杂色曲霉素诱导GES-1细胞周期G_2期阻滞可能机制的研究
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摘要
杂色曲霉素(sterigmatocystin, ST)是杂色曲霉、构巢曲霉等曲霉菌属真菌所产生的一种低分子量代谢产物,广泛存在于自然界,是动物和人类食物中最常见的污染物之一。研究表明ST具有致癌性,可诱导不同的实验动物发生肝癌、肺癌、间皮瘤等肿瘤。ST还具有遗传毒性,可直接损伤细胞DNA、与DNA形成加合物,引起基因突变。本研究室前期实验发现,ST可诱发体外培养人胚胃黏膜细胞发生恶性转化以及抑癌基因P53的突变、过表达;长期灌胃ST可以引起小鼠腺胃黏膜的肠上皮化生和腺上皮不典型增生。
细胞周期调控是细胞最重要的生物学过程之一,细胞周期的紊乱可导致细胞的癌变。研究证明ST可以影响靶细胞的细胞周期,谢同欣等研究发现,ST可诱导小鼠胚胎纤维母细胞细胞周期发生G2/M期阻滞。本研究室近期实验也证实,ST可以诱导体外培养胃黏膜上皮细胞GES-1细胞周期阻滞在G2期。但ST诱导细胞G2期阻滞的可能机制还不明确。
cyclins是细胞周期进程调控的关键因子,可以激活它们的结合蛋白——细胞周期蛋白依赖性蛋白激酶(CDKs),进而靶向作用于各自的下游蛋白。CDK通过调节靶蛋白特定位点的磷酸化,介导细胞周期通过周期调控点。有丝分裂激酶Cdc2 (Cdk1)的激活是G2期调控点的重要的调控因素,它也受到多种因素的调控,例如与CyclinB的周期性结合、可逆的磷酸化作用和胞内移位等。细胞周期蛋白B1 (CyclinB1)是细胞周期G2期的周期蛋白,与其激酶Cdc2形成的复合物是启动有丝分裂的关键因子,可促使细胞周期由G2期进入M期。CyclinB1蛋白表达在G2期达到高峰,与Cdk1形成复合物,进入细胞核,此过程也受到磷酸化作用的调节。蛋白磷酸酶Cdc25C能够使Cdc2去磷酸化,从而促进细胞周期的进程;而磷酸化Cdc25C可丧失酶活性,引起Cdc2磷酸化程度增高,进而导致细胞周期阻滞在G2期。eIF4E是蛋白质合成的重要调节因子,可与其结合蛋白4E-BP1结合,调节细胞周期蛋白D1 (CyclinD1)等蛋白质的合成,eIF4E磷酸化水平增高可增强其促进蛋白质合成的作用。研究表明,eIF4E和4E-BP1也与细胞周期紊乱有关,在多种恶性肿瘤中表达异常。本研究室近期实验发现,ST不仅可以抑制胃黏膜上皮细胞(GES-1)增殖,诱导GES-1细胞周期发生G2期阻滞,还可以影响G2期调控点关键因子——CyclinB1的表达以及Cdc2激酶和Cdc25C磷酸酶的活性,同时,ST还可以抑制eIF4E的翻译起始功能。
JNK、ERK和p38是MAPK信号转导通路中最重要的成员。霉菌毒素等环境致癌物、化疗药物等多种体内外因素均可通过影响JNK、ERK和p38信号转导通路诱导靶细胞内相关基因表达,进而引起机体和细胞的相应生物学性状变化。PI3K是Her2家族重要信号转导通路之一,可通过调节下游核因子mTOR进而调控细胞增殖及蛋白质的合成,并且与肿瘤的侵袭、转移、预后及化疗耐受有关。
为进一步探讨ST诱导体外培养人胃粘膜上皮细胞周期阻滞的可能机制。本研究在前期实验研究的基础上,观察了不同浓度ST对体外培养永生化胃粘膜上皮细胞GES-1中JNK、ERK、p38和PI3K信号转导通路的影响情况,同时探讨了JNK、ERK和PI3K信号转导通路的激活在ST诱导的GES-1细胞周期G2期阻滞中的作用。
本研究论文共分为三个部分:
1杂色曲霉素对GES-1细胞中MAPK和PI3K信号转导通路的影响研究
目的:探讨一次性给予不同浓度ST对体外培养胃黏膜上皮细胞GES-1中JNK、ERK、p38和PI3K/mTOR信号转导通路的影响情况。
方法:取对数生长期GES-1细胞,随机分为6组:溶剂对照组和对照组分别给予DMSO (0.2 ml/L)和生理盐水处理。实验组给予DMSO溶解并以生理盐水稀释的ST,使其终浓度分别为100、500、1000、2000μg/L,处理24 h后,采用Western blot和RT-PCR方法检测GES-1中JNK/p-JNK、ERK/p-ERK、p38/p-p38和PI3K/mTOR/p-mTOR表达的变化情况。
另取对数生长期GES-1细胞,分别给予1μM SP600125 (JNK信号通路特异性阻断剂)、50μM PD98059 (ERK信号通路特异性阻断剂)、0.5μM SB203580 (p38信号通路特异性阻断剂)和1μM LY-294002 (PI3K特异性阻断剂)预处理。实验分为5组,即对照组、溶剂对照组、ST处理组、阻断剂组和阻断剂+ST处理组。细胞培养24 h后,更换2%低血清DMEM培养基。阻断剂组分别加入SP600125 (1μM)、PD98059 (50μM)、SB203580 (0.5μM)和LY-294002(1μM)。30 min后ST处理组和阻断剂+ST处理组分别给予1000μg/L ST,溶剂对照组和对照组分别给予DMSO (0.1ml/L)和生理盐水处理。细胞处理24 h后离心收细胞,采用Western blot方法检测目的基因在蛋白水平上表达变化。
结果:
1.1 ST对JNK信号转导通路的影响
1.1.1不同浓度ST对JNK信号转导通路的影响
Western blot结果显示,ST 100、500、1000、2000μg/L处理后细胞内JNK蛋白相对表达量与溶剂对照组比较差异无显著性(P>0.05)。而ST 500、1000、2000μg/L处理后JNK磷酸化水平明显高于溶剂对照组(P<0.05),并且,在0～2000μg/L ST浓度范围内,JNK磷酸化水平与ST的处理浓度有明显剂量依赖关系(r=0.925, P<0.01, n=3)。RT-PCR方法检测发现,各ST处理组JNK mRNA相对表达量与溶剂对照组(P>0.05)相比,没有明显差别。
1.1.2 SP600125预处理对ST作用后GES-1细胞JNK磷酸化水平的影响
Western blot方法检测结果表明,SP600125组细胞JNK磷酸化水平明显低于溶剂对照组(P<0.05),SP600125预处理+ST处理组JNK磷酸化水平较ST单独处理组明显降低,但仍高于溶剂对照组(P<0.05),表明给予JNK阻断剂SP600125对JNK蛋白激酶具有特异阻断作用,但只能部分阻断ST对JNK信号转导通路的激活作用。
1.2 ST对ERK信号转导通路的影响
1.2.1不同浓度ST对ERK信号转导通路的影响
Western blot结果显示,各ST处理组细胞ERK蛋白相对表达量与溶剂对照组比较无明显差异(P>0.05)。而ST 500、1000、2000μg/L处理后细胞内ERK磷酸化水平明显高于溶剂对照组(P<0.05),并且,在0～2000μg/L ST浓度范围内,ERK磷酸化水平与ST的处理浓度有明显剂量依赖关系(r=0.887, P<0.01, n=3)。
RT-PCR方法检测发现,各ST处理组细胞中ERK mRNA相对表达量与溶剂对照组相比,没有明显差别(P>0.05)。
1.2.2 PD98059预处理对ST作用后GES-1细胞ERK磷酸化水平的影响
Western blot方法检测,结果表明PD98059组细胞ERK磷酸化水平明显低于溶剂对照组(P<0.05),给予PD98059预处理后,PD98059+ST处理组ERK的磷酸化水平较ST单独处理组明显降低,但仍高于溶剂对照组(P<0.05)。表明给予ERK阻断剂PD98059对ERK蛋白激酶具有特异阻断作用,但只能部分阻断ST对ERK信号转导通路的激活作用。
1.3 ST对p38信号转导通路的影响
1.3.1不同浓度ST对p38信号转导通路的影响
Western blot结果显示,在0～2000μg/L ST浓度范围内,不同浓度ST处理后p38蛋白相对表达量与溶剂对照组比较均无明显差异(P>0.05)。ST 500、1000、2000μg/L处理后相对p38磷酸化水平明显低于溶剂对照组(P<0.05),并且,在0～2000μg/L ST浓度范围内,p38磷酸化水平与ST的处理浓度呈明显负相关关系(r=-0.843, P<0.01, n=3)。
RT-PCR方法检测发现,各ST处理组细胞内p38 mRNA相对表达量与溶剂对照组相比,均没有明显差别(P>0.05)。
1.3.2 SB203580预处理对ST作用后GES-1细胞p38磷酸化水平的影响
Western blot检测结果表明,SB203580处理组细胞相对p38磷酸化水平均明显低于溶剂对照组(P<0.05),SP600125+ST处理组p38磷酸化水平较ST单独处理组明显降低(P<0.05),表明p38阻断剂SB203580对p38的磷酸化水平具有特异阻断作用,给予p38特异性阻断剂SB203580预处理,对ST诱导细胞p38磷酸化水平降低有明显的协同作用。
1.4 ST对PI3K/mTOR信号转导通路的影响
1.4.1不同浓度ST对PI3K/mTOR信号转导通路的影响
Western blot结果显示,各ST处理组细胞PI3K蛋白相对表达量与溶剂对照组相比没有明显差异(P>0.05)。1000、2000μg/L ST处理组细胞mTOR蛋白相对表达量明显高于溶剂对照组(P<0.05),并且在0～2000μg/L ST浓度范围内,mTOR的蛋白表达与ST的处理浓度有明显剂量依赖关系(r=0.831, P<0.01, n=3)。同时,ST也可剂量依赖性地上调mTOR的磷酸化水平(r=0.868, P<0.01, n=3),其中500、1000和2000μg/L ST处理组mTOR的磷酸化水平升高更明显(P<0.05)。
RT-PCR方法检测结果发现,与溶剂对照组相比,不同浓度ST处理24 h对细胞内PI3K mRNA相对表达量没有明显影响(P>0.05),但可以明显上调mTOR在mRNA水平上的表达,mTOR mRNA的相对表达量与ST的处理浓度呈明显正相关关系(r=0.831, P<0.01, n=3)。
1.4.2 LY-294002预处理对ST作用后GES-1细胞mTOR基因表达及其磷酸化水平的影响
Western blot方法检测,LY-294002组细胞mTOR及其磷酸化水平与溶剂对照组相比没有明显差别(P>0.05),LY-294002+ST处理组mTOR磷酸化水平较ST单独处理组明显降低(P<0.05),与溶剂对照组相比没有明显差别(P>0.05);而mTOR蛋白的表达则明显高于溶剂对照组(P<0.05),与ST单独处理组没有差别(P>0.05)。
RT-PCR结果显示,LY-294002组细胞mTOR mRNA相对表达量与溶剂对照组相比没有明显差别(P>0.05),LY-294002+ST处理组mTOR mRNA相对表达量明显高于溶剂对照组(P<0.05),而与ST单独处理组相比没有明显差别(P>0.05)。
结果表明,给予PI3K阻断剂LY-294002对PI3K/mTOR信号转导通路具有特异阻断作用,可以阻断ST诱导mTOR磷酸化水平升高的作用,但对mTOR在蛋白和mRNA水平上的表达没有明显影响。
2 JNK、ERK和PI3K信号转导通路的激活在杂色曲霉素诱导GES-1细胞G2期阻滞中的作用研究
目的:探讨JNK、ERK和PI3K信号转导通路特异性阻断剂预处理对ST处理24 h诱导体外培养GES-1细胞增殖抑制、G2期阻滞及其相关因子表达的影响。
方法:取对数生长期GES-1细胞,分别给予1μM SP600125 (JNK信号通路特异性阻断剂)、50μM PD98059 (ERK信号通路特异性阻断剂)和1μM LY-294002 (PI3K特异性阻断剂)预处理细胞。按1×104个/L接种于96孔板,待细胞进入对数生长期后更换培养基,实验分为5组,即对照组、溶剂对照组、ST处理组、阻断剂组和阻断剂+ST处理组。每孔终体积为200μl,同时设空白对照孔、对照孔和溶剂对照孔,细胞培养24 h后,更换2%低血清DMEM培养基,阻断剂组分别加入SP600125 (1μM)、PD98059 (50μM)和LY-294002 (1μM)。30 min后ST处理组和阻断剂+ST处理组分别给予1000μg/L ST,溶剂对照组和对照组分别给予DMSO (0.1ml/L)和生理盐水处理。细胞处理24 h后,采用MTT检测细胞存活率。
另取对数生长期GES-1细胞,分为5组,即对照组、溶剂对照组、ST处理组、阻断剂组和阻断剂+ST处理组(处理同上)。细胞处理24 h后离心收细胞,采用FCM检测细胞周期分布情况,采用Western blot和RT-PCR方法观察周期调控关键因子——CyclinB1蛋白的表达、Cdc2激酶和Cdc25C磷酸酶活性的变化情况。
结果:
2.1 JNK信号转导通路在ST诱导GES-1细胞G2期阻滞中的作用
2.1.1 SP600125预处理对ST诱导的GES-1细胞存活率降低的影响
MTT结果表明,SP600125+ST组细胞存活率明显高于ST单独处理组(P<0.05),但仍明显低于溶剂对照组(P<0.05)。表明SP600125预处理可部分阻断ST对GES-1细胞增殖的抑制作用。
2.1.2 SP600125预处理对ST诱导的GES-1细胞G2期阻滞的影响
FCM检测结果显示,与ST 1 000μg/L处理组相比,SP600125预处理+ST 1000μg/L处理组G0/G1期所占比例明显升高;而G2/M期细胞比例则明显降低(P<0.05),但仍明显高于溶剂对照组(P<0.05)。表明SP600125预处理可部分阻断ST诱导的细胞G2/M期比例增高作用。
2.1.3 SP600125预处理对ST作用后GES-1细胞G2期相关因子的影响
2.1.3.1 SP600125预处理对CyclinB1表达的影响
Western blot检测结果显示,SP600125预处理+ST 1000μg/L处理组CyclinB1蛋白的表达明显低于溶剂对照组,同时显著低于ST 1000μg/L处理组(P<0.05)。
RT-PCR结果发现,SP600125预处理+ST组细胞内CyclinB1 mRNA的表达明显低于ST 1000μg/L处理组(P<0.05),而与溶剂对照组相比没有差别。
表明SP600125预处理可阻断ST对GES-1细胞内CyclinB1在蛋白和mRNA水平表达的升高作用。
2.1.3.2 SP600125预处理对Cdc2激酶的影响
Western blot检测结果显示,与ST 1000μg/L处理组细胞相比,SP600125预处理+ST 1000μg/L处理组内Cdc2的去磷酸化水平明显升高,同时其磷酸化水平明显降低(P<0.05),而与溶剂对照组相比无明显差别。
RT-PCR结果表明,SP600125预处理+ST组细胞内Cdc2 mRNA的表达与ST 1000μg/L处理组相比没有明显差别(P>0.05)。
表明SP600125预处理可阻断ST对GES-1细胞内Cdc2去磷酸化水平和Cdc2磷酸化水平的影响,但对Cdc2 mRNA没有明显影响。
2.1.3.3 SP600125预处理对Cdc25C磷酸酶的影响
Western blot检测结果显示,与溶剂对照组相比,SP600125预处理+ST 1000μg/L处理组内Cdc25C蛋白的表达明显升高,同时明显高于ST 1000μg/L处理组;而Cdc25C磷酸化水平则较ST 1000μg/L处理组明显降低(P<0.05),与溶剂对照组相比没有明显差别。
RT-PCR结果表明,SP600125预处理+ST组细胞Cdc25C mRNA的表达与ST 1000μg/L处理组相比没有明显差别(P>0.05)。表明SP600125预处理可阻断ST对GES-1细胞内Cdc25C的蛋白表达和Cdc25C磷酸化水平的影响,但对Cdc25C mRNA没有明显影响。
