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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.
AuthorsKoji Ohara, Masayuki Kohno, Koji Kawakami
JournalJournal of peptide science : an official publication of the European Peptide Society (J Pept Sci) Vol. 19 Issue 8 Pg. 511-5 (Aug 2013) ISSN: 1099-1387 [Electronic] England
PMID23813780 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
Chemical References
  • Amino Acid Chloromethyl Ketones
  • Antineoplastic Agents
  • Caspase Inhibitors
  • EGFR-lytic peptide
  • Reactive Oxygen Species
  • Recombinant Fusion Proteins
  • benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
  • Doxorubicin
  • EGFR protein, human
  • ErbB Receptors
Topics
  • Amino Acid Chloromethyl Ketones (pharmacology)
  • Antineoplastic Agents (metabolism, pharmacology)
  • Apoptosis
  • Caspase Inhibitors (pharmacology)
  • Cell Line, Tumor
  • Cell Survival (drug effects)
  • Doxorubicin (pharmacology)
  • ErbB Receptors (metabolism)
  • Humans
  • Immunohistochemistry
  • Microscopy, Confocal
  • Mitochondria (metabolism)
  • Mitochondrial Membranes (metabolism)
  • Mitochondrial Swelling
  • Pancreatic Neoplasms
  • Protein Transport
  • Reactive Oxygen Species (metabolism)
  • Recombinant Fusion Proteins (metabolism, pharmacology)

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