Obesity and the Endometrium: Adipocyte-Secreted Proinflammatory TNF α Cytokine Enhances the Proliferation of Human Endometrial Glandular Cells

Abstract

Obesity, a state of chronic inflammation, is associated with poor fertility and low implantation rates and is a well-documented risk factor for endometrial cancer. Adipokines, such as tumor necrosis factor alpha, play an important role in initiation of endometrial cancer. The aim of this study is to evaluate in vitro effects of human adipocyte cells (SW872) on growth of endometrial glandular epithelial cells (EGE). Methods. We measured cell proliferation and expression of cell-growth proteins-proliferating cell nuclear antigen, cyclin D1, cyclin-dependent kinase-1, and apoptotic markers (BCL-2 and BAK) in human EGE cells cocultured with SW872 cells. EGE cells were also evaluated in SW872-conditioned media neutralized with anti-TNF α antibody. Results. A significant increase in EGE cell proliferation was observed in both SW872-conditioned media and in coculture (P < 0.05). We observed an upregulation of proliferation markers PCNA, cyclin D1, CDK-1, and BCL-2 and decrease in BAK (P < 0.05). Neutralization of SW872-conditioned media using anti-TNF α antibodies reversed EGE cell proliferation as indicated by BCL-2 expression. Conclusions. Adipocytes have potent proliferative paracrine effect on EGE cells which may be, in part, mediated via TNF α . Further understanding of the role of obesity in endometrial carcinogenesis should lead to better preventative and therapeutic strategies.

Figure 1

Effect of SW872-conditioned media on proliferation of EGE cells. EGE cells were cultured in 96-well cell culture plate and treated with conditioned media which were diluted from 2- to 10-fold concentrations. Cell proliferation in EGE treated with and without dilutions of SW872-conditioned media was assessed using CyQuant assay as described in Methods. Results are expressed as means ± SE from 3 separate experiments. *Significantly different from the control (P < 0.05).

Figure 2

EGE cell proliferation with and without SW872 coculture. EGE cells were cocultured with SW872 cells for days 2, 4, and 6. Comparison in cell proliferation is made with EGE cells grown without SW872 coculture. Proliferation in EGE cells was measured using CyQuant assay. Results are expressed as means ± SE from 3 separate experiments. *Significantly different from the control (P < 0.05).

Figure 3

Western blot analysis of PCNA, BCL-2, cyclin D1, CDK-1, and BAK in EGE cells with and without coculture. EGE and SW872 cells were in coculture for 6 days. Lysates prepared from control and cocultured cells were analyzed by Western blotting with (a) anti-PCNA, (b) anti-BCL-2, (c) anticyclin D1, (d) anti-CDK-1, and (e) anti-BAK antibodies. The intensity of each protein signal was quantified and normalized with corresponding β-actin. Results shown represent three separate experiments with comparable results. *P < 0.05 compared with control.

Figure 4

Effect of different concentrations of TNFα on EGE cell proliferation. Cell proliferation in EGE cell treated with different concentrations of TNFα was assessed using CyQuant assay as described in Methods. Results are expressed as means ± SE from 3 separate experiments. *Significantly different from the control (P < 0.05).

Figure 5

Effects of anti-TNFα neutralizing antibodies on EGE cell proliferation. Conditioned media collected from SW872 cells grown to more than 80 percent confluence were centrifuged, filtered, and diluted to 1%. These conditioned media were treated with 1 ng/mL of anti-TNFα antibody for an hour at 37°C. The neutralized conditioned media were then added to EGE cells and cell proliferation measured using CyQuant assay. Results shown represent three separate experiments with comparable results. *P < 0.05 (mean ± SE; n = 3).

Figure 6

Effects of anti-TNFα neutralized conditioned media on the expression of anti-apoptotic BCL-2 in EGE cells. Conditioned media collected from SW872 cells grown to more than 80 percent confluence were centrifuged, filtered, diluted to 1%, and neutralized for an hour with 1 ng/mL of anti-TNFα antibody for an hour at 37°C. Lysates prepared from control and treated EGE cells were analyzed by Western blotting with anti-BCL-2 antibody. The intensity of each protein signal was quantified and normalized with corresponding β-actin. *P < 0.05 compared with control (mean ± SE; n = 3).