新型四氢吡咯衍生物PDF64的抗肿瘤作用及其机制的初步研究

发布时间：2018-01-20 18:13

本文关键词： 肽脱甲酰基酶 PDF64 细胞增殖细胞凋亡自噬 Akt/mTOR通路　出处：《华东师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:在过去几十年中,细菌肽脱甲酰酶(PDF)在致病性微生物中被认为是抗菌药物理想的潜在靶标,并且有很多的细菌PDF抑制剂被广泛报道。然而,最近研究发现肽脱甲酰酶也存在于真核生物中,HsPDF(人线粒体PDF)由细胞核合成而在线粒体中发挥功能,同样催化新生多肽的去甲酰化。HsPDF在乳腺癌、结肠癌和肺癌等多种癌组织中高表达,其抑制剂表现出良好的体内外抗肿瘤活性,HsPDF有望成为极具潜力的新型抗肿瘤靶标。本课题组以目前处于临床试验的细菌PDF靶标化合物LBM415结构为基础,设计合成了一系列结构类似的四氢吡咯类衍生物,初步的研究发现这些化合物存在不同程度的细菌PDF体外抑制活性(IC50=2～12 nM)及肿瘤细胞增殖抑制活性。目的:本论文主要通过体内外实验对这一系列化合物的抗肿瘤活性进行评价,并对其作用机制进行初步研究。体外实验:首先我们通过CCK-8法对本课题组合成的一系列LBM415结构类似物进行抗肿瘤活性筛选及IC50值的测定,最终得到活性较好的化合物PDF64。接着我们运用CCK-8法测定PDF64对不同组织来源肿瘤细胞的抑制活性,实验发现PDF64具有广谱的抑制肿瘤细胞生长的作用(IC50=2～11μM),并呈现一定的肿瘤细胞选择性。体内实验:我们利用HCT116细胞裸鼠荷瘤模型测定了 PDF64的体内抗肿瘤活性,结果显示,高剂量PDF64在体内表现出优异的抗肿瘤作用,但也存在较大的毒性。150 mg/kg PDF组的平均抑瘤率为85.83%,优于等剂量的阳性化合物actinonin(平均抑瘤率73.53%),但其毒性效应导致荷瘤裸鼠的死亡,最后实验组仅有两只荷瘤裸鼠存活。各组肿瘤组织细胞增殖活力指标Ki67的免疫组化检测发现,PDF64组Ki67阳性细胞数目明显低于对照组,甚至低于Actonion组。各组裸鼠心脏、肝脏、肾脏、肺、和脾脏的HE染色分析没有发现PDF64对相关脏器的明显损伤效应,因此高剂量PDF64的毒性效应还需要我们做进一步的探讨。随后我们又进行了低剂量PDF64的抗肿瘤活性研究,50mg/kgPDF64组的平均抑瘤率为83.02%,优于等剂量actinonin组(平均抑瘤率为63.47%),整个实验周期均未出现小鼠的死亡,体重也没有较明显的变化,实验结果提示荷瘤裸鼠能耐受50 mg/kg的PDF64给药剂量,且低剂量的PDF64同样具有非常优异的抗肿瘤活性。机制研究:为了探讨PDF64的抗肿瘤作用机制,我们首先分析了该化合物对细胞凋亡的影响。Annexin V-FITC凋亡实验发现PDF64对Jurkat和HCT116细胞有不同程度的凋亡诱导效应,PDF64对Jurkat细胞的凋亡诱导效应明显高于HCT116,并具有时间剂量依赖性。进一步分析发现,PDF64能明显降低Jurkat细胞线粒体膜电位,而对HCT116细胞的线粒体膜电位影响较小,类似地PDF64能诱导ATP生成量下降,但对Jurkat细胞的作用更明显。在凋亡相关标志蛋白表达的研究中发现,Jurkat细胞经PDF64作用72 h后,其抗凋亡蛋白Bcl-2的表达量明显降低,Caspase-3明显被激活,并检测出PARP-1剪切形式。而在HCT116细胞中,抗凋亡蛋白Bcl-2的表达量并没有明显变化,也没有检测出Caspase3的激活形式,但检测出PARP-1剪切形式。系列实验表明PDF64可能介导了Jurkat细胞的线粒体凋亡途径,而PDF64对HCT116细胞的抑制效应则可能与其他的途径有关。我们采用CFDA SE试剂盒检测PDF64对肿瘤细胞分裂增殖的影响,实验发现PDF64均能时间、剂量依赖性的抑制HCT116、Jurkat细胞的分裂增殖。细胞周期实验发现,PDF64对Jurkat和HCT16细胞的细胞周期分布均没有明显影响,在周期相关标志蛋白表达的研究中发现,PDF64并没有使PCNA、CyclinD1和Cyclin D3的表达量产生明显变化。PDF64能有效地诱导Jurkat细胞进入线粒体凋亡途径,而对HCT116细胞的凋亡效应则不明显,我们推测PDF64对HCT116细胞作用可能是通过自噬来实现的。通过MDC染色结合流式细胞术实验,我们发现PDF64均能有效地诱导HCT116、Jurkat细胞的自噬效应,且对HCT116细胞的自噬诱导效应更为明显。自噬标志分子LC3 Western blotting检测发现,经PDF64作用72h后,在HCT116及Jurkat细胞中LC3-Ⅱ表达量明显增加。已有的实验结果说明,PDF64诱导了HCT116和Jurkat细胞的自噬效应。MAPKs及AKT/mTOR在肿瘤的发生发展中起重要作用,为了进一步分析PDF64的抗肿瘤作用机制,我们对相关通路的蛋白及其激活形式的水平进行了Western blotting检测。结果显示,在HCT116细胞中,PDF64能明显抑制p38、mTOR、Akt等蛋白的激活,而在Jurkat细胞中,PDF64也显著抑制了AKT/mTOR信号通路中mTOR的磷酸化水平,而对其他蛋白的磷酸化及非磷酸化形式影响较小。另外PDF64诱导了转录因子c-Myc蛋白表达量呈剂量依赖性的下降。已有的数据表明AKT/mTOR,p38 MAPK信号通路及转录因子c-Myc可能参与了PDF64引起肿瘤细胞的增殖抑制、凋亡和自噬效应,但其具体机制因细胞类型的不同又略有差异。实验结论:总之,四氢吡咯类化合物PDF64具有良好的体内外抗肿瘤活性,对Jurkat肿瘤细胞具有促凋亡、细胞增殖抑制及自噬效应,对HCT116细胞则主要诱导自噬效应。其抗肿瘤作用可能与AKT/mTOR通路及c-MYC蛋白相关,但具体机制及在两种细胞中效应的异同尚需进一步研究。本研究为此类药物的设计合成提供了实验依据,为PDF抑制剂的抗肿瘤活性研发提供了理论基础。
[Abstract]:Background: in the past few decades, the bacterial peptide deformylase (PDF) in pathogenic microorganisms is considered as a potential target for antibacterial drug ideal, and there are many bacterial PDF inhibitors have been widely reported. However, recent studies found that peptide deformylase also exist in eukaryotes, HsPDF (human mitochondrial PDF) by nuclear synthesis function in mitochondria, the same catalytic hydroformylation of nascent polypeptide to.HsPDF in breast cancer, high expression of a variety of carcinoma of colon and lung, the inhibitor showed good anti-tumor activity in vitro and in vivo, HsPDF is expected to become a new anticancer target