【作者】 鞠培新；

【导师】 石文君；

【作者基本信息】 中国医科大学 ， 胸外科， 2007， 博士

【摘要】 目的肺癌是我国发病率和死亡率最高的恶性肿瘤,人们都在努力寻找新的抗癌制剂或化疗方案以期提高肺癌的治愈率。肺癌已成为大城市中最常见的恶性肿瘤之一。化学治疗目前仍然是肺癌综合治疗的主要手段,细胞毒类药物主要杀伤肿瘤群体中的敏感细胞,原发或继发耐药细胞则是肿瘤复发与/或转移的根源。国内外学者一直在寻找更有效的化疗药物以解决不断出现的耐药和复发问题。我国学者成功应用三氧化二砷（AS2O3）治疗急性早幼粒细胞白血病（AcutePmmyelocytic Leukemia,APL）,引起了国内外医学界的极大兴趣。AS2O3对于其抗癌作用的研究,已经从血液系统肿瘤进入实体肿瘤,先后相继开展了AS2O3抗消化道肿瘤、生殖系统肿瘤以及泌尿系统肿瘤作用及其机制的研究。国内外研究表明,三氧化二砷具有抑制内皮细胞增殖及血管生成,选择性抑制白血病细胞在内的多种肿瘤细胞生长,诱导肿瘤细胞分化及凋亡的作用。研究表明三氧化二砷可抑制前列腺癌细胞增殖并诱导凋亡,其机制可能与三氧化二砷活化半胱天冬酶（Caspase）-3,8,9有关。有学者报道高浓度三氧化二砷可诱导HL-60和U937细胞凋亡,bcl-2及c-myc基因表达逐渐降低,同时下调拓扑异构酶Ⅱ（TOPOⅡ）基因表达、降低TOPOⅡ的活性。三氧化二砷作用白血病ML-1细胞后,抑制细胞增殖同时引起细胞周期的G2/M期停滞。诱导肿瘤细胞凋亡是肿瘤治疗的重要目的和手段。AS2O3可诱导胆囊癌细胞凋亡蛋白下调Bcl-2的表达,Bcl-2过表达可抑制AS2O3诱导凋亡的作用。AS2O3在诱导U937凋亡过程中下调肿瘤相关基因c-myc、bcl-2的表达,同时下调拓扑异构酶Ⅱ（TOPOⅡ）表达、降低TOPOⅡ的活性,在抑制细胞增殖同时引起细胞周期的G2/M期停滞。国外研究表明AS2O3能够诱导肺癌A549细胞凋亡,下调生存素（Survivin）基因表达,AS2O3诱导K562细胞凋亡时上调Smac/DIABLO蛋白表达,降低Bcl-2/Bax比值,下调Survivin基因表达:AS2O3在诱导肺癌细胞株NCI-H157细胞凋亡过程中的对Survivin基因表达及与Caspase的影响未见报道。肿瘤细胞凋亡凋亡途径由Caspase家族蛋白酶介导和执行。Caspase家族在细胞凋亡的分子机制中作为重要的效应子执行凋亡。Caspase激活的凋亡有外源性和内源性两条途径,外源性途径通过细胞表面死亡受体介导,内源性途径即线粒体信号通路由bax/bcl-2通路的活化而启动,两条途径均导致Caspases级联激活,最终活化Caspase-3。凋亡抑制蛋白（inhibitor of apoptosis protein,lAPs）通过与Caspase相互作用来抑制细胞凋亡,Survivin是IAPs家族成员之一,在多种肿瘤细胞以及造血系统恶性肿瘤均高表达。Survivin通过直接抑制Caspase-3和Caspase-7的活性发挥抗凋亡作用。目前三氧化二砷诱导NCI-H157细胞凋亡过程中的Survivin及Caspase-3的作用目前尚未见报道。丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinases,MAPK）信号转导系统是细胞外信号引起核反应的细胞信息传递的共同通路,包括细胞外信号调节激酶（Extracellular signal-regulated kinases,ERK）、C-JUN氨基末端激酶（c-junN-terminal kinases,JNK）和p38丝裂原活化蛋白激酶（p38 Mitogen-Activated ProteinKinases,p38）三个主要成员。多种细胞外刺激均可将信号传至细胞内,引起MAPK系统激酶连锁磷酸化。已有证据显示,MAPK信号传导通路的异常与肿瘤细胞增殖、分化、凋亡关系密切。ERK1和ERK2是ERK中的两个重要成员,磷酸化的ERK（P-ERK）是其活化形式,参与调节细胞的生长、分化、凋亡等生理过程,其中Ras—Raf-1—MEK1/2—ERK1/2通路是细胞外有丝分裂信号引起细胞增殖的共同通路。国外研究表明,在一些肿瘤细胞中,MEK抑制剂PD 98059通过抑制ERK活性进而抑制肿瘤生长,抑制MEK/ERK的活力可以放大紫杉醇、顺铂等化疗药物诱导肿瘤细胞凋亡作用。目前有关三氧化二砷诱导NCI-H157细胞凋亡过程中信号转导机制的研究并无报道。为进一步明确三氧化二砷诱导肺癌细胞凋亡的确切机理,本实验拟采用三氧化二砷作用于非小细胞肺癌NCI-H157细胞,观察三氧化二砷对NCI-H157细胞增殖抑制、诱导凋亡作用,进一步研究三氧化二砷在诱导凋亡过程中对Survivin基因表达及Caspase-3的影响,阐述MAPK信号转导通路在三氧化二砷诱导细胞凋亡过程中的可能机制。方法1、细胞增殖测定:采用MTT法检测细胞生长指数,绘制增殖曲线。2、细胞凋亡的检测:吖啶橙荧光染色形态学分析及流式细胞仪进行DNA含量检测细胞凋亡。透射电镜等技术进行细胞形态学观察。3、RT-PCR:检测Survivin mRNA表达。4、Western Blot:检测Survivin、Caspase-3蛋白及p-ERK1/2蛋白。5、统计学分析所有数据用3次独立实验的平均值表示,结果进行t检验、方差分析,用SPSS11.5统计软件进行数据分析。结果1、三氧化二砷对NCl-H157细胞增殖抑制及诱导凋亡作用三氧化二砷以时间、剂量依赖的方式抑制NCI-H157细胞增殖,24h、48h和72h抑制细胞增殖50%的药物浓度(IC₅₀)分别为11.6μmol/L、8.1μmol/L及5.8μmol/L。2、三氧化二砷在诱导NCI-H157细胞凋亡过程中对Survivin基因表达、Caspase-3的影响RT-PCR结果显示,5/zmol/L三氧化二砷作用12h～48h Survivin mRNA表达逐渐下降。Western Blot解析结果显示,Survivin蛋白表达在5μmol/L三氧化二砷处理后逐渐下调,处理24h后出现17KD Capase-3的活性亚基,直至72h。3、ERK信号转导通路在三氧化二砷诱导NCI-H157细胞凋亡过程的影响（1）PD98059促进三氧化二砷对NCI-H157细胞增殖抑制作用预先用20μmol/L PD98059处理NCI-H157细胞1小时,然后加入5μmol/L的三氧化二砷继续作用24 h、48 h、72h,加用PD98059后,24 h、48 h、72h细胞的增殖率分别由77.4%、59.2%、30.2%下降至62.7%、43.6%和16.1%（P＜0.05）,提示PD98059促进了三氧化二砷对NCI-H157细胞增殖抑制作用。