杂色曲霉素诱导人胃黏膜上皮细胞(GES-1)G_2期阻滞及凋亡可能分子机制的研究
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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毒性代谢产物,广泛存在于自然界,是人类和动物食物中最常见的污染物之一。国内外研究发现,ST具有致癌性、致突变性和免疫毒性等生物学效应。同时,ST可以对很多实验动物的脏器造成急性毒性损伤,并且具有种属及器官特异性。在短期实验中,ST的遗传毒性表现为可以直接造成细胞DNA损伤,与DNA形成加合物,还能引起染色体畸变,姐妹染色单体互换等。在长期实验中,ST可诱导实验动物发生肝癌、肺癌、间皮瘤等肿瘤。ST已被国际癌症研究中心列为“可能的人类致癌物”。本室前期研究发现,ST可诱发体外培养人胚胃黏膜细胞增殖活跃及抑癌基因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。ST是我国河北省南部胃癌、食管癌高发区粮食中最常见的霉菌毒素污染物之一。大量的肿瘤病因流行病学研究表明,ST的饮食暴露可能与该区域胃癌高发有关。因此,深入研究ST对人胃粘膜上皮细胞的细胞毒性作用,可为进一步探讨ST的可能致癌机制提供必要的实验依据,且具有重要的现实意义。
细胞增殖与死亡之间的失衡常被认为是肿瘤发生过程中的重要环节。当外界刺激因素引起细胞DNA损伤时,将通过启动检测点机制而诱导细胞发生周期阻滞,这使得细胞有充足的时间修复受损DNA,但修复失败的细胞则被诱导发生凋亡。这一过程对于维持基因组的稳定性具有重要意。检测点调控紊乱引起的细胞周期和凋亡异常可能导致细胞基因组失稳、基因突变和DNA异倍体出现,甚至发生癌变。研究表明,很多致癌性毒素的早期效应多是诱发细胞周期紊乱、增殖抑制及凋亡异常等。谢同欣等发现,ST可诱导小鼠胚胎纤维母细胞细G_2/M期阻滞及p53蛋白表达上调。本室也证实,ST作用于人胃粘膜上皮细胞24小时可通过影响MAPK通路而诱导其发生G_2期阻滞。
DNA损伤检测点信号转导途径是一个高度保守的信号感应过程。ATM/ATR是能早期感应DNA损伤信号的蛋白激酶,其活性的增加构成了整个途径活化的第一步,p53是ATM的底物之一,它能够根据损伤的严重程度,或激活细胞周期检测点,诱导周期阻滞从而修复DNA,或启动细胞进入凋亡途径。
为进一步阐明ST的细胞毒性作用及可能机制,本研究以永生化人正常胃黏膜上皮细胞(GES-1)为研究对象,初步探讨了ST的致损伤作用及其相关机制。我们首先观察了ST较长期作用对GES-1细胞周期进程以及细胞增殖与凋亡的影响;接下来检测了ST对GES-1细胞DNA的损伤作用及其下游可能的损伤信号传导途径;最后以同时与DNA损伤和细胞周期阻滞、凋亡密切相关的p53-p21~（WAF1/CIP1）信号通路为研究靶点,深入探讨了ST诱导GES-1细胞G_2期阻滞及凋亡的分子机制。为揭示ST饮食暴露与人胃黏膜损伤乃至胃癌发生的可能关系奠定实验基础,对提高我国胃癌高发区居民食品安全水平具有重要意义。
本研究论文共分为三个部分:
第一部分杂色曲霉素对GES-1细胞增殖和凋亡的影响
目的:探讨杂色曲霉素对体外培养的人胃黏膜上皮细胞(GES-1)细胞周期分布以及细胞增殖与凋亡的影响。
方法:采用噻唑蓝(MTT)比色法观察不同浓度(0.03~48μM) ST作用24、48及72 h对GES-1细胞存活率的影响。流式细胞定量检测(FCM)不同时间(2~12 d)及不同浓度(0.075~3μM) ST处理后GES-1细胞周期分布情况。蛋白质免疫印迹(Western Blot)、反转录聚合酶联反应(RT-PCR)方法检测ST处理后G_2/M期关键调节分子—Cdc25C、Cdc2和CyclinB1在蛋白水平和mRNA水平上的表达情况。免疫共沉淀技术检测Cdc2-CyclinB1复合物的形成情况。PI单染法和annexin V-PI双染法检测ST对GES-1细胞凋亡的影响。Hoechst 33258染色从形态上检测ST对GES-1细胞凋亡的的影响。Western Blot检测凋亡相关蛋白Bxl-2、Bax和NF-κB的表达变化及Caspase-3的激活情况。
结果:
1.1 ST对GES-1细胞存活率的影响
MTT法检测结果显示,ST处理24 h,3~48μM浓度范围的ST处理组GES-1细胞存活率均较溶剂对照组明显降低(P<0.05),且呈剂量依赖性(r24h=-0.961, P<0.01);ST处理48及72h,在1.5~48μM浓度范围内,各ST处理组细胞存活率分别明显低于各自相应的溶剂对照组(P<0.05),且呈剂量依赖性(r48h=-0.955, r72h=-0.913, P<0.01)。在24μM和48μM两个ST处理浓度,随着ST作用时间的延长GES-1细胞的存活率均明显降低,呈时间依赖性(r24μM=-0.998,r48μM =-0.998, P<0.05)。提示ST对GES-1细胞的生长有明显的抑制作用,且呈时间、剂量依赖地方式。
1.2 ST对GES-1细胞周期分布的影响
1.2.1 ST作用不同时间对GES-1细胞周期及其关键调节因子的影响
1.2.1.1 ST作用不同时间对GES-1细胞周期分布的影响FCM检测结果表明,3μM ST 2d、4d、8d及12d处理组细胞G_2/M期细胞比例明显增加(P<0.05),且随着ST作用时间的延长,G_2/M期细胞比例先增高后降低,峰值出现在ST作用后第4天(P<0.05)。
1.2.1.2 ST作用不同时间对GES-1细胞G_2期关键调节因子的影响Western Blot结果显示,3μM ST处理2d、4d、8d及12d可以降低GES-1细胞内Cdc25C的去磷酸化水平(P<0.05)和升高其磷酸化水平(P<0.05),并且分别呈时间依赖性(r=-0.814,r=0.807,P<0.01)。各ST不同时间处理组GES-1细胞Cdc25C在mRNA水平上的表达明显低于对照组(P<0.05)。
Western Blot结果显示,3μM ST 2d、4d处理组细胞Cdc2蛋白水平较对照组明显降低(P<0.05),但ST 8d及12d处理组又逐渐恢复至对照组水平。各不同时间处理组Cdc2的磷酸化水平较对照组有明显升高(P<0.05),且呈时间依赖性(r= 0.603,P<0.01)。各处理组GES-1细胞Cdc2的mRNA表达量均明显低于溶剂对照组(P<0.05)。Western Blot结果显示,3μM ST 2d、4d、8d及12d处理组CyclinB1蛋白表达水平均较对照组明显升高(P<0.05)。各处理组GES-1细胞CyclinB1的mRNA表达量均明显高于溶剂对照组(P<0.05)。
1.2.2不同浓度ST对GES-1细胞周期及其关键调节因子的影响
1.2.2.1不同浓度ST作用4天对GES-1细胞周期及其关键调节因子的影响
1.2.2.1.1不同浓度ST作用4天对GES-1细胞周期分布的影响FCM检测结果表明,ST处理4天后,0.3μM、1.5μM、3μM ST处理组G_2/M期细胞比例均较溶剂对照组明显增加(P<0.05),并且在0～3μM浓度范围内,G_2/M期细胞比例与ST的处理浓度呈正相关关系(r=0.927,P<0.01)。
1.2.2.1.2不同浓度ST作用4天对GES-1细胞G_2期关键调节因子的影响Western Blot结果显示,ST作用4天后, 0.3μM、1.5μM和3μM ST处理组细胞的Cdc25C和Cdc2蛋白表达水平较溶剂对照组明显减少(P<0.05),且分别呈剂量依赖性(r=-0.800, r=-0.876, P<0.01);而Cdc25C和Cdc2的磷酸化水平则较溶剂对照组明显增加(P<0.05),且呈剂量依赖性(r=0.714, r=0.767, P<0.01)。各处理组Cdc25C和Cdc2的mRNA表达量均明显低于溶剂对照组(P<0.05)。
Western Blot结果显示,ST作用4天后,0.3μM、1.5μM和3μM ST处理组细胞的Cyclin B1蛋白表达水平较溶剂对照组明显增加(P<0.05),且呈剂量依赖性(r=0.929, P<0.01)。
1.2.2.1.3不同浓度ST作用4天对GES-1细胞Cdc2-CyclinB1复合物的影响
免疫共沉淀结果显示,ST作用4天后,Cdc2和CyclinB1可以以复合物的形式存在,0.075μM、0.3μM、1.5μM和3μM ST处理4天可以减少Cdc2-CyclinB1复合物的形成。
1.2.2.2不同浓度ST作用8天对GES-1细胞周期及其关键调节因子的影响
1.2.2.2.1不同浓度ST作用8天对GES-1细胞周期分布的影响FCM检测结果表明,ST处理8天后,0.3μM、1.5μM、3μM ST处理组G_2/M期细胞比例均较溶剂对照组明显增加(P<0.05),并且在0～3μM浓度范围内,G_2/M期细胞比例与ST的处理浓度呈正相关关系(r=0.910,P<0.01)。
1.2.2.2.2不同浓度ST作用8天对GES-1细胞G_2期关键调节因子的影响Western Blot结果显示,不同浓度ST作用于GES-1细胞8天后,1.5μM和3μM ST处理组细胞的Cdc25C蛋白表达水平较溶剂对照组明显减少(P<0.05),且在ST 0.3~3μM的浓度范围内呈剂量依赖性(r=-0.830,P<0.01);而Cdc25C的磷酸化水平则较溶剂对照组明显增加(P<0.01),且在ST 0.3~3μM的浓度范围内呈剂量依赖性(r=0.752, P<0.01)。各处理组Cdc25C的mRNA表达量均明显低于溶剂对照组(P<0.05)。
Western Blot结果显示,不同浓度ST处理细胞8天后,各浓度处理组中只有3μM ST处理组细胞的Cdc2蛋白表达水平较溶剂对照组下降(P<0.05);但各浓度处理组Cdc2的磷酸化水平均较溶剂对照组明显增加(P<0.05),且呈剂量依赖性(r=0.781,P<0.01)。各处理组Cdc2的mRNA表达量均明显低于溶剂对照组(P<0.05)。
Western Blot结果显示,不同浓度ST处理细胞8天后,0.3μM、1.5μM和3μM ST处理组细胞的Cyclin B1蛋白表达水平较溶剂对照组明显增加(P<0.05),且在ST 0.3~3μM的浓度范围内呈剂量依赖性(r=0.753, P<0.01)。各浓度处理组GES-1细胞CyclinB1的mRNA表达量均明显高于溶剂对照组(P<0.05)。
1.3 ST对GES-1细胞凋亡的影响
1.3.1 ST作用不同时间对GES-1细胞凋亡的影响
1.3.1.1 ST作用不同时间对GES-1细胞凋亡率的影响PI单染法检测细胞凋亡率,结果表明, GES-1细胞经3μM ST处理2d、4d、8d及12d后,各ST处理组细胞凋亡率分别为3.11±0.69%,4.07±0.71%,7.96±1.17%及3.89±0.28%,其中ST 4d、8d及12d处理组细胞的凋亡率均高于溶剂对照组2.19±0.08% (P<0.05),并且细胞凋亡的高峰出现在ST处理后第8天,12天又有明显下降(P<0.01)。表明3μM ST可以诱导GES-1细胞发生凋亡,且凋亡的高峰期出现在ST处理后8天,此时间晚于ST诱导的G_2期阻滞的高峰期4天。提示ST作用于GES-1细胞时凋亡的发生晚于其诱导的G_2期阻滞。
1.3.1.2 Hoechst 33258染色检测ST作用不同时间GES-1细胞凋亡情况3μM ST ST处理GES-1细胞经Hoechst33258染色后,荧光显微镜下观察可见细胞核染色质凝集、固缩,或核碎裂呈碎块状,颜色呈致密浓染的亮蓝色。ST作用2至8天随着ST作用时间的延长,凋亡指数(AI,%)逐渐升高(r=0.827, P<0.01, Fig. 12B),至8天达最大值,12天又有所下降(P<0.05)。
1.3.1.3 ST作用不同时间对GES-1细胞凋亡相关蛋白表达的影响
1.3.1.3.1 ST作用不同时间对GES-1细胞Bcl-2表达的影响Western Blot结果显示,3μM ST作用2d、4d、8d及12d后,细胞的Bcl-2蛋白表达水平分别较溶剂对照组明显降低(P<0.05),且其表达量随着ST作用时间的延长而减少(r=-0.557, P<0.05)。提示ST处理可以时间依赖性地下调GES-1细胞内Bcl-2蛋白的表达。
1.3.1.3.2 ST作用不同时间对GES-1细胞Bax表达的影响Western Blot结果显示,3μM ST作用2d、4d、8d及12d后,细胞的Bax蛋白表达水平分别较溶剂对照组明显升高(P<0.05),且其表达量在8 d达高峰后,12天又有明显下降(P<0.05)。提示ST处理GES-1细胞可以上调Bax蛋白的表达,且表达量的峰值出现在ST处理后8天,与细胞凋亡的高峰时间一致。
1.3.1.3.3 ST作用不同时间对GES-1细胞NF-κB表达的影响Western Blot结果显示,3μM ST作用2d、4d、8d及12d后,细胞的NF-κB蛋白表达水平分别较溶剂对照组明显下降(P<0.05),且其表达量随着ST作用时间的延长而减少(r=-0.825,P<0.01)。提示ST处理可以时间依赖性地下调GES-1细胞内NF-κB蛋白的表达。
1.3.1.3.4 ST作用不同时间对Caspase-3蛋白表达的影响Western Blot结果显示,3μM ST 2d、4d、8d及12d处理组均出现了Caspase-3的活性片段,且随ST处理作用时间的延长,阳性条带颜色逐渐加深(P<0.05),并在ST处理8 d时表达量达到峰值,之后12 d又有明显的下降(P<0.05)。表明ST处理可以激活GES-1细胞Caspase-3,且其活性片段表达水平的变化与ST处理后细胞凋亡率的变化趋势一致。
1.3.2不同浓度ST对GES-1细胞凋亡的影响
1.3.2.1不同浓度ST作用4天对GES-1细胞凋亡的影响
1.3.2.1.1不同浓度ST作用4天对GES-1细胞凋亡率的影响PI单染法检测细胞凋亡率,结果表明, GES-1细胞经不同浓度ST处理4 d后,0.075μM、0.3μM、1.5μM和3Μm ST处理组细胞凋亡率明显高于溶剂对照组(P<0.05),且随浓度的增加凋亡率逐渐升高(r=0.854, P<0.01)。
进一步应用Annexin V/PI双染法检测凋亡率,结果表明,不同浓度ST处理4天后GES-1细胞早期和晚期凋亡率都较溶剂对照组显著升高(P<0.05),并且早期和晚期凋亡率之和也相应增加(P<0.05),与PI单染法结果一致。
综合以上两种方法表明,ST作用4天可以剂量依赖性地诱导体GES-1细胞发生凋亡。
1.3.2.1.2不同浓度ST作用4天对GES-1细胞凋亡相关蛋白表达的影响
1.3.2.1.2.1不同浓度ST作用4天对GES-1细胞Bcl-2表达的影响Western Blot结果显示,ST作用4天后,0.075μM、0.3μM、1.5μM和3μM ST处理组细胞的Bcl-2蛋白表达水平较溶剂对照组明显降低(P<0.05),且其表达量随着ST浓度的增加而减少(r=-0.857, P<0.05)。提示ST处理4天可以剂量依赖性地下调GES-1细胞内Bcl-2蛋白的表达。
1.3.2.1.2.2不同浓度ST作用4天对GES-1细胞Bax表达的影响Western Blot结果显示,ST作用4天后,0.075~3μM ST处理组细胞的Bax蛋白表达水平较溶剂对照组明显升高(P<0.05),且其表达量随着ST浓度的增加而升高(r=0.907,P<0.01)。提示ST处理4天可以剂量依赖性地上调GES-1细胞内Bax蛋白的表达。
1.3.2.1.2.3不同浓度ST作用4天对GES-1细胞NF-κB表达的影响Western Blot结果显示,ST作用4天后,0.3μM、1.5μM和3μM ST处理组细胞的NF-κB蛋白表达水平较溶剂对照组明显下降(P<0.05),且其表达量随着ST浓度的增加而减少(r=-0.828, P<0.01)。提示ST处理4天可以剂量依赖性地下调GES-1细胞内NF-κB蛋白的表达。
1.3.2.1.2.4不同浓度ST作用4天对Caspase-3蛋白表达的影响Western Blot结果显示,ST作用4天后,各浓度ST处理组Caspase-3活性片段都较溶剂对照组明显增加(P<0.01),且随ST浓度增加阳性酶切片段的表达量逐渐升高(r=0.905,P<0.05)。表明ST处理4天可以激活GES-1细胞Caspase-3,且其活性片段表达水平以剂量依赖方式增加。
1.3.2.2不同浓度ST作用8天对GES-1细胞凋亡的影响
1.3.2.2.1不同浓度ST作用8天对GES-1细胞凋亡率的影响PI单染法检测细胞凋亡率,结果表明, GES-1细胞经不同浓度ST处理8 d后,0.3μM、1.5μM和3μM ST处理组细胞凋亡率均明显高于溶剂对照组(P<0.05),且随浓度的增加凋亡率逐渐升高(r=0.933, P<0.01)。提示ST作用8天可以剂量依赖性地诱导体外培养人胃黏膜上皮细胞GES-1发生细胞凋亡。
1.3.2.2.2不同浓度ST作用8天对GES-1细胞凋亡相关蛋白表达的影响
1.3.2.2.2.1不同浓度ST作用8天对GES-1细胞Bcl-2表达的影响
Western Blot结果显示,ST作用8天后,0.3μM、1.5μM和3μM ST处理组细胞的Bcl-2蛋白表达水平较溶剂对照组明显降低(P<0.05),且其表达量随着ST浓度的增加而减少(r=-0.912, P<0.05)。提示ST处理8天可以剂量依赖性地下调GES-1细胞内Bcl-2蛋白的表达。
1.3.2.2.2.2不同浓度ST作用8天对GES-1细胞Bax表达的影响
Western Blot结果显示,ST作用8天后,0.3μM、1.5μM和3μM ST处理组细胞的Bax蛋白表达水平较溶剂对照组明显升高(P<0.05),且其表达量随着ST浓度的增加而升高(r=0.901, P<0.01)。提示ST处理8天可以剂量依赖性地上调GES-1细胞内Bax蛋白的表达。
1.3.2.2.2.3不同浓度ST作用8天对GES-1细胞NF-κB表达的影响
Western Blot结果显示,ST作用8天后,0.3μM、1.5μM和3μM ST处理组细胞的NF-κB蛋白表达水平较溶剂对照组明显下降(P<0.05),且其表达量随着ST浓度的增加而减少(r=-0.803,P<0.01)。提示ST处理8天可以剂量依赖性地下调GES-1细胞内NF-κB蛋白的表达。
1.3.2.2.2.4不同浓度ST作用8天对Caspase-3蛋白表达的影响
Western Blot结果显示,ST处理8天后,0.075~3μM各ST处理组Caspase-3活性片段的表达均较溶剂对照组明显增加(P<0.01),且随着ST浓度的增加,阳性酶切片段的表达量逐渐升高(r=0.850,P<0.01)。表明ST处理8天可以激活GES-1细胞Caspase-3,且其活性片段表达水平以剂量依赖方式增加。
第二部分杂色曲霉素对GES-1细胞DNA的损伤作用及损伤通路的激活目的:探讨杂色曲霉素对人胃黏膜上皮细胞(GES-1) DNA的损伤作用及其下游损伤通路的激活情况,揭示ST诱导GES-1细胞G_2期阻滞的可能原因。
方法:采用单细胞凝胶电泳试验观察ST作用不同时间(2、4、8及12d)对GES-1细胞DNA的损伤情况;采用FCM和Western Blot方法检测ATM/ATR特异性阻断剂—caffeine预处理对ST诱导的G_2期阻滞及相关调控因子的影响。
