Studies on the anticancer properties of pleurocidins: a preclinical evaluation
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Cationic antimicrobial peptides (CAPs) are small peptides that constitute an important defence against microbial pathogens. Certain CAPs also possess anticancer properties. NRC-03 and NRC-07 are pleurocidins derived from winter and yellowtail flounder, respectively. The purpose of this investigation was to study the anticancer properties of NRC-03 and NRC-07. NRC-03 and NRC-07 killed breast cancer cells, including P-glycoprotein-overexpressing cells, in a time-dependent manner that peaked at 4 h. NRC-03 and NRC-07 lysed breast cancer cells by a mechanism that involved cell binding, mitochondrial destabilization, nuclear localization, and significant membrane damage. Interestingly, NRC-07, but not NRC-03, caused DNA fragmentation. NRC-03 and NRC-07 killed normal human epithelial cells, but did not kill endothelial cells or fibroblasts, or lyse human erythrocytes. NRC-03, and to a lesser extent NRC-07, had chemo-sensitizing properties, suggesting promise for their inclusion in combinational treatment regimens. Importantly, intratumoural injections of NRC-03 or NRC-07 inhibited tumour growth in a mouse model of breast cancer. Fetal bovine serum dose-dependently reduced cell killing by NRC-03. NRC-03 was degraded in human and mouse serum, which limited its potency. NRC-03- and NRC-07-induced cytotoxicity correlated with expression of several different negatively-charged molecules, rationalizing the generation of [D]-NRC-03, which carries the same positive charge as NRC-03, and was more potent but less selective for cancer cells than NRC-03. [D]-NRC-03 was also cytolytic and exhibited in vivo anticancer properties. To further test the clinical potential of NRC-03- and NRC-07-resistant cells were generated. NRC-03 and NRC-07 bound to resistant cells to a lesser extent than parental cells and were phenotypically distinct. Importantly, NRC-03- and NRC-07-resistant cells were killed by chemotherapeutic drugs, as well as [D]-NRC-03. These studies demonstrate that NRC-03, NRC-07, and [D]-NRC-03 are cytolytic peptides that kill breast cancer cells in vitro and in vivo. While more potent than NRC-03, [D]-NRC-03 requires further modification to minimize its toxicity toward normal cells. Although cancer cells may become resistant to NRC-03 and NRC-07 over time, resistant cells are still killed by other cytotoxic drugs, thereby reinforcing the value of adding these peptides to combinational regimens for the treatment of breast cancer.