和反应网络的评估结果令人期待。但ROS半衰期很短且反应过程很快,因此,我们通过多种手段克服了检测和评估的困难。方法:利用改进的微流控和成像技术测定ROS水平,构建氧反应网络。通过调控线粒体胞浆Ca~(2+)水平、线粒体Ca~(2+)摄取、细胞内ROS自扩增以及内在凋亡途径,确定线粒体在外源氧化压力模式中扮演的角色。结论:研究结果表明1 mmol/L H_2O_2引起细胞O_2~(·-)水平的快速增加,从而导致细胞氧化能力增加和还原能力降低。此外,研究还证实了内质网中储存的Ca~(2+)是H_2O_2诱导的线粒体Ca~(2+)爆发的主要来源。外源氧化压力反应涉及细胞器间Ca~(2+)信号的传递、ROS自身扩增、线粒体功能紊乱和半胱天冬酶依赖性凋亡途径。线粒体在外源性氧化应激影响细胞命运方面发挥着关键作用。
Objective: Reactive oxygen species(ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H_2O_2 stimuli. Methods: Modified microfluidics and imaging techniques were used to determine O_2~(·-) levels and construct an O_2~(·-) reaction network. To elucidate the consequences of increased O_2~(·-) input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca~(2+) levels, mitochondrial Ca~(2+) uptake, auto-amplification of intracellular ROS and the intrinsic apoptotic pathway. Results and conclusions: Results from a modified microchip demonstrated that 1 mmol/L H·-2 O2 induced a rapid increase in cellular O_2 levels(>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power(evaluated by ROS levels) and decreased reducing power(evaluated by glutathione(GSH) levels). In addition, we examined the dynamics of cytosolic Ca~(2+) and mitochondrial Ca~(2+) by confocal laser scanning microscopy and confirmed that Ca~(2+) stores in the endoplasmic reticulum were the primary source of H_2O_2-induced cytosolic Ca~(2+) bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca~(2+) signals between organelles,ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.
Objective: Reactive oxygen species(ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H_2O_2 stimuli. Methods: Modified microfluidics and imaging techniques were used to determine O_2~(·-) levels and construct an O_2~(·-) reaction network. To elucidate the consequences of increased O_2~(·-) input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca~(2+) levels, mitochondrial Ca~(2+) uptake, auto-amplification of intracellular ROS and the intrinsic apoptotic pathway. Results and conclusions: Results from a modified microchip demonstrated that 1 mmol/L H·-2 O2 induced a rapid increase in cellular O_2 levels(>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power(evaluated by ROS levels) and decreased reducing power(evaluated by glutathione(GSH) levels). In addition, we examined the dynamics of cytosolic Ca~(2+) and mitochondrial Ca~(2+) by confocal laser scanning microscopy and confirmed that Ca~(2+) stores in the endoplasmic reticulum were the primary source of H_2O_2-induced cytosolic Ca~(2+) bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca~(2+) signals between organelles,ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.
引文
Akopova OV,2008.The role of mitochondrial permeability transition pore in transmembrane Ca2+-exchange in mitochondria.Ukr Biokhim Zh(1999),80(3):40-47(in Ukrainian).
