【作者】 陈伟；

【导师】 邹国林；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 萜类化合物是广泛存在于自然界中的一种脂溶性次级代谢产物。动植物体内广泛存在有萜类化合物。挥发油中含有很多单萜和倍半萜,这些萜类有些具有一定的生物学活性。洋甘菊的挥发油具有抗炎解痉、抗真菌、抗病毒和抗溃疡功能,其主要成分是蓝香油奥和α-红没药醇。α-红没药醇是一种低毒性的倍半萜,近百年来已被广泛应用于香水和化妆品行业。近年来,α-红没药醇被发现具有多种其他的生物学活性,如提高细菌对抗生素和抗微生物制剂的抗性,抗突变,抑制遗传损伤,去黑色素,致凋亡及抗癌活性等而被广泛研究。肝癌是最常见的癌症之一,其全球发病率逐年增长。与发达国家相比,发展中国家的肝癌发病率要高出2-3倍。由于现有的常规治疗手段对于肝癌的治疗效率低下,研究人员都致力于开发新的治疗肝癌的药物。细胞凋亡是一种基本的生物学现象,主要是通过两条通路介导的,即外源性通路(也叫死亡受体通路)和内源性通路(也叫线粒体通路)。死亡受体通路主要是由细胞表面的死亡受体如Fas蛋白和肿瘤坏死因子(TNF)受体引起的,旦这些因子被激活,就会导致一个致死信号复合物的形成,而caspase-8蛋白也被活化。活化后的caspase-8蛋白不仅能直接激活下游的效应caspase蛋白,还可以通过切割Bid蛋白为tBid来激活Bid蛋白,而Bid蛋白不仅在Fas介导的凋亡通路中起着十分重要的作用,还可以介导这两种通路之间的相互联系。线粒体凋亡通路依赖于细胞色素c从线粒体释放到胞浆,而这是由线粒体外膜的通透造成的。线粒体外膜的通透是由Bcl-2家族蛋白调控的。Bcl-2家族主要是由促凋亡分子和抗凋亡分子组成的,其中促凋亡分子包括Bax, Bcl-Xs, Bak, Bid, Bad, Bim和Bik,而抗凋亡分子包括Bcl-2, Bcl-xL, Bcl-w, Mcl-1和A1。细胞色素c一旦从线粒体释放到胞浆,就会和Apaf-1和caspase-9的酶原组合成凋亡体,从而激活caspase-9蛋白。活化后的caspase-9蛋白可以切割并活化下游的效应caspase蛋白如caspase-3和caspase-7,导致细胞凋亡。本文研究了α-红没药醇对人肝癌细胞HepG2的凋亡作用。采用MTT法检测了α-红没药醇对几种不同的人的癌细胞(PC-3, Hela, ECA-109和HepG2)的细胞毒性,发现α-红没药醇可以有效地抑制这几种癌细胞的增殖,尤其是对HepG2细胞；细胞核形态变化、DNA片段化实验结果表明,HepG2细胞被α-红没药醇作用后呈现出典型的凋亡特征；流式细胞术分析结果显示,在被α-红没药醇作用后,凋亡细胞的比例可高达近40%。聚二磷酸腺苷核糖多聚酶及caspases家族蛋白的剪切实验结果显示,α-红没药醇可能在HepG2细胞中引起了依赖于药物剂量和作用时间的凋亡作用。免疫印记实验显示出caspase-8,-9,-3的级联激活和Fas蛋白表达量的升高,表明与Fas蛋白相关的凋亡通路可能参与到这个过程,而caspase-8在其中可能扮演着上游调控蛋白的角色。通过提取线粒体和胞浆组分和之后的免疫印记分析,发现细胞色素c从线粒体释放到胞浆,Bcl-2蛋白的表达量减少,Bax、Bak和Bid蛋白的转运等,这些实验结果表明与线粒体相关的通路也可能参与到α-红没药醇引起的HepG2细胞凋亡中。此外,细胞被α-红没药醇作用后,p53蛋白和NFkB蛋白表达量的上升也显示出这两种在多种信号通路中有着重要作用的转录决定因子也参与到了α-红没药醇对HepG2细胞的凋亡作用中。这些实验结果加深了对α-红没药醇对HepG2细胞凋亡作用的理解,并拓宽了对α-红没药醇毒性及抗癌活性的认识,增加了将α-红没药醇开发为未来一个极具前途的化疗药物的可能性。Bcl-2家族蛋白是蛋白的关键调控因子,也是开发的新一代抗癌药物的热门靶点。对这些蛋白的结构-功能的研究已经揭示了抗凋亡Bcl-2蛋白和Bcl-xL蛋白的分子表面空腔对于它们与其他促凋亡蛋白的相互作用和抑制细胞死亡信号通路的功能至关重要。已报道了能够抑制Bcl-2功能的几类小分子抑制剂：(1)合成的只含BH3结构域的多肽和其类似物,这些小分子具有传透细胞膜的能力；(2)通过随机筛选发现的天然产物；(3)基于结构和计算机辅助技术设计的有机化合物。这些与Bcl-2蛋白结合的不同类型的小分子为探究Bcl-2功能提供了强有力的工具,并且极有可能导致新的抗癌药物的开发。为进一步探究α-红没药醇的抗癌机理并探寻该分子在细胞内的可能靶点蛋白,我们选取了五个Bcl-2家族中的抗凋亡成员Al, Bcl-2, Bcl-w, Bcl-xL和Mcl-1,使用同源模建和Autodock方法对α-红没药醇分子和上述五个蛋白分子进行了对接分析,初步探索了α-红没药醇与Bcl-2家族蛋白的相互作用,分析了a-红没药醇在这些蛋白上的可能结合位点,为探究α-红没药醇的抗癌分子机理及将其开发成为新一代抗癌药物寻找一种新的途径。更多还原

【Abstract】 Terpenoid is a widespread fat-soluble secondary metabolites exist in plants and animals. Essential oil contains a lot of monoterpenes and sesquiterpenes, some of which have biological activities. Chamomile essential oil has anti-inflammatory antispasmodic, anti-fungal, anti-virus and anti-cancer function, and its main ingredient is sesame oil olympic blue andα-bisabolol. a-Bisabolol, a sesquiterpene alcohol with very low toxicity, has been widely used in fragrances and cosmetic preparations for hundreds of years. Recent years, this oily compound has been studied for its effect of increasing bacterial resistance to antibiotics and antimicrobials, antimutagenic activity, inhibitory effect on the genotoxic damage, depigmenting effect and apoptosis-inducing and anti-tumor activities.Liver cancer (LC) is one of the most common cancers with an annually increasing occurrence worldwide. LC has a two-to three-fold higher incidence rates in developing countries than in developed countries. Since most of current therapeutic treatments are ineffective and limited to treat the tumor, great effort has been making to find novel compounds to treat the tumors.Apoptosis, a basic biological phenomenon, is mostly mediated through extrinsic (death receptor) pathway and/or intrinsic (mitochondrial) pathway. The death receptor pathway is stimulated by the binding of cell surface death receptor such as Fas and tumor necrosis factor (TNF) receptor, which leads to the formation of a death-inducing signaling complex and the activation of caspase-8. Activated caspase-8 can not only directly activates downstream effector caspases, but also cleave Bid to tBid, which is important in Fas receptor pathway and can