【作者】 贾欣；

【导师】 张祥宏；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 赭曲霉毒素A（OchratoxinA,OTA）是由曲霉菌属和青霉菌属的某些菌株产生的一种真菌毒素，1993年国际癌症研究中心IARC将OTA列为“可能的人类致癌物”。目前研究已经发现OTA具有肾毒性、肝毒性、神经毒性、免疫毒性、致畸性、致突变性和致癌性等生物学效应。河北省赞皇县是我国胃癌高发区，胃癌年均死亡率超过为77.67/10万。2006年，我们对当地居民粮食中OTA的污染状况进行了现场调查，OTA检出率为45.16%，最高含量为14.25μg/kg，当地居民OTA的周暴露量为8.19μg/kg，明显超过世界卫生组织/粮农组织联合专家委员会（Joint FAO/WHO ExpertCommittee on Food Additives，JECFA）暂定的每周允许摄入量100ng/kg。有关OTA研究最为广泛的是在肾毒性或致癌性方面，本实验组前期研究发现，OTA可以诱导人胃粘膜上皮（GES-1）细胞发生氧化应激损伤以及细胞周期紊乱；OTA可以导致人外周血单个核细胞的氧化应激损伤。越来越多的体外和体内的研究证明氧化应激在OTA的毒性和致癌作用方面具有重要作用。大量研究认为氧化应激损伤是启动和促进肿瘤发生的重要因素。当各种外源或内源性因素引起的活性氧（ROS）超过体内的清除能力时，就会导致ROS水平升高，使细胞内的一些生物大分子（DNA、蛋白质、脂质等）受到损伤，即发生氧化应激。在体内或体外模型研究中对许多的分子机制进行了抗氧化作用研究通过消除OTA诱导的DNA损伤，脂质过氧化作用，以及细胞毒性进一步确认的OTA毒性和氧化损伤之间的联系。有关OTA的毒性和OTA诱导肾癌形成提出的多种机制：抑制蛋白质的合成，干涉代谢系统，增加膜脂质过氧化反应，抑制线粒体呼吸和DNA损伤。我们在前期的研究中已经发现急性染毒OTA后，人胃粘膜上皮细胞GES-1内ROS水平明显升高，引起细胞氧化DNA损伤。但是目前有关OTA是否可以诱导GES-1恶性转化及氧化应激是否参与其中并不清楚。近年来有研究显示氧化应激在Wnt信号通路的激活以及上皮细胞恶性转化中起了至关重要的作用。β-catenin一方面和E-cadherin结合形成复合体，与微丝、中间丝、肌动蛋白等细胞骨架相连，介导细胞间的黏附，参与调控细胞分化和组织发生；另一方面，β-catenin进入细胞核内扮演转录因子的角色，β-catenin磷酸化阻止其与α-catenin的结合，导致其堆积于细胞核内，此过程会降低细胞间黏附，同时β-catenin作为Wnt/β-catenin信号通路的关键分子，它的表达增加可以激活Wnt信号通路的下游分子。Wnt/β-catenin信号通路是诱导恶性转化过程中不可缺少的通路。有研究表明，饮水中砷、铬诱导大肠癌发生的潜在机制就是ROS介导的Wnt/β-catenin信号通路的激活；ROS导致基因的不稳定，进而导致了细胞恶性转化的发生。我们不禁思考一个问题：OTA诱导的人胃黏膜上皮细胞氧化应激是否参与介导了Wnt信号通路的激活以及细胞的恶性转化？鉴于此，本研究利用永生化人正常胃黏膜上皮细胞（GES-1）作为研究对象，研究和探讨OTA长期染毒对GES-1细胞迁移、侵袭和克隆形成能力等方面的影响，并通过接种裸鼠成瘤进一步证明是否发生恶性转化。利用Western blot印迹等技术探讨长期暴露OTA后对GES-1细胞上皮性蛋白表达的影响；接着以Wnt/β-catenin通路作为切入点，利用蛋白印迹等技术揭示Wnt通路在OTA诱导GES-1细胞恶性转化中的作用；然后基于抗氧化策略的实施，评价氧化应激对Wnt/β-catenin通路及细胞恶性转化的影响，从整体水平观察OTA对胃粘膜上皮细胞的损伤作用，为揭示OTA与胃癌发生的关系提供科学依据。第一部分赭曲霉毒素A长期染毒诱导人胃黏膜上皮细胞恶性转化目的：探讨赭曲霉毒素A诱导体外培养的GES-1细胞发生恶性转化的可能性，分析OTA诱导GES-1细胞恶性转化的相关机制。方法：1分组及处理：在预实验的基础上，以长期染毒方法探讨OTA处理诱导GES-1细胞恶性转化作用。实验分为OTA处理组和对照组，取对数生长期GES-1细胞，用10%DMEM调整细胞浓度为（1~2）×104个/L，接种于培养瓶，细胞培养24h后，给予2.5μmol/L OTA处理72h，一周一次，染毒直至40代。2恶性转化的评价：2.1采用形态学观察OTA处理后细胞变化，划痕实验于显微镜下观察细胞迁移情况，Transwell实验在高倍镜下计数PET膜下面侵袭的细胞数，软琼脂克隆观察细胞锚着独立性生长能力；2.2裸鼠成瘤实验:实验分为对照组(n=10)，2.5μmol/L OTA处理组(n=10)，阳性对照BGC-823组(n=3)，4-6周龄雄性BALB/C-nu裸鼠，分别将100μl1×108/ml的细胞悬液皮下注射于裸鼠右侧腋窝区，16周后处死小鼠。3成瘤标本检测：取出皮下肿瘤，HE染色结果证实为接种瘤，免疫组织化学染色结果显示为上皮来源。同时，提取裸鼠肿瘤组织总RNA和总蛋白，进行Western Blot和Real time PCR检测上皮标志物cytokeratin的表达情况。结果：1OTA长期染毒GES-1细胞形态学观察倒置显微镜下观察，对照组GES-1细胞呈单层生长，排列有序，形态为梭形，细胞核圆形其边界清晰，细胞浆结构清晰。2.5μmol/L OTA染毒40代后细胞形态发生明显变化，细胞镜下呈多角形，大小不一，细胞间排列紧密，可见多核巨细胞，并呈团块状生长，排列紊乱，很多细胞周边多呈毛刺状。2OTA对GES-1细胞迁移力的影响采用细胞划痕实验观察OTA对GES-1细胞迁移力的影响。实验结果表明，2.5μmol/L OTA染毒组在第10代与对照组相比无统计学差异，第20代时开始出现迁移速度增高，第30代细胞朝向划痕迁移的速度明显高于对照组细胞，至第40代细胞迁移速度明显增高，与对照组有统计学差异（P<0.01）。此外，采用Transwell方法观察了OTA对GES-1细胞侵袭能力的影响，于接种后72小时计数穿过膜的细胞数目并拍照记录。2.5μmol/L OTA染毒组在第40代时穿过膜的细胞数目427±12.02明显高于对照组103±7.96，差异显著（P<0.05）。3OTA对GES-1细胞独立锚着生长能力的影响采用软琼脂克隆试验检测细胞独立锚着生长能力，2.5μmol/L OTA染毒20代时细胞在软琼脂上可形成小的细胞集落，细胞以数十个左右聚集成团，但生长缓慢，不能进一步形成较大的克隆，达不到每个集落大于50个细胞的标准；至第30代时，2.5μmol/L OTA染毒组细胞能在软琼脂上形成克隆并可见光滑轮廓的集落形成，但接种裸鼠并没有成瘤；至第40代时，2.5μmol/L OTA染毒组细胞形成的克隆数目多并且大，接种裸鼠可见皮下肿瘤形成。4裸鼠成瘤实验分别收集2.5μmol/L OTA染毒40代细胞，对照组细胞，阳性对照组胃癌细胞株BGC-823细胞，将1×107个不同组别的细胞皮下注射于每只裸鼠右侧腋窝区，接种后3周时2.5μmol/L OTA染毒组10只中有6只接种部位出现可见的肿块，接种后16周时瘤块的直径达1.52-1.61cm左右，阳性对照组接种后1周全部出现肿瘤，接种后16周时瘤块的直径达1.94-2.35cm左右。与此同时，裸鼠体内并没有发现其他瘤块，对照组小鼠皮下及体内均没有发现肿瘤生成。5裸鼠成瘤组织的病理形态学特点观察接种瘤组织行福尔马林固定，石蜡包埋，4μm切片，切片进行HE染色和免疫组化检查，HE染色观察2.5μmol/L OTA处理组和阳性对照组细胞形成的皮下肿瘤组织无差异，均可见高分裂像，瘤细胞排列密集形状近于卵圆形或多边形。免疫组化显示，上皮来源标志蛋白cytokeratin呈阳性表达。提取接种瘤组织的蛋白和RNA进行Western Blot和Real time PCR检测上皮标志物cytokeratin，结果显示cytokeratin呈现阳性表达。综上结果说明2.5μmol/L OTA染毒20代时迁移侵袭能力增强，克隆形成，可能是细胞发生恶性转化的早期关键阶段；OTA染毒30代时可见光滑轮廓的集落形成，但接种裸鼠并没有成瘤；至第40代时，形成的克隆数目多并且大，接种裸鼠可见皮下肿瘤形成；证明2.5μmol/L OTA慢性染毒40代时诱导GES-1细胞发生了恶性转化，进一步证实了OTA对人类细胞的致癌性。第二部分Wnt/β-catenin信号通路在赭曲霉毒素A长期染毒诱导胃黏膜上皮细胞恶性转化中的作用目的：探讨Wnt/β-catenin信号通路在赭曲霉毒素A诱导GES-1细胞恶性转化过程中的作用。