结果:
2.1 ST对GES-1细胞DNA损伤的影响
单细胞凝胶电泳试验结果显示,3μM ST作用于GES-1细胞可引起DNA的损伤。各不同时间ST处理组与相应溶剂对照组相比,DAN损伤指数(DI)显著增高(P<0.001),且随着ST作用时间的延长DI逐渐升高,两者呈正相关关系(r=0.903, p<0.001)。
不同时间ST处理的GES-1细胞在电泳之后经软件分析出的彗星尾部DNA含量、尾长及尾距三个指标与相应的溶剂对照组相比均有显著地增加,并且存在明显的时效关系(P<0.01)。
以上结果提示,ST可以引起GES-1细胞DNA损伤,且随ST作用时间的延长DNA损伤程度相应加重,它可能参与了ST诱导的细胞G_2期阻滞。
2.2 Caffeine预处理对ST诱导的GES-1细胞G_2期阻滞及细胞周期相关蛋白的影响
2.2.1 Caffeine预处理对ST诱导的GES-1细胞G_2期阻滞的影响
FCM检测结果显示,caffeine预处理+ST 3μM处理组与ST单独处理组相比,G_2/M期细胞比例明显减少(P<0.05)。表明caffeine预处理可以阻断ST诱导的细胞G_2/M期比例增高,提示ATM/ATR信号通路的激活可能参与了ST诱导的GES-1细胞G_2期阻滞。
2.2.2 Caffeine预处理对ST作用后GES-1细胞G_2期关键调节因子表达变化的影响
2.2.2.1 Caffeine预处理对Cdc25C表达的影响
Western Blot结果显示,5 mM caffeine预处理+ST(1.5μM, 3μM)处理组Cdc25C的表达水平与ST(1.5μM, 3μM)单独处理组相比明显提高(P<0.05);而Cdc25C的磷酸化水平在caffeine预处理后较ST单独处理组有所降低(P<0.05)。提示ATM/ATR信号通路可能参与了ST诱导的Cdc25C表达降低和p-Cdc25C表达的升高。
2.2.2.2 Caffeine预处理对Cdc2表达的影响
Western Blot结果显示,5 mM caffeine预处理+ST(1.5μM , 3μM)处理组Cdc2的表达水平与ST(1.5μM, 3μM)单独处理组相比明显升高(P<0.05);而Cdc2的磷酸化水平在caffeine预处理后则有所降低(P<0.05)。提示ATM/ATR信号通路可能参与了ST诱导的Cdc2表达的降低和p-Cdc2表达的升高。
2.2.2.3 Caffeine预处理对CyclinB1表达的影响Western Blot结果显示,5 mM caffeine预处理+ST(1.5μM , 3μM)处理组CyclinB1的表达水平与ST单独处理组相比无明显变化(P>0.05)。
2.3 Caffeine预处理对p-p53表达的影响
Western Blot结果显示,与溶剂对照组相比,5mM caffeine预处理组p-p53的表达水平显著降低;1.5μM ST和3μM ST单独处理组p-p53的表达水平均明显升高(P>0.05);5 mM caffeine预处理+ST(1.5μM , 3μM)处理组p-p53的表达水平与ST(1.5μM , 3μM)单独处理组相比明显降低(P>0.05)。提示,ST作用2天激活了p53;p53是ATM/ATR信号通路中的的一员;ATM/ATR信号通路可能参与了ST诱导的p-p53表达的升高。
第三部分杂色曲霉素诱导GES-1细胞周期G_2期阻滞及凋亡的可能分子机制
目的:探讨杂色曲霉素诱导体外培养的人胃黏膜上皮细胞(GES-1)G_2期阻滞及凋亡的可能分子机制。
方法:采用Western Blot和real-time PCR技术观察ST对p53-p21WAF1/CIP1信号通路相关分子表达的影响。进一步通过p53 siRNA转染干扰p53基因表达,Western Blot方法检测p53-p21WAF1/CIP1信号通路相关分子及细胞G_2/M期调控因子的表达变化情况,FCM方法检测细胞周期的变化情况。
结果:
3.1 ST对GES-1细胞p53-p21WAF1/CIP1信号通路的影响
3.1.1 ST作用不同时间对p53-p21WAF1/CIP1信号通路的的影响
3.1.1.1 ST作用不同时间对p53蛋白的影响
Western Blot结果显示,3μM ST处理GES-1细胞2、4、8及12天后,各ST处理组p-p53(Ser15)和p53蛋白水平均较对照组明显升高(P<0.05);在ST处理8天时p-p53(Ser15)的表达量达最大值,12天又明显下降(P<0.05)。提示ST处理GES-1细胞可以时间依赖性地激活p53,并且p-p53在第8天出现峰值后12天又下降。
Real time-PCR检测结果显示, ST 2、4、8及12d处理组的p53 mRNA水平均较对照组明显升高(P<0.01),且ST作用8天时达峰值,12天时又下降(P<0.05)。提示,ST作用于GES-1细胞可以上调p53 mRNA的表达,且第8天出现峰值,之后下降。与p53在蛋白水平上的激活时间保持一致。以上p53的激活在时间上与G_2期阻滞和凋亡基本一致。
3.1.1.2 ST作用不同时间对p21~(WAF1/CIP1)蛋白的影响
Western Blot结果显示,3μM ST处理GES-1细胞2、4、8及12天后,各ST处理组p~(21WAF1/CIP1)蛋白水平均较对照组明显升高(P<0.05);在ST处理后8天时其表达量达最大值,12天又有所下降(P<0.05)。提示ST作用于GES-1细胞可以时间依赖性地激活p~(21WAF1/CIP1),并且其在第8天出现峰值后12天又下降。p~(21WAF1/CIP1)的激活在时间上在时间上与G_2期阻滞和凋亡基本一致。
3.1.2不同浓度ST对p53-p~(21WAF1/CIP1)信号通路的的影响
3.1.2.1不同浓度ST作用4天对p53-p~(21WAF1/CIP1)信号通路的的影响
3.1.2.1.1不同浓度ST作用4天对p53蛋白的影响
Western Blot结果显示,0.075μM、0.3μM、1.5μM及3μM ST处理GES-1细胞4 d后,各ST处理组p-p53(Ser15)蛋白水平均较溶剂对照组明显升高(P<0.05);除0.075μM ST处理组外,各ST处理组p53蛋白水平较溶剂对照组明显升高(P<0.05)。并且p53的去磷酸化和磷酸化水平均随ST浓度的增加而升高(r=0.893; r=0.866, P<0.01)。提示ST作用于GES-1细胞4天可以剂量依赖性地激活p53。
Real time-PCR检测结果显示,除0.075μM ST处理组外,其它各ST处理组的p53 mRNA水平均较溶剂对照组明显升高(P<0.01)。提示,不同浓度ST作用于GES-1细胞4天可以上调p53 mRNA的表达。
3.1.2.1.2不同浓度ST作用4天对p~(21WAF1/CIP1)蛋白的影响
Western Blot结果显示,0.075μM、0.3μM、1.5μM及3μM ST处理GES-1细胞4 d后,各ST处理组p21蛋白表达水平均较溶剂对照组明显升高(P<0.05),且随ST浓度的增加而升高(r=0.910, P<0.01)。提示ST作用于GES-1细胞4天可以剂量依赖性地激活p~(21WAF1/CIP1)。
3.1.2.2不同浓度ST作用8天对p53-p~(21WAF1/CIP1)信号通路的影响
3.1.2.2.1不同浓度ST作用8天对p53蛋白的影响
Western Blot结果显示,0.075μM、0.3μM、1.5μM及3μM ST处理GES-1细胞8 d后,各ST处理组p-p53(Ser15)蛋白水平均较溶剂对照组明显升高(P<0.05);除0.075μM ST处理组外,各ST处理组p53蛋白水平较溶剂对照组明显升高(P<0.05)。并且p53的去磷酸化和磷酸化水平均随ST浓度的增加而升高(r=0.836; r=0.879, P<0.01)。提示ST作用于GES-1细胞8天可以剂量依赖性地激活p53。
3.1.2.2.2不同浓度ST作用8天对p~(21WAF1/CIP1)蛋白的影响
Western Blot结果显示,0.075μM、0.3μM、1.5μM及3μM ST处理GES-1细胞8 d后,各ST处理组p21蛋白表达水平均较溶剂对照组明显升高(P<0.05),且随ST浓度的增加而升高(r=0.926, P<0.01)。提示ST作用于GES-1细胞8天可以剂量依赖性地激活p~(21WAF1/CIP1)。
3.2 p53 siRNA转染对ST作用后GES-1细胞的影响
3.2.1 p53 siRNA转染对ST作用后GES-1细胞p53-p~(21WAF1/CIP1)信号通路的影响
3.2.1.1 p53 siRNA转染对ST作用后GES-1细胞p53蛋白的影响
Real time PCR结果显示,p53 siRNA单独转染组细胞在p53的mRNA水平较溶剂对照组显著降低(P<0.01,论文中未列出);Western Blot结果显示,p53 siRNA单独转染组细胞p53的磷酸化和非磷酸化蛋白表达水平均较溶剂对照组显著降低(P<0.01)。并且p53在mRNA和蛋白水平的有效抑制率均达到70%~80%,证明p53 siRNA的转染成功地干扰了p53的表达。
Western Blot结果显示,p53 siRNA+ST (3μM)处理组p-p53(Ser15)及p53的蛋白表达水平与ST (3μM)处理组相比均明显降低(P<0.05);而与p53 siRNA处理组相比均显著升高(P<0.05)。提示,p53 siRNA的转染可以阻断ST诱导的GES-1细胞p53的激活。
3.2.1.2 p53 siRNA转染对ST作用后GES-1细胞p~(21WAF1/CIP1)蛋白的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组p~(21WAF1/CIP1)的蛋白表达水平与ST (3μM)处理组相比明显降低(P<0.05);而与p53 siRNA处理组相比均显著升高(P<0.05)。提示,p53 siRNA的转染可以阻断ST诱导的GES-1细胞p~(21WAF1/CIP1)的激活。
3.2.2 p53 siRNA转染对ST作用后GES-1细胞G_2期关键调节因子表达变化的影响
3.2.2.1 p53 siRNA转染对ST作用后GES-1细胞Cdc25C的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组与ST (3μM)处理组相比,Cdc25C的蛋白表达水平明显升高,而p-Cdc25C的水平则显著下降(P<0.05)。而p53 siRNA+ST (3μM)处理组p-Cdc25C的蛋白表达水平与p53 siRNA处理组相比有显著升高(P<0.05)。提示,p53参与了ST诱导的GES-1细胞Cdc25C表达的下调和p-Cdc25C表达的上调。
3.2.2.2 p53 siRNA转染对ST作用后GES-1细胞Cdc2的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组与ST (3μM)处理组相比,Cdc2表达增多,而p-Cdc2表达则显著减少(P<0.05);p53 siRNA+ST (3μM)处理组与p53 siRNA处理组相比,Cdc2表达水平有所降低,而p-Cdc2的表达水平则有显著升高(P<0.05)。提示,p53参与了ST诱导的GES-1细胞Cdc2表达的下调和p-Cdc2表达的上调。
3.2.2.3 p53 siRNA转染对ST作用后GES-1细胞CyclinB1的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组CyclinB1的蛋白表达水平与ST (3μM)处理组相比有显著下降,而与p53 siRNA处理组相比则有显著升高(P<0.05)。提示,p53参与了ST诱导的GES-1细胞CyclinB1蛋白表达的上调。
3.2.3 p53 siRNA转染对ST作用后GES-1细胞凋亡相关蛋白表达变化的影响
3.2.3.1 p53 siRNA转染对ST作用后GES-1细胞Bcl-2的影响
Western Blot结果显示,ST (3μM)处理组与溶剂对照组相比,Bcl-2的蛋白表达水平显著下降(P<0.05);而p53 siRNA+ST (3μM)处理组与ST (3μM)处理组相比Bcl-2水平无明显变化(P>0.05)。提示,p53 siRNA转染不足以逆转ST对GES-1细胞Bcl-2表达的下调作用。
3.2.3.2 p53 siRNA转染对ST作用后GES-1细胞Bax的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组Bax的蛋白表达水平与ST (3μM)处理组相比有显著下降,而与p53 siRNA处理组相比则有明显升高(P<0.05)。提示,p53参与了ST诱导的GES-1细胞Bax蛋白表达的上调。
3.2.3.3 p53 siRNA转染对ST作用后GES-1细胞Caspase-3的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组在17kD处的Caspase-3活性酶切片段与ST (3μM)处理组相比明显减少,而与p53 siRNA处理组相比则有明显增加(P<0.05)。提示,p53参与了ST诱导的GES-1细胞caspase-3的激活。
3.2.4 p53 siRNA转染对ST诱导的GES-1细胞G_2期阻滞的影响
FCM检测结果显示,与ST (3μM)处理组相比,p53 siRNA+ST (3μM)处理组G_2/M期细胞比例明显降低(P<0.05)。表明p53 siRNA转染可以阻断ST诱导的细胞G_2/M期比例增高,提示p53以及其下游信号转导通路的激活可能参与了ST诱导的细胞周期G_2期阻滞。
结论:
1 ST可以抑制GES-1细胞增殖及诱导其发生G_2期阻滞和凋亡,而ST可能正是通过诱导G_2期阻滞和凋亡来抑制GES-1细胞增殖的。
2 ST通过抑制Bcl-2和促进Bax蛋白表达来激活线粒体凋亡途径,随后激活caspase-3而最终诱导GES-1细胞发生凋亡。
3 ST可以引起GES-1细胞DNA的损伤并激活损伤下游ATM/ATR信号通路。ATM/ATR特异性阻断剂可以阻断ST诱导的G_2期阻滞。由此可见,ATM/ATR信号通路的激活可能参与了ST诱导的GES-1细胞G_2期阻滞,且在这一过程中ST引起的DNA损伤是一个起始事件。
4 ST可以激活p53-p~(21WAF1/CIP1)信号转导通路,并且通过siRNA干扰p53基因的表达证实了p53-p~(21WAF1/CIP1)信号通路的激活参与了ST诱导的GES-1细胞G_2期阻滞和凋亡。
5从ST的时间效应来看,ST引起的GES-1细胞凋亡的发生晚于其诱导的G_2期阻滞,它们的效应高峰分别出现在ST作用后8天和4天。并且当ST持续作用12天时,G_2期阻滞和凋亡均缓解。
6 ST持续作用较长时间时(12天),有部分细胞能够脱离由p53介导的G_2期阻滞和凋亡而继续生长,但这些细胞仍携带着严重的DNA损伤信息,而这些细胞的继续复制、增殖则可能是ST致癌作用的分子机制之一。
Sterigmatocystin (ST) is the fungal secondary metabolite produced by many Aspergillus species such as A. versicolor, A. chevalieri, etc. ST is one of the most common contaminant in different foodstuffs and environment, and it was regularly detected in grains, corn, bread, cheese and animal feed. Structurally, ST is related to a?atoxins and is generally recognized as a potential carcinogen, mutagen and teratogen. ST was found to have different toxicological, mutagenic and carcinogenic effects in animals and has been recognized as a 2B carcinogen (possible human carcinogen) by International Agency for Research on Cancer. ST could induce hepatocellular carcinoma after oral administration or intraperitoneal injection and squamous cell carcinoma after repeated application to the skin in rats, and could induce lung adenocarcinoma after oral administration in NIH mice. In short-term tests, ST, although considered less potent, is known to cause DNA damage and form DNA adducts, lead to DNA breaks, chromosome aberration, and sister-chromatid exchange in animal experiments. Our previous studies 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. In China, ST has been one of the most commonly detected mycotoxins in the maize from a high incidence area of gastric carcinoma, such as Taihang Mountains in North China. Many epidemiological studies of tumor etiology showed that diet exposure of ST may have some relationship with high incidence of gastric cancer in these areas. As gastric epithelium would be directly exposured to oral intake of ST, evaluation of the cytotoxic effects of ST on human gastric epithelial cells can be of further usage for the elucidation of the possible carcinogenic mechanism of ST.
