**Genistein Aglycone Demonstrates a Protective and Reversible Effect on the Development of Steroid-Induced Secondary Osteoporosis and Increases Bone Breaking Strength in Rats**

Alessandra Bitto1, Francesca Polito2, Bruce P. Burnett3, Herbert Marini2, Robert Levy3, Domenica Altavilla1 and Francesco Squadrito1 *1Department of Clinical and Experimental Medicine and Pharmacology Section of Pharmacology, University of Messina 2Department of Biochemical, Physiological and Nutritional Sciences Section of Physiology and Human Nutrition, University of Messina 3Primus Pharmaceuticals, Inc. Scottsdale, Arizona 1,2Italy 3USA* 

#### **1. Introduction**

Glucocorticoids are used in the treatment of inflammatory and autoimmune diseases, cancers, and following organ transplantation. Glucocorticoid-induced osteoporosis is one of the primary side effects of glucocorticoid use resulting in increased risk of fractures. In a large meta-analysis study of glucocorticoid users, for example, van Staa et al (2002) found a relative risk increase of 1.91 for any fracture, 2.86 for vertebral fracture, 1.61 for hip fracture, and 1.13 for forearm fracture. Glucocorticoid-induced osteoporosis is characterized by low bone turnover and fractures, which occur in 30-50% of patients (van Staa et al., 2000). Glucocorticoids affect predominantly cancellous or trabecular bone, increasing the risk of vertebral fractures, which may be asymptomatic and occur early during the first months of glucocorticoid treatment (Laan et al., 1993; van Staa et al., 2000) and (Angeli et al., 2006). Trabecular bone accounts for approximately 20% of the total mass of bone (Bertazzo & Bertran, 2006) and also contributes to the ability of bone to tolerate stress to avoid fracture, especially in the spine. Long-term use of glucocorticoids also increases the risk of all osteoporotic fractures.

Published reports suggest no course of glucocorticoid therapy is safe for the skeleton. Regimens of daily prednisone at doses as low as 2.5 mg have been associated with an increased risk of hip and vertebral fractures. The risk increases by 5-fold, with prednisone doses above 7.5 mg daily. A dramatic 17-fold increase in vertebral fracture incidence was observed in subjects who used prednisone continuously more than 10 mg per day for longer than 3 months with increased fracture incidence in postmenopausal females and elderly males. The risk of osteoporotic fractures was also elevated in patients undergoing cyclic corticosteroid treatment at high doses (Van Staa et al., 2006) Although variable in onset,

Genistein Aglycone Demonstrates a Protective and Reversible Effect on the Development

activity both *in vitro* and *in vivo*, compared to other isoflavones (Choi et al., 2008).

In addition to competing with endogenous estrogens for binding to the estrogen receptors at high concentrations, genistein may exert anti-estrogenic effects by several potential mechanisms (Tham et al., 1998). Genistein has both ER agonist and antagonist activity in different cell types and works in a promoter specific manner in gene activation *via* ERs. The ER domain structure is typical of nuclear receptors. The amino-terminal region is involved in trans-activation of gene expression and has been termed the activation function domain or AF1 domain. The middle region contains a two-zinc finger structure, which plays an important role in binding to specific DNA response elements and in receptor dimerization. The carboxyl-terminal region contains an activation domain, termed AF2. A ligand binding domain, lying within AF2, is crucial for binding to receptor specific ligands and also corepressors and co-activators. This binding interaction affects receptor dimerization, nuclear translocation and modulation of target gene expression by AF2. The ligand-binding domain is structurally conserved among the nuclear receptor family and consists of between 10 and 12α helices folded in a globular domain. A central hydrophobic pocket accommodates the cognate ligand, which upon binding induces a conformational change in the ligand-binding domain, exposing a coactivator-docking site on ligand binding surface. However the binding of SERMs to ERs results in a different conformation change in which the coactivator-binding site is blocked from interaction with coactivator, thereby blocking AF2 mediated transcriptional activity (Liu et al., 2003). Several classes of nuclear receptor coactivators interact with ERs receptors and are specific for either ERα or ERβ. Using specifying coactivator recruitment and fluorescence resonance energy transfer, the genistein

(Larkin et al., 2008).

of Steroid-Induced Secondary Osteoporosis and Increases Bone Breaking Strength in Rats 291

of the plant. Another widely utilized source of genistin is *Sophora japonica* L (Tian et al., 2004). The glucosidal form and acetyl- and malonyl-glucosides are the major isoforms found in soybean derivates. The conversion from the glucosides to the aglycone form occurs in the gut, where the sugar residue is removed with the generation of genistein

