Estradiol agonists inhibit human LoVo colorectal-cancer cell proliferation and migration through p53

Figure 1 Estradiol and ER selective agonists impair cell proliferation in human LoVo cells. A: Structures of 17β-estradiol, propylpyrazole-triol (PPT), a selective agonist of ERα, and diarylpropionitrile (DPN), a selective agonist of ERβ; B: LoVo cells cultured in DMEM were treated with 17β-estradiol, PPT and DPN for 24 h and 48 h and were then analyzed for cell viability by the MTT assay. ^aP < 0.05 vs control; ^bP < 0.001 vs control (means ± SE, n = 3).

Figure 2 17β-estradiol and/or ER selective agonists upregulate p53 signaling proteins to change the cell cycle progression in human LoVo colorectal cancer cells. A: LoVo cells were treated with E2 or various concentrations (10^-10 mol/L, 10^-9 mol/L and 10^-8 mol/L) of PPT or DPN for 48 h; B: LoVo cells were pretreated with various concentrations (1 μmol/L, 5 μmol/L and 10 μmol/L) of a p53 inhibitor for 1 h followed by E2 (10^-8 mol/L) treatment for 48 h; C: LoVo cells were pretreated with vehicle, the ER antagonist ICI 182780 (ICI), or a p53 inhibitor for 1h, followed by E2 administration for 48 h. Subsequently, cells were harvested and measured by Western blotting. A β-actin standard was used as a loading control for all proteins. All experiments were repeated twice with identical results.

Figure 3 17β-estradiol and ER selective agonists impair the Wnt-β-catenin signaling pathway in human LoVo colorectal cancer cells. LoVo cells were treated with E2 (10^-8 mol/L) or various concentrations (10^-10 mol/L, 10^-9 mol/L and 10^-8 mol/L) of PPT (A) or DPN for 24 h (B). Cells were harvested and measured by Western blotting. An α-tubulin standard was used as a loading control for all proteins. All experiments were repeated twice with identical results.

Figure 4 17β-estradiol and/or ER selective agonistsdown-regulate the protein levels of u-PA, t-PA and MMP-9 inLoVocells via p53. A: LoVo cells were treated with E2 or various concentrations (10^-10 mol/L, 10^-9 mol/L and 10^-8 mol/L) of PPT or DPN for 24 h; B: LoVo cells were pretreated with various concentrations (10^-10 mol/L, 10^-9 mol/L and 10^-8 mol/L) of a p53 inhibitor for 1 h, followed by E2 administration for 24 h; C: LoVo cells were pretreated with vehicle, the ER antagonist ICI 182780, or a p53 inhibitor for 1 h, followed by E2 administration for 24 h. Cells were harvested and examined by Western blotting. A β-actin or α-tubulin standard was used as a loading control for all proteins. All experiments were repeated twice with identical results.

Figure 5 17β-estradiol inhibits MMP-2/9 activity in LoVo colorectal colon cells. LoVo cells cultured in DMEM were treated with E2 (10^-8 mol/L) for 24 h, and then, the culture medium was collected. Samples were electrophoresed without reduction on 8% SDS polyacrylamide gels copolymerized with 0.1% gelatin.

Figure 6 17β-estradiol and/or ER selective agonists inhibit human LoVo colorectal cancer cell motility through p53 signaling. LoVo cells cultured in DMEM were pretreated with vehicle, ICI 182780 (ER antagonist 1 μmol/L), p53 inhibitor (10 μmol/L), MPP (ERα antagonist 1 μmol/L), or PHTPP (ERβ antagonist 1 μmol/L) for 1 h, followed by treatment with E2 (10^-8 mol/L), PPT (10^-8 mol/L) or DPN (10^-8 mol/L) administration for 48h.The migration ability of LoVo cells was analyzed by a scratch motility assay (A) and a migration assay (B). The results were observed and analyzed with a fluorescence microscope. All experiments were repeated twice with identical results. ^aP < 0.05 vs control; ^bP < 0.01 vs control; ^dP < 0.01 vs E2.