2.2 ERK信号转导通路在ST诱导GES-1细胞G2期阻滞中的作用
2.2.1 PD98059预处理对ST诱导的GES-1细胞存活率降低的影响
MTT结果表明,PD98059+ST处理组细胞存活明显高于ST单独处理组(P<0.05),而与溶剂对照组相比没有明显差别(P>0.05)。表明PD98059预处理可以阻断ST对GES-1细胞的增殖抑制作用。2.2.2 PD98059预处理对ST诱导的GES-1细胞G2期阻滞的影响FCM检测结果显示,与ST 1000μg/L处理组相比,PD98029预处理+ ST 1000μg/L处理组S期细胞比例明显升高(P<0.05);而G2/M期细胞比例明显降低(P<0.05),但仍明显高于溶剂对照组(P<0.05)。表明PD98059预处理可部分阻断ST诱导的细胞G2/M期比例增高。
2.2.3 PD98059预处理对ST作用后GES-1细胞G2期相关因子的影响
2.2.3.1 PD98059预处理对CyclinB1表达的影响
Western blot检测结果显示,PD98059预处理+ST 1000μg/L处理组CyclinB1的蛋白表达与ST 1000μg/L处理组相比显著降低(P<0.05),但仍明显高于溶剂对照组(P<0.05)。
RT-PCR结果显示,PD98059预处理+ST组细胞内CyclinB1 mRNA的表达与ST 1000μg/L处理组相比没有明显差别(P>0.05)。
表明PD98059预处理可部分阻断ST对GES-1细胞内CyclinB1蛋白表达的升高作用,但对CyclinB1 mRNA没有明显影响。
2.2.3.2 PD98059预处理对Cdc2激酶的影响
Western blot检测结果显示,PD98059预处理+ST 1000μg/L处理组细胞内Cdc2的去磷酸化水平与ST 1000μg/L处理组相比,没有明显差异(P>0.05);而其磷酸化水平则明显低于ST 1000μg/L处理组(P<0.05),与溶剂对照组相比没有明显差异(P>0.05)。
RT-PCR结果显示,PD98059预处理+ST组细胞Cdc2 mRNA的表达明显低于ST 1000μg/L处理组,但仍高于溶剂对照组(P<0.05)。
表明PD98059预处理可阻断ST对GES-1细胞内Cdc2磷酸化水平的升高作用,同时部分阻断ST对Cdc2 mRNA表达的升高作用。
2.2.3.3 PD98059预处理对Cdc25C磷酸酶的影响
Western blot检测结果显示,PD98059预处理+ST 1000μg/L处理组细胞Cdc25C蛋白表达与ST 1000μg/L处理组相比没有明显差异(P>0.05);Cdc25C磷酸化水平则较ST 1000μg/L处理组明显降低,但仍高于溶剂对照组(P<0.05)。
RT-PCR结果显示,PD98059预处理+ST组细胞内Cdc25C mRNA的表达与ST 1000μg/L处理组相比没有明显差别(P>0.05)。
提示PD98059预处理可部分阻断ST对细胞内Cdc25C磷酸化水平的升高作用,但对Cdc25C mRNA没有明显影响。
2.3 PI3K信号转导通路在ST诱导GES-1细胞G2期阻滞中的作用
2.3.1 LY-294002预处理对ST诱导的GES-1细胞存活率降低的影响
MTT结果表明,LY-294002+ST处理组细胞存活率明显低于溶剂对照组(P<0.05),而与ST单独阻断剂组相比没有明显差别(P>0.05)。表明LY-294002预处理对ST诱导的GES-1细胞增殖抑制作用没有明显影响。
2.3.2 LY-294002预处理对ST诱导的GES-1细胞G2期阻滞的影响
FCM检测结果显示,LY-294002预处理+ST 1000μg/L处理组G0/G1期和S期细胞所占比例均较ST 1000μg/L处理组明显降低,而G2/M期细胞比例则明显增加(P<0.05),表明LY-294002预处理对ST诱导的细胞G2/M期比例增高具有协同作用。
2.3.3 LY-294002预处理对ST作用后GES-1细胞G2期相关因子的影响
2.3.3.1 LY-294002预处理对CyclinB1表达的影响
Western blot检测结果显示,LY-294002预处理+ST 1000μg/L处理组CyclinB1蛋白的表达与ST 1000μg/L处理组相比显著降低,较溶剂对照组也明显降低(P<0.05)。
RT-PCR结果显示,LY-294002预处理+ST组细胞内CyclinB1 mRNA的表达明显低于ST单独处理组,同时明显低于溶剂对照组(P<0.05)。表明LY-294002预处理可阻断ST诱导GES-1细胞内CyclinB1在蛋白和mRNA水平表达的升高作用。
2.3.3.2 LY-294002预处理对Cdc2激酶的影响
Western blot检测结果显示,LY-294002预处理+ST 1000μg/L处理组细胞内Cdc2的去磷酸化水平与ST 1000μg/L处理组相比没有明显差异(P>0.05);而其磷酸化水平则明显高于ST 1000μg/L处理组(P<0.05),同时明显高于溶剂对照组(P<0.05)。
RT-PCR结果显示,LY-294002预处理+ST组细胞Cdc2 mRNA的表达明显高于溶剂对照组,同时也明显高于ST单独处理组(P<0.05)。表明LY-294002预处理对ST诱导的GES-1细胞内Cdc2的磷酸化水平及其mRNA表达升高有明显的协同作用。
2.3.3.3 LY-294002预处理对Cdc25C磷酸酶的影响
Western blot检测结果显示,LY-294002预处理+ST 1000μg/L处理组细胞内Cdc25C蛋白的表达与ST 1000μg/L处理组相比没有明显差异(P>0.05);而其磷酸化水平则较ST 1000μg/L处理组明显升高(P<0.05),同时也明显高于溶剂对照组(P<0.05)。
RT-PCR结果显示,LY-294002预处理+ST组细胞内Cdc25C mRNA的表达与ST 1000μg/L处理组相比没有明显差别(P>0.05)。
表明LY-294002预处理对ST诱导的GES-1细胞内Cdc25C磷酸化水平升高有明显的协同作用,但对Cdc25C在蛋白和mRNA水平上的表达没有明显影响。
3 JNK、ERK和PI3K信号转导通路的激活在杂色曲霉素影响GES-1细胞蛋白质合成起始因子表达的作用研究
目的:进一步探讨JNK、ERK和PI3K信号转导通路特异性阻断剂预处理,对ST处理24 h诱导体外培养GES-1细胞蛋白质合成障碍的影响。
方法:实验细胞处理、分组(同前)。继续采用Western blot和RT-PCR方法研究了分别给予JNK、ERK和PI3K信号转导通路特异性阻断剂预处理,ST诱导的体外培养胃黏膜上皮细胞GES-1细胞中eIF4E和4E-BP1的表达及其活性变化,以及CyclinD1蛋白表达的变化情况
结果:
3.1 JNK信号转导通路在ST影响GES-1细胞蛋白质合成中的作用
3.1.1 SP600125预处理对eIF4E表达的影响
Western blot检测结果显示,SP600125预处理+ST 1000μg/L处理组细胞内eIF4E蛋白的表达与溶剂对照组相比明显下降,同时显著低于ST单独处理组(P<0.05),而eIF4E磷酸化水平则明显高于溶剂对照组,同时显著高于ST单独处理组(P<0.05)。
RT-PCR结果表明,SP600125预处理+ST组细胞内eIF4E mRNA的表达明显低于ST 1000μg/L处理组,但仍明显高于溶剂对照组(P<0.05)。表明SP600125预处理可阻断ST对GES-1细胞内eIF4E的活性抑制作用,同时可部分阻断ST对eIF4E mRNA表达的升高作用。
3.1.2 SP600125预处理对4E-BP1表达的影响
Western blot检测结果显示,SP600125预处理+ST 1000μg/L处理组4E-BP1蛋白的表达明显低于ST单独处理组(P<0.05),而与溶剂对照组相比没有明显变化(P>0.05);4E-BP1磷酸化水平较溶剂对照组明显升高,同时显著高于ST单独处理组(P<0.05)。
RT-PCR结果表明,SP600125预处理+ST组细胞内4E-BP1 mRNA的表达也明显低于ST单独处理组,但仍明显高于溶剂对照组(P<0.05)。表明SP600125预处理可阻断ST对GES-1细胞内4E-BP1活性的促进作用,同时可部分阻断ST对4E-BP1 mRNA表达的升高作用。
3.1.3 SP600125预处理对CyclinD1蛋白表达的影响
SP600125预处理+ST 1000μg/L处理组CyclinD1蛋白的表达明显高于ST单独处理组,但仍明显低于溶剂对照组(P<0.05),表明SP600125预处理可部分阻断ST对GES-1细胞内CyclinD1蛋白表达的降低作用。
3.2 ERK信号转导通路在ST影响GES-1细胞蛋白质合成中的作用
3.2.1 PD98059预处理对eIF4E表达的影响
Western blot检测结果显示,PD98059预处理+ST 1000μg/L处理组内eIF4E蛋白的表达与ST 1000μg/L处理组相比没有明显差异(P>0.05),而其磷酸化水平与ST 1000μg/L处理组细胞相比明显升高(P<0.05),而与溶剂对照组没有明显差别(P>0.05)。
RT-PCR结果表明,PD98059预处理+ST组细胞内eIF4E mRNA的表达明显低于ST单独处理组(P<0.05),而与溶剂对照组相比没有差别。表明PD98059预处理对ST诱导的GES-1细胞内eIF4E蛋白的表达增高没有影响;但可阻断ST对eIF4E磷酸化水平的降低以及eIF4E mRNA表达的升高作用。
3.2.2 PD98059预处理对4E-BP1的影响
Western blot检测结果显示,PD98059预处理+ST 1000μg/L处理组4E-BP1蛋白的表达较ST单独处理组明显降低(P<0.05),而与溶剂对照组相比没有明显差别;4E-BP1磷酸化水平与ST 1000μg/L处理组细胞相比也明显升高(P<0.05),也与溶剂对照组相比没有明显差别(P>0.05)。
RT-PCR结果表明,PD98059预处理+ST组细胞4E-BP1 mRNA的表达与ST单独处理组相比明显降低,但仍明显高于溶剂对照组(P<0.05)。表明PD98059预处理可阻断ST对GES-1细胞内4E-BP1蛋白的表达升高作用及4E-BP1磷酸化水平降低作用;同时可部分阻断4E-BP1 mRNA表达升高的作用。
3.2.3 PD98059预处理对CyclinD1蛋白表达的影响
PD98059预处理+ST 1000μg/L处理组CyclinD1蛋白的表达明显高于溶剂对照组(P<0.05),同时显著高于ST 1000μg/L处理组(P<0.05),表明PD98059预处理可阻断ST诱导GES-1细胞内CyclinD1蛋白表达降低的作用。
3.3 PI3K信号转导通路在ST影响GES-1细胞蛋白质合成中的作用
3.3.1 LY-294002预处理对eIF4E表达的影响
Western blot检测结果显示,LY-294002预处理+ST 1000μg/L处理组内eIF4E蛋白的表达明显高于溶剂对照组,同时明显高于ST单独处理组(P<0.05);eIF4E的磷酸化水平较ST处理组明显降低(P<0.05),同时明显低于溶剂对照组(P<0.05)。
RT-PCR结果表明,LY-294002预处理+ST组细胞内eIF4E mRNA的表达明显高于溶剂对照组,并且也较ST单独处理组明显升高(P<0.05)。
表明LY-294002预处理对ST诱导的eIF4E在蛋白和mRNA水平的表达升高及eIF4E磷酸化水平的降低均有明显的协同作用。
3.3.2 LY-294002预处理对4E-BP1的影响
Western blot检测结果显示,LY-294002预处理+ST 1000μg/L处理组内4E-BP1蛋白的表达明显低于ST单独处理组(P<0.05),而与溶剂对照组相比没有明显差别(P>0.05);4E-BP1磷酸化水平明显低于溶剂对照组(P<0.05),同时较ST 1000μg/L处理组细胞也明显降低(P<0.05)。
RT-PCR结果表明,LY-294002预处理+ST组细胞4E-BP1 mRNA的表达与ST单独处理组相比明显降低,但仍高于溶剂对照组(P<0.05)。
表明LY-294002预处理可阻断ST对GES-1细胞内4E-BP1在蛋白和mRNA水平的表达升高作用,但对ST诱导的4E-BP1磷酸化水平降低有明显的协同作用。
3.3.3 LY-294002预处理对CyclinD1蛋白表达的影响
LY-294002预处理+ST 1000μg/L处理组CyclinD1蛋白的表达明显低于溶剂对照组(P<0.05),同时显著低于ST 1000μg/L处理组(P<0.05),表明LY-294002预处理对ST诱导的GES-1细胞内CyclinD1蛋白表达降低有明显的协同作用。
结论:
1杂色曲霉素作用于GES-1细胞24 h,可以激活JNK、ERK和PI3K信号转导通路,同时可以抑制p38信号转导通路。
2杂色曲霉素可能通过激活JNK和ERK信号转导通路影响GES-1细胞周期分布,诱导G2期阻滞;但PI3K信号转导通路的激活在杂色曲霉素诱导的G2期阻滞中可能发挥反向调节作用。
3杂色曲霉素作用于GES-1细胞可通过激活JNK、ERK和PI3K信号转导通路而影响Cdc25C -Cdc2/CyclinB1的表达和功能,可能是杂色曲霉素诱导细胞G2期阻滞的机制之一。
4 JNK、ERK和PI3K信号转导通路的激活可能均参与了ST对体外培养胃黏膜上皮细胞GES-1的影响。但PI3K信号转导通路的激活此过程中可能主要发挥反向调节作用。
Sterigmatocystin (ST) is a toxic fungal metabolite with low molecular weight. It is produced by some Aspergillus species fungi, such as, A versicolor, A nidulans,etc. ST is one of the common contaminants of the ingredients of animal feed and human food all over the world. ST has been proved to have carcinogenic potency in experimental animal models. It could induce lung adenocarcinoma, hepatoma and mesotheliomas in different experimental animals. ST is one of genotoxic agents and could form DNA adducts with target cell DNA and result in direct DNA damages. Our previous studies with human tissue showed that ST could induce malignant transformation and p53 mutation in human fetal gastric mucosa cells in vitro. Oral administration of ST for long period of time could induce intestinal metaplasia and atypical hyperplasia of glandular epithelium in mice.
