摘要
癌基因、抑癌基因和DNA修复基因并称为三大肿瘤相关基因。DNA修复基因突变、功能的异常和表达水平的改变可以引起前两者DNA损伤修复的异常,而癌基因、抑癌基因突变的积累最终可导致肿瘤的发生。DNA聚合酶β(DNApolymeraseβ,DNA polβ)是参与DNA碱基切除修复的主要聚合酶之一,高表达的polβ还具有参与DNA复制、基因重组、跨损伤合成等功能,但是polβ缺乏3′-5′的校读功能,在DNA复制中保真度最低,造成基因组的不稳定性。polβ的改变与肿瘤发生发展的关系已在一些肿瘤和研究中得到证实。我们的前期研究发现食管癌组织中存在polβ基因的突变和高表达。
肿瘤细胞中存在信号转导途径的异常,一些信号分子的过度表达或组成型激活,这将导致这些信号分子下游信号途径的增强,引起细胞的转化、增殖、抵抗细胞凋亡、促进细胞存活,与肿瘤的发生、发展密切相关。研究显示polβ的高表达是转录因子的活化和调节完成的,polβ基因启动子中含有cAMP反应元件(Cyclic-AMP response element,CRE),转录因子CREB/ATF家族成员(包括CREB、ATF1/2,CREM-1等)能识别CRE基序,促进polβ基因的转录表达。转录因子的激活既是基因表达调控的一个主要事件,又是细胞内信号通路中信号传递过程的重要环节。因此研究参与polβ表达的信号通路,探讨其在肿瘤发生、发展中的作用,寻找可能的肿瘤治疗的新靶点将具有重要价值。
互隔交链孢霉是河南省食管癌的重要发病因素,其主要代谢物互隔交链孢酚(alternariol,AOH)(又称链格孢酚)具有致癌性。研究显示AOH能够损伤DNA。一般而言,DNA损伤作为一种信号,将诱导细胞应激反应,激活DNA修复系统基因,使表达增高,以修复损伤。AOH能否激活一定的细胞信号系统,上调polβ表达,尚不清楚。
由于伦理学问题使正常食管上皮取材困难,再加上正常食管上皮原代细胞扩大培养的难度的限制,故本课题选取毒理学实验中常用的NIH3T3细胞为靶细胞,拟从观察AOH对NIH3T3细胞的毒性和DNA损伤作用及转化能力入手,寻找AOH激活NIH3T3细胞中DNA polβ表达增高的信号转导通路。在此基础上,进一步研究食管癌EC9706细胞中这些信号通路的状态,探讨在食管癌细胞中这些信号通路与polβ表达的关系。然后利用阻断信号通路的方法,降低相关信号分子的活化、减少polβ表达,观察对食管癌EC9706细胞生物学特性的影响和对化疗药物顺铂的敏感性的改变,为食管癌的治疗提供新的思路。
第一部分AOH对NIH3T3细胞生物学特性的影响和激活DNA polβ表达的信号通路研究
第一章AOH对NIH3T3细胞生物学特性的影响和诱导DNA polβ表达
方法
1.以1.0,2.0,10.0,20.0,50.0μmol/L的AOH分别作用NIH3T3细胞24 h和48 h,MTT法观察不同浓度的AOH对细胞增殖的影响;以1.0,2.0,10.0,20.0,50.0μmol/L的AOH作用NIH3T3细胞4 h,单细胞凝胶电泳实验观察AOH对DNA损伤程度;以1.0,2.0,10.0,20.0,50.0μmol/L的AOH作用NIH3T3细胞24 h,流式细胞术(FCM)检测AOH对NIH3T3细胞周期的影响;以1.0,2.0,10.0,20.0,50.0μmol/L的AOH作用NIH3T3细胞48 h,将存活细胞传代后,重复刺激48 h,将细胞接种在六孔板中(150个/孔),克隆形成率实验观察AOH对NIH3T3细胞的转化作用。
2.分别用2.0,10.0,20.0μmol/L AOH作用NIH3T3细胞16 h,采用RT-PCR、免疫细胞化学法和Western Blotting检测不同浓度的AOH对NIH3T3细胞中polβmRNA和蛋白水平表达的影响。
结果
1.一定浓度的AOH抑制NIH3T3细胞增殖
1.0μmol/LAOH作用24 h和48 h吸光度值与对照组相比无统计学差异(P＞0.05);2.0μmol/L以上各浓度组24 h和48 h的吸光度值均较对照组降低,差异有统计学意义(P＜0.05),显示明显的浓度依赖关系。
2.AOH损伤NIH3T3细胞DNA
计算出各组细胞的尾部DNA含量,发现1.0,2.0μmol/L AOH剂量组与对照组无明显差异,10.0,20.0,50.0μmol/L AOH组均高于对照组(P＜0.05),且50.0μmol/L组高于10.0,20.0μmol/L组,说明AOH可造成DNA损伤,且存在浓度依赖性。
3.AOH引起NIH3T3细胞周期G2/M期阻滞
1.0μmol/L和2.0μmol/L组细胞周期与对照组相比,无明显变化。随着AOH浓度的增加,G2/M期细胞百分比逐渐增大,而G0/G1期细胞百分比降低,且呈浓度依赖性,与对照相比差异均有统计学意义(P＜0.05),表明细胞被阻滞于G2/M期。
4.AOH处理的NIH3T3细胞克隆形成率提高
克隆形成实验结果显示,随着AOH浓度的增大,平均克隆率逐渐增高(P＜0.05),但是50.0μmol/L AOH组克隆形成率降低了。
5.AOH诱导NIH3T3细胞中polβ表达增高
RT-PCR、免疫细胞化学和Western Blotting结果表明,2.0,10.0,20.0μmol/LAOH作用NIH3T3细胞16 h后,polβ表达增高,具有浓度依赖性。
第二章AOH激活NIH3T3细胞中PKA-CREB和p38MAPK-ATF2信号通路
方法
