【作者】 郭献灵；

【导师】 吴孟超； 卫立辛；

【作者基本信息】 第二军医大学 ， 外科学， 2009， 博士

【摘要】 缺营养诱导的自噬和端粒酶在肝癌细胞对化疗药物不敏感中作用的研究肝癌(hepatocellular carcinoma,HCC)是全球常见的恶性肿瘤之一,且呈逐年上升之势,肝癌的治疗目前仍是以手术为主的综合治疗,但中晚期患者常失去手术机会。由于肝癌对化疗的不敏感,所以肝癌的化疗总体效果不佳。因此研究肝癌对化疗的不敏感性对提高肝癌的疗效以及提高病人生存率有很重要的意义。虽然目前对肝癌的耐药机制研究较多,但确切的机制仍不清楚。新近的研究表明,细胞自噬与端粒酶可能和肿瘤的耐药有关。自噬是以细胞质空泡化为特征的溶酶体依赖性的降解途径。自噬是体内长寿蛋白的降解途径,也是唯一的一个降解退化细胞器的方式。自噬广泛存在于正常的生理过程中,维护着细胞内环境的稳定。自噬通过调控过氧化物体、线粒体和内质网的更新来保持细胞内环境的稳定;自噬是细胞对外源性应激的快速适应性反应,尤其当营养和能量缺乏时;其降解产物氨基酸、核苷酸、游离脂肪酸等可供物质能量循环;当细胞遭受氧化应激和病毒感染时,自噬作为细胞的一种防御机制清除胞质内受损的细胞器、有害的蛋白质等,保护其免受损害。细胞对自噬的形成有着精细的调节,这过程有多种基因的参与,这些基因称为自噬相关基因atg(autophagy related gene),它们编码的蛋白参与自噬的诱导、产生、成熟和再循环,同时自噬还受到一些信号途径如PI-3K/AKT/mTOR及p38-MAPK等的调控。自噬的过程受一系列复杂的信号调控,自噬异常与肿瘤发生、神经退行性疾病、衰老等有重要的联系。端粒酶是一种核糖核蛋白酶复合物,负责将端粒重复序列(TTAGGG)添加到染色体末端,补偿了随细胞分裂而出现的端粒丢失,稳定了永生化细胞的端粒长度。端粒酶有三个成分组成,一种是功能性RNA(hTR)成分,它是合成端粒重复序列的模板,还有一种是端粒酶的催化亚基(hTERT),它有逆转录酶的活性;第三种是端粒酶结合蛋白。端粒酶RNA成分及相关蛋白在大多数组织中有表达,相反,端粒酶催化亚基表达在正常组织中是受抑制的,只有在永生化细胞和癌细胞上才上调;此外,在端粒酶阴性的细胞中导入hTERT即可检测到端粒酶活性,这说明hTERT的表达与端粒酶活性是平行的,hTERT的激活对肿瘤细胞中端粒酶活性的激活是“足够的和必须的”。所以在端粒酶的激活过程中,hTERT基因在细胞中的表达是决定端粒酶活性的限速步骤。在本实验室以前进行的肝癌端粒酶相关研究中,发现HCC的端粒酶阳性率为85%,明显高于癌旁组织、肝硬变组织及慢性肝炎组织,而正常肝脏组织不表达端粒酶活性,认为端粒酶的表达可能在HCC的发生发展中起到了关键的作用。本实验的目的是:研究缺营养诱导的自噬和端粒酶在肝癌细胞对化疗药物不敏感中的作用。本实验主要由以下两大部分组成:第一部分,缺营养诱导的自噬在肝癌对化疗不敏感中的作用将肝癌细胞系(HepG2、Hep3B、SMMC7721)培养在全营养和自噬诱导培养基EBSS(无氨基酸无血清)中,分别用化疗药物(5FU、顺铂)处理12h,利用cck8法检测细胞活性,用AnnxinV/PI双染色来检测细胞死亡,以全营养和缺营养情况下未用药的对照组为基准,比较不同营养情况下肝癌细胞在化疗药作用下的死亡率。结果发现缺营养情况下肝癌细胞对化疗药物的敏感性降低。由于缺血缺营养是细胞发生自噬的天然诱导剂,新近的研究表明自噬与肿瘤对化疗不敏感有关,这些研究提示我们自噬可能参与了肝癌细胞对化疗的不敏感,因此我们进一步研究肝细胞癌缺营养是否诱导了自噬的发生。将肝癌细胞系(HepG2、Hep3B、SMMC7721)培养在全营养和自噬诱导培养基EBSS(无氨基酸无血清)中,应用电子透射显微镜检测自噬小体,realtime PCR检测和比较不同营养情况下自噬基因(LC3、ATG5、ATG 7、DRAM、beclin1等)表达。同时我们也利用转染LC3-GFP质粒检测和比较不同营养情况下肝癌细胞成点状绿色荧光的数量;结果表明,肝癌细胞在缺营养情况下自噬小体的形成数量要高于全营养情况,同时成点状绿色荧光的数量也明显增多。这些结果表明肝细胞癌在缺营养情况下诱导了自噬的发生。对肝癌自噬的进一步的研究还证实抑制自噬增加肿瘤细胞在缺营养情况下的死亡率和对化疗的敏感性。将肝癌细胞系(HepG2、Hep3B、SMMC7721)培养在全营养和自噬诱导培养基EBSS(无氨基酸无血清)中,预先应用自噬抑制剂(3MA、LY294002)作用2h,分别用化疗药物(5FU、顺铂)处理12h,利用cck8法检测细胞活性,在倒置相差显微镜下观察细胞形态的变化,用AnnxinV/PI双染色以及DAPI染色来检测细胞凋亡,比较和观察在全营养、缺营养及缺营养加自噬抑制剂情况下,肿瘤细胞的死亡情况以及在化疗药物作用下的死亡情况。结果发现在抑制自噬和缺营养的情况下,肿瘤细胞死亡增加,同时肿瘤细胞对化疗药物的敏感性也增加。综合上面的实验结果,我们的研究表明自噬不但是肝癌细胞在缺营养情况下的生存机制之一,同时也有助于肝癌细胞抵抗化疗的作用。但是目前关于自噬抵抗化疗的具体机制还不是很清楚。自噬是体内唯一的一个降解细胞器的途径,所以大部分的研究认为自噬可能通过降解受损的线粒体,防止促凋亡因子如细胞色素和凋亡诱导因子(AIF)的扩散,以帮助细胞逃逸凋亡。同时研究也显示p53在肿瘤细胞的自噬发生过程起到很重要的作用。因此在p53野生型的肝癌细胞中我们关注线粒体和p53在自噬诱导肝癌细胞对化疗药物不敏感中的作用。将肝癌细胞系培养在全营养和自噬诱导培养基EBSS(无氨基酸无血清)中,抽取RNA,应用realtime PCR检测和比较不同营养情况下线粒体基因(Nd2、Nme1)的表达。同时将p53野生型的肝癌细胞系(HepG2、SMMC7721)预先应用p53抑制剂PFT-a作用1h,然后继续培养在全营养和自噬诱导培养基EBSS(无氨基酸无血清)中24h。应用电子透射显微镜检测自噬小体的形成。同时我们也利用转染LC3-GFP质粒检测和比较p53抑制和未抑制情况下肝癌细胞成点状绿色荧光的数量;结果表明,在缺营养情况下线粒体基因Nd2表达降低,表明线粒体数量减少;同时肝癌细胞在缺营养联合p53抑制情况下自噬小体的形成数量低于p53未抑制情况,同时成点状绿色荧光的数量也明显减少。进一步的研究还显示在p53抑制联合缺营养的情况下,分别用化疗药物(5FU、顺铂)处理24h,利用cck8法检测细胞活性,在倒置相差显微镜下观察细胞形态的变化,用AnnxinV/PI双染色以及DAPI染色来检测细胞凋亡,比较和观察在全营养、缺营养及缺营养加自噬抑制剂情况下,肿瘤细胞的死亡情况以及在化疗药物作用下的死亡情况。