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.

Novel Polymeric Nanoparticles for Intracellular Delivery of Peptide Cargos: Antitumor Efficacy of the BCL-2 Conversion Peptide NuBCP-9

“…NuBCP-9 was linked to the D-Arg octamer r8 for intracellular delivery, a modification that has been reported to decrease selectivity by inducing BCL-2-independent cell killing involving membrane disruption (15). In the present work, delivery of NuBCP-9 into cancer cells by the polymeric NPs was achieved without the need for the R8 PTD.…”

“…The selection of a certain CPP is of importance in that the CPP can in a cargo-dependent manner contribute to decreased serum stability, inefficient transit from the endosome to the cytosol and unanticipated toxicities (14). Indeed, for NuBCP-9, the cell-penetrating r8 used for intracellular delivery acts in synergy with the N-terminal phenylalanine of NuBCP-9 to cause membrane blebbing and cell necrosis that are independent of BCL-2 expression (15). These findings have supported the study of alternative CPPs for NuBCP-9 or other formulations that preclude the use of a CPP for intracellular delivery.…”

Synthesis and evaluation of 5-(1 H -indol-3-yl)- N -aryl-1,3,4-oxadiazol-2-amines as Bcl-2 inhibitory anticancer agents

“…To test the effect of differing Bcl-2 status, further evaluation of new compounds was carried out in the human cancer cell lines KG1a (acute myelogenous leukaemia) and Jurkat (T cell leukaemia). The Jurkat cell line has previously been characterized as Bcl-2 negative by our group 17 and others, 18 unlike the Bcl-2 expressing KG1a cell line. 19 For studies on the leukaemic cells we used the CellTiter-Blue® viability assay, appropriate for endpoint determinations in these non-adherent cells.…”

Investigation of Intracellular Delivery of NuBCP-9 by Conjugation with Oligoarginines Peptides in MDA-MB-231 Cells

“…Rn induces non-specific cytotoxicity at high doses owing to its strong electrostatic interaction with the cell membrane. 14,23) Therefore, we considered the possibility that membrane integrity may be disturbed by short-time incubation of cells with the peptides. We measured cellular LDH levels after treatment with peptides to estimate membrane disruption.…”

“…12) In general, the uptake efficiency of oligoarginines (Rn), a kind of CPP, can be affected by the number of arginines comprising it. 14) Watkins et al have demonstrated that the uptake efficiency of octaarginine (R8) can be significantly enhanced by conjugation with NuBCP-9. 15) Therefore, the uptake characteristics of Rn comprising varying numbers of arginine could be altered by conjugation with NuBCP-9.…”