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中国修复重建外科杂志 2010年第11期24卷 1367-1371页

作者：曾顺福刘杰王建第三军医大学新桥医院骨科重庆400037

目的髓核细胞凋亡可能与髓核组织代谢障碍产生缺氧,导致bnip3基因表达有关。通过观察兔退变椎间盘中髓核组织的细胞密度、细胞凋亡率及bnip3的表达,为进一步了解髓核细胞凋亡机制提供实验依据。方法健康3月龄雄性新西兰大白兔30只,体重(... 详细信息

目的髓核细胞凋亡可能与髓核组织代谢障碍产生缺氧,导致bnip3基因表达有关。通过观察兔退变椎间盘中髓核组织的细胞密度、细胞凋亡率及bnip3的表达,为进一步了解髓核细胞凋亡机制提供实验依据。方法健康3月龄雄性新西兰大白兔30只,体重(2.3±0.2)kg,随机分为实验组(n=20)及对照组(n=10)。实验组大白兔采用针刺L3、4、L4、5及L5、6椎间盘制备椎间盘退变模型;对照组仅暴露椎间盘后缝合。术后4、8周通过MRI检查评价椎间盘退变情况,采用组织学观察和TUNEL法检查椎间盘髓核组织中凋亡细胞,用免疫组织化学染色法检测兔椎间盘髓核细胞bnip3的表达。结果 MRI检查示实验组术后4、8周椎间盘髓核信号强度呈逐渐降低趋势。根据Pfirrmann分级标准,实验组术后4、8周椎间盘退变分级比较,差异均有统计学意义(P<0.05)。组织学观察及TUNEL检查示:对照组椎间盘髓核中细胞密度高,可见少量散在的凋亡细胞;实验组术后4、8周时椎间盘髓核组织内细胞密度逐渐降低,可见较多凋亡细胞。各时间点实验组细胞密度、TUNEL染色阳性细胞率与对照组比较,以及实验组各指标两时间点间比较,差异均有统计学意义(P<0.05)。对照组椎间盘髓核组织细胞中无bnip3表达;实验组术后4、8周椎间盘髓核组织细胞中bnip3表达逐渐增多,bnip3染色阳性细胞率分别为13.45%±1.16%、32.00%±1.82%,bnip3灰度值分别为194.32±4.65、117.54±2.11,各时间点间比较差异均有统计学意义(P<0.05)。结论椎间盘退变与髓核组织中细胞密度下降有关,细胞凋亡是椎间盘髓核细胞减少的原因之一,bnip3参与了椎间盘髓核细胞凋亡。

关键词：椎间盘退变髓核组织细胞凋亡 bnip3 MRI 兔

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The BH3-only bnip3 binds to the dynamin Opa1 to promote mitochondrial fragmentation and apoptosis by distinct mechanisms

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EMBO REPORTS 2010年第6期11卷 459-465页

作者： Landes, Thomas Emorine, Laurent J. Courilleau, Delphine Rojo, Manuel Belenguer, Pascale Arnaune-Pelloquin, Laetitia Univ Toulouse 3 Lab Metab Plast Mitochondries F-31062 Toulouse France Univ Toulouse CNRS UMR5241 Toulouse France Univ Victor Segalen Inst Biochim & Genet Cellulaires F-33077 Bordeaux France CNRS UMR5095 Bordeaux France

Opa1 modulates mitochondrial fusion, cristae structure and apoptosis. The relationships between these functions and autosomal dominant optic atrophy, caused by mutations in Opa1, are poorly defined. We show that bnip3 interacts with Opa1, leading to mitochondrial fragmentation and apoptosis. Fission is due to inhibition of Opa1-mediated fusion and is counteracted by Opa1 in an Mfn1-dependent manner. bnip3-Opa1 interaction is necessary to trigger Opa1 complex disruption in a Baxand/or Bak-dependent manner, ultimately leading to apoptosis. Our results uncover a direct link between Opa1 on the inner mitochondrial membrane and the apoptotic machinery on the outer membrane that modulates fusion and cristae structure by separate mechanisms. These findings might help to unravel optic atrophy aetiology as retinal ganglion cells are particularly prone to hypoxia, an inductor of bnip3 expression.

