Potential use of ellagic acid for endometriosis treatment: its effect on a human endometrial cell cycle, adhesion and migration.

Endometriosis is a common and challenging condition of reproductive-aged women that is defined as the presence of endometrial-like tissue outside the uterine cavity. Despite its prevalence, there is still no effective therapeutics; so we aim to evaluate the ellagic acid (EA) effect on the most relevant aspects that are known to be altered in endometriosis. Endometrial primary cultures from women with and without endometriosis and endometrial cell lines were incubated with EA (50 and 100 µM) for 24 and 48 h. The results demonstrated that EA arrests an endometrial stromal cell cycle on the G2/M phase, after 48 h. In addition, 100 µM EA treatment significantly decreased ECC-1 cell migration at 20 h and T-HESC cell migration at 10 h and 20 h, while 50 µM EA significantly decreased T-HESC cell migration at 20 h. On the other hand, we proved that the treatment with EA for 24 h reduces T-HESC and ECC-1 adhesion to plastic. However, we did not find an effect of EA on cell proliferation. EA has an inhibitory effect on endometrial cell adhesion, migration and cell cycle progression in vitro. These highlight the idea to investigate natural compounds as novel and promising candidates for therapeutic treatment of endometriosis.

Natural therapies assessment for the treatment of endometriosis.

STUDY QUESTION: Can resveratrol and epigallocatechin-3-gallate (EGCG) inhibit the growth and survival of endometriotic-like lesions in vivo in a BALB/c model of endometriosis, and in vitro in primary cultures of human endometrial epithelial cells (EECs)? SUMMARY ANSWER: Resveratrol and EGCG exerted a potent inhibitory effect on the development of endometriosis in a BALB/c murine model and on the survival of EECs. WHAT IS KNOWN ALREADY: Endometriosis is a common condition associated with infertility and pelvic pain in women of reproductive age. Resveratrol and EGCG are two polyphenols with anticarcinogenic and antioxidant properties that have been proposed as natural therapies to treat endometriosis. STUDY DESIGN, SIZE, DURATION: Fifty-six 2-month-old female BALB/c mice underwent surgical induction of endometriosis. Treatments with resveratrol or EGCG started 15 days post-surgery and continued for 4 weeks. Human biopsies were taken with a metal Novak curette from the posterior uterine wall from 16 patients with untreated endometriosis and 15 controls who underwent diagnostic laparoscopy for infertility. MATERIALS, SETTING, METHODS: After the treatments, animals were sacrificed and lesions were counted, measured, excised and fixed. Immunohistochemistry for proliferating cell nuclear antigen and CD34 was performed for cell proliferation and vascularization assessment in the lesions. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) technique was performed for apoptosis evaluation. Peritoneal fluid was collected to analyze vascular endothelial growth factor levels. Human EECs were purified from proliferative-phase endometrial biopsies and cultured. The effect of both polyphenols on cell proliferation was determined by a colorimetric assay using the CellTiter 96®AQueous One Solution Cell Proliferation Assay kit and on apoptosis by the TUNEL technique, using an In Situ Cell Death Detection Kit with Fluorescein. MAIN RESULTS: In the mouse model, both treatments significantly reduced the mean number (P < 0.05 versus control) and the volume of established lesions (P < 0.05 versus control). Treatments consistently statistically significantly diminished cell proliferation (resveratrol P < 0.01 and EGCG P < 0.05, versus control), reduced vascular density (resveratrol P < 0.01 and EGCG P < 0.001, versus control) and increased apoptosis within the lesions (resveratrol P < 0.01 and EGCG P < 0.05, versus control). Both compounds induced reduction in human EEC proliferation (P < 0.05 versus basal) and increased apoptosis (P < 0.05 versus basal) in primary cultures. LIMITATIONS: In vitro studies were only carried out in epithelial cells from human eutopic endometrium. WIDER IMPLICATIONS OF THE FINDINGS: The present findings are promising and will assist the development of novel natural treatments for endometriosis. STUDY FUNDING: This study was supported by ANPCYT (PICT 6384 BID 1201 OC-AR) and CONICET (PIP 5471), Argentina. None of the authors has any conflict of interest to declare.

