新型HDAC抑制剂—苯达莫司汀化合物NL-101的抗白血病作用及机制研究

发布时间：2018-05-27 02:06

本文选题：新型合成药物 + 组蛋白去乙酰化酶抑制剂　；参考：《北京协和医学院》2015年博士论文

【摘要】：研究目的：开发具有生物活性高、毒性低等特点的新药一直是肿瘤治疗研究领域的热点,其中分子合成药物由于能够克服传统化疗药物的缺陷而备受关注。NL-101是一种连接组蛋白去乙酰化酶抑制剂SAHA以及烷化剂苯达莫司汀的活性基团而合成的化合物。本文探讨这种化合物在体外和体内对于白血病细胞的杀伤作用以及相关分子机制。研究方法：采用MTT法检测NL-101、SAHA和苯达莫司汀对多种白血病细胞系增殖能力的影响,用SPSS软件计算半数抑制剂量(ICso),用CompuSyn软件计算SAHA和苯达莫司汀的药物联合指数(CI)；用流式细胞术分析药物处理后细胞的周期和凋亡变化；Western blot检测药物处理后组蛋白H3乙酰化水平,以及蛋白凋亡和DNA损伤相关蛋白包括y-H2AX、PARP、caspase-3、Bax、Bcl-2和Bcl-xL的表达；Ficoll法分离病人原代细胞,瑞氏染色观察细胞经药物处理后的形态改变。体内实验部分,通过逆转录病毒感染构建共表达AML1-ETO和C-KIT突变的移植白血病小鼠模型,给予NL-101、苯达莫司汀和SAHA注射治疗,动态监测小鼠体重以及体内白血病细胞浸润情况,记录各组小鼠生存期。研究结果：在体外实验中：(1)苯达莫司汀和SAHA在白血病细胞中具有协同作用。(2)NL-101对多种白血病细胞系的生长有明显的抑制作用,其中Kasumi-1和NB4细胞的敏感性最为显著；NL-101对正常细胞系的作用较小,显示出对肿瘤细胞作用的选择性；NL-101的抗增殖能力显著高于苯达莫司汀,与SAHA没有显著差异。(3)NL-101导致白血病细胞周期阻滞于S期,以及显著的细胞凋亡。(4)NL-101的细胞毒性作用在分子水平上表现为激活依赖caspase-3的凋亡通路以及Bcl-2家族促凋亡蛋白；NL-101能增加DNA损伤标志蛋白y-H2AX和乙酰化组蛋白H3的表达。(5)NL-101能诱导急性髓系白血病患者的原代细胞发生DNA损伤和细胞凋亡。在体内实验中：(1)成功建立共表达AML1-ETO和C-KIT突变的移植白血病小鼠模型,造血器官中有大量原始细胞(c-Kit+、Gr1-、CD11b-)浸润。(2)NL-101能抑制t(8；21)白血病细胞的增殖和浸润,显著延长白血病小鼠的生存期,具有比苯达莫司汀和SAHA更强的抗白血病活性。(3)NL-101对小鼠体重没有显著影响,小鼠的耐受性良好。研究结论：由SAHA和苯达莫司汀的活性基团合成的新型药物NL-101,在体内及体外实验中均具有抑制白血病细胞增殖以及诱导细胞凋亡的生物学效应。针对作用机制的研究显示NL-101具有抑制组蛋白去乙酰化酶和破坏DNA结构的双重活性。
[Abstract]:Objective: to develop new drugs with high bioactivity and low toxicity has been a hot topic in the field of tumor therapy. Among them, molecular synthetic drugs have attracted much attention because of their ability to overcome the defects of traditional chemotherapeutic drugs. NL-101 is a kind of compound synthesized by linking histone deacetylase inhibitor SAHA and the active group of benzamoastine. The cytotoxicity of this compound to leukemia cells in vitro and in vivo and its molecular mechanisms were investigated. Methods: the effects of NL-101 SAHA and bendamoastine on the proliferation of various leukemia cell lines were detected by MTT assay. SPSS software was used to calculate the dose of ICSU, CompuSyn software was used to calculate the combined index of SAHA and bentamoastine, and flow cytometry was used to analyze the changes of cell cycle and apoptosis after drug treatment. Western blot was used to detect the acetylation level of histone H3 after drug treatment. The expression of protein apoptosis and DNA damage related proteins, including the expression of y-H2AXPnPARPncaspase-3, Bcl-2 and Bcl-xL, was detected by Ficoll method. The morphological changes of the cells treated with drugs were observed by Rayleigh staining. In vivo experiment, the transplanted leukemia mice model which co-expressed AML1-ETO and C-KIT mutations was constructed by retrovirus infection. NL-101, bentamoastine and SAHA were injected into mice to dynamically monitor the body weight and infiltration of leukemia cells in vivo. The survival time of mice in each group was recorded. Results: in vitro, the effects of bendamolastine and SAHA on the growth of various leukemia cell lines were significantly inhibited. The sensitivity of Kasumi-1 and NB4 cells was the most significant. NL-101 had little effect on normal cell lines, which showed that the selective action of NL-101 on tumor cells was significantly higher than that of bentamoastine. There was no significant difference between NL-101 and SAHA. NL-101 resulted in cell cycle arrest in S phase. The cytotoxic effects of NL-101 on apoptosis were as follows: activation of caspase-3 dependent apoptotic pathway and increased expression of DNA damage marker protein y-H2AX and acetylated histone H3 in Bcl-2 family. It can induce DNA damage and apoptosis in primary cells of patients with acute myeloid leukemia. In vivo, the mouse model of leukemia transplanted with AML1-ETO and C-KIT mutations was successfully established. A large number of primitive cells, c-Kit Gr1-Gr1-NL-101, could inhibit the proliferation and infiltration of tn821) leukemia cells, and prolong the survival time of leukemia mice. There was no significant effect on body weight of mice with stronger antileukemia activity than bendamoastine and SAHA. The mice had good tolerance. Conclusion: NL-101, a novel drug synthesized from the active groups of SAHA and bentamoastine, has the biological effects of inhibiting the proliferation and inducing apoptosis of leukemia cells in vivo and in vitro. Studies on the mechanism of action showed that NL-101 had dual activities of inhibiting histone deacetylase and destroying the structure of DNA.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R733.7

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