Blokhina O,Fagerstedt KV,2010.Oxidative metabolism,ROSand NO under oxygen deprivation.Plant Physiol Biochem,48(5):359-373.https://doi.org/10.1016/j.plaphy.2010.01.007
Bogeski I,Kappl R,Kummerow C,et al.,2011.Redox regulation of calcium ion channels:chemical and physiological aspects.Cell Calcium,50(5):407-423.https://doi.org/10.1016/j.ceca.2011.07.006
Camello-Almaraz C,Gomez-Pinilla PJ,Pozo MJ,et al.,2006.Mitochondrial reactive oxygen species and Ca2+signaling.Am J Physiol Cell Physiol,291(5):C1082-C1088.https://doi.org/10.1152/ajpcell.00217.2006
de Marchi E,Bonora M,Giorgi C,et al.,2014.The mitochondrial permeability transition pore is a dispensable element for mitochondrial calcium efflux.Cell Calcium,56(1):1-13.https://doi.org/10.1016/j.ceca.2014.03.004
Feng W,Liu GH,Allen PD,et al.,2000.Transmembrane redox sensor of ryanodine receptor complex.J Biol Chem,275(46):35902-35907.https://doi.org/10.1074/jbc.C000523200
Gao J,Yin XF,Fang ZL,2004.Integration of single cell injection,cell lysis,separation and detection of intracellular constituents on a microfluidic chip.Lab Chip,4(1):47-52.https://doi.org/10.1039/b310552k
Gao N,Li L,Shi ZK,et al.,2007.High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel.Electrophoresis,28(21):3966-3975.https://doi.org/10.1002/elps.200700124
Giorgi C,Missiroli S,Patergnani S,et al.,2015.Mitochondriaassociated membranes:composition,molecular mechanisms,and physiopathological implications.Antioxid Redox Signal,22(12):995-1019.https://doi.org/10.1089/ars.2014.6223
Hileman EO,Liu JS,Albitar M,et al.,2004.Intrinsic oxidative stress in cancer cells:a biochemical basis for therapeutic selectivity.Cancer Chemother Pharmacol,53(3):209-219.https://doi.org/10.1007/s00280-003-0726-5
K?hler AC,Sag CM,Maier LS,2014.Reactive oxygen species and excitation-contraction coupling in the context of cardiac pathology.J Mol Cell Cardiol,73:92-102.https://doi.org/10.1016/j.yjmcc.2014.03.001
Labuschagne CF,Brenkman AB,2013.Current methods in quantifying ROS and oxidative damage in Caenorhabditis elegans and other model organism of aging.Ageing Res Rev,12(4):918-930.https://doi.org/10.1016/j.arr.2013.09.003
Liochev SI,2013.Reactive oxygen species and the free radical theory of aging.Free Radic Biol Med,60:1-4.https://doi.org/10.1016/j.freeradbiomed.2013.02.011
Lyublinskaya OG,Zenin VV,Shatrova AN,et al.,2014.Intracellular oxidation of hydroethidine:compartmentalization and cytotoxicity of oxidation products.Free Radic Biol Med,75:60-68.https://doi.org/10.1016/j.freeradbiomed.2014.07.008
Maltepe E,Saugstad OD,2009.Oxygen in health and disease:regulation of oxygen homeostasis-clinical implications.Pediatr Res,65(3):261-268.https://doi.org/10.1203/PDR.0b013e31818fc83f
Martindale JL,Holbrook NJ,2002.Cellular response to oxidative stress:signaling for suicide and survival.J Cell Physiol,192(1):1-15.https://doi.org/10.1002/jcp.10119
Moreau B,Nelson C,Parekh AB,2006.Biphasic regulation of mitochondrial Ca2+uptake by cytosolic Ca2+concentration.Curr Biol,16(16):1672-1677.https://doi.org/10.1016/j.cub.2006.06.059
Mota SI,Costa RO,Ferreira IL,et al.,2015.Oxidative stress involving changes in Nrf2 and ER stress in early stages of Alzheimer’s disease.Biochim Biophys Acta,1852(7):1428-1441.https://doi.org/10.1016/j.bbadis.2015.03.015
Orrenius S,Gogvadze V,Zhivotovsky B,2015.Calcium and mitochondria in the regulation of cell death.Biochem Biophys Res Commun,460(1):72-81.https://doi.org/10.1016/j.bbrc.2015.01.137