As we know that the control of cell cycle is one of the most imortant biological processes. Disregulation of cell cycle may result in carcinogenesis. It has be showed that ST could affect cell cycle of target cells. Xie et al found that ST could cause G2/M arrest in murine fibroblasts. Our primary study with human gastric epithelial cell line also confirmed that ST could induce G2 phase arrest in human gastric GES-1 cells in vitro. But the putative mechanisms of G2 arrest induced by ST was still not clear enough.
Cyclins are key components of the cell cycle progression machinery. They activate their partner cyclin-dependent kinases (CDKs) and possibly target them to respective substrate proteins within the cell. CDK-mediated phosphorylation of specific sets of proteins drives the cell through particular phases or checkpoints of the cell cycle. It has been established that the G2 checkpoint initiation is primarily regulated through the control of Cdc2 (Cdk1) activity, which is regulated at multiple levels, including periodic association with the B-type cyclins, reversible phosphorylation, and intracellular compartimentalization. CyclinB1 is the cylcin at stage of G2, and the compound formed with its kinase Cdc2 is the key factor for mitoschisis and play key role to precipitate cell cycle from stage of G2 to stage of M. Cyclin B1 reaches maximum levels in G2, when it enters into the nucleus to form a complex with Cdk1 in a phosphorylation-dependent manner. Protein phosphatase Cdc25C could induce dephosphorylation of Cdc2 and promote the progression of cell cycle. Phosphorylation of Cdc25C could result in the loss of enzyme activity, which in turn increase the phosphorylation activity of Cdc2 and the cell cycle may be arrested at G2 phase as a result. eIF4E is the key regulatory factor in the process of protein synthesis. In association with its binding protein- 4E-BP1, eIF4E modulates the synthesis of cap-dependent protein, such as CyclinD1. The elevated level of phosphorylation could enhance the protein synthesis of eIF4E. Studies showed that eIF4E and 4E-BP1 are associated with the disruption of cell cycle, and expressed abnormally in malignant tumor cell. Our recent experiment studies indicated that ST could inhibit the proliferation and induce human gastric GES-1 cells arrested at stage of G2 in vitro. And ST was found to have impact on the expression of CyclinB1 as well as the activity of Cdc2 kinase and Cdc25 phosphatase, which are all the key factors for the G2 regulatory point. At the same time, we found that ST could inhibit the initiated translation function of eIF4E.
JNK, ERK and p38 are the important members of MAPK signal transduction pathway. Studies showed that environmental carcinogens(such as mycotoxins)and chemotherapeutic agents could affect the expressions of the related genes and biological behaviors of the target cells by their effects on JNK, ERK and p38 signal transduction pathways. PI3K is one of the important signal transduction pathways in Her2 family. It played important roles in the control of cell proliferation, protein synthesis, etc through the regulation of the downstream nuclear factor-mTOR. In malignant tumors, PI3K was associated with the invasion, metastasis and prognosis and chemotherapy tolerance of tumor.
To further explore the putative mechanisms of G2 arrest induced by ST, the present study was carried out based on our previous studies. The effects of ST on the the activities of JNK, ERK, p38 and PI3K signal transduction pathway in human gastric epithelial cell line-GES-1 were studied. And the roles of JNK, ERK and PI3K activation on G2 arrest of GES-1 induced by ST in vitro was studied.
The following three parts are included in the study:
1 Effects of Sterigmatocystin on MAPK and PI3K signal transduction pathway in GES-1 cells in vitro
Objective: To explore the effects of different concentration of ST on JNK, ERK, p38 and PI3K/mTOR signal transduction pathway in GES-1 cells.
Methods: After initial culture for 24h, GES-1 cells were harvested, centrifuged and resuspended in fresh DMEM medium supplemented with 10% FCS at the concentration of (1~2)×104 cells/L in culture flasks (8 ml). The cells were randomly divided into 6 groups. The cells in ST groups were treated with ST at 100μg/L, ST 500μg/L, ST 1000μg/L and ST 2000μg/L, respectively, while DMSO and saline were used in solvent control and control group. Cells were harvested 24 h after ST treatment. The expression of ERK/p-ERK, p38/p-p38 and PI3K/mTOR/p-mTOR at protein level in GES-1 cells treated with ST was determined by Western blot. The expression of ERK, p38 and PI3K/mTOR mRNA in GES-1 cells was detected by RT-PCR.
GES-1 cells seeded at the concentration of (1~2)×104 cells/L in culture flasks were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L. The medium of GES-1 cells was replaced by new DMEM medium supplemented with 2% FCS 24h later. The cells of blocking agent groups were pretreated for 30 min with 1μM SP600125 (JNK inhibitor), 50μM PD98059 (ERK inhibitor ), 0.5μM SB203580 (p38 inhibitor) and 1μM LY-294002 (PI3K inhibitor) respectively. Then the cells in blocking agent +ST 1000μg/L group were treated with ST 1000μg/L, while these in solvent control and control groups were incubated with DMSO and saline respectively. Cells were harvested 24 h after ST treatment. The JNK, ERK, p38 and PI3K/mTOR activation of GES-1 cells treated with ST were determined with Western blot.
Results:
1.1 Effects of ST on JNK signal transduction pathway
1.1.1 Effects of ST with different concentrations on JNK signal transduction pathway
The results of Western blot showed that there was no significant difference in JNK protein expression in GES-1 cells between all the ST treatment groups and solvent control group (DMSO) (P>0.05), while the phosphorylation of JNK in ST 500μg/L, 1000μg/L and 2000μg/L treatment groups was significantly increased as compared with that in corresponding DMSO group (P<0.05). And there is a significant dose-dependent correlation between ST concentration and the phosphorylation of JNK (r=0.925, P<0.01, n=3).
The results of RT-PCR confirmed that no significant difference in JNK mRNA expression in GES-1 cells between all the ST treatment groups and solvent control group (DMSO) (P>0.05).
1.1.2 Effects of ST on JNK signal transduction pathway, with SP600125 pretreatment
The results of Western blot showed that the phosphorylation of JNK in GRS-1 cells in SP600125 treatment group was significantly decreased than that in corresponding DMSO group (P<0.05). And the phosphorylation of JNK in SP600125+ST treatment group was dramatically decreased compared with that in ST treatment alone, while still higher than that in solvent control (P<0.05). The result indicated that the SP600125 could specifically inhibit the activation of JNK MAP kinase, but the effect of activation of JNK in ST-treated cells was partially inhibited by the presence of SP600125.
1.2 Effects of ST on ERK signal transduction pathway
1.2.1 Effects of ST with different concentrations on ERK signal transduction pathway
The results of Western blot suggested that there was no significant difference in ERK protein expression in GES-1 cells between ST treatment groups and solvent control group (DMSO) (P>0.05). In comparison with solvent control group, the phosphorylation of ERK was significantly increased in ST treatment groups (P<0.05). Results from concentration-dependent studies indicated that ST up-regulated the phosphorylation of ERK in a dose-dependent correlation (r=0.887, P<0.01, n=3).
The results of RT-PCR showed that ST had no effect on the expression of ERK mRNA.
1.2.2 Effects of ST on ERK signal transduction pathway, with PD98059 pretreatment
The results of Western blot revealed that the phosphorylation of ERK in GRS-1 cells of PD98059 treatment group was significantly decreased compared with that in solvent control group (P<0.05). And the phosphorylation of ERK in PD98059+ST treatment group was decreased significantly compared with that in ST 1000μg/L treatment, while still higher than that in solvent control (P<0.05). The result revealed that the PD98059 could inhibit the activation of ERK MAP kinase, but the effect of activation of ERK in ST-treated cells was partially inhibited by the presence of PD98059.
1.3 Effects of ST on p38 signal transduction pathway
1.3.1 Effects of ST with different concentrations on p38 signal transduction pathway
No significant difference in p38 protein expression of GES-1 cells between all the ST treatment groups and solvent control group was found by Western blot (P>0.05), while the phosphorylation of p38 in ST treatment groups was lower compared with that in corresponding solvent control group (P<0.05). And the phosphorylation of p38 decreased with ST treatment in a dose-dependent manner (r=-0.843, P<0.01, n=3).
RT-PCR results confirmed that there was no significant difference in p38 mRNA expression in GES-1 cells between all the ST treatment groups and solvent control group (P>0.05).
1.3.2 Effects of ST on p38 signal transduction pathway, with SB203580 pretreatment
Western blot results showed that SB203580 could significantly decreased the level of phosphorylation of p38 in GES-1 cells (P<0.05). And the phosphorylation of p38 in SB203580+ST treatment group was markedly decreased compared with that in ST treatment (P<0.05). The result indicated that there might be a synergistic effect of the inhibitor of p38, SB203580 and ST on the phosphorylation of p38.
1.4 Effects of ST on PI3K/mTOR signal transduction pathway
1.4.1 Effects of ST with different concentrations on PI3K/mTOR signal transduction pathway
The results of Western blot showed that the expression of PI3K protein in GES-1 cells in both the ST treatment groups and solvent control group showed no significant differences (P>0.05), while the expression of mTOR protein in ST 1000μg/L and 2000μg/L treatment groups was higher compared with that in solvent control group (P<0.05). And there is a significant dose-dependent correlation between ST concentration and the expression of mTOR protein (r=0.831, P<0.01, n=3). Meanwhile, the phosphorylation of mTOR was significantly increased by ST in a dose-dependent way (r=0.868, n=3, P<0.01).
RT-PCR analysis suggested that there was no difference in PI3K mRNA expression in GES-1 cells between all the ST treatment groups and solvent control group (P>0.05). But the expression of mTOR mRNA was increased by ST in a dose-dependent way (r=0.831, n=3, P<0.01).
1.4.2 Effects of ST on PI3K/mTOR signal transduction pathway, with LY-294005 pretreatment
The results of Western blot showed that the expression of mTOR protein and phosphorylation in GES-1 cells in LY-294002 treatment group showed no significant difference compared with that in solvent control group (P<0.05). Compared with the group with ST only treatment, the phosphorylation of mTOR in LY-294002+ST treatment group was decreased significantly (P<0.05), while there was no difference as compared with that of solvent control (P>0.05). But the exptession of mTOR protein in LY-294002+ST treatment group was higher than that in solvent control (P<0.05), and it showed no difference between LY-294002+ST treatment group and ST treatment only (P>0.05).
The results of RT-PCR displayed that there was no difference in mTOR mRNA expression in GES-1 cells between ST treatment groups and solvent control group (P>0.05). But in comparison with solvent control group, mTOR mRNA expression in LY-294002+ST treatment group was higher (P<0.05), but was not different from that in ST treatment only groups (P>0.05).
The results indicated that the LY-294002 could inhibit the activity of PI3K/mTOR signal pathway, but it had no effect on the expression of mTOR at both protein and mRNA levels in GES-1 cells.
2 Effects of JNK, ERK and PI3K signal transduction pathway activation on the G2 arrest induced by Sterigmatocystin in GES-1 in vitro
Objective: To explore the effects of inhibitors of JNK, ERK and PI3K on the ST-induced proliferation inhibition and G2 arrest.
Methods:
GES-1 cells were harvested, centrifuged and resuspended in fresh DMEM medium supplemented with 10% FCS at the concentration of (1~2)×104 cells/L in 96-well culture plates. The cells were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L. The cells of blocking agent groups were pretreated for 30 min with 1μM SP600125 (JNK inhibitor), 50μM PD98059 (ERK inhibitor), 0.5μM SB203580 (p38 inhibitor) and 1μM LY-294002 (PI3K inhibitor) respectively. Then the cells in blocking agent +ST 1000μg/L group were treated with ST 1000μg/L, while these in solvent control and control groups were incubated with DMSO and saline respectively. Cell viability for GES-1 cells was determined by MTT assay.
For the blocking studies, GES-1 cells seeded at the concentration of (1~2)×104 cells/L in culture flasks were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L (the treatment was same as above). Cells were harvested 24 h after ST treatment. The changes of cell cycle in response to ST was measured using FCM. And the expression of CyclinB1, the activity of Cdc2 kinase and Cdc25C phosphatase in GES-1 cells were determined with Western blot and RT-PCR.
Results:
2.1 Effects of JNK signal pathway on the G2 arrest induced by ST
2.1.1 Effect of SP600125 pretreatment on the changes of survival rate in GES-1 cells induced by ST
The result of MTT showed that the cell viability in SP600125+ST treatment group was much higher than that in ST group, but it still lower than that of controls (P<0.05). The result indicated that SP600125 could partly block the inhibition of proliferation induced by ST in GES-1 cell.
2.1.2 Effect of SP600125 pretreatment on G2 arrest of GES-1 cells induced by ST
FCM analysis revealed that the G0/G1 fraction was increased and the G2/M fraction was decreased in SP600125+ST treatment group compared with ST 1000μg/L group. But the G2/M faction was still increased compared with control group. Thus, addition of SP600125 to the cells could partly decrease the ability of ST to increase the percentage of G2/M faction.
2.1.3 Effect of SP600125 pretreatment on the ST-induced changes of the key factors of G2 phase in GES-1 cells
2.1.3.1 Effect of SP600125 pretreatment on the expression of CyclinB1
The results of Western blot showed that the expression of CyclinB1 protein in SP600125+ST treatment group was significantly decreased compared with that in solvant control group and ST group (P<0.05).