方法：1分组及处理：实验分为OTA处理组和对照组，取对数生长期GES-1细胞，用10%DMEM接种于培养瓶，细胞贴壁生长至40-50%时，给予2.5μmol/L OTA处理72h，一周一次，分别取染毒10代，20代，30代和40代细胞进行实验。2利用免疫共沉淀技术检测2.5μM OTA染毒组和对照组细胞E-cadherin/β-catenin复合物表达情况。3利用激光共聚焦技术观察β-catenin的核移位情况。4利用Western blot以及real-time PCR观察Wnt/β-catenin信号通路中相关分子（Dvl、GSK3β、Wnt2、β-catenin、Tcf4、Lef1）的蛋白及mRNA表达情况。5采用Western Blot和Real time PCR方法检测β-catenin siRNA干扰对Wnt/β-catenin信号通路中转录因子及细胞周期关键调控因子的影响。6给予Wnt信号通路的抑制剂DKK-1，选用OTA染毒40代细胞，给予50nM预处理1h，观察Wnt/β-catenin信号通路中分子的表达情况。结果：1OTA长期染毒诱导GES-1恶性转化过程中E-cadherin/β-catenin复合物形成情况β-catenin最初是作为与细胞膜上钙依赖性粘附分子E-cadherin相互作用的胞内分子得以分离和克隆的，β-catenin通过与E-cacdherin形成复合物的功能主要是，参与细胞的粘附、迁徙与转移。采用免疫共沉淀技术检测E-cadherin/β-catenin复合物形成，与对照组相比，2.5μM OTA染毒30代时复合物形成降低，染毒40代时复合物形成明显降低（P<0.05）。说明在恶性转化过程中细胞膜上的β-catenin表达水平降低。2OTA长期染毒诱导GES-1恶性转化过程中β-catenin的核移位情况β-catenin不仅是细胞粘附分子，还是Wnt信号通路中重要的传递子。β-catenin出现核转位是Wnt信号通路活化的重要标志事件。通过激光扫描共聚焦显微镜下发现于2.5μM OTA染毒20代时β-catenin出现核转位，染毒30代时核转位较对照组明显增加，染毒40代时与对照组比较，具有明显统计学差异（P<0.05）。3Wnt/β-catenin信号通路的激活在OTA长期染毒诱导GES-1恶性转化过程中作用在证明OTA处理能够导致β-catenin核转位的基础上，进一步采用Real-time PCR检测了OTA处理不同时间GES-1细胞Wnt/β-catenin信号通路的重要成员wnt2、β-catenin、GSK3β、Dvl、Tcf4和Lef1在mRNA水平上表达情况，结果显示，2.5μmol/L OTA染毒20代前，上述Wnt/β-catenin信号通路关键分子的表达与对照组没有明显差异，OTA染毒30代时Wnt/β-catenin信号通路中关键分子的表达较对照组出现差异，当OTA染毒40代时Wnt/β-catenin信号通路中分子Wnt2、β-catenin、Tcf4、Lef1mRNA的表达水平均明显高于对照组，而Dvl和GSK3β mRNA的表达水平明显则明显低于对照组（P<0.05）。Western Blot检测结果显示，2.5μM OTA染毒30代时Wnt/β-catenin信号通路中具有活性的磷酸化的p-Dvl和p-GSK3β蛋白表达水平增加，OTA染毒40代时Wnt信号通路关键分子（Wnt2、β-catenin、Tcf4、Lef1）蛋白表达较对照组具有显著差异（P<0.05），而失活的Dvl，GSK3β蛋白表达较对照组明显降低（P<0.05）。表明OTA诱导GES-1细胞恶性转化过程中Wnt/β-catenin信号通路被激活。进一步证实了Wnt/β-catenin信号通路在OTA诱导的细胞恶性转化中发挥重要作用。4siRNA干扰对GES-1细胞Wnt通路的影响β-catenin siRNA以及Control siRNA转染OTA处理40代细胞48小时后收集细胞。Real-time PCR和Western blot结果显示：β-catenin siRNA可以显著降低GES-1细胞β-catenin mRNA和蛋白的表达。上述结果表明，β-catenin siRNA序列可以明显干扰β-catenin在mRNA和蛋白水平的表达。因此，后续的实验中继续选择该siRNA序列进行研究。Western Blot检测结果显示，与Control siRNA+2.5μM OTA处理组相比，β-catenin siRNA+2.5μM OTA处理组转录因子Tcf4和Lef1的蛋白表达水平明显降低（P<0.05）。同时检测Wnt通路的靶基因CyclinD1、CDK4蛋白表达较对照组亦明显降低（P<0.05），提示β-catenin siRNA干扰可以阻断OTA诱导的GES-1细胞Wnt信号通路转录因子的表达进而逆转OTA诱导的GES-1细胞增殖能力增强。综上结果表明，长期OTA染毒引起GES-1细胞恶性转化中Wnt信号通路活化，促进TCF核转录，诱导细胞周期蛋白的表达，从而使细胞获得恶性增殖的能力。5DKK-1预处理在GES-1细胞Wnt/β-catenin通路激活中的作用以上研究结果表明，OTA染毒40代时Wnt/β-catenin信号通路激活，通过给予Wnt通路特异抑制剂DKK-1预处理从正反两方面验证Wnt信号通路的活化。Western Blot结果显示，2.5μM OTA染毒40代时，给予Wnt特异性抑制剂DKK-1预处理后Wnt/β-catenin信号通路中关键分子Wnt2、β-catenin、Tcf4、Lef1的蛋白表达水平较单独OTA处理组明显下降（P<0.05）。表明DKK-1可以阻断OTA诱导的Wnt通路激活。进一步阻断证明，Wnt/β-catenin信号通路的激活参与了OTA引起GES-1细胞发生恶性转化过程。第三部分氧化应激在OTA长期染毒诱导的胃黏膜上皮细胞恶性转化中的作用及其机制目的：揭示OTA诱导的氧化应激在GES-1细胞恶性转化中的作用及分子机制。方法：1分组及处理：取对数生长期GES-1细胞，分为对照组，OTA处理组和抗氧化组，用10%DMEM调整细胞浓度为（1~2）×104个/L，接种于培养瓶，细胞培养24h后，分别给予2.5μmol/L OTA处理72h，抗氧化剂N-乙酰半胱氨酸（NAC）4mM预处理1h+2.5μmol/L OTA处理72h，一周一次，染毒直至40代。2对照组，2.5μmol/L OTA处理组，NAC+2.5μmol/L OTA处理组细胞于染毒10代，20代，30代和40代时采用荧光探针DCFH-DA和DHE检测细胞内ROS含量的变化。2采用超氧化物歧化酶（SOD）和丙二醛（MDA）测试盒检测OTA染毒10代，20代，30代和40代时细胞SOD活性及MDA含量的变化。3观察2.5μmol/L OTA处理组，NAC+2.5μmol/L OTA处理组细胞在染毒30和40代时克隆形成情况，裸鼠成瘤情况。2采用Western Blot和Real-time PCR方法检测Wnt/β-catenin信号通路相关分子的表达情况。结果：1OTA长期染毒诱导GES-1恶性转化过程中ROS水平的变化OTA的毒性和致癌性已经证实与氧化应激损伤有关，流式细胞术检测结果显示，2.5μM OTA染毒20代前DCF、DHE平均荧光强度较对照组有变化但无统计学差异，OTA染毒30代DCF、DHE平均荧光强度明显高于对照组（P<0.05）；为了进一步明确OTA通过氧化应激诱导GES-1细胞恶性转化，实验采用抗氧化剂NAC预处理来减少ROS。OTA染毒40代，抗氧化剂NAC+2.5μmol/L OTA处理组与单独OTA处理组相比，DCF、DHE平均荧光强度明显减低（P<0.05）。结果显示，OTA诱导GES-1细胞ROS生成增多，NAC缓解了OTA促ROS升高作用。2OTA对GES-1细胞SOD活性和MDA含量的影响SOD活性检测结果表明，2.5μM OTA染毒30代时SOD活性为30.58±2.59U/mg protein，显著低于对照组119.11±7.55U/mg protein（P<0.05）； NAC+OTA处理组SOD活性较单独OTA处理组明显升高（66.68±5.61vs30.58±2.59U/mg protein，P<0.05）。与对照组比较，OTA处理组MDA含量显著升高（P<0.05）, NAC+OTA处理组较单独OTA处理组明显降低（P<0.05，Fig.2B）。说明OTA可以引起GES-1细胞明显的脂质过氧化损伤。