Proteome-Wide Effect of 17-beta-Estradiol and Lipoxin A(4) in an Endometriotic Epithelial Cell Line

Endometriosis affects approximately 10% of women of reproductive age. This chronic, gynecological inflammatory disease results in a decreased quality of life for patients, with the main symptoms including chronic pelvic pain and infertility. The steroid hormone 17-beta Estradiol (E2) plays a key role in the pathology. Our previous studies showed that the anti-inflammatory lipid Lipoxin A(4) (LXA(4)) acts as an estrogen receptor-alpha agonist in endometrial epithelial cells, inhibiting certain E2-mediated effects. LXA(4) also prevents the progression of endometriosis in a mouse model via anti-proliferative mechanisms and by impacting mediators downstream of ER signaling. The aim of the present study was therefore to examine global proteomic changes evoked by E2 and LXA(4) in endometriotic epithelial cells. E2 impacted a greater number of proteins in endometriotic epithelial cells than LXA(4). Interestingly, the combination of E2 and LXA(4) resulted in a reduced number of regulated proteins, with LXA(4) mediating a suppressive effect on E2-mediated signaling. These proteins are involved in diverse pathways of relevance to endometriosis pathology and metabolism, including mRNA translation, growth, proliferation, proteolysis, and immune responses. In summary, this study sheds light on novel pathways involved in endometriosis pathology and further understanding of signaling pathways activated by estrogenic molecules in endometriotic epithelial cells

Disrupting Y-Box-Binding Protein 1 Function Using OSU-03012 Prevents Endometriosis Progression in In Vitro and In Vivo Models

The objective of the present study was to test the ability of OSU-03012 (2-amino-N-[4-[5-phenanthren-2-yl-3-(trifluoromethyl)pyrazol-1-yl]phenyl]acetamide), a novel and potent celecoxib-derivative, to impair endometriosis progression in in vitro and in vivo models based on its ability to indirectly block Y-box-binding protein 1 (YB-1) function. 12Z human endometriotic epithelial cells and sexually mature female C57BL/6J mice were treated with OSU-03012. Cellular proliferation was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assay. Expression of YB-1 and phosphorylated YB-1 in 12Z cells and endometriotic lesions was evaluated by Western blotting and immunohistochemistry (IHC). The IHC for proliferating cell nuclear antigen was performed. OSU-03012 treatment resulted in decreased YB-1 and its phosphorylated form in both in vitro and in vivo models. Endometriotic lesion size was significantly reduced in OSU-03012-treated mice (27.6 +/- 4.0 mm(3)) compared to those from the control group (50.5 +/- 6.9 mm(3), P < .0001). A significant reduction in endometriotic epithelial cell proliferation was observed in endometriotic lesions exposed to OSU-03012 treatment (P = .0346). In conclusion, targeting YB-1 via OSU-03012 showed a potent antiproliferative effect on endometriotic epithelial cells in vitro and in a mouse model of disease

L1 cell adhesion molecule as a potential therapeutic target in murine models of endometriosis using a monoclonal antibody approach.

