**4. Phytoestrogens and CRC**

Phytoestrogens are heterocyclic, non steroid phenols extracted from plants. These compounds are structurally similar and have a functional action as estrogen-agonists in mammals. Four classes of phytoestrogens can be distinguished, on the basis of their different molecular structure and different biological activities, namely isoflavones, lignans, coumestans and lactones (20-21).

**Isoflavones**, including genistein and quercitin, are the most known phytoestrogens. They are primarily found in the Fabaceae family, which includes legumes, soybean, peanut and clover.

**Lignans** were first identified in plants and later in biological fluids of mammals. These compounds are found in whole grain, seeds, fruits and vegetables but also in beverages such as coffee and tea (22). The cyclic urinary excretion of these phenolic compounds during the menstrual cycle led to investigations of their biological role, and they are now considered as a new hormone class (23).

**Coumestans** are less common in the human diet than isoflavones; they are extracted from fodder, clover, legumes and soybean.

Phytoestrogens as Nutritional Modulators in Colon Cancer Prevention 325

Finally have been demonstrated that, without any apparent toxicity, the feeding of polyphenols from silymarin suppressed the tumor growth of the human SW480 CRC, implanted in *nu/nu* mice. The inhibitory activity was associated with strong antiproliferative (β-catenin, c-Myc and cyclin D1 suppression) and pro-apoptotic effects. (36). Even lignans exert similar activity in several human cancers. For example Touillaud et al. examined associations between the risk of postmenopausal invasive breast cancer and dietary intakes of four plant lignans (pinoresinol, lariciresinol, secoisolariciresinol, and matairesinol) and estimated exposure to two enterolignans (enterodiol and enterolactone), as measured with a self-administered diet history questionnaire, among postmenopausal French women who were not taking soy isoflavone supplements. They demonstrate that high dietary intakes of plant lignans and high exposure to enterolignans were associated with reduced risks of Estrogen receptor negative and Progesteron receptor positive postmenopausal breast cancer in a Western population that does not consume a diet rich in soy (56). On the other hand Kuiisten et al. studied the associations between plasma enterolignans and the risk of colorectal adenomas in a Dutch case-control study . Colorectal adenomas are considered to be precursors of colorectal cancer. Cases with at least one histologically confirmed colorectal adenoma and controls with no history of any type of adenoma were included. Plasma enterodiol and enterolactone concentrations were measured by liquid chromatography with tandem mass spectrometry and they observed a substantial reduction in colorectal adenoma risk among subjects with high plasma

Lignans exert a similar activity in several human colon cancer cells and are easily metabolized and absorbed in the colon (38-41). Lignin is a documented absorbant of carcinogens in the intestinal lumen. Its degradation to enterolignans by human intestinal

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

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,

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

a potent ER-β agonist, with a 200-fold higher affinity than estradiol (44).

that are known to precede the development of colic adenocarcinoma (45).

concentrations of enterolignans, in particular, enterodiol (42).

microbiota could delay lignan release (42).

**5. Phytoestrogens in experimental CRC** 

**Lactones** are the least common phytoestrogens in the human diet.

Natural phytoestrogens undergo glycosidic binding to carbohydrates to produce complex molecules that are hard for the intestinal tract to absorb. For this reason, after ingestion, this glycosidic binding is broken up by glycosidases, enzymes produced by intestinal microflora. This enzymatic digestion generates ''aglycone'', a compound that is quickly absorbed and can bind ERs (24).

Phytoestrogens are characterized by a higher binding affinity to ER-β as compared to the other estrogen receptor subtype, alpha (ER-α). This biological characteristic explains why the administration of phytoestrogens does not produce the classic side effects associated to estrogen administration (cerebro- and cardiovascular attacks, a higher incidence of endometrial and breast cancer) (25-27), making these substances ideal candidates for CRC prevention.

As proposed for estrogens, genomic and non-genomic mechanisms have been also suggested for phytoestrogens to explain their biological activities (20; 28-32).

One of the most interesting compounds is Silymarin, initially extracted from Silybum marianum. It is a mixture of four flavolignans (silibinin, isosilibinin, silydianin and silychristin) and the isoflavone taxifolin. It is already used in the treatment of alcoholic liver disease and as an anti-fibrotic agent (33).

Extensive research within the last decade has shown that silymarin can suppress the proliferation of a variety of tumor cells (e.g., prostate, breast, ovary, colon, lung, bladder); this is accomplished through cell cycle arrest at the G1/S-phase, induction of cyclindependent kinase inhibitors (such as p15, p21 and p27), down-regulation of anti-apoptotic gene products (e.g., Bcl-2 and Bcl-xL), inhibition of cell-survival kinases (AKT, PKC and MAPK) and inhibition of inflammatory transcription factors (e.g., NF-kappaB). Silymarin can also down-regulate gene products involved in the proliferation of tumor cells (cyclin D1, EGFR, COX-2, TGF-beta, IGF-IR), invasion (MMP-9), angiogenesis (VEGF) and metastasis (adhesion molecules). The antiinflammatory effects of silymarin are mediated through suppression of NF-kappaB-regulated gene products, including COX-2, LOX, inducible iNOS, TNF and IL-1 (35).

Silymarin has also been shown to sensitize tumors to chemotherapeutic agents through down-regulation of the MDR protein and other mechanisms. It binds to both estrogen and androgen receptors, and down-regulates PSA. In addition to its chemopreventive effects, silymarin exhibits antitumor activity against human tumors (e.g., prostate and ovary) in rodents (35)

Seidlova-Wuttke et al. (34) have demonstrated the selective binding of silymarin to ER-β and no binding to ER-α, but, how mentioned above, beyond its specific ER-β agonism silymarin, exerts an anti 5-lipoxygenase (LOX) and anti-COX2 effect (35-36).

