2010 Annual Report
1a.Objectives (from AD-416)
Utilize microbial elicitors of isoflavonoid production in manipulation of isoflavonoid levels. Test individual or combinations of isoflavonoid compounds induced by microbial elicitors for phytoestrogenic effects in animal systems. Test effects of phytoalexins using in vitro cell systems for PPAR transcriptional activity, adipocyte differentiation, and obesity related gene expression. Evaluate in vivo gene expression from tissue samples from ongoing experiments.
1b.Approach (from AD-416)
Estrogenic and antiestrogenic activities of isoflavonoids isolated from soybean tissues/organs will be analyzed by determining their ability to support in vitro and in vivo growth of several different cancer cell lines. Compounds will be screened for estrogen activity in assays using breast cancer cells incorporating an estrogen dependent promoter coupled with a luciferase reporter gene. Compounds will be assayed in order to determine synergistic effects and to measure estrogenic potency. Also, compounds will be screened for estrogen receptor binding and breast cancer proliferation. Anitestrogenic activity will be determined for all compounds. Antiestrogenic compounds will be tested in vivo using a mouse model system with different cancer cell lines, including breast, ovarian, and prostate cells.
Glyceollins, a group of novel compounds, 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 (cancer drug). By isolating the individual glyceollin isomers (I, II, and III), we have identified the active antiestrogenic component by using competition binding assays with human estrogen receptor and in a reporter gene 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 estrogen receptor 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 commercial antiestrogens hydroxytamoxifen and ICI 182,780 (fulvestrant) in MCF-7 breast cancer cells and BG-1 ovarian cancer cells on gene expression. 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-(an inducible plant defense compound) enriched food (activated) diet in terms of chemoprevention, as well as a novel therapeutic agent for hormone dependent tumors.
We have previously reported that the three glyceollin isomers (glyceollins I–III) exhibit antiestrogenic properties, which may have significant biological effects upon human exposure. Of the three isomers, we have recently shown that glyceollin I is the most potent antiestrogen. Natural (-)-glyceollin I recently was synthesized along with its racemate and unnatural (+)-enantiomer (isomer with same structure). In this study, we compared the glyceollin I enantiomers’ ER binding affinity, ability to inhibit estrogen responsive element transcriptional (ERE) activity, and endogenous gene expression in MCF-7 cells. The results demonstrated similar binding affinities for both types of estrogen receptor. Reporter gene assays in MCF-7 cells revealed that while (+)-glyceollin I slightly stimulated ERE transcriptional activity, (-)-glyceollin I decreased activity induced by estrogen. Additionally, each glyceollin I enantiomer induced unique gene expression profiles in a polymerase chain reaction (PCR) (deoxyribonucleic acid amplification) array panel of genes commonly altered in breast cancer. Research progress was monitored through teleconferencing, frequent email communications, and reports.