Anticancer β-hairpin peptides: membrane-induced folding triggers activity

Abstract

Several cationic antimicrobial peptides (AMPs) have recently been shown to display anticancer activity via a mechanism that usually entails the disruption of cancer cell membranes. In this work, we designed an 18-residue anticancer peptide, SVS-1, whose mechanism of action is designed to take advantage of the aberrant lipid composition presented on the outer leaflet of cancer cell membranes, which makes the surface of these cells electronegative relative to the surface of noncancerous cells. SVS-1 is designed to remain unfolded and inactive in aqueous solution but to preferentially fold at the surface of cancer cells, adopting an amphiphilic β-hairpin structure capable of membrane disruption. Membrane-induced folding is driven by electrostatic interaction between the peptide and the negatively charged membrane surface of cancer cells. SVS-1 is active against a variety of cancer cell lines such as A549 (lung carcinoma), KB (epidermal carcinoma), MCF-7 (breast carcinoma), and MDA-MB-436 (breast carcinoma). However, the cytotoxicity toward noncancerous cells having typical membrane compositions, such as HUVEC and erythrocytes, is low. CD spectroscopy, appropriately designed peptide controls, cell-based studies, liposome leakage assays, and electron microscopy support the intended mechanism of action, which leads to preferential killing of cancerous cells.

Figure 1

Membrane-induced folding and subsequent interpolation of β-hairpin anti-cancer peptide (SVS-1). (A) SVS-1 exists in a random coil conformation in solution. On engaging the negatively charged membrane surface, SVS-1 folds into a bioactive, lytic β-hairpin conformation capable of membrane disruption. (B) Peptide sequences of SVS-1 and its controls. Underlined amino acids are D-isomers and all others are L-isomers; stereochemistry of turn region is specified for clarity.

Figure 2

In vitro cytotoxicity of SVS-1 toward (A) A549 lung carcinoma, KB epidermal carcinoma, MCF-7 breast adenocarcinoma, MDA-MB-436 breast adenocarcinoma, and human erythrocytes (hRBCs); (B) cytotoxicity towards A549 and human umbilical vein endothelial cells (HUVEC). The cytotoxicity of SVS-1 was assessed at 24 h post addition of peptide. Error bars represent standard deviation of at least three independent experiments for the MTT assays used to measure % cell death, and of two independent blood donors for the hemolysis assay.

Figure 3

CD spectra of 50 µM (A) SVS-1 (B) ^DSVS-1 and (C) SVS-2 in: aqueous buffer solution (50mM BTP, 150 mM NaF, pH 7.4); in the presence of neutral POPC LUVs; and negatively charged POPC:POPS LUVs (1:1).

Figure 4

Tb/DPA release from model POPC:POPS (1:1) liposomes monitored as a function of time following the addition of peptide and subsequent detergent. Tb/DPA release was monitored via fluorescence. At t=0, peptides were added and Tb/DPA release measured as a function of time. Final peptide:lipid ratio was 1:25. After equilibrium is reached, 1% OG detergent is added to obtain 100% release (arrows).

Figure 5

In vitro cytotoxicity of SVS-1 toward A549 cells and its effect on cell membrane leakage. % cell death was co-plotted with % LDH release as a function of SVS-1 concentration. Error bars represent standard deviation of at least three independent experiments.

Figure 6

Effect of SVS-1 on cell membrane integrity observed by TEM (A, D) and SEM (B, C, E and F). Membranes of untreated A549 cancer cells (A, B) and non-cancerous HUVEC (C) appeared intact. A549 cells (D, E) incubated with 8 µM SVS-1 in serum-free media. HUVEC cells (F) incubated with a large excess (80 µM) of SVS-1 for 4 h at 37°C and 5 %CO₂. Cells display leakage of cellular contents (dash arrow) and pore formation (solid arrow). Scale bar: 10 µm for SEM, 2 µm for TEM.