Localization of the anti‐cancer peptide EGFR‐lytic hybrid peptide in human pancreatic cancer BxPC‐3 cells by immunocytochemistry

Abstract

Cationic lytic-type peptides have been studied for clinical application in various infections and cancers, but their functional cellular mechanisms remain unclear. We generated anti-cancer epithelial growth factor receptor (EGFR)-lytic hybrid peptide, a 32-amino-acid peptide composed of an EGFR-binding sequence and lytic sequence. In this study, we investigated the distribution of EGFR-lytic hybrid peptide in BxPC-3 human pancreatic cancer cells by an immunocytochemical (ICC) method. Distribution of EGFR protein expression was unchanged after treatment with EGFR-lytic peptide compared with non-treated cells. In confocal laser scanning microscopy, immunostaining of EGFR-lytic peptide was observed in the cytoplasm, mostly in the form of granules. Some staining was also localized on the mitochondrial membrane. At the ultrastructure level, cells treated with EGFR-lytic peptide had a low electron density, disappearance of microvilli, and swollen mitochondria. Fragments of cell membrane were also observed in the proximity of the membrane. In immunoelectron microscopy, EGFR-lytic peptide was observed in the cell membrane and cytoplasm. A number of granules were considered swollen mitochondria. Activation of the caspase pathway as a result of mitochondrial dysfunction was also examined to determine the cytotoxic activity of EGFR-lytic peptide; however, no effect on cell death after EGFR-lytic treatment was observed, and moreover, apoptosis was not found to play a critical role in the cell death mechanism. These results suggest that EGFR-lytic peptide is localized on cell and mitochondrial membranes, with disintegration of the cell membrane contributing mainly to cell death.