The results of RT-PCR suggested that the expression of CyclinB1 mRNA in SP600125+ST treatment group was significantly decreased as compared with that in ST group (P<0.05) and there was no difference of that compared with solvent control group.
Thus, the results in this study revealed that the increase in the expression of CyclinB1 at both protein and mRNA level was blocked by the presence of SP600125 in ST-treated GES-1 cells.
2.1.3.2 Effect of SP600125 pretreatment on Cdc2 kinase
Western blot results showed that in comparison with ST treatment group, the dephosphorylation level of Cdc2 in SP600125+ST treatment group was significantly increased, meanwhile the phosphorylation level of Cdc2 was decreased (P<0.05). And no significant difference was found between SP600125+ST treatment group and solvent control group.
The results of RT-PCR displayed that the expression of Cdc2 mRNA had no difference between SP600125+ST treatment group and ST treatment group.
Thus, the results in this study suggested that alteration in dephosphorylation and phosphorylation of Cdc2 caused by ST were blocked by the JNK inhibitor SP600125, which had no effect on the expression of Cdc2 mRNA.
2.1.3.3 Effect of SP600125 pretreatment on Cdc25C phosphatase
The results of Western blot showed that the presence of the JNK inhibitor simultaneously with ST induced a significant increase in the expression of Cdc25C, as compared with that in solvent control group and ST treatment group (P<0.05). The level of phosphorylation of Cdc25C was reduced as compared with ST treatment group (P<0.05), and had no difference compared with solvent control group.
RT-PCR results confirmed that there was no difference in the expression of Cdc25C mRNA between SP600125+ST treatment group and ST treatment group.
Thus, the results in this study indicated that SP600125 could inhibit the alterations in protein expression and phosphorylation level of Cdc25C induced by ST, but had no influence on the expression of Cdc25C mRNA.
2.2 Effects of ERK signal pathway on the G2 arrest induced by ST
2.2.1 Effect of PD98059 pretreatment on the changes of GES-1 cells survival rate induced by ST
The result of MTT showed that the cell viability in PD98059+ST treatment group was much higher than that of ST group, and there was no difference of that as compared with solvant control group (P<0.05). The result indicated that the PD98059 could partly block the ST-induced antiproliferative action in GES-1 cells.
2.2.2 Effect of PD98059 pretreatment on the G2 arrest of GES-1 cells induced by ST
FCM analysis showed that the number of S faction was increased and that of G2/M faction was decreased in PD98059+ST treatment group compared with ST 1000μg/L groups. But the G2/M faction was still increased compared with solvant control group. Thus, addition of PD98059 to the cells could partly decrease the ability of ST to increase the percentage of G2/M faction.
2.2.3 Effect of PD98059 pretreatment on the changes of the key factors expression of G2 stage induced by ST in GES-1 cells
2.2.3.1 Effect of PD98059 pretreatment on the expression of CyclinB1
Western blot results showed that the expression of CyclinB1 protein in PD98059+ST treatment group was significantly decreased as compared with that in ST group, but it was still higher than that in solvant control group (P<0.05).
The results of RT-PCR showed that the expression of CyclinB1 mRNA in GES-1 cells had no difference between PD98059+ST treatment group and ST treatment group (P>0.05).
Thus, the results in this study suggested that the alterations in protein expression of CyclinB1 caused by ST were blocked by the presence of ERK inhibitor PD98059, which had no effect on the expression of CyclinB1 mRNA.
2.2.3.2 Effect of PD98059 pretreatment on Cdc2 kinase
The results of Western blot showed that no difference in Cdc2 dephosphorylation in PD98059+ST treatment group and ST group could be found. But the phosphorylation of Cdc2 in PD98059+ST treatment group was decreased as compared with that in ST group (P<0.05), and there was no difference of that compared with solvent control group (P>0.05).
The results of RT-PCR confirmed that the expression of Cdc2 mRNA in PD98059+ST treatment group was decreased as compared with that in ST treatment group, but it was still higher than that in solvent control group (P<0.05).
Thus, the results in this study revealed that the increase in the level of phosphorylation of Cdc2 was blocked by the addition of PD98059 which could partly inhibit the expression of Cdc2 mRNA in ST-treated GES-1 cells.
2.2.3.2 Effect of PD98059 pretreatment on Cdc25C phosphatase
The results of Western blot showed that, the expression of Cdc25C protein had no difference in PD98059+ST treatment group and ST group. The phosphorylation of Cdc25C in PD98059+ST treatment group was significantly decreased as compared with ST group, but it was still higher than that in solvent control group (P<0.05).
The results of RT-PCR revealed that there was no difference of the expression of Cdc25C mRNA between PD98059+ST treatment group and ST group (P>0.05).
Thus, the results in this study indicated that PD98059 could inhibit the alterations in phosphorylation of Cdc25C induced by ST, but had no effect on the expression of Cdc25C mRNA.
2.3 Effects of PI3K signal pathway on the G2 arrest induced by ST
2.3.1 Effect of LY-294002 pretreatment on the changes of GES-1 cells survival rate induced by ST
The result of MTT showed that there was no difference in the cell viability between LY-294002+ST treatment group and ST group (P>0.05), and it was much lower than that of controls (P<0.05). The result indicated that LY-294002 had no effect on the cell viability.
2.3.2 Effect of LY-294002 pretreatment on the G2 arrest of GES-1 cells induced by ST
FCM analysis showed that the number of G0/G1 and S faction were all decreased and that of G2/M faction was increased in LY-294002+ST treatment group compared with ST 1000μg/L groups (P<0.05). The result indicated that there might be a synergistic effect of LY-294002 and ST on the increase of G2/M faction of GES-1 cells.
2.3.3 Effect of LY-294002 pretreatment on the changes of the key factors expression of G2 stage induced by ST in GES-1 cells
2.3.3.1 Effect of LY-294002 pretreatment on the expression of CyclinB1.
Western blot results showed that addition of the PI3K inhibitor combined with ST induced a significant decrease of CyclinB1 protein expression, as compared with that in solvant control group and ST group (P<0.05).
The results of RT-PCR confirmed that the expression of CyclinB1 mRNA in LY-294002+ST treatment group was significantly decreased as compared with ST group and solvent control group (P<0.05).
Thus, the results in this study revealed that the increase in the expression of CyclinB1 at protein and mRNA level was blocked by the addition of LY-294002 in ST-treated GES-1 cells.
2.3.3.2 Effect of LY-294002 pretreatment on Cdc2 kinase
The results of Western blot showed that there was no difference in the level of dephosphorylation of Cdc2 in LY-294002+ST treatment group (P>0.05), meanwhile the level of phosphorylation of Cdc2 was increased as compared with that in ST group and solvent control group (P<0.05).
RT-PCR results confirmed that the expression of Cdc2 mRNA was increased in LY-294002+ST treatment group as compared with solvant control group and ST group (P<0.05).
The results in this study suggested that a synergistic effect might exist between LY-294002 and ST on the phosphorylation and mRNA expression of Cdc2.
2.3.3.2 Effect of LY-294002 pretreatment on Cdc25C phosphatase
The results of Western blot showed that the expression of Cdc25C protein in LY-294002+ST treatment group had no difference compared with ST group (P>0.05). But the level of phosphorylation of Cdc25C was higher compared with ST group and solvent control group (P<0.05).
The results of RT-PCR confirmed that there was no difference in the expression of Cdc25C mRNA in LY-294002+ST treatment group compared with ST group (P>0.05).
Thus, the results in this study suggested that LY-294002 might have synergistic effect with ST on the phosphorylation of Cdc25C, but had no effects on the expression of Cdc25C at both protein and mRNA level.
3 Effects of JNK, ERK and PI3K signal transduction pathway activation on the protein dyssynthesis of GES-1 cells induced by Sterigmatocystin in vitro
Objective: To explore the effects of inhibitors of JNK, ERK and PI3K on the ST-induced protein dyssynthesis.
Methods: GES-1 cells seeded at (1~2)×104 cells/L in culture flasks were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L (the treatment was the same as above). The expression of CyclinD1, the activity of eIF4E and 4E-BP1 in GES-1 cells were determined with Western blot and RT-PCR.
Results:
3.1 Effects of JNK signal pathway on the protein dyssynthesis induced by ST 3.1.1 Effect of SP600125 pretreatment on the expression of eIF4E The results of Western blot showed that the expression of eIF4E protein in SP600125+ST treatment group was significantly decreased as compared with that in solvant control group and ST group (P<0.05), meanwhile the phosphorylation of eIF4E in SP600125+ST treatment group was significantly increased (P<0.05).
The results of RT-PCR confirmed that the expression of eIF4E mRNA in SP600125+ST treatment group was significantly decreased as compared with that in ST group, but it was still higher than that in solvent control group (P<0.05).
Thus, the results in this study revealed that the decrease in the phosphorylation of eIF4E was blocked by the addition of SP600125, which could partly inhibit the expression of eIF4E mRNA in ST-treated GES-1 cells.
3.1.2 Effect of SP600125 pretreatment on the expression of 4E-BP1
The results of Western blot showed that in comparison with ST group, the expression of 4E-BP1 protein in SP600125+ST treatment group was significantly decreased (P<0.05), but had no difference compared with solvent control group. And the phosphorylation of 4E-BP1 in SP600125+ST treatment group was significantly increased as compared with that both in ST group and solvant control group (P<0.05).
The results of RT-PCR confirmed that the expression of 4E-BP1 mRNA in SP600125+ST treatment group was reduced compared with ST group, but it was still higher than that in solvant control group (P<0.05).
The results in this study suggested that SP600125 could inhibit the alteration of 4E-BP1 activity induced by ST. And it could partly decreased the ability of ST to increase the expression of 4E-BP1 mRNA.
3.1.3 Effect of SP600125 pretreatment on the exprssion of CyclinD1 protein
The results of Western blot showed that addition of the JNK inhibitor combined with ST induced a significant increase of CyclinD1 expression, as compared with that in ST group, but the expression of CyclinD1 was still lower than that in solvent control group (P<0.05). And the results suggested that the increase in the expression of CyclinD1 at protein level was blocked by the addition of SP600125 in ST-treated GES-1 cells.
3.2 Effects of ERK signal pathway on the protein dyssynthesis induced by ST
3.2.1 Effect of PD98059 pretreatment on the expression of eIF4E
The results of Western blot showed that there was no difference in the expression of eIF4E protein in PD98059+ST treatment group compared with ST group (P>0.05). But the phosphorylation of eIF4E in PD98059+ST treatment group was significantly increased as compared with that in solvant control and ST group (P<0.05).
The results of RT-PCR revealed that the expression of eIF4E mRNA in PD98059+ST treatment group was significantly decreased as compared with that in ST group, and had no difference as compared with that in solvent control group (P<0.05).
Thus, the results in this study suggested PD98059 could block both of the decrease in phosphorylation of eIF4E and the increase in eIF4E expression at mRNA level induced by ST.
3.2.2 Effect of PD98059 pretreatment on the expression of 4E-BP1
The results of Western blot showed that the expression of 4E-BP1 protein in PD98059+ST treatment group was significantly decreased as compared with that in ST group (P<0.05), and had no difference compared with solvent control group. And addition of the ERK inhibitor combined with ST induced a significant increase of 4E-BP1 phosphorylation, as compared with that both in ST group, and had no difference compared with solvant control group (P<0.05).
The results of RT-PCR confirmed that the expression of 4E-BP1 mRNA in PD98059+ST treatment group was decreased compared with ST group and solvant control group (P<0.05).
The results in this study suggested that the additon of PD98059 could block the ST-induced increase in 4E-BP1 protein expression and decrease in the phosphorylation of 4E-BP1, and also partly block the increase in the expression of 4E-BP1 mRNA induced by ST.
3.2.3 Effect of PD98059 pretreatment on the exprssion of CyclinD1 protein
The results of Western blot showed that addition of PD98059 combined with ST induced a significant increase of CyclinD1 expression, as compared with solvent control group and ST group (P<0.05). And the results suggested that the decrease in the expression of CyclinD1 at protein level in ST-treated GES-1 cells was blocked by the addition of PD98059. 3.3 Effects of PI3K signal pathway on the protein dyssynthesis induced by ST
3.3.1 Effect of LY-294002 pretreatment on the expression of eIF4E
The results of Western blot showed that the expression of eIF4E protein in LY-294002+ST treatment group was significantly increased as compared with that in solvant control group and ST group (P<0.05). Meanwhile the phosphorylation of eIF4E in LY-294002+ST treatment group was significantly decreased as compared with that in ST group and solvant control group (P<0.05).
The results of RT-PCR confirmed that the expression of eIF4E mRNA in LY-294002+ST treatment group was significantly increased as compared with that in ST group and solvent control group (P<0.05).
Thus, the results in this study suggested that there might be a synergistic effect between LY-294002 and ST on the expression of eIF4E at both protein and mRNA level. And there might be a synergistic effect between LY-294002 and ST on the phosphorylation of eIF4E.
3.3.2 Effect of LY-294002 pretreatment on the expression of 4E-BP1
The results of Western blot showed that in comparison with ST group, the expression of 4E-BP1 protein in LY-294002+ST treatment group was significantly decreased (P<0.05), but had no difference compared with solvent control group. And the phosphorylation of 4E-BP1 in LY-294002+ST treatment group was significantly decreased as compared with ST group and solvant control group (P<0.05).
The results of RT-PCR confirmed that the expression of 4E-BP1 mRNA in LY-294002+ST treatment group was decreased compared with ST group, but it was still higher than that in solvant control group (P<0.05). The results in this study suggested that the increase in 4E-BP1 expression at both protein and mRNA level induced by ST could be blocked by LY-294002. But LY-294002 could produce a synergistic effect with ST on the phosphorylation of 4E-BP1.
3.3.3 Effect of LY-294002 pretreatment on the exprssion of CyclinD1 protein
The results of Western blot showed that the addition of LY-294002 simultaneously with ST induced a significant increase of CyclinD1 expression, as compared with solvent control group and ST group (P<0.05). And the results in this study suggested that there might be a synergistic effect between LY-294002 and ST on the protein expression of CyclinD1.
Conclusions:
1. ST could activate JNK, ERK and PI3K signal trusduction pathway, and block p38 signal trusduction pathway at the same time.
2. ST induced G2 arrest of GES-1 cells may be through activion of JNK, ERK signal pathways. But the activation of PI3K signal pathway may play negative regulating role on G2 arrest of GES-1 cells induced by ST.
