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. 2015 Aug 4;112(31):9716-21.
doi: 10.1073/pnas.1507931112. Epub 2015 Jul 21.

Molecular and preclinical basis to inhibit PGE2 receptors EP2 and EP4 as a novel nonsteroidal therapy for endometriosis

Joe A Arosh  1 , JeHoon Lee  2 , Dakshnapriya Balasubbramanian  2 , Jone A Stanley  2 , Charles R Long  3 , Mary W Meagher  4 , Kevin G Osteen  5 , Kaylon L Bruner-Tran  5 , Robert C Burghardt  2 , Anna Starzinski-Powitz  6 , Sakhila K Banu  1
Affiliations

Molecular and preclinical basis to inhibit PGE2 receptors EP2 and EP4 as a novel nonsteroidal therapy for endometriosis

Joe A Arosh et al. Proc Natl Acad Sci U S A. .

Abstract

Endometriosis is a debilitating, estrogen-dependent, progesterone-resistant, inflammatory gynecological disease of reproductive age women. Two major clinical symptoms of endometriosis are chronic intolerable pelvic pain and subfertility or infertility, which profoundly affect the quality of life in women. Current hormonal therapies to induce a hypoestrogenic state are unsuccessful because of undesirable side effects, reproductive health concerns, and failure to prevent recurrence of disease. There is a fundamental need to identify nonestrogen or nonsteroidal targets for the treatment of endometriosis. Peritoneal fluid concentrations of prostaglandin E2 (PGE2) are higher in women with endometriosis, and this increased PGE2 plays important role in survival and growth of endometriosis lesions. The objective of the present study was to determine the effects of pharmacological inhibition of PGE2 receptors, EP2 and EP4, on molecular and cellular aspects of the pathogenesis of endometriosis and associated clinical symptoms. Using human fluorescent endometriotic cell lines and chimeric mouse model as preclinical testing platform, our results, to our knowledge for the first time, indicate that selective inhibition of EP2/EP4: (i) decreases growth and survival of endometriosis lesions; (ii) decreases angiogenesis and innervation of endometriosis lesions; (iii) suppresses proinflammatory state of dorsal root ganglia neurons to decrease pelvic pain; (iv) decreases proinflammatory, estrogen-dominant, and progesterone-resistant molecular environment of the endometrium and endometriosis lesions; and (v) restores endometrial functional receptivity through multiple mechanisms. Our novel findings provide a molecular and preclinical basis to formulate long-term nonestrogen or nonsteroidal therapy for endometriosis.