Pan H,Wang BH,Lv W,et al.,2015.Esculetin induces apoptosis in human gastric cancer cells through a cyclophilin D-mediated mitochondrial permeability transition pore associated with ROS.Chemico-Biological Interactions,242:51-60.https://doi.org/10.1016/j.cbi.2015.09.015
Pelicano H,Carney D,Huang P,2004.ROS stress in cancer cells and therapeutic implications.Drug Resist Updat,7(2):97-110.https://doi.org/10.1016/j.drup.2004.01.004
Qin Y,Chen FD,Zhou L,et al.,2009.Proliferative and antiproliferative effects of thymosinα1 on cells are associated with manipulation of cellular ROS levels.Chem Biol Interact,180(3):383-388.https://doi.org/10.1016/j.cbi.2009.05.006
Qin Y,Pan X,Tang TT,et al.,2011.Anti-proliferative effects of the novel squamosamide derivative(FLZ)on HepG2human hepatoma cells by regulating the cell cycle-related proteins are associated with decreased Ca2+/ROS levels.Chem Biol Interact,193(3):246-253.https://doi.org/10.1016/j.cbi.2011.07.004
Rhee SG,2006.Cell signaling:H2O2,a necessary evil for cell signaling.Science,312(5782):1882-1883.https://doi.org/10.1126/science.1130481
Singh DK,Kumar D,Siddiqui Z,et al.,2005.The strength of receptor signaling is centrally controlled through a cooperative loop between Ca2+and an oxidant signal.Cell,121(2):281-293.https://doi.org/10.1016/j.cell.2005.02.036
Solier S,Pommier Y,2011.MDC1 cleavage by caspase-3:a novel mechanism for inactivating the DNA damage response during apoptosis.Cancer Res,71(3):906-913.https://doi.org/10.1158/0008-5472.CAN-10-3297
Sp?t A,Pitter JG,2004.The effect of cytoplasmic Ca2+signal on the redox state of mitochondrial pyridine nucleotides.Mol Cell Endocrinol,215(1-2):115-118.https://doi.org/10.1016/j.mce.2003.11.004
Sun Y,Yin XF,2006.Novel multi-depth microfluidic chip for single cell analysis.J Chromatogr A,1117(2):228-233.https://doi.org/10.1016/j.chroma.2006.03.088
Sun Y,Yin XF,Ling YY,et al.,2005.Determination of reactive oxygen species in single human erythrocytes using microfluidic chip electrophoresis.Anal Bioanal Chem,382(7):1472-1476.https://doi.org/10.1007/s00216-005-3352-8
Tang HY,Qin Y,Li JY,et al.,2011.The scavenging of superoxide radicals promotes apoptosis induced by a novel cell-permeable fusion protein,s TRAIL:Fe SOD,in tumor necrosis factor-related apoptosis-inducing ligand-resistant leukemia cells.BMC Biol,9:18.https://doi.org/10.1186/1741-7007-9-18
Thannickal VJ,Fanburg BL,2000.Reactive oxygen species in cell signaling.Am J Physiol Lung Cell Mol Physiol,279(6):L1005-L1028.https://doi.org/10.1152/ajplung.2000.279.6.L1005
Tonks NK,2005.Redox redux:revisiting PTPs and the control of cell signaling.Cell,121(5):667-670.https://doi.org/10.1016/j.cell.2005.05.016
von Montfort C,Matias N,Fernandez A,et al.,2012.Mitochondrial GSH determines the toxic or therapeutic potential of superoxide scavenging in steatohepatitis.JHepatol,57(4):852-859.https://doi.org/10.1016/j.jhep.2012.05.024
Voronina S,Sukhomlin T,Johnson PR,et al.,2002.Correlation of NADH and Ca2+signals in mouse pancreatic acinar cells.J Physiol,539:41-52.https://doi.org/10.1113/jphysiol.2001.013134
Yu SY,Jang Y,Paik D,et al.,2015.Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species.Biochem Biophys Res Commun,465(4):845-850.https://doi.org/10.1016/j.bbrc.2015.08.098
Yuana Y,Sturk A,Nieuwland R,2013.Extracellular vesicles in physiological and pathological conditions.Blood Rev,27(1):31-39.https://doi.org/10.1016/j.blre.2012.12.002
Zhao HT,Kalivendi S,Zhang H,et al.,2003.Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium:potential implications in intracellular fluorescence detection of superoxide.Free Radic Biol Med,34(11):1359-1368.https://doi.org/10.1016/S0891-5849(03)00142-4