关键词： ADOA apoptosis bnip3 mitochondrial dynamics Opa1

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miR-145调控心肌细胞凋亡、肥大的机制研究

miR-145调控心肌细胞凋亡、肥大的机制研究

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作者：李若天南京大学

学位级别：博士

第一部分miR-145调控过氧化氢介导的心肌细胞凋亡的机制研究目的:研究miR-145对过氧化氢(H202)介导的心肌细胞凋亡的调控作用及其机制;探讨miR-145的表达与心肌缺血-再灌注损伤的相关性。方法:1、采用胰酶联合胶原酶消化的方式从新生大... 详细信息

第一部分miR-145调控过氧化氢介导的心肌细胞凋亡的机制研究目的:研究miR-145对过氧化氢(H202)介导的心肌细胞凋亡的调控作用及其机制;探讨miR-145的表达与心肌缺血-再灌注损伤的相关性。方法:1、采用胰酶联合胶原酶消化的方式从新生大鼠(1d～3d)的心脏组织中获得心肌细胞(NRCM);加入H202刺激,实时定量PCR法检测刺激不同时间点的心肌细胞中成熟miR-145的水平。2、利用雄性10周左右的C57小鼠手术诱导心肌缺血-再灌注。取缺血区周边心肌组织,实时定量PCR法检测其中成熟miR-145的水平。3、体外心肌细胞中利用腺病毒诱导预先过表达miR-145,加入H202刺激,检测凋亡有关的指标如 DNA ladder 和 TUNEL;Western blot 检测 cleaved caspase3、bnip3、Cytochrome C等与凋亡相关的信号分子水平;透射电镜下观察心肌细胞线粒体的形态;DCFH-DA荧光染色观察心肌细胞中活性氧自由基(ROS)的水平。4、分别构建表达bnip3CDS及bnip3CDS+3'UTR的质粒载体,用于转染过表达miR-145的心肌细胞,加入H202刺激,检测DNA ladder、TUNEL;电镜下观察心肌细胞线粒体形态;荧光染色观察ROS水平。5、Western blot检测miR-145对bnip3表达水平的影响;构建包含miR-145预测结合序列及其突变序列的bnip3 3'UTR的荧光素酶报告基因载体,利用荧光素酶报告基因试验验证miR-145对bnip3的靶向调控作用。结果:1、分离得到的NRCMs,在50μMH2O2刺激0.5、1、2、4、8小时成熟miR-145的水平均显著低于静息状态。2、缺血-再灌注1小时及3小时的小鼠心肌组织中miR-145的表达水平较假手术组均显著降低。3、DNA ladder及TUNEL荧光染色均显示过表达miR-145显著降低了 H2O2刺激下心肌细胞的凋亡率;western blot结果显示,除cleaved caspase3变化不明显外,p-p53、Apaf-1及线粒体bnip3水平均可被miR-145剂量依赖性地下调,过表达miR-145可抑制cytochrome C从线粒体向胞质释放;电镜见在H2O2刺激下miR-145能显著保护心肌细胞线粒体的结构破坏;DCFH-DA荧光染色见H2O2刺激下miR-145能显著降低心肌细胞中ROS水平。4、在过表达miR-145及H202刺激的心肌细胞中预先过表达bnip3CDS能显著增加细胞的凋亡率、ROS的水平;而预先过表达bnip3CDS+3’UTR对上述指标无明显影响;过表达bnip3CDS促进了心肌细胞线粒体的形态结构紊乱。5、Western blot结果显示miR-145剂量依赖性地下调bnip3的表达水平;荧光素酶报告基因试验显示bnip3的3'UTR受到miR-145的靶向调控。结论:miR-145可通过下调线粒体凋亡诱导因子bnip3的表达,抑制体外H2O2诱导的心肌细胞凋亡,降低心肌细胞氧化应激损伤,减轻线粒体的结构破坏;miR-145的表达异常与心肌缺血-再灌注损伤相关。第二部分miR-145调控异丙肾上腺素诱导的心肌细胞肥大的机制研究目的:研究miR-145对异丙肾上腺素(ISO)诱导的心肌细胞肥大的调控作用及其机制;探讨miR-145的表达与后负荷增高引起的心肌肥大的相关性。方法:1、分离新生大鼠(ld～3d)的心肌细胞(NRCM)。加入ISO处理,检测刺激不同时间点的心肌细胞中成熟miR-145的水平;检测上述时间点的心肌细胞中GATA6以及转录因子p53、C/EBP-β、FOX03a等蛋白水平。2、手术缩窄小鼠主动脉弓段以构造后负荷增高导致的心肌肥大动物模型(TAC),检测手术后1周、4周模型和对照小鼠左心室组织中miR-145表达水平。3、利用腺病毒载体在体外心肌细胞中高表达miR-145,用ISO刺激,实时定量RCR检测与肥大相关的胚胎基因ANF、BNP及β-MHC的表达;同位素硫35标记半胱氨酸检测细胞摄取氨基酸水平,反映心肌细胞蛋白合成能力;用心肌特异性的sarcomeric actin的抗体对细胞行荧光染色,观察心肌细胞的体积。4、构建GATA6过表达质粒载体,转染心肌细胞,观察miR-145对ISO诱导的胚胎基因表达、蛋白合成增加以及细胞体积增大的作用能否被过表达GATA6逆转。5、Western blot检测与心肌细胞肥大相关的经典信号通路Akt-mTOR、Akt-GSK3β、ERK1/2、JNK、p38通路中重要信号分子及其磷酸化产物的水平,寻找受miR-145调控的信号通路。6、细胞免疫荧光观察miR-145对GATA6在心肌细胞中的定位的影

