GnRH agonists induce endometrial epithelial cell apoptosis via GRP78 down-regulation

Abstract

Background: Endometriosis is a benign chronic gynecological disease that affects women of reproductive age, characterized by the presence of functional endometrial tissues outside the uterine cavity. GnRH agonists exhibit anti-proliferative and apoptosis-enhancing activities and have long been used for the treatment of endometriosis. There is a critical need to identify the signaling modules involving GnRH agonist therapy for the treatment of endometriosis. In this study, we compared the proteomic profiles of endometriosis in patients before and after GnRH agonist therapy to identify proteins that might provide further information concerning the mechanisms underlying the functions of GnRH agonists.

Methods: A total of 55 protein spots with different abundances were observed using Difference Gel Electrophoresis (DIGE), and 26 of these proteins were assigned clear identities through Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Tandem Mass Spectroscopy (MALDI-TOF/TOF MS).

Results: We validated four of these proteins through Western blotting and immunohistochemistry using human endometrial tissue. We also characterized the effect of Leuprolide acetate (LA) on the apoptosis of eutopic endometrial epithelial cells. LA treatment significantly promoted the apoptosis of eutopic endometrial epithelial cells and inhibited the expression of the anti-apoptotic factor GRP78. GRP78 knockdown enhanced LA-induced cell apoptosis, whereas, the overexpression of GRP78 in eutopic endometrial epithelial cells suppresses LA-induced apoptosis.

Conclusion: These results suggest that GnRH agonists induce endometrial epithelial cell apoptosis via GRP78 down-regulation. This study might provide an important molecular framework for further evaluation of GnRH agonist therapy.

Figure 1

Triptorelin acetate induces eutopic endometrial epithelial cell apoptosis in patients with endometriosis. A. Western blot analysis of cleaved Caspase 3 in eutopic endometrial epithelial cells from endometriosis patients after Triptorelin acetate treatment. B. TUNEL assay to detect apoptosis in eutopic endometrial epithelial cells from endometriosis patients after Triptorelin acetate treatment. *P <0.05.

Figure 2

2D-DIGE of eutopic endometrial proteins. Each individual sample (before and after GnRHa treatment) and internal reference sample was labeled with Cy5, Cy3 and Cy2, respectively, mixed, and separated on a 2D-PAGE gel. The gels were scanned, and Cy5 (A), Cy3 (B) and Cy2 (C) images were obtained from each gel. An overlay of three dye-scanned images was also obtained (D).

Figure 3

GRP78, PPA1, EFHA2, and TGM2 proteins expression in tissues before and after GnRHa treatment. A: Western blot analysis of 4 identified proteins (GRP78, PPA1, EFHA2, and TGM2) in eutopic endometrial epithelial tissues before and after GnRHa treatment (n = 10). The statistical analysis of the protein expression levels in eutopic endometrial epithelial tissues is shown on the right. B: Immunohistochemical detection of GRP78, PPA1, EFHA2, and TGM2 proteins in eutopic endometrial epithelial tissues before and after GnRHa treatment (n = 10). *P <0.05.

Figure 4

Leuprolide acetate induces eutopic endometrial epithelial cell apoptosis. A: Leuprolide acetate (100 ng/mL) up-regulated the expression of cleaved Caspase 3. B: Apoptosis detected through TUNEL staining. Compared with the normal group (untreated), the rate of apoptosis in eutopic endometrial epithelial cells after treatment with 25, 50 and 100 ng/mL Leuprolide acetate was statistically significant. *P <0.05.

Figure 5

Influence of GRP78 knockdown and overexpression on eutopic endometrial epithelial cells apoptosis. A: GRP78 expression in separated eutopic endometrial epithelial cells before and after GnRHa treatment was detected through Western blotting. The statistical analysis of the two groups showed obvious differences. *P <0.05 B: The GRP78 knockdown cells exhibited lower GRP78 expression than the control cells. (Left) The knockdown of GRP78 did not affect normal endometrial cell apoptosis, while Leuprolide acetate treatment increased the percentage of apoptosis in the GRP78 knockdown cells compared with the control cells (Right). Ctrl, cells without LA treatment; NC, cells transfected with scrambled control; siRNA-1, cells transfected with siRNA-1 that targeting GRP78; siRNA-2, cells transfected with siRNA-2 that targeting GRP78. *P <0.05 vs NC within the Ctrl goup, #P <0.05 vs NC within the LA goup. C: Comparison of the GRP78 protein levels in GRP78 overexpression and control groups. (Left); GRP78 overexpression did not affect normal endometrial cell apoptosis, while GRP78 overexpression protected the cells from Leuprolide acetate-induced apoptosis (Right). Ctrl, cells without LA treatment; NC, cells transfected with pCMV-3X Flag; GRP78, cells transfected with pCMV-3X Flag-GRP78. *P <0.05 vs NC within the Ctrl goup, #P <0.05 vs NC within the LA goup.