Old Polyanionic Drug Suramin Suppresses Detrimental Cytotoxicity of the Host Defense Peptide LL-37

Abstract

The host defense peptide LL-37 is the only human cathelicidin, characterized by pleiotropic activity ranging from immunological to anti-neoplastic functions. However, its overexpression has been associated with harmful inflammatory responses and apoptosis. Thus, for the latter cases, the development of strategies aiming to reduce LL-37 toxicity is highly desired as these have the potential to provide a viable solution. Here, we demonstrate that the reduction of LL-37 toxicity might be achieved by the impairment of its cell surface binding through interaction with small organic compounds that are able to alter the peptide conformation and minimize its cell penetration ability. In this regard, the performed cell viability and internalization studies showed a remarkable attenuation of LL-37 cytotoxicity toward colon and monocytic cells in the presence of the polysulfonated drug suramin. The mechanistic examinations of the molecular details indicated that this effect was coupled with the ability of suramin to alter LL-37 secondary structure via the formation of peptide-drug complexes. Moreover, a comparison with other therapeutic agents having common features unveiled the peculiar ability of suramin to optimize the binding to the peptide sequence. The newly discovered suramin action is hoped to inspire the elaboration of novel repurposing strategies aimed to reduce LL-37 cytotoxicity under pathological conditions.

Figure 1

Chemical structures of the studied compounds. Helical wheel representations of peptides (a) LL-37 and (b) FK-16 drawn with HELIQUEST. N and C indicate the N- and C-termini of the peptides. The underlined letters highlight the position of FK-16 sequence within that of LL-37. The arrows indicate the peptide hydrophobic face. The C-terminal of the peptide sequence is amidated. (c) Chemical structure of suramin.

Figure 2

Effect of suramin on LL-37 and FK-16 cytotoxicity. (a and c) Cytotoxicity (4 h of treatment) and (b and d) cytostasis (24 h of treatment) of LL-37 and FK-16 in the presence and absence of suramin (Sur) measured on HT-29 cells and presented by the mean IC₅₀ values measured in three parallel experiments (***: p < 0.001). The results are presented as mean ± SEM. The data indicated as “>50 μM” are characterized by IC₅₀ values higher than 50 μM. The samples labeled as “+Sur x:x” correspond to the combination peptide + suramin at the specified peptide to drug molar ratio. (e) Microscopic images of HT-29 cells treated with 17 μM LL-37 or 33 μM FK-16 in the presence and absence of suramin at different peptide to drug molar ratios (2:1, 1:1, and 1:2). Cells were incubated with the studied peptides for 1.5 h at 37 °C.

Figure 3

Peptide internalization. Cellular uptake of HT-29 cells assessed by flow cytometry using Cf-labeled LL-37 and FK-16 at 1, 5, and 10 μM concentrations in the presence and absence of suramin (Sur) at 2:1, 1:1, and 2:1 peptide to drug molar ratios. Percentage of cells containing Cf-peptide (Cf-positive cells, detected on FITC channel) and mean fluorescence intensity (MFI) values obtained for (a and c) Cf-LL-37 and (b and d) Cf-FK-16. The results are presented as mean ± SEM.

Figure 4

Cellular localization of LL-37. Confocal laser scanning microscopy visualizations of HT-29 cells incubated with LL-37 (1 μM) in the absence and in combination with suramin at 2:1 and 1:2 peptide to drug molar ratios. Nuclei were stained with Hoechst 33342 (blue), the peptide was Cf-labeled (green), and lysosomes were stained with LysoTracker Deep Red (red). Scale bar: 20 μM.

Figure 5

Structural changes of LL-37 and FK-16 driven by interaction with suramin. Left panels in a–d: far-UV CD spectra of peptides in the presence and absence of suramin in (a and c) Tris HCl buffer (10 mM, pH 7.4) or (b and d) PBS. Right panels in a–d: representative ATR-IR spectra in the amide region of peptides in the presence and absence of suramin in (a and c) Tris buffer or (b and d) PBS. Insets in a–d: in-phase S–O stretching of the sulfonyl groups (∼1041 cm^–1) characteristic of the complex formation peptide–suramin. Peptide spectra are normalized by the intensity at 1654 cm^–1 in the amide region and at 1060 or 1080 cm^–1 in the insets. Second derivatives are shown below and were used for peak identification.

Figure 6

Influence of BBG, SBPC, and CTT on LL-37 conformation and cytotoxicity. (a) Far-UV CD spectra of LL-37 in Tris HCl buffer (pH 7.4) upon stepwise addition of BBG. (b and c) Far-UV CD spectra of LL-37 in Tris HCl buffer (pH 7.4) and in the presence of 20 or 80 and 120 μM SBPC or CTT. (d) Cytotoxicity (gray bars) and cytostasis (orange bars), presented by the IC₅₀ values of LL-37 alone and in the presence of BBG at 1:1 and 1:3 peptide/BBG ratios measured on HT-29 cells. (e and f) Cytotoxicity (gray bars) and cytostasis (orange bars) of LL-37 alone and in the presence of SBPC or CTT at an 1:3 peptide to drug ratio measured on MonoMac6 cells.