结果发现在抑制p53和缺营养的情况下,肿瘤细胞死亡增加,同时肿瘤细胞对化疗药物的敏感性也增加。这个结果表明线粒体的减少以及p53的激活可能参与了肝癌对化疗的不敏感。第二部分,端粒酶在肝癌对化疗不敏感中的作用端粒酶重新激活是癌变细胞一个带有普遍性意义的生物学标志,被认为是细胞癌变的一个重要细胞生物学异常事件,是细胞永生化及肿瘤发生的关键步骤。近年来大量的研究资料表明,端粒酶除了有延长端粒长度的功能外还有抗凋亡作用,由于大多数化疗药物作用于肿瘤细胞是通过凋亡而实现,因此端粒酶的抗凋亡效应在肿瘤对化疗的不敏感性中的作用愈来愈引起大家的关注。在这一部分研究中我们首先发现低浓度化疗药物顺铂诱导肝癌细胞端粒酶上调和hTERT表达的升高。肝癌细胞7721培养在含不同浓度化疗药物顺铂的培养基中24h,抽提蛋白,利用TRAP法(端粒重复扩增法)检测和比较不同浓度顺铂作用的SMMC7721细胞的端粒酶活性。Western blot和RT-PCR(逆转录-PCR)方法检测和比较细胞中hTERT的表达情况;同时我们也利用已构建好的hTERT基因启动子荧光素报告基因(hTERT promoter-luciferase reporter system)系统来检测和比较SMMC7721细胞的端粒酶hTERT基因启动子活性;结果我们发现不同浓度化疗药物顺铂对肝癌细胞SMMC7721其端粒酶的活性和hTERT基因的表达水平的诱导有一个剂量依赖的关系。而且研究结果还表明药物诱导肝癌细胞端粒酶的上调,其上调可能是由于端粒酶hTERT组分表达量的增加所致。端粒酶hTERT基因的表达是受多种转录因子调控的。进一步深入的研究发现低浓度顺铂能上调NF-кB活性以及p65组分的表达,抑制NF-кB会损害低浓度顺铂对端粒酶hTERT基因表达的上调作用。这表明转录因子NF-кB参与了低浓度顺铂对端粒酶hTERT基因表达的上调作用。在本实验室以前进行的肝癌端粒酶相关研究中,发现HCC的端粒酶阳性率为85%,明显高于癌旁组织、肝硬变组织及慢性肝炎组织,而正常肝脏组织不表达端粒酶活性,认为端粒酶的表达可能在HCC的发生发展中起到了关键的作用。由于端粒酶具有抗凋亡效应,同时结合肝癌对化疗的不敏感,因此我们猜想低浓度顺铂诱导的端粒酶的升高参与肝癌对化疗的不敏感。肝癌细胞7721培养在含低浓度(1μg/ml)化疗药物顺铂的培养基中24h,随后用5-FU (65μg/ml)和表柔比星(1μg/ml)分别处理诱导的和未诱导的7721细胞24小时;在倒置相差显微镜下观察细胞形态的变化,Western blot检测PARP来指示细胞凋亡,结果发现诱导过的肝癌细胞7721的存活率比未诱导的高,细胞出现变小、变圆和透亮等形态变化的较少;同时PARP表达减少说明凋亡率减少。这些结果提示经低浓度顺铂诱导过的SMMC7721细胞比未诱导过的细胞显示出了更低的化疗药物的敏感性。近一步的实验还证实抑制hTERT的表达提高肿瘤细胞对化疗药物的敏感性。在体外的研究中发现针对hTERT的siRNA能明显抑制hTERT蛋白的表达和hTERT启动子的活性;抑制端粒酶hTERT能显著提高肿瘤细胞对化疗药物5-FU、表柔比星和顺铂的敏感性,表现在细胞hTERT抑制后,在药物作用下细胞死亡增加,DAPI染色显示核固缩、碎裂增多。接着我们在体内实验中发现了相似的现象,我们将肝癌细胞系7721和HepG2种植BALB/c裸鼠皮下成瘤,然后直接将腺病毒注射到肿瘤内,并联合腹腔注射顺铂,每隔5天一次,共4次,结果显示联合hTERT抑制和顺铂作用能有效的抑制种植瘤的生长。综上所述,本课题得出以下结论:1、营养缺乏能诱导肝癌细胞激活自噬,自噬能诱导肝癌细胞对化疗不敏感,抑制自噬能提高肿瘤细胞在缺营养情况下的死亡率以及对化疗的敏感性。2、p53参与了营养缺乏情况下肝癌细胞自噬的激活,抑制p53能提高肿瘤细胞在营养缺乏情况下的死亡率以及对化疗的敏感性。3、低浓度的顺铂能诱导肝癌细胞端粒酶上调和hTERT表达的升高,这种升高受到NF -κB的调控。4、低浓度顺铂能诱导肝癌细胞对化疗药物的不敏感。抑制端粒酶hTERT的表达,能提高肝癌细胞对化疗药物的敏感性,而能明显抑制肝癌细胞在体内的生长。总之,我们的研究表明,自噬和端粒酶参与了肝癌细胞对化疗的不敏感,,自噬和端粒酶的激活可能是肝癌抵抗化疗的机制之一。更多还原

【Abstract】 The role of nutrient-free induced autophagy and telomerase in insensitivity of hepatocellular carcinoma to chemotherapyHepatocellular carcinoma (HCC) is one of the most common malignancies; the incident rate of it is growing every year in our country. The most common way to cure it is undergoing combined therapy including chemotherapy. But the curative chemotherapy is far from satisfaction, because hepatocellular carcinoma is more resistant to chemotherapeutics. How to increase hepatocellular carcinoma sensitive to chemotherapy is more significant to hepatocellular carcinoma (HCC) therapy and improving patients’survival. Althoμgh there are a lot of drμg-resistant mechanisms about HCC, it is still out of fully understanding. Recently, growing evidence show that autophagy and telomerase were involved in tumor drμg resistance.Autophagy is a regulated lysosomal degradation pathway。