Effect of sulphation on the oestrogen agonist activity of the phytoestrogens genistein and daidzein in MCF-7 human breast cancer cells

Abstract

The phytoestrogens genistein, daidzein and the daidzein metabolite equol have been shown previously to possess oestrogen agonist activity. However, following consumption of soya diets, they are found in the body not only as aglycones but also as metabolites conjugated at their 4'- and 7-hydroxyl groups with sulphate. This paper describes the effects of monosulphation on the oestrogen agonist properties of these three phytoestrogens in MCF-7 human breast cancer cells in terms of their relative ability to compete with [(3)H]oestradiol for binding to oestrogen receptor (ER), to induce a stably transfected oestrogen-responsive reporter gene (ERE-CAT) and to stimulate cell growth. In no case did sulphation abolish activity. The 4'-sulphation of genistein reduced oestrogen agonist activity to a small extent in whole-cell assays but increased the relative binding affinity to ER. The 7-sulphation of genistein, and also of equol, reduced oestrogen agonist activity substantially in all assays. By contrast, the position of monosulphation of daidzein acted in an opposing manner on oestrogen agonist activity. Sulphation at the 4'-position of daidzein resulted in a modest reduction in oestrogen agonist activity but sulphation of daidzein at the 7-position resulted in an increase in oestrogen agonist activity. Molecular modelling and docking studies suggested that the inverse effects of sulphation could be explained by the binding of daidzein into the ligand-binding domain of the ER in the opposite orientation compared with genistein and equol. This is the first report of sulphation enhancing activity of an isoflavone and inverse effects of sulphation between individual phytoestrogens.

Figure 1

Comparison of the chemical structures of 17β-oestradiol, genistein, genistein-4′-sulphate, genistein-7-sulphate, daidzein, daidzein-4′-sulphate, daidzein-7-sulphate, equol and equol-7-sulphate. Dotted arrows indicate the orientation of binding of 17β-oestradiol (Brzozowski et al. 1997) and genistein (Manas et al. 2004) into the LBD of ERα and ERβ respectively, as determined by X-ray crystallography.

Figure 2

Competitive binding of monosulphated and non-sulphated forms of (A) genistein, (B) daidzein and (C) equol to ER from MCF-7 human breast cancer cells. In single-point competitive binding assays, 4×10⁻¹⁰ M [2,4,6,7-³H]oestradiol was incubated with cytosol plus the stated molar excess of unlabelled genistein (solid square, solid line), genistein-4′-sulphate (open square, dotted line), genistein-7-sulphate (open diamond, dotted line), daidzein (solid circle, solid line), daidzein-4′-sulphate (open circle, dotted line), daidzein-7-sulphate (open star, dotted line), equol (solid triangle, solid line) and equol-7-sulphate (open triangle, dotted line). Error bars represent the mean±s.e.m. of triplicate assays.

Figure 3

Regulation by monosulphated and non-sulphated forms of (A) genistein, (B) daidzein and (C) equol of CAT gene expression from a stably transfected ERE-CAT gene in MCF-7 human breast cancer cells. The cells were grown in RPMI 1640 medium/5% DCFCS for 7 days, and then in the same medium for a further 24 h with genistein (solid square, solid line), genistein-4′-sulphate (open square, dotted line), genistein-7-sulphate (open diamond, dotted line), daidzein (solid circle, solid line), daidzein-4′-sulphate (open circle, dotted line), daidzein-7-sulphate (open star, dotted line), equol (solid triangle, solid line) and equol-7-sulphate (open triangle, dotted line) at the molar concentrations indicated. The results are presented graphically as the % of the induction with 10⁻⁸ M 17β-oestradiol. Bars represent the mean±s.e.m. of triplicate assays both with 17β-oestradiol and with phytoestrogen.

Figure 4

Effects of monosulphated and non-sulphated forms of (A) genistein, (B) daidzein and (C) equol on the proliferation of MCF-7 human breast cancer cells in monolayer culture. The cells were grown for 7 days in phenol red-free RPMI 1640 medium/5% DCFCS with no further addition (given at zero molar concentration) or with genistein (solid square, solid line), genistein-4′-sulphate (open square, dotted line), genistein-7-sulphate (open diamond, dotted line), daidzein (solid circle, solid line), daidzein-4′-sulphate (open circle, dotted line), daidzein-7-sulphate (open star, dotted line), equol (solid triangle, solid line) and equol-7-sulphate (open triangle, dotted line) at the molar concentrations indicated. The results are presented graphically as the % number of doublings with the phytoestrogen compared with the number of doublings with 10⁻⁸ M 17β-oestradiol in that same assay. Error bars are the standard error of all nine values from triplicate dishes with 10⁻⁸ M 17β-oestradiol and triplicate dishes with phytoestrogen.

Figure 5

Effects of monosulphated and non-sulphated forms of (A) genistein, (B) daidzein and (C) equol on the proliferation of MCF-7 human breast cancer cells in monolayer culture. The cells were grown for 14 days in phenol red-free RPMI 1640 medium/5% DCFCS with no further addition (given at zero molar concentration) or with genistein (solid square, solid line), genistein-4′-sulphate (open square, dotted line), genistein-7-sulphate (open diamond, dotted line), daidzein (solid circle, solid line), daidzein-4′-sulphate (open circle, dotted line), daidzein-7-sulphate (open star, dotted line), equol (solid triangle, solid line) and equol-7-sulphate (open triangle, dotted line) at the molar concentrations indicated. The results are presented graphically as the % number of doublings with the phytoestrogen compared with the number of doublings with 10⁻⁸ M 17β-oestradiol in that same assay. Error bars are the standard error of all nine values from triplicate dishes with 10⁻⁸ M 17β-oestradiol and triplicate dishes with phytoestrogen.

Figure 6

Competitive binding of monosulphated and non-sulphated forms of (A) genistein, (B) daidzein and (C) equol to recombinant human ERα (Invitrogen). In single-point competitive binding assays, 0·8 nM [2,4,6,7-³H]oestradiol was incubated with 0·8 nM recombinant receptor protein plus the stated molar excess of unlabelled genistein (solid square, solid line), genistein-4′-sulphate (open square, dotted line), genistein-7-sulphate (open diamond, dotted line), daidzein (solid circle, solid line), daidzein-4′-sulphate (open circle, dotted line), daidzein-7-sulphate (open star, dotted line), equol (solid triangle, solid line) and equol-7-sulphate (open triangle, dotted line). Error bars represent the mean±s.e.m. of triplicate assays.

Figure 7

Molecular modelling and docking of (A) genistein, (B) equol and (C) daidzein into the crystal structure of human LBD of ERα (1A52). The ligands are shown in bold outline with neighbouring amino acid residues numbered according to 1A52. Hydrogen bonds (depicted by dashed lines) are shown between the ligand and binding site residues with the bond lengths given in Angstroms (Å) for the energetically favoured conformation. The docked energies for genistein, equol and daidzein were −16·38, −17·60 and −14·91 kcal/mol respectively. The number of van der Waals interactions was 42, 48 and 41 for genistein, equol and daidzein respectively.