Effects of a selective cyclooxygenase-2 inhibitor on endometrial epithelial cells from patients with endometriosis.

BACKGROUND: Celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, also has anti-proliferative properties and pro-apoptotic effects on different in vivo and in vitro models, two actions that may be efficacious in therapy for endometriosis. We evaluated the effects of celecoxib on apoptosis and proliferation, and vascular endothelial growth factor (VEGF) production and COX-2 expression and activity in endometrial epithelial cells (EECs). METHODS AND RESULTS: Thirty-two endometriosis and 13 control women were included in the study. EECs from eutopic endometrium and control biopsies were cultured with different doses of celecoxib. Celecoxib at 50, 75 and 100 microM (versus vehicle control) inhibited EEC proliferation in cultures from controls (P < 0.05, P < 0.01 and P < 0.01, respectively) and patients with endometriosis (P < 0.05, P < 0.01 and P < 0.01), as assessed by (3)H-thymidine uptake. Celecoxib at 50, 75 and 100 microM induced apoptosis in EEC from controls (P < 0.05, P < 0.001 and P < 0.001) and patients with endometriosis (P < 0.001, P < 0.001 and P < 0.01), as revealed by the Acridine Orange-Ethidium Bromide technique. Western blot analysis showed that celecoxib was effective at increasing COX-2 protein at 100 microM in EEC from endometriosis patients (P < 0.05). In EEC from endometriosis patients, celecoxib at 25, 50 and 100 microM was also effective in reducing COX-2 activity, reflected in the reduction of prostaglandin E(2) (PGE(2)) synthesis (P < 0.001), and VEGF secretion (P < 0.001; P < 0.05 and P < 0.001), assessed by enzyme-linked immunosorbent assay. Exogenous PGE(2) did not reverse celecoxib-induced growth inhibition. CONCLUSIONS: This study suggests a direct effect of celecoxib on reduction of endometrial growth and supports further research on selective COX-2 inhibition as a novel therapeutic modality in endometriosis.

Effect of GnRH analogues on apoptosis and expression of Bcl-2, Bax, Fas and FasL proteins in endometrial epithelial cell cultures from patients with endometriosis and controls.

BACKGROUND: Our purpose was to evaluate the effect of the GnRH agonist (GnRHa), leuprolide acetate (LA), and the GnRH antagonist (GnRHant), Antide, on apoptosis and expression of apoptosis-related proteins in endometrial epithelial cell (EEC) cultures from patients with endometriosis and controls (infertile women without endometriosis). METHODS: Biopsy specimens of eutopic endometrium were obtained from 22 patients with endometriosis and from 14 women that served as controls. Apoptosis was examined in EEC after incubation with LA and Antide. Bax, Bcl-2, Fas and FasL expression was evaluated after exposure to LA, Antide or a combination of both. The percentage of apoptotic cells (%ApC) was assessed by the acridine orange-ethidium bromide technique, and protein expression was evaluated by western blot and immunocytochemistry. RESULTS: LA 100 and 1000 ng/ml increased the %ApC in EEC from patients with endometriosis (both P < 0.05) and controls (p < 0.05 and P < 0.01, respectively). Antide 10(-5) M increased the %ApC in EEC from patients with endometriosis and controls (P < 0.01). In EEC from women with endometriosis, Bax expression increased after treatment with LA, Antide and LA + Antide (P < 0.05, P < 0.001 and P < 0.001), whereas Bcl-2 expression decreased after exposure to LA and Antide (P < 0.001 and P < 0.01). FasL expression increased after LA, Antide and LA + Antide treatments (P < 0.01, P < 0.001 and P < 0.01). No significant changes were observed on Fas expression. CONCLUSIONS: GnRH analogues enhanced apoptosis in EEC, and this was accompanied by an increase in expression of the pro-apoptotic proteins Bax and FasL and a decrease in expression of the anti-apoptotic protein Bcl-2.