3NAC预处理对OTA长期染毒GES-1细胞独立锚着生长能力的影响软琼脂克隆试验证明，OTA染毒30代时2.5μmol/L OTA组细胞能在软琼脂上形成克隆，NAC+2.5μmol/L OTA处理组与2.5μmol/L OTA处理组相比，形成的集落明显减少，OTA染毒40代时，与2.5μmol/L OTA处理组比较，NAC+2.5μmol/L OTA处理组形成的克隆数目及光滑轮廓的集落形成减小。4NAC预处理对OTA诱导恶性转化细胞荷瘤裸鼠模型的影响分别收集2.5μmol/L OTA染毒40代细胞，NAC+2.5μmol/L OTA染毒40代细胞，对照组细胞，将1×107个不同组别的细胞皮下注射于每只裸鼠右侧腋窝区，接种后3周时OTA处理组10只中有6只接种部位可见皮下瘤块，接种后5周时NAC+OTA处理组10只中有3只接种部位出现瘤块，接种后16周时OTA处理组瘤块的直径达1.52-1.61cm左右，NAC+OTA处理组瘤块的直径在0.58-0.67cm左右。5NAC预处理对OTA诱导的恶性转化细胞Wnt/β-catenin信号通路的影响大量研究显示致癌物的致癌机制之一就是氧化应激介导的Wnt信号通路的激活。Western Blot方法显示，2.5μmol/L OTA处理组相比， NAC+OTA染毒40代时Wnt/β-catenin信号通路相关分子的水平明显降低(P均<0.05)。Real-time PCR检测结果显示，与2.5μmol/L OTA染毒相比，NAC+染毒组均具有统计学意义(P均<0.05)。6DKK-1预处理在OTA引起氧化应激介导的GES-1细胞Wnt通路激活中的作用Western Blot结果显示，NAC+OTA染毒40代时，给予Wnt信号通路特异性抑制剂DKK-1预处理后Wnt/β-catenin信号通路中关键分子Wnt2、β-catenin、P-Gsk3β的蛋白表达水平较单独OTA处理组明显下降（P <0.05）。表明DKK-1可以阻断氧化应激介导的Wnt/β-catenin信号通路激活。综上结果表明，应激介导Wnt/β-catenin信号通路活化进而参与OTA慢性染毒诱导的GES-1细胞发生恶性转化。结论：1OTA慢性染毒能够诱导GES-1细胞发生恶性转化，细胞迁移、侵袭和克隆形成能力增强，并可致裸鼠成瘤。2OTA慢性染毒能够诱导GES-1细胞Wnt/β-catenin信号通路的激活。3Wnt/β-catenin信号通路活化促进核转录，诱导细胞周期蛋白的表达，从而使细胞获得恶性增殖的能力。4氧化应激参与了OTA慢性染毒诱导GES-1细胞发生恶性转化过程。5给予氧化应激拮抗剂NAC可以降低OTA诱导GES-1细胞发生恶性转化作用。6氧化应激通过介导Wnt/β-catenin信号通路的激活参与细胞恶性转化。更多还原

【Abstract】 Ochratoxin a (OTA), a mycotoxin mainly produced by ubiquitousAspergillus and Penicillium, OTA had been classified as “possiblycarcinogenic to humans” by the International Agency for Research on Cancersince1993. OTA exhibits a wide range of toxic activities includingnephrotoxicity, hepatotoxicity, neurotoxicity, immunotoxicity, teratogenecity,mutagenicity and carcinogenicity. Zanhuang County is one of the highincidence areas of gastric cancer in north China with an annual gastric cancermortality being77.67/100,000/year. Our previous study showed thatmycotoxin Ochratoxin a (OTA) in wheat samples reached to8.19μg/kg in thisarea, which was significantly higher than that of provisional tolerable weeklyintake allocated by the Joint FAO/WHO Expert Committee on Food Additives(JECFA). Our previous study showed that OTA could induce G2phase arrestand oxidative damage in immortalized human gastric epithelial cells (GES-1).The putative toxicological effects and relate mechanism of OTA on humanperipheral blood mononuclear cells (hPBMC). The in vitro and in vivoevidence compatible with a role for oxidative stress in OTA carcinogenicityhas been collected and described.It looks mostly accepted that oxidative damage is a critical event in theinitiation and development of carcinoma. A number of studies havedemonstrated that OTA could result in oxidative stress associated with theproduction of ROS in different cells through various direct and indirectmechanisms. A number of molecules with various antioxidant properties weretested, using in vivo or in vitro models. Protection against OTA-induced DNAdamage, lipid peroxidation, several mechanisms have been proposed for OTAtoxicity and OTA renal tumor formation: Inhibition of protein synthesis,interference with metabolic systems, Promotion of membrane lipid peroxidation, inhibition of mitochondrial respiration and DNA damage. Agrowing number of in vitroand in vivo studies has been collected and describeevidence compatible with a role for oxidative stress in OTA toxicity andcarcinogenicity.Thus based on our previous study, the current study firstevaluated the effects of OTA in ROS production and DNA damage in GES-1cells. However, it is not clear that OTA-induced malignant transformationmight contribute to oxidative stress.In recent years, studies have shown that oxidative stress in the activationand malignant transformation of epithelial cells in the Wnt signaling pathwayplays a crucial role. β-catenin and E-cadherin hand combine to formcomplexes with the actin filaments, intermediate