【作者】 张须龙；

【导师】 田志刚； 张建；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2008， 博士

【摘要】 研究目的:癌症作为一种恶性疾病严重威胁着人类身体健康,总死亡率为23%左右且半个多世纪以来居高不下。据美国癌症研究协会2008最新统计,2008年预计肺癌发病率占总肿瘤发病率15%,但是肺癌的死亡率远远高于其他肿瘤,占总死亡率的29%。另据2005年新英格兰医学杂志报导,癌症已成为中国男性第一位、女性第三位死亡原因,肺癌死亡率为肿瘤致死中的第一位。在临床上肺癌主要分为小细胞肺癌（13%）和非小细胞肺癌（87%）。虽然现在治疗手段很多,例如手术治疗、放疗、化疗和靶向生物疗法以及联合治疗,但是肺癌的1年生存率约为41%,而5年生存率只有15%,被认为是目前对人类健康和生命威胁最大的恶性肿瘤。肺癌放疗、化疗在临床上广泛应用,但他们都缺少靶向性,给患者带来很大的副作用。近年来,许多有效的肿瘤基因治疗靶点的发现为肺癌基因治疗提供了新思路。信号转导子和转录激活子（Signal Transducers and Activators ofTranscription,STAT）家族是潜在的胞质转录因子,在细胞中起到传递胞浆信号和启动核内基因转录的双重功能,尤其是STAT3,在胚胎发育和细胞增殖分化中起重要作用。在正常组织中,STAT3的活化是瞬时的并受到严格调控。然而,在许多血液性和实体肿瘤组织中STAT3是持续活化的,其中包括白血病、多发性骨髓瘤、前列腺癌、乳腺癌、肺癌、卵巢癌和胃癌等。IL-6、EGF等细胞因子与受体结合后酪氨酸激酶被激活,从而激活STAT3。磷酸化STAT3分子相互作用形成二聚体,然后转位到核内,与靶基因启动子区域特异DNA反应元件结合,启动目的基因转录。持续激活的STAT3调控多种基因的表达,包括负调免疫应答的因子（如IL-10、VEGF、TGF-β等）以及细胞增殖、凋亡、周期相关的基因（bcl-xl、cyclinD1、mcl-1、survivin、cyclinE等）,促使肿瘤增殖、抑制细胞凋亡、诱导血管生成和逃逸免疫监视,STAT3被认定是癌基因。阻断核转录因子与DNA结合区（主要为promoter）的结合是目前靶向基因群进行干预的有效方法之一。阻断核转录因子与DNA结合多采用转录因子诱骗（TFD）方法,该方法为人工合成的20-30bp双链DNA,与转录因子作用元件序列相同且与转录因子有高亲和力的寡核苷酸片段（Decoy-ODN）,Decoy-ODN被导入目的细胞后,可作为“诱骗”顺式作用元件与靶基因顺式作用元件竞争结合转录因子,改变核转录因子所调控靶基因表达。与其它方法相比,“诱骗”技术具有独到的优点,例如序列短、易进入细胞、特异性高、可实现基因的群谱控制等,具有很大的应用潜力。诱骗寡核苷酸技术已广泛用作基因功能研究的工具,已有大量临床研究评估它们疾病治疗的潜在价值。化疗一直是临床肿瘤治疗的常用方法,但多药耐药（MDR）是有效治疗癌症中最大的难题,其中P-糖蛋白（P-gp）的过表达为MDR的主要原因。目前为止,已发现了很多MDR的逆转剂,但这些药物在临床上的应用却不尽人意。因此,发展一种新的能够阻止P-gp介导的MDR药物或者策略就显得十分重要。诱导肿瘤细胞P-gp的表达和获得多药耐药的机制尚不是十分清楚,那么鉴定这个机制和寻找诱导因子就显得十分急需。因为持续活化的STAT3在许多恶性肿瘤中对化疗诱导的凋亡有抑制作用,而抑制STAT3活性则能提高癌细胞的化疗敏感性:在很多耐药细胞中STAT3的mRNA较敏感细胞是过表达的,其活性也是特异性提升;同时还发现,在临床上恶性程度高的肿瘤通常展现对化疗药物的不敏感性,而且它们的STAT3也是高度活化的。所有这些结果都提示STAT3可能参与了肿瘤细胞的多药耐药,与MDR间有一定关联。本研究采用STAT3 decoy ODN在体内外阻断人肺癌细胞中活化的STAT3,观察对肺癌细胞增殖的抑制作用,并进一步阐述了作用机制;在此基础上,我们研究了阻断STAT3增强白血病耐药细胞对化疗药物的敏感性,探索靶向STAT3逆转肿瘤细胞耐药性的可行性,为肿瘤治疗提供新的思路和途径。研究方法Western印迹和凝胶电泳迟滞实验（EMSA）方法检测肺巨细胞癌细胞系PG和非小细胞肺癌细胞系A549中STAT3的表达和磷酸化活化情况及DNA结合能力。细胞计数法观察JAK2特异性抑制剂AG490对细胞增殖的影响。脂质体转染dccoy-ODN或scramble-ODN,流式细胞仪及荧光显微镜观察其转染效率及在细胞中的定位。荧光素酶报告基因实验分析STAT3 decoy ODN对肺癌细胞STAT3活性的特异性影响。计数法及³H掺入法检测转染decoy ODN后对细胞增殖的影响,流式细胞仪Annexin V和PI双染法检测细胞凋亡的变化。RT-PCR及Western印迹检测decoy ODN对下游细胞周期和抗凋亡相关基因表达的变化。建立A549裸鼠肿瘤异种移植模型,瘤内注射decoy ODN观察对肿瘤生长变化影响。TUNEL分析decoy ODN在体内对细胞凋亡的影响。免疫组化分析decoyODN在体内对下游细胞周期和抗凋亡相关基因表达变化。Western印迹和荧光素酶报告基因实验分析耐药细胞K562/A02和敏感细胞K562中STAT3活性及下游基因表达。Western印迹分析JSI-124对K562/A02细胞中STAT3磷酸化的时间和浓度依赖性抑制关系。CCK-8及细胞计数检测抑制STAT3活化后K562/A02细胞对阿霉素的敏感性,并利用流式细胞仪检测对细胞凋亡的影响和胞内阿霉素的蓄积变化。荧光定量PCR检测JSI-124对K562/A02细胞中MDR1及其他MDR相关基因转录水平的影响。Western印迹检测JSI-124对K562/A02细胞中P-gp表达的影响。选择Jak2特异性抑制剂AG490为对照,western检测对STAT3磷酸化的影响。CCK-8法检测AG490增加K562/A02细胞对阿霉素的敏感性。荧光定量PCR检测AG490对K562/A02细胞中MDR1及其他MDR相关基因转录水平的变化。Western印迹检测AG490对K562/A02细胞中P-gp表达的影响。计算机辅助生物信息学分析MDR1启动子序列中STAT3的可能结合位点。结果一1、根据文献报道及转录因子结合位点数据库,我们设计了具有与STAT3强结合能力的诱骗序列,经生物信息学进一步分析显示该诱骗寡核苷酸序列具有强的与STAT3结合能力。2、持续激活的STAT3对肺癌细胞的增殖起关键作用。Western印迹结果显示,肺巨细胞癌细胞系PG和非小细胞肺癌细胞系A549中STAT3高表达并持续磷酸化（Try705,Ser727）。EMSA结果显示,³²p标记的STAT3 decoy ODN与核蛋白抽提物孵育后有寡核苷酸与蛋白的结合带,而³²p标记的随机序列（Scramble）核苷酸没有结合带,进一步说明胞内有活化的STAT3,同时证明我们设计的诱骗序列具有强的与STAT3结合的能力。特异性抑制剂AG490阻断Jak2后能显著抑制细胞增殖,说明Jak2/STAT3信号通路在PG和A549增殖中的关键作用。3、decoy-ODN能高效转染入细胞并大多数定位于细胞核中。脂质体转染ODN-FITC后,流式细胞仪检测结果显示ODN能有效进入细胞,并且转染的量与ODN浓度成正比。转染后DAPI染细胞核,荧光显微镜观察发现ODN—FITC大多数定位于细胞核中。