活性氧激活内质网应激和线粒体凋亡通路介导亚硒酸钠诱导NB4细胞凋亡的研究

【作者】 关丽英；

【导师】 许彩民；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 硒是人体所必需微量元素,具有抗癌作用能够诱导多种肿瘤细胞凋亡。大量研究结果表明:微量元素硒将成为抗肿瘤药物研究领域的一个研究热点。我们前期的研究表明,20μM亚硒酸钠能够有效抑制人急性早幼粒白血病NB4细胞的生长增殖并诱发细胞凋亡,其详细作用机制尚不清楚。同时发现硒对细胞的作用呈浓度依赖性,营养剂量即低浓度的硒对细胞的生长与增殖有保护作用,而超营养剂量即高浓度的硒则可明显地诱导细胞凋亡。本文对亚硒酸钠诱导NB4细胞凋亡过程中内质网应激和线粒体凋亡通路信号调控的分子机制进行了深入的研究。发现亚硒酸钠诱导NB4细胞凋亡启动了ER应激介导的细胞凋亡以及P53转录非依赖的线粒体凋亡通路,而且发现这两条凋亡途径均依赖于亚硒酸钠诱导凋亡过程中ROS的产生。同时发现ER应激所介导的促生存和促凋亡信号介导了不同浓度亚硒酸钠对肿瘤细胞的不同作用。所获结果如下:发现亚硒酸钠可诱导Caspase的激活,而CaspaSe广谱抑制剂z-VAD-fm能够部分的阻断亚硒酸钠诱导的NB4细胞的凋亡,表明亚硒酸钠激活了Caspase依赖的细胞凋亡。发现亚硒酸钠特异性的激活了与线粒体凋亡通路相关的Caspase-9以及与ER应激凋亡相关的Caspase-4。ER应激介导的细胞凋亡是近年新发现的凋亡途径,未折叠/错误折叠蛋白在ER的积累或Ca²+稳态的破坏均可引起ER应激。本研究发现亚硒酸钠诱导了内质网应激介导的凋亡,亚硒酸钠作用早期明显诱导了未折叠蛋白响应（UPR）三条信号通路的激活,即PERK-eIF2α-ATF4,IRE1-XBP-1S和ATF6。UPR的激活促进了内质网对蓄积在腔内的错误折叠或未折叠蛋白的处理,有利于维持细胞的正常功能并使之存活。随着亚硒酸钠作用时间的延长,ER应激加剧,UPR的激活被抑制,细胞启动凋亡信号,激活了GADD153蛋白的表达。GADD153是内质网应激特异的促凋亡转录因子,应用RNA干扰技术,发现GADD153特异的siRNA阻断了亚硒酸钠诱导的细胞凋亡,表明GADD153在亚硒酸钠诱导的ER应激介导的细胞凋亡中发挥着重要作用。进一步的研究发现GADD153特异的siRNA干扰后能够阻断亚硒酸钠诱导的AKT的去磷酸化失活,而AKT则可以通过对Bad的磷酸化修饰而诱导线粒体凋亡通路,我们发现GADD153在亚硒酸钠诱导NB4细胞凋亡过程中充当AKT的负调控子将凋亡信号从ER传递到线粒体,成为沟通ER应激与线粒体凋亡通路的关键节点蛋白。本实验室的前期研究已经发现亚硒酸钠诱导NB4细胞的凋亡启动了线粒体凋亡通路,而对于其上游机制尚不清楚。本研究发现亚硒酸钠通过诱导P53转位到线粒体启动线粒体凋亡通路。NB4细胞表达一种转录功能缺失的突变P53,应用P53特异的siRNA和抑制剂pifithrin-α（PFT）均可有效抑制亚硒酸钠诱导的细胞凋亡,免疫细胞化学染色和Western-blot结果显示亚硒酸钠诱导了P53向线粒体转位,表明P53以转录非依赖的方式参与亚硒酸钠引起的细胞凋亡。P53特异的抑制剂PFT预处理细胞,提取线粒体蛋白,Western-blot分析发现PFT抑制了亚硒酸钠引起的P53的线粒体转位,同时也抑制了亚硒酸钠引起的BAX的线粒体转位、线粒体膜电位的下降、细胞色素C的释放以及Caspases的激活,表明亚硒酸钠诱导P53转位到线粒体进而启动了线粒体凋亡通路。同时发现P38 MAPK特异抑制剂SB203580抑制了亚硒酸钠诱导的P53 Ser15的磷酸化及其线粒体转位。以上结果表明亚硒酸钠诱导了ER应激和线粒体介导凋亡通路,而ER和线粒体是细胞中直接参与Ca²⁺储存和调控的两个重要细胞器,本研究发现亚硒酸钠引起了细胞内Ca²⁺稳态的变化。亚硒酸钠作用早期引起胞质[Ca²⁺]的增加,随着作用时间的延长,胞质[Ca²⁺]逐渐降低,进一步用激光共聚焦显微镜观察发现胞质[Ca²⁺]减少的同时线粒体内[Ca²⁺]增加。本实验室前期研究已经证明亚硒酸钠诱导NB4细胞凋亡能够引起ROS的产生。本研究发现抗氧化剂MnSOD的模拟物MnTMPyP完全阻断了亚硒酸钠诱导的NB4细胞凋亡,表明ROS尤其是超氧阴离子在亚硒酸钠诱导NB4细胞凋亡过程中发挥着极其重要的作用。进一步研究发现MnTMPyP能够延缓亚硒酸钠引起的UPR的激活;抑制亚硒酸钠诱导的促凋亡蛋白ATF4和GADD153的激活;抑制亚硒酸钠诱导的P53的线粒体转位以及由此引起的线粒体损伤;同时也参与了亚硒酸钠引起的细胞内Ca²⁺稳态的调节。表明亚硒酸钠诱导产生的ROS是ER应激与线粒体凋亡的上游信号,启动了ER应激及P53依赖的线粒体凋亡通路。ROS的产生与清除之间的平衡对维持细胞的氧化还原状态非常重要。抗氧化酶是清除ROS引起的氧化损伤的一个主要机制,MnSOD是线粒体中重要的抗氧化酶,能够歧化超氧阴离子（O₂^-）形成过氧化氢（H_O2）。我们的研究发现MnSOD的模拟物MnTMPyP完全阻断了亚硒酸钠诱导的NB4细胞凋亡,而进一步应用RNA干扰技术降低MnSOD水平诱导了细胞凋亡,表明MnSOD是一个抗凋亡蛋白。本研究首次发现亚硒酸钠诱导NB4细胞凋亡过程中引起了MnSOD由线粒体向胞质的释放,而MnSOD由线粒体释放到胞质中就不能及时清除由线粒体呼吸链上产生的O₂^-,从而引起ROS在细胞中的积累。因此亚硒酸钠通过对MnSOD的亚细胞定位的改变介导了细胞内ROS的积累。不同浓度硒对肿瘤细胞的作用不同,MTT和流式细胞术对细胞增殖和凋亡的分析发现,低浓度（2μM）的亚硒酸钠处理48 h诱导了细胞的增殖,促进细胞生存,高浓度（5-20μM）的亚硒酸钠则引起细胞凋亡。推测ER应激所激活的促生存和促凋亡信号介导了不同浓度硒对肿瘤细胞的不同作用。本研究发现低浓度亚硒酸钠激活了UPR,而对促凋亡信号分子没有明显的激活,随着亚硒酸钠浓度的升高（高于5μM）UPR信号分子的激活被抑制,激活了促凋亡分子GADD153、P53以及Caspases等。因此亚硒酸钠在营养剂量即低浓度诱导轻微的ER应激,激活UPR以帮助细胞处理ER应激引起的损伤,恢复细胞的正常功能,促进细胞生存。然而这种保护能力是非常有限的,随着亚硒酸钠浓度的升高,UPR的激活被抑制,而凋亡相关的信号分子被激活。我们的研究证明细胞最终是生存还是凋亡取决于这两种信号之间的平衡。更多还原

【Abstract】 Selenium,an essential trace element possessing anti-carcinogenic properties,can induce apoptosis in cancer cells,raising a new idea for its clinical application.Previous studies discovered that 20μM selenite markedly inhibited the proliferation and induced apoptosis of acute promyelocytic leukemia NB4 cells in a time-dependent manner. Selenium at low concentrations has a chemopreventive role against cancer,while at high concentrations,it exerts a direct antitumor effect.cDNA