filaments, actin cytoskeleton,such as connected-mediated cell-cell adhesion, involved in the regulation ofcell differentiation and tissue. the other hand, β-catenin into the nucleus playsa role in transcription factors, β-catenin phosphorylation prevents binding withα-catenin, leading to its accumulation in the nucleus, this process will reducethe adhesion between cells, At the same time as the Wnt/β-catenin signal keymolecular pathway, increasing the expression of its downstream moleculescan activate Wnt signaling pathway. Wnt/β-catenin signaling pathway isinduced malignant transformation indispensable pathway. Studies have shownthat the underlying mechanisms of arsenic in drinking water,chromium-induced colorectal cancer is the ROS-mediated activation of Wntsignaling pathway, ROS cause genetic instability, leading to the occurrence ofmalignant transformation of cells. As well as cytotoxicity were observed wecan not help but ponder the question: whether the OTA-induced oxidativestress in human gastric epithelial cells involved in mediating the activation ofthe Wnt signaling pathway and malignant transformation of cells?In view, this study immortalized normal human gastric epithelial cells(GES-1) as the object of study, observable OTA induced cell migration,invasion and colony formation capacity enhancement, which may lead totumor formation. By Western blot blotting technique to explore the impact onprotein expression in epithelial GES-1cells after long-term exposure to OTA; followed by Wnt/β-catenin pathway as a starting point, the use of technologiessuch as Western blot revealed Wnt pathway in GES-1cells induced by OTAmalignant transformation in the role; then, based on the implementation ofanti-oxidant strategies, evaluation of oxidative stress in the Wnt pathway andcell malignant transformation, the overall level of injury observed from OTAto gastric epithelial cells and to reveal OTA the relationship between gastriccancer and provide scientific basis.Part Ⅰ Chronic exposure OTA-induced GES-1cells malignanttransformation and tumorigenesisObjective: To explore effects and mechanisms of OTA-Induced GES-1cells malignant transformationMethods:1Group and Treatment: Based on preliminary experiments toinvestigate the long-term exposure OTA induced GES-1cells malignanttransformation. Cells were divided into OTA treatment group and controlgroup. logarithmic growth phase GES-1cells, with10%DMEM adjust thecell concentration (1~2)×104/L, seeded in culture flasks,24h after cell culture,2.5μmol/L OTA treatment72h, once a week, until40passages exposure.2Evaluate malignant transformation.2.1by cell morphology changes after OTAtreatment, cell migration scratch experimentally observed under themicroscope, Transwell experiments at high magnification count PET filmfollowing the invasion of cells, the cells were observed in soft agar anchorageindependent growth.2.2To evaluate the tumorigenieity of transformed GES-1cells in vivo. The experiment is divided into a control group (n=10),2.5μmol/L OTA treatment group (n=10), positive control BGC-823group(n=3). Cells collected in suspended saline at a concentration of1×108per mlof100μL were inoculated into the the right hind limbs of Four-tosix-week-old male Balb/c nu-nu mice mice (n=10).The BALB/C mices weresacrificed and the subcutaneous tumors were harvested at16weeksPost-injeetion.3tumor specimens: Remove the subcutaneous tumorspecimens. HE staining to identify the cellular origin of the neoplasm,immunohistochemical staining results indicated that epithelial origin. Meanwhile, the total RNA and protein were extracted from tumor tissue todetect cytokeratin expression in the