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United States Department of Agriculture

Agricultural Research Service

Research Project: PHYTOESTROGENIC EFFECTS OF ELICITOR INDUCED ISOFLAVONOIDS IN LEGUMES
2011 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.


3.Progress Report
This serves as the final report and is also the In-house Research project of the following cooperative agreements involved in like research: Tulane University, 6435-53000-002-01S; Toledo University, 6435-53000-001-02S. This past year, several new advancements were made in the area of stress-induced phytoestrogens in soybean namely glyceollins. New research examined the ability of the glyceollins to enhance glucose uptake in fat cells. The ability of the glyceollins to stimulate glucose uptake, similar to insulin, would enable a glyceollin-enriched food to ameliorate the symptoms of diabetes and assist in the management of insulin resistance. Also, other research showed glyceollins lowered plasma cholesterol levels in vivo and regulated genes involved in cholesterol transport. This year other legumes were examined and the red kidney bean phytoalexins kievitone and phaseollin displayed both estrogenic and antiestrogenic activities. In further cancer research with glyceollins, the glyceollins 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 and also estrogen receptor (ER)-negative breast cancer. 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 from natural sources. 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.


4.Accomplishments
1. Glyceollins enhance glucose uptake in vitro. Much effort has been ongoing to search for dietary components that improve diabetes. Agricultural Research Service scientists in New Orleans, LA, and Tulane University scientists in New Orleans, LA, (with industry partner NuMe Health) found glyceollins stimulated glucose uptake (basal and insulin stimulated) in 3T3L1 adipocytes (fat cells). Preliminary data suggest that the glyceollins regulate glucose through a GLUT (glucose transporter) receptor. The ability of the glyceollins to stimulate glucose uptake, similar to insulin, would enable a glyceollin-enriched food to ameliorate the symptoms of diabetes and assist in the management of insulin resistance. Separate studies have also shown the glyceollins regulate genes involved in cholesterol transport. In a hamster animal model, the glyceollins lowered harmful cholesterol levels. These results point to additional health benefits of the glyceollins for overcoming diabetic conditions.

2. Glyceollins identified as antiestrogens with anticancer activity. Soybean and other plants produce higher levels of certain compounds during periods of stress that may be beneficial to human health. Research by Tulane University in New Orleans, LA, and Agricultural Research Service in New Orleans, LA, has 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. Compared with the plant compounds genistein and daidzein, normally found in soy, purified glyceollins have displayed greater inhibition of estrogen’s effects on proliferation and estrogen receptor signaling in breast cancer cells. These findings suggest 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 may enhance the antiestrogenic properties of standard soy foods in reproductive tissues, and could be used as food supplements to prevent certain types of cancer.

3. Glyceollin-enriched soy foods produced. Post harvest treatment of soy may enhance health benefits. 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 by the Agricultural Research Service scientists in New Orleans, LA. Other glyceollin-enriched soy foods being tested are soy sprouts and roasted soybeans. Further research has led to unique methods to produce a soy isoflavone extract containing the glyceollins from seeds grown under conditions of stress. Several ongoing studies are examining the potential health benefits, including anticancer activities, of this soy extract. Newer glyceollin-enriched soy foods would benefit human health; such products could enhance the value of soy.

4. Glyceollin I and III as selective estrogen receptor alpha versus estrogen receptor beta regulating ligands. Anticancer agents can be harmful in other organs by activating estrogen receptor (ER). Examining ERalpha versus ERbeta selective activation has identified a unique estrogenic activity of glyceollin III on ERbeta not observed for the other glyceollins. Based upon this Tulane University in New Orleans, LA, and the Agricultural Research Service scientists in New Orleans, LA, have continued investigating the selective ERbeta activity of glyceollin III and its potential human health implications. Given the unique expression of ERbeta the potential exists that a selective ERbeta agonistic activity by glyceollin III may have a role in prostate cancer, diabetes/obesity and inflammation. The glyceollins may have selective antiestrogenic properties useful in the treatment of prostate cancer.

5. Generation of novel glyceollin-mimetic analogs with anti-estrogenic activity. There is a continual need for new and effective anti-estrogenic agents that target cancer. Based upon GLY I structural features and known binding data Xavier University collaborators in New Orleans, LA, sought to generate a series of analogs that maximized the anti-estrogenic activity of the glyceollin series while simultaneously enhancing estrogen receptor (ER) binding activity. We generated a series of polar analogs mimicking the hydrophobic regions of glyceollin using a daidzein backbone. These studies in collaboration with Agricultural Research Service in New Orleans, LA, and Xavier University were published. These studies have also generated a patent application on the anti-estrogenic and anti-cancer activity of the analogs series. This research has identified the structure of new compounds that could have significant potential for the treatment of cancer.

6. Glyceollins identified as anticancer activity independent of estrogen receptor (ER). Much effort has been ongoing to search for anticancer agents that are active independent of ER. Tulane collaborators in New Orleans, LA, found that glyceollins suppressed cancer growth in the absence of estrogen stimulation, suggesting unique anticancer activity using a kinase pathway. Building upon the kinase targeting activity of glyceollins the Agricultural Research Service scientists in New Orleans, LA, along with Tulane collaborators have begun to investigate their activity in ER-negative breast cancer cell systems. Ongoing studies have demonstrated that the glyceollins can suppress tumorigenesis and metastasis of ER-negative breast carcinoma cell systems. Studies have further demonstrated the ability of glyceollins to suppress in vitro invasion/migration. These data provide evidence that the glyceollins may represent a more universally useful anticancer agent and could be used as a food supplement.


Review Publications
Khupse, R.S., Sarver, J.G., Trendel, J.A., Ellis, N.R., Reese, M.D., Wiese, T.E., Boue, S.M., Burow, M.E., Cleveland, T.E., Bhatnagar, D., Erhardt, P.W. 2011. Biomimetic synthesis and antiproliferative properties of racemic, natural(-), and unnnatural(+) glyceollin I. Journal of Medicinal Chemistry. 54(10):3506-3523.

Jiang, Q., Payton-Stewart, F., Elliott, S., Driver, J., Rhodes, L.V., Zhang, Q., Zheng, S., Bhatnagar, D., Boue, S.M., Collins-Burow, B.M., Sridhar, J., Stevens, C., Mclachlan, J.A., Wiese, T.E., Burow, M.E., Wang, G. 2010. Effects of 7-O substitutions on estrogenic and anti-estrogenic activities of daidzein analogues in MCF-7 breast cancer cells. Journal of Medicinal Chemistry. 53(16):6153-6163.

Boue, S.M., Burow, M.E., Wiese, T.E., Shih, B.Y., Elliott, S., Carter Wientjes, C.H., Mclachlan, J.A., Bhatnagar, D. 2011. Estrogenic and antiestrogenic activities of phytoalexins from red kidney bean (Phaseolus vulgaris L.). Journal of Agricultural and Food Chemistry. 59:112-120.

Last Modified: 10/20/2014
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