Identification of a human estrogen receptor α tetrapeptidic fragment with dual antiproliferative and anti-nociceptive action

Abstract

The synthetic peptide ERα17p (sequence: PLMIKRSKKNSLALSLT), which corresponds to the 295-311 region of the human estrogen receptor α (ERα), induces apoptosis in breast cancer cells. In mice and at low doses, it promotes not only the decrease of the size of xenografted triple-negative human breast tumors, but also anti-inflammatory and anti-nociceptive effects. Recently, we have shown that these effects were due to its interaction with the seven-transmembrane G protein-coupled estrogen receptor GPER. Following modeling studies, the C-terminus of this peptide (sequence: NSLALSLT) remains compacted at the entrance of the GPER ligand-binding pocket, whereas its N-terminus (sequence: PLMI) engulfs in the depth of the same pocket. Thus, we have hypothesized that the PLMI motif could support the pharmacological actions of ERα17p. Here, we show that the PLMI peptide is, indeed, responsible for the GPER-dependent antiproliferative and anti-nociceptive effects of ERα17p. By using different biophysical approaches, we demonstrate that the NSLALSLT part of ERα17p is responsible for aggregation. Overall, the tetrapeptide PLMI, which supports the action of the parent peptide ERα17p, should be considered as a hit for the synthesis of new GPER modulators with dual antiproliferative and anti-nociceptive actions. This study highlights also the interest to modulate GPER for the control of pain.

Figure 1

Kinetics of formation of ERα17p amyloid fibrils by ThT fluorescence spectroscopy, at various peptide concentrations (in μM) and pH values. Experiments are tested in water at four peptide concentrations, i.e. at 10 (black curve), 25 (grey curve), 50 (black dashed line) and 100 μM (black dotted lines), in the presence of ThT 10 μM. (a) pH 3.4 (0.2 M glycine HCl in water), (b) pH 7.4 (0.2 M KH₂PO₄/0.2 M K₂HPO₄ in water) and (c) pH 9.1 (0.2 M glycine NaOH in water). Excitation and emission wavelengths are 440 and 485 nm, respectively. Fluorescence intensity is expressed in arbitrary units (a.u.) and time in hours. Data are the means of experiments performed in triplicate. Each experiment is carried out over 40 h.

Figure 2

Evolvement of the intensity of the ERα17p aliphatic proton signals as a function of time, by 1D ¹H-NMR (500 MHz). Aliphatic proton signals of highest intensity (0.80, 0.90, 1.50 and 3.60 ppm) are used to follow aggregation. Each experiment (8 scans/15 min.: 258 points over 65 h) is performed at (a) pH 3.4 (0.2 M glycine HCl in water), (b) 7.4 (0.2 M KH₂PO₄/0.2 M K₂HPO₄ in water) and (c) 9.1 (0.2 M glycine NaOH in water), at 298 K and with peptide samples of 100 μM in water. (d) Evolvement of the intensity of the proton signals as a function of time, at pH 3.4, 7.4 and 9.1.

Figure 3

TEM images of ERα17p amyloid fibrils. ERα17p is incubated over 48 h at the concentration of 100 μM at the pH values of (a) 3.4 (glycine HCl 0.2 M in water), (b) and (c) 7.4 (0.2 M KH₂PO₄/0.2 M K₂HPO₄ in water) and (d) 9.1 (0.2 M glycine NaOH in water).

Figure 4

Study of the formation of peptide 1 fibrils. (a) Kinetics of formation of fibrils from peptide 1 (black dashed line) and peptide 2 (black dotted line) by ThT fluorescence spectroscopy. Experiments are carried out in water at the concentration of 100 μM and at pH 9.1 (glycine NaOH 0.2 M). ThT is used at the concentration of 10 μM. ERα17p (black line) is the reference. Excitation and emission wavelengths are 440 and 485 nm, respectively. Fluorescence intensity and time are expressed in arbitrary units (a.u.) and hours, respectively. Data are the means of experiments performed in triplicate. Each experiment is carried out over 40 h. (b) Kinetics of formation of peptide 1 aggregates recorded by CD. Experiments are performed after an equilibration period of 5 min, at 25 °C and pH 9.1, within the wavelength range 190 and 260 nm and at different incubation times, i.e., at 0 h (grey curve), 5 h (grey dotted lines), 22 h (black curve) and 28 h (black dotted line). CD unit is expressed as mean residue ellipticity [θ]_MRE (in deg.cm².dmol⁻¹). (c) TEM image of peptide 1 fibrils at the concentration of 100 μM, after 48 h incubation and at pH 9.1 (glycine NaOH 0.2 M in water).

Figure 5

Concentration-dependent effects of ERα17p (-■-) and peptide 2 (-○-) on cells viability (in %), in (a) MCF-7 and (b) HS578T breast cancer cells. Concentrations are expressed in μM. Each experiment is performed in triplicate (N = 3). Standard errors of the means (± SEM) are specified.

Figure 6

(a) Schematic illustration of the sgRNA sequence cloned into the pX459 vector used to generate GPER knockout (KO) in MDA-MB-231 cells. (b) Protein expression levels of GPER in GPER (WT) and GPER (KO) MDA-MB-231 cells, as evaluated by western blotting analysis. β-Actin served as loading control. Immunoblots shown are representative of three independent experiments. Original blots are presented in Supplementary Fig. 1. GPER (WT) and GPER (KO) (c) MDA-MB-231 cells are treated for 72 h with either vehicle or increasing concentrations of ERα17p and peptide 2. The growth of cells receiving vehicle is set as 100%, upon which the viability of cells treated with ERα17p and peptide 2 is calculated. Values shown are mean ± SD of three independent experiments performed in triplicate. (*) p < 0.05.

Figure 7

Effects of ERα17p and peptide 2 on tactile hypersensitivity in the murine CFA model by von Frey test. 50% paw withdrawal threshold (PWT) is determined using an adaptation of the Dixon up–down method. Seven days after CFA injection, anti-hyperalgesic actions of (a) ERα17p and (b) peptide 2 are evaluated by measuring dose-dependent effects. Von Frey test is assessed before CFA injection (baseline) and after vehicle injection (saline solution, control), ERα17p (1.25, 2.5 and 10 mg/kg), peptide 2 (1.25, 2.5, 10 and 20 mg/kg) or morphine (1 mg/kg, reference) treatments (i.p 10 mL/kg). The nine treatments of each experimental series (n = 8 mice per condition) are done in the same frame. Each treatment corresponds to a time-course curve. (c) Area under the time-course (0–180 min) of PWT variations from (a) and (b). Data are shown as mean ± SEM (n = 8 per group). *p < 0.05, **p < 0.01, ***p < 0.001, compared with the vehicle group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; two way ANOVA followed by Tukey test for the dose–response and Kruskal–Wallis followed by Dunn's test for AUC means comparison.