3. The changes in the expression and function of Cdc25C-Cdc2/CyclinB1 by the activation of JNK, ERK and PI3K signal transduction pathways may be the putative mechanisms of G2 arrest of GES-1 cells induced by ST in vitro.
4. The activation of JNK, ERK and PI3K signal transduction pathways were all involved in the effects of ST on GES-1 cells in vitro. But the activation of PI3K signal pathway may mainly play the
细胞周期调控是细胞最重要的生物学过程之一,细胞周期的紊乱可导致细胞的癌变。研究证明ST可以影响靶细胞的细胞周期,谢同欣等研究发现,ST可诱导小鼠胚胎纤维母细胞细胞周期发生G2/M期阻滞。本研究室近期实验也证实,ST可以诱导体外培养胃黏膜上皮细胞GES-1细胞周期阻滞在G2期。但ST诱导细胞G2期阻滞的可能机制还不明确。
cyclins是细胞周期进程调控的关键因子,可以激活它们的结合蛋白——细胞周期蛋白依赖性蛋白激酶(CDKs),进而靶向作用于各自的下游蛋白。CDK通过调节靶蛋白特定位点的磷酸化,介导细胞周期通过周期调控点。有丝分裂激酶Cdc2 (Cdk1)的激活是G2期调控点的重要的调控因素,它也受到多种因素的调控,例如与CyclinB的周期性结合、可逆的磷酸化作用和胞内移位等。细胞周期蛋白B1 (CyclinB1)是细胞周期G2期的周期蛋白,与其激酶Cdc2形成的复合物是启动有丝分裂的关键因子,可促使细胞周期由G2期进入M期。CyclinB1蛋白表达在G2期达到高峰,与Cdk1形成复合物,进入细胞核,此过程也受到磷酸化作用的调节。蛋白磷酸酶Cdc25C能够使Cdc2去磷酸化,从而促进细胞周期的进程;而磷酸化Cdc25C可丧失酶活性,引起Cdc2磷酸化程度增高,进而导致细胞周期阻滞在G2期。eIF4E是蛋白质合成的重要调节因子,可与其结合蛋白4E-BP1结合,调节细胞周期蛋白D1 (CyclinD1)等蛋白质的合成,eIF4E磷酸化水平增高可增强其促进蛋白质合成的作用。研究表明,eIF4E和4E-BP1也与细胞周期紊乱有关,在多种恶性肿瘤中表达异常。本研究室近期实验发现,ST不仅可以抑制胃黏膜上皮细胞(GES-1)增殖,诱导GES-1细胞周期发生G2期阻滞,还可以影响G2期调控点关键因子——CyclinB1的表达以及Cdc2激酶和Cdc25C磷酸酶的活性,同时,ST还可以抑制eIF4E的翻译起始功能。
JNK、ERK和p38是MAPK信号转导通路中最重要的成员。霉菌毒素等环境致癌物、化疗药物等多种体内外因素均可通过影响JNK、ERK和p38信号转导通路诱导靶细胞内相关基因表达,进而引起机体和细胞的相应生物学性状变化。PI3K是Her2家族重要信号转导通路之一,可通过调节下游核因子mTOR进而调控细胞增殖及蛋白质的合成,并且与肿瘤的侵袭、转移、预后及化疗耐受有关。
为进一步探讨ST诱导体外培养人胃粘膜上皮细胞周期阻滞的可能机制。本研究在前期实验研究的基础上,观察了不同浓度ST对体外培养永生化胃粘膜上皮细胞GES-1中JNK、ERK、p38和PI3K信号转导通路的影响情况,同时探讨了JNK、ERK和PI3K信号转导通路的激活在ST诱导的GES-1细胞周期G2期阻滞中的作用。
本研究论文共分为三个部分:
1杂色曲霉素对GES-1细胞中MAPK和PI3K信号转导通路的影响研究
目的:探讨一次性给予不同浓度ST对体外培养胃黏膜上皮细胞GES-1中JNK、ERK、p38和PI3K/mTOR信号转导通路的影响情况。
方法:取对数生长期GES-1细胞,随机分为6组:溶剂对照组和对照组分别给予DMSO (0.2 ml/L)和生理盐水处理。实验组给予DMSO溶解并以生理盐水稀释的ST,使其终浓度分别为100、500、1000、2000μg/L,处理24 h后,采用Western blot和RT-PCR方法检测GES-1中JNK/p-JNK、ERK/p-ERK、p38/p-p38和PI3K/mTOR/p-mTOR表达的变化情况。
另取对数生长期GES-1细胞,分别给予1μM SP600125 (JNK信号通路特异性阻断剂)、50μM PD98059 (ERK信号通路特异性阻断剂)、0.5μM SB203580 (p38信号通路特异性阻断剂)和1μM LY-294002 (PI3K特异性阻断剂)预处理。实验分为5组,即对照组、溶剂对照组、ST处理组、阻断剂组和阻断剂+ST处理组。细胞培养24 h后,更换2%低血清DMEM培养基。阻断剂组分别加入SP600125 (1μM)、PD98059 (50μM)、SB203580 (0.5μM)和LY-294002(1μM)。30 min后ST处理组和阻断剂+ST处理组分别给予1000μg/L ST,溶剂对照组和对照组分别给予DMSO (0.1ml/L)和生理盐水处理。细胞处理24 h后离心收细胞,采用Western blot方法检测目的基因在蛋白水平上表达变化。
结果:
1.1 ST对JNK信号转导通路的影响
1.1.1不同浓度ST对JNK信号转导通路的影响
Western blot结果显示,ST 100、500、1000、2000μg/L处理后细胞内JNK蛋白相对表达量与溶剂对照组比较差异无显著性(P>0.05)。而ST 500、1000、2000μg/L处理后JNK磷酸化水平明显高于溶剂对照组(P<0.05),并且,在0～2000μg/L ST浓度范围内,JNK磷酸化水平与ST的处理浓度有明显剂量依赖关系(r=0.925, P<0.01, n=3)。RT-PCR方法检测发现,各ST处理组JNK mRNA相对表达量与溶剂对照组(P>0.05)相比,没有明显差别。
1.1.2 SP600125预处理对ST作用后GES-1细胞JNK磷酸化水平的影响
Western blot方法检测结果表明,SP600125组细胞JNK磷酸化水平明显低于溶剂对照组(P<0.05),SP600125预处理+ST处理组JNK磷酸化水平较ST单独处理组明显降低,但仍高于溶剂对照组(P<0.05),表明给予JNK阻断剂SP600125对JNK蛋白激酶具有特异阻断作用,但只能部分阻断ST对JNK信号转导通路的激活作用。
1.2 ST对ERK信号转导通路的影响
1.2.1不同浓度ST对ERK信号转导通路的影响
Western blot结果显示,各ST处理组细胞ERK蛋白相对表达量与溶剂对照组比较无明显差异(P>0.05)。而ST 500、1000、2000μg/L处理后细胞内ERK磷酸化水平明显高于溶剂对照组(P<0.05),并且,在0～2000μg/L ST浓度范围内,ERK磷酸化水平与ST的处理浓度有明显剂量依赖关系(r=0.887, P<0.01, n=3)。
RT-PCR方法检测发现,各ST处理组细胞中ERK mRNA相对表达量与溶剂对照组相比,没有明显差别(P>0.05)。
1.2.2 PD98059预处理对ST作用后GES-1细胞ERK磷酸化水平的影响
Western blot方法检测,结果表明PD98059组细胞ERK磷酸化水平明显低于溶剂对照组(P<0.05),给予PD98059预处理后,PD98059+ST处理组ERK的磷酸化水平较ST单独处理组明显降低,但仍高于溶剂对照组(P<0.05)。表明给予ERK阻断剂PD98059对ERK蛋白激酶具有特异阻断作用,但只能部分阻断ST对ERK信号转导通路的激活作用。
1.3 ST对p38信号转导通路的影响
1.3.1不同浓度ST对p38信号转导通路的影响
Western blot结果显示,在0～2000μg/L ST浓度范围内,不同浓度ST处理后p38蛋白相对表达量与溶剂对照组比较均无明显差异(P>0.05)。ST 500、1000、2000μg/L处理后相对p38磷酸化水平明显低于溶剂对照组(P<0.05),并且,在0～2000μg/L ST浓度范围内,p38磷酸化水平与ST的处理浓度呈明显负相关关系(r=-0.843, P<0.01, n=3)。
RT-PCR方法检测发现,各ST处理组细胞内p38 mRNA相对表达量与溶剂对照组相比,均没有明显差别(P>0.05)。
1.3.2 SB203580预处理对ST作用后GES-1细胞p38磷酸化水平的影响
Western blot检测结果表明,SB203580处理组细胞相对p38磷酸化水平均明显低于溶剂对照组(P<0.05),SP600125+ST处理组p38磷酸化水平较ST单独处理组明显降低(P<0.05),表明p38阻断剂SB203580对p38的磷酸化水平具有特异阻断作用,给予p38特异性阻断剂SB203580预处理,对ST诱导细胞p38磷酸化水平降低有明显的协同作用。
1.4 ST对PI3K/mTOR信号转导通路的影响
1.4.1不同浓度ST对PI3K/mTOR信号转导通路的影响
Western blot结果显示,各ST处理组细胞PI3K蛋白相对表达量与溶剂对照组相比没有明显差异(P>0.05)。1000、2000μg/L ST处理组细胞mTOR蛋白相对表达量明显高于溶剂对照组(P<0.05),并且在0～2000μg/L ST浓度范围内,mTOR的蛋白表达与ST的处理浓度有明显剂量依赖关系(r=0.831, P<0.01, n=3)。同时,ST也可剂量依赖性地上调mTOR的磷酸化水平(r=0.868, P<0.01, n=3),其中500、1000和2000μg/L ST处理组mTOR的磷酸化水平升高更明显(P<0.05)。
RT-PCR方法检测结果发现,与溶剂对照组相比,不同浓度ST处理24 h对细胞内PI3K mRNA相对表达量没有明显影响(P>0.05),但可以明显上调mTOR在mRNA水平上的表达,mTOR mRNA的相对表达量与ST的处理浓度呈明显正相关关系(r=0.831, P<0.01, n=3)。
1.4.2 LY-294002预处理对ST作用后GES-1细胞mTOR基因表达及其磷酸化水平的影响
Western blot方法检测,LY-294002组细胞mTOR及其磷酸化水平与溶剂对照组相比没有明显差别(P>0.05),LY-294002+ST处理组mTOR磷酸化水平较ST单独处理组明显降低(P<0.05),与溶剂对照组相比没有明显差别(P>0.05);而mTOR蛋白的表达则明显高于溶剂对照组(P<0.05),与ST单独处理组没有差别(P>0.05)。
RT-PCR结果显示,LY-294002组细胞mTOR mRNA相对表达量与溶剂对照组相比没有明显差别(P>0.05),LY-294002+ST处理组mTOR mRNA相对表达量明显高于溶剂对照组(P<0.05),而与ST单独处理组相比没有明显差别(P>0.05)。
结果表明,给予PI3K阻断剂LY-294002对PI3K/mTOR信号转导通路具有特异阻断作用,可以阻断ST诱导mTOR磷酸化水平升高的作用,但对mTOR在蛋白和mRNA水平上的表达没有明显影响。
2 JNK、ERK和PI3K信号转导通路的激活在杂色曲霉素诱导GES-1细胞G2期阻滞中的作用研究
目的:探讨JNK、ERK和PI3K信号转导通路特异性阻断剂预处理对ST处理24 h诱导体外培养GES-1细胞增殖抑制、G2期阻滞及其相关因子表达的影响。
方法:取对数生长期GES-1细胞,分别给予1μM SP600125 (JNK信号通路特异性阻断剂)、50μM PD98059 (ERK信号通路特异性阻断剂)和1μM LY-294002 (PI3K特异性阻断剂)预处理细胞。按1×104个/L接种于96孔板,待细胞进入对数生长期后更换培养基,实验分为5组,即对照组、溶剂对照组、ST处理组、阻断剂组和阻断剂+ST处理组。每孔终体积为200μl,同时设空白对照孔、对照孔和溶剂对照孔,细胞培养24 h后,更换2%低血清DMEM培养基,阻断剂组分别加入SP600125 (1μM)、PD98059 (50μM)和LY-294002 (1μM)。30 min后ST处理组和阻断剂+ST处理组分别给予1000μg/L ST,溶剂对照组和对照组分别给予DMSO (0.1ml/L)和生理盐水处理。细胞处理24 h后,采用MTT检测细胞存活率。
另取对数生长期GES-1细胞,分为5组,即对照组、溶剂对照组、ST处理组、阻断剂组和阻断剂+ST处理组(处理同上)。细胞处理24 h后离心收细胞,采用FCM检测细胞周期分布情况,采用Western blot和RT-PCR方法观察周期调控关键因子——CyclinB1蛋白的表达、Cdc2激酶和Cdc25C磷酸酶活性的变化情况。
结果:
2.1 JNK信号转导通路在ST诱导GES-1细胞G2期阻滞中的作用
2.1.1 SP600125预处理对ST诱导的GES-1细胞存活率降低的影响
MTT结果表明,SP600125+ST组细胞存活率明显高于ST单独处理组(P<0.05),但仍明显低于溶剂对照组(P<0.05)。表明SP600125预处理可部分阻断ST对GES-1细胞增殖的抑制作用。
2.1.2 SP600125预处理对ST诱导的GES-1细胞G2期阻滞的影响
FCM检测结果显示,与ST 1 000μg/L处理组相比,SP600125预处理+ST 1000μg/L处理组G0/G1期所占比例明显升高;而G2/M期细胞比例则明显降低(P<0.05),但仍明显高于溶剂对照组(P<0.05)。表明SP600125预处理可部分阻断ST诱导的细胞G2/M期比例增高作用。
2.1.3 SP600125预处理对ST作用后GES-1细胞G2期相关因子的影响
2.1.3.1 SP600125预处理对CyclinB1表达的影响
Western blot检测结果显示,SP600125预处理+ST 1000μg/L处理组CyclinB1蛋白的表达明显低于溶剂对照组,同时显著低于ST 1000μg/L处理组(P<0.05)。
RT-PCR结果发现,SP600125预处理+ST组细胞内CyclinB1 mRNA的表达明显低于ST 1000μg/L处理组(P<0.05),而与溶剂对照组相比没有差别。
表明SP600125预处理可阻断ST对GES-1细胞内CyclinB1在蛋白和mRNA水平表达的升高作用。
2.1.3.2 SP600125预处理对Cdc2激酶的影响
Western blot检测结果显示,与ST 1000μg/L处理组细胞相比,SP600125预处理+ST 1000μg/L处理组内Cdc2的去磷酸化水平明显升高,同时其磷酸化水平明显降低(P<0.05),而与溶剂对照组相比无明显差别。
RT-PCR结果表明,SP600125预处理+ST组细胞内Cdc2 mRNA的表达与ST 1000μg/L处理组相比没有明显差别(P>0.05)。
表明SP600125预处理可阻断ST对GES-1细胞内Cdc2去磷酸化水平和Cdc2磷酸化水平的影响,但对Cdc2 mRNA没有明显影响。
2.1.3.3 SP600125预处理对Cdc25C磷酸酶的影响
Western blot检测结果显示,与溶剂对照组相比,SP600125预处理+ST 1000μg/L处理组内Cdc25C蛋白的表达明显升高,同时明显高于ST 1000μg/L处理组;而Cdc25C磷酸化水平则较ST 1000μg/L处理组明显降低(P<0.05),与溶剂对照组相比没有明显差别。
RT-PCR结果表明,SP600125预处理+ST组细胞Cdc25C mRNA的表达与ST 1000μg/L处理组相比没有明显差别(P>0.05)。表明SP600125预处理可阻断ST对GES-1细胞内Cdc25C的蛋白表达和Cdc25C磷酸化水平的影响,但对Cdc25C mRNA没有明显影响。