4、STAT3 decoy ODN特异性阻断STAT3转录活性.STAT3 decoy ODN对pGL3-STAT3-TK-luciferase荧光素酶的表达抑制作用呈浓度依赖性降低,当转染decoy ODN浓度越高时,STAT3诱导表达的荧光素酶活性越低,而scrambleODN对荧光素酶活性几乎没有影响。5、STAT3 decoy ODN能有效抑制PG和A549细胞的增殖。转染不同浓度的decoy-ODN后分别于24h、48h、72h检测细胞数。结果显示decoy-ODN能显著抑制细胞增殖,50nM decoy-ODN对PG的抑制率达到69%,而25nMdecoy-ODN对A549抑制率达64%。³H掺入法检测结果表明,decoy ODN可以显著降低PG和A549细胞对³H的摄取,抑制掺入率分别达到61%和66%（P＜0.01）,而STAT3 scramble ODN与对照比较没有显著变化（P＞0.05）。同时,decoy ODN对STAT3不活化的NIH/3T3细胞增殖没有显著影响。6、decoy-ODN诱导细胞凋亡。转染decoy-ODN 12h后,流式细胞仪Annexin-V/PI双染法检测细胞凋亡情况。结果表明,25nM decoy-ODN能显著诱导A549细胞凋亡,凋亡率从6.41%升高到29%。而scramble组凋亡率升高不明显。7、STAT3 decoy ODN下调STAT3下游细胞周期及抗凋亡相关基因的表达。分别以50nM和25nM decoy-ODN剂量转染PG和A549细胞,24h后检测STAT3的靶基因,RT-PCR结果显示STAT3 decoy ODN转染细胞后bcl-xl、cyclinD1、mcl-1、survivin、cyclinE等mRNA表达水平显著低于空白对照组细胞,westernblot也表明bcl-xl、cyclinD1的蛋白表达受到明显抑制。而scramble-ODN对细胞以上各种基因的表达水平无明显差异。8、STAT3 decoy ODN抑制裸鼠体内肿瘤生长并诱导凋亡。在裸鼠皮下接种A549细胞建立了A549裸鼠异种移植肿瘤模型,瘤内注射ODN或溶媒对照PBS,结果发现decoy ODN能有效抑制肿瘤体内生长,30天后瘤组织体积抑制率达到69.5%（p＜0.01）,瘤重抑制率达到52.7%。肿瘤组织冷冻切片,TUNEL法检测肿瘤组织凋亡细胞,发现decoy ODN处理组凋亡阳性数显著高于PBS处理组,增加约20倍（p＜0.001）,而scramble ODN处理组无明显差异。9、STAT3 decoy ODN通过下调其下游基因表达抑制裸鼠体内肿瘤生长。免疫组化检测肿瘤组织中bcl-xl和cyclin D1的表达变化,结果显示decoy组bcl-xl和cyclin D1表达均比对照组和scramble组有明显下降（p＜0.05）。结果二1、STAT3的活性在耐阿霉素的K562/A02细胞中比其敏感K562细胞中高。首先western blotting发现耐药细胞株K562/A02细胞中STAT3表达及活化水平明显比K562细胞中高,并且强表达与耐药相关的蛋白P-gp。荧光素酶报告基因实验结果显示,在耐药K562/A02细胞中有较高的STAT3依赖的转录活性。2、JsI-124（Cueurbitaein I）剂量和时间依赖性地抑制K562/A02细胞中STAT3的活化。JSI-124是一种高度特异的STAT3抑制剂,以不同剂量JSI-124处理K562/A02细胞后,经western检测磷酸化STAT3的水平,结果显示JSI-124能够剂量和时间依赖性地抑制STAT3活化,当以1μM JSI-124处理24小时即能明显抑制STAT3活化。3、阻断STAT3增加K562/A02细胞对阿霉素的敏感性并促进阿零素诱导的细胞凋亡。当在含有1μg/ml阿霉素的正常培养基中加入不同浓度JSI-124后发现,JSI-124能够剂量和时间依赖性的抑制K562/A02增殖。对K562/A02和K562细胞用不同浓度的阿霉素和1μM JSI-124联合或单独处理48h时,发现JSI-124能显著增加K562/A02细胞对阿霉素的敏感性,IC50降低约8倍左右。但是这种降低效应没有在药物敏感的K562细胞中发现。流式细胞仪检测凋亡结果显示,用JSI-124和阿霉素联合处理能显著提升K562/A02细胞的凋亡率,凋亡率从2.72±0.53%提高到11.67±2.8%。4、JSI-124部分通过增加K562/A02对阿霉素的摄取诱导细胞凋亡。JSI-124能剂量依赖性增加阿霉素在耐药细胞的蓄积（P＜0.05）,但是这个效应在药物敏感细胞中却不明显。当用JSI-124处理后,胞内罗丹明123荧光强度增高,说明JSI-124可以抑制P-gp介导的罗丹明123的外排作用。5、JsI-124下调K562/A02细胞中MDR1及其他MDR相关基因的转录和表达。荧光定量PCR结果显示,JSI-124可以剂量依赖性的降低K562/A02细胞中MDRI的转录。当用1.25μM JSI-124处理K562/A02细胞后,其MDR1下降到对照组细胞的38%,2.5μM JSI-124处理组只有对照组的25%,均具有统计学显著性差异（P＜0.01）;同时,MRP1和BCRP1的表达也显著降低。JSI-124能剂量依赖性的降低P-gp表达的丰度,用1.25μM和2.5μM JSI-124处理K562/A02细胞36小时后,P-gp的表达分别降低到对照DMSO处理组的47%和29%。6、Jak2特异性抑制剂AG490通过下调P-gp增强K562/A02对药物敏感度。AG490能够剂量依赖性地抑制K562/A02细胞中STAT3活性,并显著性增加K562/A02细胞对阿霉素的敏感性,100μM AG490能降低K562/A02细胞对阿霉素的IC50约8-16倍。AG490也能下调MDR1的转录,100μM AG490处理K562/A02细胞,MDR1的转录抑制率达到约57%（P＜0.01）,同时P-gp的表达到对照的58%。7、STAT3参与P-gp的表达调节。用TFSEARCH和TRANSFAC数据库,我们利用计算机辅助搜索P-gp启动子区域潜在的STAT3结合位点,搜索结果显示,在启动子区域中含有一个与STAT3结合的DNA序列,这个序列位于P-gp启动子中P-gp起始编码位点上游1014bp处,并具有较强的结合能力。结论及意义本研究证实肺癌细胞中STAT3是高度持续活化的,并在肺癌细胞增殖中起关键作用,STAT3 decoy ODN能特异性阻断肺癌细胞中活化的STAT3,在体内外抑制肺癌细胞增殖和诱导细胞凋亡,并进一步阐述了作用机制与下调STAT3靶向的抗凋亡基因、细胞周期相关基因有关;初步证明运用decoy ODN抑制STAT3通路作为肺癌基因治疗具有可行性。在此基础上,我们研究了阻断STAT3对白血病耐药细胞株对化疗药物阿霉素敏感性的影响。实验证明耐药细胞株中STAT3活性高于敏感细胞株,JSI-124抑制STAT3活化后逆转耐药细胞对化疗药物的耐药性、促进化疗药物诱导的凋亡,其机制为:抑制STAT3活化可降低胞内P-gp的表达,从而增加胞内阿霉素的蓄积,抑制细胞增殖。上述研究为探索靶向STAT3逆转肿瘤细胞耐药性的可行性、应用STAT3抑制剂和化疗药物联合用药治疗肿瘤细胞的多药耐药提供了理论和实验基础,证明靶向肿瘤细胞中活化的STAT3,在抑制肿瘤细胞增殖的同时逆转肿瘤多药耐药状态,有可能是肿瘤治疗的新靶点、新途径。更多还原