关键词：心肌细胞缺血-再灌注损伤细胞凋亡 miR-145 bnip3 线粒体凋亡通路心肌细胞肥大 β肾上腺素能受体核移位 miR-145 GATA 家族转录因子 Akt通路

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低氧对神经干细胞中miR-210的表达调控及功能研究

低氧对神经干细胞中miR-210的表达调控及功能研究

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作者：王飞中国人民解放军军事医学科学院

学位级别：博士

神经干细胞（Neural stem cells, NSCs）是存在于胚胎期神经系统和成体脑组织中的一类具有自我更新和分化成神经元、星形胶质细胞和少突胶质细胞能力的干细胞。神经干细胞不仅对胚胎神经系统发育至关重要，也为各种中枢神经系统的损伤... 详细信息

神经干细胞（Neural stem cells, NSCs）是存在于胚胎期神经系统和成体脑组织中的一类具有自我更新和分化成神经元、星形胶质细胞和少突胶质细胞能力的干细胞。神经干细胞不仅对胚胎神经系统发育至关重要，也为各种中枢神经系统的损伤和疾病的治疗带来了希望。通过移植神经干细胞可以在动物或人体中治疗帕金森病、亨廷顿舞蹈症、肌萎缩侧索硬化症、阿兹海默症、多发性硬化症、中风和脊髓损伤等神经系统疾病。目前，神经干细胞的体外培养和扩增通常是在常氧即20%的氧浓度条件下进行的，而胚胎发育和成体脑组织中的氧浓度只有3-5%，甚至更低，这种现象被称作生理性低氧。近年来，越来越多的实验证据表明低氧对于神经干细胞的增殖和分化具有重要的调节作用。本实验室的研究结果表明，适度低氧能促进成年动物脑内和离体培养神经干细胞的增殖和分化。因此，研究低氧对神经干细胞存活、增殖和分化的调控具有重要的意义。低氧诱导因子（Hypoxia-inducible factor, HIF）作为细胞和分子水平的低氧感知分子是低氧信号通路中关键的调控分子之一。低氧条件下，细胞中的HIF-1α稳定并转移至细胞核，通过与低氧反应元件（Hypoxia responsive element, HRE）结合调控多种下游靶基因的表达，从而调控细胞存活、增殖和分化等生物学过程。最新的研究发现microRNAs参与HIF信号通路的调控。低氧下HIF-1α能上调多种miRNAs的表达如miR-23，miR-24，miR-26，miR-107，miR-210和miR-373等，而miR-199和miR-20b则能调控HIF-1α的表达。miR-210在现有报道的所有低氧处理的细胞中均一致表达上调，而且上调最为显著，被认为是低氧标志性microRNA。现有研究证明HIF-1能够与miR-210转录起始位点上游40bp处的低氧反应元件结合，进而调控miR-210的表达。miR-210通过其靶基因参与对细胞存活、凋亡、线粒体代谢、DNA损伤修复和血管生成等生物学过程的调控。