DNA拓扑异构酶Ⅱ抑制剂以依赖LXR的方式诱导巨噬细胞ABCA1表达及胆固醇外输

发布时间：2018-08-11 13:32

【摘要】：动脉粥样硬化斑块的形成是心血管疾病的主要病理之一。富集大量胆固醇和脂质的巨噬细胞(亦称为泡沫细胞)是动脉粥样硬化斑块的重要组成成分。因此,维持巨噬细胞内胆固醇的动态平衡对于阻止动脉粥样硬化斑块及心血管疾病的发生发展至关重要。胆固醇逆转运是体内胆固醇代谢的重要过程,能有效地防止冠心病的发生。 ATP结合盒转运蛋白Al1(ATP-binding cassette transporter A1, ABCA1)对心血管系统的保护作用已在人类和动物模型中得到验证。ABCA1利用ATP水解能量,主动、单向地将自由胆固醇和磷脂从巨噬细胞内运输到细胞外胆固醇接收提,是胆固醇外流与逆向转运过程的关键第一步,对初生态HDL的形成及其代谢过程中起着至关重要的作用。细胞中ABCA1的水平主要通过转录来调节,特别是通过配体激活的肝X受体,LXR(一种重要的转录因子)。 Teniposide和etoposide是目前已上市的两种DNA拓扑异构酶Ⅱ的抑制剂。两者的作用机制类似,均促使酶-DNA断裂复合物的形成,使平衡反应趋向于酶-DNA断裂复合物,延长其半衰期并使其稳定。可断裂复合物的形成及稳定存在促进了DNA的异常重组,从而启动细胞凋亡程序而导致细胞死亡,因此,此类抑制剂临床上可应用与肿瘤肿瘤。DNA拓扑异构酶Ⅱ与其抑制剂在肿瘤学和免疫学得到深入的研究,但是否在其他领域发挥功能尚不清晰。有报道发现etoposide可以显著减缓兔中动脉硬化进程,但其机理不明,我们认为DNA拓扑异构酶Ⅱ的抑制剂可通过调节巨噬细胞胆固醇外流及相关分子的表达,从而抑制巨噬细胞泡沫化并发挥抗动脉粥样硬化功能。在此文中,我们通过实验证实DNA拓扑异构酶Ⅱ抑制剂teniposide和etoposide可以有效地诱导巨噬细胞自由胆固醇外流,这种诱导作用与巨噬细胞ABCA1mRNA和蛋白上调相一致。teniposide和etoposide可以诱导ABCA1启动子活性,这种诱导作用与ABCA1启动子上的LXRE密切相关,是以依赖LXR的方式上调ABCA1启动子活性。同时EMSA实验结果证实,teniposide和etoposide有效增强了LXRE-LXR/RXR复合体的形成。启动子实验及EMSA实验结果证明teniposide和etoposide在转录水平上以LXR依赖的方式诱导ABCA1表达。同时,我们也证实了teniposide和etoposide可以诱导其它LXR靶基因ABCG1和FASN的表达。体内实验证明teniposide和etoposide可以有效地诱导体内胆固醇由巨噬细胞向体外的逆转运。在本文中,我们还发现另外一类抗肿瘤药物MEK1/2抑制剂PD98059和U0126亦可以很好地诱导体外和体内巨噬细胞ABCG1的表达和体内胆固醇外流。U0126亦可以很好地诱导动脉粥样硬化斑块处巨噬细胞中ABCG1的表达。综上所述,我们的研究结果证实teniposide和etoposide及其他抗肿瘤药物可以诱导巨噬细胞胆固醇外流分子的表达及胆固醇外输,抑制巨噬细胞内胆固醇的积累,细胞泡沫化与动脉粥样硬化的发展。我们的工作一方面揭示了抗肿瘤药物的新功能,另一方面为这类药物潜在的新功能发挥提供了理论依据。
[Abstract]:The formation of atherosclerotic plaques is one of the major pathologies of cardiovascular diseases. Macrophages rich in cholesterol and lipids (also known as foam cells) are important components of atherosclerotic plaques. Therefore, maintaining the dynamic balance of cholesterol in macrophages can prevent atherosclerotic plaques and cardiovascular diseases. Reverse cholesterol transport is an important process of cholesterol metabolism and can effectively prevent coronary heart disease.
The protective effect of ATP-binding cassette transporter A1 (ABCA1) on cardiovascular system has been demonstrated in human and animal models. ABCA1 actively and unilaterally transports free cholesterol and phospholipids from macrophages to extracellular cholesterol receiving and extracting by using ATP hydrolysis energy, which is cholesterol outflow and reverse. The key first step in the transport process plays a crucial role in the formation and metabolism of primary HDL. The level of ABCA1 in cells is regulated mainly through transcription, especially through ligand-activated liver X receptor, LXR (an important transcription factor).
Teniposide and etoposide are currently listed as two inhibitors of DNA topoisomerase II. Their mechanisms of action are similar, both of them promote the formation of enzyme-DNA breaking complex, make the equilibrium reaction tend to enzyme-DNA breaking complex, prolong its half-life and stabilize it. The formation and stabilization of breakable complex promote the abnormal weight of DNA. DNA topoisomerase II and its inhibitors have been extensively studied in oncology and immunology, but it is not clear whether etoposide can play a role in other fields. It has been reported that etoposide can significantly slow down the rabbit middle artery. We believe that the inhibitor of DNA topoisomerase II can inhibit macrophage foaming and play an anti-atherosclerotic role by regulating the cholesterol efflux and the expression of related molecules in macrophages. In this paper, we confirm that the inhibitors of DNA topoisomerase II, teniposide and etopo, can inhibit macrophage foaming and play an anti-atherosclerotic role. Both teniposide and etoposide can induce the activity of ABCA1 promoter, which is closely related to the LXRE of ABCA1 promoter and up-regulates the activity of ABCA1 promoter in a LXR-dependent manner. Teniposide and etoposide enhanced the formation of LXRE-LXR/RXR complex. Promoter and EMSA experiments showed that teniposide and etoposide induced ABCA1 expression in a LXR-dependent manner at the transcriptional level. Teniposide and etoposide also demonstrated that teniposide and etoposide could induce other LXR target genes ABCG1 and FASN. Experiments in vivo showed that teniposide and etoposide could effectively induce the reversal of cholesterol transport from macrophages in vivo to in vitro.
In this study, we also found that PD98059 and U0126, inhibitors of MEK1/2, could induce the expression of ABCG1 in macrophages in vitro and in vivo and cholesterol efflux in vivo. U0126 could also induce the expression of ABCG1 in macrophages in atherosclerotic plaques.
To sum up, our results confirm that teniposide and etoposide and other antineoplastic drugs can induce macrophage cholesterol efflux molecule expression and cholesterol efflux, inhibit macrophage cholesterol accumulation, cellular foaming and atherosclerosis. Our work reveals new antineoplastic drugs on the one hand. Function, on the other hand, it provides a theoretical basis for the potential new functions of these drugs.
【学位授予单位】：南开大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96

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