title: Co-operative membrane disruption between cell-penetrating peptide and cargo: implications for the therapeutic use of the Bcl-2 converter peptide D-NuBCP-9-r8.
abstract: Delivering apoptosis inducing peptides to cells is an emerging area in cancer and molecular therapeutics. Here, we have identified an alternative mechanism of action for the proapoptotic chimeric peptide D-NuBCP-9-r8. Integral to D-NuBCP-9-r8 is the Nur-77-derived D-isoform sequence fsrslhsll that targets Bcl-2, and the cell-penetrating peptide (CPP) octaarginine (r8) that is required for intracellular delivery. We find that the N-terminal phenylalanine of fsrslhsll acts in synergy with the cell-penetrating moiety to enhance peptide uptake at low nontoxic levels and cause rapid membrane blebbing and cell necrosis at higher (IC(50)) concentrations. These effects were not observed when a single phenylalanine-alanine mutation was introduced at the N-terminus of D-NuBCP-9-r8. Using primary samples from chronic lymphocytic leukemia (CLL) patients and cancer cell lines, we show that NuBCP-9-r8 induced toxicity, via membrane disruption, is independent of Bcl-2 expression. Overall, this study demonstrates a new mechanism of action for this peptide and cautions its use as a highly specific entity for targeting Bcl-2. For delivery of therapeutic peptides the work emphasizes that key amino acids in cargo, located several residues away from the cell-penetrating sequence, can significantly influence their cellular uptake and mode of action.
authors.affiliation: Welsh School of Pharmacy, Cardiff University, Cardiff, UK.
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Watkins
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Sayers
authors.forename: Chris
authors.initials: C
authors.lastname: Allender
authors.forename: David
authors.initials: D
authors.lastname: Barrow
authors.forename: Christopher
authors.initials: C
authors.lastname: Fegan
authors.forename: Paul
authors.initials: P
authors.lastname: Brennan
authors.forename: Arwyn T
authors.initials: AT
authors.lastname: Jones
mesh_terms: D017209:Apoptosis; D001692:Biological Transport; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D057846:Cell-Penetrating Peptides / Q000493:pharmacokinetics / Q000627:therapeutic use*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1 / Q000378:metabolism*; D009842:Oligopeptides / Q000493:pharmacokinetics / Q000627:therapeutic use*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D057846:Cell-Penetrating Peptides; C539821:NR4A1 protein, human; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; C000590397:phenylalanyl-seryl-arginyl-seryl-leucyl-histidyl-seryl-leucyl-leucine