【Abstract】 Object:Malignant cancer which as a serious disease had great threat to human health,it accounted for 23%of all deaths,and had no downward trend in the past 50 years. According to the latest statistics of American Cancer Association in 2008,estimated new lung cancer cases accounted for 15%of cancer diagnoses,but it was the most cancer related deaths in both men and women,accounting for 29%of total cancer deaths.Meanwhile,in 2005,New England Journal of Medicine reported cancer has become the first in male and the third in female causes of death in China,and lung cancer was the first reason in cancer mortality.Lung cancer is classified clinically as small cell（13%）or non-small cell（87%）for the purposes of treatment.Although there are many treatment methods,such as surgery,radiotherapy,chemotherapy, targeted biological therapy,as well as combination treatment,the 1-year survival rate of lung cancer is about 41%,and 5-year survival rate only 15%.It was considered to be the most malignant tumor and had the greatest threat to the life.Radiotherapy and chemotherapy were widely used in clinical treatment of lung cancer,but they had no targeting and gave great side effects to patients.In recent years,many effective cancer gene therapy targets were discovered and provided new idea of lung cancer gene therapy.Signal Transducers and Activators of Transcription （STAT）family are potential cytoplasmic transcription factors,which played dual functions of transferring signal and initiating transcription.Especially,STAT3 plays important roles in embryo development and cell proliferation.The activation of STAT3 is instantaneous and strictly regulated in normal tissue.However,it was constitutively activated in many hematopoietic and solid tumors including leukemia, multiple myelomas,as well as prostate,breast,lung,ovarian,gastric cancers.The activated STAT3 could regulate the expression of many genes including negative factors for immune response（IL-10,VEGF and TGF-β）and genes associated with proliferation,apoptosis and cell cycle（bcl-xl,cyclinD1,mcl-1,surviving,cyclin E）.It could enhance proliferation,inhibit apoptosis,induce angiogenesis and invade immune surveillance.STAT3 was therefore being considered as oncogene.Some cytokines such as IL-6 and EGF can bind to their receptor and activate tyrosine kinase, and then activate STAT3,two p-STAT3 molecules dimmerized and translocated to nucleus,and then bindeded to specific DNA response element in the promoter region and initiated gene transcription.It was one of the effective methods to inhibit a cluster of genes by blocking the binding of transcription factor with their DNA response element,of which, transcription factor decoy（TFD）was mostly used.The transcription factor decoy oligodeoxynucleotide（decoy-ODN）which had the same sequence with DNA response element and had high affinity to transcription factor was synthesized,and the decoy ODN were transfected into cells and competited binding to transcription factor with their own response element.They were used as decoy molecular to alter gene transcription.Compared with other methods,"Decoy" technology