Figure 4.

Dienogest induces cell cycle arrest in EMOsis cc/TERT cells and CRL-4003 cells by promoting p21 production and affects MET expression. (A) Inhibition of cell cycle progression by decorin and dienogest. EMOsis cc/TERT cells and CRL-4003 cells were exposed to 100 or 500 nmol/l dienogest or 3 or 6 μg/ml decorin overnight, or were left untreated, and then processed for PI flow cytometry. The cell cycle characteristics (G0–G1: S: G2–M%) of EMOsis cc/TERT cells were as follows: control (52.64±0.67: 29.45±0.92: 17.91±1.08), 100 nM dienogest (66.39±0.18*: 19.33±0.36*: 14.28±1.81), 500 nM dienogest (80.38±0.57*: 12.14±0.32*: 7.48±0.37*), 3 mg/ml decorin (67.59±0.95*: 19.8±0.04*: 12.61±0.83), and 6 mg/ml decorin (77.21±0.26*: 14.51±0.64*: 8.28±0.56*) and CRL-4003 cells: control (57.23±0.10: 20.46±0.63: 22.31±0.71), 100 nM dienogest (66.57±0.38*: 15.23±0.09: 18.2±0.26), 500 nM dienogest (78.07±0.69*: 13.61±0.17*: 8.32±0.91*), 3 mg/ml decorin (65.05±0.50*: 15.22±0.75: 19.73±0.27), and 6 mg/ml decorin (75.31±0.47*: 15.28±1.08*: 9.41±0.29*). The data are expressed as the means±s.d. (N=5), and * indicates a significant (P<0.05) difference compared with the untreated control. The panel shows the proportion of cells in the G0/G1 phase (black), S phase (gray), and G2/M phase (white). (B) The expression of p21 was increased and the expression of MET was decreased by dienogest and progesterone in a decorin-dependent manner. Proteins were extracted from CRL-4003 cells and EMOsis cc/TERT cells after treatment with 100 nmol/l dienogest, 100 nmol/l progesterone, or 100 nmol/l dienogest with or without 5 μg/ml of a decorin-neutralizing antibody for 24 h. The western blot analysis was performed with anti-Met antibodies, anti-p21 antibodies, and anti-β-actin antibodies. The lower panel shows the densitometric quantification of the western blot analysis normalized to the β-actin expression and expressed as a fold-increase relative to the basal transcription level in the control. The mean±s.d. of three determinations is shown. *P<0.05 (C) Decorin and p21 expression is increased and MET expression is decreased by dienogest and progesterone in a decorin-dependent manner. Proteins were extracted from CRL-4003 cells, EMOsis cc/TERT cells, and EMOsis cc/TERT cells transfected with DCN siRNA cultured with or without progesterone or dienogest for 24 h. The western blot analysis was performed with anti-decorin antibodies, anti-p21 antibodies, anti-Met antibodies, and anti-β-actin antibodies. The lower panel shows the densitometric quantification of the western blot analysis normalized to the β-actin expression and expressed as a fold-increase relative to the basal transcription level in the control. The mean±s.d. of three determinations is shown. *P<0.05