Autophagy is the cell’s major regulated mechanism for degrading long-lived proteins and the only known pathway for degrading organelles. Autophagy widely exists in the normal physiological process, and maintains the intracellular homeostatisis .autophagy is the rapid adaptive response of cells to exogenous stress. Autophagy is rapidly upregulated when cells need to generate intracellular nutrients and energy (e.g., during starvation or trophic factor withdrawal), or rid themselves of damaging cytoplasmic components (e.g., during oxidative stress, infection, and accumulation of protein aggregates). There are many gene products essential for autophagy and related pathways referred to as the ATG genes .The ATG genes encode proteins needed for the induction of autophagy, and the generation, maturation, and recycling of autophagosomes. Several pathways are involved in regulation of autophagy, for example PI-3K-AKT-mTOR or p38-MAPK.The deregulation of autophagy is related to many human diseases, including infections, cancer, neurodegeneration, aging, and heart disease.Telomerase is a rib nucleoprotein enzymatic complex that adds repeated telomere sequences (TTAGGG) onto the chromosome ends, compensates for the telomeric loss that occurs with cell division and stabilizes. telomerase consists of three components: telomerase reverse transcriptase (hTERT), an RNA template—telomerase RNA component (hTR), an other subunit is telomerase associate protein (TEP1) .the hTR and TEP1 is ubiquitously exist in both normal and malignant tissues and cells, but the hTERT component is undetectable in most normal human tissues and somatic cell but almost expressed in human cancer cell/immortally cell line and malignant tissues. So the expression of hTERT is thoμght to be paralleled with telomerase activity and considered as a rate-limiting determinant of enzymatic activity. Data of our lab revealed more than 85% HCC with much stronger telomerase activity than cirrhosis. Based on the other spurring observation that normal hepatocellular tissue without telomerase activity, many researcher propose that telomerase plays an important role in the initiation and development of HCC. In the preliminary experiment, we find that low concentrate cisplatin not only can’t kill HCC cell line but also up-regulate telomerase and induce drμg insensitivity to cisplatin. So our object is to study the role of autophagy and telomerase in insensitivity of hepatocellular carcinoma to chemotherapy.PartI: The role of nutrient-free induced autophagy in insensitivity of hepatocellular carcinoma to chemotherapyHCC cell lines (HepG2、Hep3B、SMMC7721) were cultured in the full nutrient and nutrient free media ,and then were treated with chemotherapeutic agents (5FU and cisplatin) for12h. Cell viability was measured by cck8 assay, cell morphological change was detected under phase contrast microscope and apoptosis cell was stained with Annixin V/PI double staining. The results showed that cells