Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro
Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro
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DOI：
10.1152/ajpheart.00795.2008
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发表时间：
2009-05-01
影响因子：
4.8
通讯作者：
Pacher, Pal
中科院分区：
文献类型：
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作者：
Mukhopadhyay, Partha;Rajesh, Mohanraj;Pacher, Pal
MukHopadhyay P，Rajesh M，Batkai S，Yoshihiro K，Hasko G，Liaudet L，Szabo C，Pacher P.超氧化物、一氧化氮和过氧亚硝酸盐在阿霉素诱导的体内和体外细胞死亡中的作用。Am J Physiol心圈Physiol 296：H1466-H1483,2009。首次发表于2009年3月13日；DOI：10.1152/ajpheart.00795.2008。-阿霉素(DOX)是一种有效的抗肿瘤药物；然而，由于其心脏毒性，其临床应用受到限制。细胞死亡是DOX心脏毒性的关键成分，但其机制尚不清楚。在这里，我们使用体内和体外心脏毒性模型来探讨超氧化物、一氧化氮(NO)和过氧亚硝酸盐在DOX诱导的细胞死亡中的作用。采用Western印迹分析、实时定量聚合酶链式反应、免疫组织化学、流式细胞仪、荧光显微镜、生化分析等方法检测细胞的凋亡/坏死指标、一氧化氮和超氧化物歧化物质的来源及其产生。用压力-容量系统测量左心功能。我们发现心肌细胞凋亡(caspase-3裂解/活性，细胞色素c释放和TUNEL)增加，诱导型一氧化氮合酶(INOS)表达，线粒体超氧化物生成，3-硝基酪氨酸(NT)形成，基质金属蛋白酶(MMP2/MMP9)基因表达，多(ADP-核糖)聚合酶激活[在NAD(P)H氧化酶亚型1，NAD(P)H氧化酶亚型1，NAD(P)H氧化酶亚型2，P22(PHX)，p40(PHX)，P47(PHX)，P67(PHX)，黄嘌呤氧化酶，内皮和神经元型NOS表达减少]和谷胱甘肽过氧化物酶活性在DOX处理后5天。DOX的上述作用均可被过氧亚硝酸盐清除剂明显减弱。阿霉素剂量依赖性地增加心肌H9c2细胞线粒体超氧化物歧化、NT生成和细胞凋亡/坏死。DOX或过氧亚硝酸盐诱导的细胞凋亡/坏死与细胞内NT的形成呈正相关，过氧亚硝酸盐清除剂可将其消除。选择性诱导型一氧化氮合酶抑制剂或诱导型一氧化氮合酶基因敲除小鼠也可减轻DOX诱导的细胞死亡和NT形成。不同的NO供体与DOX联合给药时，显着增加DOX诱导的细胞死亡，同时增加NT的形成。DOX诱导的细胞死亡也可被细胞通透性的SOD减弱，但不能被细胞通透性的过氧化氢酶、黄嘌呤氧化酶抑制剂别嘌醇或NADPH氧化酶抑制剂apocynine或二苯碘所减弱。因此，过氧亚硝酸盐在体内和体内都是DOX诱导细胞死亡的主要触发因素，调节导致其产生或有效中和的途径可能具有显著的治疗益处。
Mukhopadhyay P, Rajesh M, Batkai S, Yoshihiro K, Hasko G, Liaudet L, Szabo C, Pacher P. Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. Am J Physiol Heart Circ Physiol 296: H1466-H1483, 2009. First published March 13, 2009; doi:10.1152/ajpheart.00795.2008.-Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P) H oxidase isoform 1, NAD(P) H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.