potential. This topic in group is currently in the bacterial PDF compounds with LBM415 structure of clinical trials based on a series of four similar structural tetrahydropyrrole derivatives were designed and synthesized, a preliminary study found that these compounds are not the same degree of bacterial PD In vitro inhibitory activity of F (IC50=2 ~ 12 nM) inhibitory activity and the proliferation of tumor cells. Objective: To evaluate the in vitro and in vivo to a series of anticancer activity, and preliminary study on its mechanism. In vitro: first we through the CCK-8 method on this subject are combined into a series of LBM415 structural analogs were determined to study the antineoplastic activity and IC50 value, finally get the compound PDF64. good activity and then we use CCK-8 method for suppressing the activity of PDF64 was measured on tumor cells in different tissues, we found that PDF64 has a broad spectrum of growth inhibition of tumor cells (IC50=2 ~ 11 M), and presents a selection of tumor cells the in vivo experiment: we use tumor model of HCT116 cells in nude mice by PDF64 in vivo antitumor activity, results showed that high dose of PDF64 in vivo showed excellent resistance Tumor effect, but there are also large.150 mg/kg PDF toxicity group average inhibition rate was 85.83%, better than the dose of compound actinonin (average positive inhibition rate 73.53%), but the toxic effects of lead in nude mice death, finally the experimental group only two nude mice survival. Immunohistochemical tumor cell proliferation in each group Ki67 activity index were found in PDF64 group the number of Ki67 positive cells was significantly lower than the control group, even lower than the Actonion group. The nude mice heart, liver, kidney, lung, analysis there is no obvious damage effect of PDF64 on the discovery and related dirty spleen stained with HE, so the toxic effects of high doses of PDF64 we need to make further discussion. Then we studied the antitumor activity of low dose PDF64, 50mg/kgPDF64 group the average inhibitory rate of tumor was 83.02% better than the dose of actinonin group (the average inhibition rate of 63.47%), the The death of mice were not found in the experimental period, body weight did not change significantly. The experimental results suggested that nude mice can tolerate 50 mg/kg PDF64 dose, and low dose of PDF64 also has a very excellent antitumor activity. The mechanism of antitumor effect: in order to investigate the mechanism of PDF64, we firstly analyze the compound effects on apoptosis of.Annexin V-FITC apoptosis PDF64 showed that apoptosis inducing effect of different degree of Jurkat and HCT116 cells, PDF64 induced apoptosis of Jurkat cells was significantly higher than that of HCT116, and has a time dose dependent. Further analysis showed that PDF64 can significantly reduce the mitochondrial membrane potential of Jurkat cells, and mitochondrial membrane potential of HCT116 cells less influence, similar PDF64 can induce the formation of ATP decreased, but the effects on Jurkat cells is more obvious. In the sign of apoptosis related protein expression The study found that Jurkat cells were treated with PDF64 after 72 h, the expression of anti apoptosis protein Bcl-2 decreased