Ruth MacDonald - Colon Cancer Research Projects

There is increasing evidence that estrogen plays a role in colon cancer risk. For example, women who received hormone replacement therapy after menopause were found to have lower risk of colon cancer than women not treated with estrogens. To examine this response, we designed an experiment with estrogen receptor a-knockout mice (ERaKO), and normal littermate (wild type, WT) that were obtained from Dr. Dennis Lubahn Funding for this project is a grant from the American Institute for Cancer Research, in collaboration with Drs. Maurice Bennink, Michigan State University and Andreas Constantinou, University of Illinois. Because we bred the mice in-house, we were able to control the exposure to phytoestrogens throughout life.

The primary investigators of this project was Ju-yuan Guo, currently a Post-Doctoral Fellow at the University of Missouri Ellis Fischel Cancer Institute with the assistance of Tracy Rode, Chemistry major. All mice were maintained on an isoflavone-free diet (AIN-93G) and females pups were weaned to one of 5 experimental diets (Table 1). The diets were designed to include a casein control and a soy protein control (soy protein stripped of isoflavones). The other three diets were made using the stripped soy protein, to which genistein (250 mg/kg), NovaSoy (1.04 gm/kg; a commercial mixture of isoflavones provided by the Archer Daniels Midland Company, Decatur IL) or estrone (0.1 mg/kg). We included estrone as a dietary treatment as a way of comparing to human exposure to hormone replacement therapy. The mice underwent ovariectomy at 7 weeks of age to control endogenous estrogen levels. They were then treated with azoxymethane once per week for 6 weeks given by intraperitoneal injection. The animals were euthanized at 35 weeks of age and colon tumors quantified. When the data were analyzed there was no difference in response between the WT and ERaKO mice, hence the effect was not dependent on expression of estrogen receptor a, and we have combined the data. The results have been published in the Journal of Nutrition.

As shown in Figure 1, the tumor incidence (% of mice with tumors) was less in mice fed estrone compared to either the casein or soy control diets. Tumor incidence was less in mice fed NovaSoy compared to the casein control, but not the soy protein control. Genistein was had no effect on tumor incidence.
We also observed that soy protein, independent of isoflavone or estrone, reduced tumor weight and tumor burden (Figure 2). This response may have been associated with an overall effect on colon cell proliferation, as the total weight of the colon was significantly reduced in mice fed soy protein containing diets compared to casein (Table 2). The effect was specific for colon as no effect on duodenal weight was observed. There was also an apparent effect of the dietary treatments on abdominal fat, as mice fed genistein, NovaSoy or estrone had more fat than mice fed casein (Table 2). In ongoing work, we are using microarray to identify potentially important genes that are altered by the dietary treatments, and are quantifying the ERa, ERb and IGF-I receptors in colon using real time PCR. Dr. Bennink will be quantifying cell proliferation kinetics in the colon and Dr. Constantinou is using immunohistochemistry to quantify ERa and ERb in the colon.
Another project we are working on is to characterize components of soy that may influence colon cancer risk. The components of soy that we are interested in are soy protein, isoflavones and saponins. Saponins are found in high concentration in soybeans and are present in extracts, such as NovaSoy. In collaboration with Dr. Mark Berhow, USDA National Center for Agricultural Utilization Research, Peoria, IL, and Dr. George Rottinghaus, University of Missouri Veterinary Medical Diagnostic Laboratory we have obtained a USDA grant to examined the effects of each of these compounds and their interactions in colon cancer. In preliminary work, we have examined the effects of purified soy saponins on rat colon morphology and found no changes in colon with up to 3% saponin in the diet (Figure 3).
However, genistein and daidzein were effective inhibitors of cell proliferation in these cells (Figure 5). The growth inhibition of these cells is therefore potentially mediated through effects on signaling pathways within the cell. We are currently quantifying several intracellular proteins in Caco-2 cells following exposure to the isoflavones. The principal investigator on this project is Sam Cole, Nutritional Sciences major.

Related Publications:

• Thornton WH, J., MacDonald RS. Insulin-like growth factor-II receptor expression in normal and N-methyl-N'-nitro-nitroso guanidine exposed cell lines: Assessment by flow cytometry. In Vitro, 29A:114-116, 1993.
• Thornton WH, Jr., MacDonald RS. Dietary fat quantity and composition alter colon cell kinetics in growing rats. Ann Nutr Metab., 38:270-280, 1994.
• Ganjam LS, Thornton,WH Jr., Marshall RT, MacDonald RS. Antiproliferative effects of yogurt fractions obtained by membrane dialysis on cultured mammalian intestinal cells. Journal of Dairy Science 80:2325-2329, 1997.
• Thornton WH, Jr., MacDonald RS. Dietary fat quantity and composition induce changes in proliferation and membrane lipids in rat colon cells. Annals Nutr. Metab. 41:260-268, 1997.
• Zhang W, Thornton WH, Jr., MacDonald RS. Insulin-like growth factor-I and II receptor expression in rat colon are affected by dietary lipid intake. Journal of Nutrition 128:158-165, 1998
• Guo, J-Y, Li, X, Browning, JD, Rottinghaus, GE, MacDonald, RS. Soy isoflavones and estrone prevent colon cancer in mice. J. Nutrition, 134:179-182, 2004.

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