1.分别用2.0,10.0,20.0μmol/L AOH作用NIH3T3细胞1 h,检测NIH3T3细胞中PKA的活化水平;作用2 h,检测CREB的活化水平。20.0μmol/L AOH作用细胞0,30,60和120 min,检测NIH3T3细胞中PKA和CREB的活化水平。PKA特异性抑制剂H89预处理细胞1 h,再加入20.0μmol/LAOH作用1 h,检测PKA活性变化;作用2 h,检测CREB的活性变化。
2.分别用2.0,10.0,20.0μmol/L AOH作用NIH3T3细胞2 h,Western Blotting方法检测细胞中p38和ATF2的磷酸化水平;20.0μmol/L AOH作用细胞0,1,2和4h,检测p38和ATF2的磷酸化水平;利用p38特异性抑制剂SB203580预处理细胞1 h,再加入20.0μmol/L AOH作用2 h,检测p38和ATF2磷酸化水平的变化。
3.JNK特异性抑制剂SP600125预处理细胞1 h后,再加入20.0μmol/L AOH作用细胞2 h,同时设20.0μmol/L AOH直接作用组,Western Blotting检测AOH对NIH3T3细胞中JNK1/2的磷酸化作用和对ATF2磷酸化水平的影响。
4.p38特异性抑制剂SB203580预处理细胞1 h,再加入20.0μmol/L AOH作用细胞2h,Western Blotting检测细胞中磷酸化CREB表达水平的变化。
结果
1.AOH激活NIH3T3细胞中PKA-CREB信号通路
1.1 AOH诱导PKA激活并发生核转位
Western Blotting的结果表明,2.0μmol/L AOH对PKA的活化作用不明显(P＞0.05),而10.0,20.0μmol/L的AOH能够强烈激活PKA,与对照组相比差异有显著性(P＜0.05),显示浓度依赖关系。免疫细胞化学法检测结果与之一致。免疫荧光结果表明,PKA活化并转位入核。
20.0μmol/L的AOH作用NIH3T3细胞不同时间,Western Blotting的结果表明,随着作用时间的延长,PKA的活化水平逐渐增高,在60 min时达到高峰,显示时间依赖关系。
抑制实验的Western Blotting结果显示,H89预处理组活化的PKA的表达水平低于AOH直接处理组(P＜0.05),免疫细胞化学法和免疫荧光检测结果与之一致。
1.2 AOH诱导转录因子CREB激活
Western Blotting的结果表明,2.0μmol/L AOH对CREB磷酸化水平增加不明显(P＞0.05),而10.0,20.0μmol/L的AOH能够强烈激活CREB,与对照组相比差异有显著性(P＜0.05),显示浓度依赖关系。免疫细胞化学法、免疫荧光检测结果与之一致。
20.0μmol/L的AOH作用NIH3T3细胞不同时间,Western Blotting的结果表明,随着作用时间的延长,CREB的磷酸化水平逐渐增高,在2 h达到高峰,显示时间依赖关系。
抑制剂H89预处理细胞后,Western Blotting的结果表明,H89部分阻断AOH诱导的CREB的活化,H89预处理组磷酸化CREB的表达水平低于AOH直接处理组(P＜0.05),免疫细胞化学法和免疫荧光检测结果与之一致。提示AOH处理细胞后,CREB的磷酸化一定程度上依赖于上游PKA的激活。
2.AOH激活NIH3T3细胞中p38MAPK-ATF2信号通路
2.1 AOH诱导p38MAPK激活
Western Blotting的结果表明,2.0,10.0,20.0μmol/L的AOH均能够增加p38的磷酸化水平,与对照组相比差异有显著性(P＜0.05),并显示浓度依赖关系。
20.0μmol/L的AOH作用NIH3T3细胞不同时间,Western Blotting的结果表明,随着作用时间的延长,p38的活化水平逐渐增高(P＜0.05),在2 h时达到高峰,显示时间依赖关系。
用p38特异性抑制剂SB203580预处理细胞,Western Blotting的结果表明,SB203580明显抑制AOH诱导的p38的活化,SB203580预处理组p38的磷酸化水平低于AOH直接处理组(P＜0.05)。
2.2 AOH诱导转录因子ATF2激活
Western Blotting的结果表明,2.0μmol/LAOH对ATF2磷酸化水平增加不明显(P＞0.05),而10.0,20.0μmol/L的AOH能够强烈激活ATF2,与对照组相比差异有显著性(P＜0.05),显示浓度依赖关系。
20.0μmol/L的AOH作用NIH3T3细胞不同时间,Western Blotting的结果表明,随着作用时间的延长,ATF2的活化水平逐渐增高(P＜0.05),在2 h时达到高峰,与p38的磷酸化一致,并显示时间依赖关系。
用p38特异性抑制剂SB203580预处理细胞,Western Blotting的结果表明,SB203580明显抑制AOH诱导的ATF2的活化,SB203580预处理组ATF2的磷酸化水平低于AOH直接处理组(P＜0.05)。提示AOH处理细胞后,ATF2的磷酸化依赖于上游p38MAPK的激活。
3.JNK通路未被AOH激活
JNK是MAPK家族成员之一,介导多种刺激引起的细胞应激反应。WesternBlotting方法检测DNA损伤剂AOH能否激活JNK通路,结果显示,AOH未能增加NIH3T3细胞中的JNK磷酸化水平,JNK抑制剂未能降低AOH对ATF2的激活作用。
4.在AOH诱导下,p38MAPK促进CREB的磷酸化
Western Blotting结果显示,p38特异性抑制剂SB203580预处理细胞后,结果显示,与AOH激活组相比,SB203580预处理组中磷酸化CREB的水平降低(P＜0.05)。
第三章AOH诱导NIH3T3细胞中DNA polβ表达依赖PKA-CREB和p38MAPK-ATF2通路的激活
