【作者】 张云；

【导师】 倪京满；

【作者基本信息】 兰州大学 ， 基础医学·中西医结合临床， 2014， 博士

【摘要】 在癌症治疗中,传统化疗药的频繁使用导致多药耐药现象频繁出现,使化疗药物丧失疗效,严重威胁着人类健康和生命安全。因此,发展新型高效的抗肿瘤药以抵御耐药癌细胞显得至关重要。在天然免疫系统中发挥重要作用的抗菌肽(AMPs)由于具有高效的抗肿瘤活性、对传统耐药机制敏感性低的特点,被人们广泛认为是一种能抵御多药耐药现象的候选抗癌药物。另外,与抗菌肽结构特征相类似、被广泛用以药物输送的细胞穿膜肽(CPPs)对改善传统抗癌药的治疗效果表现出很大的潜力。所以AMPs和CPPs可以作为优选的研究模板用以研究开发新型高效的抗肿瘤药物。本论文分别以新型抗菌肽NS、TAT与喜树碱共辄物TAT-CPT/2CPT、[Cys-CPT2,9]penetratin为研究对象,通过一系列研究考察了它们的抗肿瘤活性及作用特点,对其在抗肿瘤应用中的潜在价值进行评价。1.新型抗菌肽NS在抗肿瘤应用中的多重功能特点。在本研究中我们评价了新型杂合抗菌肽NS的抗肿瘤活性,并对其作用机制进行了研究。实验结果表明,NS能通过扰动细胞膜和抑制细胞内DNA合成的双重作用机制迅速地抑制和杀死肿瘤细胞。另外,与许多已经报道的穿膜肽一样,NS经N末端的18烷基化修饰后能有效地输送质粒进入细胞;当N/P比率为3:1和4:1时,其转染效率比单独质粒转染水平高出数倍数量级,这表明NS还可发展为潜在药物载体用以肿瘤基因治疗。因此,NS有效的肿瘤细胞杀伤作用与质粒输送能力的结合使它可以作为肿瘤治疗的候选药物加以深入研究,使其开发成为抵御多药耐药现象的抗肿瘤药物。2.TAT-CPT/2CPT共辄物的抗肿瘤活性研究。本文通过可裂解的二硫键连接子将喜树碱(CPT)连接到经典的细胞穿膜肽TAT的N末端合成了共轭物TAT-CPT和TAT-2CPT,并评价了共轭物的抗肿瘤活性和相应的作用机制。实验结果表明:TAT共轭连接后可提高喜树碱的抗肿瘤活性,其中以共轭物TAT-2CPT活性最高。有意思的是,共轭物不仅可以通过上调caspase3/7活性水平杀死肿瘤细胞,它还能类似于抗菌肽,在短时间内干扰并破坏肿瘤细胞膜快速地发挥抗肿瘤活性;另外,通过分子模拟动力学研究显示共辄物可以明显升高膜脂质分子层饱和SN1链走向参数(+0.0043,+0.0143),进一步证实了共轭物与膜脂质分子层存在的干扰作用。因此,具有细胞膜靶向和胞内作用的共辄物在肿瘤治疗中更具有优势。3.具有抗肿瘤活性的高效穿膜肽[Cys-CPT2,9]penetratin的设计。在前期研究的基础上,我们以细胞穿膜肽penetratin为母体采用扩大疏水性结构域的方法,将Cys-CPT(半胱氨酸-喜树碱)引入到penetratin氨基酸序列中第2、9位,合成了 penetratin类似物[Cys-CPT2,9]penetratin,旨在探索具有抗肿瘤活性且穿膜活性强的新型细胞穿膜肽。细胞摄取实验结果表明[Cys-CPT2,9]penetratin能通过网格蛋白、巨胞饮介导的内吞途径内化进细胞;与母体penetratin相比,它的穿膜效率明显提高5倍。另外,[Cys-CPT2,9]penetratin还能有效地抑制和杀死肿瘤细胞,显示明显的抗肿瘤活性。因此,与penetratin相比,[Cys-CPT2,9]penetratin高效的穿膜能力和显著的抗肿瘤活性明显增加其在抗肿瘤应用中的潜力。总之,本文通过一系列实验研究对新型抗菌肽NS、细胞穿膜肽TAT与喜树碱共轭物、[Cys-CPT2,9]penetratin功能特点进行分析,考察了它们在肿瘤治疗中的潜在价值,为建立新型的肿瘤治疗策略进行有益的探索。更多还原