grown in nutrient free media demonstrated more resistant to chemotherapeutic agents than that in full nutrient media.It has been demonstrated that nutrient starvation is the natural inducer of autophagy. Autophagy was also reported to be contributing to chemoresistance in tumor. Thus we want to investigate whether autophagy was invovled in hepatocarcinoma insensitivity to chemotheraputic agents.HCC cell lines (HepG2、Hep3B、SMMC7721) were cultured in the full nutrient and nutrient free media. Electron microscope was performed to detect autophagysome and realtime PCR assays were used to detect the expression of autophagy related gene (LC3、ATG5、ATG 7、DRAM、beclin1). LC3-GFP plasmid was transfected into HCC cell lines, and the green dot was detected and measured. The results demonstrated that the expression of autophagy related gene was significant upregulated and the green dots were also increased during nutrient starvation which suggested that HCC cells showed more activated autophagy. Futher reseach suggested that inhibition of autophagy increased cell death and chemosensitivity during nutrient starvation.HCC cell lines (HepG2、Hep3B、SMMC7721) were cultured in the full nutrient and nutrient free media. cells were pretreated with autophagy inhibitor(3MA、LY294002) for 2h and then were incubated with chemotherapeutic agents (5FU and cisplatin) for another 12h. Cell viability was measured by cck8 assay, cell morphological change was detected under phase contrast microscope and apoptosis cell was stained with Annixin V/PI double staining and DAPI staining. The results showed that cells treated with autophagy inhibitor demonstrated more insensitivity to chemotherapeutic during nutrient starvation.Taken together our data suggested that autophagy was one of the suvival mechnism of hepatocarcinoma during nutrient starvation. Meanwhile, autophagy was also contributed to chemotherapy resistance. But the mechnism of how autophagy contributed to chemotherapy resistance is largely unknown.it has been reported that mitochondria and p53 were connected with autophagy, thus we investigated the possible role of mitochondria and p53 in heaptocarcinoma autophagy. HCC cell lines (HepG2、SMMC7721) were pretreated with p53 inhibitor(PFT-a) for 1h, and then were cultured in the full nutrient and nutrient free media for another 24h. Electron microscope was performed to detect autophagysome and realtime PCR assays were used to detect the expression of mitocondria gene (Nd2、Nme1). LC3-GFP plasmid was transfected into HCC cell lines, and the green dot was detected and measured. The results demonstrated that the expression of mitocondria gene Nd2 was significant downregulated and the autophagysome and green dots were also decreased during nutrient starvation when p53 was inhibited. To further investigate the role of p53 in autophagy activation,p53 