has unique advantages and more application potential,such as cluster control,short sequence, easy to transfection,high specificity,et al.By now,decoy ODN method was widely used as a tool to investigate the gene function,and more and more clinical study were done to evaluate their potential value in clinic.Chemotherapy is the common method to treat tumor in clinic,resistance to chemotherapy which termed multidrug resistance（MDR）is considered one of the major obstacles to successful cancer chemotherapy.The overexpression of P-glycoprotein（P-gp）is the main reason in MDR.By now,many MDR modulators were investigated;however,the use of efflux-pump modulators in clinical cancer treatments had proved disappointing.Thus,it is of great importance to develop new compounds or strategies that are capable of circumventing P-gp-mediated MDR with improved clinical characteristics.The mechanisms to induce the expression of P-gp and to acquire drug resistance were unclear.Identification of the mechanisms and the inducing factors were a high priority.Constitutive STAT3 has been shown to confer resistance to chemotherapy-induced apoptosis in some malignancies.Inhibition of STAT3 activity enhances chemo-sensitivity in carcinoma.Meanwhile,it was found that STAT3 mRNA was overexpression in drug-resistant cancer cells than in sensitive cells,STAT3 activity was also specifically elevated in drug-resistant cells.In clinic, STAT3 is highly activated in advanced tumors which present non-sensitivity to chemotherapy.All the results suggested that STAT3 activity might be associated with drug resistance and there might be some relation between STAT3 and MDR.In this study,we used STAT3 decoy ODN to inhibit the activity of STAT3 in lung cancer cells in vitro and in vivo,and observed their effect on proliferation and apoptosis,and then investigated their mechanisms.We also studied inhibition of STAT3 enhanced the sensitivity of drug-resistant leukemia cells to chemotherapy,and the roles and mechanisms of STAT3 in reversing multidrug resistance were also investigated.It provided new idea and method for cancer gene therapy.Methods:Western blotting and EMSA assays were used to detect the expression and activity of STAT3 and its DNA binding ability in pulmonary giant cell carcinoma cell line PG and non-small-cell lung cancer A549 cell line.Cell counting method was used to observe the effect of AG490,a specific inhibitor of Jak2,on the proliferation of the cells.The ODN transfection rate and its location in cells were assayed by flow cytometry and fluorescence microscopy after transfected with lipofectamine.The inhibition effect of STAT3 decoy ODN on STAT3 activity in lung cancer cells was analyzed by luciferase reporter construct assay.Cell counting and ³H incorporation methods were used to detect the influence of decoy ODN on cell growth.The apoptosis was assayed using Annexin V and PI by flow cytometry.The expression of STAT3 target genes related cell cycle and anti-apoptosis were detected using RT-PCR and western blotting.We established the A549 nude mice xenograft and intratumorally injected decoy or scramble ODN and vehicle control.The tumor