【作者】 刘振兴；

【导师】 曾申明；

【作者基本信息】 中国农业大学 ， 动物遗传育种与繁殖， 2014， 博士

【摘要】 咪唑是一种生物弱碱,它是生物体内组胺、组氨酸发挥生理作用的重要基团。咪唑及其衍生物广泛应用在不同类型的药物中,显示出不同的药理活性,如抗菌、抗真菌、抗病毒、抗寄生虫、拮抗组胺受体等。尤其作为抗癌药物,咪唑显示了广阔的应用前景。该领域已经取得了许多重要成果,其中多个咪唑类化合物已作为抗癌药物应用于临床。然而,咪唑类药物的抗癌机制尚不完全清楚。细胞自噬和凋亡与癌症的发生密切相关,目前,绝大多数化疗药物主要是通过抑制自噬和促进凋亡来实现癌症治疗目的。因此,本论文以人子宫内膜腺上皮癌细胞(HEC-1B)为材料,系统地研究分析咪唑对细胞自噬和凋亡的影响,旨在揭示咪唑类药物抗癌作用的分子机制。在自噬方面：(1)较低浓度的咪唑(5-25mM)能够在6h内引起HEC-1B细胞空泡化,通过Western blotting检测发现咪唑能够明显地升高自噬体标志物LC3-II的含量,进一步利用透射电镜观察发现咪唑诱导胞内产生大量自噬体样囊泡(AVs),表明咪唑影响细胞的自噬过程；(2)Western blotting检测显示咪唑并未对自噬启动相关激酶及分子产生明显影响,表明咪唑处理并未启动细胞的自噬；(3)在咪唑处理的细胞中EGFP-LC3呈点状分布,游离EGFP蛋白水平显著下降,进一步通过Western blotting检测表明咪唑处理显著增加p62蛋白水平,并且BafA1抑制并未提高咪唑处理细胞内LC3-II的水平,表明咪唑阻断自噬物的降解；(4)利用激光共聚焦检测mRFP-GFP-LC3的亚细胞定位发现咪唑能够增加自噬体的数量,但未检测到自噬溶酶体数量增加,表明咪唑抑制自噬体成熟为自噬溶酶体。在凋亡方面：(1)较高浓度的咪唑(50-100mM)能够在12-48h显著地抑制HEC-1B细胞存活,Western blotting检测显示咪唑处理引起caspase9和caspase3断裂,表明咪唑能够诱导细胞凋亡；(2)利用RT-PCR和Western blotting检测促凋亡蛋白Bim的表达,结果表明咪唑处理导致Bim mRNA和蛋白水平表达显著升高;(3)通过Western blotting和蛋白超表达检测咪唑对转录因子Fox03a的影响,结果显示,咪唑能够诱导FoxO3a蛋白上调并促进其入核；(4)进一步研究发现siRNA介导的FoxO3a基因沉默显著地抑制咪唑引起的Bim上调,并降低细胞的死亡率,表明咪唑通过激活Fox03a上调Bim的表达。综上所述,咪唑通过抑制自噬体成熟从而阻断自噬体的降解：同时,咪唑通过FoxO3a-Bim通路诱导细胞凋亡。本研究结果明晰了咪唑对HEC-1B细胞自噬和凋亡作用的分子机制,并且为进一步研发咪唑类抗癌药物提供了有价值的科学依据。更多还原

【Abstract】 Imidazole, an organic compound with the formula C3H4N2, is classified as an alkaloid. It is incorporated into many important biological molecules; the most widespread is the amino acid histidine, which has an imidazole side-chain. Imidazole is an important pharmacophore in drug discovery. Its derivatives have diverse pharmaceutical effects, including histamine-H3antagonist, anti-inflammatory, gastroprotective, and antioxidant. Importantly, several imidazole derivatives had anticancer activity against a variety of malignant cells. However, mechanisms by which tumor cells respond to these stimuli remain to be elucidated. Autophagy and apoptosis are both cellular degradation pathways essential for organismal homeostasis. Therefore, it is not surprising that both autophagy and apoptosis have been implicated in protecting organisms against a variety of diseases, especially cancer. As autophagy removes damaged proteins and organelles, it limits their cumulative deleterious effects inside cells. Therefore, it is not surprising that autophagy defects are found in many human tumors. In contrast to these tumor-suppressor roles for autophagy, stress-activated autophagy may promote survival of tumor cells, especially when apoptosis is defective. Apoptosis is a tumor suppressor pathway. Deregulation of apoptosis leads to accumulation of "unwanted" cells and contributes to cancer development. Autophagy and apoptosis have key roles in tumorigenesis and tumor treatment. In this study, we systematically examined autophagic events induced by imidazole in HEC-1B cells. Accumulation of autophagic vacuoles in imidazole-treated cells was verified by conversion of LC3protein, as well as confocal and transmission electron microscopy. Furthermore, imidazole blocked autophagic degradation by impairing maturation of autophagosomes into autolysosomes. Concurrently, imidazole treatment induced apoptosis in HEC-1B cells, accompanied by activation of caspase9and caspase3. The proapoptotic effect was mediated by increased expression of the BH3-only protein Bim. Moreover, imidazole upregulated the protein level of transcription factor Foxo3a and induced its increased nuclear localisation. In addition, siRNA-mediated silencing of FoxO3a effectively attenuated imidazole-induced Bim upregulation and cell death, indicating direct involvement of this pathway in the imidazole-induced apoptosis. Taken together, our data provided a molecular link between imidazole drugs and anticancer therapies; understanding of these properties of imidazole is essential for future development of effective cancer therapeutics using imidazoles.更多还原