Blokhina O,Fagerstedt KV,2010.Oxidative metabolism,ROSand NO under oxygen deprivation.Plant Physiol Biochem,48(5):359-373.https://doi.org/10.1016/j.plaphy.2010.01.007
Bogeski I,Kappl R,Kummerow C,et al.,2011.Redox regulation of calcium ion channels:chemical and physiological aspects.Cell Calcium,50(5):407-423.https://doi.org/10.1016/j.ceca.2011.07.006
Camello-Almaraz C,Gomez-Pinilla PJ,Pozo MJ,et al.,2006.Mitochondrial reactive oxygen species and Ca2+signaling.Am J Physiol Cell Physiol,291(5):C1082-C1088.https://doi.org/10.1152/ajpcell.00217.2006
de Marchi E,Bonora M,Giorgi C,et al.,2014.The mitochondrial permeability transition pore is a dispensable element for mitochondrial calcium efflux.Cell Calcium,56(1):1-13.https://doi.org/10.1016/j.ceca.2014.03.004
Feng W,Liu GH,Allen PD,et al.,2000.Transmembrane redox sensor of ryanodine receptor complex.J Biol Chem,275(46):35902-35907.https://doi.org/10.1074/jbc.C000523200
Gao J,Yin XF,Fang ZL,2004.Integration of single cell injection,cell lysis,separation and detection of intracellular constituents on a microfluidic chip.Lab Chip,4(1):47-52.https://doi.org/10.1039/b310552k
Gao N,Li L,Shi ZK,et al.,2007.High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel.Electrophoresis,28(21):3966-3975.https://doi.org/10.1002/elps.200700124
Giorgi C,Missiroli S,Patergnani S,et al.,2015.Mitochondriaassociated membranes:composition,molecular mechanisms,and physiopathological implications.Antioxid Redox Signal,22(12):995-1019.https://doi.org/10.1089/ars.2014.6223
Hileman EO,Liu JS,Albitar M,et al.,2004.Intrinsic oxidative stress in cancer cells:a biochemical basis for therapeutic selectivity.Cancer Chemother Pharmacol,53(3):209-219.https://doi.org/10.1007/s00280-003-0726-5
K?hler AC,Sag CM,Maier LS,2014.Reactive oxygen species and excitation-contraction coupling in the context of cardiac pathology.J Mol Cell Cardiol,73:92-102.https://doi.org/10.1016/j.yjmcc.2014.03.001
Labuschagne CF,Brenkman AB,2013.Current methods in quantifying ROS and oxidative damage in Caenorhabditis elegans and other model organism of aging.Ageing Res Rev,12(4):918-930.https://doi.org/10.1016/j.arr.2013.09.003
Liochev SI,2013.Reactive oxygen species and the free radical theory of aging.Free Radic Biol Med,60:1-4.https://doi.org/10.1016/j.freeradbiomed.2013.02.011
Lyublinskaya OG,Zenin VV,Shatrova AN,et al.,2014.Intracellular oxidation of hydroethidine:compartmentalization and cytotoxicity of oxidation products.Free Radic Biol Med,75:60-68.https://doi.org/10.1016/j.freeradbiomed.2014.07.008
Maltepe E,Saugstad OD,2009.Oxygen in health and disease:regulation of oxygen homeostasis-clinical implications.Pediatr Res,65(3):261-268.https://doi.org/10.1203/PDR.0b013e31818fc83f
Martindale JL,Holbrook NJ,2002.Cellular response to oxidative stress:signaling for suicide and survival.J Cell Physiol,192(1):1-15.https://doi.org/10.1002/jcp.10119
Moreau B,Nelson C,Parekh AB,2006.Biphasic regulation of mitochondrial Ca2+uptake by cytosolic Ca2+concentration.Curr Biol,16(16):1672-1677.https://doi.org/10.1016/j.cub.2006.06.059
Mota SI,Costa RO,Ferreira IL,et al.,2015.Oxidative stress involving changes in Nrf2 and ER stress in early stages of Alzheimer’s disease.Biochim Biophys Acta,1852(7):1428-1441.https://doi.org/10.1016/j.bbadis.2015.03.015
Orrenius S,Gogvadze V,Zhivotovsky B,2015.Calcium and mitochondria in the regulation of cell death.Biochem Biophys Res Commun,460(1):72-81.https://doi.org/10.1016/j.bbrc.2015.01.137
