基于色氨酸代谢通路的抗肿瘤小分子化合物的筛选评价

发布时间：2018-07-11 14:27

本文选题：色氨酸 + 犬尿氨酸　；参考：《南京大学》2015年博士论文

【摘要】：L-色氨酸是人体体内不能合成的8种必需氨基酸之一,在体内代谢途径包括由色氨酸羟基化酶(tryptophan hydroxylase)等介导的芳香环羟基化反应,是合成褪黑素和抑制性神经递质5-羟色胺的重要途径;而由吲哚胺-2,3-双加氧酶(indoleamine-2,3-dioxygenase,IDO)介导的降解途径,是合成烟酸/维生素B3等生理物质的重要途径。色氨酸摄入不足或者色氨酸代谢异常可以导致婴幼儿发育迟缓以及精神抑郁等。色氨酸代谢主要发生在小肠和中枢神经系统,但是在肿瘤细胞中异常活跃,与某些肿瘤的发生以及肿瘤免疫逃逸密切相关。色氨酸在肿瘤细胞中主要通过IDO降解,其降解产物之一犬尿氨酸(kynurenine,Kyn)不但可以抑制树突状细胞(dendritic cell,DC)功能,还可以与芳香族化合物受体(aryl hydrocarbon receptor,AHR)结合,激活AHR通路,从而促进肿瘤细胞生长和迁移。临床试验证明,选择性抑制IDO具有明显治疗作用。本研究建立了以Kyn-AHR为评价系统的小分子化合物筛选体系,以脑胶质瘤模型评价了这类化合物的抗肿瘤活性以及作用机制。首先,我们利用最新的基因敲除技术CRISPR/Cas9建立了 AHR基因缺陷的脑胶质瘤稳定细胞系,发现AHR缺失的细胞系与野生型细胞系相比生长速度减慢,并且肿瘤集落形成能力显著降低。AHR作为细胞内一个重要的转录因子,与配体结合后进入细胞核内行使转录调控功能,我们发现AHR基因敲除后不仅能够显著抑制其下游基因CYP1B1的表达,同时还可以降低其他促进肿瘤生长的炎症因子的水平如IL-8和IL-6等。在脑胶质瘤细胞中,AHR敲除后不仅能够降低细胞本身炎症因子mRNA的表达并且能够显著减少TNF-α诱导的炎症因子的表达。通过建立小鼠的皮下肿瘤模型,我们进一步确认了 AHR敲除细胞系在体内的肿瘤生长能力显著低于野生型细胞系。基于敲除AHR基因可以显著降低肿瘤的生长能力,我们利用荧光素酶报告基因方法筛选出了两个能够抑制Kyn-AHR通路的小分子化合物,并利用免疫印迹技术对其抑制活性进行了验证。肿瘤集落形成实验证明候选化合物能够抑制肿瘤细胞的生长,而利用核磁共振成像技术显示小分子化合物抑制AHR活性可以在体内显著抑制脑胶质瘤的生长以及提高小鼠的生存率。进一步的研究发现,小分子化合物主要通过抑制AHR的入核来抑制AHR的功能从而发挥抗肿瘤活性。综上所述,我们利用CRISPR/Cas9基因敲除技术证明了抑制AHR的功能可以抑制脑胶质瘤的生长。通过建立体外筛选模型,从天然产物和合成的小分子化合物中筛选出犬尿氨酸激活AHR通路的抑制剂,利用生物化学方法和小鼠动物模型系统证明了这些抑制剂具有抗脑胶质瘤作用,并进一步阐明小分子化合物抑制色氨酸代谢和抗胶质瘤作用的分子机理,探讨抑制Kyn-AHR信号通路作为抗脑胶质瘤治疗方案的可能性,有望发现新的候选药物靶点分子。
[Abstract]:L-tryptophan is one of the eight essential amino acids that cannot be synthesized in human body. The metabolic pathways in vivo include aromatic cyclohydroxylation mediated by tryptophan hydroxylase (tryptophan hydroxylase) and so on. It is an important way to synthesize melatonin and 5-hydroxytryptamine, and the degradation pathway mediated by indoleamine-2o 3-dioxygenase (IDO) is an important pathway for the synthesis of physiological substances such as nicotinic acid / vitamin B3. Inadequate tryptophan intake or abnormal tryptophan metabolism can lead to stunting and depression in infants and young children. Tryptophan metabolism mainly occurs in the small intestine and central nervous system, but it is very active in tumor cells, which is closely related to the occurrence of some tumors and tumor immune escape. Tryptophan is mainly degraded by IDO in tumor cells. Kynurenine kyn, one of the degradation products of tryptophan, can not only inhibit the function of dendritic cells (dendritic cells), but also bind to (aryl hydrocarbon receptor AHR, and activate the AHR pathway. Thus promoting the growth and migration of tumor cells. Clinical trials have proved that selective inhibition of IDO has obvious therapeutic effect. In this study, a screening system of small molecular compounds with Kyn-AHR as evaluation system was established, and the antitumor activity and mechanism of these compounds were evaluated by glioma model. First of all, we used the latest gene knockout technique CRISPR-Cas9 to establish a stable glioma cell line with AHR gene deficiency. We found that the AHR deficient cell line was slower than the wild-type cell line. The ability of tumor colony formation was significantly reduced. AHR, as an important transcriptional factor in cells, entered the nucleus to perform transcriptional regulation after binding with ligands. We found that the knockout of AHR gene not only inhibited the expression of CYP1B1, but also decreased the levels of other inflammatory factors such as IL-8 and IL-6. AHR knockout in glioma cells not only decreased the expression of inflammatory factor mRNA but also significantly reduced the expression of TNF- 伪 -induced inflammatory factor in glioma cells. By establishing a subcutaneous tumor model in mice, we further confirmed that the tumor growth ability of AHR knockout cell line was significantly lower than that of wild-type cell line in vivo. Based on the fact that knockout of AHR gene can significantly reduce tumor growth ability, two small molecular compounds which can inhibit Kyn-AHR pathway were screened by luciferase reporter gene method, and their inhibitory activity was verified by Western blotting. The tumor colony formation assay showed that the candidate compounds could inhibit the growth of tumor cells. Magnetic resonance imaging showed that the inhibition of AHR activity by small molecular compounds could significantly inhibit the growth of gliomas and improve the survival rate of mice in vivo. Further studies have shown that small molecular compounds inhibit the function of AHR by inhibiting the nucleation of AHR and thus play an important role in antitumor activity. In conclusion, we use CRISPRR / Cas9 gene knockout technique to prove that inhibiting the function of AHR can inhibit the growth of glioma. An in vitro screening model was established to screen out the inhibitors of canine uric acid activating the AHR pathway from natural products and synthetic small molecular compounds. The biochemistry method and mouse animal model system showed that these inhibitors had anti-glioma effect, and further elucidated the molecular mechanism of the inhibition of tryptophan metabolism and anti-glioma action by small molecular compounds. To explore the possibility of inhibiting Kyn-AHR signaling pathway as an antiglioma therapy, we hope to find new candidate drug target molecules.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R73-36

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