growth was observed by volume calculated using the length and wide.The tumor was weighed after the final injection,and froze it quickly to do frozen section.The apoptosis induced by decoy ODN in vivo were detected using TUNEL.The expression of STAT3 target genes related cell cycle and anti-apoptosis were detected by immunohistochemistry after decoy ODN treatment in vivo.The activity of STAT3 and expression of its target genes were analyzed by western blotting and luciferase reporter plasmid assay in multidrug resistance K562/A02 cells and drug sensitive K562 cells.The inhibition of STAT3 activity by JSI-124 was detected by western blotting.CCK-8 kit and cell counting were used to detect whether inhibition of STAT3 reversed MDR of K562/A02 to adfiamycin.The apoptosis was assayed by flow cytometry.The accumulation of intracellular adriamycin was detected by flow cytometry after treated with JSI-124.The transcripts of MDR1 and other MDR associated genes were detected by real time PCR in K562/A02 cells which treated with JSI-124 or not,and the expression of P-gp was assayed using western blotting.Meanwhile,another STAT3 signal pathway inhibitor, AG490（Jak2 specific inhibitor）,was chosen.The phosphorylation of STAT3 was assayed by western after treated with AG490.CCK-8 kit was used to detect whether inhibition STAT3 by AG490 increased the sensitivity of K562/A02 cells to adriamycin.The transcripts of MDR1 and other MDR associated genes were detected by real time PCR in K562/A02 cells after treated with AG490 or not,and the expression of P-gp was assayed using western blotting.The possible STAT3 binding sites in the promoter of MDR1 were searched by computer assisted bioinformatics analysis.Result:part 11.Bioinformatics analysis suggested that the designed decoy ODN had strong binding ability with STAT3.According to the obvious reports and the database of transcription factor binding sequence,we designed the decoy ODN sequence which had high affinity with STAT3.2.The constitutively activated STAT3 played important roles in the proliferation of lung cancer cells.Western blotting indicated that STAT3 was highly expressed and was constant phosphorylation（Try705,Ser727）in pulmonary giant cell carcinoma cell line PG and non-small-cell lung cancer A549 cell line.The results of EMSA demonstrated that ³²P-labeled STAT3 decoy ODN could bind with activated STAT3,but scramble ODN could not.The results further suggested STAT3 in lung cancer cells was activated and had DNA binding ability,and our designed decoy ODN sequence could bind with activated STAT3.Blocking signal pathway using Jak2 specific inhibitor AG490 could significantly inhibit the cell growth;all the results demonstrated the critical role of Jak2/STAT3 signal pathway in the proliferation of PG and A549 cells.3.Decoy ODN could be transfected into cells efficiently and most of them located in nucleus.To detect the transfection rate,FITC labeled ODN were transfected with lipofectamine 2000 and detected by flow cytometry,the results demonstrated that ODN could be dose dependently transfected into cells.After cell nucleus stained with DAPI and observed with fluorescence