2.2 ERK信号转导通路在ST诱导GES-1细胞G2期阻滞中的作用
2.2.1 PD98059预处理对ST诱导的GES-1细胞存活率降低的影响
MTT结果表明,PD98059+ST处理组细胞存活明显高于ST单独处理组(P<0.05),而与溶剂对照组相比没有明显差别(P>0.05)。表明PD98059预处理可以阻断ST对GES-1细胞的增殖抑制作用。2.2.2 PD98059预处理对ST诱导的GES-1细胞G2期阻滞的影响FCM检测结果显示,与ST 1000μg/L处理组相比,PD98029预处理+ ST 1000μg/L处理组S期细胞比例明显升高(P<0.05);而G2/M期细胞比例明显降低(P<0.05),但仍明显高于溶剂对照组(P<0.05)。表明PD98059预处理可部分阻断ST诱导的细胞G2/M期比例增高。
2.2.3 PD98059预处理对ST作用后GES-1细胞G2期相关因子的影响
2.2.3.1 PD98059预处理对CyclinB1表达的影响
Western blot检测结果显示,PD98059预处理+ST 1000μg/L处理组CyclinB1的蛋白表达与ST 1000μg/L处理组相比显著降低(P<0.05),但仍明显高于溶剂对照组(P<0.05)。
RT-PCR结果显示,PD98059预处理+ST组细胞内CyclinB1 mRNA的表达与ST 1000μg/L处理组相比没有明显差别(P>0.05)。
表明PD98059预处理可部分阻断ST对GES-1细胞内CyclinB1蛋白表达的升高作用,但对CyclinB1 mRNA没有明显影响。
2.2.3.2 PD98059预处理对Cdc2激酶的影响
Western blot检测结果显示,PD98059预处理+ST 1000μg/L处理组细胞内Cdc2的去磷酸化水平与ST 1000μg/L处理组相比,没有明显差异(P>0.05);而其磷酸化水平则明显低于ST 1000μg/L处理组(P<0.05),与溶剂对照组相比没有明显差异(P>0.05)。
RT-PCR结果显示,PD98059预处理+ST组细胞Cdc2 mRNA的表达明显低于ST 1000μg/L处理组,但仍高于溶剂对照组(P<0.05)。
表明PD98059预处理可阻断ST对GES-1细胞内Cdc2磷酸化水平的升高作用,同时部分阻断ST对Cdc2 mRNA表达的升高作用。
2.2.3.3 PD98059预处理对Cdc25C磷酸酶的影响
Western blot检测结果显示,PD98059预处理+ST 1000μg/L处理组细胞Cdc25C蛋白表达与ST 1000μg/L处理组相比没有明显差异(P>0.05);Cdc25C磷酸化水平则较ST 1000μg/L处理组明显降低,但仍高于溶剂对照组(P<0.05)。
RT-PCR结果显示,PD98059预处理+ST组细胞内Cdc25C mRNA的表达与ST 1000μg/L处理组相比没有明显差别(P>0.05)。
提示PD98059预处理可部分阻断ST对细胞内Cdc25C磷酸化水平的升高作用,但对Cdc25C mRNA没有明显影响。
2.3 PI3K信号转导通路在ST诱导GES-1细胞G2期阻滞中的作用
2.3.1 LY-294002预处理对ST诱导的GES-1细胞存活率降低的影响
MTT结果表明,LY-294002+ST处理组细胞存活率明显低于溶剂对照组(P<0.05),而与ST单独阻断剂组相比没有明显差别(P>0.05)。表明LY-294002预处理对ST诱导的GES-1细胞增殖抑制作用没有明显影响。
2.3.2 LY-294002预处理对ST诱导的GES-1细胞G2期阻滞的影响
FCM检测结果显示,LY-294002预处理+ST 1000μg/L处理组G0/G1期和S期细胞所占比例均较ST 1000μg/L处理组明显降低,而G2/M期细胞比例则明显增加(P<0.05),表明LY-294002预处理对ST诱导的细胞G2/M期比例增高具有协同作用。
2.3.3 LY-294002预处理对ST作用后GES-1细胞G2期相关因子的影响
2.3.3.1 LY-294002预处理对CyclinB1表达的影响
Western blot检测结果显示,LY-294002预处理+ST 1000μg/L处理组CyclinB1蛋白的表达与ST 1000μg/L处理组相比显著降低,较溶剂对照组也明显降低(P<0.05)。
RT-PCR结果显示,LY-294002预处理+ST组细胞内CyclinB1 mRNA的表达明显低于ST单独处理组,同时明显低于溶剂对照组(P<0.05)。表明LY-294002预处理可阻断ST诱导GES-1细胞内CyclinB1在蛋白和mRNA水平表达的升高作用。
2.3.3.2 LY-294002预处理对Cdc2激酶的影响
Western blot检测结果显示,LY-294002预处理+ST 1000μg/L处理组细胞内Cdc2的去磷酸化水平与ST 1000μg/L处理组相比没有明显差异(P>0.05);而其磷酸化水平则明显高于ST 1000μg/L处理组(P<0.05),同时明显高于溶剂对照组(P<0.05)。
RT-PCR结果显示,LY-294002预处理+ST组细胞Cdc2 mRNA的表达明显高于溶剂对照组,同时也明显高于ST单独处理组(P<0.05)。表明LY-294002预处理对ST诱导的GES-1细胞内Cdc2的磷酸化水平及其mRNA表达升高有明显的协同作用。
2.3.3.3 LY-294002预处理对Cdc25C磷酸酶的影响
Western blot检测结果显示,LY-294002预处理+ST 1000μg/L处理组细胞内Cdc25C蛋白的表达与ST 1000μg/L处理组相比没有明显差异(P>0.05);而其磷酸化水平则较ST 1000μg/L处理组明显升高(P<0.05),同时也明显高于溶剂对照组(P<0.05)。
RT-PCR结果显示,LY-294002预处理+ST组细胞内Cdc25C mRNA的表达与ST 1000μg/L处理组相比没有明显差别(P>0.05)。
表明LY-294002预处理对ST诱导的GES-1细胞内Cdc25C磷酸化水平升高有明显的协同作用,但对Cdc25C在蛋白和mRNA水平上的表达没有明显影响。
3 JNK、ERK和PI3K信号转导通路的激活在杂色曲霉素影响GES-1细胞蛋白质合成起始因子表达的作用研究
目的:进一步探讨JNK、ERK和PI3K信号转导通路特异性阻断剂预处理,对ST处理24 h诱导体外培养GES-1细胞蛋白质合成障碍的影响。
方法:实验细胞处理、分组(同前)。继续采用Western blot和RT-PCR方法研究了分别给予JNK、ERK和PI3K信号转导通路特异性阻断剂预处理,ST诱导的体外培养胃黏膜上皮细胞GES-1细胞中eIF4E和4E-BP1的表达及其活性变化,以及CyclinD1蛋白表达的变化情况
结果:
3.1 JNK信号转导通路在ST影响GES-1细胞蛋白质合成中的作用
3.1.1 SP600125预处理对eIF4E表达的影响
Western blot检测结果显示,SP600125预处理+ST 1000μg/L处理组细胞内eIF4E蛋白的表达与溶剂对照组相比明显下降,同时显著低于ST单独处理组(P<0.05),而eIF4E磷酸化水平则明显高于溶剂对照组,同时显著高于ST单独处理组(P<0.05)。
RT-PCR结果表明,SP600125预处理+ST组细胞内eIF4E mRNA的表达明显低于ST 1000μg/L处理组,但仍明显高于溶剂对照组(P<0.05)。表明SP600125预处理可阻断ST对GES-1细胞内eIF4E的活性抑制作用,同时可部分阻断ST对eIF4E mRNA表达的升高作用。
3.1.2 SP600125预处理对4E-BP1表达的影响
Western blot检测结果显示,SP600125预处理+ST 1000μg/L处理组4E-BP1蛋白的表达明显低于ST单独处理组(P<0.05),而与溶剂对照组相比没有明显变化(P>0.05);4E-BP1磷酸化水平较溶剂对照组明显升高,同时显著高于ST单独处理组(P<0.05)。
RT-PCR结果表明,SP600125预处理+ST组细胞内4E-BP1 mRNA的表达也明显低于ST单独处理组,但仍明显高于溶剂对照组(P<0.05)。表明SP600125预处理可阻断ST对GES-1细胞内4E-BP1活性的促进作用,同时可部分阻断ST对4E-BP1 mRNA表达的升高作用。
3.1.3 SP600125预处理对CyclinD1蛋白表达的影响
SP600125预处理+ST 1000μg/L处理组CyclinD1蛋白的表达明显高于ST单独处理组,但仍明显低于溶剂对照组(P<0.05),表明SP600125预处理可部分阻断ST对GES-1细胞内CyclinD1蛋白表达的降低作用。
3.2 ERK信号转导通路在ST影响GES-1细胞蛋白质合成中的作用
3.2.1 PD98059预处理对eIF4E表达的影响
Western blot检测结果显示,PD98059预处理+ST 1000μg/L处理组内eIF4E蛋白的表达与ST 1000μg/L处理组相比没有明显差异(P>0.05),而其磷酸化水平与ST 1000μg/L处理组细胞相比明显升高(P<0.05),而与溶剂对照组没有明显差别(P>0.05)。
RT-PCR结果表明,PD98059预处理+ST组细胞内eIF4E mRNA的表达明显低于ST单独处理组(P<0.05),而与溶剂对照组相比没有差别。表明PD98059预处理对ST诱导的GES-1细胞内eIF4E蛋白的表达增高没有影响;但可阻断ST对eIF4E磷酸化水平的降低以及eIF4E mRNA表达的升高作用。
3.2.2 PD98059预处理对4E-BP1的影响
Western blot检测结果显示,PD98059预处理+ST 1000μg/L处理组4E-BP1蛋白的表达较ST单独处理组明显降低(P<0.05),而与溶剂对照组相比没有明显差别;4E-BP1磷酸化水平与ST 1000μg/L处理组细胞相比也明显升高(P<0.05),也与溶剂对照组相比没有明显差别(P>0.05)。
RT-PCR结果表明,PD98059预处理+ST组细胞4E-BP1 mRNA的表达与ST单独处理组相比明显降低,但仍明显高于溶剂对照组(P<0.05)。表明PD98059预处理可阻断ST对GES-1细胞内4E-BP1蛋白的表达升高作用及4E-BP1磷酸化水平降低作用;同时可部分阻断4E-BP1 mRNA表达升高的作用。
3.2.3 PD98059预处理对CyclinD1蛋白表达的影响
PD98059预处理+ST 1000μg/L处理组CyclinD1蛋白的表达明显高于溶剂对照组(P<0.05),同时显著高于ST 1000μg/L处理组(P<0.05),表明PD98059预处理可阻断ST诱导GES-1细胞内CyclinD1蛋白表达降低的作用。
3.3 PI3K信号转导通路在ST影响GES-1细胞蛋白质合成中的作用
3.3.1 LY-294002预处理对eIF4E表达的影响
Western blot检测结果显示,LY-294002预处理+ST 1000μg/L处理组内eIF4E蛋白的表达明显高于溶剂对照组,同时明显高于ST单独处理组(P<0.05);eIF4E的磷酸化水平较ST处理组明显降低(P<0.05),同时明显低于溶剂对照组(P<0.05)。
RT-PCR结果表明,LY-294002预处理+ST组细胞内eIF4E mRNA的表达明显高于溶剂对照组,并且也较ST单独处理组明显升高(P<0.05)。
表明LY-294002预处理对ST诱导的eIF4E在蛋白和mRNA水平的表达升高及eIF4E磷酸化水平的降低均有明显的协同作用。
3.3.2 LY-294002预处理对4E-BP1的影响
Western blot检测结果显示,LY-294002预处理+ST 1000μg/L处理组内4E-BP1蛋白的表达明显低于ST单独处理组(P<0.05),而与溶剂对照组相比没有明显差别(P>0.05);4E-BP1磷酸化水平明显低于溶剂对照组(P<0.05),同时较ST 1000μg/L处理组细胞也明显降低(P<0.05)。
RT-PCR结果表明,LY-294002预处理+ST组细胞4E-BP1 mRNA的表达与ST单独处理组相比明显降低,但仍高于溶剂对照组(P<0.05)。
表明LY-294002预处理可阻断ST对GES-1细胞内4E-BP1在蛋白和mRNA水平的表达升高作用,但对ST诱导的4E-BP1磷酸化水平降低有明显的协同作用。
3.3.3 LY-294002预处理对CyclinD1蛋白表达的影响
LY-294002预处理+ST 1000μg/L处理组CyclinD1蛋白的表达明显低于溶剂对照组(P<0.05),同时显著低于ST 1000μg/L处理组(P<0.05),表明LY-294002预处理对ST诱导的GES-1细胞内CyclinD1蛋白表达降低有明显的协同作用。
结论:
1杂色曲霉素作用于GES-1细胞24 h,可以激活JNK、ERK和PI3K信号转导通路,同时可以抑制p38信号转导通路。
2杂色曲霉素可能通过激活JNK和ERK信号转导通路影响GES-1细胞周期分布,诱导G2期阻滞;但PI3K信号转导通路的激活在杂色曲霉素诱导的G2期阻滞中可能发挥反向调节作用。
3杂色曲霉素作用于GES-1细胞可通过激活JNK、ERK和PI3K信号转导通路而影响Cdc25C -Cdc2/CyclinB1的表达和功能,可能是杂色曲霉素诱导细胞G2期阻滞的机制之一。
4 JNK、ERK和PI3K信号转导通路的激活可能均参与了ST对体外培养胃黏膜上皮细胞GES-1的影响。但PI3K信号转导通路的激活此过程中可能主要发挥反向调节作用。
Sterigmatocystin (ST) is a toxic fungal metabolite with low molecular weight. It is produced by some Aspergillus species fungi, such as, A versicolor, A nidulans,etc. ST is one of the common contaminants of the ingredients of animal feed and human food all over the world. ST has been proved to have carcinogenic potency in experimental animal models. It could induce lung adenocarcinoma, hepatoma and mesotheliomas in different experimental animals. ST is one of genotoxic agents and could form DNA adducts with target cell DNA and result in direct DNA damages. Our previous studies with human tissue showed that ST could induce malignant transformation and p53 mutation in human fetal gastric mucosa cells in vitro. Oral administration of ST for long period of time could induce intestinal metaplasia and atypical hyperplasia of glandular epithelium in mice.