The imbalance between cell proliferation and death is regularly considered to be an important event in carcinogenesis. At key transitions during eukaryotic cell cycle progression, signaling pathways monitor the successful completion of upstream events prior to proceeding to the next phase and these regulatory pathways are commonly referred to as cell cycle checkpoints. In mammalian cells, proliferation is controlled by factors that regulate the transition at cell cycle checkpoints, which are responsible for the initiation and completion of DNA replication and control of cell division respectively. Cell cycle progression is regulated by the checkpoint controls that function to allow time for the DNA repair and result in activation of pathways leading to apoptosis if cellular damage cannot be properly repaired. Defects in cell cycle checkpoints can result in cell genomic instability, gene mutations, and aneuploidy, all of which can contribute to carcinogenesis. Many studies have shown apoptosis, proliferation inhibition and cell cycle arrest are among the early effects of carcinogenic toxins on animal cells. Xie, et al. found that ST could cause G_2/M arrest and overexpression of p53 protein in murine fibroblasts. Our primary study confirmed that ST treatment for 24 hours could induce the G_2 arrest by affecting the MAPK signaling pathway in GES-1 cells in vitro.
DNA damage checkpoint signal pathway is a highly conserved signal induction process. ATM/ATR is a early sensor of protein kinase to DNA damage signal and the increase of its activity constitutes the first step in the whole activation pathway. p53 is a substrate of ATM, which can either activate cell cycle checkpoints and induce cycle arrest to repair damaged DNA, or activate cells into the apoptosis pathway, according to severity of injury. To further evaluate the effect of ST treatment on cell cycle and apoptosis and to explore their corresponding mechanism, in this study, we used a human gastric epithelial cell line (GES-1) and carried out the following studies. First, we observed the effect of ST treatment on GES-1 cell cycle distribution, proliferation and apoptosis. And then, we investigated the effect of ST treatment on DNA damage of GES-1 cells and the changes in DNA damage downstream signaling pathway. Finally, we chose p53-p21WAF1/CIP1 signaling pathway, which were closely related with DNA damage and cell cycle arrest, and further explored the molecular mechanism of ST-induced G_2 arrest. The aim of this study is to lay an experimental foundation for revealing the possible relationship between diet exposure of ST and gastric mucosa damage as well as the possible effects on gastric carcinogenesis.. The following three parts are included in the study
PartⅠThe effect of ST on the distribution of cell cycle, proliferation and apoptosis in GES-1 cells in vitro
Objective: To further confirm the effect of ST on the regulation of the cell cycle distribution, proliferation and apoptosis of human gastric epithelium cells (GES-1) in vitro.
Methods: The effect of ST treatment at different concentrations (0.03~48μM) for 24, 48 and 72 h on GES-1 cells survival rates was tested by MTT. The distribution of cell cycle after ST treatment was analyzed by FCM. Western Blot and RT-PCR were used for the determination of expression of cell cycle key factors (Cdc25C, Cdc2 and CyclinB1) at protein and mRNA level respectively. The effect of ST on the Cdc2-CyclinB1 complex was evaluated with immunoprecipitation. The effect of ST on apoptosis was measured by flow cytometry after Annexin V-PI staining and PI staining and detected morphologically by Hoechst 33258 staining. Western Blot was used for the expression of apoptosis related factor Bcl-2, Bax, NF-κB, and Caspase-3.
Results:
1.1 Effects of ST on survival rates in GES-1 cells
MTT assay showed that ST had a potent inhibitory effect on the growth of GES-1 cells. When GES-1 cells were treated by ST for 24 hours, the cell survival rates in ST treated groups ranging from 3 to 48μM displayed a significant decrease (P<0.05) in a dose-dependent manner (r24h=-0.961, P<0.01) and treated for 48 and 72 hours, the cell survival rates in ST treated groups ranging from 1.5 to 48μM respectively displayed a significant decrease (P<0.05) in a dose-dependent manner (r48h=-0.955, r72h=-0.913, P<0.01). When treated at 24μM and 48μM, the cell survival rates in ST treated groups significantly decreased in a time-dependent manner (r24μM=-0.998, r48μM=-0.998, P<0.05).
1.2 Effects of ST on the distribution of cell cycle in GES-1 cells
1.2.1 Effects of ST on cell cycle and G_2 phase key regulatory factor in GES-1 cells at various time points
1.2.1.1 Effects of ST on cell cycle in GES-1 cells at various time points Flow Cytometric results showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the proportion of cells in G_2/M phase in all ST treatment groups was significantly increased as compared with that in control group (P<0.05). The peak value of the proportion of cells in G_2/M phase was reached appeared at the 4th day after exposure to 3μM ST. Subsequently, this G_2 delay was sustained till day 8 of ST treatment (P<0.05).
1.2.1.2 Effects of ST on G_2 phase key regulatory factor in GES-1 cells at various time points
Western Blot analysis showed that the expression of the key G_2 phase regulatory proteins Cdc25C was significantly decreased after 2d, 4d, 8d and 12d in 3μM ST treatment cells (P<0.05) in a time-dependent manner (r=-0.814, P<0.01). At the same time, the phosphorylation level of Cdc25C was increased in ST treatment groups (P<0.05) time-dependently(r=0.807, P<0.01). RT-PCR analysis showed that the expression of Cdc25C mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).
3μM ST treatment significantly decreased the key G_2 phase regulatory proteins Cdc2 expression from 2 to 4d after treatment, When the treatment continued for 8 and 12days, the expression of Cdc2 was gradually recovered to level of the control group In the time range from 2 to 8 days, the phosphorylation levels of Cdc2 were increased at all ST treatment groups in time-dependent manner (r= 0.603,P<0.01). Different from the expression at protein level, RT-PCR analysis showed that the expression of Cdc2 mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).
Western Blot analysis and RT-PCR analysis showed that the expression of Cyclin B1 at protein and mRNA level were all significantly increased in all ST treatment groups as compared with that in control group (P<0.05).
1.2.2 Effects of ST treatment with different concentrations on cell cycle and G_2 phase key regulatory factor in GES-1 cells
1.2.2.1 Effects of ST treatment with different concentrations for 4 days on cell cycle and G_2 phase key regulatory factor in GES-1 cells
1.2.2.1.1 Effects of ST treatment with different concentrations for 4 days on cell cycle in GES-1 cells Flow Cytometric results showed that when GES-1 cells were treated by ST for 4 days, the proportions of cells in G_2/M phase in 0.3μM, 1.5μM, 3μM ST treatment groups were all significantly increased as compared with that in solvent control group in a dose-dependent manner (r=0.927, P<0.01).
1.2.2.1.2 Effects of ST treatment with different concentrations for 4 days on G_2 phase key regulatory factor in GES-1 cells
Western Blot analysis results showed that 4 days after ST treatment at 0.3μM, 1.5μM, 3μM, the expressions of Cdc25C and Cdc2 were all significantly decreased as compared with that in solvent control group (P<0.05) and a dose effects could be seen (r=-0.800, r=-0.876, P<0.01). At the same time, the phosphorylation level of Cdc25C and Cdc2 was increased at all ST treatment groups dose-dependently (r=0.714, r=0.767, P<0.01). Similar to the expression at protein level, RT-PCR analysis showed that the expression of Cdc25C and Cdc2 mRNA in ST treatment groups was also significantly decreased as compared with that in control group (P<0.05).
Four days after ST treatment, the expressions of Cyclin B1 at protein and mRNA level were all significantly increased in 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=0.929, P<0.01).
1.2.2.1.3 Effects of ST treatment with different concentrations for 4 days on Cdc2-CyclinB1 complex in GES-1 cells Immunoprecipitation results suggested that when treated by ST for 4 days, ST could induce the decrease of Cdc2-CyclinB1 complex of GES-1 cells in 0.075μM, 0.3μM, 1.5μM and 3μM ST treatment groups.
1.2.2.2 Effects of ST treatment with different concentrations for 8 days on cell cycle and G_2 phase key regulatory factor in GES-1 cells
1.2.2.1.1 Effects of ST treatment with different concentrations for 8 days on cell cycle in GES-1 cells
Flow Cytometry showed that when GES-1 cells were treated by ST for 8 days, the proportion of cells in G_2/M phase in 0.3μM to 3μM ST treatment groups for 8 days was significantly increased (P<0.05) as compared with that in solvent control group in a dose-dependent manner (r=0.910, P<0.01).
1.2.2.2.2 Effects of ST treatment with different concentrations for 8 days on G_2 phase key regulatory factor in GES-1 cells
Western Blot analysis showed that when GES-1 cells were treated by ST for 8 days, the expression of Cdc25C was significantly decreased in 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner ranging from 0.3 to 3μM (r=-0.830, P<0.01). Conversely, the phosphorylation level of Cdc25C was increased in 1.5μM, 3μM ST treatment groups in dose-dependent manner (r=0.714, r=0.767, P<0.01). RT-PCR analysis showed that the expression of Cdc25C mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).
Western Blot analysis showed that when GES-1 cells were treated by ST for 8 days, the expression of Cdc2 was significantly decreased in only 3μM ST treatment groups as compared with that in solvent control group (P<0.05). Conversely, the phosphorylation level of Cdc2 was increased at all ST treatment groups in dose-dependent manner (r=0.781,P<0.01). RT-PCR analysis showed that the expression of Cdc2 mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).
Western Blot analysis showed that when GES-1 cells were treated by ST for 8 days, the expression of Cyclin B1 was significantly increased in 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.753, P<0.01). RT-PCR analysis showed that the expression of Cyclin B1 mRNA in ST treatment groups was significantly increased as compared with that in control group (P<0.05).
1.3 Effects of ST on apoptosis in GES-1 cells
1.3.1 Effects of ST on apoptosis in GES-1 cells at various time points
1.3.1.1 Effects of ST on apoptosis rate in GES-1 cells
The PI staining result showed that when the GES-1 cells were treated by 3μM ST for 4, 8 and 12d, the apoptosis rate in all ST treatment groups (4.07±0.71%,7.96±1.17% and 3.89±0.28%) was significantly increased compared with that in control group (2.19±0.08%, P<0.05). Exposure to 3μM ST, the peak value of apoptosis rate appeared at the 8th day and subsequently decreased after ST treatment for 12 days(P<0.05). It is worth noting that the peak value of apoptosis rate was later four days than that of G_2 arrest.
1.3.1.2 Apoptosis was detected in GES-1 cells with ST treatment at various time points by staining
Cells treated with 3μM ST were stained by Hoechst 33258 and observed through fluorescence microscope. The typical morphological changes, such as condensation of chromatin and nuclear fragmentations appeared in GES-1 cells treated with ST at different time points. Apoptotic index of 2d, 4d and 8d ST treatment groups was significantly increased as compared with that in control group (r=0.827, P<0.01) and the peak value appeared at the 8th day, subsequently decreased (P<0.05) .
1.3.1.3 Effects of ST on apoptosis related proteins in GES-1 cells at various time points
1.3.1.3.1 Effects of ST on expression of Bcl-2 in GES-1 cells at various time points
Western Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of Bcl-2 was significantly decreased in all ST treatment groups(P<0.05) as compared with that in control group in time-dependent manner (r=-0.557, P<0.05).
1.3.1.3.2 Effects of ST on expression of Bax in GES-1 cells at various time points
Western Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of Bax was significantly increased in all ST treatment groups as compared with that in control group(P<0.05). Protein expression peaked at 8th day and decreased at 12th day (P<0.05), which consistented with the change of cell cycle arrest.
1.3.1.3.3 Effects of ST on expression of NF-κB in GES-1 cells at various time points
Western Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of NF-κB was significantly decreased in all ST treatment groups as compared with that in control group(P<0.05) in time-dependent manner(r=-0.825,P<0.01).
1.3.1.3.4 Effects of ST on activation of caspase-3 in GES-1 cells at various time points
Western Blot analysis showed that ST could activate caspase-3 (P<0.05). When the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the peak value of the cleaved (activated) form of Caspase-3 could be seen in 8 day ST treated groups and decreased in 12 day ST treated groups (P<0.05), which consistented with the changes of apoptosis rate.
1.3.2 Effects of ST treatment with different concentrations on apoptosis in GES-1 cells
1.3.2.1 Effects of ST treatment with different concentrations for 4 days on apoptosis in GES-1 cells
1.3.2.1.1 Effects of ST treatment with different concentrations for 4 days on apoptosis rate in GES-1 cells
The PI staining result showed that after ST treatment for 4 days the apoptosis rate in 0.075μM, 0.3μM, 1.5μM and 3μM ST treatment groups was significantly increased as compared with that in solvent control group(P<0.05) in dose-dependent manner (r=0.854, P<0.01).
Annexin V-PI staining showed that the apoptosis percentage of cells including early and late apoptotic cells was significantly increased in different concentrations ST treatment groups as compared with that in solvent control group (P<0.05), which consistented with the result of PI staining.
Based on the above two methods shows that ST treatment for 4 days could induce apoptosis in GES-1 cells in dose-dependent manner.
1.3.2.1.2 Effects of ST treatment with different concentrations for 4 days on apoptosis related proteins in GES-1 cells
1.3.2.1.2.1 Effects of ST treatment with different concentrations for 4 days on Bcl-2 in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of Bcl-2 was significantly decreased in 0.075μM, 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.857, P<0.05).
1.3.2.1.2.2 Effects of ST treatment with different concentrations for 4 days on Bax in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of Bax was significantly increased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.907, P<0.01).
1.3.2.1.2.3 Effects of ST treatment with different concentrations for 4 days on NF-κB in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of NF-κB was significantly decreased in 0.3μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.828, P<0.01).
1.3.2.1.2.4 Effects of ST treatment with different concentrations for 4 days on Caspase-3 in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of the cleaved fragment of Caspase-3 was significantly decreased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=0.905, P<0.05).
1.3.2.2 Effects of ST treatment with different concentrations for 8 days on apoptosis in GES-1 cells
1.3.2.2.1 Effects of ST treatment with different concentrations for 8 days on apoptosis rate in GES-1 cells
The PI staining result showed that after ST treatment for 8 days the apoptosis rate in 0.3μM, 1.5μM and 3μM ST treatment groups was significantly increased as compared with that in solvent control group(P<0.05) in dose-dependent manner (r=0.933, P<0.01).
1.3.2.2.2 Effects of ST treatment with different concentrations for 8 days on apoptosis related proteins in GES-1 cells
1.3.2.1.2.1 Effects of ST treatment with different concentrations for 8 days on Bcl-2 in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of Bcl-2 was significantly decreased in 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.912, P<0.05).
1.3.2.2.2.2 Effects of ST treatment with different concentrations for 8 days on Bax in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of Bax was significantly increased in 0.3μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.901, P<0.01).
1.3.2.1.2.3 Effects of ST treatment with different concentrations for 8 days on NF-κB in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of NF-κB was significantly decreased in 0.3μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.803,P<0.01).
1.3.2.1.2.4 Effects of ST treatment with different concentrations for 8 days on Caspase-3 in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of the cleaved fragment of Caspase-3 was significantly decreased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=0.850, P<0.01).