microarray analysis showed that different gene expression induced by 2μM and 20μM sodium selenite respectively.However,the mechanisms behind these effects remain elusive.In this article,we delineated the apoptotic signaling pathways activated by sodium selenite in NB4 cells.We found that sodium selenite induces NB4 cell apoptosis through induction of ER stress and p53-dependent mitochondrial dysfunction,both of which act as the downstream events of ROS.In addition,sodium selenite at low concentrations activated pro-survival pathways,whereas high concentrations of selenite induced activation of pro-apoptotic molecules.The findings are shown blow:Selenite specifically activated caspase-9 and -4 rather than caspase-8 in NB4 cells, suggested the possible involvement of both mitochondria and ER-dependent pathways in selenite-induced NB4 cell apoptosis.ER stress triggers the UPR to protect cells against ER stress.The present work showed that three UPR transducer pathways, PERK-eIF2a,IRE1-XBP1S and ATF6,were activated very rapidly following selenite exposure,although their activation receded at later stages.These UPR signaling pathways initiate cell protective mechanisms to protect cells against ER stress-induced damage.However,if the stress level is too severe to be repaired by cells,the apoptotic signals will be triggered.CHOP,also known as growth-arrest and DNA-damage inducible gene 153（GADD153）,is a key pro-apoptosis transcription factor that is closely related with ER stress.Selenite induced GADD153 expression,and GADD153-specific siRNA reduced cell apoptosis induced by selenite,suggesting a causal role of GADD153 in selenite-induced NB4 cells apoptosis.The down-stream effectors of GADD153 are poorly understood.Further study found that selenite suppressed the activation of anti-apoptotic kinase AKT,while silencing of GADD153 gene expression by siRNA prevented selenited-induced the inactivation of AKT.These data suggest that GADD153 acts as a critical negative regulator of AKT,therefore pushing the cell in the direction of apoptosis.Since activated AKT can induce the phosphorylation of Bad and caused a decrease of cytochrome c release.Thus GADD153 induction may transmit the signal from ER to mitochondria and execution of death by dephosphorylation of AKT and Bad.Selenite-induced mitochondrial apoptotic pathway has been documented in other studies.In this study,we demonstrated that selenite-induced mitochondrial apoptotic pathway is dependent on p53.P53 can induce cell apoptosis in transcription-dependent and transcription-independent manner.NB4 cells express a mutant form of p53 that is incapable of binding DNA.Inhibition of p53 by PFT prevented selenite-cauced mitochondrial membrane permeabilization and cytotoxicity,but had no effect on Bax protein expression,indicated that this transactivation-deficient mutant of p53 in NB4 cells participates in the apoptotic process in a transcription-independent manner. Immunofluorescence result and western-blot analysis provided evidence of p53 mitochondrial translocation after selenite treatment.PFT suppressed p53 mitochondrial translocation along with Bax mitochondrial translocation that resulted in mitochondrial membrane permeabilization,cytochrome c release