microscope,we found most of ODN located in nucleus.4.STAT3 decoy ODN specifically inhibited the transcription activity of STAT3. STAT3 decoy ODN dose dependently decreased the expression of luciferase induced by STAT3.The more STAT3 decoy ODN were transfected,the lower activity of luciferase were induce by STAT3.However,STAT3 scramble ODN had no effect on the expression of luciferase.The transcription activity of STAT3 was significantly decreased when 50nmol/L decoy ODN were transfected into PG and A549 cells （p＜0.01）,but scramble ODN didn’t show detectable effect（p＞0.05）.In a word, STAT3 decoy ODN could effectively and specifically inhibit the transcription activity of STAT3.5.STAT3 decoy ODN could effectively inhibit the proliferation of PG and A549 cells.To investigate the effect of decoy ODN on cell growth,different concentration of decoy ODN were transfected,and incubated for 24h,48h and 72h respectively, then cell number were counted.The results demonstrated that STAT3 decoy ODN could significantly inhibit the proliferation of cells,the inhibition rate was 69%for PG cells when treated with 50nM decoy ODN,and inhibition rate was 64%for A549 cells when treated with 25nM decoy ODN.To further establish the inhibition effect, ³HTdR incorporation method was used.The results suggested that decoy ODN could also significantly inhibit the incorporation of ³HTdR,with the inhibition rate 61%and 66%for PG and A549 cells respectively（p＜0.01）.However,STAT3 scramble ODN didn’t decrease the proliferation compared with vehicle control.Meanwhile,STAT3 decoy ODN had no obviously effect on proliferation of NIH/3T3 cells which had no activated STAT3.6.STAT3 decoy ODN induced cell apoptosis.To investigate the effect of decoy ODN on apoptosis,annexin-V/PI were used to detect the apoptosis by flow cytometry when transfected with ODN for 12h.The results demonstrated that apoptosis was significantly increased when A549 cells were treated with 25nM decoy ODN,the apoptosis rate rose from 6.41%to 29%,but scramble ODN had no obviously changes on cell apoptosis.7.STAT3 decoy ODN down-regulated the expression of STAT3 targeted genes related with cell cycle and anti-apoptosis.To investigate the mechanism of STAT3 decoy ODN on cell proliferation,the expression of STAT3 target genes were detected after transfected with 50nM and 25nM decoy ODN for PG and A549 cells respectively.Reverse transcription PCR results demonstrated that the mRNA levels of bcl-xl,cyclinD1,mcl-1,survivin and cyclinE were down regulated after transfected with STAT3 decoy ODN compared with vehicle control.The expression of bcl-xl and cyclinD1 were also significantly inhibited when detected by western blotting. However,STAT3 scramble ODN had no obvious effect on the expression of the target genes including mRNA levels and protein levels.8.STAT3 decoy ODN inhibited growth and induced apoptosis of A549 cells in xenograft.A549 nude mice xenograft model were established by inoculating A549 cells in nude mice subcutaneously,and ODN or vehicle control PBS were intratumor injected.The results showed that STAT3 decoy ODN effectively inhibited the growth of A549 cells in