探究新型端粒酶抑制剂Nilo 22对白血病细胞的杀伤作用与机制  

The Effect and Mechanism of Novel Telomerase Inhibitor Nilo 22 on Leukemia Cells

作　　者：尹晶晶 唐倩 谷佳礼 李亚芳 高慧儿 何媚 杨铭 张文姗 徐惠 王超群 李迎辉 白翠改[2] 高瀛岱 YIN Jing-Jing;TANG Qian;GU Jia-Li;LI Ya-Fang;GAO Hui-Er;HE Mei;YANG Ming;ZHANG Wen-Shan;XU Hui;WANG Chao-Qun;LI Ying-Hui;BAI Cui-Gai;GAO Ying-Dai(State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Institute of Hematology&Blood Disease Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Tianjin 300020,China;High-throughput Molecular Drug Discovery Center,Tianjin International Joint Academy of Biomedicine,Tianjin 300457,China;Department of Pharmacy,Tianjin First Central Hospital,School of Medicine,Nankai University,Tianjin 300192,China)

机构地区：[1]中国医学科学院血液病医院(中国医学科学院血液学研究所),实验血液学国家重点实验室,国家血液系统疾病临床医学研究中心,天津300020 [2]天津国际生物医药联合研究院高通量分子药物筛选中心,天津300457 [3]南开大学医学院天津市第一中心医院药剂科,天津300192

出　　处：《中国实验血液学杂志》2021年第4期1056-1064,共9页Journal of Experimental Hematology

基　　金：国家自然科学基金(NSFC 81970105,81870083和81800165);天津科技计划(18ZXXYSY00010)。

摘　　要：目的:探讨一系列人工合成的小分子化合物对白血病细胞的杀伤作用及其作用机制。方法:采用MTT法检测28个小分子化合物对K562、KA、KG、HA及32D细胞系的杀伤作用,筛选出安全有效的化合物Nilo 22。采用流式细胞术检测该化合物对MLL-AF9小鼠白血病细胞(GFP+细胞)凋亡和周期的影响。集落形成实验(CFU)检测Nilo 22对小鼠白血病细胞集落形成能力的影响,利用体内移植实验进一步验证该化合物对小鼠白血病细胞的杀伤作用。端粒酶活性检测实验以及C-circle实验检测化合物对细胞端粒延长机制的影响,RT-PCR检测经化合物处理后细胞端粒长度的变化。结果:筛选到能有效杀伤白血病细胞系且对32D细胞系相对安全的小分子化合物Nilo 22;相对于尼洛替尼,Nilo 22诱导小鼠白血病细胞凋亡的作用更加显著,轻微地阻滞细胞周期于G0/G1期,同时显著抑制白血病细胞的集落形成能力;体内移植实验结果表明,该化合物显著延缓MLL-AF9白血病小鼠的发病进展;化合物Nilo 22通过抑制端粒酶活性和端粒选择性延长(ALT)机制来抑制端粒的延长。结论:Nilo 22对白血病细胞有显著的杀伤作用,尤其对耐药细胞有很好的杀伤效果,有望成为治疗白血病的先导化合物以解决目前临床常见的白血病耐药和复发问题。Objective: To investigate the cytotoxic effect and its mechanism of the micromolecule compound on the leukemia cells. Methods: The cytotoxic effects of 28 Nilotinib derivatives on K562,KA,KG,HA and 32 D cell lines were detected by MTT assays,and the compound Nilo 22 was screen out. Cell apoptosis and cell cycle on leukemia cells were detected by flow cytometry. The effect of compound screened out on leukemogenesis potential of MLL-AF9 leukemia mice GFP + cells was tested by colony-forming units assays( CFU). The cytotoxic effect was further detected by transplant assays ex vivo. Telomerase activity assay,C-circle assay were used to measure the effects of compound on the length mechanism of telomere,RT-PCR was used to detected the changes of telomere. Results: Nilo 22 serves as the most outstanding candidate out of 28 Nilotinib derivatives,which impairs leukemia cell lines,but spares normal hematopoietic cell line. Comparing with Nilotinib,Nilo 22 could induce the apoptosis of GFP + cells significantly,slightly arrests the cell cycle at G0/G1 phase,and significantly inhibits colony formation and prolong the progression in MLL-AF9 leukemia mice model. The expression showed that the compound could slow the disease progression in MLL-AF9 leukemia mice significantly. Mechanistically,Nilo 22 could reduce the length of telomere by inhibiting telomerase activity and alternative lengthening of telomere( ALT). Conclusion: Nilo 22 shows a significant cytotoxic effect on mice and human leukemia cells,especially for drug resistance cells. Nilo 22 is a promising anti-leukemia agent to solve the common clinical problems of drug resistance and relapse of leukemia.

关 键 词：小分子化合物 白血病干细胞 细胞凋亡 端粒 

分 类 号：R733.7[医药卫生—肿瘤]