widetype HCC cell lines (HepG2、SMMC7721) were cultured in the full nutrient and nutrient free media. Cells were pretreated with p53 inhibitor (PFT-a) for 1h or transfected with si-p53 plasmid, and then were incubated with chemotherapeutic agents (5FU and cisplatin) for another 24h. Cell viability was measured by cck8 assay, cell morphological change was detected under phase contrast microscope and apoptosis cell was stained with Annixin V/PI double staining and DAPI staining. The results showed that cells treated with p53 inhibitor demonstrated more insensitivity to chemotherapeutic during nutrient starvation. Thus,our results suggested that mitocondria and p53 were involved in chemotherpy resistance in hepatocarcinoma. PartII: The role of telomerase in insensitivity of hepatocellular carcinoma to chemotherapyTelomerase reactivation is considered to be a landmarker of cancer. Telomerase was also reported to have antiapotosis function otherwise the role of maintainance of telomere. In this section we found that Low-dose cisplatin induces up-regulation of telomerase activity and hTERT expressionHuman HCC cells SMMC7721 were treated with various concentrations of cisplatin for 24 h. TRAP assay was used to test telomerase activity. Western blotting analysis and semi-quantitative RT–PCR assays were performed to examine whether activation of telomerase by cisplatin was due to up-regulation of the hTERT expression. Telomerase activity was found to reach a plateau at 1μg/ml, and declined thereafter.Treatment of SMMC7721cells with indicated concentrations of cispaltin for 24 h led to up-regulations of hTERT protein and hTERT mRNA. To examine the effect of cisplatin on the transcriptional activity of the hTERT promoter, luciferase assay in which hTERT luciferase reporter plasmid (pGL3B-TRTP) was transfected into SMMC7721cells was performed. Cisplatin increased the transcriptional activity of the hTERT promoter for up to 3.5-fold. These results sμggest that cisplatin can induce the hTERT expression at transcriptional level. hTERT expression was regulated by lots of transcriptional factors. Cispaltin was reported to activate the NF-кB. Thus, we investigated whether NF-кB is involved in the induction of hTERT expression by cisplatin. Our results showed cisplatin can activate NF-кB and inhibition of NF-кB can reduce the hTERT expression induced by cisplatin.Data of our lab revealed more than 85% HCC with much stronger telomerase activity than cirrhosis. Based on the other spurring observation that normal hepatocellular tissue without telomerase activity, many researcher propose that telomerase plays an important role in the initiation and development of HCC. Based on the antiapoptosis role of telomerase and hepatocarcinoma insensetivity to chemotherapy, we hypothsis that telomerase is invovled in chemotherapy insensetivity