BACKGROUND/AIMS: The neural cell adhesion molecule L1CAM is a transmembrane glycoprotein abnormally expressed in tumors and previously associated with cell proliferation, adhesion and invasion, as well as neurite outgrowth in endometriosis. Being an attractive target molecule for antibody-based therapy, the present study assessed the ability of the monoclonal anti-L1 antibody (anti-L1 mAb) to impair the development of endometriotic lesions in vivo and endometriosis-associated nerve fiber growth. METHODS AND RESULTS: Endometriosis was experimentally induced in sexually mature B6C3F1 (n=34) and CD-1 nude (n=21) mice by autologous and heterologous transplantation, respectively, of endometrial fragments into the peritoneal cavity. Transplantation was confirmed four weeks post-surgery by in vivo magnetic resonance imaging and laparotomy, respectively. Mice were then intraperitoneally injected with anti-L1 mAb or an IgG isotype control antibody twice weekly, over a period of four weeks. Upon treatment completion, mice were sacrificed and endometrial implants were excised, measured and fixed. Endometriosis was histologically confirmed and L1CAM was detected by immunohistochemistry. Endometriotic lesion size was significantly reduced in anti-L1-treated B6C3F1 and CD-1 nude mice compared to mice treated with control antibody (P<0.05). Accordingly, a decreased number of PCNA positive epithelial and stromal cells was detected in autologously and heterologously induced endometriotic lesions exposed to anti-L1 mAb treatment. Anti-L1-treated mice also presented a diminished number of intraperitoneal adhesions at implantation sites compared with controls. Furthermore, a double-blind counting of anti-neurofilament L stained nerves revealed significantly reduced nerve density within peritoneal lesions in anti-L1 treated B6C3F1 mice (P=0.0039). CONCLUSIONS: Local anti-L1 mAb treatment suppressed endometriosis growth in B6C3F1 and CD-1 nude mice and exerted a potent anti-neurogenic effect on induced endometriotic lesions in vivo. The findings of this preliminary study in mice provide a strong basis for further testing in in vivo models

Mediators implicated in estrogen production and cellular proliferation in endometriotic lesions and PFCs are inhibited by LXA₄ treatment.

<p>mRNA was extracted from endometriotic lesions and PFCs and subjected to qPCR analysis to assess transcript levels of <b>A.</b> CYP19a1, <b>B.</b> ERα, <b>C.</b> GREB1, <b>D.</b> CCND1, <b>E.</b> c-Myc and <b>F.</b> ERα, <b>G.</b> CCND1, <b>H.</b> c-Myc, respectively. Results were normalized to GAPDH. Ten mice were used per group (Sham, Veh-, LXA₄-treatment) and sacrificed 21 days after surgical induction of endometriosis. Data are presented as mean ± SEM. (***p<0.001 compared to Veh; ζζζ p<0.001 compared to Sham). <b>I.</b> Immunohistochemical staining for ERα was performed on transverse sections of endometriotic lesions from Veh- (left panel) and LXA₄-treated (right panel) mice. The negative control is shown in the inset.</p

Treatment with LXA₄ decreases endometriotic lesion growth.

<p><b>A.</b> Representative pictures of a mouse with endometriotic lesions treated with vehicle (Veh, left panel) or LXA₄ (right panel) for 21 days. Six uterine biopsies were implanted on the peritoneal wall, 3 on each side, in a minimum of 20 mice which were randomly divided into two groups: Veh- and LXA₄-treated. Implanted tissues grew into enlarged cyst-like lesions by 21 days after endometriosis induction in the Veh group, while LXA₄ treatment significantly reduced lesion size. <b>B</b>. Graphical representation of endometriotic lesions volume, in mm³ ± SEM (**p<0.01). <b>C.</b> Morphology of formalin-fixed, paraffin-embedded lesion sections stained with HE. Upper panels a. and b. Vehicle-treated mouse with endometriosis. Lower panels c. and d. LXA₄-treated mouse with endometriosis. Images are representative of ten biological replicates.</p

LXA₄ reduces peritoneal fluid cytokine and PGE₂ levels as well as the expression of COX-2 in endometriotic lesions.

<p>Peritoneal fluid from mice was collected at sacrifice (Sham, Veh-, LXA₄; 3–5 mice/group) and ELISAs performed to quantify <b>A.</b> IL-1β, <b>B.</b> VEGF and <b>C.</b> PGE₂ levels. Results are expressed as pg mediator/ml for 50 μg of total peritoneal fluid protein and presented as mean ± SEM (*p<0.05, ***p<0.001 compared to Veh; ζ<0.05 compared to Sham). <b>D.</b> Immunohistochemical staining for COX-2 was performed on transverse paraffin-embedded sections of lesions from Veh- and LXA₄-treated mice. Representative stainings are shown with the negative control in the inset.</p