**3. Estrogens in colorectal cancer**

Phytoestrogens (heterocyclic non steroid phenols) are plant-derived compounds with a structural and functional action as estrogen agonists in mammals. To understand their

Phytoestrogens as Nutritional Modulators in Colon Cancer Prevention 323

In the last few years, numerous epidemiological, clinical and experimental studies have explored the role of estrogens in intestinal carcinogenesis, suggesting their protective role and potential use in CRC prevention (10-14). In particular, estrogen protective activities are thought to be related to their receptor subtype beta (ER-β), suggesting the use of selective

Since the early 80s, the role of a progressive silencing of Estrogen Receptor beta (ERβ) expression in intestinal cells, as a pathogenetic factor involved in intestinal tumorigenesis and its progression to an overt cancerous phenotype, has been studied in both animal

There is some evidence supporting ER-β as a prognostic factor in sporadic adenocarcinoma, and suggesting its role as a relevant surrogate biomarker in the follow-up of intestinal

In the ApcMin/+ mouse, that represents the animal model equivalent to FAP in humans, the loss of apoptotic control also occurs in non adenomatous (normal) mucosa, again depending upon a decreased ER-β expression and related decreased TUNEL and caspase-3 expression. In intact male ApcMin/+ mice it has been demonstrated that supplementing the diet with selected, weak but specific ER-β agonists reversed the hyperproliferative behavior in non adenomatous mucosa, and reduced the number and the degree of polyp dysplasia in

In human sporadic polyps, a progressive, significant decrease of ER-β expression has been demonstrated, a finding confirmed in subjects affected by Familial Adenomatous Polyposis (FAP) (4). In these patients, in fact, a progressive, significant decrease of ER-β expression was observed in the different stages of the disease, correlated with apoptosis (r=0.76, p<

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,

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

**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

**Coumestans** are less common in the human diet than isoflavones; they are extracted from

ER-β agonists in primary CRC prevention .

neoplasia development and dysplastic severity (15-18).

0.001), and inversely correlated with cell proliferation.

models and clinical settings (11-14).

adenomatous mucosa (19).

**4. Phytoestrogens and CRC** 

coumestans and lactones (20-21).

considered as a new hormone class (23).

fodder, clover, legumes and soybean.

clover.

biological activities and the possible interactions between phytoestrogens and colorectal cancer, a knowledge of some fundamental data on estrogens is essential.

Estrogen biological activities are mainly mediated by their binding with two specific receptors: estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β). Both of these estrogen receptors (ERs) belong to the steroid/thyroid hormone receptor superfamily of nuclear receptors, which are activated upon binding of the ligand. After binding, activated ERs are able to interact directly with cis-regulatory elements of target genes by binding to estrogen-response elements (EREs), or indirectly through interaction with another DNAbound transcription factor, such as activator protein 1 (AP-1), thus facilitating the assembly of basal transcription factors into a stable pre-initiation complex, followed by increased transcription rates for target mRNAs (5).

Both ERs consist of three main regions: 1) a hypervariable N-terminal, that contributes to the transactivation function, 2) a highly conserved DNA-binding domain, responsible for specific DNA-binding and dimerization and 3) a C-terminal domain, involved in ligandbinding (LBD) and nuclear localization, as well as ligand-dependent transactivation functions. ER-α and ER-β are produced by different genes located on different chromosomes (6).

In mammals, both ER-α and ER-β have conserved DNA binding domains (96%) but they have different LBD showing only 58% homology. ER-α has two distinct transcriptional activation functions (AF): AF-1 and AF-2. AF-1, located at the N-terminal, is ligandindependent, constitutively active and contributes to the transcriptional activity of the receptor by recruiting co-activator proteins such as GRIP1 and SRC-1 and the histone acetyltransferases (HAT) p300/CBP and pCAF. The AF-2 domain is under the control of ligands in both ER-α and ER-β.

Variations observed in the phenotypes of knock-out mice lacking ER-α or ER-β suggest that these two proteins have different biological activities. This view has been further supported by in vitro and in vivo studies in ER-β knock-out mice, indicating that ER-β is a modulator of ER-α activity as it is able to reverse the effects of ER-α and to inhibit estradiol (E2) dependent proliferation (7). In addition, it is known that ER-α and ER-β have a different distribution in the various organs and apparatuses. ER-α is essentially expressed in the breast, bone, cardiovascular tissue, urogenital tract and central nervous system, while ER-β is the prevalent form in the gut. Both receptors bind E2 but they activate promoters in different ways. Studies on breast and prostate carcinogenesis suggest an opposite role of ERα and ER-β in the proliferation and differentiation of target tissues, a hypothesis described as the ying/yang relationship (8).