Pan H,Wang BH,Lv W,et al.,2015.Esculetin induces apoptosis in human gastric cancer cells through a cyclophilin D-mediated mitochondrial permeability transition pore associated with ROS.Chemico-Biological Interactions,242:51-60.https://doi.org/10.1016/j.cbi.2015.09.015
Pelicano H,Carney D,Huang P,2004.ROS stress in cancer cells and therapeutic implications.Drug Resist Updat,7(2):97-110.https://doi.org/10.1016/j.drup.2004.01.004
Qin Y,Chen FD,Zhou L,et al.,2009.Proliferative and antiproliferative effects of thymosinα1 on cells are associated with manipulation of cellular ROS levels.Chem Biol Interact,180(3):383-388.https://doi.org/10.1016/j.cbi.2009.05.006
Qin Y,Pan X,Tang TT,et al.,2011.Anti-proliferative effects of the novel squamosamide derivative(FLZ)on HepG2human hepatoma cells by regulating the cell cycle-related proteins are associated with decreased Ca2+/ROS levels.Chem Biol Interact,193(3):246-253.https://doi.org/10.1016/j.cbi.2011.07.004
Rhee SG,2006.Cell signaling:H2O2,a necessary evil for cell signaling.Science,312(5782):1882-1883.https://doi.org/10.1126/science.1130481
Singh DK,Kumar D,Siddiqui Z,et al.,2005.The strength of receptor signaling is centrally controlled through a cooperative loop between Ca2+and an oxidant signal.Cell,121(2):281-293.https://doi.org/10.1016/j.cell.2005.02.036
Solier S,Pommier Y,2011.MDC1 cleavage by caspase-3:a novel mechanism for inactivating the DNA damage response during apoptosis.Cancer Res,71(3):906-913.https://doi.org/10.1158/0008-5472.CAN-10-3297
Sp?t A,Pitter JG,2004.The effect of cytoplasmic Ca2+signal on the redox state of mitochondrial pyridine nucleotides.Mol Cell Endocrinol,215(1-2):115-118.https://doi.org/10.1016/j.mce.2003.11.004
Sun Y,Yin XF,2006.Novel multi-depth microfluidic chip for single cell analysis.J Chromatogr A,1117(2):228-233.https://doi.org/10.1016/j.chroma.2006.03.088
Sun Y,Yin XF,Ling YY,et al.,2005.Determination of reactive oxygen species in single human erythrocytes using microfluidic chip electrophoresis.Anal Bioanal Chem,382(7):1472-1476.https://doi.org/10.1007/s00216-005-3352-8
Tang HY,Qin Y,Li JY,et al.,2011.The scavenging of superoxide radicals promotes apoptosis induced by a novel cell-permeable fusion protein,s TRAIL:Fe SOD,in tumor necrosis factor-related apoptosis-inducing ligand-resistant leukemia cells.BMC Biol,9:18.https://doi.org/10.1186/1741-7007-9-18
Thannickal VJ,Fanburg BL,2000.Reactive oxygen species in cell signaling.Am J Physiol Lung Cell Mol Physiol,279(6):L1005-L1028.https://doi.org/10.1152/ajplung.2000.279.6.L1005
Tonks NK,2005.Redox redux:revisiting PTPs and the control of cell signaling.Cell,121(5):667-670.https://doi.org/10.1016/j.cell.2005.05.016
von Montfort C,Matias N,Fernandez A,et al.,2012.Mitochondrial GSH determines the toxic or therapeutic potential of superoxide scavenging in steatohepatitis.JHepatol,57(4):852-859.https://doi.org/10.1016/j.jhep.2012.05.024
Voronina S,Sukhomlin T,Johnson PR,et al.,2002.Correlation of NADH and Ca2+signals in mouse pancreatic acinar cells.J Physiol,539:41-52.https://doi.org/10.1113/jphysiol.2001.013134
Yu SY,Jang Y,Paik D,et al.,2015.Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species.Biochem Biophys Res Commun,465(4):845-850.https://doi.org/10.1016/j.bbrc.2015.08.098
Yuana Y,Sturk A,Nieuwland R,2013.Extracellular vesicles in physiological and pathological conditions.Blood Rev,27(1):31-39.https://doi.org/10.1016/j.blre.2012.12.002
Zhao HT,Kalivendi S,Zhang H,et al.,2003.Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium:potential implications in intracellular fluorescence detection of superoxide.Free Radic Biol Med,34(11):1359-1368.https://doi.org/10.1016/S0891-5849(03)00142-4