Involvement of cyclin B1 in progesterone-mediated cell growth inhibition, G2/M cell cycle arrest, and apoptosis in human endometrial cell

Abstract

Background: Progesterone plays an important role in the proliferation and differentiation of human endometrial cells (hECs). Large-dose treatment with progesterone has been used for treatment of endometrial proliferative disorders. However, the mechanisms behind remain unknown.

Methods: To investigate the role of cyclin B1 in proliferation and differentiation of hECs in menstrual cycle, the expression of cyclin B1 throughout the menstrual cycle was evaluated in hECs. To determine the effects of progesterone on the proliferation, cell cycle progression and apoptosis of hECs and to test if cyclin B1 is involved in these effects, progesterone and/or Alsterpaullone (Alp, a specific inhibitor of Cyclin B1/Cdc2) were added to primary hECs. Cellular proliferation was evaluated with MTT test, cell cycle with propidium iodide (PI) staining and flow cytometry, apoptosis with FITC-Annexin V and the expression of cyclin B1 with Western blotting.

Results: The expression level of cyclin B1 in secretory endometria was significantly lower than in proliferative endometria (p < 0.01). Progesterone significantly inhibited the growth of hECs in a concentration-dependent manner (P < 0.01). The treatment with progesterone significantly decreased the expression of cyclin B1, increased the proportions of cell in G2/M phase, and apoptotic cells (P < 0.05 for all). The presence of Alp significantly enhanced the effects of progesterone on cyclin B1 down-regulation, G2/M cell cycle arrest and induction of apoptosis (P < 0.01 for all).

Conclusion: Our findings suggest that cyclin B1 is a critical factor in proliferation and differentiation of hECs. Progesterone may inhibit cell proliferation, mediate G2/M cell cycle arrest and induce apoptosis in hECs via down-regulating Cyclin B1.

Figure 1

Expression of cyclin B1 in human endometrium detected by Western blot. The relative expression of cyclin B1 in human endometrium at the secretory phase is significantly lower than the proliferative phase (**P < 0.01).

Figure 2

Inhibition of cell growth on hECs by progesterone. Human endometrial cells were treated with progesterone at concentrations of 1 × 10^-9, 1 × 10^-8, 1 × 10^-7 or 1 × 10^-6 M and cell growth was evaluated with MTT. Progesterone inhibited hECs growth in a dose-dependent manner. (*P < 0.05, **P < 0.01, compared with the control cells).

Figure 3

Decreased expression of Cyclin B1 induced by progesterone and/or Alp. Human endometrial cells were treated with progesterone and/or Alp. The expression of cyclin B1 was significantly decreased by the treatment of progesterone or progesterone and Alp, but not Alp alone (*P < 0.05, **P < 0.01, compared with the control cells). The levels of cyclin B1 was significantly decreased in cells treated with progesterone and Alp compared with those treated with progesterone or Alp alone (#P < 0.01).

Figure 4

Arrest of cell cycle in G2/M phase induced by progesterone and Alp. Human endometrial cells were treated with progesterone alone or in combination with Alp. Cell cycles were analyzed by flow cytometry. A: Cell cycle analyzed with propidium iodide (PI) staining followed by flow cytometry. B: Comparison of the proportions of cells in G2/M phase. The proportion of cells in G2/M phase was significantly increased after hECs were treated with progesterone or progesterone and Alp (*P < 0.05, **P < 0.01, compared with the control cells). There was also significant difference between treatment with progesterone alone and progesterone plus Alp (#P < 0.05).

Figure 5

Apoptosis of hECs induced by progesterone and Alp. Human endometrial cells were treated with progesterone alone or in combination with Alp. Apoptosis was indexed by the detection of Annexin V. A: Apoptotic cells as labeled with Annexin V and followed by flow cytometry. B: Comparison of the proportions of apoptotic cells. The amount of apoptotic cells was significantly increased with the treatment of progesterone alone or progesterone plus ALP (*P < 0.05, **P < 0.01, compared with the control cells). There was also significant difference between treatment with progesterone alone and progesterone plus Alp (#P < 0.01).