然而， miR-210在神经干细胞中的表达及调节机制尚未见报道。因此，在该研究中我们探讨了低氧对神经干细胞中miR-210的表达调控和作用机制。主要研究结果如下：一、低氧对神经干细胞中miR-210的表达调控及功能研究 1.将离体NSCs置于三个氧浓度下培养1、2、3天，然后用Realtime-PCR检测NSCs中miR-210表达量，发现3%和0.3%低氧能显著促进NSCs中miR-210的表达，并且随着低氧时间的延长和氧浓度的降低miR-210的表达量逐渐增高，提示miR-210在NSCs中可能具有重要的生物学作用。 2.用DNA甲基化抑制剂AZA抑制DNA甲基化，能在常氧（20%）和低氧（3%，0.3%）下显著上调NSCs中miR-210的表达，说明DNA甲基化可能参与了miR-210的表达调控，而且这一调控通路不依赖HIF-1通路。 3.通过在神经干细胞中过表达或抑制miR-210然后检测其对细胞存活、凋亡和增殖的影响，结果显示miR-210可以显著抑制缺氧（0.3%）引起的细胞凋亡，促进细胞存活，而不影响细胞增殖。 4.为了进一步探讨miR-210作用的分子机制，通过生物信息学筛选与细胞存活和凋亡相关的靶基因，发现促凋亡蛋白bnip3的编码区和3’UTR有两个可以和miR-210结合的种子序列，可能是miR-210的靶基因。 5.通过过表达和抑制miR-210，发现miR-210能显著抑制bnip3蛋白的表达。将bnip3mRNA编码区和3’UTR的两个可能与miR-210结合的互补位点构建到荧光素酶报告载体，发现过表达miR-210能显著抑制这两个质粒的荧光素酶活性，而将这两个位点突变后能反转miR-210的抑制作用，说明bnip3是miR-210的靶基因。 6.通过bnip3siRNA特异抑制bnip3能显著减少低氧下miR-210inhibitor导致的细胞凋亡，说明miR-210—bnip3在低氧下NSCs中发挥了关键的抗凋亡作用。 7.通过核浆分离Western Blot和免疫荧光实验证明miR-210能够抑制促凋亡蛋白AIF入核，进而抑制了缺氧下神经干细胞的凋亡。二、低氧对循环miR-210的分泌调控及其初步的功能研究最新的研究发现miRNAs可以被细胞分泌到胞外，进入细胞培养液和体液循环系统。这种被膜结构和蛋白包裹的分泌形式的miRNAs被称为循环miRNAs。由于血浆中的循环miRNA具有稳定、易于获得和便于检测的特点，使其成为理想的疾病诊断的标志分子而受到广泛的专注。然而，循环miRNAs的功能研究目前少有报

关键词：低氧 MiR-210 bnip3 细胞凋亡循环miRNAs

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Mutant Huntingtin induces activation of the Bcl-2/adenovirus E1B 19-kDa interacting protein (bnip3)

