声动力疗法诱导S180腹水瘤细胞凋亡的信号转导通路及机制研究

【作者】 汤薇；

【导师】 刘全宏；

【作者基本信息】 陕西师范大学 ， 动物学， 2010， 博士

【摘要】 2008年末,世界卫生组织发布报告称,2010年癌症将超越心脏病成为危害人类健康的头号杀手,而到2030年,全球可能将有2700万人罹患癌症,其中1700万人将死亡。基于这一严峻形势,科学家们日以继夜地探索新的抗癌方法,以期能够找到更多治疗癌症的有效途径。目前临床上常用的癌症治疗方法有外科手术、化疗、放疗等,对多种恶性肿瘤起到一定治愈作用。但这些方法都有自身的不足之处,甚至有较强的副作用,特别是对于深层组织肿瘤的治疗常常效果不佳。随着研究不断深入,学者们发现多因素综合治疗能够有效杀死肿瘤细胞。1989年,Yumita和Umemura等发现超声能够激活血卟啉产生抗癌作用,并将这一现象定义为声动力疗法(Sonodynamic Therapy,SDT)。超声能够穿透深层组织并聚焦于特定区域来激活肿瘤组织富集并长时间潴留的声敏药物,显著增强定位区域药物的细胞毒作用,而对周围正常组织的损伤降到最小,在非侵入性治疗深层组织肿瘤方面具有独特的优势。血卟啉在正常人体中为线粒体酶系催化乙酸和甘氨酸合成血红素的中间产物,在肿瘤组织中选择性聚积并能够长时间潴留。血卟啉的这一特性使声动力疗法的靶向性和针对性大大增加,且肿瘤细胞对血卟啉不产生耐受性,可接受多次声动力处理。因此,声动力疗法是一种十分有应用前景的抗癌方法。自声动力疗法提出至今,超声和各种声敏剂的协同杀伤效应已在多种细胞系中得到证实,并有学者初步开展了临床试验。但目前在国内外已报道的研究中,还没有人对声动力诱导细胞凋亡进行较系统的研究,声动力疗法产生细胞毒作用的确切化学机制和细胞学机制目前仍无定论。本研究应用频率为1.75 MHz、强度ISATA为1.4 W的超声,采用流式细胞仪、激光共聚焦显微镜、免疫印迹、单细胞凝胶电泳、反转录-聚合酶链式反应以及DNA测序等技术,研究了超声结合血卟啉处理腹水型S180肿瘤细胞后,细胞膜受体和线粒体凋亡通路的激活；讨论了Bcl-2家族促凋亡蛋白Bax, Bid以及抑凋亡蛋白Bcl-2在凋亡中的作用；分析了p53肿瘤抑制蛋白在凋亡中的活化及其与S180、H-22、EAC肿瘤细胞声动力敏感性之间的关系；并探讨了超声和声动力诱导S180细胞凋亡的机制。目前得到研究结果如下：1.本研究证实单纯超声和超声结合血卟啉均可诱导S180细胞凋亡,但在相同声照参数下20μg/ml血卟啉能够显著增强超声的细胞毒作用,超声和血卟啉对S180细胞具有协同杀伤效应。2..实验发现膜受体-和线粒体-caspase凋亡通路在声动力处理后的S180细胞中活化。声动力处理后,膜受体通路中的FADD和caspase-8转定位至细胞膜并且caspase-8被剪切活化。同时线粒体膜通透性改变、外膜去极化,cytochrome-c由线粒体释放至胞质内,caspase-9被剪切。活化的caspase-8和-9进而剪切caspase-3,caspase-3进入细胞核将PARP剪切,促使细胞凋亡。另外,膜受体凋亡通路活化是由声动力处理直接引起,而不是线粒体凋亡通路信号放大的结果,也没有通过Bid蛋白将凋亡信号传导至线粒体。3.声动力处理还可激活caspase非依赖模式的凋亡通路。AIF在声动力处理后由胞质转定位至核内,直接发挥促凋亡作用。4.本研究首次发现声动力处理能够激活凋亡相关蛋白表达。声动力处理后,死亡受体Fas的mRNA和蛋白表达上调；Bcl-2家族抑凋亡蛋白Bcl-2的mRNA和蛋白表达下调而促凋亡蛋白Bax转定位至线粒体,并且表达量上调,抑凋亡蛋白和促凋亡蛋白之间的平衡被打破,确保凋亡进行。5.在研究中首次发现肿瘤抑制蛋白p53在声动力诱导S180和H-22细胞凋亡中活化,通过转定位至线粒体开启转录非依赖模式和激活下游靶基因PUMA、Bax和Fas表达的转录依赖模式加速凋亡过程。而在EAC细胞中没有发现p53的转定位以及p53及其下游靶蛋白的活化。6.首次提出细胞的声动力敏感性与p53蛋白的活化相关,而与p53基因的状态无直接关系。S180细胞对声动力损伤的敏感性最高,p53最先活化,表达量上调并转定位至线粒体促进凋亡发生,同时激活下游靶基因PUMA、Bax和Fas的表达。H-22细胞的敏感性次之,其p53在声动力处理后表达量上调,并激活其下游多个蛋白,一段时间后p53转至线粒体。而EAC对声动力损伤敏感性最弱,其p53蛋白没有明显的表达量变化和转定位现象发生,其下游蛋白表达也没有显著改变。经p53基因测序,三种细胞株p53基因均为野生型,没有发现碱基缺失和突变,因此细胞的声敏性与p53基因的状态可能无直接关系。7.对声动力疗法和单纯超声诱导细胞凋亡机制的研究指出,声动力诱导S180细胞凋亡中存在多种可诱导凋亡途径同时发挥作用。声动力处理后,富集于线粒体的血卟啉被激活产生大量单线态氧,导致线粒体内膜通透性增加,外膜去极化,使线粒体内的促凋亡蛋白释放,启动caspase级联反应介导凋亡；另外,实验发现细胞膜也是损伤位点之一,caspase-8被活化启动下游死亡程序。本实验首次发现NF-κB在超声诱导S180细胞凋亡中起重要的调节作用。超声诱导S180细胞凋亡时,NF-κB快速活化,mRNA水平迅速升高,蛋白表达上调,并从胞质转定位至细胞核,而活性氧在这一过程中没有明显作用。本研究首次系统的对声动力疗法诱导肿瘤细胞凋亡进行分析探讨,在细胞和分子水平初步阐明凋亡信号转导过程,为声动力学抗癌理论的形成和发展,及临床治疗、仪器研制积累资料。更多还原