There is strong positive correlation has been recently established between 5-LOX overexpression and the appearance of typical high-risk factors for malignant transformation of polyps, such as histological epithelial localization, increased polyp size, villous and tubulovillous adenomas, high grade of intraepithelial neoplasia, and patient age because both inflammatory enzymes are up-regulated in colon carcinogenesis and involved in silencing apoptosis (37).

Natural phytoestrogens undergo glycosidic binding to carbohydrates to produce complex molecules that are hard for the intestinal tract to absorb. For this reason, after ingestion, this glycosidic binding is broken up by glycosidases, enzymes produced by intestinal microflora. This enzymatic digestion generates ''aglycone'', a compound that is quickly absorbed and

Phytoestrogens are characterized by a higher binding affinity to ER-β as compared to the other estrogen receptor subtype, alpha (ER-α). This biological characteristic explains why the administration of phytoestrogens does not produce the classic side effects associated to estrogen administration (cerebro- and cardiovascular attacks, a higher incidence of endometrial and breast cancer) (25-27), making these substances ideal candidates for CRC

As proposed for estrogens, genomic and non-genomic mechanisms have been also

One of the most interesting compounds is Silymarin, initially extracted from Silybum marianum. It is a mixture of four flavolignans (silibinin, isosilibinin, silydianin and silychristin) and the isoflavone taxifolin. It is already used in the treatment of alcoholic liver

Extensive research within the last decade has shown that silymarin can suppress the proliferation of a variety of tumor cells (e.g., prostate, breast, ovary, colon, lung, bladder); this is accomplished through cell cycle arrest at the G1/S-phase, induction of cyclindependent kinase inhibitors (such as p15, p21 and p27), down-regulation of anti-apoptotic gene products (e.g., Bcl-2 and Bcl-xL), inhibition of cell-survival kinases (AKT, PKC and MAPK) and inhibition of inflammatory transcription factors (e.g., NF-kappaB). Silymarin can also down-regulate gene products involved in the proliferation of tumor cells (cyclin D1, EGFR, COX-2, TGF-beta, IGF-IR), invasion (MMP-9), angiogenesis (VEGF) and metastasis (adhesion molecules). The antiinflammatory effects of silymarin are mediated through suppression of NF-kappaB-regulated gene products, including COX-2, LOX, inducible

Silymarin has also been shown to sensitize tumors to chemotherapeutic agents through down-regulation of the MDR protein and other mechanisms. It binds to both estrogen and androgen receptors, and down-regulates PSA. In addition to its chemopreventive effects, silymarin exhibits antitumor activity against human tumors (e.g., prostate and ovary) in

Seidlova-Wuttke et al. (34) have demonstrated the selective binding of silymarin to ER-β and no binding to ER-α, but, how mentioned above, beyond its specific ER-β agonism

There is strong positive correlation has been recently established between 5-LOX overexpression and the appearance of typical high-risk factors for malignant transformation of polyps, such as histological epithelial localization, increased polyp size, villous and tubulovillous adenomas, high grade of intraepithelial neoplasia, and patient age because both inflammatory enzymes are up-regulated in colon carcinogenesis and involved in

silymarin, exerts an anti 5-lipoxygenase (LOX) and anti-COX2 effect (35-36).

suggested for phytoestrogens to explain their biological activities (20; 28-32).

**Lactones** are the least common phytoestrogens in the human diet.

can bind ERs (24).

prevention.

disease and as an anti-fibrotic agent (33).

iNOS, TNF and IL-1 (35).

silencing apoptosis (37).

rodents (35)

Finally have been demonstrated that, without any apparent toxicity, the feeding of polyphenols from silymarin suppressed the tumor growth of the human SW480 CRC, implanted in *nu/nu* mice. The inhibitory activity was associated with strong antiproliferative (β-catenin, c-Myc and cyclin D1 suppression) and pro-apoptotic effects. (36).

Even lignans exert similar activity in several human cancers. For example Touillaud et al. examined associations between the risk of postmenopausal invasive breast cancer and dietary intakes of four plant lignans (pinoresinol, lariciresinol, secoisolariciresinol, and matairesinol) and estimated exposure to two enterolignans (enterodiol and enterolactone), as measured with a self-administered diet history questionnaire, among postmenopausal French women who were not taking soy isoflavone supplements. They demonstrate that high dietary intakes of plant lignans and high exposure to enterolignans were associated with reduced risks of Estrogen receptor negative and Progesteron receptor positive postmenopausal breast cancer in a Western population that does not consume a diet rich in soy (56). On the other hand Kuiisten et al. studied the associations between plasma enterolignans and the risk of colorectal adenomas in a Dutch case-control study . Colorectal adenomas are considered to be precursors of colorectal cancer. Cases with at least one histologically confirmed colorectal adenoma and controls with no history of any type of adenoma were included. Plasma enterodiol and enterolactone concentrations were measured by liquid chromatography with tandem mass spectrometry and they observed a substantial reduction in colorectal adenoma risk among subjects with high plasma concentrations of enterolignans, in particular, enterodiol (42).

Lignans exert a similar activity in several human colon cancer cells and are easily metabolized and absorbed in the colon (38-41). Lignin is a documented absorbant of carcinogens in the intestinal lumen. Its degradation to enterolignans by human intestinal microbiota could delay lignan release (42).