tumor.Results:1Cells morphologic observationUnder an inverted microscope, the control group GES-1cells were grownas monolayers, orderly, and morphology of the spindle, the boundary clearround nuclei and clear cytoplasm structure.2.5μmol/L OTA infected cellmorphogenesis significant changes after40passages, the cells were polygonalmicroscope, sizes, between tightly packed cells, multinucleated giant cells,and lumps growing disorder, many cells surrounding mostly burr-like.2Effects of OTA exposure on cell migrationExperimental results show that,2.5μmol/L OTA exposure group was nosignificant difference, began the migration speed increased, the30passages ofcell migration speed toward scratches20passages in the10passagescompared with the control group was significantly higher than the controlgroup of cells to40passages was significantly higher migration rate, asignificant difference (P<0.01). In addition, Boyden chamber assay was usedto observe the effect of GES-1cells migration.72hours after inoculationcounting the number of cells through the membrane and photographed.2.5μmol/L OTA exposure group through the40passages in the number of cellmembranes were427±12.02vs103±7.96significantly higher, the differencewas significant (P <0.05).3Soft agar assay and isolation of OTA-transformed clonesFor evaluation of anchorage-independent growth, cells were assayed bysoft agar assay.2.5μmol/L OTA infected cells in soft agar can form a smallcolony for20passages, the cells around dozens gathered into groups, but slowgrowth, not further to form larger clones reach each colony than the standard50cells.30passages,2.5μmol/L OTA exposure groups were able to formcolonies in soft agar and smooth contours visible colony formation, however,no tumor formation in nude mice inoculated.40passages, the multiple numberof clones2.5μmol/L OTA exposure group cell formation and large, visible subcutaneous tumor formation in nude mice.4Preparation nude mice with translating tumorCells were collected2.5μmol/L OTA exposed to40passages, controlcells, the gastric cancer cell line BGC-823cells,1×107cells were injectedsubcutaneously in nude mice each right armpit area,2.5μmol/L OTA exposuregroup six of ten mice injected had tumors (size at16weeks,1.52-1.61).BGC-823group three of three mice injected had tumors (size at16weeks,1.94-2.35). Meanwhile, the nude mice and found no other tumor mass,subcutaneous and control mice were not found in vivo tumor formation.5Tumorigenic specimensTumors were4-μm-thick sections from formalin-fixed,paraffin-embedded tissue blocks were stained with H&E staining andimmunohistochemical examination, HE staining of subcutaneous tumors2.5μmol/L OTA treatment group and positive control cells formed nodifferences were seen high mitotic tumor cells arranged in dense shape nearlyoval or polygonal. Immunohistochemistry showed thart epithelial originfactors of cytokeratin protein was positive expression. Extraction of tissueprotein and RNA Western Blot and Real time PCR detection cytokeratinshowed high expression.Overall, results indicated2.5μmol/L OTA exposure during20passagesmigration and invasion capacity enhancement, cloning, may be an earlycritical stage of malignant transformation of cells. visible cloning formingsmooth contour OTA exposure during30passages, but nude mice inoculatedand no tumor.40passages, the number of colony-forming and multi-big,visible subcutaneous tumor formation in nude mice inoculated.2.5μmol/LOTA chronic exposure induced malignant transformation during40passagesGES-1cells occurred.PartⅡ The effect of Wnt/β-catenin signaling pathway by the ochratoxinA-induced malignant transformation in human gastric epithelium