硒诱导白血病细胞自噬和凋亡的分子机制

【作者】 史可鉴；

【导师】 许彩民；

【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2014， 博士

【摘要】 研究目的实验室前期研究结果证实,超营养剂量的亚硒酸盐通过诱导白血病NB4细胞中保护性自噬向凋亡的转化促进细胞死亡,而具体机制不清,且亚硒酸盐在其他白血病细胞系中的作用机理仍不十分明确。本研究以亚硒酸钠为抗癌药物,研究其作用下白血病NB4细胞中保护性自噬下调的分子生物学机制,同时探索亚硒酸钠诱导其他白血病细胞(HL60细胞)的凋亡机理,在两株细胞的异种移植瘤裸鼠模型中验证硒的治疗效果及相关蛋白分子在瘤组织中的变化情况,为阐明蛋白分子位于自噬和凋亡信号网络的分子地位以及硒的临床应用提供实验依据。研究方法采用免疫印迹、间接免疫荧光标记LC3后进行点状聚集分析以及透射电镜观察自噬泡的方法检测细胞中自噬水平的变化。采用流式细胞术检测细胞的凋亡率和细胞周期进程变化。通过活细胞计数试剂盒-8检测细胞活力。免疫共沉淀、GST pull-down以及荧光共定位的方法证明蛋白质间相互作用。采用染色质免疫共沉淀的方法确定转录因子与靶基因启动子的相互结合。通过免疫印迹实验检测相关蛋白表达及磷酸化水平的变化。采用反转录PCR的方法检测相关蛋白转录水平的变化。通过特异性化学抑制剂的使用和转染双链小分子RNA及质粒的方法探究蛋白质的功能。构建异种移植瘤模型并通过TUNEL和苏木精-伊红染色的方法证明硒于体内的治疗效果。采用免疫组化及免疫印迹方法检测动物模型中相关蛋白的变化趋势。研究内容本研究以亚硒酸钠为抗癌药物,研究其作用下白血病细胞的死亡机理。探索无机硒处理NB4细胞后survivin-2B, IKK alpha, P73及UVRAG蛋白水平的变化趋势,确定这些蛋白分子本身对于细胞自噬和凋亡的调控方式以及这些蛋白之间的作用关系,通过探讨硒作用下NB4细胞中自噬和凋亡的转化关系,揭示survivin-2B及其相关通路在硒诱导NB4细胞死亡中所发挥的作用。最后,在实验室前期构建的NB4细胞的异种移植瘤裸鼠模型中验证硒在动物模型中的治疗效果及相关蛋白在细胞水平的变化趋势。与此同时,鉴于亚硒酸钠在其他白血病细胞中的作用机理仍不十分明确。我们采用另一种白血病细胞系(HL60细胞)为研究对象,发现硒还可以通过抑制白血病HL60细胞中微管的动态变化影响细胞周期进程,通过细胞周期激酶的活性变化调节凋亡相关分子,并最终诱导HL60细胞凋亡,最后,在新构建的HL60细胞的异种移植瘤裸鼠模型中,我们进一步验证硒的治疗效果、硒对微管的抑制作用及其对Mcl-1和cyclin Bl的调控方式。研究结果1、survivin-2B/IKK alpha/P73/UVRAG通路参与NB4细胞保护性自噬的调控。首先,我们验证NB4细胞中自噬水平发生下调的同时凋亡相关蛋白的活性明显上升,抑制自噬过程可以有效促进亚硒酸钠诱导的NB4细胞凋亡。与此同时,我们发现survivin-2B, IKK alpha,P73及UVRAG在硒处理的NB4细胞中均发生下调。进一步研究发现survivin-2B与IKK alpha之间存在相互作用并借此将IKK alpha稳定在细胞核内。细胞核内的IKK alpha可以进一步调控转录因子P73的稳定性并最终促进自噬起始蛋白UVRAG的表达。在NB4细胞的异种移植瘤模型实验中,我们证实硒具有较为明显的治疗效果,并且发现亚硒酸钠引起的survivin-2B, IKK alpha,P73及UVRAG变化与它们在体外实验的变化趋势基本一致。至此,我们的结果说明了survivin-2B在自噬网络的调节机理,同时也为亚硒酸钠的临床应用提供了理论基础。2、硒通过影响微管动态变化影响HL60细胞凋亡。我们首先在人白血病HL60细胞中确定了亚硒酸钠的生长抑制作用及凋亡诱导功能,明确20μM亚硒酸钠可有效诱导HL60细胞凋亡,且该凋亡过程与线粒体凋亡途径有关。进一步研究发现硒在细胞水平可有效抑制HL60细胞中微管的聚合并借此将HL60细胞捕获在细胞周期的G2/M期。在此过程中,G2/M期标志性蛋白梒---cyclin B1与激酶CDK1的相互作用增强,且转染cyclin B1特异性干扰序列以及抑制CDK1的激酶活性均可以明显抑制线粒体抗凋亡蛋白Mcl-1的下调。最后,我们在HL60细胞异种移植瘤裸鼠模型中,从裸鼠组织水平验证硒的治疗效果、硒对于微管的抑制功能及cyclin B1和Mcl-1在细胞水平的变化趋势。至此,我们分别从体内及体外证实亚硒酸钠的微管解聚作用是其诱导HL60细胞死亡的重要原因。研究结论1、survivin-2B通过核内积累IKK alpha进而调控P73稳定性及自噬起始蛋白UVRAG的表达,最终影响硒作用下NB4细胞中的自噬变化。2、亚硒酸钠可以调节HL60细胞中微管的动态变化,抑制HL60细胞的周期进程,通过上调周期蛋白cyclin B1进一步促进抗凋亡蛋白Mcl-1的减少,最终引发HL60细胞凋亡。更多还原

