N-脂酰化对抗菌肽CM4选择性抗癌的影响与分子机制研究

As an important anticancer resource, development of antimicrobial peptides(AMPs) as functional anticancer bio-therapeutics with high anti-cancer activity and low toxicity to normal host cells would represent great progress in cancer treatment. Many reported cancer-selective AMPs either have potent anticancer activity but relatively low selectivity or relativity low anticancer activity but high selectivity, which hampers their use in cancer therapy directly. Thus despite numerous anticancer peptides reported, none of the AMPs has so far made it on to the market. Recently, conjugation of a fatty acid to AMPs has been explored as a method to modulate the antimicrobial activity and selectivity. However, whether anticancer activity and selectivity of AMPs is affected by fatty acid conjugation is still unclear. We have identified that N-acylation (C8-C14) of CM4, a natural cationic amphipathic antimicrobial peptide, enhanced its anticancer activity greatly. At the same time, the toxicity to normal cells and erythrocytes also increased together with the increase of anticancer activity, but the impact on cancer cells was greater than that on normal cells (including normal mammalian cells and erythrocytes). Thus N-acylated CM4 showed higher toxicity and selectivity to cancer cells. Then, at least two questions arise. One is how about the effect of other chain length of fatty acids modification on the anticancer activity and selectivity of CM4? The other is what is the caner-selective toxicity mechanism of N-acylated CM4 complex? Based on an amount of researches, structural moieties in a variety of glycoproteins, glycolipids, phosphoplids, cholesterol-riched lipid raft have been shown to act as membrane-based factors that contribute to the cancer-selective toxicity. We hypothesized that different chain length of fatty acids has different effect in modulating the relationship of anticancer activity and selectivity, which may resulted in different properties and effect on the structure of N-acylated CM4 hybrid compounds. And these changes modulated the interaction on plasma membrane and mitochondrial membrane both in the cancer cells and normal cells. We hypothesized that the effective charges influenced by acylation was still the key factor to cancer-selective binding by electrostatic interactions. Based these hypothesis, we decided to conjugate different chain length acyl(C2-C16) to the N terminus of antimicrobial peptide CM4, then focus on the effect of acyl on the plasma membrane binding, penetrating, trans-membrane transportation and mitochondrial targeting between cancer cells and normal cell. We hope to explore the rules of the N-acylation on the relationship of anticancer activity and selectivity, to deeply understand the molecular mechanism of cancer-selective toxicity of N-acylated CM4. Our research will provide important theoretical foundation to develop acylated antimicrobial peptides as therapeutic anticancer compounds in the future.

具有选择性抗癌活性的抗菌肽因能克服癌细胞耐药性等优点而成为新一类抗癌资源。近年来发现脂酰化修饰能够改变抗菌肽的抗菌活性与选择性，然而脂酰化修饰是否影响抗菌肽的抗癌活性与选择性未见报道。我们已证实不同脂链修饰能极显著提高抗菌肽CM4的抗癌活性，脂酰化CM4表现出较好的选择性，并且不同脂链对抗癌选择性作用存在差异。我们推测脂酰基对抗癌活性及选择性的影响与脂酰基引起的生化性质与分子结构的改变密切相关，这些改变影响与肿瘤细胞、正常细胞质膜以及线粒体膜的结合与作用，是脂酰化CM4肽选择性抗癌作用的重要机制。围绕以上假设，本项目拟从不同链长脂酰化修饰CM4与不同质膜的结合、跨膜转运以及胞内线粒体定位与作用进行研究，探寻脂酰基对抗癌活性与选择性间关系的影响规律，阐明脂-CM4肽抗癌选择性的分子机制。项目成果对于深化抗菌肽选择性抗癌的分子机制；并为发展脂酰化修饰在抗菌肽抗癌领域的应用提供重要的理论基础。

抗菌肽作为一类重要的抗癌资源，由于抗癌活性不高或缺乏对癌细胞的选择性，迄今尚未应用于临床。本研究对抗菌肽CM4进行C4-C16脂酰链长的N-脂酰化修饰，旨在探索并发展新一类高效低毒的抗癌药物。开展如下研究：（1）N-脂酰化CM4理化性质分析；（2）不同链长N-脂酰化CM4对乳腺癌细胞抗癌活性与选择性的影响规律分析；（3）N-脂酰化CM4抗癌高效性与选择性的细胞膜机制分析；（4）N-脂酰化CM4与线粒体膜相互作用与效应分析；（5）高效低毒N-脂酰化CM4对白血病与脑瘤细胞作用分析。取得主要研究结果：（1）N-脂酰化CM4的疏水性、α-螺旋含量（螺旋度）及对胰蛋白酶的稳定性随脂酰基链长增加而增加，C12-C16长链修饰能显著提高N-脂酰化CM4对胰蛋白酶的稳定性；（2）中长链修饰能提高CM4抗癌活性；且随链长增加，N-脂酰化CM4对乳腺癌细胞的毒性增强、克隆形成能力降低、细胞周期阻滞能力增强；红细胞溶血性和正常细胞毒性也增强；然而仍表现为腺癌细胞的选择性杀伤；（3）脂酰化CM4通过正电荷与乳腺癌细胞表面负电荷分子的电荷作用是N-脂酰化CM4差异性结合于肿瘤细胞与正常细胞的主要因素，是N-脂酰化CM4选择性抗癌的重要机制；（4）N-脂酰化CM4通过增强的疏水性和α-螺旋度促进与乳腺癌质膜脂质双分子层的作用，当疏水性达到C12的阈值时，可以介导N-脂酰化CM4跨膜转运至乳腺癌细胞胞内；（5）长链（C12-C16）脂酰化CM4在乳腺癌细胞中可以靶向线粒体，影响线粒体膜势，增加线粒体膜通透性，诱导ROS的产生进而影响线粒体功能，并上调促凋亡/抗凋亡蛋白和阴离子通道蛋白VDAC，释放CytC启动线粒体依赖的凋亡途径；（6）C14-CM4对白血病细胞以及C12-CM4对脑瘤细胞都具有很好的杀伤活性，且都可以通过靶向线粒体诱导线粒体依赖的凋亡。本研究全面揭示不同链长脂酰化对N-脂酰化CM4选择性抗癌的分子机制，发现C12-C16长链修饰（尤其是C12），无论是抗癌活性还是对正常细胞的毒副作用，都表现出很好的应用前景，为发展脂酰化抗菌肽应用于抗癌药物提供了重要的理论基础。

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