cellsObjective: The effect of mechanism of OTA-induced malignanttransformation Methods:1Group and Treatment: cells were divided into OTA treatmentgroup and control group, logarithmic growth phase GES-1cells were seededin10%DMEM culture flasks, adherent cells were grown to40-50%whengiven2.5μmol/L OTA treatment72h, once a week,10,20,30and40passageswere used.2To detect the expression of E-cadherin/β-catenin complex on2.5μM OTA exposure group and control group by co-immunoprecipitationtechnique3observed nuclear translocation of β-catenin by confocal laser.4Western blot and real-time PCR were observed Wnt/β-catenin signalingpathway related molecules (Dvl, GSK3β, Wnt2, β-catenin, Tcf4, Lef1) proteinand mRNA expression.5Western Blot and Real time PCR results showed thattranscription factor and key regulator of cell cycle on Wnt/β-catenin signalingpathway by β-catenin siRNA interference.6gives the Wnt signaling pathwayinhibitor DKK-1, selected OTA exposed to40passages cells, giving50nMpretreatment1h, observe the expression of Wnt/β-catenin signaling pathwaymolecules.Results:1Effect of OTA exposure on E-cadherin/β-catenin complex expressionβ-catenin was originally developed as a membrane molecule andintracellular calcium dependent adhesion molecule E-cadherin interactions canbe isolated and cloned, β-catenin through functional complexes formed withthe main E-cacdherin sticky involved in cell attachment, migration andmetastasis. Co-immunoprecipitation technique to detect E-cadherin/β-catenincomplex formation, compared with the control group,2.5μM OTA exposureduring30passages complex formation decreased, exposed to40passages wassignificantly lower (P<0.05).2Effect of OTA exposure on β-catenin nuclear translocationβ-catenin is not only cell adhesion molecule, or a Wnt signaling pathwayis important in the sub-transmission. β-catenin nuclear translocation occurs anevent is an important symbol of the Wnt signaling pathway. Laser scanningconfocal microscopy2.5μM OTA exposure20passages occur when β-cateninnuclear translocation,30passages when exposed to nuclear translocation was significantly increased compared with the control group,40passages wheninfected with the control group, with a significant statistical difference(P<0.05).3Wnt signaling pathway is activated and involved in OTA-inducedmalignant transformationOTA treatment can lead to prove the basis of β-catenin nucleartranslocation, and further using Real-time PCR to detect an important memberof the OTA for different time GES-1cells Wnt/β-catenin signaling pathwaywnt2, β-catenin, GSK3β, Dvl, Tcf4and Lef1expression at the mRNA level,the results show,2.5μM OTA exposure20generations, there is no significantdifference in these Wnt/β-catenin signaling pathway molecules expression andthe control group, OTA exposure30generations when the expression ofWnt/β-catenin signaling pathway key molecules there was a difference,Real-time PCR analysis showed that,2.5μmol/L OTA exposure40passageswhen Wnt/β-catenin signaling pathway molecules (Wnt2, β-catenin, Tcf4,Lef1) mRNA expression levels higher, Dvl and GSK3β mRNA the expressionlevel was significantly lower than the control group (P<0.05). Show thatWnt/β-catenin signaling pathway plays an important role in cell OTA-inducedmalignant transformation. Western Blot results showed that when exposed to2.5μM OTA30passages express Wnt/β-catenin signaling pathway molecules(p-Dvl, p-GSK3β, Wnt2, β-catenin, Tcf4, Lef1) protein increases,40passagesprotein expression when compared with the control group with significantdifference (P<0.05). The Dvl, GSK3β protein expression was significantlylower than the control group (P <0.05).Overall, our study showed that