The Effects Of Membrane-active Peptides Temporin-1CEa From Chinese Brown Frog(Rana Chensinensis) On Three Breast Cancer Cells

Membrane-active peptides (MAPs) are usually short, cationic and amphipathic peptides, and displays broad-spectrum antibacterial and anti-cancer activity. Anticancer MAPs exert selective cytotoxicity to cancer cells, but almost no or less toxicity to the human body, therefore MAPs have the potential to be developed as novel type of anticancer agents. Antimicrobial peptides from amphibian assume generally an amphiphilic a-helix structure, in which polar/charged residues are segregated on one side of the helix and hydrophobic residues on the other side. The anticancer activity of MAPs seems to stem from their electrostatic interactions with the charged head groups of membrane lipids, whereas their hydrophobicities are apparently important for insertion into membranes.In this study, temporin-1CEa, a novel amphiphilic MAP consisting17amino acids (nine hydrophobic and seven hydrophilic amino acids), was extracted from the Chinese brown frog skin secretions. CD spectra result showed that temporin-1CEa adopted an unordered structure in the aqueous solution, but had a spectrum with double maxima at208and220nm in solution of50%TFE, which indicated that temporin-1CEa adopted a-helix conformation in lipid membranes and a-helix content was87.59%. MTT assay indicated that temporin-1CEa inhibited the proliferation of the human breast cancer MCF-7, Bcap-37and MDA-MB-231cells lines and showed rapid anti-tumor activity in60min in a dose-dependent manner but not time-dependent manner. The results of hemolysis experiment showed that temporin-1CEa had low cytotoxicity to human red blood cells under the anticancer dose range.Scanning and transmission electron microscopy results showed that the morphological changes could be observed in three breast cancer cells with increasing concentrations of temporin-1CEa,. The surface of breast cancer cells had depression in varying degrees, the cell membrane microvilli reduced significantly, the cell membrane had been damaged, cytoplasm was vacuolization.The results of laser scanning confocal microscopy showed that temporin-1CEa began to influx into the test cancer cells after5min they were co-cultured with cancer cells, and finally entered into cell nucleus in60min, as shown by the increased green fluorescent intensity of FITC-labeled temporin-1CEa. Our results indicated that FITC-temporin-1CEa first binded to cells membrane by electrostatic interaction, then entered into cytoplasm and nucleus, ultimately led to cell death.