**5. Phytoestrogens in experimental CRC**

Mice with the Apc gene (ApcMin/+) mutation are highly susceptible to spontaneous intestinal adenoma formation and are therefore considered the most suitable model for experimental CRC studies (43). A recent experimental study demonstrated that in ovariectomized ApcMin/+ female mice, the administration of a diet enriched with the phytoestrogen cumestrol induced a reduction of the number of polyps and an increased enterocyte migration as compared to control animals. Cumestrol was chosen in this study because it is a potent ER-β agonist, with a 200-fold higher affinity than estradiol (44).

Seidlova-Wuttke et al. (34) compared the effect of silymarin and estradiol in ovariectomized female mice and confirmed the selective binding of silymarin to ER-β by in vitro experiments. In another study, conducted by Khono et al., a silymarin-enriched diet significantly reduced azoxymethane-induced intestinal carcinogenesis in male mice. This effect was dose-dependent and determined a reduction of the number of cryptic adenomas, that are known to precede the development of colic adenocarcinoma (45).

The effect of a 0.02*%* silymarin-enriched diet on tumor development was also tested in intact ApcMin/+ male mice, i.e. in physiological conditions. Intestinal polyp development was evaluated together with ER-β expression, as well as other biological parameters influencing tumor growth (epithelial cell proliferation, apoptosis and migration), following the addition

Phytoestrogens as Nutritional Modulators in Colon Cancer Prevention 327

patients affected by sporadic adenopolyposis, presenting with a similar cell proliferation rate. We found that the normal mucosa of an APC-mutated intestinal environment is prone to polyp development and recurrence because of an altered proliferation-apoptosis ratio, related to a decreased ER-β expression. Moreover, we demonstrated that ER-β dependent apoptosis can be restored by administering specific phytoestrogens supplements with a selective action on ERβ, in a similar manner to what we had previously observed in ApcMin/+ mice. This randomized, double-blind placebo-controlled study showed that sylimarin, lignans and lignin can positively affect the ERβ-driven apoptotic control of colon epithelial turnover, by increasing ER-β expression in the normal mucosa of sporadic adenopolyposis patients prone to polyp recurrence. This is achieved via an increased ERβ content and was demonstrated in all patients, regardless of whether they were free from

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**7. References** 

of a combination of the ER-β-selective agonist silymarin and/or lignin to a high-fat/lowfiber diet, which has been shown to foster tumor growth. The addition of silymarin or lignin to the diet and, to an even greater extent, the specific combination of the two, significantly counteracted intestinal tumorigenesis and increased ER-β mRNA and protein levels. Cell proliferation and apoptosis were rebalanced and cell migration accelerated, restoring values similar to those observed in wild type animals. These results further support a protective effect of ER-β in CRC, suggesting that dietary supplementation with the combination of silymarin and lignin could be a potential approach to CRC prevention (46).