vivo,and the inhibition rate reached 69.5%after thirty days treatment. The tumor weight was also decreased,and inhibition rate reached to 52.7%.The tumor tissues were sectioned,and TUNEL was used to detect the apoptosis.The results showed that the apoptosis positive cells were significantly higher in STAT3 decoy ODN treatment group than that in PBS treatment control group,which increased about 20 folds（p＜0.001）.However,STAT3 scramble ODN treatment group had similar apoptosis positive cells with control group.9.STAT3 decoy ODN inhibited the proliferation of lung cancer cells in vivo via down regulating its target genes.The expression of bcl-xl and cyclinD1 were assayed using frozen section by immunohistochemistry.The results demonstrated that the expression of bcl-xl and cyclinD1 in STAT3 decoy ODN treatment group were much lower than that in scramble ODN treatment group or vehicle control group （p＜0.05）.Result:part 21.STAT3 is more active in adriamycin-resistant K562/A02 cells than its parental K562 cells.The expression and activity of STAT3 were higher in drug resistant K562/A02 ceils than that in drug sensitive K562 cells which detected by western blotting.The P-gp which is related with MDR was highly expressed in K562/A02 cells but not in K562 cells.The luciferase reporter assay also showed that the transcription activity of STAT3 was higher in K562/A02 cells than that in K562 cells.2.JSI-124（Cucurbitacin I）inhibited the STAT3 activated in K562/A02 cells dose and time dependently.JSI-124 was highly selective inhibitor for JAK/STAT3 and did not inhibit other oncogenic and tumor survival pathways.The western blot results demonstrated that JSI-124 could inhibit the activity of STAT3 dose and time dependently,and with significant inhibition at 1μM for 24h.3.Blocking STAT3 increased the sensitivity of K562/A02 cells to adriamycin and promoted its apoptosis under the treatment of adriamycin.JSI-124 inhibited the proliferation of K562/A02 cells in dose and time dependent manner,which were normally cultured in medium containing 1μg/ml adriamycin.To further investigate whether JSI-124 could reverse the adriamycin resistance in K562/A02 cells, K562/A02 and K562 cells were treated with different concentrations of adriamycin lonely or combined with 1μM JSI-124,after 48h,the proliferation were observed. JSI-124 significantly increased the sensitivity of K562/A02 cells to adriamycin. Blocking STAT3 made about 8-fold reduction in adriamycin resistance as determined by fold-change in IC50.However,this effect was not seen in drug sensitivity K562 cells.Treatment of K562/A02 with both drugs resulted in significantly greater cell apoptosis than either drug alone.Combination treatment of both drugs increased the apoptosis from 2.72±0.53%to 11.67±2.8%.4.JSI-124 promoted apoptosis through increasing the up-take of adriamyein in K562/A02 cells partly.Compared with their control,JSI-124 dose dependently increased accumulation of adriamycin in drug resistant cells（p＜0.05）.But this effect was not obviously in drug sensitivity cells.The fluorescence of rhodamine-123 was higher when treatment with JSI-124,which indicated that JSI-124 