| Anticancer peptides are peptides with anticancer activity. They are from animal innate immunity, which exhibit rapid and broad spectrum of activity. Anticancer peptides are generally composed of 5 to 50 amino acid residues, containing certain positively charged residues such as lysine and arginine and substantial hydrophobic residues (-30% or more). These innate immunity host defense peptides play a significant role in the antimicrobial regulation of organism. Most species would release these small molecular peptides when invaded by microorganism. Recently studies have shown that many antimicrobial peptides indicated anticancer activity, which could kill cancer cells in a mode of action similar to antimicrobial mechanism. Although the sources of anticancer peptides are widespread, the function of anticancer peptides depends on a number of physicochemical properties, such as the amino acid sequence, net charge, hydrophobicity, amphipathicity, peptide structure interacting with membrane and self-association, etc. The generally accepted mechanism of anticancer peptides is that the cationic peptides bind to the anionic cell membrane via electrostatic interacton at first, then the peptides insert into the hydrophobic core of cell membrane by hydrophobic interactions, which resulted in cell lysis or induced apoptosis. Current anticancer chemodrugs disrupt normal eukaryotic cells while killing cancer cells, causing different side effects. In addition, cancer cells can reduce the therapeutic effect of chemdrugs by pumping the drugs out of cells using multi-drug-resistant proteins. Anticancer peptides are independent on cell metabolic activity and targeting on cell membrane, which could solving the resistance problem. Anticaner peptides may be promising candidates for clinical practices.The secondary structure of anticancer peptides include three major classes, α-helix,β-sheet, and random coil. When peptides interacting with the membrane, the secondary structure of the peptides could change. There are several proposed mechanisms of action of anticaner peptide, such as cell necrosis, apoptosis, and other signal transduction, etc. However, the interaction between peptide and cell membrane is the essential prerequisite for anticancer activity. Membrane differences between cancer cell and normal eukaryotic cells play a key role in the selectivity of anticancer peptides. For example, The outer membrane of cancer cells contains only a small amount of negatively charged phosphatidylserine (3-9% of the total membrane phospholipids), making only slightly more negative than that of normal eukaryotic cells. Cancer cells exhibit greater transmembrane potential and more membrane fluidity. The alteration of glycosylation of membrane associated glycoproteins and glycolipids on cancer cells creates additional negative charges on the cancer cell's surface. According to the differences between cancerous and eukaryotic cells, we may design peptides with strong anticancer activity and weak hemolysis, thus enhance the selectivity of the anticancer peptides.Objectives:We design a series of peptides with similar sequence on the basis of the amphipathic a-helical antimicrobial peptide V13K (model peptide P) to investigate the effect of hydrophobicity on secondary structure and self-association of peptides, to study the role of peptide hydrophobicity on the anticancer activity against HeLa cells and hemolysis actitivity, and to explore the anticancer mechanism of anticancer peptides.Methods:The interactions between amphipathic a-helical peptides and cell membranes are related with the net charge, hydrophobicity and helicity of the peptides. In the present study, we focus on the hydrophobicity of peptides, using the alanine or leucine substitutions on the non-polar face of the peptides to investigate the relationship of peptide hydrophobicity and anticancer activity. Peptides were synthesized by solid phase peptide synthesis approach and purified by RP-HPLC. The molecular weight of the peptides were verified by ESI-MS. Single-point external standard method on amino acid analysis were executed to quantitate the peptides. The relative hydrophobicity of the anticancer peptides were determined by the peptide retention time using RP-HPLC. Temperature profiling methods were used to monitor the retention behavior of the peptides on RP-HPLC and evaluate the self-association ability of the peptides. The secondary structures of the peptide under aqueous and hydrophobic environments were detected with CD spectra. The anticancer activity and hemolytic activity were measured by 50% inhibitory concentration (IC50) and minimal hemolytic concentration, respectively. The anticancer peptide A12L/A20L was labeled with fluorescein isothiocyanate (FITC) to study on the localization of the peptides on cancer cells. Confocal fluorescence microscopy, flow cytometry, and scanning electron microscopy were used to study the target and mode of action of anticancer peptides.Result:This thesis systematically indicates the role of peptide hydrophobicity on the biological activity of anticancer peptides. We designed peptides with relative hydrophobicity ranging from 32.2 to 49.94 min. The peptides showed major random coil structure in solution and could be induced into different degrees of a-helical structure in 50% TFE-KP buffer which mimicking the environmet of cell membrane. The peptide self-association ability was measured by the parameter PA, greater PA values showing stronger ability of peptide self-association in solution. The order of the peptide hydrophobicity in this study is L6A/L17A< L17A/L21A< L17A< L21A < L6A< P< A12L< A20L< A12L/A20L< A12L/A20L/A23L, which is generally consisitent with the order of anticancer activity, except that the leucine substituted peptides with higher hydrophobiciy than that of peptide P showed no further enhanced anticancer activity (IC50 from 1.2 to 2.0μM). In contrast, increasing the peptide hydrophobicity would enhance the hemolytic activity. Peptide A12L/A20L killed HeLa cells via necrosis with pore forming on the cell surface and resulting in cell membrane lysis.Conclusion:The hydrophobicity of peptides can be altered in two ways:first, by replaceing amino acids, the intrinsic hydrophobicity of the amino acid side chain was changed; and second, peptide hydrophobicity is influenced by the number of i→i+3 and i→i+4 hydrophobic interactions, which affects the continuity of the hydrophobic surface on the non-polar face of the peptide. The peptide self-association ability is influenced dramatically by the number of i→i+3 and i→i+4 hydrophobic interactions, which indicates that the peptide self-association was formed by the hydrophobic interaction of the non-polar face of peptides molecules.As the peptide hydrophobicity increased, the anticancer activity increase consistently. However, when the peptide hydrophobicity reach a threshold, the anticancer activity stays stable. In contrast, increasing peptide hydrophobicity caused the enhanced hemolytic activity. There is no hydrophobic threshold for hemolytic activity. These differences between anticancer and hemolytic activity induced by peptide hydrophobicity change could guide the searching and designing of anticancer peptides with high selectivity.In this study, the amphipathicα-helical anticancer peptides interact with the cell membrane via their non-polar face, forming pores on the surface of the cell membrane and increasing the permeability of the membrane, which causes the cell necrosis. Through our preliminary study, we explained the anticancer mechanism of peptides, thereby lighted up the way for further study on anticancer peptide associated signal transduction, metabolic pathway, and high selectivity design.
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