ApoG2增强放射线诱导鼻咽癌细胞自噬性死亡及其分子机制

发布时间：2018-02-12 16:49

  本文关键词： ApoG2 放射增敏 鼻咽癌 Bcl-2 自噬 裸鼠　出处：《暨南大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的：程序性细胞死亡包括凋亡和自噬性死亡,越来越多的研究表明自噬在肿瘤中起重要作用。Beclin 1与Ⅲ型PI3K结合启动自噬的发生,Bcl-2/Bcl-xL结合Beclin 1阻断其自噬诱导功能。Bcl-2/Bcl-xL等在鼻咽癌中高表达且与其预后呈负相关。鼻咽癌以放疗为主,放疗除了诱导凋亡还能诱导自噬,在抗肿瘤中起重要作用。Apogossypolone (ApoG2)是经过改造的棉酚衍生物,它是一种新型的抗凋亡Bcl-2家族蛋白的小分子抑制剂。我们研究发现ApoG2能与Bcl-2/Bcl-xl结合,在鼻咽癌细胞中阻断Bcl-2的功能,并在体内外增强化疗敏感性；还发现抗癌药物、射线等调控Beclin 1的表达诱导鼻咽癌细胞自噬性死亡。本课题拟进一步研究ApoG2抑制Bcl-2/Bcl-xL与Beclin 1的结合,阻断Beclin 1诱导自噬的功能,体内外增强放射线诱导鼻咽癌细胞自噬性死亡以及放疗敏感性,为Bcl-2小分子抑制剂类型的抗肿瘤药物联合放射治疗鼻咽癌提供实验依据,同时为将ApoG2研发成新型的抗肿瘤药物奠定基础。 方法：体外培养人鼻咽癌CNE1、CNE2和SUNE1细胞及鼻咽上皮细胞NP69,MTT法检测不同浓度Bcl-2抑制剂ApoG2对人鼻咽癌CNE1、CNE2和SUNE1细胞的抑制作用及对鼻咽上皮细胞NP69的细胞毒作用,计算各自的IC50值,选择低于IC50的药物浓度作为后续实验浓度。克隆形成实验检测不同剂量放射线加或不加ApoG2对人鼻咽癌CNE1、CNE2和SUNE1细胞生存曲线的影响,计算其存活率,绘制细胞存活曲线。免疫共沉淀法检测ApoG2对Beclin 1和Bcl-2结合的影响。慢病毒感染CNE2细胞,Knock down Atg5,建立稳定细胞株,观察自噬的缺失对ApoG2引起的鼻咽癌细胞放疗增敏的影响。GFP-LC3质粒转染肿瘤细胞,荧光显微镜观察自噬标志物LC3的聚集。透射电镜检测细胞自噬形态。免疫印迹法(Western blot)检测自噬相关蛋白LC3、P62的表达。建立裸鼠移植瘤模型,观察ApoG2口服、单独放疗及两者联合对鼻咽癌细胞移植肿瘤的生长抑制作用；免疫组化检测移植瘤组织中LC3表达的变化。 结果： 1. ApoG2可增强CNE1、CNE2、SUNE1细胞的放射敏感性 MTT结果显示Bcl-2抑制剂ApoG2对人鼻咽癌CNE1、CNE2、SUNE1细胞均具有增殖抑制作用,呈剂量依赖性,IC50值分别为2.84、2.18、5.64μM。对正常鼻咽上皮细胞无细胞毒作用。体外克隆形成实验分析显示,CNE2细胞中放疗+ApoG2不同浓度组的剂量效应因子(DEF)值均大于1,分别为1.92、1.52和1.13。ApoG2可以提高放射增敏作用,并且放射增敏效果呈药物剂量依赖性。 2. ApoG2可促使Bcl-2与Beclin 1解离 Beclin 1是一种BH3-only蛋白家族成员,起初被鉴定为Bcl-2相互作用蛋白,也是第一个被发现的哺乳动物自噬相关基因,在肿瘤的发生发展中起关键作用。免疫共沉淀检测结果显示ApoG2可与Beclin 1竞争性抑制Bcl-2,从而促使Bcl-2与Beclin 1解离。 3. ApoG2可诱导鼻咽癌细胞发生自噬 激光共聚焦显微镜观察可见ApoG2及放射处理后的转染GFP-LC3质粒的CNE2细胞内,LC3呈现明显的点状聚集,且ApoG2联合放射可以增加LC3的点状聚集,对照组胞浆内LC3呈均匀弥散分布；透射电镜观察2Gy放射联合20μMApoG2组照射48 h后可观察到自噬小体的形成。Western blot检测可见放疗组和ApoG2组均出现LC3的表达,而ApoG2联合放疗可以显著增加LC3的表达；并呈时间剂量依赖性。提示ApoG2联合放疗可进一步诱导鼻咽癌细胞发生自噬。流式细胞术检测细胞坏死,结果显示ApoG2联合放疗处理可以诱导CNE2细胞发生凋亡和坏死。 4. knock down Atg5可抑制CNE2细胞自噬的发生,并提高ApoG2+放疗组的克隆形成率 建立Atg5 knock down CNE2细胞株,Western blot检测到与VEC组(转染空载体)相比shAtg5组(转染质粒shAtg5)中Atg5的表达被抑制。Western blot检测ApoG2不能诱导shAtg5细胞发生自噬。克隆形成实验检测放疗、ApoG2处理及两者联合处理VEC组和shAtg5组细胞的细胞存活率,shAtg5组细胞存活率明显高于VEC组,细胞死亡率平均从67%升至82%。 5. ApoG2可抑制裸鼠移植瘤的生长 建立荷人鼻咽癌细胞的裸鼠放疗增敏模型,与对照组对比,ApoG2联合放射线可减小裸鼠的瘤体积及瘤重。ApoG2组、放疗组、ApoG2+放疗组对CNE 2裸鼠移植瘤生长的抑制率分别是46.89%、19.34%和61.64%。免疫组化结果显示ApoG2联合放射可增加瘤组织中LC3的表达。 结论： 1. ApoG2可诱导鼻咽癌细胞发生自噬。 2. ApoG2可增强鼻咽癌细胞对放疗的敏感。 3. ApoG2可促使Bcl-2与Beclin 1解离,游离出的Beclin 1活化下游自噬信号通路,这可能是ApoG2提高放射增敏作用的机制。 4. ApoG2联合放疗可显著抑制裸鼠(CNE2细胞)移植瘤的生长。
