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. 2012 Sep;97(9):3224-30.
doi: 10.1210/jc.2012-1538. Epub 2012 Jul 3.

Mullerian inhibiting substance induces apoptosis of human endometrial stromal cells in endometriosis

Jeong Namkung  1 , Jae Yen Song, Hyun Hee Jo, Mee Ran Kim, Young Oak Lew, Patricia K Donahoe, David T MacLaughlin, Jang Heub Kim
Affiliations

Mullerian inhibiting substance induces apoptosis of human endometrial stromal cells in endometriosis

Jeong Namkung et al. J Clin Endocrinol Metab. 2012 Sep.

Abstract

Context: Müllerian inhibiting substance (MIS) is produced in Sertoli cells of fetal testis and causes regression of müllerian ducts in male embryos. MIS also can induce the cell cycle arrest and apoptosis in müllerian duct-derived tumors in vivo and in vitro.

Objective: Our objective was to investigate the expression of MIS type II receptor (MISR II) and whether MIS can inhibit the proliferation and induce apoptosis in primary cultures of endometrial stromal cells (ESC) of endometriosis.

Design and settings: In vitro experiments were performed in the university research laboratory.

Participants: Tissue samples from 12 patients who had undergone evisceration for ovarian endometrial cysts were included in this study.

Interventions and main outcome measures: The expression of MISR II in ESC was investigated by immunohistochemistry. The cell viability and apoptosis in ESC treated with MIS was measured by methylthiazoletetrazolium assay and annexin V analysis. The expression of regulatory proteins in ESC treated with MIS was shown by Western blotting.

Results: ESC showed specific immunostaining for the MISR II. ESC treated with MIS exhibited 32% growth inhibition (P = 0.0001). The changes in cell cycle distribution after MIS exposure at 72 h demonstrated that S and G(2)M phases were decreased; G(0)G(1) and sub-G(0)G(1) phases were increased. ESC treated with MIS showed 13.72% annexin V-fluorescein isothiocyanate positivity. In the ESCs, which contain defective p16, MIS increased the expression of pocket proteins p107 and p130 and decreased E2F transcription factor 1.

Conclusions: The results support a central role for MIS in endometriosis. Although the precise mechanism of MIS-mediated inhibition of ESC growth has not been fully defined, these data suggest that MIS has activity against ESC in vitro and may also be an effective targeted therapy for endometriosis.

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Figures

Fig. 1.
Fig. 1.
ESC express the MISR II by immunohistochemistry with rabbit polyclonal antihuman MISR II antiserum. In this figure and all others that follow, the right lower panel is a higher magnification of the boxed area (×400). Chromogen is 3-amino-9-ethylcarbazole. Magnification, ×200.
Fig. 2.
Fig. 2.
Effect of MIS on the viability in ESC. Cells were treated with 7.14 nm MIS at 24 h after plating and MTT added at various times later, and the absorbance was read at 550 nm. Results are presented as percentage of control, which was calculated using the following equation: (mean absorbance of treated cells/mean absorbance of control cells) × 100. Data are expressed as mean ± sd from five independent experiments. *, P < 0.05 compared with corresponding vehicle control cells.
Fig. 3.
Fig. 3.
Cell cycle distribution after exposure of ESC to 7.14 nm MIS for 72 h. Cells were trypsinized, fixed in 100% methanol, washed, and exposed to PI/ribonuclease solution. Histograms of cellular DNA content were obtained by flow cytometry.
Fig. 4.
Fig. 4.
Induction of early and late apoptosis by MIS in ESC. Cells were treated with 7.14 nm MIS for 72 h. For apoptosis, the externalization of phosphatidylserine was assessed by measuring annexin-V-FITC binding using PI as a counterstain. Quadrant rectangular dot grams from a representative of three independent experiments are shown.
Fig. 5.
Fig. 5.
Western blot analysis for the expression of cell-cycle-related proteins in ESC treated with 7.14 nm MIS or vehicle for 72 h. The protein level of the negative control was set at 100%. Data means ± sd of the OD were normalized to actin. *, P < 0.05 compared with vehicle controls.
Fig. 6.
Fig. 6.
Western blot analysis for the expression of apoptosis-related proteins in ESC treated with 7.14 nm MIS or vehicle for 72 h. The OD of the stained protein of the vehicle control was set at 100%. Data means ± sd of the OD were normalized to actin. *, P < 0.05 compared with vehicle controls.
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