【Abstract】 ObjectivePrevious study had discovered super-nutritional selenite induced leukemia NB4cells death through switching protective autophagy to apoptosis and this mechanism was unclear, while in other leukemia cell lines the anti-tumor effect of selenite was still unknown. In this investigation, selenite was used as an anti-tumor agent and the mechanisms by which selenite inhibited autophagy in NB4cells were explored. Additionally, we also determined the mechanisms by which selenite induced apoptosis in other leukemia cell lines. Finally, we established two leukemia cells bearing nude mice models and confirmed selenite therapeutic effects and some proteins alterations in vitro. All of the results helped us explore the regulatory status of some molecules in autophagy network and also supported the theoretical and experimental basis of selenite clinical application.MethodsWestern blots, indirectly labeling LC3and calculating the ratio of dots positive cells and transmission electron microscopy were used to detect the alteration of autophagy. Flow cytometry was used to analyze the change of apoptosis ratio and cell cycle distribution. To measure the cell viability, cell counting kit-8was purchased. Protein interaction was determined with immunoprecipitation, GST pull-down assay and immunofluorescence. To investigation the interaction between transcription factor and DNA sequence, chromatin immunoprecipitation was performed. Revers transcription-polymerase chain reaction was used to test the change of mRNA level. Special inhibitors uses and transfection of siRNA and plasmids were used to investigate the protein function. Leukemia cells bearing nude mice model was established to detect selenite therapeutic effects through TUNEL technology and hematoxylin-eosin staining assay. Immunohistochemistry and western blots were carried out to detect alterations of some proteins in vivo.ContentsIn this study, we investigated the mechanisms of selenite-induced leukemia cells death. On the one hand, we detected the alterations of survivin-2B, IKK alpha, P73and UVRAG, the relationship between these proteins and the role of them in autophagy process in selenite-treated NB4cells. And then, we concluded their function in cell fate determination through exploring the relationship between autophagy and apoptosis. Finally, we confirmed selenite anti-tumor activity and alterations of these proteins in a nude mice model which had been demonstrated previously.Additionally, because effects of selenite on other leukemia cells were unknown, we investigated the apoptotic mechanisms in HL60cells. In this study, we also found selenite could affect the dynamics of microtubule and further inhibited HL60cycle distribution. Further experiments indicated the alteration of the CDK activity should be responsible for the change of apoptosis-related proteins. Finally, we also established an HL60cells bearing nude mice model to confirm selenite anti-tumor activity, its microtubule inhibitory effects and alterations of cyclin Bl and Mcl-1.ResultsSunivin2B/IKK alpha/P73/UVRAG axis involved in the regulation of protective autophagy in NB4cells.First of all, we confirmed autophagy was down-regulated in selenite-treated NB4cells and inhibition of autophagy could promote selenite-induced apoptosis. Further experiments found survivin-2B interacted with IKK aloha and further accumulated it in the nucleus. The nuclear IKK alpha stabilized P73which promoted the expression of UVRAG, known as the initiator of autophagy. Finally, selenite was proved to have anti-tumor activity and alter these proteins just as it was indicated in vitro. Collectively, our results discovered the regulatory status of survivin-2B in the network of autophagy and provided a experimental basis for the selenite clinical application.Selenite induced apoptosis in HL60cells through altering microtubule dynamics At first, we determined the growth inhibitory effects and apoptosis induction energy of selenite in HL60cells and made sure that20μM selenite induced apparent apoptosis in HL60cells which was related with mitochondrial apoptotic pathway. Then, we also discovered selenite prevented microtubule assembly and arrested HL60cells at G2/M phase. In this process, increases of cyclin B1interacted with CDK1. And either transfection of siRNA targeting cyclin B1or inactivation of CDK1could reverse the down-regulation of anti-apoptotic Mcl-1.Finally, in HL60cells bearing nude mice model, selenite anti-tumor activity, its microtubule inhibitory effects and the alterations of cyclin B1and Mcl-1were confirmed. Collectively, both of in vitro and in vivo experiments confirmed selenite depolymerized microtubule by which selenite induced HL60cells death.Conclusions1、Survivin-2B accumulated IKK alpha in the nucleus which further regulated the stability of P73and UVRAG expression by which selenite regulated autophagy in selenite-treated NB4cells.2、Selenite regulated the microtubule dynamics and inhibited cell cycle distribution which finally resulted in the cyclin B1-dependent degradation of Mcl-1and apoptosis in HL60cells.更多还原