（2）PD98059增强三氧化二砷对NCI-H157细胞凋亡作用①单用20μmol/L的PD98059作用NCI-H157细胞24h,形态学可见细胞生长良好,未见到凋亡改变。单用5μmol/L的三氧化二砷作用NCI-H157细胞24h始,形态学观察可见细胞凋亡改变,用20μmol/L的PD98059预先处理NCI-H157细胞1h,然后加入5μmol/L的三氧化二砷继续共同孵育24h,同单用三氧化二砷组比,形态学凋亡改变更加明显。流式细胞仪分析提示,同单用三氧化二砷组比较,联合应用组处理NCI-H157细胞24h,,亚G₁期细胞比例由20.5%增加至39.3%（P＜0.05）。②三氧化二砷5μmol/L分别处理NCI-H157细胞5min～72h,Western Blot解析结果显示,p-ERK1/2蛋白在5 min即可出现,表达持续增强至3 h,后逐渐减弱,至72 h后消失。20μmol/L PD98059预先处理NCI-H157细胞1h后,加入三氧化二砷5μmol/L继续处理至24 h,未检测到p-ERK1/2 MAPK蛋白的表达,提示三氧化二砷可以活化ERK信号转导通路。③20μmol/L PD98059预先处理NCI-H157细胞1 h后加入三氧化二砷5μmol/L共同孵育至24 h,Survivin mRNA表达由单用三氧化二砷组59.4%,降至联合应用的29.6%。Western Blot解析结果显示,Survivin蛋白的表达水平由单用三氧化二砷组的56.8%下调至联合组22.8%（P＜0.01）。非激活状态32KD的proCaspase-3由单用三氧化二砷组的46.5%下调至12.8%,17KD的活性亚基明显增加（P＜0.01）,表明PD98059阻断ERK信号转导通路促进了三氧化二砷诱导细胞凋亡时的Survivin蛋白表达下调和Caspase-3活化。研究表明,三氧化二砷可选择性抑制多种人类肿瘤细胞生长,诱导肿瘤细胞凋亡与分化,高浓度的三氧化二砷处理HL60、K562和U937细胞,可见凋亡的形态学改变,低浓度下诱导这三种细胞株向单核/巨噬细胞样细胞分化,并下调肿瘤相关基因如c-myc、bcl-2、WT1等基因的表达。Yeh报道三氧化二砷可以抑制前列腺癌增殖并诱导细胞凋亡,其机制可能与三氧化二砷活化Caspase-3,8,9有关,进一步研究表明三氧化二砷诱导HL-60细胞凋亡下调bcl-2、Survivin基因表达,活化Caspase-3。本实验结果显示,三氧化二砷以时间、剂量依赖的方式抑制NCI-H157细胞增殖。三氧化二砷单独应用,在高于5μmol/L诱导NCI-H157细胞凋亡。5μmol/L三氧化二砷作用Survivin mRNA及蛋白表达表达逐渐下降,逐渐出现17KD Caspase-3的活性亚基。首次证实三氧化二砷诱导NCI-H157细胞凋亡过程中下调Survivin基因及蛋白表达、活化Caspase-3。国外研究表明,MEK抑制剂PD 98059可以抑制ERK的活性,抑制MEK/ERK信号转导通路可以放大多种化疗药物诱导肿瘤细胞凋亡的作用,应用MEK抑制剂可增强紫杉醇诱导的凋亡作用,ERK激活的同时具有介导细胞凋亡的作用。有学者报道MEK1抑制剂还可以明显的促进三氧化二砷诱导K562细胞凋亡。研究表明,ERK信号途径的激活对三氧化二砷诱导U937细胞的凋亡是必须的,我们应用ERK抑制剂PD98059协同三氧化二砷处理NCI-H157细胞,其杀伤细胞的作用明显高于三氧化二砷的单独作用。这说明ERK信号通路可能由三氧化二砷介导激活,而PD98059部分阻断ERK的活化后,能够促进三氧化二砷诱导NCI-H157凋亡,进一步检测磷酸化ERK1/2蛋白,结果证实三氧化二砷作用短时间内即活化了ERK1/2通路。基于已有的研究证实ERK信号传导系统在促进细胞增殖及抗凋亡方面起重要作用,由此我们推测,被三氧化二砷激活的ERK信号通路可能保护了NCI-H157细胞,使其具有一定的抗凋亡作用,启动了细胞凋亡的防御机制。本研究用PD98059预处理NCI-H157细胞与三氧化二砷共同培养,Survivin mRNA及蛋白的表达水平进一步下调,Caspase-3酶原表达水平明显下降,而17KD的Caspase-3活性亚基表达水平明显增加,提示抑制ERK信号促进三氧化二砷诱导NCI-H157细胞Survivin表达进一步下调和Caspase-3活化。首次证实三氧化二砷诱导NCI-H157细胞凋亡过程中激活了ERK信号转导通路,并下调Survivin表达,活化Caspase-3,提示二者均位于ERK信号的下游。结论1.三氧化二砷以时间、剂量依赖的方式抑制NCI-H157细胞增殖,诱导NCI-H157细胞凋亡。2.三氧化二砷诱导NCI-H157细胞凋亡过程中下调Survivin mRNA及蛋白表达,并活化Caspases-3,提示三氧化二砷诱导的NCI-H157细胞凋亡可能与下调Survivin基因表达及活化Caspase-3有关。3.抑制ERK信号转导通路增强三氧化二砷诱导NCI-H157细胞凋亡,并进一步下调Survivin表达、活化Caspase-3,提示ERK途径可能参与了三氧化二砷诱导NCI-H157细胞凋亡的信号转导系统,抑制该途径,可起到增强三氧化二砷诱导凋亡作用。更多还原

【Abstract】 Lung cancer is a malignancy with a predominant morbidity and mortality.It gets to be a menace not only to the health but also to the existance of human being because of the fact that the elevation of the amplitude of its mortality being the first among all the tumors.The development of lung cancer is a complicated process,most likely it is the result of interaction between a group of environmental and hereditary factors.A vast amount of studies