方法
1.PKA特异性抑制剂H89预处理NIH3T3细胞1 h,再加入20.0μmol/L AOH作用细胞16 h,免疫细胞化学法和Western Blotting检测polβ的表达,同时设20.0μmol/LAOH直接作用组和溶剂对照组。
2.p38特异性抑制剂SB203580预处理细胞1 h,再加入20.0μmol/L AOH作用细胞16 h,Western Blotting检测polβ的表达,同时设20.0μmol/LAOH直接作用组和溶剂对照组。
3.H89和SB203580共同预处理细胞1 h,再加入20.0μmol/L AOH作用细胞16 h,Western Blotting检测PKA和p38MAPK双通路阻断对AOH诱导的NIH3T3细胞中polβ表达的影响。
4.JNK特异性抑制剂SP600125预处理细胞1 h,再加入20.0μmol/L AOH作用细胞16 h,Western Blotting检测AOH诱导的polβ表达,观察JNK信号通路在DNA polβ基因表达中的作用。
结果
1.PKA特异性抑制剂H89部分降低AOH诱导的polβ表达
为探讨AOH诱导的DNA polβ蛋白表达增加是否通过PKA-CREB信号通路,我们应用PKA特异性抑制剂H89预处理细胞,再用Western Blotting方法检测AOH对DNA polβ蛋白表达的影响。结果显示,H89可以部分抑制AOH诱导的DNA polβ蛋白表达,H89预处理组polβ表达水平低于AOH直接处理组(P＜0.05)。
2.p38特异性的抑制剂SB203580部分抑制AOH诱导的polβ表达
为探讨AOH诱导的DNA polβ蛋白表达增加是否通过p38-ATF2信号通路,应用p38特异性抑制剂SB203580预处理细胞,再用Western Blotting方法检测AOH对DNA polβ蛋白表达的影响。结果显示,SB203580可以部分抑制AOH诱导的DNApolβ蛋白表达,SB203580预处理组polβ表达水平低于AOH直接处理组(P＜0.05)。
3.PKA和p38MAPK双通路阻断明显降低polβ表达
Western Blotting结果显示,与单独使用H89和SB203580预处理组相比,H89、SB203580联合作用明显降低AOH诱导的polβ表达(P＜0.05)。
4.JNK通路未在NIH3T3细胞中AOH诱导的polβ表达中起作用
JNK是MAPK家族成员之一,介导多种刺激引起的细胞应激反应。WesternBlotting方法检测DNA损伤剂AOH能否激活JNK通路参与polβ表达。结果表明,JNK抑制剂未能降低AOH诱导的polβ的表达,提示JNK通路未参与AOH诱导的polβ表达。
第二部分PKA-CREB和p38MAPK-ATF2通路激活调节DNA polβ表达在食管癌细胞EC9706中的作用
第一章食管癌EC9706细胞中PKA-CREB和p38MAPK-ATF2通路激活调节DNA polβ表达
方法
1.利用免疫细胞化学和Western Blotting方法检测未处理的食管癌EC9706细胞中PKA催化亚基、CREB的磷酸化状态,同时用PKA特异性的抑制剂H89处理细胞2 h,观察活化的PKA、CREB的改变。
2.利用免疫细胞化学和Western Blotting方法检测未处理的食管癌EC9706细胞中p38MAPK、ATF2的磷酸化状态,同时用p38抑制剂SB203580处理细胞2 h,观察活化的p38、ATF2的改变。
3.PKA特异性的抑制剂H89和p38抑制剂SB203580单独和联合处理EC9706细胞16 h,利用免疫细胞化学和Western Blotting方法观察DNA polβ表达的变化。
4.提取未处理EC9706细胞、H89和SB203580分别处理16 h的EC9706细胞的核蛋白,经凝胶电泳阻滞实验(EMSA),分析EC9706细胞中活化的CREB、ATF2与DNA polβ启动子中CRE元件的结合活性。
结果
1.EC9706细胞中PKA-CREB通路处于激活状态
Western Blotting与免疫细胞化学结果显示,未处理的EC9706细胞中含有活化的PKA和CREB,H89可以降低其磷酸化水平。
2.EC9706细胞中p38-ATF2通路处于激活状态
Western Blotting与免疫细胞化学结果显示,未处理的EC9706细胞中含有活化的p38和ATF2,抑制剂SB203580可以降低其磷酸化水平。
3.EC9706细胞中激活状态的PKA和p38MAPK通路参与调节DNA polβ的表达
为证实EC9706细胞中PKA、p38MAPK通路的激活与DNA polβ的表达存在一定的关系,我们采用阻断信号通路的方法,分别单独和联合使用PKA抑制剂H89和p38抑制剂SB203580作用EC9706细胞16 h,观察DNA polβ的表达的变化。免疫细胞化学和免疫印迹实验结果显示,未处理的EC9706细胞中有一定水平的DNA polβ的表达,H89和SB203580可以部分降低细胞中polβ的表达,两者联合使用,polβ的表达降低更明显。EMSA结果显示,EC9706细胞中活化的CREB、ATF2与polβ启动子中的CRE元件有较强的结合活性。
第二章阻断DNA polβ表达的信号通路对食管癌EC9706细胞生物学特性和对顺铂敏感性的影响
方法
1.PKA特异性抑制剂H89和p38MAPK特异性抑制剂SB203580单独和联合处理EC9706细胞24 h,用倒置显微镜观察细胞形态学变化;用Annexin V-FITC和PI双染色,流式细胞仪检测细胞凋亡率;水溶性四氮唑(WST-8)测定抑制剂作用24和48 h细胞增殖改变。