[Abstract]:Objective: programmed cell death, including apoptosis and autophagic cell death, a growing number of studies suggest that autophagy plays an important role in.Beclin 1 and type PI3K in tumors with start autophagy, Bcl-2/Bcl-xL combined with Beclin 1 to block the function of.Bcl-2/Bcl-xL in autophagy induced high expression in nasopharyngeal carcinoma and its prognosis was negatively related to nasopharyngeal carcinoma radiotherapy. In addition, radiotherapy induced apoptosis can induce autophagy,.Apogossypolone plays an important role in anti-tumor (ApoG2) after gossypol derivative transformation, it is a kind of novel small molecule inhibitors of anti apoptotic Bcl-2 family proteins. We found that ApoG2 can bind to Bcl-2/Bcl-xl, blocking the function of Bcl-2 in nasopharyngeal carcinoma cells, and enhance the chemosensitivity in vitro and in vivo; also found anticancer drugs, radiation and other expression regulation of Beclin 1 induced autophagic cell death in nasopharyngeal carcinoma. This article intends to With the further study of ApoG2 inhibition of Bcl-2/Bcl-xL and Beclin 1, Beclin 1 blockade induced autophagy function induced by radiotherapy in nasopharyngeal carcinoma cells and enhance the radiosensitivity of autophagic cell death in vitro and in vivo antitumor drugs combined with radiotherapy for nasopharyngeal carcinoma and provide experimental evidence of small molecule inhibitors of Bcl-2 type, and ApoG2 will be developed into new anticancer drugs laid the foundation.
Methods: human nasopharyngeal carcinoma CNE1 in vitro, CNE2 and SUNE1 cells and nasopharyngeal epithelial cells NP69, detection of different concentrations of Bcl-2 inhibitor ApoG2 MTT on human nasopharyngeal carcinoma CNE1, inhibition of CNE2 and SUNE1 cells of nasopharyngeal epithelial cells and the cytotoxicity of NP69, to calculate their IC50 value, choose a lower drug concentration of IC50 as a follow-up experiment concentration. Colony formation assay of different doses of radiation with or without ApoG2 CNE1 on human nasopharyngeal carcinoma, effect of CNE2 and SUNE1 cell survival curve, calculate the survival rate, cell survival curve. Co immunoprecipitation method was used to detect the effect of Beclin 1 and ApoG2 Bcl-2 binding. Lentivirus infected CNE2 cells, Knock down Atg5. To establish a stable cell line, NPC cells lack of observation of radiotherapy autophagy induced by ApoG2 sensitized.GFP-LC3 plasmid was transfected into tumor cells, fluorescence microscope was used to observe autophagy marker LC3 Aggregation. Transmission electron microscope was used to detect autophagy morphology. Immunoblotting (Western blot) detection of autophagy related protein LC3, P62 expression. Nude mice, observe ApoG2 orally, radiotherapy alone or in combination on nasopharyngeal carcinoma cell transplantation tumor growth inhibition; expression of LC3 immunohistochemical detection of tumor tissues.
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