【Abstract】 In 2008, World Health Organization reported that cancer will become the number one killer of human health in 2010. Even in 2030,27 million people may have cancer all over the world, and 17 million people will die for this disease. Therefore, scientists are exploring new anticancer therapy day after night in order to find out more effective ways. At present, clinical therapies in common use include surgical, chemotherapy and physiotherapy. These anticancer strategies can cure some kind of cancer, but their own shortages and side-effects restrict the application especially for cancer in deep.With the research carrying on, scholars find multifactor treatments can kill cancer cells in effect. In 1989, Yumita and Umemura discovered that ultrasound could activate hematoporphyrin to anticancer, and named this treatment as "Sonodynamic Therapy, SDT". Ultrasound can penetrate deep tissue and focus on certain area. This is a unique advantage when compared to electromagnetic modalities such as laser beams in the noninvasive treatment of nonsuperficial tumors in clinical treatment. Hematoporphyrin is a intermediate product in haemachrome synthesize in normal human body. It can selectively accumulate in tumor tissue and be reserved for a long time. SDT is a promising anticancer therapy with more pertinency and efficiency. Although the synergistic anticancer effect of ultrasound and sonosensitizers has been proved, and the clinical experiment has been carried out, there is little report about the SDT inducing apoptosis. The exact chemical and mechanism of ultrasonic cytotoxic effect is still unclear.In this research,1.75 MHz continuous-wave ultrasound was used to investigate that(1)whether death receptor-and mitochondria apoptosis signaling pathway are activated; (2) the regulation effect of Bcl-2 family members (Bax, Bid and Bcl-2) in SDT induced S180 cells apoptosis; (3) activation of tumor suppressor protein p53 and the relation between p53 and sonosensitivity of tumor cells;(4) the mechanism of ultrasound-and SDT-induced apoptosis. To analyze the protein and mRNA, flow cytometer, laser scanning confocal microscope, single cell gel electrophoresis, western blotting, RT-PCR and DNA sequencing were used. The results are as follow:(1) The ultrasound alone and SDT can induce S180 cells apoptosis at same condition, whereas 20μg/ml hematoporphyrin significantly enhances the cytotoxic effect of ultrasound. Ultrasound and hematoporphyrin have a synergistic antitumor effect.(2) The death receptor and mitochondria apoptosis signaling pathway are activated in SDT-induced S180 cells apoptosis. In this process, FADD and caspase-8 translocated from cytoplasm to plasma membrane, and caspase-8 was activated. At the same time, mitochondrial depolarization and permeabilization of the inner mitochondrial membrane were found. Cytochrome-c was released to cytoplasm to promote cleavage of caspase-9. Activated caspase-8 and-9 in turn cleave caspase-3, and PARP.The activation of death receptor signaling pathway was a direct consequence after SDT treatment but not a signaling magnification of mitochondria signaling pathway. The Bid which can transmit apoptosis signaling between death receptor and mitochondria signaling pathway did not be activated.(3)The SDT can also initiate caspase-independent pathway to mediate apoptosis. AIF was released and translocated to nuclear to promote apoptosis.(4) The SDT can regulate apoptosis-relative protein expression. After SDT treatment, the protein and mRNA level of Fas and Bax increased to promote apoptosis, whereas the level of apoptosis-inhibitor Bcl-2 declined.(5) In the SDT-induced S180 and H-22 cells apoptosis, tumor suppressor protein p53 expressed and translocated to mitochondria to accelerate apoptosis by transcription-dependent and transcription-independent way. The downstream protein PUMA,Bax and Fas were also activated. P53 was not involved in EAC cells apoptosis after SDT treatment.(6) There is a relationship between p53 protein activation and sonosensitivity of tumor cells. The S180 cells are the most sensitivity to SDT treatment. The level of p53 increased and p53 translocated to mitochondria. The sonosensitivity of H-22 cells was in the middle.After SDT treatment, p53 expressed, and translocated after a period of time. The p53 was not activated in the EAC cells, and the EAC cells were resistant to SDT treatment.(7) The mechanism of SDT-inducing apoptosis included multiform apoptosis pathway and death mode. After SDT, the death receptor and mitochondria apoptosis signaling pathway are activated, and initiate caspase cascade to mediate apoptosis. The nuclear factor NF-κB may play pivotal role in regulating ultrasound-induced apoptosis. When S180 cells underwent sonication,NF-κB expressed immediately and translocated to nuclear.In the present research, for the first time, we systemicly studied the apoptotic signaling pathway in SDT-induced tumor cell apoptosis and discussed the mechanism underline ultrasound-and SDT-induced apoptosis to provide a basis for explaining the synergistic effect of ultrasound and Hematoporphyrin and facilitate SDT application in clinical anticancer treatment.更多还原