2.在EC9706细胞中加入H89和SB203580单独和联合预处理1 h,再加入顺铂培养24 h,用倒置显微镜观察细胞形态学变化;用Annexin V-FITC和PI双染色,流式细胞仪检测细胞凋亡率;WST-8测定抑制剂作用24和48 h细胞增殖改变。
结果
1.阻断PKA和p38信号通路、降低DNA polβ表达对食管癌EC9706细胞生物学特性的影响
H89和SB203580单独作用EC9706细胞,部分细胞出现细胞形态缩小,细胞增殖减慢;当H89和SB203580联合作用,EC9706细胞出现细胞形态缩小,胞质凝缩,细胞漂浮数明显增加,细胞数量下降,增殖受到抑制。
细胞增殖实验结果显示,与对照组相比,H89组和SB203580组细胞的24 h、48 h存活率均下降(P＜0.05),当H89与SB203580共同作用于细胞时,细胞存活率进一步下降(P＜0.05)。
Annexin V-FITC和PI双染色,细胞凋亡检测结果显示,EC9706在H89和SB203580单独作用24 h,均表现为促进细胞凋亡;当H89和SB203580联合使用时,促进细胞凋亡和坏死作用更加显著。
2.阻断PKA和p38信号通路、降低DNA polβ表达增加食管癌EC9706细胞对顺铂的敏感性
化疗药顺铂单独作用EC9706细胞,出现细胞形态缩小,漂浮细胞数量增加,细胞增殖较慢;当H89和SB203580分别与顺铂联合作用,EC9706细胞出现细胞形态缩小,胞质凝缩,细胞漂浮数明显增加,细胞数量下降,增殖明显受到抑制,而H89、SB203580共同联合顺铂使用时,这种变化尤为明显。
细胞增殖实验结果显示,与单用顺铂相比,当H89或SB203580与顺铂联合作用于细胞时,细胞存活率下降,细胞的增殖受到抑制(P＜0.05),增加了对顺铂的化疗敏感性;当三者联合,抑制更明显。
细胞凋亡检测结果显示,与单独使用顺铂的对照组相比,当H89或SB203580联合顺铂使用时,细胞总凋亡率增加(P＜0.05);尤其是H89、SB203580和顺铂三者联合时更明显。
结论
1.一定浓度的AOH能够产生抑制细胞增殖、引起细胞周期阻滞、造成DNA损伤等急性反应和和克隆形成率增加,并能够诱导DNA polβ表达增高。
2.AOH诱导NIH3T3细胞中的PKA-CREB信号通路和p38MAPK-ATF2信号通路激活,上调DNA polβ基因表达。
3.在食管癌细胞EC9706中PKA-CREB信号通路和p38MAPK-ATF2信号通路处于激活状态,两者共同促进DNA polβ的表达。
4.阻断PKA和p38MAPK信号途径,降低DNA polβ表达,改变EC9706细胞生物学特性,增加对化疗药顺铂的敏感性,有可能成为食管癌治疗的辅助治疗方法。
In many carcinoma related genes,oncogene,tumor suppressor genes and DNA repair gene play a major role in cancerogenesis.The DNA repair genes can not correctly to repair DNA damage when they are mutational or their expression are abnormal.DNA polymeraseβ(polβ) is involved in base-excision repair(BER) for DNA maintenance.It also may be involved in replication,recombination,and drug resistance in eukaryotic cells.However,polβlacks any intrinsic 3' to 5' exonuclease activity and is incapable of proofreading,polβcan lead to DNA repair synthesis errors and confers to cells a mutator phenotype,which has been confirmed in some tumor. Our previous studies have shown that polβgene were mutational and its mRNA expression wers increased in Esophageal carcinoma specimens.It is necessary to research deeply on the relation of polβoverexpression and tumorigenesis and it may be novel idea of oncotherapy.
Abnomrality of cell signal transduction and regulation is intimately related to the pathogenesis and promotion of tumor.Tumor cells have developed various mechanisms to achieve constitutive activation and overexpression of signal members, to lead to cell malignant transformation,survival,proliferation,resisting apoptosis. polβgene expression is a precisely regulated process in mammalian cells that is controlled,in part,through cis-elements(CRE) in promoters and their cognate DNA-binding factors.It is important to know the pathway of polβexpression and regulate its level and understand its role in tumor.