indicate that many gene.AS₂O₃ can inhibit the growth of various lines of human cancer cells,due at least in part to its specific effect on topoisomerase（topo）Ⅱ.A variety of differentiation-inducing agents,such as camptothecin,VP16,geranylacetone and all-trans-retinoic acid,were shown to induce apoptosis in tumor and normal cells.AS₂O₃ alone is only a modest inducer of differentiation of AML cells in primary culture.AS₂O₃ restored RA sensitivity to previously resistant NCI-H157 cells. Therefore,AS₂O₃ seems to act as a potent differentiation- and apoptosis-inducing agent in cancer cells.U937 cells.When U937 cells were treated with AS₂O₃ in the absence of serum,mitogen-activated protein（MAP） kinase activity was markedly increased after the start of treatment and elevated.Prior to the activation of MAP kinase,increased activities of Ras,Raf-1,and MAP kinase kinase were found,but these enzymes were transiently activated by the treatment with AS₂O₃.These results suggest that the signal was transmitted sequentially from Ras,Raf-1,and MAP kinase kinase to MAP kinase. That a pathway with the persistent activation of MAP kinase in U937 cells in response to AS203 is at least one of the signal transduction pathways involved in the induction of apoptosis.It has been reported that AS₂O₃ utilizes the extracellular signal-regulated kinase（ERK） cascade for signal transduction,leading not only to cell differentiation but also to apoptosis.It is thus postulated that the ERK cascade may control AS₂O₃ induced cell differentiation and apoptosis simultaneously.Caspases are cysteine proteases as the executioners of apoptosis.They activated, initiator Caspases activate downstream Caspases,resulting in an amplification of the Caspase cascade.Active Caspases cleave critical cellular protein substrates and systematically dismantle the cell.The availability and activation of Caspases are crucial steps in the commitment of a cell to die,are known to survive for the life of the organism,inhibition of apoptosis is a primary consideration in setting up cellular survival mechanisms.When in close proximity Caspases can autoactivate. Baculoviruses harbor yet another type of Caspase inhibitor,the inhibitor of apoptosis proteins（IAPs）.These proteins are characterized by the presence of a homologous domain named the baculoviral IAP repeat（BIR） domain and IAPs.Survivin,a single BLR domain containing IAP,is upregulated in many common human tumors, implicating that the deregulation of IAPs contributes to human disease.Understanding of the mechanism by which IAPs inhibit apoptosis has come with the demonstration that XIAP can physically interact with and block Caspase-3,-7,and -9.Survivin,a novel inhibitor of apoptosis,expressing in a cell cycle-dependent manner,regulates the