LAUSR

Antimicrobial peptides (AMPs) are important anticancer resources, and exploring AMP conjugates as highly effective and selective anticancer agents would represent new progress in cancer treatment. In this study, we synthesized C4–C16 fatty-acyl-conjugated AMP CM4 and investigated its physiochemical properties and cytotoxicity activity in breast cancer cells. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase high-performance liquid chromatography (RP-HPLC) showed that long-chain fatty acyl (≥C12) conjugation prevented N-acyl-CM4 from trypsin hydrolysis. RP-HPLC and circular dichroism (CD) spectra showed that the hydrophobicity and helical content of N-acyl-CM4 increased with the acyl length. The acyl chain length was positively related to the cytotoxicity of C8–C16 conjugates, and C12–C16 fatty acyl conjugates exhibited significant cytotoxicity against MX-1, MCF-7, and MDA-MB-231 cells, with IC50 values <8 μM. Flow cytometry and confocal laser scanning microscopy results showed that N-acylated conjugation significantly increased the membrane affinity in breast cancer cells, and C12–C16 acyl conjugates were capable of translocating to the intracellular space, thereby targeting mitochondria and inducing apoptosis. N-acyl-CM4 showed low cytotoxicity against normal mammalian cells and erythrocytes, especially ≤C12 fatty acyl conjugates, exhibiting selective cytotoxicity to breast cancer cells. The current work indicated that increasing hydrophobicity by attaching long fatty acyl (≥C12) to AMPs may be an effective method to improve the anticancer activity, together with selectivity and resistance to trypsin hydrolysis. This finding provides a good strategy to develop AMPs as effective anticancer agents in the future.