Alternariol(AOH),a secondary metabolite produced by various species of the genera Alternaria,is often found in wheat,grain and fruits.A long-term exposure to low levels of AOH is associated with esophageal cancer.AOH has DNA toxicity.In general,DNA damage,a stimulated signal,induces stress reaction in cells,and increases the expression of DNA repair genes to maintain DNA stabilization.It is unknown that AOH induces DNA polβexpression via some singnal pathway.
In this study,we investigated the cytotoxicity and ability of colony formation of AOH on NIH/3T3 Cells.AOH up-regulated DNA polβexpression via the activated PKA-CREB and p38MAPK-ATF2 pathway in NIH3T3 cells.We discussed the relation of overexpression of polβ,the activated pathway of PKA and p38,and cell transformation.In addition we detected the activation status of PKA-CREB and p38MAPK-ATF2 signaling pathway in the EC9706 cells and found that the expression of polβwas dependent on the activation of PKA and p38MAPK pathway. Treatment of EC9706 cells with PKA inhibitor H89 and/or p38 inhibitor SB203580 reduced cell viability and increased apoptosis compared with the untreated control. While combination with cisplatin,the results were more prominent.This may be a target for the development of novel therapeutic strategies.
PartⅠAOH affects NIH3T3 cells biological characteristics and activates the signaling pathway of DNA polβexpression
Chapter 1 AOH affects NIH3T3 cells biological characteristics and induces the expression of DNA polβ
Methods
1.NIH3T3 cells in logarithmic growth phase were treated at the dosage of 1.0,2.0, 10.0,20.0,50.0μmol/L AOH and treated with DMSO(0.25%) for 24 h,then the effects of AOH on cell proliferations were assessed by morphologic observasion,and cell cycle distributions were detected by flow cytometric assay(FCM).Cell viability was detected by MTT with the same concentrion of AOH treatment for 24h and 48h. Single Cell Gel Electrophoresis(SCGE) assay was used to examine DNA damage induced by AOH for 4h.The ability of AOH-induced transformation of NIH3T3 cells were by clony formation assay.