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CELL DEATH & DISEASE 2010年第1期1卷 e7-e7页

作者： Sassone, J. Colciago, C. Marchi, P. Ascardi, C. Alberti, L. Di Pardo, A. Zippel, R. Sipione, S. Silani, V. Ciammola, A. Univ Milan IRCCS Ist Auxol Italiano Dept Neurol I-20149 Milan Italy Univ Milan IRCCS Ist Auxol Italiano Lab Neurosci Ctr Dino Ferrari I-20149 Milan Italy Univ Milan Dept Biomol Sci & Biotechnol I-20149 Milan Italy Univ Alberta Dept Pharmacol Edmonton AB Canada Univ Insubria Dept Struct & Funct Biol Varese Italy Univ Insubria Ctr Neurosci Varese Italy IRCCS Ist Auxol Italiano Unit Metab Dis & Diabet Milan Italy

Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive neuronal death in the basal ganglia and cortex. Although increasing evidence supports a pivotal role of mitochondrial dysfunction in the death of patients' neurons, the molecular bases for mitochondrial impairment have not been elucidated. We provide the first evidence of an abnormal activation of the Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (bnip3) in cells expressing mutant Huntingtin. In this study, we show an abnormal accumulation and dimerization of bnip3 in the mitochondria extracted from human HD muscle cells, HD model cell cultures and brain tissues from HD model mice. Importantly, we have shown that blocking bnip3 expression and dimerization restores normal mitochondrial potential in human HD muscle cells. Our data shed light on the molecular mechanisms underlying mitochondrial dysfunction in HD and point to bnip3 as a new potential target for neuroprotective therapy in HD. Cell Death and Disease (2010) 1, e7;doi:10.1038/cddis.2009.6;published online 14 January 2010

关键词： Huntington's disease mitochondria bnip3

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耐力运动对衰老小鼠骨骼肌线粒体功能和自噬相关基因影响的研究

耐力运动对衰老小鼠骨骼肌线粒体功能和自噬相关基因影响的研究

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作者：郭晶晶天津体育学院

学位级别：硕士

研究目的：衰老是生物必然面对的生理过程，对衰老机制的研究，有利于人类延长寿命保持机体功能。线粒体作为细胞内的能量工厂，是维持细胞功能的主要动力来源。当线粒体功能异常时，细胞的生存状态就会改变，进而影响组织器官的功能，... 详细信息

