Gonadotropin-releasing hormone (GnRH)-I and GnRH-II induce cell growth inhibition in human endometrial cancer cells: involvement of integrin beta3 and focal adhesion kinase

Abstract

Endometrial carcinoma is the most common neoplasm of the female genital tract, accounting for nearly one half of all gynecologic cancers in the Western world. Although intensive research on pathological phenomena of endometrial cancer is currently going on, but exact cause and biological aspects of this disease are not well described yet. In addition to well-documented roles of gonadotropin-releasing hormone (GnRH) in hypopituitary ovarian (HPO) axis, the agonistic or antagonistic analogs (or both) of GnRH have been shown to inhibit the proliferation of a variety of human gynecologic cancers. Thus, in the present study, we further examined the possibility that GnRH induces integrin beta3 and activation of focal adhesion kinase (FAK) through mitogen-activated protein kinases (MAPKs), ERK1/2 and p38, to inhibit the growth of HEC1A endometrial cancer cell line. As a result, both GnRH-I and GnRH-II resulted in a significant increase in integrin beta3 expression and evoked the activation of FAK in a time-dependent manner in these cells. In addition, these analogs induced an activation of ERK1/2 and p38 MAPK in a time-dependent manner as downstream pathways of FAK. It appears that GnRH-II has much greater effect on the activation of FAK, ERK1/2 and p38 compared to GnRH-I in these cells. Further, we demonstrated that the growth inhibition of HEC1A cells by GnRH-I or GnRH-II is involved in the activation of integrin-FAK and ERK1/2 and p38 MAPK pathways. Taken together, these results suggest that GnRH may be involved in the inhibition of endometrial cancer cell growth via activation of integrin beta3 and FAK as a direct effect. This knowledge could contribute to a better understanding of the mechanisms implicated in the therapeutic action of GnRH and its biomedical application for the treatment against endometrial cancer.

Figure 1

Effect of GnRH-I and GnRH-II on the cell growth. HEC1A cells were treated with GnRH-I or GnRH-II analog (10 nM or 100 nM) for 24 h in serum-free medium. Cell proliferation was determined by ³H-thymidin incorporation assay following treatment with GnRHs. Values are the means of cell number (± S.E.) in triplicates of three independent experiments. a, indicates p < 0.05 vs. control (cont).

Figure 2

Effect of GnRH-I and GnRH-II on the expression of integrin β3 subunit. HEC1A cells were treated with GnRH-I or GnRH-II analog (100 nM) for 24 h in serum-free medium. The protein level of integrin β3 subunit was determined and measured by immunoblot analysis. Data are shown as means of three individual experiments and presented as the mean ± S.D. a, indicates p < 0.05 vs. control (cont).

Figure 3

Effect of GnRH-I and GnRH-II on the activation of FAK. HEC1A cells were treated with GnRH-I or GnRH-II (100 nM) in a time-dependent manner. Phosphorylation of FAK was determined by Western blot analysis following treatment with GnRHs. Data are shown as means of three individual experiments and presented as the mean ± S.D. a, indicates p < 0.05 vs. control (cont). The white bars indicate GnRH-I treatment while gray bars indicate GnRH-II treatment.

Figure 4

Effect of GnRH-I and GnRH-II on the activation of ERK1/2. HEC1A cells were treated with GnRH-I or GnRH-II (100 nM) in a time-dependent manner. Phosphorylation of ERK1/2 was determined by Western blot analysis following treatment with GnRHs. The error bars represent the mean ± S.D. from three independent experiments. a, indicates p < 0.05 vs. control (cont).). The white bars indicate GnRH-I treatment while gray bars indicate GnRH-II treatment.

Figure 5

Effect of GnRH-I and GnRH-II on the activation of p38. HEC1A cells were treated with GnRH-I or GnRH-II (100 nM) in a time-dependent manner. Phosphorylation of ERK1/2 was determined by Western blot analysis following treatment with GnRHs. The error bars represent the mean ± S.D. from three independent experiments. a, indicates p < 0.05 vs. control (cont).). The white bars indicate GnRH-I treatment while gray bars indicate GnRH-II treatment.

Figure 6

Effect of SB202190 on the GnRH-I-induced or GnRH-II-induced p38 activation. HEC1A cells were treated with GnRH-I or GnRH-II for 20 min in serum-free medium in the absence or presence of SB202190 (100 nM), a specific inhibitor of p38, to further elucidate the direct effect of GnRH-I or -II on the activation of p38 in HEC1A cells.

Figure 7

Effect of SB202190 on the GnRH-I- or GnRH-II-induced inhibition of cell growth. HEC1A cells were treated with GnRH-I or GnRH-II for 24 h in serum-free medium in the absence or presence of SB202190 (100 nM), a specific inhibitor of p38. Cell proliferation was determined by ³H-thymidin incorporation assay following treatment with GnRHs. Values are the mean cell number (± S.E.) from three independent experiments. Values are the means of cell number (± S.E.) in triplicates of three independent experiments. a, indicates p < 0.05 vs. control (cont); b, indicates p < 0.05 vs. GnRH-I or GnRH-II treatment only.