2.The expression of DNA polβevoked by different concentrations of AOH in NIH3T3 cells was investigated with reverse transcription-polymerase chain reaction (RT-PCR),immunocytochemistry and Western Blotting.
Results
1.The cells treated with AOH presented morlogic changes and the inhibition of cell proliferation occurred(P＜0.05).There were significant comet in the intermediate and high dosage group(ranging from 10.0 to 50.0μmol/L) in the SCGE assay(P＜0.05). In comparison with the control group,the percents of G2/M and S phase cells were increased after treatment of 10.0,20.0,50.0μmol/L AOH for 24 h(P＜0.05).
2.The results of RT-PCR,immunocytochemistry and Western Blotting indicated that the expression of polβincreased with treatment of 2.0,10.0,20.0μmol/LAOH for 16h in the NIH3T3 cells,which was a concentration-dependent manner.
Chaper 2 AOH activates the PKA-CREB and p38MAPK-ATF2 pathway in NIH3T3 cells
Methods
1.NIH3T3 cells were treated by by 2.0,10.0,20.0μmol/LAOH for 1h,then activation of PKA were investigated with immunocytochemistry,immunofluorescence and Western Blotting.The cells were exposed respectively by 20.0μmol/L AOH for 0, 30,60,120min,then activation of PKA were investigated with Western Blotting.In addition,the cells were pretreated with an inhibitor of PKA(H89) for 1h,then exposed to 20.0μmol/L AOH for 1h,and activation of PKA were detected by the same methods.
2.NIH3T3 cells were treated by by 2.0,10.0,20.0μmol/LAOH for 2 h,then phosphorylation of CREB were investigated with immunocytochemistry, immunofluorescence and Western Blotting.The cells were exposed respectively by 20.0μmol/L AOH for 0,1,2,4h,then activation of CREB were investigated with Western Blotting.In addition,the cells were pretreated with an inhibitor of PKA(H89) for 1h,then exposed to 20.0μmol/L AOH for 2h,and activation of CREB were detected by the same methods.
3.NIH3T3 cells were treated by by 2.0,10.0,20.0μmol/L AOH for 2h,then phosphorylation of p38MAPK and ATF2 were investigated with Western Blotting. The cells were exposed respectively by 20.0μmol/L AOH for 0,1,2,4h,then activation of p38MAPK and ATF2 were investigated with Western Blotting.In addition,the cells were pretreated with an inhibitor of p38(SB203580) for 1h,then exposed to 20.0μmol/L AOH for 2h,and activation of p38MAPK and ATF2 were detected by the same methods.
4.Phosphorylation of JNK1/2 in NIH3T3 cells induced by 20.0μmol/L AOH was investigated with Western Blotting.NIH3T3 cells were pretreated with an inhibitor of JNK(SP600125) for 1h,then exposed to 20.0μmol/L AOH for 2h.Phosphorylation of JNK1/2 and ATF2 were investigated with Western Blotting.
5.NIH3T3 cells were pretreated with an inhibitor of p38(SB203580) for 1h,then exposed to 20.0μmol/L AOH for 2h.Phosphorylation of CREB was investigated with Western Blotting.
Result
1.AOH -induced activation of PKA and nuclear translocation
In the results of Western blot,2.0μmol/L AOH-elicited activation of PKA was not obvious,but it was significantly increased elicited by 10.0,20.0μmol/L AOH compared with the control,which was a concentration-dependent manner.The results of immunocytochemistry coincided with Western Blotting.Application of AOH increased nuclear accumulation of PKA in immunofluorescence assay.
Activation of PKA was increased in time-dependent manner andhad reached maximal levels by 1h after AOH treatment.
The results of immunocytochemistry,immunofluorescence and Western Blotting indicated that H89 blocked the AOH-induced activation of PKA.
2.AOH -induced activation of CREB
In the results of Western blot,2.0μmol/L AOH-elicited phosphorylated CREB was not obvious,but it was significantly increased elicited by 10.0,20.0μmol/L AOH compared with the control in a concentration-dependent manner.The results of immunocytochemistry and immunofluorescence coincided with Western Blotting.
Phosphorylation of CREB was increased in time-dependent manner andhad reached maximal levels by 2h after AOH treatment,which was latter than the time at which the level of p-PKAhad increased. The results of immunocytochemistry,immunofluorescence and Western Blotting indicated that H89 partly blocked the AOH-induced phosphorylation of CREB.