研究目的：衰老是生物必然面对的生理过程，对衰老机制的研究，有利于人类延长寿命保持机体功能。线粒体作为细胞内的能量工厂，是维持细胞功能的主要动力来源。当线粒体功能异常时，细胞的生存状态就会改变，进而影响组织器官的功能，导致机体的功能异常，如，衰老、癌症等。有研究表明，自噬作为细胞自身清除和保护机制，可能对衰老有直接的影响。线粒体自噬特异性地清除功能异常的线粒体，避免细胞凋亡或者坏死。有研究报道，在动物实验中线粒体自噬参与了红细胞成熟，神经退行性疾病等发生过程。本研究以15月龄衰老小鼠骨骼肌为研究对象，观察耐力运动后线粒体力能学、自噬及线粒体自噬的情况，探讨运动如何影响线粒体功能、自噬及线粒体自噬水平，以及线粒体自噬发生对于衰老小鼠骨骼肌线粒体功能的影响，并为运动延缓衰老提供理论依据。研究方法：6-8周龄雄性C57BL/6小鼠20只，随机分为安静组（C组）和运动组（E组）。采用耐力跑台运动，小鼠13月龄进行8周耐力跑台训练。每周训练5天，每天40分钟，12米/分（75%VO2max）。运动24小时后处死。每组10只。1）取腓肠肌制备新鲜的线粒体，采用Clark氧电极法检测腓肠肌线粒体呼吸态3（S3）、态4（S4）和呼吸控制比（RCR），荧光素－荧光素酶发光法检测线粒体ATP酶合成速率，荧光分光光度计检测线粒体膜电位以及线粒体ROS生成速率;2）实时荧光定量PCR检测Beclin1、Bcl-2、Bax、bnip3、Nix、HIF-1α mRNA表达;3）Western-blotting法检测Beclin1、Bcl-2、Bax蛋白水平。研究结果：1）实验前、后，C组和E组间小鼠体重变化无显著性;2）E组线粒体呼吸态3、态4、RCR及ATP酶合成活力较C组显著升高（分别：P<0.05，P<0.05，P<0.01，P<0.05）;3）E组线粒体膜电位MP较C组有显著升高（P<0.05）;4）E组ROS生成速率较C组显著降低（P<0.05）;5）E组Beclin1mRNA表达和蛋白水平较C组显著下降（分别：P<0.05，P<0.01）;E组Bcl-2mRNA表达和蛋白水平较C组显著升高（分别：P<0.01，P<0.05）;6）E组Bax mRNA表达和蛋白水平较C组无显著变化，E组Bcl-2/Bax蛋白比值较C组显著增高（P<0.01）;7）E组bnip3mRNA和Nix mRNA表达较C组无显著变化;8）E组HIF-1α mRNA表达无显著变化。研究结论：1）8周耐力训练提高衰老小鼠骨骼肌线粒体呼吸功能和膜电位，显著改善线粒体整体功能;2）8周耐力训练降低衰老小鼠骨骼肌线粒体ROS生成水平和自噬水平，推测ROS生成的减少降低了对自噬的诱导作用;3）8周耐力训练未影响衰老小鼠骨骼肌线粒体自噬水平，可能与线粒体功能适应性提高有关。

关键词：衰老自噬线粒体自噬 Beclin1 Bcl-2 Bax bnip3 Nix HIF-1α

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Autophagy and Heart Failure: A Possible Role for Homocysteine

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CELL BIOCHEMISTRY AND BIOPHYSICS 2012年第1期62卷 1-11页

作者： Vacek, Thomas P. Vacek, Jonathan C. Tyagi, Neetu Tyagi, Suresh C. Univ Louisville Dept Physiol & Biophys Sch Med Louisville KY 40202 USA

Autophagy is a process used for intracellular digestion of organelles and proteins and has special relevance to the long-lived cardiomyocytes in heart disease. The pathway for autophagy and all its mediators remain to be elucidated, but involve such proteins as Atg, Beclin-1, LAMP-2, BH3, Bcl2, PI3K Kinase as well as a plethora of others. It is still not entirely clear whether autophagy is destructive or beneficial to the cell;evidence suggests that the answer is case-specific. For instance, autophagy appears to preserve cell life under cases of ischemia in I/R injury, but is detrimental during reperfusion. High levels of homocysteine (Hcy), a sulfur-containing amino acid, have been shown to be an independent risk factor for chronic heart failure. There are several links to induction and repression of autophagy and Hcy;the following connections to Hcy and autophagy have been made: intracellular nitrous oxide production, intracellular calcium production, and reactive oxygen species production. Further work remains to be elucidated concerning the specific mechanisms under which autophagy occurs and possible Hcy-mediated connections. Moreover, the therapeutic implications might be of some promise to patients.

关键词： Beclin-1 bnip3 Calpain-1 Ischemia/reperfusion injury Mitochondrial pore transition Oxidative stress

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DNase activation by hypoxia-acidosis parallels but is independent of programmed cell death

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LIFE SCIENCES 2012年第7-8期91卷 223-229页

作者： Thompson, John W. Graham, Regina M. Webster, Keith A. Univ Miami Sch Med Dept Mol & Cellular Pharmacol Miami FL 33136 USA Univ Miami Sch Med Vasc Biol Inst Miami FL 33136 USA