PartⅡThe study on the effect of DNA damage and the activation of damage pathway in GES-1 cells induced by ST in vitro
Objective: To explore the effect of ST on DNA damage and the activation of damage pathway in human gastric epithelium cells (GES-1). And to reveal that the possible reason for ST-induced G_2 phase arrest in GES-1 cells.
Methods: The situation of DNA damage was measured by single cell gel electrophoresis assay after different time (2, 4, 8 and 12d) of ST treatment. Western Blot detected the expression of cell cycle key factors (Cdc25C, Cdc2 and CyclinB1). The distribution of cell cycle after ST treatment was detected by FCM.
Results:
2.1 Effects of ST on DNA damage in GES-1 cells
Single cell gel electrophoresis assay result showed that 3μM ST treatment could induce DNA damage. Damage Index of different time ST treatment groups were significantly higher than that of corresponding solvent control groups (P<0.001) in time-dependent manner (r=0.903, p<0.001).
The analysis of DNA content of the comet tail, tail length and tail moment by the software after ST treatment at different time in GES-1 cells after electrophoresis were significantly increased as compared with the corresponding solvent control groups(P<0.01) in a time-dependent manner.
The result suggested that ST could induce GES-1 cells DNA damage, which was possible reason on ST-induced G_2 phase arrest.
2.2 Effects of ST on G_2 arrest and cell cycle related proteins in GES-1 cells, with caffeine pretreatment
2.2.1 Effects of ST on cell cycle distribution in GES-1 cells, with caffeine pretreatment
Flow Cytometry showed that the proportion of cells in G_2/M phase in caffeine pretreatment group was reduced as compared with that in 3μM ST treatment group (P<0.05). And caffeine could inhibit ST-induced G_2 phase arrest in GES-1 cells. The results indicated that ST induced G_2 phase arrest in GES-1 cells involving the activation of ATM/ATR and its related signaling pathway.
2.2.2 Effects of ST on cell cycle related proteins in GES-1 cells, with caffeine pretreatment
2.2.2.1 Effects of ST on Cdc25C in GES-1 cells, with caffeine pretreatment
The results of Western Blot showed that the expression of Cdc25C in caffeine pretreatment group were significantly increased as compared with that in ST (1.5μM, 3μM) treatment groups (P<0.05) and the phosphorylation level of Cdc25C was opposite (P<0.05). The results suggested that ATM/ATR signaling pathway may be involved in the changes of Cdc25C and p-Cdc25C ST-induced.
2.2.2.2 Effects of ST on Cdc2 in GES-1 cells, with caffeine pretreatment
The results of Western Blot showed that the expression of Cdc2 in caffeine pretreatment group were significantly increased as compared with that in ST (1.5μM, 3μM) treatment groups (P<0.05) and the phosphorylation level of Cdc2 was opposite (P<0.05). The results suggested that ATM/ATR signaling pathway may be involved in the changes of Cdc2 and p-Cdc2 ST-induced.
2.2.2.3 Effects of ST on CyclinB1 in GES-1 cells, with caffeine pretreatment
The results of Western Blot showed that no significant difference was found between caffeine + ST treatment groups and ST treatment groups (P>0.05).
2.3 Effects of ST on p-p53 in GES-1 cells, with caffeine pretreatment
The results of Western Blot showed that the expression of p-p53 in caffeine pretreatment group was significantly decreased and in 1.5μM and 3μM ST treatment groups were significantly increased as compared with that in solvent control groups (P<0.05). The expression of p-p53 in caffeine pretreatment group were significantly decreased as compared with that in ST (1.5μM, 3μM) treatment groups (P<0.05). The results suggested that after ST treatment for 2 days, ST could activate p53; p53 is a downstream protein of ATM/ATR signaling pathway and ATM/ATR signaling pathway may be involved in the changes of p-p53 ST-induced.
PartⅢThe study on the molecular mechanisms of G_2 Arrest in GES-1 cells induced by ST in vitro
Objective: To explore the possible molecular mechanisms on ST induced G_2 phase arrest in GES-1cells in vitro.
Methods: The expression of p53-p~(21WAF1/CIP1) signaling pathway after ST treatment was analysized with Western Blot and real-time PCR. GES-1 cells were transfected with p53 siRNA, Western Blot detected the expression of signaling pathway related molecule and G_2/M phase key regulatory factors.
The cell cycle distribution of GES-1cells after p53 siRNA transfection was detected with FCM. Results:
3.1 Effects of ST on p53-p~(21WAF1/CIP1) signaling pathway in GES-1 cells
3.1.1 Effects of ST on p53-p~(21WAF1/CIP1) in GES-1 cells at various time points
3.1.1.1 Effects of ST on p53 in GES-1 cells at various time points
Western Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of p53 and p-p53 was significantly decreased in all ST treatment groups (P<0.05). The peak value of p-p53 appeared at 8th day, subsequently decreased at 12th day(P<0.05). The results suggested that ST treatment could activate p53 and the peak value of p-p53 appeared at 8th day.
Real time-PCR analysis showed that the expression of p53 mRNA was significantly increased after different time ST treatment(P<0.01), while the peak value appeared at 8th day which was consistent with expression of p53 at protein level.
Based on the above, the activation of p53 was basically consistent in time with G_2 arrest and apoptotsis.
3.1.1.2 Effects of ST on p~(21WAF1/CIP1) in GES-1 cells at various time points Western Blot analysis showed that when cells were treated by 3μM ST from 2 to 12d, the expression of p21 was significantly decreased in all ST treatment groups (P<0.05). The peak value of p21 appeared at 8th day, subsequently decreased at 12th day(P<0.05). The results suggested that ST treatment could activate p53 and the peak value of p-p53 appeared at 8th day. The activation of p21 was basically consistent in time with G_2 arrest and apoptotsis.
3.1.2 Effects of ST treatment with different concentrations on p53-p~(21WAF1/CIP1) signaling pathway in GES-1 cells
3.1.2.1 Effects of ST treatment with different concentrations for 4 days on p53-p~(21WAF1/CIP1) signaling pathway in GES-1 cells
3.1.2.1.1 Effects of ST treatment with different concentrations for 4 days on p53 in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of p-p53 was significantly increased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.893, P<0.01). The expression of p53 was significantly increased in 0.3μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.866, P<0.01).
Real time-PCR analysis showed that the expression of p53 mRNA was significantly increased after different concentrations ST treatment(P<0.01),
3.1.2.1.2 Effects of ST treatment with different concentrations for 4 days on p~(21WAF1/CIP1) in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of p21 was significantly increased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.910, P<0.01).
3.1.2.2 Effects of ST treatment with different concentrations for 8 days on p53-p~(21WAF1/CIP1) signaling pathway in GES-1 cells
3.1.2.1.1 Effects of ST treatment with different concentrations for 8 days on p53 in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of p-p53 was significantly increased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.836, P<0.01). The expression of p53 was significantly increased in 0.3μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.879, P<0.01).
Real time-PCR analysis showed that the expression of p53 mRNA was significantly increased after different concentrations ST treatment(P<0.01),
3.1.2.1.2 Effects of ST treatment with different concentrations for 8 days on p~(21WAF1/CIP1) in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of p21 was significantly increased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.926, P<0.01).
3.2 Effects of ST on GES-1 cells with p53 siRNA transfection
3.2.1 Effects of ST on p53-p~(21WAF1/CIP1) signaling pathway in GES-1 cells with p53 siRNA transfection
3.2.1.1 Effects of ST on p53 in GES-1 cells with p53 siRNA transfection
Real time-PCR analysis showed that the expression of p53 mRNA was significantly decreased after p53 siRNA transfection for 2 days (P<0.01,data not given). Western Blot analysis showed that the expression of p53 and p-p53 was significantly increased in p53 siRNA transfection group as compared with that in solvent control group (P<0.05). The inhibition rate of p53 at mRNA and protein level reached 70%~80% which proved p53 siRNA transfection was successful in interfering with the expression of p53.
Western Blot analysis showed that after ST treatment for 2 days, the expression of p53 and p-p53 was significantly decreased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while increased in p53 siRNA treatment group (P<0.05). The results suggested that p53 siRNA transfection could inhibit ST-induced the activition of p53.
3.2.1.2 Effects of ST on p~(21WAF1/CIP1) in GES-1 cells with p53 siRNA transfection
Western Blot analysis showed that after ST treatment for 2 days, the expression of p53 and p-p53 was significantly decreased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while increased in p53 siRNA treatment group (P<0.05). The results suggested that p53 siRNA transfection could inhibit ST-induced the activition of p~(21WAF1/CIP1).
3.2.2 Effects of ST on the key regulatory factors of G_2 phase in GES-1 cells with p53 siRNA transfection
3.2.2.1 Effects of ST on Cdc25C in GES-1 cells with p53 siRNA transfection
Western Blot analysis showed that the expression of Cdc25C was significantly increased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while that of p-Cdc25C decreased in ST treatment group (P<0.05). The results suggested that p53 was involved in the ST-induced decreasement of Cdc25C and increasement of p-Cdc25C.
3.2.2.2 Effects of ST on Cdc2 in GES-1 cells with p53 siRNA transfection Western Blot analysis showed that the expression of Cdc2 was increased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while that of p-Cdc2 significantly decreased in ST treatment group (P<0.05). The results suggested that p53 was involved in ST-induced the decreasement of Cdc2 and increasement of p-Cdc2.
3.2.2.3 Effects of ST on CyclinB1 in GES-1 cells with p53 siRNA transfection
Western Blot analysis showed that the expression of CyclinB1 was significantly decreased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while significantly increased as compared with that in p53 siRNA treatment group (P<0.05). The results suggested that p53 was involved in the ST-induced increasement of CyclinB1.
3.2.3 Effects of ST on apoptosis related proteins in GES-1 cells with p53 siRNA transfection
3.2.3.1 Effects of ST on Bcl-2 in GES-1 cells with p53 siRNA transfection
Western Blot analysis showed that the expression of Bcl-2 was significantly decreased in ST treatment group as compared with that in solvent control group (P<0.05), but no significant difference was found between p53 siRNA + ST (3?
细胞增殖与死亡之间的失衡常被认为是肿瘤发生过程中的重要环节。当外界刺激因素引起细胞DNA损伤时,将通过启动检测点机制而诱导细胞发生周期阻滞,这使得细胞有充足的时间修复受损DNA,但修复失败的细胞则被诱导发生凋亡。这一过程对于维持基因组的稳定性具有重要意。检测点调控紊乱引起的细胞周期和凋亡异常可能导致细胞基因组失稳、基因突变和DNA异倍体出现,甚至发生癌变。研究表明,很多致癌性毒素的早期效应多是诱发细胞周期紊乱、增殖抑制及凋亡异常等。谢同欣等发现,ST可诱导小鼠胚胎纤维母细胞细G_2/M期阻滞及p53蛋白表达上调。本室也证实,ST作用于人胃粘膜上皮细胞24小时可通过影响MAPK通路而诱导其发生G_2期阻滞。