decreased rhodamine-123 efflux medicated by P-gp.5.MDR1 and other MDR associated genes transcripted in K562/A02 cells were down regulated by JSI-124.The real-time PCR results showed that the transcript of MDR1 was decreased by JSI-124 dose dependently.There were only～38%of control when treated with 1.25μM JSI-124（p＜0.01）,and only～25%when treated with 2.5μM（p＜0.01）.Other MDR associated genes such as MRP1 and BCRP1 were also decreased.JSI-124 decreased the abundance of P-gp in a concentration-dependent manner.P-gp was reduced to～47%and～29%of the control level after treated with 1.25μM and 2.5μM JSI-124 for 36h,respectively.6.Jak2 specific inhibitor AG490 enhanced the drug sensitivity of K562/A02 via down-regulating P-gp.The activity of STAT3 was inhibited by AG490 dose dependently,while the sensitivity of K562/A02 cells to adriamycin was significantly increased.About 8～16-fold in adriamycin resistance was reduced at 100μM AG490 as determined by fold-change in IC50.The transcription of MDR1 was down-regulated by AG490,and inhibition ratio reached to 57%at 100μM（p＜0.01）.The amount of P-gp was reduced to 58%of the control level when treating with AG490.7.STAT3 takes part in the regulation of P-gp.Computer assisted searches of potential STAT3 binding sites within the entire P-gp promoter region by TFSEARCH and TRANSFAC database revealed only one potential site which containing putative STAT3 DNA binding elements.This region of the P-gp promoter is located approximately 1014 base pairs upstream of the initiation coding for P-gp,and has strong binding activity with STAT3. Conclusion:In this study,we demonstrated that STAT3 was highly constitutively activated in human lung cancer cells,and played important roles in cell proliferation.STAT3 decoy ODN could specificily block the activity of STAT3 in lung cancer cells,which resulted in proliferation inhibition and apoptosis promotion in vitro and in vivo.Their mechanisms were also investigated that it was related with the down-regulating of STAT3 targeted anti-apoptosis and cell cycle genes.These results demonstrated that it is feasibility for lung cancer gene therapy by using STAT3 decoy ODN to inhibit STAT3 signal pathway.We next studied the feasibility of reversing multidrug resistance by inhibiting STAT3 activity,and investigated their mechanisms.We found that the activity of STAT3 in MDR cells was higher than that in sensitivity cells. Inhibition of STAT3 by JSI-124 reversed multidrug resistance of MDR cells to chemotherapy and increased the apoptosis induced by chemotherapy,which were associated with the down-regulation of MDR1 and P-gp levels.At the same time,the accumulation of adriamycin in cells treated with JSI-124 could be increased which inhibited the cell proliferation further.It provided the feasibility of reversing multidrug resistance by blocking STAT3 and the foundation for combination STAT3 inhibitor with chemotherapy drug in treatment of multidrug resistance.Therefore, targeting activated STAT3 in cancer cells not only inhibits tumor growth but also reverses multidrug resistance.It provided an excellent new target and method for cancer gene therapy.更多还原