OTA GES-1cells induced malignanttransformation process Wnt/β-catenin signaling pathway is activated.Further confirmed the Wnt/β-catenin signaling pathway plays an importantrole in OTA induced malignant transformation.4Effects of siRNA interference GES-1cells on Wnt pathwayAt48h post transfection, the40passages cells were harvested andprocessed for analysis of Western Blot and Real-time PCR. The results showed that the siRNAs dramatically reduced β-catenin expressions at boththe mRNA and protein levels. These data indicated that the siRNA sequencescould knockdown the expression of β-catenin in GES-1cells. Therefore,β-catenin siRNA were selected to be used in the subsequent experiments.The Western Blot results showed that transcription factors in β-cateninsiRNA treatment group have no changed compared with the Control siRNAgroup (P<0.05). The expressions of transcription factors was significantlydecreased in β-catenin siRNA+2.5μM OTA treatment group compared withControl siRNA+2.5μM OTA treatment group (P<0.05), but still higher thanthat in Control siRNA treatment group (P<0.05). CyclinD1and CDK4weresignificantly decreased in β-catenin siRNA+2.5μM OTA treatment groupcompared with Control siRNA+2.5μM OTA treatment group (P<0.05). Itindicated that β-catenin siRNA prevented the OTA-induced up-regulation ofthe transcription factors proteins and reversed the OTA-induced up-regulationof the CyclinD1and CDK4proteins.All the above results showed that Wnt signaling pathways activated inOTA-induced GES-1cells malignant transformation, promoting TCF nucleartranscription and induced cell cycle proteins, enhanced cell proliferationability.5Effects of DKK-1pretreatment on Wnt signaling pathway in GES-1cellsThe above results show that, OTA exposure Wnt/β-catenin signalingpathway activated40generations, the pathway specific inhibitor DKK-1activation of the Wnt signaling pathway pretreatment verification from bothpositive and negative by giving specific inhibitors.Western Blot resultsshowed that,2.5μM OTA when exposed to40passages, giving Wnt inhibitorDKK-1Wnt2, β-catenin, Tcf4, Lef1protein levels than single OTA treatmentgroup was significantly decreased (P<0.05). It indicated that DKK-1can blockOTA-induced activation of Wnt signaling pathway in GES-1cells.Further block show that the activation of Wnt signaling pathwaysinvolved in GES-1cells malignant transformation. PartⅢ The effect of oxidative stress on the ochratoxin A-induced humangastric epithelium cells malignant transformationObjective: To explore the oxidative stress involved in OTA-inducedmalignant transformation.Methods:1Group and Treatment: logarithmic growth phase GES-1cellswere divided into control group, OTA-treated group and antioxidant group,with10%DMEM adjust the cell concentration (1~2)×104个/L, seeded inculture flasks, cells cultured for24h, were given2.5μmol/L OTA treatment72h, the antioxidant N-acetylcysteine (NAC)4mM pretreatment1h+2.5μmol/L OTA treatment72h, once a week, until exposure40th generations.2control group,2.5μmol/L OTA treatment group, NAC+2.5μmol/L OTAtreated cells.10,20,30and40passages cells using the fluorescent probeDCFH-DA and DHE detect the generation of ROS changes.2usingsuperoxide dismutase (SOD) and malondialdehyde (MDA) test kit to detectOTA exposure for10,20,30and40passages.3Observed2.5μmol/L OTAtreatment group, NAC+2.5μmol/L OTA exposed to30and40passages forclone formation, tumor formation in nude mice.2Western Blot and Real-timePCR were used to detect the expression of Wnt/β-catenin signaling pathwayrelated molecules.Results:1Effect of OTA exposure on intracellular ROS levelOTA toxicity and carcinogenicity has been confirmed with oxidativestress