靶向FOXM1c多肽先导药物P201对肝癌细胞的杀伤作用及优化设计

发布时间：2018-07-03 20:59

本文选题：FoxM1 + 肝癌　；参考：《西南交通大学》2016年硕士论文

【摘要】：肝癌是一种常见的严重危害公众健康的恶性肿瘤,我国每年约来13万人死于肝癌,成为受肝癌影响最大的国家之一。FoxM1作为Fox家族的特殊成员,在多种人类癌症细胞中过量表达,与癌症晚期、癌细胞转移、高增殖、化疗耐药性以及预后不良等密切相关,还参与多种癌症相关基因的表达及信号传导途径。因此,FoxM1被认为是抗癌药物治疗与干预的重要药靶与标记。以FOXM1cDNA结合域为靶标,通过噬菌体展示肽库筛选出的高亲和力多肽P201可能成为未来靶向抗肿瘤治疗药物。本研究通过一系列细胞、分子生物学实验和计算机辅助药物设计方法,初步探讨P201多肽对肝癌细胞的杀伤作用和死亡途径,再对多肽进行氨基酸水平的优化设计,并检验优化后多肽对肝癌细胞的杀伤作用,发现多肽的关键氨基酸。MTT细胞活力测定和形态观察显示,P201多肽能够显著抑制人肝癌HepG2细胞的活力,60μg/mL浓度作用48h抑制率达到96%,计算IC50为25.16μg/mL。P201多肽对MCF-7和293T细胞增殖也具有一定杀伤作用,60μg/mL浓度作用48小时抑制率分别为64.1%和61.9%,形态上变化明显,并且在一定浓度范围内具有时间剂量依赖性。在此基础上,进一步定性实验AO-EB细胞双染以及定量实验流式细胞术,发现P201多肽对HepG2细胞的杀伤作用与诱导细胞凋亡有关,但可能存在多种死亡途径,需进一步研究证明。在实验探讨P201多肽的抗癌活性和可能机制基础上,基于虚拟筛选对P201多肽进行优化,模拟丙氨酸突变,运用LibDock、CDOCKER和MVD等分子对接方法,发现第10位氨基酸残基为多肽优化位点,并确定第10位氨基酸优化为天冬氨酸；结合模体序列搜索、反义氨基酸分析、虚拟丙氨酸突变及分子对接的结果,确定第1、2、4、7、9位5个P201关键氨基酸。最后通过CCK-8法检测发现优化后的多肽M10aa-D对HepG2细胞杀伤作用显著低于P201多肽,100μg/mL浓度作用48h抑制率仅26.3%,在低浓度下甚至促进细胞增殖。多肽对FOXM1的亲和力与其对细胞的杀伤作用呈相关性。综上所述,P201多肽一方面具有成为靶向抗肿瘤治疗药物的潜力,同时尚待进一步分子作用机理研究和其他水平的优化,以满足抗癌多肽生物技术药物研发的要求。
[Abstract]:Liver cancer is a common malignant tumor that seriously endangers public health. About 130000 people die of liver cancer every year in China. FoxM1, as a special member of the Fox family, has become one of the countries most affected by liver cancer, and it is overexpressed in many kinds of human cancer cells. It is closely related to advanced cancer metastasis, high proliferation, chemotherapeutic resistance and poor prognosis. It is also involved in the expression and signal transduction pathway of many kinds of cancer-related genes. Therefore, FoxM1 is regarded as an important drug target and marker for anticancer drug therapy and intervention. Using FOXM1 cDNA binding domain as the target, P201, a high affinity peptide screened from phage display peptide library, may be used as a target antitumor drug in the future. In this study, a series of cell, molecular biological experiments and computer-aided drug design methods were used to study the killing effect and death pathway of P201 polypeptide on hepatoma cells, and then to optimize the amino acid level of the peptide. The killing effect of the optimized polypeptide on hepatoma cells was tested. It was found that the activity of the key amino acid, MTT cell, and morphological observation showed that P201 polypeptide could significantly inhibit the activity of HepG2 cells. The inhibitory rate of P201 peptide on MCF-7 and 293T cell proliferation was calculated to be 25.16 渭 g / mL.P201 polypeptide for 48 h after exposure to 60 渭 g / mL concentration. The inhibitory rates of 60 渭 g / mL for 48 hours were 64.1% and 61.9%, respectively. And in a certain concentration range of time and dose dependent. On this basis, further qualitative experiments of AO-EB cell double staining and quantitative flow cytometry showed that the killing effect of P201 polypeptide on HepG2 cells was related to the induction of apoptosis, but there may be many death pathways, which need to be further studied and proved. On the basis of the experimental study on the anticancer activity and possible mechanism of P201 polypeptide, the P201 polypeptide was optimized based on virtual screening, and the alanine mutation was simulated. The 10th amino acid residue was found to be the optimal peptide site by using the molecular docking methods such as LibDocktl CDOCKER and MVD. The 10th amino acid was optimized as aspartic acid, combined with motif sequence search, antisense amino acid analysis, virtual alanine mutation and molecular docking results, 5 P201 key amino acids were determined at the 9th position. Finally, CCK-8 assay showed that the cytotoxicity of the optimized polypeptide M10aa-D to HepG2 cells was significantly lower than that of P201 polypeptide 100 渭 g / mL for 48 h. The inhibition rate of M10aa-D on HepG2 cells was only 26.3g / mL at low concentration, and even promoted the proliferation of HepG2 cells at low concentration. The affinity of polypeptide to FOXM1 was correlated with its cytotoxicity. In conclusion, P201 polypeptide has the potential to be a target antitumor drug, and needs further molecular mechanism research and other optimization to meet the requirements of anti-cancer polypeptide biotechnology drug development.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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