significantly, Caspase-3 was activated obviously, and detect the PARP-1 shear form. In HCT116 cells, the expression of the anti apoptotic protein Bcl-2 and did not change significantly, also did not detect the activation of Caspase3, but the detection of PARP-1 splicing forms. A series of experiments show that the PDF64 might be mediated by the mitochondrial apoptosis pathway of Jurkat cells, and the inhibitory effect of PDF64 on HCT116 cells may be associated with the other way. We use CFDA SE kit for detecting PDF64 proliferation of tumor cells, we found that PDF64 was time dose dependent inhibition of HCT116 the Jurkat cell proliferation. Cell cycle experiments, cell cycle distribution of PDF64 on Jurkat and HCT16 cells had no obvious effect on the cycle, related marker protein expression The study found that PDF64 did not make PCNA, expression of CyclinD1 and Cyclin D3 produced significant changes in.PDF64 can effectively induce Jurkat cells into the mitochondrial apoptosis pathway. Apoptosis effect on HCT116 cells is not obvious, we speculate that the effect of PDF64 on HCT116 cells may be realized through autophagy. Combined with the flow cytometry experiments by MDC staining, we found that PDF64 could effectively induce HCT116, autophagy effector Jurkat cells, and HCT116 cells autophagy induction effect is more obvious. LC3 Western blotting showed that the molecular markers of autophagy detection, by PDF64 72h, in HCT116 and Jurkat cells showed significantly increased expression of LC3- II. The experimental results PDF64,.MAPKs and AKT/mTOR induced autophagy effector HCT116 and Jurkat cells play an important role in the occurrence and development of tumor, in order to further analyze the anti-tumor effect of PDF64 We are related to mechanism, pathway activation protein and form the level of the Western blotting test. The results showed that in HCT116 cells, PDF64 inhibited p38, mTOR, Akt protein activation, whereas in Jurkat cells, PDF64 can significantly inhibit the AKT/mTOR signaling pathway in mTOR phosphorylation, and for other protein phosphorylation and non phosphorylation form has little effect. In addition PDF64 induced transcription factor c-Myc protein expression decreased with dose dependence. The existing data show that AKT/mTOR, p38 and MAPK signaling pathways and transcription factor c-Myc might be involved in tumor cell proliferation inhibition of PDF64, apoptosis and autophagy, but its specific because the mechanism of cell types and different slightly different. Conclusion: in short, four hydrogen pyrrole compound PDF64 has good anti-tumor activity in vitro and in vivo, can promote the apoptosis of Jurkat tumor cells, fine Cell proliferation and autophagy of HCT116 cells, mainly induced autophagy effect. Its anti-tumor effect may be related to AKT/mTOR pathway and c-MYC protein, but the specific mechanism and the similarities and differences in the two kinds of cells effect still need further study. The research for the design and synthesis of this kind of drugs provides the experimental basis, provides a theoretical basis for for the development of antitumor activity of PDF inhibitors.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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