3.AOH -induced activation of p38MAPK
In the results of Western blot,AOH-elicited phosphorylated p38 was significantly increased compared with the control(P＜0.05) in a concentrationdependent manner.
Phosphorylation of p38 was increased in time-dependent manner and had reached maximal levels by 2h after AOH treatment.
The results of Western Blotting indicated that SB203580 blocked the AOH-induced phosphorylation of p38.
4.AOH -induced activation of ATF2
In the results of Western blot,AOH-elicited phosphorylated ATF2 was significantly increased compared with the control in a concentration- dependent manner.
Phosphorylation of ATF2 was increased in time-dependent manner and had reached maximal levels by 2h after AOH treatment,which coincided with the time at which the level of p-p38 kinasehad increased.
The results of Western Blotting indicated that SB203580 decreased the AOH-induced phosphorylation of ATF2.
5.The effect of AOH on phosphorylation of JNK
In general,the JNK pathways are preferentially activated by genotoxic agents and cytokines,and tend to mediate the stress response.The results of Western Blotting indicated that AOH failed to increase phosphorylation of JNK.Pretreatment of NIH3T3 cells with JNK inhibitor SP600125 failed to decrease AOH-induced ATF-2 phosphorylation at all.These results showed that ATF-2 phosphorylation was catalyzed mainly by p38 kinase but not by JNK.
6.Phosphorylation of CREB partly dependent on p38 activation
Exposure of NIH3T3 cells to AOH resulted in CREB phosphorylation which was significantly decreased after reduction of p38 activity using the specific inhibitor SB203580.
Chaper 3 AOH-induced DNA polβexpression is dependent on the activated PKA-CREB and p38MAPK-ATF2 pathway in NIH3T3 cells
Methods
1.To study whether AOH-elicited expression of polβwas dependent of PKA-CREB pathway,NIH3T3 cells were pretreated with an inhibitor of PKA(H89) for 1h,then exposed to 20.0μmol/L AOH for 16h.The expression of polβwas investigated with immunocytochemistry and Western Blotting.
2.To study whether AOH-elicited expression of polβwas dependent of p38MAPK -ATF2 pathway,NIH3T3 cells were pretreated with an inhibitor of p38(SB203580) for 1h,then exposed to 20.0μmol/L AOH for 16h.The expression of polβwas detected with Western Blotting.
3.NIH3T3 cells were pretreated with H89 combination with SB203580 for 1h,then exposed to 20.0μmol/L AOH for 16h.The expression of polβwas investigated with Western Blotting.
4.NIH3T3 cells were pretreated with an inhibitor of JNK(SP600125) for 1h,then exposed to 20.0μmol/L AOH.The expression of polβwas investigated with Western Blotting.
Results
1.AOH up-regulated polβexpression via the activated PKA-CREB pathway in NIH3T3 cells
The expression of DNA polβin NIH3T3 cells induced by AOH was significantlyhigher than that in the control group(P＜0.05).However,H89 decreased the AOH-induced DNA polβexpression.The inhibitory effect of H89 was incomplete,however,it indicated that PKA activation was not the only mechanism by which AOH induced DNA polβexpression.
2.AOH up-regulated polβexpression via the activated p38-ATF2 pathway in NIH3T3 cells
SB203580 partly blocked the AOH-induced DNA polβexpression which was significantly lower than that in theAOH-induced directly group(P＜0.05).The inhibitory effect of SB203580 was incomplete,however,it indicated that p38 activation was not the only mechanism by which AOH induced DNA polβexpression.
3.H89 and SB203580 cooperated to decrease AOH-induced DNA polβexpression
The Western Blotting results showed H89 combination with SB203580 blocked the AOH-induced DNA polβexpression which was significantly lower than that H89 or SB203580 alone group(P＜0.05).
4.The AOH-induced expression of Polβwere independent on JNK pathway
In general,the JNK pathways are preferentially activated by genotoxic agents and cytokines,and tend to mediate the stress response.Pretreatment of NIH3T3 cells with JNK inhibitor SP600125 failed to decrease AOH-induced polβexpression.
PartⅡThe role of activated of PKA-CREB and p38MAPK-ATF2 pathway regulating the expression of DNA polβin EC9706 cells
Chapter 1 Activation of PKA-CREB and p38MAPK-ATF2 pathway controls the expression of DNA polβin EC9706 cells
Methods
1.The activation status of PKA-CREB signaling pathway in EC9706 cells was investigated with immunocytochemistry and Western Blotting.In addition,cells were treated with an inhibitor of PKA(H89) for 2h,and activation of PKA and CREB were detected by the same methods.
2.The activation status of p38MAPK-ATF2 signaling pathway in EC9706 cells was investigated with immunocytochemistry and Western Blotting.In addition,cells were treated with an inhibitor of p38(SB203580) for 2h,and activation of p38 and ATF2 were detected by the same methods.