DNA损伤检测点信号转导途径是一个高度保守的信号感应过程。ATM/ATR是能早期感应DNA损伤信号的蛋白激酶,其活性的增加构成了整个途径活化的第一步,p53是ATM的底物之一,它能够根据损伤的严重程度,或激活细胞周期检测点,诱导周期阻滞从而修复DNA,或启动细胞进入凋亡途径。
为进一步阐明ST的细胞毒性作用及可能机制,本研究以永生化人正常胃黏膜上皮细胞(GES-1)为研究对象,初步探讨了ST的致损伤作用及其相关机制。我们首先观察了ST较长期作用对GES-1细胞周期进程以及细胞增殖与凋亡的影响;接下来检测了ST对GES-1细胞DNA的损伤作用及其下游可能的损伤信号传导途径;最后以同时与DNA损伤和细胞周期阻滞、凋亡密切相关的p53-p21~（WAF1/CIP1）信号通路为研究靶点,深入探讨了ST诱导GES-1细胞G_2期阻滞及凋亡的分子机制。为揭示ST饮食暴露与人胃黏膜损伤乃至胃癌发生的可能关系奠定实验基础,对提高我国胃癌高发区居民食品安全水平具有重要意义。
本研究论文共分为三个部分:
第一部分杂色曲霉素对GES-1细胞增殖和凋亡的影响
目的:探讨杂色曲霉素对体外培养的人胃黏膜上皮细胞(GES-1)细胞周期分布以及细胞增殖与凋亡的影响。
方法:采用噻唑蓝(MTT)比色法观察不同浓度(0.03~48μM) ST作用24、48及72 h对GES-1细胞存活率的影响。流式细胞定量检测(FCM)不同时间(2~12 d)及不同浓度(0.075~3μM) ST处理后GES-1细胞周期分布情况。蛋白质免疫印迹(Western Blot)、反转录聚合酶联反应(RT-PCR)方法检测ST处理后G_2/M期关键调节分子—Cdc25C、Cdc2和CyclinB1在蛋白水平和mRNA水平上的表达情况。免疫共沉淀技术检测Cdc2-CyclinB1复合物的形成情况。PI单染法和annexin V-PI双染法检测ST对GES-1细胞凋亡的影响。Hoechst 33258染色从形态上检测ST对GES-1细胞凋亡的的影响。Western Blot检测凋亡相关蛋白Bxl-2、Bax和NF-κB的表达变化及Caspase-3的激活情况。
结果:
1.1 ST对GES-1细胞存活率的影响
MTT法检测结果显示,ST处理24 h,3~48μM浓度范围的ST处理组GES-1细胞存活率均较溶剂对照组明显降低(P<0.05),且呈剂量依赖性(r24h=-0.961, P<0.01);ST处理48及72h,在1.5~48μM浓度范围内,各ST处理组细胞存活率分别明显低于各自相应的溶剂对照组(P<0.05),且呈剂量依赖性(r48h=-0.955, r72h=-0.913, P<0.01)。在24μM和48μM两个ST处理浓度,随着ST作用时间的延长GES-1细胞的存活率均明显降低,呈时间依赖性(r24μM=-0.998,r48μM =-0.998, P<0.05)。提示ST对GES-1细胞的生长有明显的抑制作用,且呈时间、剂量依赖地方式。
1.2 ST对GES-1细胞周期分布的影响
1.2.1 ST作用不同时间对GES-1细胞周期及其关键调节因子的影响
1.2.1.1 ST作用不同时间对GES-1细胞周期分布的影响FCM检测结果表明,3μM ST 2d、4d、8d及12d处理组细胞G_2/M期细胞比例明显增加(P<0.05),且随着ST作用时间的延长,G_2/M期细胞比例先增高后降低,峰值出现在ST作用后第4天(P<0.05)。
1.2.1.2 ST作用不同时间对GES-1细胞G_2期关键调节因子的影响Western Blot结果显示,3μM ST处理2d、4d、8d及12d可以降低GES-1细胞内Cdc25C的去磷酸化水平(P<0.05)和升高其磷酸化水平(P<0.05),并且分别呈时间依赖性(r=-0.814,r=0.807,P<0.01)。各ST不同时间处理组GES-1细胞Cdc25C在mRNA水平上的表达明显低于对照组(P<0.05)。
Western Blot结果显示,3μM ST 2d、4d处理组细胞Cdc2蛋白水平较对照组明显降低(P<0.05),但ST 8d及12d处理组又逐渐恢复至对照组水平。各不同时间处理组Cdc2的磷酸化水平较对照组有明显升高(P<0.05),且呈时间依赖性(r= 0.603,P<0.01)。各处理组GES-1细胞Cdc2的mRNA表达量均明显低于溶剂对照组(P<0.05)。Western Blot结果显示,3μM ST 2d、4d、8d及12d处理组CyclinB1蛋白表达水平均较对照组明显升高(P<0.05)。各处理组GES-1细胞CyclinB1的mRNA表达量均明显高于溶剂对照组(P<0.05)。
1.2.2不同浓度ST对GES-1细胞周期及其关键调节因子的影响
1.2.2.1不同浓度ST作用4天对GES-1细胞周期及其关键调节因子的影响
1.2.2.1.1不同浓度ST作用4天对GES-1细胞周期分布的影响FCM检测结果表明,ST处理4天后,0.3μM、1.5μM、3μM ST处理组G_2/M期细胞比例均较溶剂对照组明显增加(P<0.05),并且在0～3μM浓度范围内,G_2/M期细胞比例与ST的处理浓度呈正相关关系(r=0.927,P<0.01)。
1.2.2.1.2不同浓度ST作用4天对GES-1细胞G_2期关键调节因子的影响Western Blot结果显示,ST作用4天后, 0.3μM、1.5μM和3μM ST处理组细胞的Cdc25C和Cdc2蛋白表达水平较溶剂对照组明显减少(P<0.05),且分别呈剂量依赖性(r=-0.800, r=-0.876, P<0.01);而Cdc25C和Cdc2的磷酸化水平则较溶剂对照组明显增加(P<0.05),且呈剂量依赖性(r=0.714, r=0.767, P<0.01)。各处理组Cdc25C和Cdc2的mRNA表达量均明显低于溶剂对照组(P<0.05)。
Western Blot结果显示,ST作用4天后,0.3μM、1.5μM和3μM ST处理组细胞的Cyclin B1蛋白表达水平较溶剂对照组明显增加(P<0.05),且呈剂量依赖性(r=0.929, P<0.01)。
1.2.2.1.3不同浓度ST作用4天对GES-1细胞Cdc2-CyclinB1复合物的影响
免疫共沉淀结果显示,ST作用4天后,Cdc2和CyclinB1可以以复合物的形式存在,0.075μM、0.3μM、1.5μM和3μM ST处理4天可以减少Cdc2-CyclinB1复合物的形成。
1.2.2.2不同浓度ST作用8天对GES-1细胞周期及其关键调节因子的影响
1.2.2.2.1不同浓度ST作用8天对GES-1细胞周期分布的影响FCM检测结果表明,ST处理8天后,0.3μM、1.5μM、3μM ST处理组G_2/M期细胞比例均较溶剂对照组明显增加(P<0.05),并且在0～3μM浓度范围内,G_2/M期细胞比例与ST的处理浓度呈正相关关系(r=0.910,P<0.01)。
1.2.2.2.2不同浓度ST作用8天对GES-1细胞G_2期关键调节因子的影响Western Blot结果显示,不同浓度ST作用于GES-1细胞8天后,1.5μM和3μM ST处理组细胞的Cdc25C蛋白表达水平较溶剂对照组明显减少(P<0.05),且在ST 0.3~3μM的浓度范围内呈剂量依赖性(r=-0.830,P<0.01);而Cdc25C的磷酸化水平则较溶剂对照组明显增加(P<0.01),且在ST 0.3~3μM的浓度范围内呈剂量依赖性(r=0.752, P<0.01)。各处理组Cdc25C的mRNA表达量均明显低于溶剂对照组(P<0.05)。
Western Blot结果显示,不同浓度ST处理细胞8天后,各浓度处理组中只有3μM ST处理组细胞的Cdc2蛋白表达水平较溶剂对照组下降(P<0.05);但各浓度处理组Cdc2的磷酸化水平均较溶剂对照组明显增加(P<0.05),且呈剂量依赖性(r=0.781,P<0.01)。各处理组Cdc2的mRNA表达量均明显低于溶剂对照组(P<0.05)。
Western Blot结果显示,不同浓度ST处理细胞8天后,0.3μM、1.5μM和3μM ST处理组细胞的Cyclin B1蛋白表达水平较溶剂对照组明显增加(P<0.05),且在ST 0.3~3μM的浓度范围内呈剂量依赖性(r=0.753, P<0.01)。各浓度处理组GES-1细胞CyclinB1的mRNA表达量均明显高于溶剂对照组(P<0.05)。
1.3 ST对GES-1细胞凋亡的影响
1.3.1 ST作用不同时间对GES-1细胞凋亡的影响
1.3.1.1 ST作用不同时间对GES-1细胞凋亡率的影响PI单染法检测细胞凋亡率,结果表明, GES-1细胞经3μM ST处理2d、4d、8d及12d后,各ST处理组细胞凋亡率分别为3.11±0.69%,4.07±0.71%,7.96±1.17%及3.89±0.28%,其中ST 4d、8d及12d处理组细胞的凋亡率均高于溶剂对照组2.19±0.08% (P<0.05),并且细胞凋亡的高峰出现在ST处理后第8天,12天又有明显下降(P<0.01)。表明3μM ST可以诱导GES-1细胞发生凋亡,且凋亡的高峰期出现在ST处理后8天,此时间晚于ST诱导的G_2期阻滞的高峰期4天。提示ST作用于GES-1细胞时凋亡的发生晚于其诱导的G_2期阻滞。
1.3.1.2 Hoechst 33258染色检测ST作用不同时间GES-1细胞凋亡情况3μM ST ST处理GES-1细胞经Hoechst33258染色后,荧光显微镜下观察可见细胞核染色质凝集、固缩,或核碎裂呈碎块状,颜色呈致密浓染的亮蓝色。ST作用2至8天随着ST作用时间的延长,凋亡指数(AI,%)逐渐升高(r=0.827, P<0.01, Fig. 12B),至8天达最大值,12天又有所下降(P<0.05)。
1.3.1.3 ST作用不同时间对GES-1细胞凋亡相关蛋白表达的影响
1.3.1.3.1 ST作用不同时间对GES-1细胞Bcl-2表达的影响Western Blot结果显示,3μM ST作用2d、4d、8d及12d后,细胞的Bcl-2蛋白表达水平分别较溶剂对照组明显降低(P<0.05),且其表达量随着ST作用时间的延长而减少(r=-0.557, P<0.05)。提示ST处理可以时间依赖性地下调GES-1细胞内Bcl-2蛋白的表达。
1.3.1.3.2 ST作用不同时间对GES-1细胞Bax表达的影响Western Blot结果显示,3μM ST作用2d、4d、8d及12d后,细胞的Bax蛋白表达水平分别较溶剂对照组明显升高(P<0.05),且其表达量在8 d达高峰后,12天又有明显下降(P<0.05)。提示ST处理GES-1细胞可以上调Bax蛋白的表达,且表达量的峰值出现在ST处理后8天,与细胞凋亡的高峰时间一致。
1.3.1.3.3 ST作用不同时间对GES-1细胞NF-κB表达的影响Western Blot结果显示,3μM ST作用2d、4d、8d及12d后,细胞的NF-κB蛋白表达水平分别较溶剂对照组明显下降(P<0.05),且其表达量随着ST作用时间的延长而减少(r=-0.825,P<0.01)。提示ST处理可以时间依赖性地下调GES-1细胞内NF-κB蛋白的表达。
1.3.1.3.4 ST作用不同时间对Caspase-3蛋白表达的影响Western Blot结果显示,3μM ST 2d、4d、8d及12d处理组均出现了Caspase-3的活性片段,且随ST处理作用时间的延长,阳性条带颜色逐渐加深(P<0.05),并在ST处理8 d时表达量达到峰值,之后12 d又有明显的下降(P<0.05)。表明ST处理可以激活GES-1细胞Caspase-3,且其活性片段表达水平的变化与ST处理后细胞凋亡率的变化趋势一致。
1.3.2不同浓度ST对GES-1细胞凋亡的影响
1.3.2.1不同浓度ST作用4天对GES-1细胞凋亡的影响
1.3.2.1.1不同浓度ST作用4天对GES-1细胞凋亡率的影响PI单染法检测细胞凋亡率,结果表明, GES-1细胞经不同浓度ST处理4 d后,0.075μM、0.3μM、1.5μM和3Μm ST处理组细胞凋亡率明显高于溶剂对照组(P<0.05),且随浓度的增加凋亡率逐渐升高(r=0.854, P<0.01)。
进一步应用Annexin V/PI双染法检测凋亡率,结果表明,不同浓度ST处理4天后GES-1细胞早期和晚期凋亡率都较溶剂对照组显著升高(P<0.05),并且早期和晚期凋亡率之和也相应增加(P<0.05),与PI单染法结果一致。
综合以上两种方法表明,ST作用4天可以剂量依赖性地诱导体GES-1细胞发生凋亡。
1.3.2.1.2不同浓度ST作用4天对GES-1细胞凋亡相关蛋白表达的影响
1.3.2.1.2.1不同浓度ST作用4天对GES-1细胞Bcl-2表达的影响Western Blot结果显示,ST作用4天后,0.075μM、0.3μM、1.5μM和3μM ST处理组细胞的Bcl-2蛋白表达水平较溶剂对照组明显降低(P<0.05),且其表达量随着ST浓度的增加而减少(r=-0.857, P<0.05)。提示ST处理4天可以剂量依赖性地下调GES-1细胞内Bcl-2蛋白的表达。
1.3.2.1.2.2不同浓度ST作用4天对GES-1细胞Bax表达的影响Western Blot结果显示,ST作用4天后,0.075~3μM ST处理组细胞的Bax蛋白表达水平较溶剂对照组明显升高(P<0.05),且其表达量随着ST浓度的增加而升高(r=0.907,P<0.01)。提示ST处理4天可以剂量依赖性地上调GES-1细胞内Bax蛋白的表达。
1.3.2.1.2.3不同浓度ST作用4天对GES-1细胞NF-κB表达的影响Western Blot结果显示,ST作用4天后,0.3μM、1.5μM和3μM ST处理组细胞的NF-κB蛋白表达水平较溶剂对照组明显下降(P<0.05),且其表达量随着ST浓度的增加而减少(r=-0.828, P<0.01)。提示ST处理4天可以剂量依赖性地下调GES-1细胞内NF-κB蛋白的表达。
1.3.2.1.2.4不同浓度ST作用4天对Caspase-3蛋白表达的影响Western Blot结果显示,ST作用4天后,各浓度ST处理组Caspase-3活性片段都较溶剂对照组明显增加(P<0.01),且随ST浓度增加阳性酶切片段的表达量逐渐升高(r=0.905,P<0.05)。表明ST处理4天可以激活GES-1细胞Caspase-3,且其活性片段表达水平以剂量依赖方式增加。
1.3.2.2不同浓度ST作用8天对GES-1细胞凋亡的影响
1.3.2.2.1不同浓度ST作用8天对GES-1细胞凋亡率的影响PI单染法检测细胞凋亡率,结果表明, GES-1细胞经不同浓度ST处理8 d后,0.3μM、1.5μM和3μM ST处理组细胞凋亡率均明显高于溶剂对照组(P<0.05),且随浓度的增加凋亡率逐渐升高(r=0.933, P<0.01)。提示ST作用8天可以剂量依赖性地诱导体外培养人胃黏膜上皮细胞GES-1发生细胞凋亡。
1.3.2.2.2不同浓度ST作用8天对GES-1细胞凋亡相关蛋白表达的影响
1.3.2.2.2.1不同浓度ST作用8天对GES-1细胞Bcl-2表达的影响
Western Blot结果显示,ST作用8天后,0.3μM、1.5μM和3μM ST处理组细胞的Bcl-2蛋白表达水平较溶剂对照组明显降低(P<0.05),且其表达量随着ST浓度的增加而减少(r=-0.912, P<0.05)。提示ST处理8天可以剂量依赖性地下调GES-1细胞内Bcl-2蛋白的表达。
1.3.2.2.2.2不同浓度ST作用8天对GES-1细胞Bax表达的影响
Western Blot结果显示,ST作用8天后,0.3μM、1.5μM和3μM ST处理组细胞的Bax蛋白表达水平较溶剂对照组明显升高(P<0.05),且其表达量随着ST浓度的增加而升高(r=0.901, P<0.01)。提示ST处理8天可以剂量依赖性地上调GES-1细胞内Bax蛋白的表达。
1.3.2.2.2.3不同浓度ST作用8天对GES-1细胞NF-κB表达的影响
Western Blot结果显示,ST作用8天后,0.3μM、1.5μM和3μM ST处理组细胞的NF-κB蛋白表达水平较溶剂对照组明显下降(P<0.05),且其表达量随着ST浓度的增加而减少(r=-0.803,P<0.01)。提示ST处理8天可以剂量依赖性地下调GES-1细胞内NF-κB蛋白的表达。
1.3.2.2.2.4不同浓度ST作用8天对Caspase-3蛋白表达的影响
Western Blot结果显示,ST处理8天后,0.075~3μM各ST处理组Caspase-3活性片段的表达均较溶剂对照组明显增加(P<0.01),且随着ST浓度的增加,阳性酶切片段的表达量逐渐升高(r=0.850,P<0.01)。表明ST处理8天可以激活GES-1细胞Caspase-3,且其活性片段表达水平以剂量依赖方式增加。
第二部分杂色曲霉素对GES-1细胞DNA的损伤作用及损伤通路的激活目的:探讨杂色曲霉素对人胃黏膜上皮细胞(GES-1) DNA的损伤作用及其下游损伤通路的激活情况,揭示ST诱导GES-1细胞G_2期阻滞的可能原因。
方法:采用单细胞凝胶电泳试验观察ST作用不同时间(2、4、8及12d)对GES-1细胞DNA的损伤情况;采用FCM和Western Blot方法检测ATM/ATR特异性阻断剂—caffeine预处理对ST诱导的G_2期阻滞及相关调控因子的影响。
结果:
2.1 ST对GES-1细胞DNA损伤的影响
单细胞凝胶电泳试验结果显示,3μM ST作用于GES-1细胞可引起DNA的损伤。各不同时间ST处理组与相应溶剂对照组相比,DAN损伤指数(DI)显著增高(P<0.001),且随着ST作用时间的延长DI逐渐升高,两者呈正相关关系(r=0.903, p<0.001)。
不同时间ST处理的GES-1细胞在电泳之后经软件分析出的彗星尾部DNA含量、尾长及尾距三个指标与相应的溶剂对照组相比均有显著地增加,并且存在明显的时效关系(P<0.01)。
以上结果提示,ST可以引起GES-1细胞DNA损伤,且随ST作用时间的延长DNA损伤程度相应加重,它可能参与了ST诱导的细胞G_2期阻滞。
2.2 Caffeine预处理对ST诱导的GES-1细胞G_2期阻滞及细胞周期相关蛋白的影响
2.2.1 Caffeine预处理对ST诱导的GES-1细胞G_2期阻滞的影响
FCM检测结果显示,caffeine预处理+ST 3μM处理组与ST单独处理组相比,G_2/M期细胞比例明显减少(P<0.05)。表明caffeine预处理可以阻断ST诱导的细胞G_2/M期比例增高,提示ATM/ATR信号通路的激活可能参与了ST诱导的GES-1细胞G_2期阻滞。
2.2.2 Caffeine预处理对ST作用后GES-1细胞G_2期关键调节因子表达变化的影响
2.2.2.1 Caffeine预处理对Cdc25C表达的影响
Western Blot结果显示,5 mM caffeine预处理+ST(1.5μM, 3μM)处理组Cdc25C的表达水平与ST(1.5μM, 3μM)单独处理组相比明显提高(P<0.05);而Cdc25C的磷酸化水平在caffeine预处理后较ST单独处理组有所降低(P<0.05)。提示ATM/ATR信号通路可能参与了ST诱导的Cdc25C表达降低和p-Cdc25C表达的升高。
2.2.2.2 Caffeine预处理对Cdc2表达的影响
Western Blot结果显示,5 mM caffeine预处理+ST(1.5μM , 3μM)处理组Cdc2的表达水平与ST(1.5μM, 3μM)单独处理组相比明显升高(P<0.05);而Cdc2的磷酸化水平在caffeine预处理后则有所降低(P<0.05)。提示ATM/ATR信号通路可能参与了ST诱导的Cdc2表达的降低和p-Cdc2表达的升高。
2.2.2.3 Caffeine预处理对CyclinB1表达的影响Western Blot结果显示,5 mM caffeine预处理+ST(1.5μM , 3μM)处理组CyclinB1的表达水平与ST单独处理组相比无明显变化(P>0.05)。
2.3 Caffeine预处理对p-p53表达的影响
Western Blot结果显示,与溶剂对照组相比,5mM caffeine预处理组p-p53的表达水平显著降低;1.5μM ST和3μM ST单独处理组p-p53的表达水平均明显升高(P>0.05);5 mM caffeine预处理+ST(1.5μM , 3μM)处理组p-p53的表达水平与ST(1.5μM , 3μM)单独处理组相比明显降低(P>0.05)。提示,ST作用2天激活了p53;p53是ATM/ATR信号通路中的的一员;ATM/ATR信号通路可能参与了ST诱导的p-p53表达的升高。
第三部分杂色曲霉素诱导GES-1细胞周期G_2期阻滞及凋亡的可能分子机制
目的:探讨杂色曲霉素诱导体外培养的人胃黏膜上皮细胞(GES-1)G_2期阻滞及凋亡的可能分子机制。
方法:采用Western Blot和real-time PCR技术观察ST对p53-p21WAF1/CIP1信号通路相关分子表达的影响。进一步通过p53 siRNA转染干扰p53基因表达,Western Blot方法检测p53-p21WAF1/CIP1信号通路相关分子及细胞G_2/M期调控因子的表达变化情况,FCM方法检测细胞周期的变化情况。
结果:
3.1 ST对GES-1细胞p53-p21WAF1/CIP1信号通路的影响
3.1.1 ST作用不同时间对p53-p21WAF1/CIP1信号通路的的影响
3.1.1.1 ST作用不同时间对p53蛋白的影响
Western Blot结果显示,3μM ST处理GES-1细胞2、4、8及12天后,各ST处理组p-p53(Ser15)和p53蛋白水平均较对照组明显升高(P<0.05);在ST处理8天时p-p53(Ser15)的表达量达最大值,12天又明显下降(P<0.05)。提示ST处理GES-1细胞可以时间依赖性地激活p53,并且p-p53在第8天出现峰值后12天又下降。
Real time-PCR检测结果显示, ST 2、4、8及12d处理组的p53 mRNA水平均较对照组明显升高(P<0.01),且ST作用8天时达峰值,12天时又下降(P<0.05)。提示,ST作用于GES-1细胞可以上调p53 mRNA的表达,且第8天出现峰值,之后下降。与p53在蛋白水平上的激活时间保持一致。以上p53的激活在时间上与G_2期阻滞和凋亡基本一致。
3.1.1.2 ST作用不同时间对p21~(WAF1/CIP1)蛋白的影响
Western Blot结果显示,3μM ST处理GES-1细胞2、4、8及12天后,各ST处理组p~(21WAF1/CIP1)蛋白水平均较对照组明显升高(P<0.05);在ST处理后8天时其表达量达最大值,12天又有所下降(P<0.05)。提示ST作用于GES-1细胞可以时间依赖性地激活p~(21WAF1/CIP1),并且其在第8天出现峰值后12天又下降。p~(21WAF1/CIP1)的激活在时间上在时间上与G_2期阻滞和凋亡基本一致。
3.1.2不同浓度ST对p53-p~(21WAF1/CIP1)信号通路的的影响
3.1.2.1不同浓度ST作用4天对p53-p~(21WAF1/CIP1)信号通路的的影响
3.1.2.1.1不同浓度ST作用4天对p53蛋白的影响
Western Blot结果显示,0.075μM、0.3μM、1.5μM及3μM ST处理GES-1细胞4 d后,各ST处理组p-p53(Ser15)蛋白水平均较溶剂对照组明显升高(P<0.05);除0.075μM ST处理组外,各ST处理组p53蛋白水平较溶剂对照组明显升高(P<0.05)。并且p53的去磷酸化和磷酸化水平均随ST浓度的增加而升高(r=0.893; r=0.866, P<0.01)。提示ST作用于GES-1细胞4天可以剂量依赖性地激活p53。
Real time-PCR检测结果显示,除0.075μM ST处理组外,其它各ST处理组的p53 mRNA水平均较溶剂对照组明显升高(P<0.01)。提示,不同浓度ST作用于GES-1细胞4天可以上调p53 mRNA的表达。
3.1.2.1.2不同浓度ST作用4天对p~(21WAF1/CIP1)蛋白的影响
Western Blot结果显示,0.075μM、0.3μM、1.5μM及3μM ST处理GES-1细胞4 d后,各ST处理组p21蛋白表达水平均较溶剂对照组明显升高(P<0.05),且随ST浓度的增加而升高(r=0.910, P<0.01)。提示ST作用于GES-1细胞4天可以剂量依赖性地激活p~(21WAF1/CIP1)。
3.1.2.2不同浓度ST作用8天对p53-p~(21WAF1/CIP1)信号通路的影响
3.1.2.2.1不同浓度ST作用8天对p53蛋白的影响
Western Blot结果显示,0.075μM、0.3μM、1.5μM及3μM ST处理GES-1细胞8 d后,各ST处理组p-p53(Ser15)蛋白水平均较溶剂对照组明显升高(P<0.05);除0.075μM ST处理组外,各ST处理组p53蛋白水平较溶剂对照组明显升高(P<0.05)。并且p53的去磷酸化和磷酸化水平均随ST浓度的增加而升高(r=0.836; r=0.879, P<0.01)。提示ST作用于GES-1细胞8天可以剂量依赖性地激活p53。
3.1.2.2.2不同浓度ST作用8天对p~(21WAF1/CIP1)蛋白的影响
Western Blot结果显示,0.075μM、0.3μM、1.5μM及3μM ST处理GES-1细胞8 d后,各ST处理组p21蛋白表达水平均较溶剂对照组明显升高(P<0.05),且随ST浓度的增加而升高(r=0.926, P<0.01)。提示ST作用于GES-1细胞8天可以剂量依赖性地激活p~(21WAF1/CIP1)。
