靶向阻断神经激肽受体抑制白血病细胞生长的分子机制及其在白血病治疗中的潜在价值

Neurokinin receptors (NK1R and NK2R) are the members of G protein-coupled receptors family involved in the regulation of multiple physiological activities in mammals. In certain types of maligancies, the high expression levels of NK1R and NK2R have been shown to be closely related to tumor formation and progression, but the molecular mechanisms of their actions are unclear. Our previous studies have demonstrated that the expression levels of NK1R and NK2R in the circulated leukocytes of leukemia patients were markedly up-regulated, and targeting NK1R and NK2R significantly inhibited proliferation and induce apoptosis in a panel of leukemia cell lines. In this project, we will extend these data to characterize the functionsal roles of NK1R and NK2R in the growth of leukemia cells; to elucidate the molecular mechanisms of targeting NK1R and NK2R to suppress leukemia cell growth; to identify downstream molecules mediating NK1R and NK2R functions; to determine the inhibitory effects of targeting NK1R and NK2R on leukemia cell growth in vivo; as well as to investigate the relationship among the expression levels of NK1R and NK2R in the circulation and the survival rates and metastasis of leukemia patients. The studies proposed here will support to design NK1R and NK2R based targeting therapeutics including monoclonal antibodies and small molecular inhibitors with high selectivity and low toxic and side effects. Meanwhile, NK1R and NK2R downstream target proteins identified in our present project may become the novel targets for the diagnosis and treatment of leukemia.

神经激肽受体是一种 G 蛋白偶联受体，参与哺乳动物多种生理活性调节。研究表明，神经激肽受体高表达与一些肿瘤发展密切相关，但分子机制尚不明确。申请者前期研究结果显示，白血病患者白细胞中神经激肽受体的表达明显上调，靶向阻断神经激肽受体能显著抑制多种白血病细胞增殖，诱导细胞凋亡。本项目拟在前期结果基础上，通过RNAi等方法确立神经激肽受体在白血病细胞生长过程中的功能；阐明靶向阻断神经激肽受体抑制白血病细胞生长的分子机制，筛选并鉴定相关靶点蛋白；利用白血病荷瘤裸鼠模型和Eμ-Myc白血病小鼠模型，研究靶向阻断神经激肽受体抗白血病的效应；分析白血病患者白细胞中神经激肽受体及靶点蛋白的表达与患者预后之间的关系。本项目的完成将为开发基于神经激肽受体的单克隆抗体或高选择性、低毒副作用的靶向治疗白血病的小分子药物提供基础实验数据支持。同时，本项目研究鉴定出的相关靶点蛋白有可能成为白血病诊治过程的新靶点。

白血病是一类严重危害人类健康的造血系统恶性肿瘤。神经激肽受体（ Neurokinin Receptors， NKRs）是一种 G 蛋白偶联受体，参与哺乳动物体内多种生理活性调节，已成为临床前药靶研究的热点。在本项目中，我们分别用神经激肽受体（NK1R和NK2R）拮抗剂SR140333和SR48968处理粒性白血病细胞后发现拮抗剂能剂量和时间依赖地抑制粒性白血病细胞增殖，同时阻滞细胞周期于G0/G1期，并诱导白血病细胞发生凋亡；拮抗剂抑制白血病细胞增殖的机制与调控周期相关蛋白如Cyclin D1、Cyclin D2、Cyclin D3、Cyclin E1、Cyclin B1、CDK4、CDK5、CDK7、Cdc25A、p15、p16、p18、p21的表达密切相关，同时也与调控凋亡相关蛋白如PARP/Cleaved PARP、Bcl-2、Bcl-xL、Cleaved capsese 3、Cleaved capsese 3、Bax、Bim的表达相关；在动物水平，拮抗剂能抑制荷瘤裸鼠白血病的增殖，同时也能有效缓解白血病骨癌痛小鼠的相关疼痛行为；与正常供者外周血白细胞相比，粒性白血病病人外周血白细胞中神经激肽受体呈现显著高表达，且拮抗剂对正常小鼠骨髓细胞的增殖无显著影响，也不引起正常人外周血红细胞出现溶血现象；蛋白质组学和转录组学研究结合生物信息学分析鉴定出了拮抗剂处理粒性白血病细胞后的数百个差异表达蛋白及差异表达基因，以及差异乙酰化修饰蛋白，生物信息学分析显示这些差异表达蛋白和基因富集于粒性白血病细胞增殖的代谢通路；流逝细胞术检测活性氧，激光共聚焦检测活性氧，seahorse检测线粒体功能，免疫印迹检测信号通路蛋白的表达变化等实验结果显示拮抗剂处理后剂量依赖地引起了粒性白血病细胞基础OCR的快速降低，ATP的产生和质子泄漏，内源性线粒体活性氧水平的增加，该DNA损伤反应是通过ATM和ATR信号通路所介导，表明拮抗剂诱导凋亡是通过活性氧激活了DDR反应。这些研究结果为临床粒性白血病的诊断和治疗提供了新的有效的靶点和新药选择的可能。最后我要特别感谢国家自然科学基金给予的课题资助。