of hepatocarcinoma. SMMC7721cells were preincubated in 1μg/ml cisplatin for 24 h, and then treated with 5-FU (65μg/ml) and epirubicin (1μg/ml) for another 24 h respectively. The morphology of cells was observed, and pictures were taken under the microscope. Then, the cells were harvested and used to prepare cell lysates. The lysates were subjected to SDS–PAGE and blotted with anti-PARP antibody.the result revealed that control cells, but not preincubated cells, show significantly cell death. Control cells revealed morphologic features typical of apoptosis, including cytoplasmic condensation, nuclear fragmentation, and blebbing. These results were confirmed by immunoblotting, where control cells resulted in increased level of cleaved PARP. Thus these data sμggested Low concentrate cisplatin induce insensitivity of HCC to chemotherapeutic agents. Furthermore, we also demonstrated Inhibition of hTERT can enhance the chemosensitivity of SMMC-7721 cellsSMMC7721cells were infected with Ad-si/hTERT virus for 24 h, and then preincubated in 1μg/ml cisplatin for another 24 h. After preincubation, cells were treated with 5-FU (65μg/ml) and epirubicin (1μg/ml) for 24 h respectively.WST-8 assay and DAPI staining was performed to detected cell death. The results suggested Ad-si/hTERT can significantly inhibit hTERT expression and telomerase activity. Meanwhile, SMMC7721cells infected with Ad-si/hTERT showed dramatically sensitivity to chemotherapeutic agents.To determine the effect of Ad-si/hTERT plus cisplatin on tumor growth in vivo, SMMC-7721 and HepG2 cells were injected into the right back of BALB/c nude mice. After 4 days, the xenograft tumor started to grow and after about 15 days the model was successfully established to initiate treatment. Ad-si/hTERT and empty vector Ad-null (1x 109/30 ul/time, separately) was directly injected into the tumor, and cisplatin (3 g/Kg/100 ul/time) was intraperitoneally injected. All types of treatment were administered at the same time every 5 days for 4 administrations. Tumor sizes were subsequently measured daily. The results indicate that combination Ad-si/hTERT with cisplatin can significantly inhibit tumor growth in vivo.To be conclued, our results sμggested1、Nutrient starvation can activate autophagy in hepatocarcinoma cells, and autophagy contributes to chemoresistance; inhibtion of autophagy increased the senstivity to chemotherpeutic agents of hepatocarcinoma during nutrient starvation.2、P53 was invovled in autophagy activation of hepatocarcinoma during nutrient starvation. Inhibition of p53 increased cell death and chemosensitivity during nutrient starvation.3、NF-кB is involved in low-dose cisplatin inducd up-regulation of telomerase activity and hTERT expression Inhibition of hTERT enhances the chemosensitivity of hepatocarcinoma cells to chemotherpeutic agents更多还原