G（2）/M phase of the cell cycle by associating with mitotic spindle microtubules;it directly inhibits Caspase-3 and Caspase-7activity.During tumorigenesis,Survivin expression is inversely correlated with apoptosis and is positively correlated with proliferation and angiogenesis.Survivin expression up-regulation predicts short survival and poor prognosis in human cancers.Survivin targeting antisense nucleotide and Survivin mutants induce apoptosis,reduce tumor growth potential,and Sensitize tumor cells to chemotherapeutic drugs and X-irradiation in vitro and in vivo.These results suggest that Survivin may has the potential function as a new target for the diagnosis and treatment of cancer. Mitogen-activated protein kinases（MAPKs） participate in signaling cascades from transmission of extracellular signals into their intracellular targets,which regulate important biologic activities.The MAP kinase family members have been implicated in events necessary for proliferation,differentiation,apoptosis,and certain kinds of stress responses.Three major groups of Map kinases exist:the p38 Map kinase family,the extracellular signal-regulated kinase（ERk） family,and the c-Jun NH2-terminal kinase （JNK） kinase.Map kinase signaling cascades are activated by a variety of different cellular stimuli（stress,cytokine） and mediate diverse responses,The ERK pathway is activated in response to several cytokines and growth factors,and primarily mediates mitogenic and antiapoptotic signals.It has been suggested that the excessive activation of the ERK-kinase cascade,which is generally known to play a role in cell survival,is an event necessary for AS₂O₃-mediated apoptosis.Such a hyperexcitation of the ERK-kinase cascade is due to the activation of an upper signal component Ras and the concomitant downregulation of the protein kinase A activity that may negatively regulate the Raf-1 function.However,signal transduction leading to AS₂O₃ mediated cell differentiation has not been explored.In order to characterize the mechanisms of apoptosis-inducing effect of AS₂O₃ on NCI-H157 cells,we examined the expression of Survivin and role of ERK pathway in AS₂O₃-induced apoptosis.Methods1、Assays for the proliferationThe NCI-H157 cell proliferation was determined by MTT.2、Assay for apoptosisThe apoptosis of NCI-H157 cell was assessed by morphological analysis.Cell Cycle Analysis Using a Becton Dickinson FACScan flow cytometer（Cambridge,MA） using a commercially available software program.3、Western Blot analysis Survivin,Caspase-3 and p-ERK were analyzed by Western blot 4、RT-PCRSurvivin mRNA mRNA expression was detected by semi-quantitative RT-PCR.5、Statistical Analysis.The significance of differences between experimental conditions was determined using the two-tailed Student t