Aims: Hypoxia, acidosis and programmed cell death are each hallmarks of acute myocardial infarction (AMI). We previously described a death pathway of cardiac myocytes mediated by hypoxia-acidosis that was characterized by activation of the Bcl2-family protein bnip3 and programmed necrosis. The pathway included extensive DNA fragmentation that was sensitive to inhibition of the mitochondrial permeability transition pore (mPTP) and calpain inhibitors, but not caspase inhibitors. We did not identify the DNases responsible for DNA cleavage. Main methods: Neonatal rat cardiomyocytes were subjected to hypoxia with and without concurrent acidosis, and the cellular localization of apoptosis-inducing factor (AIF), DNase II and caspase-dependent DNase (CAD) were determined. Key findings: Here we report the occurrence of biphasic pH-dependent translocations of AIF and DNase II but no change in CAD or its inhibitor ICAD. AIF co-localized with the mitochondria under aerobic and hypoxia-neutral conditions but translocated to the nucleus at pH similar to 6.7 coincident with a decrease of the mitochondrial membrane potential. DNase II co-localized with lysosomes under normoxia and hypoxia-neutral conditions, and translocated to the nucleus at pH similar to 6.1 coincident with the appearance of single strand DNA cuts. Inhibition of the mPTP pore with BH4-TAT peptide, calpain inhibition with PD150606, or knockdown (MD) of bnip3 failed to prevent nuclear translocation of these DNase although bnip3 MD blocked mitochondrial fission. Significance: These results suggest that caspase-independent DNA fragmentation is precisely regulated and occurs in parallel but independently from programmed necrosis mediated by hypoxia-acidosis. (C) 2012 Elsevier Inc. All rights reserved.

关键词： Hypoxia DNase II Cardiac myocyte Ischemia Apoptosis inducing factor Mitochondrial fission bnip3 Calpains

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Concanavalin-A-induced autophagy biomarkers requires membrane type-1 matrix metalloproteinase intracellular signaling in glioblastoma cells

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GLYCOBIOLOGY 2012年第9期22卷 1245-1255页

作者： Pratt, Jonathan Roy, Rene Annabi, Borhane Ctr Rech BioMED Lab Oncol Mol Quebec City PQ Canada Univ Quebec Dept Chim Ctr Rech PharmaQAM Montreal PQ H3C 3P8 Canada

Pre-clinical trials for cancer therapeutics support the antineoplastic properties of the lectin from Canavalia ensiformis [Concanavalin-A, ConA) in targeting apoptosis and autophagy in a variety of cancer cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP), a plasma membrane-anchored matrix metalloproteinase, is a glycoprotein strongly expressed in radioresistant and chemoresistant glioblastoma that mediates pro-apoptotic signalling in brain cancer cells, we investigated whether MT1-MMP could also signal autophagy. Among the four lectins tested, we found that the mannopyranoside/glucopyranoside-binding ConA, which is also well documented to trigger MT1-MMP expression, increases autophagic acidic vacuoles formation as demonstrated by Acridine Orange cell staining. Although siRNA-mediated MT1-MMP gene silencing effectively reversed ConA-induced autophagy, inhibition of the MT1-MMP extracellular catalytic function with Actinonin or Ilomastat did not. Conversely, direct overexpression of the recombinant Wt-MT1-MMP protein triggered proMMP-2 activation and green fluorescent protein-microtubule-associated protein light chain 3 puncta indicative of autophagosomes formation, while deletion of MT1-MMP's cytoplasmic domain disabled such autophagy induction. ConA-treated U87 cells also showed an upregulation of bnip3 and of autophagy-related gene members autophagy-related protein 3, autophagy-related protein 12 and autophagy-related protein 16-like 1, where respective inductions were reversed when MT1-MMP gene expression was silenced. Altogether, we provide molecular evidence supporting the pro-autophagic mechanism of action of ConA in glioblastoma cells. We also highlight new signal transduction functions of MT1-MMP within apoptotic and autophagic pathways that often characterize cancer cell responses to chemotherapeutic drugs.