3.2 p53 siRNA转染对ST作用后GES-1细胞的影响
3.2.1 p53 siRNA转染对ST作用后GES-1细胞p53-p~(21WAF1/CIP1)信号通路的影响
3.2.1.1 p53 siRNA转染对ST作用后GES-1细胞p53蛋白的影响
Real time PCR结果显示,p53 siRNA单独转染组细胞在p53的mRNA水平较溶剂对照组显著降低(P<0.01,论文中未列出);Western Blot结果显示,p53 siRNA单独转染组细胞p53的磷酸化和非磷酸化蛋白表达水平均较溶剂对照组显著降低(P<0.01)。并且p53在mRNA和蛋白水平的有效抑制率均达到70%~80%,证明p53 siRNA的转染成功地干扰了p53的表达。
Western Blot结果显示,p53 siRNA+ST (3μM)处理组p-p53(Ser15)及p53的蛋白表达水平与ST (3μM)处理组相比均明显降低(P<0.05);而与p53 siRNA处理组相比均显著升高(P<0.05)。提示,p53 siRNA的转染可以阻断ST诱导的GES-1细胞p53的激活。
3.2.1.2 p53 siRNA转染对ST作用后GES-1细胞p~(21WAF1/CIP1)蛋白的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组p~(21WAF1/CIP1)的蛋白表达水平与ST (3μM)处理组相比明显降低(P<0.05);而与p53 siRNA处理组相比均显著升高(P<0.05)。提示,p53 siRNA的转染可以阻断ST诱导的GES-1细胞p~(21WAF1/CIP1)的激活。
3.2.2 p53 siRNA转染对ST作用后GES-1细胞G_2期关键调节因子表达变化的影响
3.2.2.1 p53 siRNA转染对ST作用后GES-1细胞Cdc25C的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组与ST (3μM)处理组相比,Cdc25C的蛋白表达水平明显升高,而p-Cdc25C的水平则显著下降(P<0.05)。而p53 siRNA+ST (3μM)处理组p-Cdc25C的蛋白表达水平与p53 siRNA处理组相比有显著升高(P<0.05)。提示,p53参与了ST诱导的GES-1细胞Cdc25C表达的下调和p-Cdc25C表达的上调。
3.2.2.2 p53 siRNA转染对ST作用后GES-1细胞Cdc2的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组与ST (3μM)处理组相比,Cdc2表达增多,而p-Cdc2表达则显著减少(P<0.05);p53 siRNA+ST (3μM)处理组与p53 siRNA处理组相比,Cdc2表达水平有所降低,而p-Cdc2的表达水平则有显著升高(P<0.05)。提示,p53参与了ST诱导的GES-1细胞Cdc2表达的下调和p-Cdc2表达的上调。
3.2.2.3 p53 siRNA转染对ST作用后GES-1细胞CyclinB1的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组CyclinB1的蛋白表达水平与ST (3μM)处理组相比有显著下降,而与p53 siRNA处理组相比则有显著升高(P<0.05)。提示,p53参与了ST诱导的GES-1细胞CyclinB1蛋白表达的上调。
3.2.3 p53 siRNA转染对ST作用后GES-1细胞凋亡相关蛋白表达变化的影响
3.2.3.1 p53 siRNA转染对ST作用后GES-1细胞Bcl-2的影响
Western Blot结果显示,ST (3μM)处理组与溶剂对照组相比,Bcl-2的蛋白表达水平显著下降(P<0.05);而p53 siRNA+ST (3μM)处理组与ST (3μM)处理组相比Bcl-2水平无明显变化(P>0.05)。提示,p53 siRNA转染不足以逆转ST对GES-1细胞Bcl-2表达的下调作用。
3.2.3.2 p53 siRNA转染对ST作用后GES-1细胞Bax的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组Bax的蛋白表达水平与ST (3μM)处理组相比有显著下降,而与p53 siRNA处理组相比则有明显升高(P<0.05)。提示,p53参与了ST诱导的GES-1细胞Bax蛋白表达的上调。
3.2.3.3 p53 siRNA转染对ST作用后GES-1细胞Caspase-3的影响
Western Blot结果显示,p53 siRNA+ST (3μM)处理组在17kD处的Caspase-3活性酶切片段与ST (3μM)处理组相比明显减少,而与p53 siRNA处理组相比则有明显增加(P<0.05)。提示,p53参与了ST诱导的GES-1细胞caspase-3的激活。
3.2.4 p53 siRNA转染对ST诱导的GES-1细胞G_2期阻滞的影响
FCM检测结果显示,与ST (3μM)处理组相比,p53 siRNA+ST (3μM)处理组G_2/M期细胞比例明显降低(P<0.05)。表明p53 siRNA转染可以阻断ST诱导的细胞G_2/M期比例增高,提示p53以及其下游信号转导通路的激活可能参与了ST诱导的细胞周期G_2期阻滞。
结论:
1 ST可以抑制GES-1细胞增殖及诱导其发生G_2期阻滞和凋亡,而ST可能正是通过诱导G_2期阻滞和凋亡来抑制GES-1细胞增殖的。
2 ST通过抑制Bcl-2和促进Bax蛋白表达来激活线粒体凋亡途径,随后激活caspase-3而最终诱导GES-1细胞发生凋亡。
3 ST可以引起GES-1细胞DNA的损伤并激活损伤下游ATM/ATR信号通路。ATM/ATR特异性阻断剂可以阻断ST诱导的G_2期阻滞。由此可见,ATM/ATR信号通路的激活可能参与了ST诱导的GES-1细胞G_2期阻滞,且在这一过程中ST引起的DNA损伤是一个起始事件。
4 ST可以激活p53-p~(21WAF1/CIP1)信号转导通路,并且通过siRNA干扰p53基因的表达证实了p53-p~(21WAF1/CIP1)信号通路的激活参与了ST诱导的GES-1细胞G_2期阻滞和凋亡。
5从ST的时间效应来看,ST引起的GES-1细胞凋亡的发生晚于其诱导的G_2期阻滞,它们的效应高峰分别出现在ST作用后8天和4天。并且当ST持续作用12天时,G_2期阻滞和凋亡均缓解。
6 ST持续作用较长时间时(12天),有部分细胞能够脱离由p53介导的G_2期阻滞和凋亡而继续生长,但这些细胞仍携带着严重的DNA损伤信息,而这些细胞的继续复制、增殖则可能是ST致癌作用的分子机制之一。
Sterigmatocystin (ST) is the fungal secondary metabolite produced by many Aspergillus species such as A. versicolor, A. chevalieri, etc. ST is one of the most common contaminant in different foodstuffs and environment, and it was regularly detected in grains, corn, bread, cheese and animal feed. Structurally, ST is related to a?atoxins and is generally recognized as a potential carcinogen, mutagen and teratogen. ST was found to have different toxicological, mutagenic and carcinogenic effects in animals and has been recognized as a 2B carcinogen (possible human carcinogen) by International Agency for Research on Cancer. ST could induce hepatocellular carcinoma after oral administration or intraperitoneal injection and squamous cell carcinoma after repeated application to the skin in rats, and could induce lung adenocarcinoma after oral administration in NIH mice. In short-term tests, ST, although considered less potent, is known to cause DNA damage and form DNA adducts, lead to DNA breaks, chromosome aberration, and sister-chromatid exchange in animal experiments. Our previous studies 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. In China, ST has been one of the most commonly detected mycotoxins in the maize from a high incidence area of gastric carcinoma, such as Taihang Mountains in North China. Many epidemiological studies of tumor etiology showed that diet exposure of ST may have some relationship with high incidence of gastric cancer in these areas. As gastric epithelium would be directly exposured to oral intake of ST, evaluation of the cytotoxic effects of ST on human gastric epithelial cells can be of further usage for the elucidation of the possible carcinogenic mechanism of ST.
The imbalance between cell proliferation and death is regularly considered to be an important event in carcinogenesis. At key transitions during eukaryotic cell cycle progression, signaling pathways monitor the successful completion of upstream events prior to proceeding to the next phase and these regulatory pathways are commonly referred to as cell cycle checkpoints. In mammalian cells, proliferation is controlled by factors that regulate the transition at cell cycle checkpoints, which are responsible for the initiation and completion of DNA replication and control of cell division respectively. Cell cycle progression is regulated by the checkpoint controls that function to allow time for the DNA repair and result in activation of pathways leading to apoptosis if cellular damage cannot be properly repaired. Defects in cell cycle checkpoints can result in cell genomic instability, gene mutations, and aneuploidy, all of which can contribute to carcinogenesis. Many studies have shown apoptosis, proliferation inhibition and cell cycle arrest are among the early effects of carcinogenic toxins on animal cells. Xie, et al. found that ST could cause G_2/M arrest and overexpression of p53 protein in murine fibroblasts. Our primary study confirmed that ST treatment for 24 hours could induce the G_2 arrest by affecting the MAPK signaling pathway in GES-1 cells in vitro.
DNA damage checkpoint signal pathway is a highly conserved signal induction process. ATM/ATR is a early sensor of protein kinase to DNA damage signal and the increase of its activity constitutes the first step in the whole activation pathway. p53 is a substrate of ATM, which can either activate cell cycle checkpoints and induce cycle arrest to repair damaged DNA, or activate cells into the apoptosis pathway, according to severity of injury. To further evaluate the effect of ST treatment on cell cycle and apoptosis and to explore their corresponding mechanism, in this study, we used a human gastric epithelial cell line (GES-1) and carried out the following studies. First, we observed the effect of ST treatment on GES-1 cell cycle distribution, proliferation and apoptosis. And then, we investigated the effect of ST treatment on DNA damage of GES-1 cells and the changes in DNA damage downstream signaling pathway. Finally, we chose p53-p21WAF1/CIP1 signaling pathway, which were closely related with DNA damage and cell cycle arrest, and further explored the molecular mechanism of ST-induced G_2 arrest. The aim of this study is to lay an experimental foundation for revealing the possible relationship between diet exposure of ST and gastric mucosa damage as well as the possible effects on gastric carcinogenesis.. The following three parts are included in the study
PartⅠThe effect of ST on the distribution of cell cycle, proliferation and apoptosis in GES-1 cells in vitro
Objective: To further confirm the effect of ST on the regulation of the cell cycle distribution, proliferation and apoptosis of human gastric epithelium cells (GES-1) in vitro.
Methods: The effect of ST treatment at different concentrations (0.03~48μM) for 24, 48 and 72 h on GES-1 cells survival rates was tested by MTT. The distribution of cell cycle after ST treatment was analyzed by FCM. Western Blot and RT-PCR were used for the determination of expression of cell cycle key factors (Cdc25C, Cdc2 and CyclinB1) at protein and mRNA level respectively. The effect of ST on the Cdc2-CyclinB1 complex was evaluated with immunoprecipitation. The effect of ST on apoptosis was measured by flow cytometry after Annexin V-PI staining and PI staining and detected morphologically by Hoechst 33258 staining. Western Blot was used for the expression of apoptosis related factor Bcl-2, Bax, NF-κB, and Caspase-3.
Results:
1.1 Effects of ST on survival rates in GES-1 cells
MTT assay showed that ST had a potent inhibitory effect on the growth of GES-1 cells. When GES-1 cells were treated by ST for 24 hours, the cell survival rates in ST treated groups ranging from 3 to 48μM displayed a significant decrease (P<0.05) in a dose-dependent manner (r24h=-0.961, P<0.01) and treated for 48 and 72 hours, the cell survival rates in ST treated groups ranging from 1.5 to 48μM respectively displayed a significant decrease (P<0.05) in a dose-dependent manner (r48h=-0.955, r72h=-0.913, P<0.01). When treated at 24μM and 48μM, the cell survival rates in ST treated groups significantly decreased in a time-dependent manner (r24μM=-0.998, r48μM=-0.998, P<0.05).
1.2 Effects of ST on the distribution of cell cycle in GES-1 cells
1.2.1 Effects of ST on cell cycle and G_2 phase key regulatory factor in GES-1 cells at various time points
1.2.1.1 Effects of ST on cell cycle in GES-1 cells at various time points Flow Cytometric results showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the proportion of cells in G_2/M phase in all ST treatment groups was significantly increased as compared with that in control group (P<0.05). The peak value of the proportion of cells in G_2/M phase was reached appeared at the 4th day after exposure to 3μM ST. Subsequently, this G_2 delay was sustained till day 8 of ST treatment (P<0.05).
1.2.1.2 Effects of ST on G_2 phase key regulatory factor in GES-1 cells at various time points
Western Blot analysis showed that the expression of the key G_2 phase regulatory proteins Cdc25C was significantly decreased after 2d, 4d, 8d and 12d in 3μM ST treatment cells (P<0.05) in a time-dependent manner (r=-0.814, P<0.01). At the same time, the phosphorylation level of Cdc25C was increased in ST treatment groups (P<0.05) time-dependently(r=0.807, P<0.01). RT-PCR analysis showed that the expression of Cdc25C mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).
3μM ST treatment significantly decreased the key G_2 phase regulatory proteins Cdc2 expression from 2 to 4d after treatment, When the treatment continued for 8 and 12days, the expression of Cdc2 was gradually recovered to level of the control group In the time range from 2 to 8 days, the phosphorylation levels of Cdc2 were increased at all ST treatment groups in time-dependent manner (r= 0.603,P<0.01). Different from the expression at protein level, RT-PCR analysis showed that the expression of Cdc2 mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).
Western Blot analysis and RT-PCR analysis showed that the expression of Cyclin B1 at protein and mRNA level were all significantly increased in all ST treatment groups as compared with that in control group (P<0.05).