Keywords: PGE2 signaling; endometriosis; infertility; pain pathways; pelvic pain.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Effects of inhibition of EP2 and EP4 (EP2/4-I) on growth and survival of endometriosis lesions. Fluorescence microscopy of (A1) human endometriotic epithelial cells 12Z-GFP and (A2) stromal cells 22B-RFP grown in coculture and imaged with red or green channels and (A3) overlay of both channels. (B) A mixture of epithelial cells 12Z-GFP and stromal cells 22B-RFP suspension was injected into the peritoneal cavity of nude mice, peritoneal endometriosis was induced (day 1), and necropsied on day 29 as detailed in Materials and Methods. Gross examination of white (1) and bleeding red (2) endometriosis lesions phenotypes with adhesions (ad). (C1–C3) Fluorescence zoomstereo microscopy examination of dissemination of 12Z-GFP and 22B-RFP cells of endometriosis lesions in the peritoneal cavity. Histomorphology of endometriosis lesions, (D1) 12Z-GFP cells formed the glands (GLE), (D2) 22B-RFP formed the stroma (STR), and (D3) established communicating glands and stroma. (E) Endometriosis nude mice were treated with EP2 (AH6809) and EP4 (AH23848) inhibitors (EP2/4-I) at 5, 10, or 25 mg/kg, i.p, at 24h intervals from days 15–28 of xenograft. Growth of endometriosis lesions was imaged in vivo real-time on days 1 (before), 7, 14, 21, and 28 of xenograft. Representative in vivo images for (F) control and (G) EP2/4-I at 25 mg/kg on day 28 are shown. (H) Gross number and volume of endometriosis lesions. (I) Fluorescence zoomstereo microscopy examination of peritoneal endometriosis lesions and (J) quantity of 12Z-GFP and 22B-RFP cells in these lesions. (K) Plasma biochemical parameters. *Control vs. EP2/4-I, P < 0.05, n = 6.
Fig. 2.
Fig. 2.
Effects of inhibition of EP2 and EP4 (EP2/4-I) on induction of apoptosis and activation of caspase-3 and PARP proteins in endometriosis lesions. (A) TUNNEL assay. (B) The 22B-RFP stromal cells (STR) were labeled with Alexa 594 (red) and cl-caspase3 protein was labeled with Alexa 488 (green) antibodies. (C) The 12Z-GFP epithelial cells (GLE) were labeled with Alexa 488 (green), and cl-PARP protein was labeled with Alexa 594 (red) antibodies. Nuclei were stained with DAPI (blue). (D) IgG controls. Peritoneal endometriosis was induced in nude mice, treated with EP2 (AH6809) and EP4 (AH23848) inhibitors (EP2/4-I) at 25 mg/kg body weight, and necropsied as detailed in Fig. 1. *Control vs. EP2/4-I, P < 0.05, n = 8.
Fig. 3.
Fig. 3.
Effects of inhibition of EP2 and EP4 (EP2/4-I) on regulation of key proteins involved in inflammation, survival, invasion, angiogenesis, and biosynthesis and signaling of PGE2, E2, and P4 in endometriosis lesions. (A1–A3) COX-2. (B1–B3) EP2. (C1–C3) EP4. (D1–D3) IL1β. (E1–E3) TNFα. (F1–F3) IL6. (G1–G3) p-AKT. (H1–H3) p-ERK1/2. (I1–I3) β-catenin. (J1–J3) MMP2. (K1–K3) MMP9. (L1–L3) p450 aromatase. (M1–M3) ERα. (N1–N3) ERβ. (O1–O3) PR. (P1–P3) SF-1. (Q1–Q3) VEGF. (R1–R3) vWF proteins. (S1–S2) IgG controls. Nuclei were stained with DAPI (blue), and each protein was stained using Alexa 488 (green) or Alexa 594 (red) secondary antibodies. Peritoneal endometriosis was induced in nude mice, treated with EP2 (AH6809) and EP4 (AH23848) inhibitors (EP2/4-I) at 25 mg/kg, and necropsied as detailed in Fig. 1. *Control vs. EP2/4-I, P < 0.05, n = 8.
Fig. 4.
Fig. 4.
Effects of inhibition of EP2 and EP4 (EP2/4-I) on innervation of endometriosis lesions, regulation of proinflammatory machinery proteins in DRG, and nociception of pelvic pain in endometriosis. (A) Growth of endometriosis lesions measured by gross examination and morphometry (A and B) and fluorescence zoomstereo microscopy (C and D). (B) Pelvic floor referred hyperalgesia using von-Frey test. (C) Expression of neuronal markers PGP9.5, CGRP, SP, TRPV1, and VMAT proteins in endometriosis lesions. Expression of COX-2 (D), EP2 (E), EP4 (F), ILβ (G), TNFα (H), and IL6 (I) proteins in DRG neurons L1, L2, L3, L4, L5, and S1. (J) IgG controls. GLE, glandular epithelium; STR, stromal cells. Nuclei stained with DAPI (blue), each protein stained with Alexa 488 (green) or Alexa 594 (red) secondary antibodies. *Control vs. EP2/4-I, P < 0.05, n = 8. After 3 wk of disease, endometriosis Rag2g(c) mice were treated with EP2 (AH6809) and EP4 (AH23848) inhibitors (EP2/4-I) at 25 mg/kg for 2 wk. At the end of 5 wk, the mice were necropsied at the E2 versus P4 phase of the estrous cycle as confirmed by vaginal cytology, and data from E2 phase shown.
Fig. 5.
Fig. 5.
Effects of inhibition of EP2 and EP4 (EP2/4-I) on regulation of key proteins involved in biosynthesis and signaling of PGE2, E2, and P4, and proinflammation in eutopic endometrium in endometriosis. (A1–A3) COX-2. (B1–B3) EP2. (C1–C3) EP4. (D1–D3) ILβ. (E1–E3) TNFα. (F1–F3) IL6. (G1–G3) p450 aromatase (p450arom). (H1–H3) ERα. (I1–I3) ERβ. (J1–J2) PR protein. (K1–K2) IgG controls. Nuclei were stained with DAPI (blue), each protein was stained with Alexa 488 (green) secondary antibody. GLE, glandular epithelium; LE, luminal epithelium; STR, stromal cells. *Control vs. EP2/4-I, P < 0.05, n = 8. Peritoneal endometriosis was induced in Rag2g(c) mice, treated with EP2 (AH6809) and EP4 (AH23848) inhibitors (EP2/4-I) at 25 mg/kg, necropsied as detailed in Fig. 4, and data from E2 phase shown.
Fig. S1.
Fig. S1.
Proposed mechanism through which selective inhibition of EP2/EP4 induces apoptosis of endometriosis lesions, decreases pelvic pain, and improves endometrial receptivity in endometriosis. Endometriosis Lesions: (1) Inhibition of EP2/EP4 inhibits PGE2-EP2/EP4 signaling in (2) epithelial and stromal cells of endometriosis lesions; (3) suppresses survival and invasion and increases intrinsic apoptosis pathways; (4) inhibits endometriosis-induced neoangiogenesis; (5) decreases production of proinflammatory mediators; (6) decreases E2 biosynthesis and signaling and suppresses E2-dominance; (7) increases PR signaling, decreases P4 resistance, and restores P4-responsive state; and together lead to (8) apoptosis of epithelial and stromal cells and regression of endometriosis lesions. Pelvic Pain: Inhibition of EP2/EP4 by either (9) suppressing the growth of peritoneal endometriosis lesions and their secretory products or (10) directly acting on the nociceptors and DRG neurons (11) decreases afferent/sensory and efferent/sympathetic innervation of endometriosis lesions; (12) decreases proinflammatory microenvironment of DRG (L1-S1); and (13) suppresses peripheral nociception pain pathways and decreases endometriosis-induced pelvic pain. Eutopic Endometrium: Inhibition of EP2/EP4 by either (9) suppressing the growth of peritoneal endometriosis lesions and their secretory products or (14) directly acting on the endometrial epithelial and stromal cells (15) inhibits production of proinflammatory mediators; (16) decreases E2 biosynthesis and signaling and suppresses E2 dominance; (17) increases PR signaling and decreases P4 resistance and restores P4-responsive state; and (18) improves endometrial receptivity machinery.
Fig. S2.
Fig. S2.
GFP and RFP vector map.
See this image and copyright information in PMC

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