Genistein is freely absorbed from the intestine and a large fraction is converted to the 7β-Oglucuronide as it crosses the brush border and ultimately enters the portal vein (Sfakianos et al., 1997), a process that is influenced by intestinal bacteria (Setchell et al., 2002; Day et al., 1998). Recent *ex vivo* data using isolated human gastrointestinal tract tissue also suggest that genistein may also undergo sulfonation in the small intestine, though the extent to which these sulfonates are absorbed following dietary intake is unknown (Ronis et al., 2006). The exact percentages of glucuronidated and sulfonated metabolites after crossing the lumen are also unknown, although it is clear that only a small percentage of the parent molecule remains as unconjugated genistein once it reaches the liver. Once in the liver, genistein undergoes additional biotransformation *via* CYP450-mediated hydroxylation (Hu et al., 2003) followed by glucuronidation and sulfonation by UDP-glucuronosyl transferase and sulphotransferases, respectively (Sfakianos et al. 1997). Genistein 7β-O-glucuronide can be recovered from bile after infusion of genistein into the small bowel of rats (Prasain et al., 2006). As much as 70% of the recovered genistein from bile is in the form of glucuronidated conjugates with smaller amounts reappearing in the distal duodenum and jejunum (Sfakianos et al., 1997). The vast majority of circulating genistein in serum has been found to be in the form of glucuronidated and sulfonated conjugates which represent excretion forms of the molecule (Setchell et al., 2002). Little is known about the bioactivity of conjugated isoflavones. Genistein is composed of two benzene rings (A and B) linked through a heterocyclic pyrane C ring. The hydroxyl group on the A ring confers to genistein a greater

fracture risk decreases after discontinuation of oral corticosteroids (Vestergaard et al., 2008). The risk of osteoporosis associated with inhaled glucocorticoids or with budesonide, a topical steroid used in inflammatory bowel disease, is small because their absorption is limited. Due to the high risk of fracture in glucocorticoid-administered patients, treatment guidelines exist.

Treatment guidelines for the use of glucocorticoids have been established which advise that if prednisolone is administered at ≥ 5 mg per day for three months or longer requires regular monitoring of bone mineral density (BMD) and treatment to prevent osteoporosis must be initiated (American college of Rheumatology, 2001; Adler & Hochberg, 2003; National Osteoporosis Society, 1998). Vitamin D and calcium are also recommended for the management of all patients treated with glucocorticoids. Bisphosphonates should be considered for the prevention and treatment of this disorder, because they can prevent the initial loss of bone mass from glucocoriticoids. Alendronate, risedronate, and zoledronic acid were shown to prevent and reverse the loss of BMD in glucocorticoid-induced osteoporosis with greater effects than those observed with vitamin D and calcium (Doga et al., 2008) and (Amin et al., 2002). In fact, bisphosphonates induce improvement of BMD that is 2-fold greater than that observed during vitamin D treatment alone (4.6% vs. 2.0%, respectively) (Amin et al., 2002). Anabolic therapy is also utilized for treatment of glucocorticoid-induced osteoporosis. Teriparatide causes a greater increase in BMD than alendronate and greater reduction in the risk of vertebral fractures (Saag et al., 2007). Even with these evidentiary clinical trials and guidelines, patient bone loss is, in general, poorly managed (Eastell et al., 1998; Walsh et al., 1996; Gudbjornsson et al., 2002). In glucocorticoidinduced osteoporosis, fractures also occur at higher BMDs than in postmenopausal osteoporosis in untreated women (van Staa et al., 2003). Consequently, guidelines for the treatment of postmenopausal osteoporosis are not applicable to glucocorticoid-induced osteoporosis, and patients should be treated at BMD T-scores of ≤−1.0 to −1.5 (Compston, 2004). In addition, vertebral fractures may be asymptomatic and often require radiological diagnosis before treatment. Since the incidence of fractures is higher in the spine compared to other areas of the body, glucocorticoid therapy appears to affect rapid bone remodelling.

During the initial phases of glucocorticoid exposure bone resorption is increased (van Staa et al., 2000). Glucocorticoids inhibit the formation of mature osteoblasts, but also activate apoptosis in these cell types (Jones & Sambrook, 1994; Canalis et al., 2007). Osteoprotegrin (OPG) expression, a key factor involved in modulating maturation of osteoclasts, is reduced also by glucocorticoids resulting in increased osteoclastogenesis (Orcel, 2005). Therefore, the combination of reduced osteoblast formation, increased osteoclast maturation leads to accelerated bone loss while on glucocorticoid therapy. Therapies are needed which modulate osteoclast as well as osteoblast activity to restore a more normal balance to the bone remodeling process in glucocorticoid treated patients.