As we know that the control of cell cycle is one of the most imortant biological processes. Disregulation of cell cycle may result in carcinogenesis. It has be showed that ST could affect cell cycle of target cells. Xie et al found that ST could cause G2/M arrest in murine fibroblasts. Our primary study with human gastric epithelial cell line also confirmed that ST could induce G2 phase arrest in human gastric GES-1 cells in vitro. But the putative mechanisms of G2 arrest induced by ST was still not clear enough.
Cyclins are key components of the cell cycle progression machinery. They activate their partner cyclin-dependent kinases (CDKs) and possibly target them to respective substrate proteins within the cell. CDK-mediated phosphorylation of specific sets of proteins drives the cell through particular phases or checkpoints of the cell cycle. It has been established that the G2 checkpoint initiation is primarily regulated through the control of Cdc2 (Cdk1) activity, which is regulated at multiple levels, including periodic association with the B-type cyclins, reversible phosphorylation, and intracellular compartimentalization. CyclinB1 is the cylcin at stage of G2, and the compound formed with its kinase Cdc2 is the key factor for mitoschisis and play key role to precipitate cell cycle from stage of G2 to stage of M. Cyclin B1 reaches maximum levels in G2, when it enters into the nucleus to form a complex with Cdk1 in a phosphorylation-dependent manner. Protein phosphatase Cdc25C could induce dephosphorylation of Cdc2 and promote the progression of cell cycle. Phosphorylation of Cdc25C could result in the loss of enzyme activity, which in turn increase the phosphorylation activity of Cdc2 and the cell cycle may be arrested at G2 phase as a result. eIF4E is the key regulatory factor in the process of protein synthesis. In association with its binding protein- 4E-BP1, eIF4E modulates the synthesis of cap-dependent protein, such as CyclinD1. The elevated level of phosphorylation could enhance the protein synthesis of eIF4E. Studies showed that eIF4E and 4E-BP1 are associated with the disruption of cell cycle, and expressed abnormally in malignant tumor cell. Our recent experiment studies indicated that ST could inhibit the proliferation and induce human gastric GES-1 cells arrested at stage of G2 in vitro. And ST was found to have impact on the expression of CyclinB1 as well as the activity of Cdc2 kinase and Cdc25 phosphatase, which are all the key factors for the G2 regulatory point. At the same time, we found that ST could inhibit the initiated translation function of eIF4E.
JNK, ERK and p38 are the important members of MAPK signal transduction pathway. Studies showed that environmental carcinogens(such as mycotoxins)and chemotherapeutic agents could affect the expressions of the related genes and biological behaviors of the target cells by their effects on JNK, ERK and p38 signal transduction pathways. PI3K is one of the important signal transduction pathways in Her2 family. It played important roles in the control of cell proliferation, protein synthesis, etc through the regulation of the downstream nuclear factor-mTOR. In malignant tumors, PI3K was associated with the invasion, metastasis and prognosis and chemotherapy tolerance of tumor.
To further explore the putative mechanisms of G2 arrest induced by ST, the present study was carried out based on our previous studies. The effects of ST on the the activities of JNK, ERK, p38 and PI3K signal transduction pathway in human gastric epithelial cell line-GES-1 were studied. And the roles of JNK, ERK and PI3K activation on G2 arrest of GES-1 induced by ST in vitro was studied.
The following three parts are included in the study:
1 Effects of Sterigmatocystin on MAPK and PI3K signal transduction pathway in GES-1 cells in vitro
Objective: To explore the effects of different concentration of ST on JNK, ERK, p38 and PI3K/mTOR signal transduction pathway in GES-1 cells.
Methods: After initial culture for 24h, GES-1 cells were harvested, centrifuged and resuspended in fresh DMEM medium supplemented with 10% FCS at the concentration of (1~2)×104 cells/L in culture flasks (8 ml). The cells were randomly divided into 6 groups. The cells in ST groups were treated with ST at 100μg/L, ST 500μg/L, ST 1000μg/L and ST 2000μg/L, respectively, while DMSO and saline were used in solvent control and control group. Cells were harvested 24 h after ST treatment. The expression of ERK/p-ERK, p38/p-p38 and PI3K/mTOR/p-mTOR at protein level in GES-1 cells treated with ST was determined by Western blot. The expression of ERK, p38 and PI3K/mTOR mRNA in GES-1 cells was detected by RT-PCR.
GES-1 cells seeded at the concentration of (1~2)×104 cells/L in culture flasks were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L. The medium of GES-1 cells was replaced by new DMEM medium supplemented with 2% FCS 24h later. The cells of blocking agent groups were pretreated for 30 min with 1μM SP600125 (JNK inhibitor), 50μM PD98059 (ERK inhibitor ), 0.5μM SB203580 (p38 inhibitor) and 1μM LY-294002 (PI3K inhibitor) respectively. Then the cells in blocking agent +ST 1000μg/L group were treated with ST 1000μg/L, while these in solvent control and control groups were incubated with DMSO and saline respectively. Cells were harvested 24 h after ST treatment. The JNK, ERK, p38 and PI3K/mTOR activation of GES-1 cells treated with ST were determined with Western blot.
Results:
1.1 Effects of ST on JNK signal transduction pathway
1.1.1 Effects of ST with different concentrations on JNK signal transduction pathway
The results of Western blot showed that there was no significant difference in JNK protein expression in GES-1 cells between all the ST treatment groups and solvent control group (DMSO) (P>0.05), while the phosphorylation of JNK in ST 500μg/L, 1000μg/L and 2000μg/L treatment groups was significantly increased as compared with that in corresponding DMSO group (P<0.05). And there is a significant dose-dependent correlation between ST concentration and the phosphorylation of JNK (r=0.925, P<0.01, n=3).
The results of RT-PCR confirmed that no significant difference in JNK mRNA expression in GES-1 cells between all the ST treatment groups and solvent control group (DMSO) (P>0.05).
1.1.2 Effects of ST on JNK signal transduction pathway, with SP600125 pretreatment
The results of Western blot showed that the phosphorylation of JNK in GRS-1 cells in SP600125 treatment group was significantly decreased than that in corresponding DMSO group (P<0.05). And the phosphorylation of JNK in SP600125+ST treatment group was dramatically decreased compared with that in ST treatment alone, while still higher than that in solvent control (P<0.05). The result indicated that the SP600125 could specifically inhibit the activation of JNK MAP kinase, but the effect of activation of JNK in ST-treated cells was partially inhibited by the presence of SP600125.
1.2 Effects of ST on ERK signal transduction pathway
1.2.1 Effects of ST with different concentrations on ERK signal transduction pathway
The results of Western blot suggested that there was no significant difference in ERK protein expression in GES-1 cells between ST treatment groups and solvent control group (DMSO) (P>0.05). In comparison with solvent control group, the phosphorylation of ERK was significantly increased in ST treatment groups (P<0.05). Results from concentration-dependent studies indicated that ST up-regulated the phosphorylation of ERK in a dose-dependent correlation (r=0.887, P<0.01, n=3).
The results of RT-PCR showed that ST had no effect on the expression of ERK mRNA.
1.2.2 Effects of ST on ERK signal transduction pathway, with PD98059 pretreatment
The results of Western blot revealed that the phosphorylation of ERK in GRS-1 cells of PD98059 treatment group was significantly decreased compared with that in solvent control group (P<0.05). And the phosphorylation of ERK in PD98059+ST treatment group was decreased significantly compared with that in ST 1000μg/L treatment, while still higher than that in solvent control (P<0.05). The result revealed that the PD98059 could inhibit the activation of ERK MAP kinase, but the effect of activation of ERK in ST-treated cells was partially inhibited by the presence of PD98059.
1.3 Effects of ST on p38 signal transduction pathway
1.3.1 Effects of ST with different concentrations on p38 signal transduction pathway
No significant difference in p38 protein expression of GES-1 cells between all the ST treatment groups and solvent control group was found by Western blot (P>0.05), while the phosphorylation of p38 in ST treatment groups was lower compared with that in corresponding solvent control group (P<0.05). And the phosphorylation of p38 decreased with ST treatment in a dose-dependent manner (r=-0.843, P<0.01, n=3).
RT-PCR results confirmed that there was no significant difference in p38 mRNA expression in GES-1 cells between all the ST treatment groups and solvent control group (P>0.05).
1.3.2 Effects of ST on p38 signal transduction pathway, with SB203580 pretreatment
Western blot results showed that SB203580 could significantly decreased the level of phosphorylation of p38 in GES-1 cells (P<0.05). And the phosphorylation of p38 in SB203580+ST treatment group was markedly decreased compared with that in ST treatment (P<0.05). The result indicated that there might be a synergistic effect of the inhibitor of p38, SB203580 and ST on the phosphorylation of p38.
1.4 Effects of ST on PI3K/mTOR signal transduction pathway
1.4.1 Effects of ST with different concentrations on PI3K/mTOR signal transduction pathway
The results of Western blot showed that the expression of PI3K protein in GES-1 cells in both the ST treatment groups and solvent control group showed no significant differences (P>0.05), while the expression of mTOR protein in ST 1000μg/L and 2000μg/L treatment groups was higher compared with that in solvent control group (P<0.05). And there is a significant dose-dependent correlation between ST concentration and the expression of mTOR protein (r=0.831, P<0.01, n=3). Meanwhile, the phosphorylation of mTOR was significantly increased by ST in a dose-dependent way (r=0.868, n=3, P<0.01).
RT-PCR analysis suggested that there was no difference in PI3K mRNA expression in GES-1 cells between all the ST treatment groups and solvent control group (P>0.05). But the expression of mTOR mRNA was increased by ST in a dose-dependent way (r=0.831, n=3, P<0.01).
1.4.2 Effects of ST on PI3K/mTOR signal transduction pathway, with LY-294005 pretreatment
The results of Western blot showed that the expression of mTOR protein and phosphorylation in GES-1 cells in LY-294002 treatment group showed no significant difference compared with that in solvent control group (P<0.05). Compared with the group with ST only treatment, the phosphorylation of mTOR in LY-294002+ST treatment group was decreased significantly (P<0.05), while there was no difference as compared with that of solvent control (P>0.05). But the exptession of mTOR protein in LY-294002+ST treatment group was higher than that in solvent control (P<0.05), and it showed no difference between LY-294002+ST treatment group and ST treatment only (P>0.05).
The results of RT-PCR displayed that there was no difference in mTOR mRNA expression in GES-1 cells between ST treatment groups and solvent control group (P>0.05). But in comparison with solvent control group, mTOR mRNA expression in LY-294002+ST treatment group was higher (P<0.05), but was not different from that in ST treatment only groups (P>0.05).
The results indicated that the LY-294002 could inhibit the activity of PI3K/mTOR signal pathway, but it had no effect on the expression of mTOR at both protein and mRNA levels in GES-1 cells.
2 Effects of JNK, ERK and PI3K signal transduction pathway activation on the G2 arrest induced by Sterigmatocystin in GES-1 in vitro
Objective: To explore the effects of inhibitors of JNK, ERK and PI3K on the ST-induced proliferation inhibition and G2 arrest.
Methods:
GES-1 cells were harvested, centrifuged and resuspended in fresh DMEM medium supplemented with 10% FCS at the concentration of (1~2)×104 cells/L in 96-well culture plates. The cells were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L. The cells of blocking agent groups were pretreated for 30 min with 1μM SP600125 (JNK inhibitor), 50μM PD98059 (ERK inhibitor), 0.5μM SB203580 (p38 inhibitor) and 1μM LY-294002 (PI3K inhibitor) respectively. Then the cells in blocking agent +ST 1000μg/L group were treated with ST 1000μg/L, while these in solvent control and control groups were incubated with DMSO and saline respectively. Cell viability for GES-1 cells was determined by MTT assay.
For the blocking studies, GES-1 cells seeded at the concentration of (1~2)×104 cells/L in culture flasks were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L (the treatment was same as above). Cells were harvested 24 h after ST treatment. The changes of cell cycle in response to ST was measured using FCM. And the expression of CyclinB1, the activity of Cdc2 kinase and Cdc25C phosphatase in GES-1 cells were determined with Western blot and RT-PCR.
Results:
2.1 Effects of JNK signal pathway on the G2 arrest induced by ST
2.1.1 Effect of SP600125 pretreatment on the changes of survival rate in GES-1 cells induced by ST
The result of MTT showed that the cell viability in SP600125+ST treatment group was much higher than that in ST group, but it still lower than that of controls (P<0.05). The result indicated that SP600125 could partly block the inhibition of proliferation induced by ST in GES-1 cell.
2.1.2 Effect of SP600125 pretreatment on G2 arrest of GES-1 cells induced by ST
FCM analysis revealed that the G0/G1 fraction was increased and the G2/M fraction was decreased in SP600125+ST treatment group compared with ST 1000μg/L group. But the G2/M faction was still increased compared with control group. Thus, addition of SP600125 to the cells could partly decrease the ability of ST to increase the percentage of G2/M faction.
2.1.3 Effect of SP600125 pretreatment on the ST-induced changes of the key factors of G2 phase in GES-1 cells
2.1.3.1 Effect of SP600125 pretreatment on the expression of CyclinB1
The results of Western blot showed that the expression of CyclinB1 protein in SP600125+ST treatment group was significantly decreased compared with that in solvant control group and ST group (P<0.05).
The results of RT-PCR suggested that the expression of CyclinB1 mRNA in SP600125+ST treatment group was significantly decreased as compared with that in ST group (P<0.05) and there was no difference of that compared with solvent control group.