mediates cross-talk between these two pathways. The mitochondrial pathway is dependent on the release of cytochrome c from the mitochondria by mitochondrial outer membrane permeabilization (MOMP). MOMP is controlled by the BCL-2 family, which is composed of both pro-apoptotic molecules (Bax, Bcl-Xs, Bak, Bid, Bad, Bim and Bik) and antiapoptotic molecules (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Al). Once released to cytosol, cytochrome c combines with Apaf-1 and procaspase-9 to form the apoptosome. Activated caspase-9 can cleave and activate downstream effector caspases, such as caspase-3 and-7, leading to apoptotic cell death.In this study, the apoptotic effect of a-bisabolol against human liver carcinoma cell line HepG2 was investigated. MTT assay showed a-bisabolol could effectively induce cytotoxicity in several human cancer cell lines (PC-3, Hela, ECA-109 and HepG2), especially in HepG2 cells. The results of nuclei morphology examination, DNA fragmentation detection indicated HepG2 cells exhibited typical apoptotic features after treated with a-bisabolol. The results of flow cytometry analysis showed up to nearly 40%. The analysis of the cleavage of poly(ADP ribose) polymerase and caspases indicated a-bisabolol might induce dose-and time-dependent apoptosis in HepG2 cells. Western blot data also showed a cascade activation of caspase-8,-9,-3 and an evidently promoted expression of Fas, implying caspase-8 might function as an upstream regulator, and the Fas-related pathway might be involved in this process. Preparation of mitochondrial/cytosol fraction followed with immunoblot analysis showed the release of chromosome c from mitochondria, down-regulated expression of Bcl-2 and translocation of Bax, Bak and Bid, suggesting the mitochondrial-related pathway might be involved in a-bisabolol-induced apoptosis either. Detection of accumulation of nuclear wild-type p53 and up-regulated expression of NFkB after cells were treated with a-bisabolol indicated these two key regulator with transcriptional decision-making function in various signaling pathways might also play a role in a-bisabolol-induced apoptosis in HepG2 cells. These results provide further insight into a-bisabolol-induced apoptosis and deepen our previous cognition of the toxicity and anticancer activity of a-bisabolol, and can increase the possibility of developing a-bisabolol to be a promising future chemotherapeutic agent.Bcl-2 family proteins are key regulatory factors, and also are hot targets of a new generation of anti-cancer drugs in developing. Studies on the structure-function of these proteins have revealed the critical role of the surface cavity of anti-apoptotic Bcl-2 and Bcl-xL in the interaction with pro-apoptotic proteins and in the inhibition of cell death signaling pathway. Several classes of small molecule inhibitors of Bcl-2 have been reported:(1) synthesized BH3 peptides and mimic small molecules which could penetrate cell membrane; (2) natural products identified by random screening methods; (3) organic compounds designed by structrue-based and computer-aided technique. These various small molecules binding with Bcl-2 provides a powerful tool to explore the function of Bcl-2, and very likely lead to the develpoment of new anti-cancer drugs. To further explore the anti-cancer mechanism of a-bisabolol and to search for the potential target of which in cell, we selected five anti-apoptotic members of Bcl-2 family(Al, Bcl-2, Bcl-w, Bcl-xL and Mcl-1), using computer modeling and Autodock to dock a-bisabolol with these five proteins. We performed a preliminary study on the potential binding site of a-bisabolol on these five Bcl-2 family proteins, and try to find a new way to study the molecular mechanisms of the anti-cancer effect of a-bisabolol and to explore a new generation of anticancer drugs.更多还原