testResults1.AS₂O₃ induced apoptosis in a dose and time- dependent fashion in NCI-H157 cells.2.Proliferation of NCI-H157 cells was inhibited by AS₂O₃ and the IC₅0 at 24h, 48h and 72h were 11.6μmol/L、8.1μmol/L and 5.8μmol/L,respectively.Apoptosis of NCI-H157 cells were induced when the cells were treated with AS₂O₃ at concentrations of 5μmol/L and higher.cell hypodiploid percentage at 24h,48h and 72h was 13.7 %,20.5%及37.3%by flow cytometry analyses.respectively Compared with control,treatment with AS₂O₃ at 5μmol/L for 12 h,24 h and 48h resulted in decrease of Survivin protein expression.The cleaved active subunits of Caspase-3 were observed following AS₂O₃ treatment from 24h to 72h.3.We found that in NCI-H157 acute promyelocytic cell line treatment with the MEK inhibitors PD98059 greatly enhances apoptotic cell death induced by AS₂O₃ alone.Combined treatment results in downregulation of the Survivin in NCI-H157 cell line.The levels of p-ERK were determined by Western Blot analysis at 5 minute after treatment with AS₂O₃ alone.DiscussionsSomeone reports that AS₂O₃ induced apoptosis of HL60,ML1 and U937 cell in high concentration,and induced differentiation toward monocyte/macrophage-like cells in low concentration.Our results showed that AS₂O₃ alone induced apoptosis of NCI-H157 cells and NCI-H157 in the concentration of more than 5μmol/L,but there is no apoptosis in less than 5μmol/L.Some observations were reported that MEK1/2 inhibitors potentiate the antitumor activity of various cytotoxic agents,including ara-C, cisplatin,and paclitaxel,suggests a possible role for MEK1/2 inhibitors in the treatment of human malignancies.At present,mechanism of AS₂O₃ is still unknown.It possibly actived APK,inhibit TopoⅡactivity,and up-regulated Tiaml or down-regulated the express of c-myc and bcl-2 mRNA to induce apoptosis.To explore details involved in the process,apoptosis-related protein Survivin was determined by Western Blot and RT-PCR,Caspase-3 also was tested by Western Blot. We found that exposure of NCI-H157 cells to AS₂O₃ resulted in down-regulation of Survivin and Caspase-3 protein,down-regulation of Survivin mRNA was also detected.Furthermore,Survivin downregulation and activation of Caspase-3 became more marked in coadministration of AS₂O₃ with PD98059,the results indicates MEK/ERK signaling pathway mediates progess of AS₂O₃-induced apoptosis involved in Survivin downregulation in the post-transcription level.Conclusions1.AS₂O₃ inhibited NCI-H157 cell proliferation in a time- and dose-dependent manner and induced NCI-H157 cell apoptosis2.AS₂O₃ induced NCI-H157 cell apoptosis accompany with Survivin downregulation and activation of Caspase-3.3.MEK/ERK signaling pathway is negative regulation in AS₂O₃- induced apoptosis in NCI-H157 cell.更多还原