Membrane Active And DNA Binding Related Dual Mechanism Of Anticancer And Antibacterial Activity Of NK-18, The Core Region Of Mammalian NK-lysin

Cancer is one of the important social issues throughout the world. Although cancer chemotherapy has made great progress with the development of technology, the increasing phenomenon of multi-drug resistance of cancer cells to chemotherapy drugs has become a major obstacle in cancer treatment. Many cancer patients undergoing chemotherapy treatment become insensitive to chemotherapy drugs due to multi-drug resistance and have to face the consequences of treatment failure. Similar to the predicament of multi-drug resistant cancer cells, a large number of multi-drug resistant bacteria have evolved due to the misuse of traditional antibiotics in recent years. Therefore, it became increasingly urgent to find and develop novel chemotherapy drugs and antimicrobial agents against multi-drug resistant cancer cells and bacteria.Antimicrobial peptides have been found to kill pathogenic microorganisms rapidly in vivo, have broad spectrum biological activities, active against many clinical multi-drug resistant cancer cells and bacteria, and do not seem to induce resistant mutants quickly in the targeted cancer cells or bacteria in vitro. Hence, they are widely recognized as effective candidates against multi-drug resistance. NK-lysin, a 78-amino acid protein isolated from mammals, is an effector protein of cytotoxic T cells and the neutral killer cells, and possesses potent anticancer and antibacterial activity. However, the formidable length of the peptide not only causes technical difficulties in synthesis but also makes it costly, both of which limited its further application. Therefore, the aims of this research are to develop antimicrobial peptides which could be potential clinical drugs based on the NK-lysin template and to investigate the mechanism of their biological activity.In this study, a suit of derivatives, named NK-18, NK-13 and NK-9, were designed and synthesized by gradual omission of the a-helices from the C-terminal of NK-2, the core region of NK-lysin, to find the minimum core functional fragment. By comparing the anticancer activities of these derivatives, NK-18 was found to be the minimum core functional fragment of NK-lysin. The action mechanism of NK-18 against cancer was also investigated. It was found that NK-18 exhibited potent anticancer activity against a series of cancer cells, including multidrug-resistant cancer cells, by disrupting the integrity of cell membrane. Meanwhile, NK-18 had negligible effect on erythrocytes and low cytotoxicity on mouse fibroblast NIH3T3, human umbilical vein endothelial cells HUVEC, and human embryonic kidney cells HEK293, three normal cell lines. In addition, the structure-activity relationship studies showed that NK-18 adopted different secondary conformations in different environments and the a-helix content affected their anticancer activity directly.The antibacterial activity of NK-18 and the corresponding action mechanism were further investigated. It was found that NK-18 could inhibit or kill both Gram-negative and -positive bacteria quickly in a dose-dependent manner. NK-18 exhibited its antimicrobial activity by a dual mechanism of disturbing and interfering with the cell membrane outside the bacteria cell and binding with DNA in the bacterial cytoplasm. In this process, the cationic characteristic of NK-18 is the basis of its selective effect against the bacteria. Besides the direct antibacterial activity, NK 18 could also bind with lipopolysaccharides (LPS) released from dead bacteria and reduce the cytotoxicity of LPS to prevent and suppress inflammatory reaction caused by LPS, the production of which could induce intracellular NO signaling molecules and inflammation.In all, based on this work, NK-18, the minimum core functional fragment of NK-lysin, has potent anticancer and antibacterial activities. Compared with NK-lysin, NK-18 maintained the rapid mode of action, but has unique dual action mechanism and much shorter amino acid sequence, hence lowered the cost for chemical synthesis. All these features of NK-18 make it a promising lead drug template in developing novel drugs against multi-drug resistance.