1.2.2 Effects of ST treatment with different concentrations on cell cycle and G_2 phase key regulatory factor in GES-1 cells
1.2.2.1 Effects of ST treatment with different concentrations for 4 days on cell cycle and G_2 phase key regulatory factor in GES-1 cells
1.2.2.1.1 Effects of ST treatment with different concentrations for 4 days on cell cycle in GES-1 cells Flow Cytometric results showed that when GES-1 cells were treated by ST for 4 days, the proportions of cells in G_2/M phase in 0.3μM, 1.5μM, 3μM ST treatment groups were all significantly increased as compared with that in solvent control group in a dose-dependent manner (r=0.927, P<0.01).
1.2.2.1.2 Effects of ST treatment with different concentrations for 4 days on G_2 phase key regulatory factor in GES-1 cells
Western Blot analysis results showed that 4 days after ST treatment at 0.3μM, 1.5μM, 3μM, the expressions of Cdc25C and Cdc2 were all significantly decreased as compared with that in solvent control group (P<0.05) and a dose effects could be seen (r=-0.800, r=-0.876, P<0.01). At the same time, the phosphorylation level of Cdc25C and Cdc2 was increased at all ST treatment groups dose-dependently (r=0.714, r=0.767, P<0.01). Similar to the expression at protein level, RT-PCR analysis showed that the expression of Cdc25C and Cdc2 mRNA in ST treatment groups was also significantly decreased as compared with that in control group (P<0.05).
Four days after ST treatment, the expressions of Cyclin B1 at protein and mRNA level were all significantly increased in 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=0.929, P<0.01).
1.2.2.1.3 Effects of ST treatment with different concentrations for 4 days on Cdc2-CyclinB1 complex in GES-1 cells Immunoprecipitation results suggested that when treated by ST for 4 days, ST could induce the decrease of Cdc2-CyclinB1 complex of GES-1 cells in 0.075μM, 0.3μM, 1.5μM and 3μM ST treatment groups.
1.2.2.2 Effects of ST treatment with different concentrations for 8 days on cell cycle and G_2 phase key regulatory factor in GES-1 cells
1.2.2.1.1 Effects of ST treatment with different concentrations for 8 days on cell cycle in GES-1 cells
Flow Cytometry showed that when GES-1 cells were treated by ST for 8 days, the proportion of cells in G_2/M phase in 0.3μM to 3μM ST treatment groups for 8 days was significantly increased (P<0.05) as compared with that in solvent control group in a dose-dependent manner (r=0.910, P<0.01).
1.2.2.2.2 Effects of ST treatment with different concentrations for 8 days on G_2 phase key regulatory factor in GES-1 cells
Western Blot analysis showed that when GES-1 cells were treated by ST for 8 days, the expression of Cdc25C was significantly decreased in 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner ranging from 0.3 to 3μM (r=-0.830, P<0.01). Conversely, the phosphorylation level of Cdc25C was increased in 1.5μM, 3μM ST treatment groups in dose-dependent manner (r=0.714, r=0.767, P<0.01). RT-PCR analysis showed that the expression of Cdc25C mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).
Western Blot analysis showed that when GES-1 cells were treated by ST for 8 days, the expression of Cdc2 was significantly decreased in only 3μM ST treatment groups as compared with that in solvent control group (P<0.05). Conversely, the phosphorylation level of Cdc2 was increased at all ST treatment groups in dose-dependent manner (r=0.781,P<0.01). RT-PCR analysis showed that the expression of Cdc2 mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).
Western Blot analysis showed that when GES-1 cells were treated by ST for 8 days, the expression of Cyclin B1 was significantly increased in 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.753, P<0.01). RT-PCR analysis showed that the expression of Cyclin B1 mRNA in ST treatment groups was significantly increased as compared with that in control group (P<0.05).
1.3 Effects of ST on apoptosis in GES-1 cells
1.3.1 Effects of ST on apoptosis in GES-1 cells at various time points
1.3.1.1 Effects of ST on apoptosis rate in GES-1 cells
The PI staining result showed that when the GES-1 cells were treated by 3μM ST for 4, 8 and 12d, the apoptosis rate in all ST treatment groups (4.07±0.71%,7.96±1.17% and 3.89±0.28%) was significantly increased compared with that in control group (2.19±0.08%, P<0.05). Exposure to 3μM ST, the peak value of apoptosis rate appeared at the 8th day and subsequently decreased after ST treatment for 12 days(P<0.05). It is worth noting that the peak value of apoptosis rate was later four days than that of G_2 arrest.
1.3.1.2 Apoptosis was detected in GES-1 cells with ST treatment at various time points by staining
Cells treated with 3μM ST were stained by Hoechst 33258 and observed through fluorescence microscope. The typical morphological changes, such as condensation of chromatin and nuclear fragmentations appeared in GES-1 cells treated with ST at different time points. Apoptotic index of 2d, 4d and 8d ST treatment groups was significantly increased as compared with that in control group (r=0.827, P<0.01) and the peak value appeared at the 8th day, subsequently decreased (P<0.05) .
1.3.1.3 Effects of ST on apoptosis related proteins in GES-1 cells at various time points
1.3.1.3.1 Effects of ST on expression of Bcl-2 in GES-1 cells at various time points
Western Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of Bcl-2 was significantly decreased in all ST treatment groups(P<0.05) as compared with that in control group in time-dependent manner (r=-0.557, P<0.05).
1.3.1.3.2 Effects of ST on expression of Bax in GES-1 cells at various time points
Western Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of Bax was significantly increased in all ST treatment groups as compared with that in control group(P<0.05). Protein expression peaked at 8th day and decreased at 12th day (P<0.05), which consistented with the change of cell cycle arrest.
1.3.1.3.3 Effects of ST on expression of NF-κB in GES-1 cells at various time points
Western Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of NF-κB was significantly decreased in all ST treatment groups as compared with that in control group(P<0.05) in time-dependent manner(r=-0.825,P<0.01).
1.3.1.3.4 Effects of ST on activation of caspase-3 in GES-1 cells at various time points
Western Blot analysis showed that ST could activate caspase-3 (P<0.05). When the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the peak value of the cleaved (activated) form of Caspase-3 could be seen in 8 day ST treated groups and decreased in 12 day ST treated groups (P<0.05), which consistented with the changes of apoptosis rate.
1.3.2 Effects of ST treatment with different concentrations on apoptosis in GES-1 cells
1.3.2.1 Effects of ST treatment with different concentrations for 4 days on apoptosis in GES-1 cells
1.3.2.1.1 Effects of ST treatment with different concentrations for 4 days on apoptosis rate in GES-1 cells
The PI staining result showed that after ST treatment for 4 days the apoptosis rate in 0.075μM, 0.3μM, 1.5μM and 3μM ST treatment groups was significantly increased as compared with that in solvent control group(P<0.05) in dose-dependent manner (r=0.854, P<0.01).
Annexin V-PI staining showed that the apoptosis percentage of cells including early and late apoptotic cells was significantly increased in different concentrations ST treatment groups as compared with that in solvent control group (P<0.05), which consistented with the result of PI staining.
Based on the above two methods shows that ST treatment for 4 days could induce apoptosis in GES-1 cells in dose-dependent manner.
1.3.2.1.2 Effects of ST treatment with different concentrations for 4 days on apoptosis related proteins in GES-1 cells
1.3.2.1.2.1 Effects of ST treatment with different concentrations for 4 days on Bcl-2 in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of Bcl-2 was significantly decreased in 0.075μM, 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.857, P<0.05).
1.3.2.1.2.2 Effects of ST treatment with different concentrations for 4 days on Bax in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of Bax was significantly increased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.907, P<0.01).
1.3.2.1.2.3 Effects of ST treatment with different concentrations for 4 days on NF-κB in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of NF-κB was significantly decreased in 0.3μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.828, P<0.01).
1.3.2.1.2.4 Effects of ST treatment with different concentrations for 4 days on Caspase-3 in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of the cleaved fragment of Caspase-3 was significantly decreased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=0.905, P<0.05).
1.3.2.2 Effects of ST treatment with different concentrations for 8 days on apoptosis in GES-1 cells
1.3.2.2.1 Effects of ST treatment with different concentrations for 8 days on apoptosis rate in GES-1 cells
The PI staining result showed that after ST treatment for 8 days the apoptosis rate in 0.3μM, 1.5μM and 3μM ST treatment groups was significantly increased as compared with that in solvent control group(P<0.05) in dose-dependent manner (r=0.933, P<0.01).
1.3.2.2.2 Effects of ST treatment with different concentrations for 8 days on apoptosis related proteins in GES-1 cells
1.3.2.1.2.1 Effects of ST treatment with different concentrations for 8 days on Bcl-2 in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of Bcl-2 was significantly decreased in 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.912, P<0.05).
1.3.2.2.2.2 Effects of ST treatment with different concentrations for 8 days on Bax in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of Bax was significantly increased in 0.3μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.901, P<0.01).
1.3.2.1.2.3 Effects of ST treatment with different concentrations for 8 days on NF-κB in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of NF-κB was significantly decreased in 0.3μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.803,P<0.01).
1.3.2.1.2.4 Effects of ST treatment with different concentrations for 8 days on Caspase-3 in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of the cleaved fragment of Caspase-3 was significantly decreased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=0.850, P<0.01).
PartⅡThe study on the effect of DNA damage and the activation of damage pathway in GES-1 cells induced by ST in vitro
Objective: To explore the effect of ST on DNA damage and the activation of damage pathway in human gastric epithelium cells (GES-1). And to reveal that the possible reason for ST-induced G_2 phase arrest in GES-1 cells.
Methods: The situation of DNA damage was measured by single cell gel electrophoresis assay after different time (2, 4, 8 and 12d) of ST treatment. Western Blot detected the expression of cell cycle key factors (Cdc25C, Cdc2 and CyclinB1). The distribution of cell cycle after ST treatment was detected by FCM.
Results:
2.1 Effects of ST on DNA damage in GES-1 cells
Single cell gel electrophoresis assay result showed that 3μM ST treatment could induce DNA damage. Damage Index of different time ST treatment groups were significantly higher than that of corresponding solvent control groups (P<0.001) in time-dependent manner (r=0.903, p<0.001).
The analysis of DNA content of the comet tail, tail length and tail moment by the software after ST treatment at different time in GES-1 cells after electrophoresis were significantly increased as compared with the corresponding solvent control groups(P<0.01) in a time-dependent manner.
The result suggested that ST could induce GES-1 cells DNA damage, which was possible reason on ST-induced G_2 phase arrest.
2.2 Effects of ST on G_2 arrest and cell cycle related proteins in GES-1 cells, with caffeine pretreatment
2.2.1 Effects of ST on cell cycle distribution in GES-1 cells, with caffeine pretreatment
Flow Cytometry showed that the proportion of cells in G_2/M phase in caffeine pretreatment group was reduced as compared with that in 3μM ST treatment group (P<0.05). And caffeine could inhibit ST-induced G_2 phase arrest in GES-1 cells. The results indicated that ST induced G_2 phase arrest in GES-1 cells involving the activation of ATM/ATR and its related signaling pathway.
2.2.2 Effects of ST on cell cycle related proteins in GES-1 cells, with caffeine pretreatment
2.2.2.1 Effects of ST on Cdc25C in GES-1 cells, with caffeine pretreatment
The results of Western Blot showed that the expression of Cdc25C in caffeine pretreatment group were significantly increased as compared with that in ST (1.5μM, 3μM) treatment groups (P<0.05) and the phosphorylation level of Cdc25C was opposite (P<0.05). The results suggested that ATM/ATR signaling pathway may be involved in the changes of Cdc25C and p-Cdc25C ST-induced.
2.2.2.2 Effects of ST on Cdc2 in GES-1 cells, with caffeine pretreatment
The results of Western Blot showed that the expression of Cdc2 in caffeine pretreatment group were significantly increased as compared with that in ST (1.5μM, 3μM) treatment groups (P<0.05) and the phosphorylation level of Cdc2 was opposite (P<0.05). The results suggested that ATM/ATR signaling pathway may be involved in the changes of Cdc2 and p-Cdc2 ST-induced.
2.2.2.3 Effects of ST on CyclinB1 in GES-1 cells, with caffeine pretreatment
The results of Western Blot showed that no significant difference was found between caffeine + ST treatment groups and ST treatment groups (P>0.05).
2.3 Effects of ST on p-p53 in GES-1 cells, with caffeine pretreatment
The results of Western Blot showed that the expression of p-p53 in caffeine pretreatment group was significantly decreased and in 1.5μM and 3μM ST treatment groups were significantly increased as compared with that in solvent control groups (P<0.05). The expression of p-p53 in caffeine pretreatment group were significantly decreased as compared with that in ST (1.5μM, 3μM) treatment groups (P<0.05). The results suggested that after ST treatment for 2 days, ST could activate p53; p53 is a downstream protein of ATM/ATR signaling pathway and ATM/ATR signaling pathway may be involved in the changes of p-p53 ST-induced.
PartⅢThe study on the molecular mechanisms of G_2 Arrest in GES-1 cells induced by ST in vitro
Objective: To explore the possible molecular mechanisms on ST induced G_2 phase arrest in GES-1cells in vitro.
Methods: The expression of p53-p~(21WAF1/CIP1) signaling pathway after ST treatment was analysized with Western Blot and real-time PCR. GES-1 cells were transfected with p53 siRNA, Western Blot detected the expression of signaling pathway related molecule and G_2/M phase key regulatory factors.
The cell cycle distribution of GES-1cells after p53 siRNA transfection was detected with FCM. Results:
3.1 Effects of ST on p53-p~(21WAF1/CIP1) signaling pathway in GES-1 cells
3.1.1 Effects of ST on p53-p~(21WAF1/CIP1) in GES-1 cells at various time points
3.1.1.1 Effects of ST on p53 in GES-1 cells at various time points
Western Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of p53 and p-p53 was significantly decreased in all ST treatment groups (P<0.05). The peak value of p-p53 appeared at 8th day, subsequently decreased at 12th day(P<0.05). The results suggested that ST treatment could activate p53 and the peak value of p-p53 appeared at 8th day.
Real time-PCR analysis showed that the expression of p53 mRNA was significantly increased after different time ST treatment(P<0.01), while the peak value appeared at 8th day which was consistent with expression of p53 at protein level.
Based on the above, the activation of p53 was basically consistent in time with G_2 arrest and apoptotsis.
3.1.1.2 Effects of ST on p~(21WAF1/CIP1) in GES-1 cells at various time points Western Blot analysis showed that when cells were treated by 3μM ST from 2 to 12d, the expression of p21 was significantly decreased in all ST treatment groups (P<0.05). The peak value of p21 appeared at 8th day, subsequently decreased at 12th day(P<0.05). The results suggested that ST treatment could activate p53 and the peak value of p-p53 appeared at 8th day. The activation of p21 was basically consistent in time with G_2 arrest and apoptotsis.
3.1.2 Effects of ST treatment with different concentrations on p53-p~(21WAF1/CIP1) signaling pathway in GES-1 cells
3.1.2.1 Effects of ST treatment with different concentrations for 4 days on p53-p~(21WAF1/CIP1) signaling pathway in GES-1 cells
3.1.2.1.1 Effects of ST treatment with different concentrations for 4 days on p53 in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of p-p53 was significantly increased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.893, P<0.01). The expression of p53 was significantly increased in 0.3μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.866, P<0.01).
Real time-PCR analysis showed that the expression of p53 mRNA was significantly increased after different concentrations ST treatment(P<0.01),
3.1.2.1.2 Effects of ST treatment with different concentrations for 4 days on p~(21WAF1/CIP1) in GES-1 cells
Western Blot analysis showed that after ST treatment for 4 days, the expression of p21 was significantly increased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.910, P<0.01).
3.1.2.2 Effects of ST treatment with different concentrations for 8 days on p53-p~(21WAF1/CIP1) signaling pathway in GES-1 cells
3.1.2.1.1 Effects of ST treatment with different concentrations for 8 days on p53 in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of p-p53 was significantly increased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.836, P<0.01). The expression of p53 was significantly increased in 0.3μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.879, P<0.01).
Real time-PCR analysis showed that the expression of p53 mRNA was significantly increased after different concentrations ST treatment(P<0.01),
3.1.2.1.2 Effects of ST treatment with different concentrations for 8 days on p~(21WAF1/CIP1) in GES-1 cells
Western Blot analysis showed that after ST treatment for 8 days, the expression of p21 was significantly increased in 0.075μM~3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.926, P<0.01).
3.2 Effects of ST on GES-1 cells with p53 siRNA transfection
3.2.1 Effects of ST on p53-p~(21WAF1/CIP1) signaling pathway in GES-1 cells with p53 siRNA transfection
3.2.1.1 Effects of ST on p53 in GES-1 cells with p53 siRNA transfection
Real time-PCR analysis showed that the expression of p53 mRNA was significantly decreased after p53 siRNA transfection for 2 days (P<0.01,data not given). Western Blot analysis showed that the expression of p53 and p-p53 was significantly increased in p53 siRNA transfection group as compared with that in solvent control group (P<0.05). The inhibition rate of p53 at mRNA and protein level reached 70%~80% which proved p53 siRNA transfection was successful in interfering with the expression of p53.
Western Blot analysis showed that after ST treatment for 2 days, the expression of p53 and p-p53 was significantly decreased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while increased in p53 siRNA treatment group (P<0.05). The results suggested that p53 siRNA transfection could inhibit ST-induced the activition of p53.
