Pleurocidin-family cationic antimicrobial peptides are cytolytic for breast carcinoma cells and prevent growth of tumor xenografts.
BACKGROUND
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 (EC50) 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.
Department of Microbiology & Immunology, Dalhousie University, 5850 College St., Halifax, B3H 4R2, Canada.
Ashley L
AL
Hilchie
Carolyn D
CD
Doucette
Devanand M
DM
Pinto
Aleksander
A
Patrzykat
Susan
S
Douglas
David W
DW
Hoskin
D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000473:pathology; D002277:Carcinoma / Q000188:drug therapy / Q000473:pathology; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured; D002945:Cisplatin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D005260:Female; D005347:Fibroblasts / Q000187:drug effects; D029941:Fish Proteins / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D051336:Mitochondrial Membranes / Q000187:drug effects; D008969:Molecular Sequence Data; D017382:Reactive Oxygen Species / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D023041:Xenograft Model Antitumor Assays
D023181:Antimicrobial Cationic Peptides; D000970:Antineoplastic Agents; D029941:Fish Proteins; D017382:Reactive Oxygen Species; C105851:pleurocidin; D002945:Cisplatin