2010 Annual Report
1a.Objectives (from AD-416)
1. Induce, purify, and characterize isoflavonoids in soybean seed, root, plant, and cell culture induced with food grade elicitors including fungi.
2. Determine anticancer, hormonal, and antioxidant activities of individual, and combinations of, induced isoflavonoids with in vitro bioassays. Effects of phytoalexins using in vitro cell systems for PPAR transcriptional activity, adipocyte differentiation, and obesity related gene expression will be tested. In vivo gene expression from tissue samples from ongoing experiments will be evaluated. Computer modeling of potential phytoalexin structures for PPAR agonist activity will be performed.
3. Determine hormonal and anticancer effects of individual and combinations of induced isoflavonoids with in vivo animal model systems.
4. Develop methods for the organic syntheses of the glyceollins I, II, and III. Also, precursors and synthetic analogues of the glyceollins will be produced and characterized for beneficial hormonal activities. Computer modeling methods will be developed to assist in the development of structure-activity relationships that point toward the most active forms of the molecule.
1b.Approach (from AD-416)
The long-term objective of this project is to determine the health effects of induced isoflavones in both in vitro and in vivo model systems. Preliminary data indicate that the induced isoflavone glyceollins from soybean have anticancer activity against several different forms of cancer. This project will focus on natural induced isoflavones and foods containing these compounds. Foods containing these induced isoflavones called “activated foods” will be developed and tested for beneficial health promoting properties. Although the focus of this project is on natural compounds, synthetic compounds based on these natural compounds will also be utilized for the prevention of cancer and safety testing.
This Agricultural Research Service (ARS) research project is also the in-house project of the following cooperative agreements involved in like research: McLachlan/Tulane University/6435-53000-002-01S; Erhardt/Toledo University/6435-53000-002-02S. This past year, several new advancements were made in the area of stress-induced phytoestrogens (plant compounds that mimic estrogen activity in animals) in soybean. Further investigations using lab assays and animal testing have confirmed the active stress-induced soy compound to be glyceollin I (from a mixture of glyceollins I, II, and III). Also, the glyceollins have demonstrated inhibitory activity in animal testing against several breast cancer cell lines, including two cancer cell lines that do not react to the cancer agent tamoxifen. In contrast to tamoxifen, the glyceollins had no harmful effects and partially blocked the effects of female estrogen. These findings identify glyceollins as antiestrogenic agents that may be useful in the prevention or treatment of breast and ovarian carcinoma without harmful effects. In order to further study the glyceollins activity in animal models, we have successfully developed a synthetic scheme to produce glyceollin I and II. A synthetic route is desirable since the glyceollins (a mixture of glyceollin I, II, and III) have been difficult to purify as individual components. A large scale synthesis of glyceollin I beginning from nearly 250 grams of starting material has been completed. Fortuitously, our novel synthetic step may also be useful for the production of related natural products that have already been advanced into clinical studies by other investigators. Taking advantage of this finding, we are now including these types of utilities in our chemical patent applications. At this point, we have firmly established a solid patent base around the intellectual property associated with the specific chemical production of the glyceollins, as well as providing further coverage for the various therapeutic applications that we have previously identified.
Glyceollin I is Active Component of Glyceollin Mixture. The glyceollins, a group of novel phytoalexins isolated from activated soy, consist of a mixture of 3 compounds. We have shown that the glyceollin mixture is effective as a potential antiestrogenic, therapeutic agent that prevents estrogenstimulated tumorigenesis and displays a differential pattern of gene expression from tamoxifen, the current drug used in therapy. We identified glyceollin I as the active component of the combined glyceollin mixture. Further comparison of the effects of glyceollin I to the antiestrogens, -hydroxytamoxifen and ICI 182,780 (fulvestrant), in MCF-7 breast cancer cells and BG-1 ovarian cancer cells on estradiol-stimulated expression of progesterone receptor and stromal derived factor established 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.
Glyceollin-Enriched Soy Foods Produced. The production of soy foods containing the glyceollins is needed. Preliminary results indicate that several different elicitors can be used to induce glyceollins to high concentrations in different soy foods. A glyceollin-enriched soy protein isolate was first produced in our laboratory. Other glyceollin-enriched soy foods being tested are soy sprouts and roasted soybeans and a soy isoflavone extract containing the glyceollins from seeds grown under conditions of stress. These foods offer potential health benefits, including anticancer activities and could therefore be included in diets.
Glyceollins Induced in Soy Roots During Root Cyst Nematode Infestations. The glyceollins can be produced in living soybean plants, but research needed to identify effective production methods. University of Toledo collaborators have analyzed the effect of root cyst nematode infections on soybean plants after having detected glyceollins in this type of stressed root tissue. The major impact of this research could lead to alternative or value-added uses of induced phytoestrogen containing food and health products.
Glyceollins Identified as Antiestrogens With Anticancer Activity. New effective treatments for breast cancer utilize antiestrogens. Agricultural Research Service researchers at New Orleans, Louisiana in collaboration with scientists at Tulane University have identified the compounds called glyceollins I, II, and III in soybean plants grown under conditions of stress as therapeutic antiestrogens that inhibit the growth of estrogen-dependent cancers. Glyceollins are not strong plant estrogens which differ from the strong estrogens genistein and daidzein normally found in soy, which suggests that soy foods enriched in glyceollins may have distinct estrogen-modulating properties compared to standard soy foods. Initial animal studies demonstrated that the glyceollins inhibited breast and ovarian cancer cell proliferation. These findings suggest that glyceollins in diets or as a pharmaceutical may enhance the antiestrogenic properties of standard soy foods in reproductive tissues and prevent cancers.
Zimmermann, M., Tilghman, S.L., Boue, S.M., Salvo, V.A., Elliott, S., Williams, K., Skripnikova, E.V., Ashe, H., Payton-Stewart, F., Vanhoy-Rhodes, L., Fonseca, J., Corbitt, C., Collins-Burow, B.M., Howell, M.H., Lacey, M., Shih, B.Y., Carter Wientjes, C.H., Cleveland, T.E., Mclachlan, J.A., Wiese, T.E., Beckman, B.S., Burow, M.E. 2010. Glyceollin I, A Novel Antiestrogenic Phytoalexin Isolated from Activated Soy. Journal of Pharmacology and Experimental Therapeutics. 332(1):35-45.