Pleurocidin-family cationic antimicrobial peptides are cytolytic for breast carcinoma cells and prevent growth of tumor xenografts

Introduction: Cationic antimicrobial peptides (CAPs) defend against microbial pathogens; however, certain CAPs also exhibit anticancer activity. The purpose of this investigation was to determine the effect of the pleurocidin-family CAPs, NRC-03 and NRC-07, on breast cancer cells. Methods: MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) and acid phosphatase cell-viability assays were used to assess NRC-03- and NRC-07-mediated killing of breast carcinoma cells. Erythrocyte lysis was determined with hemolysis assay. NRC-03 and NRC-07 binding to breast cancer cells and normal fibroblasts was assessed with fluorescence microscopy by using biotinylated-NRC-03 and -NRC-07. Lactate dehydrogenase-release assays and scanning electron microscopy were used to evaluate the effect of NRC-03 and NRC-07 on the cell membrane. Flow-cytometric analysis of 3,3'-dihexyloxacarbocyanine iodide- and dihydroethidium-stained breast cancer cells was used to evaluate the effects of NRC-03 and NRC-07 on mitochondrial membrane integrity and reactive oxygen species (ROS) production, respectively. Tumoricidal activity of NRC-03 and NRC-07 was evaluated in NOD SCID mice bearing breast cancer xenografts. Results: NRC-03 and NRC-07 killed breast cancer cells, including drug-resistant variants, and human mammary epithelial cells but showed little or no lysis of human dermal fibroblasts, umbilical vein endothelial cells, or erythrocytes. Sublethal doses of NRC-03 and, to a lesser extent, NRC-07 significantly reduced the median effective concentration (EC 50) of cisplatin for breast cancer cells. NRC-03 and NRC-07 bound to breast cancer cells but not fibroblasts, suggesting that killing required peptide binding to target cells. NRC-03- and NRC-07-mediated killing of breast cancer cells correlated with expression of several different anionic cell-surface molecules, suggesting that NRC-03 and NRC-07 bind to a variety of negatively-charged cell-surface molecules. NRC-03 and NRC-07 also caused significant and irreversible cell-membrane damage in breast cancer cells but not in fibroblasts. NRC-03- and NRC-07-mediated cell death involved, but did not require, mitochondrial membrane damage and ROS production. Importantly, intratumoral administration of NRC-03 and NRC-07 killed breast cancer cells grown as xenografts in NOD SCID mice. Conclusions: These findings warrant the development of stable and targeted forms of NRC-03 and/or NRC-07 that might be used alone or in combination with conventional chemotherapeutic drugs for the treatment of breast cancer.Peer reviewed: YesNRC publication: Ye

Isolation, design, and biological properties of fish-derived cationic antimicrobial peptides

Cationic antimicrobial peptides are components of the host innate immune response against infections in almost all species. Although innate defenses in fish are particularly important due to a frequently late onset and short duration of the secondary immune response in these animals, few fish peptides have been studied to date. This research identifies a novel Hl-histone derived cationic peptide (HSDF-1) from coho salmon (Oncorhynchus kisutch), and describes the design, as well as the antibacterial, membrane, and intracellular activities of several other fish-based synthetic peptides of potential importance in aquaculture. HSDF-1, a 26 residue species identical to the N-terminal segment of trout HI histone, was isolated from the serum and mucus of disease-challenged coho salmon. The purified fractions inhibited the growth of antibiotic-supersusceptible S.typhimurium, as well as A. salmonicida and V. anguillarum. Synthetic HSDF-1 and its derivative HSDF-2 were inactive in antimicrobial assays, but they potentiated the antimicrobial activities of the flounder peptide pleurocidin, lysozyme, and crude lysozyme-containing extracts from coho salmon. Although the peptides inserted specifically into anionic lipid monolayers, synergy with pleurocidin did not appear to occur at the cytoplasmic membrane level. The effects of pleurocidin and several of its derivatives on bacterial membranes and intracellular functions were further studied to gain insight into the mode of action of these fish-derived peptides. All peptides tested at their minimal inhibitory concentrations (MICs) inhibited macromolecular synthesis in bacteria, causing a decrease in the incorporation of [3H]thymidine, [3H]uridine, or [3H]histidine into DNA, RNA, and proteins, respectively. However, many of the peptides applied at their MICs showed limited or negligible ability to damage bacterial cytoplasmic membranes, as observed using a fluorescent dye (diSC35) assay. The inhibition of macromolecular synthesis may thus be a result of the peptides entering bacterial cells and acting intracellularly in a direct fashion, as suggested by the fact that when two peptides were tested, they were able to translocate into liposomes without lysing them. A combined approach of isolating antimicrobial peptides from nature and designing them from existing templates, should eventually provide the medical and veterinary clinicians with a useful arsenal of weapons in the face of developing resistance to conventional antibiotics.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Antimicrobial peptides: Cooperative approaches to protection

Reports of cationic antimicrobial peptides (CAPs) have become standard fare in research literature. But with several hundred peptides described to date, the investigator who tries to navigate the proposed models of their activity is only treated to a generous serving of incongruencies. Rather than acting in isolation as antimicrobial molecules, CAPs also may synergize with other molecules of innate immunity and modulate both innate and adaptive immune systems, thus providing a link between the various mechanisms that result in host protection. - See more at: http://www.eurekaselect.com/79082/article#sthash.8JDjRl98.dpufPeer reviewed: YesNRC publication: Ye

Isolation and characterization of two antimicrobial peptides from haemocytes of the American lobster Homarus americanus

Two antimicrobial peptides from haemocytes of the American lobster, Homarus americanus H. Milne Edwards 1837, were isolated and partially characterised \u2013 the first such description for this species. CAP-2, an approximately 12 kDa peptide, contained amino acid sequences corresponding to the predicted sequence for Hoa-crustin. Crustins are whey acidic protein (WAP) domain - containing peptides isolated from crustacean haemocytes. CAP-2 did not have any activity towards the Gram positive coccus Aerococcus viridans unlike carcinin, a crustin from Carcinus maenas haemocytes, which may partially explain the lobster's susceptibility to this bacterium. A second peptide, CAP-1, was a multimer composed of 4\u20136 kDa subunits with similarities to amphibian temporins. CAP-1 may represent a novel group of antimicrobial peptides for marine invertebrates and has been tentatively named \u2018homarin\u2019. Homarin had bacteriostatic activity against some Gram negative bacteria and both protozoastatic and protozoacidal activity against two cultured scuticociliate parasites Mesanophrys chesapeakensis and Anophryoides haemophila, the latter a significant pathogen of H. americanus.Peer reviewed: YesNRC publication: Ye