The Antitumor Peptide ERα17p Exerts Anti-Hyperalgesic and Anti-Inflammatory Actions Through GPER in Mice

Abstract

Persistent inflammation and persistent pain are major medical, social and economic burdens. As such, related pharmacotherapy needs to be continuously improved. The peptide ERα17p, which originates from a part of the hinge region/AF2 domain of the human estrogen receptor α (ERα), exerts anti-proliferative effects in breast cancer cells through a mechanism involving the hepta-transmembrane G protein-coupled estrogen receptor (GPER). It is able to decrease the size of xenografted human breast tumors, in mice. As GPER has been reported to participate in pain and inflammation, we were interested in exploring the potential of ERα17p in this respect. We observed that the peptide promoted anti-hyperalgesic effects from 2.5 mg/kg in a chronic mice model of paw inflammation induced by the pro-inflammatory complete Freund's adjuvant (CFA). This action was abrogated by the specific GPER antagonist G-15, leading to the conclusion that a GPER-dependent mechanism was involved. A systemic administration of a Cy5-labeled version of the peptide allowed its detection in both, the spinal cord and brain. However, ERα17p-induced anti-hyperalgesia was detected at the supraspinal level, exclusively. In the second part of the study, we have assessed the anti-inflammatory action of ERα17p in mice using a carrageenan-evoked hind-paw inflammation model. A systemic administration of ERα17p at a dose of 2.5 mg/kg was responsible for reduced paw swelling. Overall, our work strongly suggests that GPER inverse agonists, including ERα17p, could be used to control hyperalgesia and inflammation.

Keywords: ERα17p; GPER; hyperalgesia; inflammation; pain.

Figure 1

GPER-dependent action of ERα17p in tactile hypersensitivity in a CFA model. The Von Frey test was performed to assess the impact of ERα17p on CFA-induced mechanical hypersensitivity in inflammatory pain. The 50% paw withdrawal threshold (PWT) was determined with a modified version of the Dixon up–down method. (A) The anti-hyperalgesic action of ERα17p was determined by measuring dose-dependent effects. The Von Frey test was assessed before injection of CFA (baseline) and after that of vehicle (saline solution) or ERα17p (1.25, 2.5, and 10 mg/kg, i.p.) 7 days after CFA injection. (C) Involvement of GPER was determined using ERα17p with or without G-15. Mice were i.p. pre-treated with vehicle (5% DMSO, 5% Tween80 in saline solution, reference) or G-15 (0.3 mg/kg) 15 min before administration of vehicle (saline) or ERα17p (2.5 mg/kg, i.p.). (B, D) Area under the time-course AUC (0–180 min) of PWT variations obtained from (A, C), respectively. Data are expressed as mean ± SEM (n = 8–10 per group). *p < 0.05, **p < 0.005, ***p < 0.001, when compared to the vehicle group (or G-15+ERα17p group, as mentioned in D); two-way ANOVA followed by Dunnett post hoc test for time comparison or Kruskal-Wallis test for AUC mean comparison.

Figure 2

CNS distribution of the Cy5-labeled ERα17p peptide. Upper views of brain (A) and spinal cord (B) sampled from three mice 30 min after an i.p. injection of H₂N-ERα17p-Pra(Cy5)-COOH (2 mg/kg).

Figure 3

Involvement of supraspinal GPER in ERα17p action in the CFA model. (A, C) Area under the time-course AUC (0–180 min) of PWT variations from (B, D), respectively. (B) Time-course effect of an i.c.v. administration of vehicle (saline solution, 2 µl/mice) or ERα17p (1, 2.5 and 5 µg/mice) on mechanical hypersensitivity in CFA mice model. (D) Involvement of supraspinal GPER in the action of ERα17p with or without G-15 i.c.v. Mice were i.c.v. pre-treated with vehicle (5% DMSO, 5% Tween80 in saline solution, 2 µl/mice) or G-15 (5 µg/mice) 20 min before administration of vehicle (saline solution, reference) or ERα17p (2.5 mg/kg, i.p.). Data are expressed as mean ± SEM (n = 8–9 per group). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle group; two-way ANOVA followed by Dunnett post hoc test for time comparison or Kruskal-Wallis test for AUC mean comparison.

Figure 4

Spinal GPER is not involved in the action of ERα17p in the CFA model. (A) Time-course effect of the intrathecal administration of vehicle (saline solution, reference, 2 µl), ERα17p (1, 2.5 and 5 µg/mice) on mechanical hypersensitivity in CFA mice. (C) Evaluation of the effect of intrathecally administered ERα17p (5 µg/mice) or vehicle 20 min after G-15 (5 µg/mice, i.t.) or vehicle administration. (E) The involvement of spinal GPER in the mechanism of action of systemic ERα17p is investigated by testing ERα17p i.p. with or without G-15 i.t. Mice were i.t. pre-treated with vehicle (saline solution, 2 µl/mice, reference) or G-15 (5 µg/mice) 20 min before an administration of vehicle (saline solution, reference) or ERα17p (2.5 mg/kg, i.p.). (B, D, F) Area under the time-course (AUC, 0–180 min) of PWT variations from (A, C, E), respectively. Data are expressed as mean ± SEM (n = 8–9 per group). *p < 0.05, **p < 0.01, ***p < 0.001, compared with the vehicle group; two-way ANOVA followed by Dunnett post hoc test for time comparison or Kruskal-Wallis test for AUC mean comparison.

Figure 5

GPER involvement in the anti-inflammatory action of ERα17p in the carrageenan model. (A) Ankle diameter of mice was measured before (baseline) and 4 h after carrageenan injection. The involvement of GPER in the mechanism of action of ERα17p was investigated with or without G-15. Mice were i.p. pretreated with vehicle (5% DMSO, 5% Tween80 in saline solution, 10 ml/kg, reference) or G-15 (0.3 mg/kg), 20 min before the administration of vehicle (saline solution, reference) or ERα17p (2.5 mg/kg, 10 ml/kg, i.p.). (B) Effect of an intra-plantar (i.pl.) injection of vehicle (saline solution, 10 µl, reference) or of ERα17p (20 µg) on edema measured by ankle diameter (in cm) induced by carrageenan. Data are expressed as mean ± SEM (n = 10–12 per group). Two-way ANOVA followed by Dunnett post hoc test (A) or Sidak post hoc test (B). *p < 0.05, **p < 0.01 compared with the vehicle group.