Glyceollin I, a novel antiestrogenic phytoalexin isolated from activated soy

Abstract

Glyceollins, a group of novel phytoalexins isolated from activated soy, have recently been demonstrated to be novel antiestrogens that bind to the estrogen receptor (ER) and inhibit estrogen-induced tumor progression. Our previous publications have focused specifically on inhibition of tumor formation and growth by the glyceollin mixture, which contains three glyceollin isomers (I, II, and III). Here, we show the glyceollin mixture is also effective as a potential antiestrogenic, therapeutic agent that prevents estrogen-stimulated tumorigenesis and displays a differential pattern of gene expression from tamoxifen. By isolating the individual glyceollin isomers (I, II, and III), we have identified the active antiestrogenic component by using competition binding assays with human ERalpha and in an estrogen-responsive element-based luciferase reporter assay. We identified glyceollin I as the active component of the combined glyceollin mixture. Ligand-receptor modeling (docking) of glyceollin I, II, and III within the ERalpha ligand binding cavity demonstrates a unique type II antiestrogenic confirmation adopted by glyceollin I but not isomers II and III. We further compared the effects of glyceollin I to the antiestrogens, 4-hydroxytamoxifen and ICI 182,780 (fulvestrant), in MCF-7 breast cancer cells and BG-1 ovarian cancer cells on 17beta-estradiol-stimulated expression of progesterone receptor and stromal derived factor-1alpha. Our results establish a novel inhibition of ER-mediated gene expression and cell proliferation/survival. Glyceollin I may represent an important component of a phytoalexin-enriched food (activated) diet in terms of chemoprevention as well as a novel therapeutic agent for hormone-dependent tumors.

Fig. 1.

Effects of the glyceollin mixture on breast tumor growth in vivo. MCF-7 cells were injected in the mammary fat pad. Tumors were allowed to form over 10 days, and mice were randomized to four treatment groups with five mice per group: Con, E₂ + vehicle, Gly, and E₂ + Gly. The glyceollin mixture was suspended in a solution of DMSO (one-third volume) and propylene glycol (two-thirds volume) and was given intraperitoneally at 20 mg/kg/mouse/day for 15 days to Gly and E₂ + Gly groups starting after tumors were measurable. Control and E₂ groups were injected with vehicle daily for 15 days. Tumor size was measured every 2 days. Treatment, trial day, and treatment by trial day interactions are statistically significant for tumors (p < 0.001), and the tumors of all groups are significantly smaller than those of the E₂ only group on every treatment day measured.

Fig. 2.

Effects of glyceollin mixture on ERα, SDF-1, and PgR mRNA gene expression in MCF-7 cells. E₂ treatment (1 nM) increased PgR and SDF-1 expression, whereas 10 μM glyceollin and tamoxifen treatment decreased PgR and SDF-1 gene expression ± E₂ treatment.

Fig. 3.

Flavonoid pathway showing the biosynthetic route from daidzein to glyceollins I to III.

Fig. 4.

Glyceollin I Inhibits the ER transcriptional activity in MCF-7 cells. Cells were transfected with pGL2-ERE2x-TK-luciferase plasmid. After 5 h, the transfection medium was removed and replaced with phenol red-free DMEM supplemented with 5% CS-FBS containing vehicle, glyceollin I, glyceollin II, glyceollin III, and glyceollin mixture (A) and vehicle, E₂ + glyceollin I, E₂ + glyceollin II, E₂ + glyceollin III, and E₂ + glyceollin mixture (B). After 18 h, the medium was removed and 100 μl of lysis buffer was added per well and incubated for 15 min at room temperature.

Fig. 5.

Competition binding curves of glyceollin I, II, III, and glyceollin mixture to ERα. Increasing concentrations of glyceollin I, II, III, and the glyceollin mixture were added to the ERα complex and compared with E₂. Data points and error bars represent the mean ± S.E.M. of at least three experiments (n = 3) for each concentration tested. (p < 0.05).

Fig. 6.

A and B, 4-hydroxytamoxifen and glyceollin I in the binding cavity of the ligand binding domain of ERα interacting with histidine 524, arginine 394, and glutamate 353; glyceollin I (atom is colored yellow) (A) and glyceollin II (magenta) and glyceollin III (red) in the binding cavity of the ligand binding domain of the ERα interacting with histidine 524, arginine 394, and glutamate 353 (B).

Fig. 7.

PgR and SDF-1 expression. Total RNA was isolated from MCF-7 and BG-1 cells, reverse-transcribed into cDNA, and subjected to real-time RT-PCR analysis for quantitation. Treatment on MCF-7 cells was as follows: DMSO (vehicle), E₂ alone, E₂ + ICI, E₂ + 4-OH-tamoxifen (TAM) and glyceollin I (0.1, 1, and 10 μM with stimulation (A and B). Treatment on BG-1 cells was as follows: DMSO (vehicle), E₂ alone, E₂ + ICI 182,780 (ICI), E₂ + 4-OH TAM, and glyceollin I (10 μM + E₂) (C and D). Results are expressed as the mean unit ± S.E.M. (***, p < 0.001; **, p < 0.01; and *, p < 0.05), where “a” represents treatments compared with control and “b” represents treatments compared with estrogen.

Fig. 8.

Effects of glyceollin I on colony formation on MCF-7 and BG-1 cells. MCF-7 (A) and BG-1 (B) cells were placed in phenol red-free DMEM supplemented with 5% dextran-coated charcoal-treated FBS for 48 h before plating. MCF-7 or BG-1 cells (1000) were plated in six-well plates. Forty-eight hours later, cells were treated with DMSO (vehicle) or E₂ + glyceollin I (0.1–10 μM). Colonies of ≥50 cells were counted as positive. Results are normalized to percentage of clonogenic survival from DMSO control cells. Results are expressed as the mean unit ± S.E.M. (***, p < 0.001; **, p < 0.01; and *, p < 0.05), where “a” represents treatments compared with control and “b” represents treatments compared with estrogen.