3.EC9706 cells were treated with H89,SB203580 or combinations thereof for 16h, and the expression of polβwas detected with immunocytochemistry and Western Blotting.The CRE-DNA binding activation of CREB or ATF2 was determined by electrophoretic mobility-shift assays(EMSA).
Results
1.Activated PKA-CREB pathway in EC9706 cells
Immunocytochemistry and Western Blotting showed that EC9706 cellshad activation of PKA and CREB.H89 reduced phosphorylation of both PKA and CREB.
2.Activated p38-ATF2 pathway in EC9706 cells
Immunocytochemistry and Western Blotting showed that EC9706 cellshad activation of p38 and ATF2.SB203580 reduced phosphorylation of both p38 and ATF2.
3.Activated PKA-CREB and p38MAPK-ATF2 pathway up-regulated the expression of DNA polβin EC9706 cells
Immunocytochemistry and Western Blotting showed that untreated EC9706 cells had expression of polβ.H89,SB203580 alone or combinations thereof reduced expression of polβcompared with the control group.However,The polβexpression of H89 plus SB203580 group was lower than those of H89 group and SB203580 group alone(P＜0.05).The results indicaed that PKA and p38MAPK acted co-operatively to control DNA polβexpression in EC9706 cells.
Chapter 2 The effect of blocking the signal pathway of polβexpression on the biological characteristics and the sensitivity to cisplatin in EC9706 cells
Methods
1.EC9706 cells were treated with H89,SB203580 or combinations thereof for 24h in order to block PKA and p38MAPK pathway and decrease the expression of polβ. Then the number of viable cells were determined by WST-8 and the morphologic change of the cells were observed by a inverted microscope,and the cells were stained with FITC-annexin V and PI for analyzing cell apoptosis by flow cytometry in Cell Quest acquisition and analysis programs.
2.EC9706 cells were treated with H89 or/and SB203580 combination with cisplatin for 24h in order to block PKA and p38MAPK pathway and decrease the expression of polβ.Then the number of viable cells were determined by WST-8 and the morphologic change of the cells were observed by a inverted microscope.The cells were stained with FITC-annexin V and PI for analyzing cell apoptosis by flow cytometry in Cell Quest acquisition and analysis programs.
Results
1.The effect of blocking PKA and p38MAPK pathway and decreasing the expression of polβon biological characteristics of EC9706 cells
Treatment with H89 or SB203580,EC9706 cells growth were slower compared with the control,and some cells became smaller.When H89 plus SB203580,these changes were more significant.
Treatment with H89 or SB203580 for 24h or 48h,EC9706 cells growth were inhibited compared with the control group(P＜0.05).When H89 plus SB203580,these changes were more significant(P＜0.05).
H89 or SB203580 alone could increase cell apoptosis(P＜0.05).While H89 combination with SB203580,the results were more prominent(P＜0.05).
2.The increasing sensitivity of EC9706 to cisplatin when blocking PKA and p38MAPK pathway and decreasing the expression of polβ
H89 or SB203580 combination with cisplatin -treated cells showed an decrease in the speed of cell growth,and the number of floating cells increased.While combination with all,the results were more prominent.
Treatment with H89 or SB203580 plus cisplatin,the EC9706 cells growth were inhibited drastically compared with cisplatin alone(P＜0.05).While H89 and SB203580 in combination with cisplatin,the results were more prominent(P＜0.05). H89 or SB203580 in combination with cisplatin could increase cell apoptosis (P＜0.05).While H89 and SB203580 in combination with cisplatin,the results were more prominent(P＜0.05).The results likely indicated that H89 and SB203580 enhanced sensitivity of the EC9706 cells to cisplatin.
Conclusions
1.AOH inhibits NIH3T3 cells proliferation,induces G2/M phase arrest and DNA damage and increases clony formation.AOH also induces the expression of DNA polβ,which may be one of important mechanisms being involved in AOH carcinogenicity.
2.The results have shown that AOH-elicited expression of polβare dependent of the activated PKA-CREB and p38MAPK-ATF2 pathway in NIH3T3 cells.
3.There is a constitutively activated PKA-CREB and p38MAPK-ATF2 signaling pathway in EC9706 cell lines.They play a cooperation role in the expression of DNA polβ.H89 and SB203580 blocks the two pathway and downregulate the expression of DNA polβ,which causes the changes of biological characteristics of EC9706 cells. H89 and/or SB203580 in combination with cisplatin,can obviously inhibit cell proliferation and induce apoptosis and have synergistic anti-tumor efficacy,which may be become a novel therapeutic strategies of esophageal carcinoma.
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