and subsequent caspase-9 activation. These suites of results suggest that p53 mediates mtiochondrial apoptotic pathway by direct activation of Bax mtiochondrial translocation.Since selenite induced ER stress and mitochondrial dysfunction,ER and mitochondria are two major sites that are directly involved in the storage and regulation of intracellular Ca²⁺.Time-course analysis in NB4 cells showed a significantly increased[Ca²⁺]c within initial hours of selenite treatment and decreased with time. Further studies showed that Ca²⁺ accumulated in mitochondria,which may promote the loss of mitochondrial permeability.Selenite has been reported to increase the generation of ROS,which is an important signal molecule during apoptosis.A cell-permeable superoxide scavenger, MnSOD mimic,MnTMPyp,abrogated the increase in ROS and simultaneity completely inhibited cell apoptosis induced by selenite,suggesting that ROS plays an important role in selenite-induced cell apoptosis.Antioxidant MnTMPyP attenuated the activation of UPR as well as abolished the expression of ATF4 and GADD153 induced by selenite. Further study found that removal of ROS by MnTMPyP completely prevented p53 translocation to mitochondria,in return blocked Bax mitochondrial translocation, mitochondrial membrane permeabilization,cytochrome c release from mitochondria and caspases activation.ROS also regulates intracellular Ca²⁺ homeostasis.In conclusion,these results show that selenite induces apoptosis by producing ROS to initiate ER stress and p53-dependent mitochondrial dysfunction in NB4 cells.Antioxidant enzyme plays a vital role in controlling the redox status of the cell. Manganese superoxide dismutase（MnSOD） is an essential primary antioxidant enzyme, which converts superoxide radical to hydrogen peroxide within the mitochondrial matrix.MnSOD mimic,MnTMPyp and MnSOD-specific siRNA inhibited and increased selenite-induced cell apoptosis respectively,suggest that MnSOD plays a prominent role in protection against selenite-induced cell apoptosis.In this study,we first found that selenite induced MnSOD translocation from mitochondria to cytosol, which leads to a reduction of superoxide scavenging by MnSOD and subsequent accumulation of ROS.Selenium at low concentrations has a chemopreventive role against cancer,while at high concentrations,selenite exerts a direct antitumor effect.We speculated that ER stress-induced pro-survival and pro-apoptotic pathways probably mediate the chemopreventive and antitumor effects of selenite.MTT assay and flow cytometry analysis show that selenite above 5μM reduces NB4 cell viability and induces apoptosis,while 2μM selenite increases cell viability and has no effect on apoptosis.In this study,we found that low concentrations of selenite elicited mild ER stress and mediated cell survival by activating UPR signaling whereas high concentrations of selenite induced severe ER stress and caused cell death by activation of the pro-apoptotic transcription factors GADD153,p53 and caspase.The net balance between these signaling cascades probably governs cell survival or apoptosis.These findings uncover the molecular mechanisms of the chemopreventive and antitumor effects of different concentrations of selenite.更多还原