咪唑对人子宫内膜腺上皮癌细胞（HEC-1B）自噬及凋亡的影响

发布时间：2019-02-25 21:37

【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。
[Abstract]:Imidazole is a weak alkaloid. It is an important group of histamine and histidine in organism. Imidazole and its derivatives are widely used in different types of drugs, showing different pharmacological activities, such as antibacterial, antifungal, anti-virus, anti-parasite, antagonistic histamine receptor and so on. Especially as an anticancer drug, imidazole has shown a broad application prospect. Many important achievements have been made in this field, and many imidazole compounds have been used as anticancer drugs in clinic. However, the anti-cancer mechanism of imidazole drugs is not completely clear. Autophagy and apoptosis are closely related to the occurrence of cancer. At present, the majority of chemotherapeutic drugs are mainly through inhibiting autophagy and promoting apoptosis to achieve the purpose of cancer treatment. In this study, the effects of imidazole on autophagy and apoptosis of human endometrial adenocarcinoma cell line (HEC-1B) were systematically studied in order to reveal the molecular mechanism of anti-cancer effect of imidazole drugs. In autophagy: (1) low concentration of imidazole (5-25mM) could induce vacuolation of HEC-1B cells within 6 hours. Western blotting assay showed that imidazole could significantly increase the content of LC3-II, a marker of autophagy. Furthermore, transmission electron microscopy (TEM) showed that imidazole induced a large number of autophagy-like vesicles (AVs), indicating that imidazole affected the autophagy process of cells. (2) Western blotting assay showed that imidazole did not significantly affect autophagy-priming-related kinases and molecules, indicating that imidazole-treated cells did not initiate autophagy. (3) in the cells treated with imidazole, EGFP-LC3 distributed in dot shape and the level of free EGFP protein decreased significantly. Further Western blotting analysis showed that the level of p62 protein was significantly increased by imidazole treatment. The inhibition of BafA1 did not increase the level of LC3-II in the cells treated with imidazole, which indicated that imidazole blocked the degradation of autophagy. (4) Laser confocal detection of subcellular localization of mRFP-GFP-LC3 showed that imidazole could increase the number of autophages, but no increase in the number of autophagy lysosomes was detected, indicating that imidazole inhibited autophagy maturation to autophagy lysosomes. In the aspect of apoptosis: (1) High concentration of imidazole (50-100mM) could significantly inhibit the survival of HEC-1B cells at 12 h., Western blotting assay showed that caspase9 and caspase3 breaks were induced by imidazole treatment, indicating that imidazole could induce apoptosis of HEC-1B cells. (2) RT-PCR and Western blotting were used to detect the expression of pro-apoptotic protein Bim. The results showed that the expression of Bim mRNA and protein was significantly increased by imidazole treatment. (3) the effect of imidazole on transcription factor Fox03a was detected by Western blotting and protein overexpression. The results showed that imidazole could induce the upregulation of FoxO3a protein and promote its entry into nucleus. (4) further studies showed that siRNA-mediated FoxO3a gene silencing significantly inhibited Bim upregulation induced by imidazole and reduced cell mortality, suggesting that imidazole up-regulated Bim expression by activating Fox03a. In conclusion, imidazole blocks autophagy degradation by inhibiting autophagy maturation; at the same time, imidazole induces apoptosis through FoxO3a-Bim pathway. The results of this study have clarified the molecular mechanism of imidazole on autophagy and apoptosis of HEC-1B cells and provided valuable scientific basis for further research and development of imidazolium anticancer drugs.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33

【共引文献】