Thus, the results in this study revealed that the increase in the expression of CyclinB1 at both protein and mRNA level was blocked by the presence of SP600125 in ST-treated GES-1 cells.
2.1.3.2 Effect of SP600125 pretreatment on Cdc2 kinase
Western blot results showed that in comparison with ST treatment group, the dephosphorylation level of Cdc2 in SP600125+ST treatment group was significantly increased, meanwhile the phosphorylation level of Cdc2 was decreased (P<0.05). And no significant difference was found between SP600125+ST treatment group and solvent control group.
The results of RT-PCR displayed that the expression of Cdc2 mRNA had no difference between SP600125+ST treatment group and ST treatment group.
Thus, the results in this study suggested that alteration in dephosphorylation and phosphorylation of Cdc2 caused by ST were blocked by the JNK inhibitor SP600125, which had no effect on the expression of Cdc2 mRNA.
2.1.3.3 Effect of SP600125 pretreatment on Cdc25C phosphatase
The results of Western blot showed that the presence of the JNK inhibitor simultaneously with ST induced a significant increase in the expression of Cdc25C, as compared with that in solvent control group and ST treatment group (P<0.05). The level of phosphorylation of Cdc25C was reduced as compared with ST treatment group (P<0.05), and had no difference compared with solvent control group.
RT-PCR results confirmed that there was no difference in the expression of Cdc25C mRNA between SP600125+ST treatment group and ST treatment group.
Thus, the results in this study indicated that SP600125 could inhibit the alterations in protein expression and phosphorylation level of Cdc25C induced by ST, but had no influence on the expression of Cdc25C mRNA.
2.2 Effects of ERK signal pathway on the G2 arrest induced by ST
2.2.1 Effect of PD98059 pretreatment on the changes of GES-1 cells survival rate induced by ST
The result of MTT showed that the cell viability in PD98059+ST treatment group was much higher than that of ST group, and there was no difference of that as compared with solvant control group (P<0.05). The result indicated that the PD98059 could partly block the ST-induced antiproliferative action in GES-1 cells.
2.2.2 Effect of PD98059 pretreatment on the G2 arrest of GES-1 cells induced by ST
FCM analysis showed that the number of S faction was increased and that of G2/M faction was decreased in PD98059+ST treatment group compared with ST 1000μg/L groups. But the G2/M faction was still increased compared with solvant control group. Thus, addition of PD98059 to the cells could partly decrease the ability of ST to increase the percentage of G2/M faction.
2.2.3 Effect of PD98059 pretreatment on the changes of the key factors expression of G2 stage induced by ST in GES-1 cells
2.2.3.1 Effect of PD98059 pretreatment on the expression of CyclinB1
Western blot results showed that the expression of CyclinB1 protein in PD98059+ST treatment group was significantly decreased as compared with that in ST group, but it was still higher than that in solvant control group (P<0.05).
The results of RT-PCR showed that the expression of CyclinB1 mRNA in GES-1 cells had no difference between PD98059+ST treatment group and ST treatment group (P>0.05).
Thus, the results in this study suggested that the alterations in protein expression of CyclinB1 caused by ST were blocked by the presence of ERK inhibitor PD98059, which had no effect on the expression of CyclinB1 mRNA.
2.2.3.2 Effect of PD98059 pretreatment on Cdc2 kinase
The results of Western blot showed that no difference in Cdc2 dephosphorylation in PD98059+ST treatment group and ST group could be found. But the phosphorylation of Cdc2 in PD98059+ST treatment group was decreased as compared with that in ST group (P<0.05), and there was no difference of that compared with solvent control group (P>0.05).
The results of RT-PCR confirmed that the expression of Cdc2 mRNA in PD98059+ST treatment group was decreased as compared with that in ST treatment group, but it was still higher than that in solvent control group (P<0.05).
Thus, the results in this study revealed that the increase in the level of phosphorylation of Cdc2 was blocked by the addition of PD98059 which could partly inhibit the expression of Cdc2 mRNA in ST-treated GES-1 cells.
2.2.3.2 Effect of PD98059 pretreatment on Cdc25C phosphatase
The results of Western blot showed that, the expression of Cdc25C protein had no difference in PD98059+ST treatment group and ST group. The phosphorylation of Cdc25C in PD98059+ST treatment group was significantly decreased as compared with ST group, but it was still higher than that in solvent control group (P<0.05).
The results of RT-PCR revealed that there was no difference of the expression of Cdc25C mRNA between PD98059+ST treatment group and ST group (P>0.05).
Thus, the results in this study indicated that PD98059 could inhibit the alterations in phosphorylation of Cdc25C induced by ST, but had no effect on the expression of Cdc25C mRNA.
2.3 Effects of PI3K signal pathway on the G2 arrest induced by ST
2.3.1 Effect of LY-294002 pretreatment on the changes of GES-1 cells survival rate induced by ST
The result of MTT showed that there was no difference in the cell viability between LY-294002+ST treatment group and ST group (P>0.05), and it was much lower than that of controls (P<0.05). The result indicated that LY-294002 had no effect on the cell viability.
2.3.2 Effect of LY-294002 pretreatment on the G2 arrest of GES-1 cells induced by ST
FCM analysis showed that the number of G0/G1 and S faction were all decreased and that of G2/M faction was increased in LY-294002+ST treatment group compared with ST 1000μg/L groups (P<0.05). The result indicated that there might be a synergistic effect of LY-294002 and ST on the increase of G2/M faction of GES-1 cells.
2.3.3 Effect of LY-294002 pretreatment on the changes of the key factors expression of G2 stage induced by ST in GES-1 cells
2.3.3.1 Effect of LY-294002 pretreatment on the expression of CyclinB1.
Western blot results showed that addition of the PI3K inhibitor combined with ST induced a significant decrease of CyclinB1 protein expression, as compared with that in solvant control group and ST group (P<0.05).
The results of RT-PCR confirmed that the expression of CyclinB1 mRNA in LY-294002+ST treatment group was significantly decreased as compared with ST group and solvent control group (P<0.05).
Thus, the results in this study revealed that the increase in the expression of CyclinB1 at protein and mRNA level was blocked by the addition of LY-294002 in ST-treated GES-1 cells.
2.3.3.2 Effect of LY-294002 pretreatment on Cdc2 kinase
The results of Western blot showed that there was no difference in the level of dephosphorylation of Cdc2 in LY-294002+ST treatment group (P>0.05), meanwhile the level of phosphorylation of Cdc2 was increased as compared with that in ST group and solvent control group (P<0.05).
RT-PCR results confirmed that the expression of Cdc2 mRNA was increased in LY-294002+ST treatment group as compared with solvant control group and ST group (P<0.05).
The results in this study suggested that a synergistic effect might exist between LY-294002 and ST on the phosphorylation and mRNA expression of Cdc2.
2.3.3.2 Effect of LY-294002 pretreatment on Cdc25C phosphatase
The results of Western blot showed that the expression of Cdc25C protein in LY-294002+ST treatment group had no difference compared with ST group (P>0.05). But the level of phosphorylation of Cdc25C was higher compared with ST group and solvent control group (P<0.05).
The results of RT-PCR confirmed that there was no difference in the expression of Cdc25C mRNA in LY-294002+ST treatment group compared with ST group (P>0.05).
Thus, the results in this study suggested that LY-294002 might have synergistic effect with ST on the phosphorylation of Cdc25C, but had no effects on the expression of Cdc25C at both protein and mRNA level.
3 Effects of JNK, ERK and PI3K signal transduction pathway activation on the protein dyssynthesis of GES-1 cells induced by Sterigmatocystin in vitro
Objective: To explore the effects of inhibitors of JNK, ERK and PI3K on the ST-induced protein dyssynthesis.
Methods: GES-1 cells seeded at (1~2)×104 cells/L in culture flasks were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L (the treatment was the same as above). The expression of CyclinD1, the activity of eIF4E and 4E-BP1 in GES-1 cells were determined with Western blot and RT-PCR.
Results:
3.1 Effects of JNK signal pathway on the protein dyssynthesis induced by ST 3.1.1 Effect of SP600125 pretreatment on the expression of eIF4E The results of Western blot showed that the expression of eIF4E protein in SP600125+ST treatment group was significantly decreased as compared with that in solvant control group and ST group (P<0.05), meanwhile the phosphorylation of eIF4E in SP600125+ST treatment group was significantly increased (P<0.05).
The results of RT-PCR confirmed that the expression of eIF4E mRNA in SP600125+ST treatment group was significantly decreased as compared with that in ST group, but it was still higher than that in solvent control group (P<0.05).
Thus, the results in this study revealed that the decrease in the phosphorylation of eIF4E was blocked by the addition of SP600125, which could partly inhibit the expression of eIF4E mRNA in ST-treated GES-1 cells.
3.1.2 Effect of SP600125 pretreatment on the expression of 4E-BP1
The results of Western blot showed that in comparison with ST group, the expression of 4E-BP1 protein in SP600125+ST treatment group was significantly decreased (P<0.05), but had no difference compared with solvent control group. And the phosphorylation of 4E-BP1 in SP600125+ST treatment group was significantly increased as compared with that both in ST group and solvant control group (P<0.05).
The results of RT-PCR confirmed that the expression of 4E-BP1 mRNA in SP600125+ST treatment group was reduced compared with ST group, but it was still higher than that in solvant control group (P<0.05).
The results in this study suggested that SP600125 could inhibit the alteration of 4E-BP1 activity induced by ST. And it could partly decreased the ability of ST to increase the expression of 4E-BP1 mRNA.
3.1.3 Effect of SP600125 pretreatment on the exprssion of CyclinD1 protein
The results of Western blot showed that addition of the JNK inhibitor combined with ST induced a significant increase of CyclinD1 expression, as compared with that in ST group, but the expression of CyclinD1 was still lower than that in solvent control group (P<0.05). And the results suggested that the increase in the expression of CyclinD1 at protein level was blocked by the addition of SP600125 in ST-treated GES-1 cells.
3.2 Effects of ERK signal pathway on the protein dyssynthesis induced by ST
3.2.1 Effect of PD98059 pretreatment on the expression of eIF4E
The results of Western blot showed that there was no difference in the expression of eIF4E protein in PD98059+ST treatment group compared with ST group (P>0.05). But the phosphorylation of eIF4E in PD98059+ST treatment group was significantly increased as compared with that in solvant control and ST group (P<0.05).
The results of RT-PCR revealed that the expression of eIF4E mRNA in PD98059+ST treatment group was significantly decreased as compared with that in ST group, and had no difference as compared with that in solvent control group (P<0.05).
Thus, the results in this study suggested PD98059 could block both of the decrease in phosphorylation of eIF4E and the increase in eIF4E expression at mRNA level induced by ST.
3.2.2 Effect of PD98059 pretreatment on the expression of 4E-BP1
The results of Western blot showed that the expression of 4E-BP1 protein in PD98059+ST treatment group was significantly decreased as compared with that in ST group (P<0.05), and had no difference compared with solvent control group. And addition of the ERK inhibitor combined with ST induced a significant increase of 4E-BP1 phosphorylation, as compared with that both in ST group, and had no difference compared with solvant control group (P<0.05).
The results of RT-PCR confirmed that the expression of 4E-BP1 mRNA in PD98059+ST treatment group was decreased compared with ST group and solvant control group (P<0.05).
The results in this study suggested that the additon of PD98059 could block the ST-induced increase in 4E-BP1 protein expression and decrease in the phosphorylation of 4E-BP1, and also partly block the increase in the expression of 4E-BP1 mRNA induced by ST.
3.2.3 Effect of PD98059 pretreatment on the exprssion of CyclinD1 protein
The results of Western blot showed that addition of PD98059 combined with ST induced a significant increase of CyclinD1 expression, as compared with solvent control group and ST group (P<0.05). And the results suggested that the decrease in the expression of CyclinD1 at protein level in ST-treated GES-1 cells was blocked by the addition of PD98059. 3.3 Effects of PI3K signal pathway on the protein dyssynthesis induced by ST
3.3.1 Effect of LY-294002 pretreatment on the expression of eIF4E
The results of Western blot showed that the expression of eIF4E protein in LY-294002+ST treatment group was significantly increased as compared with that in solvant control group and ST group (P<0.05). Meanwhile the phosphorylation of eIF4E in LY-294002+ST treatment group was significantly decreased as compared with that in ST group and solvant control group (P<0.05).
The results of RT-PCR confirmed that the expression of eIF4E mRNA in LY-294002+ST treatment group was significantly increased as compared with that in ST group and solvent control group (P<0.05).
Thus, the results in this study suggested that there might be a synergistic effect between LY-294002 and ST on the expression of eIF4E at both protein and mRNA level. And there might be a synergistic effect between LY-294002 and ST on the phosphorylation of eIF4E.
3.3.2 Effect of LY-294002 pretreatment on the expression of 4E-BP1
The results of Western blot showed that in comparison with ST group, the expression of 4E-BP1 protein in LY-294002+ST treatment group was significantly decreased (P<0.05), but had no difference compared with solvent control group. And the phosphorylation of 4E-BP1 in LY-294002+ST treatment group was significantly decreased as compared with ST group and solvant control group (P<0.05).
The results of RT-PCR confirmed that the expression of 4E-BP1 mRNA in LY-294002+ST treatment group was decreased compared with ST group, but it was still higher than that in solvant control group (P<0.05). The results in this study suggested that the increase in 4E-BP1 expression at both protein and mRNA level induced by ST could be blocked by LY-294002. But LY-294002 could produce a synergistic effect with ST on the phosphorylation of 4E-BP1.
3.3.3 Effect of LY-294002 pretreatment on the exprssion of CyclinD1 protein
The results of Western blot showed that the addition of LY-294002 simultaneously with ST induced a significant increase of CyclinD1 expression, as compared with solvent control group and ST group (P<0.05). And the results in this study suggested that there might be a synergistic effect between LY-294002 and ST on the protein expression of CyclinD1.
Conclusions:
1. ST could activate JNK, ERK and PI3K signal trusduction pathway, and block p38 signal trusduction pathway at the same time.
2. ST induced G2 arrest of GES-1 cells may be through activion of JNK, ERK signal pathways. But the activation of PI3K signal pathway may play negative regulating role on G2 arrest of GES-1 cells induced by ST.
3. The changes in the expression and function of Cdc25C-Cdc2/CyclinB1 by the activation of JNK, ERK and PI3K signal transduction pathways may be the putative mechanisms of G2 arrest of GES-1 cells induced by ST in vitro.
4. The activation of JNK, ERK and PI3K signal transduction pathways were all involved in the effects of ST on GES-1 cells in vitro. But the activation of PI3K signal pathway may mainly play the
引文
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