title: A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells.
abstract: BACKGROUND
  Transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth. Recent studies have shown the elevated expression levels of TfR on cancer cells compared with normal cells. The elevated expression levels of this receptor in malignancies, which is the accessible extracellular protein, can be a fascinating target for the treatment of cancer. We have recently designed novel type of immunotoxin, termed "hybrid peptide", which is chemically synthesized and is composed of target-binding peptide and lytic peptide containing cationic-rich amino acids components that disintegrates the cell membrane for the cancer cell killing. The lytic peptide is newly designed to induce rapid killing of cancer cells due to conformational change. In this study, we designed TfR binding peptide connected with this novel lytic peptide and assessed the cytotoxic activity in vitro and in vivo.
  
  
  METHODS
  In vitro: We assessed the cytotoxicity of TfR-lytic hybrid peptide for 12 cancer and 2 normal cell lines. The specificity for TfR is demonstrated by competitive assay using TfR antibody and siRNA. In addition, we performed analysis of confocal fluorescence microscopy and apoptosis assay by Annexin-V binding, caspase activity, and JC-1 staining to assess the change in mitochondria membrane potential. In vivo: TfR-lytic was administered intravenously in an athymic mice model with MDA-MB-231 cells. After three weeks tumor sections were histologically analyzed.
  
  
  RESULTS
  The TfR-lytic hybrid peptide showed cytotoxic activity in 12 cancer cell lines, with IC(50) values as low as 4.0-9.3 μM. Normal cells were less sensitive to this molecule, with IC(50) values > 50 μM. Competition assay using TfR antibody and knockdown of this receptor by siRNA confirmed the specificity of the TfR-lytic hybrid peptide. In addition, it was revealed that this molecule can disintegrate the cell membrane of T47D cancer cells just in 10 min, to effectively kill these cells and induce approximately 80% apoptotic cell death but not in normal cells. The intravenous administration of TfR-lytic peptide in the athymic mice model significantly inhibited tumor progression.
  
  
  CONCLUSIONS
  TfR-lytic peptide might provide a potent and selective anticancer therapy for patients.
authors.affiliation: Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Kyoto, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Kawamoto
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Horibe
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kohno
authors.forename: Koji
authors.initials: K
authors.lastname: Kawakami
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D006801:Humans; D000922:Immunotoxins / Q000378:metabolism / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007668:Kidney / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D010455:Peptides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D034622:RNA Interference; D011990:Receptors, Transferrin / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013154:Spleen / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D023041:Xenograft Model Antitumor Assays
chemical_list: D000922:Immunotoxins; D010455:Peptides; D011990:Receptors, Transferrin