injury, To further clarify the OTA by oxidative stress-induced malignanttransformation of GES-1cells, NAC pretreatment experiments usingantioxidants to reduce ROS. Flow cytometry showed that,2.5μM OTAexposure during30passages DCF, DHE mean fluorescence intensity weresignificantly higher (P<0.05). NAC+2.5μmol/L OTA treatment groups DCF,DHE mean fluorescence intensity was significantly lower (P<0.05). Theresults showed that, OTA-induced GES-1cells generation ROS increased,NAC can block the OTA induced ROS increased.2Effect of OTA exposure on SOD activity and MDA contentSOD activity was significantly decreased in the OTA-exposed30 passages groups (P<0.05). OTA-induced the decrease of SOD was preventedby NAC pretreatment (66.68±5.61vs30.58±2.59U/mg protein, P<0.05).NAC inhibited OTA-induced MDA activity increase. OTA-induced GES-1cells oxidative stress.3NAC pretreatment on anchorage independent growthFor evaluation of anchorage-independent growth, cells were assayed bysoft agar assay.2.5μmol/L OTA treatment cells able to form colonies in softagar, NAC+2.5μmol/L OTA treatment group compared with2.5μmol/L OTAtreatment group, colony formation reduction, OTA treatment40passages,compared to2.5μmol/L OTA treatment group, the NAC+2.5μmol/L OTAtreatment group number of clones were significantly reduced.4NAC pretreatment on OTA induced malignant transformationControl cells,2.5μmol/L OTA treatment40passages, NAC+2.5μmol/LOTA treatment40passages, different groups cells were collected the1×107cells were injected subcutaneously in nude mice each right armpit area, OTAexposure group six of ten mice injected had tumors (size at16weeks,1.52to1.61). NAC+OTA exposure group three of ten mice injected had tumors (sizeat16weeks,0.58to0.67).5Wnt signaling pathway is activated and involved in OTA-inducedmalignant transformation by oxidative stressNumerous studies have shown the activation of carcinogens iscarcinogenic mechanism of oxidative stress-mediated Wnt signaling pathway.Western Blot results showed,30passages, NAC+OTA treatment groupcompared with OTA treatment group the Wnt/β-catenin signaling pathwayrelated molecules were significantly decrease (P<0.05). Real-time PCRanalysis showed that NAC+OTA treatment group compared with OTAtreatment group Wnt signaling pathway related molecules mRNA level weredecrease significantly (P <0.05).6Effects of DKK-1pretreatment on Wnt signaling pathway in GES-1cells40passages, Western Blot results showed that the levels of Wnt2,β-catenin, P-Gsk3β, Gsk3β in DKK-1pretreatment for1h before2.5μM OTA or pre-treated with4mM NAC+2.5μM OTA treatment group was decreasedsignificantly compared with OTA treatment group (P<0.05). DKK-1can blockoxidative stress-mediated Wnt/β-catenin signaling pathway activation.It indicated that DKK-1can block OTA-induced activation of Wntsignaling pathway in GES-1cells.Conclusions:1OTA could induce GES-1cells malignant transformation, promoted cellmigration and invasion, resulted in tumor formation in the xenografts ofnude mice.2The activation of Wnt signaling pathways was involved in theOTA-induced cell malignant transformation.3The activation of Wnt signaling pathways was induction of cell cycleprotein expression through the activation of TCF, and enhance ofproliferation of GES-1cells.4Oxidative stress involved in chronic OTA exposure induced GES-1cellsmalignant transformation process.5OTA-induced oxidative stress damage, Addition of Pre-treatment withNAC (a direct ROS scavenger) could protection against OTA induced cellmalignant transformation and tumor formation.6Wnt signaling pathway is activated and involved in OTA-inducedmalignant transformation by oxidative stress.更多还原