关键词： Autophagy bnip3 Concanavalin-A Glioblastoma MT1-MMP

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Autophagy and senescence in cancer-associated fibroblasts metabolically supports tumor growth and metastasis, via glycolysis and ketone production

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CELL CYCLE 2012年第12期11卷 2285-2302页

作者： Capparelli, Claudia Guido, Carmela Whitaker-Menezes, Diana Bonuccelli, Gloria Balliet, Renee Pestell, Timothy G. Goldberg, Allison F. Pestell, Richard G. Howell, Anthony Sneddon, Sharon Birbe, Ruth Tsirigos, Aristotelis Martinez-Outschoorn, Ubaldo Sotgia, Federica Lisanti, Michael P. Thomas Jefferson Univ Kimmel Canc Ctr Jefferson Stem Cell Biol & Regenerat Med Ctr Philadelphia PA 19107 USA Thomas Jefferson Univ Kimmel Canc Ctr Dept Stem Cell Biol & Regenerat Med Philadelphia PA 19107 USA Thomas Jefferson Univ Kimmel Canc Ctr Dept Canc Biol Philadelphia PA 19107 USA Univ Calabria Dept Cellular Biol I-87036 Cosenza Italy Thomas Jefferson Univ Dept Surg Philadelphia PA 19107 USA Thomas Jefferson Univ Kimmel Canc Ctr Dept Med Oncol Philadelphia PA 19107 USA Univ Manchester Manchester Acad Hlth Sci Ctr Manchester Breast Ctr Paterson Inst Canc ResSch Canc Manchester M13 9PL Lancs England Univ Manchester Manchester Acad Hlth Sci Ctr Paterson Inst Canc ResSch Canc Breakthrough Breast Canc Res Unit Manchester M13 9PL Lancs England Thomas Jefferson Univ Dept Pathol Anat & Cell Biol Philadelphia PA 19107 USA IBM Thomas J Watson Res Ctr Computat Genom Grp Yorktown Hts NY USA

Senescent fibroblasts are known to promote tumor growth. However, the exact mechanism remains largely unknown. An important clue comes from recent studies linking autophagy with the onset of senescence. Thus, autophagy and senescence may be part of the same physiological process, known as the autophagy-senescence transition (AST). To test this hypothesis, human fibroblasts immortalized with telomerase (hTERT-BJ1) were stably transfected with autophagy genes (bnip3, CTSB or ATG16L1). Their overexpression was sufficient to induce a constitutive autophagic phenotype, with features of mitophagy, mitochondrial dysfunction and a shift toward aerobic glycolysis, resulting in L-lactate and ketone body production. Autophagic fibroblasts also showed features of senescence, with increased p21(WAF1/CIP1), a CDK inhibitor, cellular hypertrophy and increased beta-galactosidase activity. Thus, we genetically validated the existence of the autophagy-senescence transition. Importantly, autophagic-senescent fibroblasts promoted tumor growth and metastasis, when co-injected with human breast cancer cells, independently of angiogenesis. Autophagic-senescent fibroblasts stimulated mitochondrial metabolism in adjacent cancer cells, when the two cell types were co-cultured, as visualized by MitoTracker staining. In particular, autophagic ATG16L1 fibroblasts, which produced large amounts of ketone bodies (3-hydroxy-butyrate), had the strongest effects and promoted metastasis by up to 11-fold. Conversely, expression of ATG16L1 in epithelial cancer cells inhibited tumor growth, indicating that the effects of autophagy are compartment-specific. Thus, autophagic-senescent fibroblasts metabolically promote tumor growth and metastasis, by paracrine production of high-energy mitochondrial fuels. Our current studies provide genetic support for the importance of "two-compartment tumor metabolism" in driving tumor growth and metastasis via a simple energy transfer mechanism. Finally, beta-galactosid

关键词： tumor stroma cancer-associated fibroblasts glycolysis autophagy senescence cancer metabolism bnip3 bnip3L beclin1 ATG16L1 cathepsin B

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