Nannan Yang

Relative Spatial Positions of Tryptophan and Cationic Residues in Helical Membrane-active Peptides Determine Their Cytotoxicity

Background: Tryptophan side chains can influence the binding of amphipathic peptides to biological membranes. Results: The cytotoxic activity of model helical amphipathic peptides was markedly influenced by the positions of tryptophan residues in the sequence. Conclusion: Tryptophan residues located adjacent to a hydrophobic helical portion created the most potent cytotoxic peptides. Significance: More potent anticancer helical peptides can now be designed. The cytotoxic activity of 10 analogs of the idealized amphipathic helical 21-mer peptide (KAAKKAA)3, where three of the Ala residues at different positions have been replaced with Trp residues, has been investigated. The peptides cytotoxic activity was found to be markedly dependent upon the position of the Trp residues within the hydrophobic sector of an idealized α-helix. The peptides with Trp residues located opposite the cationic sector displayed no antitumor activity, whereas those peptides with two or three Trp residues located adjacent to the cationic sector exhibited high cytotoxic activity when tested against three different cancer cell lines. Dye release experiments revealed that in contrast to the peptides with Trp residues located opposite the cationic sector, the peptides with Trp residues located adjacent to the cationic sector induced a strong permeabilizing activity from liposomes composed of a mixture of zwitterionic phosphatidylcholine and negatively charged phosphatidylserine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)) (2:1) but not from liposomes composed of zwitterionic phosphatidylcholine, POPC. Fluorescence blue shift and quenching experiments revealed that Trp residues inserted deeper into the hydrophobic environment of POPC/POPS liposomes for peptides with high cytotoxic activity. Through circular dichroism studies, a correlation between the cytotoxic activity and the α-helical propensity was established. Structural studies of one inactive and two active peptides in the presence of micelles using NMR spectroscopy showed that only the active peptides adopted highly coiled to helical structures when bound to a membrane surface.

The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells

Host defense peptides (HDPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line defense against intruding pathogens, and several HDPs have been shown to possess anticancer activity. Structure-activity relationship studies on the HDP bovine lactoferricin revealed a de novo design of a nonamer peptide LTX-315, with oncolytic properties. In the present study, we investigated the oncolytic activity of LTX-315 in human melanoma cells (A375). LTX-315 induced a rapid plasma membrane disruption and cell death within 2 hours. At a low concentration, fluorescence-labeled LTX-315 was internalized and accumulated in cytoplasmic vacuoles in close proximity to the mitochondria. The mitochondrial membrane potential was shown to depolarize as a consequence of LTX-315 treatment and at ultrastructural level, the mitochondria morphology was significantly altered. Release of danger signals (DAMPs) such as ATP, Cytochrome C and HMGB1 into the cell supernatant of cultured cells was evident minutes after peptide treatment. The oncolytic effect of LTX-315 involving perturbation of both the cell membrane and the mitochondria with subsequent release of DAMPs may highlight the ability of LTX-315 to induce complete regression and long-term protective immune responses as previously reported in experimental animal models.

Abstract 2589: In vitro activities of the cationic antitumoral peptide LTX-315 against human melanoma cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The LTX-315 peptide is a lactoferricin-derived short antitumor peptide which was designed to treat solid tumors with intra-tumoral injections through a two-stage mode of action. First, the collapse of the tumor itself, then the released DAMPs from the dying tumor cell, could induce a subsequent immune protection against recurrences and metastastis. In the present study, we investigate the mechanisms behind the killing of A375 melanoma cells by LTX-315. The peptide kills the tumor cells within a few minutes by direct plasma membrane attack, but at lower concentration internalizes- and can be associated with mitochondria, ultimately leading to a lytic cell death. We show that a fluorescence-labeled peptide was internalized and localized in cytoplasmic vacuoles and accumulated around the mitochondria with subsequent rapid lysis of cells as shown by confocal live cell imaging and transmission electron microscopy. Furthermore we detected the secretion of DAMPs into the extracellular compartment minutes after peptide exposure using ELISA assay, ATP luciferase assay and western blot. Taken together, these findings suggest that LTX-315 has the ability to cause immunogenic cell death in cancer cells, and thus might have a potential as an in situ therapeutic vaccination agent Citation Format: Liv-Marie Eike, Nannan Yang, Oystein Rekdal, Baldur Sveinbjornsson. In vitro activities of the cationic antitumoral peptide LTX-315 against human melanoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2589. doi:10.1158/1538-7445.AM2014-2589