Estrogens regulate cellular function also through non-genomic pathways. In fact, after palmitoylation ERs can localize at the plasma membrane, associate to caveolin-1 and, upon estrogens stimulation, activate rapid signals. In the case of ER-α, palmitoylation stimulates proliferation, while ER-β localization at the plasma membrane and its association with caveolin-1 activates p38 (a member of the MAPK family), that promotes apoptosis (9). This finding is confirmed by the observation, in the tumor tissue, of a reduction of ER-β and an increased alpha/beta ratio, that is related to a reduction of apoptosis and an increased rate of proliferation.

biological activities and the possible interactions between phytoestrogens and colorectal

Estrogen biological activities are mainly mediated by their binding with two specific receptors: estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β). Both of these estrogen receptors (ERs) belong to the steroid/thyroid hormone receptor superfamily of nuclear receptors, which are activated upon binding of the ligand. After binding, activated ERs are able to interact directly with cis-regulatory elements of target genes by binding to estrogen-response elements (EREs), or indirectly through interaction with another DNAbound transcription factor, such as activator protein 1 (AP-1), thus facilitating the assembly of basal transcription factors into a stable pre-initiation complex, followed by increased

Both ERs consist of three main regions: 1) a hypervariable N-terminal, that contributes to the transactivation function, 2) a highly conserved DNA-binding domain, responsible for specific DNA-binding and dimerization and 3) a C-terminal domain, involved in ligandbinding (LBD) and nuclear localization, as well as ligand-dependent transactivation functions. ER-α and ER-β are produced by different genes located on different chromosomes

In mammals, both ER-α and ER-β have conserved DNA binding domains (96%) but they have different LBD showing only 58% homology. ER-α has two distinct transcriptional activation functions (AF): AF-1 and AF-2. AF-1, located at the N-terminal, is ligandindependent, constitutively active and contributes to the transcriptional activity of the receptor by recruiting co-activator proteins such as GRIP1 and SRC-1 and the histone acetyltransferases (HAT) p300/CBP and pCAF. The AF-2 domain is under the control of

Variations observed in the phenotypes of knock-out mice lacking ER-α or ER-β suggest that these two proteins have different biological activities. This view has been further supported by in vitro and in vivo studies in ER-β knock-out mice, indicating that ER-β is a modulator of ER-α activity as it is able to reverse the effects of ER-α and to inhibit estradiol (E2) dependent proliferation (7). In addition, it is known that ER-α and ER-β have a different distribution in the various organs and apparatuses. ER-α is essentially expressed in the breast, bone, cardiovascular tissue, urogenital tract and central nervous system, while ER-β is the prevalent form in the gut. Both receptors bind E2 but they activate promoters in different ways. Studies on breast and prostate carcinogenesis suggest an opposite role of ERα and ER-β in the proliferation and differentiation of target tissues, a hypothesis described

Estrogens regulate cellular function also through non-genomic pathways. In fact, after palmitoylation ERs can localize at the plasma membrane, associate to caveolin-1 and, upon estrogens stimulation, activate rapid signals. In the case of ER-α, palmitoylation stimulates proliferation, while ER-β localization at the plasma membrane and its association with caveolin-1 activates p38 (a member of the MAPK family), that promotes apoptosis (9). This finding is confirmed by the observation, in the tumor tissue, of a reduction of ER-β and an increased alpha/beta ratio, that is related to a reduction of apoptosis and an increased rate

cancer, a knowledge of some fundamental data on estrogens is essential.

transcription rates for target mRNAs (5).

ligands in both ER-α and ER-β.

as the ying/yang relationship (8).

of proliferation.

(6).

In the last few years, numerous epidemiological, clinical and experimental studies have explored the role of estrogens in intestinal carcinogenesis, suggesting their protective role and potential use in CRC prevention (10-14). In particular, estrogen protective activities are thought to be related to their receptor subtype beta (ER-β), suggesting the use of selective ER-β agonists in primary CRC prevention .

Since the early 80s, the role of a progressive silencing of Estrogen Receptor beta (ERβ) expression in intestinal cells, as a pathogenetic factor involved in intestinal tumorigenesis and its progression to an overt cancerous phenotype, has been studied in both animal models and clinical settings (11-14).

There is some evidence supporting ER-β as a prognostic factor in sporadic adenocarcinoma, and suggesting its role as a relevant surrogate biomarker in the follow-up of intestinal neoplasia development and dysplastic severity (15-18).

In the ApcMin/+ mouse, that represents the animal model equivalent to FAP in humans, the loss of apoptotic control also occurs in non adenomatous (normal) mucosa, again depending upon a decreased ER-β expression and related decreased TUNEL and caspase-3 expression. In intact male ApcMin/+ mice it has been demonstrated that supplementing the diet with selected, weak but specific ER-β agonists reversed the hyperproliferative behavior in non adenomatous mucosa, and reduced the number and the degree of polyp dysplasia in adenomatous mucosa (19).

In human sporadic polyps, a progressive, significant decrease of ER-β expression has been demonstrated, a finding confirmed in subjects affected by Familial Adenomatous Polyposis (FAP) (4). In these patients, in fact, a progressive, significant decrease of ER-β expression was observed in the different stages of the disease, correlated with apoptosis (r=0.76, p< 0.001), and inversely correlated with cell proliferation.