【Abstract】 In cancer treatment,the constant use of traditional chemotherapeutical agents causes a dramatic increase in the emergence of multidrug resistant tumors and many chemotherapeutical agents are becoming increasingly ineffective,which seriously threaten public health and life safety.Therefore,development of alternative agents becomes more and more critical to fight against resistant tumors.The antimicrobial peptides(AMPs)play an essential part in the innate immune response.Nowadays,they are considered as a novel class of antitumor candidate with characteristics including potent activity against tumor cells and low susceptibility to conventional mechanisms.In addition,cell-penetrating peptides(CPPs)share the common structural features with AMPs.They are widely used to deliver various molecular into cells because of their membrane translocating ability.In cancer therapy,they also could play a promising role in improving therapeutic efficiency of chemotherapeutical agents.Therefore,AMPs and CPPs can be regarded as ideal templates for the development of better antitumor drugs with potent activity.In this paper,we synthesized a series of new antitumor agents,including novel antimicrobial peptide NS,TAT-CPT/2CPT conjugates constructed by combining camptothecin with cell penetrating peptides TAT,and[Cys-CPT2,9]penetratin.In order to evaluate their potential in antitumor application,we systematically studied their antitumor activity and functional properties.1.Multiple functional properties of novel antimicrobial peptide NS in antitumor application.In this study,we evaluated its cytotoxicity on tumor cells and studied the possible mechanism of action.The results showed that NS could efficiently kill tumor cells by disrupting membrane and inhibiting DNA synthesis.In addition,it was found that NS could efficiently deliver plasmids into cell after N-terminal stearyl modification,like many reported cell-penetrating peptides.Stearyl-NS at 3:1 and 4:1 N/P ratios exhibited several orders of magnitude higher transfection efficiency than plasmid only,suggesting that NS could work as a potential vector for tumor gene therapy.Taken together,the ability of NS to kill tumor cells and deliver plasmid indicates that it has the potential to develop into a new antitumor agent to defend against multidrug-resistant tumor.2.Antitumor activity of conjugates TAT-CPT/2CPT.In this part,we introduced CPT group to the N-terminal of TAT via a releasable disulfide carbonate linker and synthesized TAT-CPT and TAT-2CPT conjugates.Additionally,their antitumor activity and detailed action mode were further determined and analysized.It has been shown that the linked TAT could enhance antitumor activity of CPT;the conjugate TAT-2CPT dispiayed more effective killing activity against tumor cells.Interestingly,conjugates could effectively kill tumor cells via rapid membrane disruption,similar to antimicrobial peptides.In addition,they could also exhibit antitumor activity via higher level of activation of caspase3/7.Furthermore,the results of molecular dynamics(MD)simulations showed that conjugates could lead to obvious orientation changes of saturated SN1 chain(+0.0043,+0.0143),further clarifying the perturbation of conjugates and membrane bilayers.The results reveal that membrane targeting and intracellular interaction make conjugates highly promising for future antitumor application.3.Design of[Cys-CPT2,9]penetratin with high membrane penetrating ability and antitumor activity.Based on the previous research,a analogue of penetratin,[Cys-CPT2,9]penetratin,was designed and synthesized by substitution of Gln2 and Asn9 residue with Cys-CPT.We hoped to develop a more intriguing cell penetrating peptide with potent membrane penetrating ability and effective antitumor activity by enlargiing hydrophobic patch.Cell uptake results indicated that the internalization of[Cys-CPT2,9]penetratin was mainly mediated by clathrin and macropinocytosis-mediated endocytosis.Its translocating efficiency was 5 times higher in comparison with the level of penetratin.In addition,[Cys-CPT2,9]penetratin could effectively inhibit and kill tumor cells,exhibiting remarkable antitumor activity.Thus,[Cys-CPT2,9]penetratin has more appealing potential in future antitumor therapy because of its efficient penetrating ability and higher antitumor activity.In the present study,we systematically studied the functional properties of NS,TAT-CPT/2CPT conjugates and[Cys-CPT2,9]penetratin,and evaluated their potential to be used as promising antitumor candidates.The findings obtained from our study would contribute to the establishment of novel strategy for tumor treatment.更多还原