3.2.1.2 Effects of ST on p~(21WAF1/CIP1) in GES-1 cells with p53 siRNA transfection
Western Blot analysis showed that after ST treatment for 2 days, the expression of p53 and p-p53 was significantly decreased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while increased in p53 siRNA treatment group (P<0.05). The results suggested that p53 siRNA transfection could inhibit ST-induced the activition of p~(21WAF1/CIP1).
3.2.2 Effects of ST on the key regulatory factors of G_2 phase in GES-1 cells with p53 siRNA transfection
3.2.2.1 Effects of ST on Cdc25C in GES-1 cells with p53 siRNA transfection
Western Blot analysis showed that the expression of Cdc25C was significantly increased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while that of p-Cdc25C decreased in ST treatment group (P<0.05). The results suggested that p53 was involved in the ST-induced decreasement of Cdc25C and increasement of p-Cdc25C.
3.2.2.2 Effects of ST on Cdc2 in GES-1 cells with p53 siRNA transfection Western Blot analysis showed that the expression of Cdc2 was increased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while that of p-Cdc2 significantly decreased in ST treatment group (P<0.05). The results suggested that p53 was involved in ST-induced the decreasement of Cdc2 and increasement of p-Cdc2.
3.2.2.3 Effects of ST on CyclinB1 in GES-1 cells with p53 siRNA transfection
Western Blot analysis showed that the expression of CyclinB1 was significantly decreased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while significantly increased as compared with that in p53 siRNA treatment group (P<0.05). The results suggested that p53 was involved in the ST-induced increasement of CyclinB1.
3.2.3 Effects of ST on apoptosis related proteins in GES-1 cells with p53 siRNA transfection
3.2.3.1 Effects of ST on Bcl-2 in GES-1 cells with p53 siRNA transfection
Western Blot analysis showed that the expression of Bcl-2 was significantly decreased in ST treatment group as compared with that in solvent control group (P<0.05), but no significant difference was found between p53 siRNA + ST (3?
引文
1 Tian H, Liu X. Survey and analysis on sterigmatocystin contaminated in grains in China. Wei Sheng Yan Jiu, 2004, 33(5): 606-608
2 Fujii, K., Kurata, H., Shigeyoshi, O., Hatsuda, Y., Turmor induction by a single subcutaneous injection of sterigmatocystin in newborn mice. Cancer Res, 1976, 36: 1615-1618
3 Purchase, I. F. H., Van der Watt, J. J., Carcinogenicity of sterigmatocystin to rat skin. Toxicol. Appl. Pharmacol., 1973, 26: 274-281
4 Huang XH, Zhang XH, Li YH, et al. Carcinogenic effects of sterigmatocystin and deoxynivalenol in NIH mice. Zhonghua Zhong Liu Za Zhi, 2004, 26(12): 705-708
5 Dong, L.M., Brennan, P., Karami, S., et al. An analysis of growth, differentiation and apoptosis genes with risk of renal cancer. PLoS, 2009, One 4, e4895
6 Weinert, T., A DNA damage checkpoint meets the cell cycle engine. Science, 1997, 277: 1450-1451
7 Orren, D.K., Petersen, L.N., Bohr, V.A., Persistent DNA damage inhibits S-phase and G2 progression, and results in apoptosis. Mol. Biol. Cell, 1997, 8: 1129-1142
8 Cui J, Xing L, Li Z, Wu S, et al. 2010. Ochratoxin A induces G2 phase arrest in human gastric epithelium GES-1 cells in vitro. Toxicol Lett, 2010 193(2): 152-158
9 Abid-Essefi, S., Ouanes, Z., Hassen, W., et al. Cytotoxicity, inhibition of DNA and protein syntheses and oxidative damage in cultured cells exposed to zearalenone. Toxicol In Vitro, 2004, 18(4): 467-474.
10刘晋红,张祥宏,左连富等.单次灌胃杂色曲霉素对小鼠大脑细胞的影响.中国药理学与毒理学杂志, 2004, 18(1):57-61
11黄向华,张祥宏,左连富等.杂色曲霉素对小鼠肾脏细胞凋亡与增殖的影响.中国病理生理杂志, 2004, 20(7):1302-1303,1321
12黄向华,张祥宏,左连富等.杂色曲霉素对小鼠肝脏细胞凋亡与增殖影响的研究.中国实验动物学杂志, 2002, 12(2):109-112
13曹文军,孙旭明,张祥宏等.杂色曲霉素诱导体外培养人外周血淋巴细胞凋亡的研究.中国病理生理杂志, 1996, 15(1):33-35
14谢同欣,张祥宏,严霞等.杂色曲霉素对人胃黏膜的致癌作用.肿瘤防治研究, 1996, 23(6):341-343
15邢欣,邢凌霄,李月红等.杂色曲霉素诱导胃黏膜上皮细胞细胞周期G2期阻滞的研究.细胞生物学杂志, 2009, 31(1):1329-1331
16 Terao K. Mesotheliomas induced by sterigmatocystin in Wistar rats. Gann, 1978, 69(2): 237-247
17 Enomoto M, Hatanaka J, Igarashi S,et al. High incidence of angiosarcomas in brown-fat tissue and livers of mice fed sterigmatocystin. Food & Chemical Toxicology, 1982, 20(5): 547-556
18 Sreemannarayana O, Frohlich AA, Marquardt RR, et al.Effects of repeated intra-abdominal injections of sterigmatocystin on relative organ weights, concentration of serum and liver constituents, and histopathology of certain organs of the chick. Poultry Science, 1988, 67(3): 502-509
19 Zhang XH, Xie TX, Li SS, et al. Preventive detection of fungi and mycotoxins in corn from high risk area of esophageal cancer in Cixian County. Chin J Cancer Res, 1995, 7(3):172-176
20王凤荣,张祥宏,严霞等.赞皇县胃癌霉菌病因研究简介.中国肿瘤, 2002, 11(7): 389-290
21 Hengartner MO. The biochemistry of apoptosis. Nature, 2000, 407(6805): 770-776
22 Yang H, Chung DH, Kim YB, et al. Ribotoxic mycotoxin deoxynivalenol induces G2/M cell cycle arrest via p21Cip/WAF1 mRNA stabilization in human epithelial cells. Toxicology, 2008, 243(1-2): 145-154
23 Saxena N, Ansari KM, Kumar R, et al. Patulin causes DNA damage leading to cell cycle arrest and apoptosis through modulation of Bax, p(53) and p(21/WAF1) proteins in skin of mice. Toxicol Appl Pharmacol, 2009, 234(2): 192-201
24 Xie, T.X., Misumi, J., Aoki, K., et al. Absence of p53-mediated G1 arrest with induction of MDM2 in sterigmatocystin-treated cells. Int J Oncol, 2000, 17(4): 737-742
25 Smits VA, Medema RH. Checking out the G(2)/M transition. Biochim Biophys Acta, 2001, 1519(1-2): 1-12
26 Lindqvist A, van Zon W, Karlsson Rosenthal C, et al. Cyclin B1-Cdk1 activation continues after centrosome separation to control mitotic progression. PLoS Biol, 2007, 5(5): e123
27 Perdiguero E, Nebreda AR. Regulaion of Cdc25C activity during the meiotic G2/M transition. Cell Cycle, 2004, 3(6): 733-737
28 Fletcher L , Cheng Y, Muschel R J. Abolishment of the Tyr-15 inhibitory phosphorylation site on cdc2 reduces the Radiation-induced G2 delay, revealing a potential checkpoint in early. Cancer Res, 2002, 62(1): 241-250
29 Peter M E, Heufflder A E, Hengartner M O. Advances in apoptosis research. Proc Natl Acad Sci USA, 1997, 94(24):12736-12737
30 Thernberry N A, Laxebnid Y. Caspases: enemies within. Science, 1998, 281(5381):1312-1316
31 Reed JC. Mechanisms of apoptosis. Am J Pathol, 2000, 157(5): 1415-1430
32 Arajuskovic G, Orolicki SV, Cerovic S, et al. Bcl–2 and Bax protein interaction in B–lymphocyteu of peripheral blood in patients with chronic lymphocytic leukemia. Vojnosantit Pregl, 2005, 62(5): 357-363
33 Filippovich I V, Sorokina NI, Lisbona A, et al. Radianon–induced apoptosis in human myeloma cell line increases BCL–2/BAX dimer formation and does not result in BAX/BAX homodimerization. Int J Cancer, 2001, 92(5):651-660
34 Dutta J, Fan Y, Gupta N, Fan G, Gelinas C. Current insights into the regulation of programmed cell death by NF-kappa B. Oncogene, 2006, 25(51): 6800-6816
35 Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature, 1995, 376(6536): 167-170
36 Doi TS, Marino MW, Takahashi T, et al. Absence of tumor necrosis factor rescues RelA-deficient mice from embryonic lethality. Proc Natl Acad Sci USA, 1999, 96(6): 2994-2999
37 Chaisson ML, Brooling JT, Ladiges W, et al. Hepatocyte-specific inhibition of NF-kappa B leads to apoptosis after TNF treatment, but not after partial hepatectomy. J Clin Invest, 2002; 110(2): 193-202
38 Kucharczak J, Simmons MJ, Fan Y, Gelinas C. To be, or not to be: NF-kappa B is the answer-role of Rel/NF-kappaB in the regulation of apoptosis. Oncogene, 2003, 22(56): 8961-8982
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3 Curry PT, Reed RN, Martino RM and Kitchin RM. Induction of sister-chromatid exchanges in vivo in mice by the mycotoxins sterigmatocystin and griseofulvin. Mutat Res, 1984, 137(2-3): 111-115,
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12 Lazzaro F, Giannattasio M, Puddu F, et al. Checkpoint mechanisms at the intersection between DNA damage and repair. DNA Repair (Amst), 2009, 8(9): 1055-1067
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2 Fujii, K., Kurata, H., Shigeyoshi, O., Hatsuda, Y., Turmor induction by a single subcutaneous injection of sterigmatocystin in newborn mice. Cancer Res, 1976, 36: 1615-1618
3 Purchase, I. F. H., Van der Watt, J. J., Carcinogenicity of sterigmatocystin to rat skin. Toxicol. Appl. Pharmacol., 1973, 26: 274-281
4 Huang XH, Zhang XH, Li YH, et al. Carcinogenic effects of sterigmatocystin and deoxynivalenol in NIH mice. Zhonghua Zhong Liu Za Zhi, 2004, 26(12): 705-708
5 Dong, L.M., Brennan, P., Karami, S., et al. An analysis of growth, differentiation and apoptosis genes with risk of renal cancer. PLoS, 2009, One 4, e4895
6 Weinert, T., A DNA damage checkpoint meets the cell cycle engine. Science, 1997, 277: 1450-1451
7 Orren, D.K., Petersen, L.N., Bohr, V.A., Persistent DNA damage inhibits S-phase and G2 progression, and results in apoptosis. Mol. Biol. Cell, 1997, 8: 1129-1142
8 Cui J, Xing L, Li Z, Wu S, et al. 2010. Ochratoxin A induces G2 phase arrest in human gastric epithelium GES-1 cells in vitro. Toxicol Lett, 2010 193(2): 152-158
9 Abid-Essefi, S., Ouanes, Z., Hassen, W., et al. Cytotoxicity, inhibition of DNA and protein syntheses and oxidative damage in cultured cells exposed to zearalenone. Toxicol In Vitro, 2004, 18(4): 467-474.
10刘晋红,张祥宏,左连富等.单次灌胃杂色曲霉素对小鼠大脑细胞的影响.中国药理学与毒理学杂志, 2004, 18(1):57-61
11黄向华,张祥宏,左连富等.杂色曲霉素对小鼠肾脏细胞凋亡与增殖的影响.中国病理生理杂志, 2004, 20(7):1302-1303,1321
12黄向华,张祥宏,左连富等.杂色曲霉素对小鼠肝脏细胞凋亡与增殖影响的研究.中国实验动物学杂志, 2002, 12(2):109-112
13曹文军,孙旭明,张祥宏等.杂色曲霉素诱导体外培养人外周血淋巴细胞凋亡的研究.中国病理生理杂志, 1996, 15(1):33-35
14谢同欣,张祥宏,严霞等.杂色曲霉素对人胃黏膜的致癌作用.肿瘤防治研究, 1996, 23(6):341-343
15邢欣,邢凌霄,李月红等.杂色曲霉素诱导胃黏膜上皮细胞细胞周期G2期阻滞的研究.细胞生物学杂志, 2009, 31(1):1329-1331
16 Terao K. Mesotheliomas induced by sterigmatocystin in Wistar rats. Gann, 1978, 69(2): 237-247
17 Enomoto M, Hatanaka J, Igarashi S,et al. High incidence of angiosarcomas in brown-fat tissue and livers of mice fed sterigmatocystin. Food & Chemical Toxicology, 1982, 20(5): 547-556
18 Sreemannarayana O, Frohlich AA, Marquardt RR, et al.Effects of repeated intra-abdominal injections of sterigmatocystin on relative organ weights, concentration of serum and liver constituents, and histopathology of certain organs of the chick. Poultry Science, 1988, 67(3): 502-509
19 Zhang XH, Xie TX, Li SS, et al. Preventive detection of fungi and mycotoxins in corn from high risk area of esophageal cancer in Cixian County. Chin J Cancer Res, 1995, 7(3):172-176
20王凤荣,张祥宏,严霞等.赞皇县胃癌霉菌病因研究简介.中国肿瘤, 2002, 11(7): 389-290
21 Hengartner MO. The biochemistry of apoptosis. Nature, 2000, 407(6805): 770-776
22 Yang H, Chung DH, Kim YB, et al. Ribotoxic mycotoxin deoxynivalenol induces G2/M cell cycle arrest via p21Cip/WAF1 mRNA stabilization in human epithelial cells. Toxicology, 2008, 243(1-2): 145-154
23 Saxena N, Ansari KM, Kumar R, et al. Patulin causes DNA damage leading to cell cycle arrest and apoptosis through modulation of Bax, p(53) and p(21/WAF1) proteins in skin of mice. Toxicol Appl Pharmacol, 2009, 234(2): 192-201
24 Xie, T.X., Misumi, J., Aoki, K., et al. Absence of p53-mediated G1 arrest with induction of MDM2 in sterigmatocystin-treated cells. Int J Oncol, 2000, 17(4): 737-742
25 Smits VA, Medema RH. Checking out the G(2)/M transition. Biochim Biophys Acta, 2001, 1519(1-2): 1-12
26 Lindqvist A, van Zon W, Karlsson Rosenthal C, et al. Cyclin B1-Cdk1 activation continues after centrosome separation to control mitotic progression. PLoS Biol, 2007, 5(5): e123
27 Perdiguero E, Nebreda AR. Regulaion of Cdc25C activity during the meiotic G2/M transition. Cell Cycle, 2004, 3(6): 733-737
28 Fletcher L , Cheng Y, Muschel R J. Abolishment of the Tyr-15 inhibitory phosphorylation site on cdc2 reduces the Radiation-induced G2 delay, revealing a potential checkpoint in early. Cancer Res, 2002, 62(1): 241-250
29 Peter M E, Heufflder A E, Hengartner M O. Advances in apoptosis research. Proc Natl Acad Sci USA, 1997, 94(24):12736-12737
30 Thernberry N A, Laxebnid Y. Caspases: enemies within. Science, 1998, 281(5381):1312-1316
31 Reed JC. Mechanisms of apoptosis. Am J Pathol, 2000, 157(5): 1415-1430
32 Arajuskovic G, Orolicki SV, Cerovic S, et al. Bcl–2 and Bax protein interaction in B–lymphocyteu of peripheral blood in patients with chronic lymphocytic leukemia. Vojnosantit Pregl, 2005, 62(5): 357-363
33 Filippovich I V, Sorokina NI, Lisbona A, et al. Radianon–induced apoptosis in human myeloma cell line increases BCL–2/BAX dimer formation and does not result in BAX/BAX homodimerization. Int J Cancer, 2001, 92(5):651-660
34 Dutta J, Fan Y, Gupta N, Fan G, Gelinas C. Current insights into the regulation of programmed cell death by NF-kappa B. Oncogene, 2006, 25(51): 6800-6816
35 Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature, 1995, 376(6536): 167-170
36 Doi TS, Marino MW, Takahashi T, et al. Absence of tumor necrosis factor rescues RelA-deficient mice from embryonic lethality. Proc Natl Acad Sci USA, 1999, 96(6): 2994-2999
37 Chaisson ML, Brooling JT, Ladiges W, et al. Hepatocyte-specific inhibition of NF-kappa B leads to apoptosis after TNF treatment, but not after partial hepatectomy. J Clin Invest, 2002; 110(2): 193-202
38 Kucharczak J, Simmons MJ, Fan Y, Gelinas C. To be, or not to be: NF-kappa B is the answer-role of Rel/NF-kappaB in the regulation of apoptosis. Oncogene, 2003, 22(56): 8961-8982
1 Gopalakrishnan S, Liu X, Patel DJ. Solution structure of the covalent sterigmatocystin-DNA adduct. Biochemistry, 1992, 31(44):10790-10801
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