Ecteinascidins简易结构类似物抗肿瘤作用研究

发布时间：2018-01-18 06:07

本文关键词：Ecteinascidins简易结构类似物抗肿瘤作用研究　出处：《北京协和医学院》2014年硕士论文　论文类型：学位论文

【摘要】：Ecteinascidins (ETs)是一类四氢异喹啉生物碱类海洋天然产物,能够与DNA共价结合,具有显著的抗肿瘤活性和独特的肿瘤类型选择性。其代表化合物ET-743,已在欧洲批准上市,用于软组织肉瘤和卵巢癌的治疗。但其天然产量极低,合成路线繁琐,因此,寻求简易高效的合成类似物成为研究热点。GJ7-1和GJ7-2是经过结构优化合成的ETs简易结构类似物,具有一定的抗肿瘤活性,但与ETs相比,细胞毒性降低了约两个数量级。本文对两个化合物GJ7-1和GJ7-2进行了药理效应特征及DNA靶点专一性的观察,以期提供这类化合物抗肿瘤活性降低的可能药理学基础。 GJ7-1和GJ7-2对体外培养的15种肿瘤细胞都显示一定的生长抑制作用,GJ7-1IC50约为01μmol/L-0.3μmol/L, GJ7-2IC50约为0.05μmol/L-0.16μmol/L, GJ7-2活性稍高于GJ7-1,二者都没有明显的肿瘤类型选择性。在小鼠体内移植肉瘤模型中,GJ7-2(20mg/kg和40mg/kg)每隔一天腹腔给药,10d后与溶剂对照组相比,对EHS肉瘤生长无明显的抑制作用,瘤重和小鼠体重也无明显变化。琼脂糖凝胶电泳及DNA结合荧光竞争实验检测化合物与DNA的结合。结果显示GJ7-1和GJ7-2浓度增加到1mmol/L对DNA迁移阻滞仍无明显影响；100μmol/L两个化合物对嵌入结合DNA的荧光探针以及具有小沟结合作用的探针与DNA结合后的荧光强度都无明显的影响。提示,GJ7-1和GJ7-2与DNA无明显结合。1μmol/L两个化合物作用24h和48h对A549细胞周期有一定影响,但不以对G2/M期阻滞为主；低中高剂量(0.01μmol/L、0.1μmol/L和1μmol/L)化合物作用24h、48h和72h都不能诱导该细胞明显的凋亡。 GJ7-1和GJ7-2对A549细胞热诱导的HSP70的表达具有抑制作用并具有剂量依赖性,这虽然与ET-743作用类似,但ET-743对HSP70诱导表达的抑制作用是通过结合DNA后影响基因转录调控过程引起的,显然诱导抑制机制并不同。极高浓度(5μmol/L)两个化合物作用8h才对A549细胞微管形态和分布产生影响,IC50剂量下无明显影响。细胞毒作用的发挥应与对微管的影响关系不大。综上,GJ7-1和GJ7-2与ETs相比,体内外活性明显降低,且无明显的肿瘤类型选择性。由于DNA靶点特异性作用地丧失,其特征性的细胞周期阻滞和凋亡诱导作用也基本丧失。这可能是由于化合物结构上关键基团的改变造成。
[Abstract]:Ecteinascidins is a kind of marine natural products of tetrahydroisoquinoline alkaloids, which can bind to DNA covalently. ET-743, a representative compound, has been approved for European use in the treatment of soft tissue sarcoma and ovarian cancer, but its natural production is extremely low. Therefore, the search for simple and efficient synthetic analogs has become a research hotspot. GJ7-1 and GJ7-2 are simple structural analogues of ETs synthesized by structural optimization. It has some antitumor activity, but compared with ETs. The cytotoxicity was reduced by about two orders of magnitude. The pharmacological effects of GJ7-1 and GJ7-2 and the specificity of DNA targets were observed. To provide a possible pharmacological basis for the decrease of anti-tumor activity of these compounds. Both GJ7-1 and GJ7-2 showed certain growth inhibitory effects on 15 kinds of tumor cells cultured in vitro. The IC50 of GJ7-1IC50 was about 01 渭 mol/L-0.3 渭 mol/L. GJ7-2IC50 was about 0. 05 渭 mol/L-0.16 渭 mol / L, GJ7-2 activity was slightly higher than that of GJ7-1, neither of them had obvious tumor type selectivity. In mice transplanted sarcoma model, 20 mg / kg and 40 mg / kg GJ7-2G / kg were given intraperitoneally every other day and compared with the solvent control group after 10 days of intraperitoneal administration. There was no obvious inhibition on the growth of EHS sarcoma and no significant changes in tumor weight and body weight of mice. Agarose gel electrophoresis and DNA combined with fluorescence competition assay were used to detect the binding of the compounds to DNA. The results showed that the concentration of GJ7-1 and GJ7-2 increased to 1 mmol / L and still had no effect on DNA migration. Obvious influence; There was no obvious effect of 100 渭 mol/L on the fluorescence intensity of the fluorescent probe embedded with DNA or the probe with small groove binding with DNA, indicating that the two compounds had no significant effect on the fluorescence intensity after binding to DNA. GJ7-1, GJ7-2 and DNA did not bind to 1 渭 mol/L for 24 h and 48 h to affect the cell cycle of A549 cells. But there was no G _ 2 / M phase block. 0. 1 渭 mol / L 0. 1 渭 mol/L and 1 渭 mol / L) compound at low, medium and high doses could not induce the apoptosis of the cells at 24 h for 48 h or 72 h. GJ7-1 and GJ7-2 inhibited the expression of HSP70 induced by heat in A549 cells in a dose-dependent manner, which was similar to that of ET-743. However, the inhibitory effect of ET-743 on the expression of HSP70 was induced by binding to the process of gene transcription regulation after DNA, and obviously the mechanism of the inhibition was not the same. The microtubule morphology and distribution of A549 cells were affected by two compounds at very high concentration (5 渭 mol / L) for 8 h. The effect of cytotoxicity on microtubules was not related to the effect of IC50 on microtubules. Compared with ETs, the activity of GJ7-1 and GJ7-2 decreased significantly in vivo and in vitro, and the tumor type selectivity was not obvious. The specific target of DNA was lost due to the specific effect of GJ7-1 and DNA. The characteristic cell cycle arrest and apoptosis-inducing effect were also basically lost, which may be due to the change of the key groups in the structure of the compound.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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