#### **2. Glucocorticoid-induced osteoporosis: Is there a role for genistein?**

Among the anabolic compounds tested in recent years genistein aglycone seems a promising agent able to stimulate bone formation and to reduce bone resorption, acting *via* a genomic as well as a non-genomic pathways. Genistein is an isoflavone found in small quantities in certain legumes throughout the plant kingdom. Soybeans are a particularly rich source of genistin, the glucosidal precursor of genistein, although the concentration varies with the strain, location and environmental conditions of cultivation

fracture risk decreases after discontinuation of oral corticosteroids (Vestergaard et al., 2008). The risk of osteoporosis associated with inhaled glucocorticoids or with budesonide, a topical steroid used in inflammatory bowel disease, is small because their absorption is limited. Due to the high risk of fracture in glucocorticoid-administered patients, treatment

Treatment guidelines for the use of glucocorticoids have been established which advise that if prednisolone is administered at ≥ 5 mg per day for three months or longer requires regular monitoring of bone mineral density (BMD) and treatment to prevent osteoporosis must be initiated (American college of Rheumatology, 2001; Adler & Hochberg, 2003; National Osteoporosis Society, 1998). Vitamin D and calcium are also recommended for the management of all patients treated with glucocorticoids. Bisphosphonates should be considered for the prevention and treatment of this disorder, because they can prevent the initial loss of bone mass from glucocoriticoids. Alendronate, risedronate, and zoledronic acid were shown to prevent and reverse the loss of BMD in glucocorticoid-induced osteoporosis with greater effects than those observed with vitamin D and calcium (Doga et al., 2008) and (Amin et al., 2002). In fact, bisphosphonates induce improvement of BMD that is 2-fold greater than that observed during vitamin D treatment alone (4.6% vs. 2.0%, respectively) (Amin et al., 2002). Anabolic therapy is also utilized for treatment of glucocorticoid-induced osteoporosis. Teriparatide causes a greater increase in BMD than alendronate and greater reduction in the risk of vertebral fractures (Saag et al., 2007). Even with these evidentiary clinical trials and guidelines, patient bone loss is, in general, poorly managed (Eastell et al., 1998; Walsh et al., 1996; Gudbjornsson et al., 2002). In glucocorticoidinduced osteoporosis, fractures also occur at higher BMDs than in postmenopausal osteoporosis in untreated women (van Staa et al., 2003). Consequently, guidelines for the treatment of postmenopausal osteoporosis are not applicable to glucocorticoid-induced osteoporosis, and patients should be treated at BMD T-scores of ≤−1.0 to −1.5 (Compston, 2004). In addition, vertebral fractures may be asymptomatic and often require radiological diagnosis before treatment. Since the incidence of fractures is higher in the spine compared to other areas of the body, glucocorticoid therapy appears to affect rapid bone remodelling. During the initial phases of glucocorticoid exposure bone resorption is increased (van Staa et al., 2000). Glucocorticoids inhibit the formation of mature osteoblasts, but also activate apoptosis in these cell types (Jones & Sambrook, 1994; Canalis et al., 2007). Osteoprotegrin (OPG) expression, a key factor involved in modulating maturation of osteoclasts, is reduced also by glucocorticoids resulting in increased osteoclastogenesis (Orcel, 2005). Therefore, the combination of reduced osteoblast formation, increased osteoclast maturation leads to accelerated bone loss while on glucocorticoid therapy. Therapies are needed which modulate osteoclast as well as osteoblast activity to restore a more normal balance to the

bone remodeling process in glucocorticoid treated patients.

**2. Glucocorticoid-induced osteoporosis: Is there a role for genistein?** 

Among the anabolic compounds tested in recent years genistein aglycone seems a promising agent able to stimulate bone formation and to reduce bone resorption, acting *via* a genomic as well as a non-genomic pathways. Genistein is an isoflavone found in small quantities in certain legumes throughout the plant kingdom. Soybeans are a particularly rich source of genistin, the glucosidal precursor of genistein, although the concentration varies with the strain, location and environmental conditions of cultivation

guidelines exist.

of the plant. Another widely utilized source of genistin is *Sophora japonica* L (Tian et al., 2004). The glucosidal form and acetyl- and malonyl-glucosides are the major isoforms found in soybean derivates. The conversion from the glucosides to the aglycone form occurs in the gut, where the sugar residue is removed with the generation of genistein (Larkin et al., 2008).

Genistein is freely absorbed from the intestine and a large fraction is converted to the 7β-Oglucuronide as it crosses the brush border and ultimately enters the portal vein (Sfakianos et al., 1997), a process that is influenced by intestinal bacteria (Setchell et al., 2002; Day et al., 1998). Recent *ex vivo* data using isolated human gastrointestinal tract tissue also suggest that genistein may also undergo sulfonation in the small intestine, though the extent to which these sulfonates are absorbed following dietary intake is unknown (Ronis et al., 2006). The exact percentages of glucuronidated and sulfonated metabolites after crossing the lumen are also unknown, although it is clear that only a small percentage of the parent molecule remains as unconjugated genistein once it reaches the liver. Once in the liver, genistein undergoes additional biotransformation *via* CYP450-mediated hydroxylation (Hu et al., 2003) followed by glucuronidation and sulfonation by UDP-glucuronosyl transferase and sulphotransferases, respectively (Sfakianos et al. 1997). Genistein 7β-O-glucuronide can be recovered from bile after infusion of genistein into the small bowel of rats (Prasain et al., 2006). As much as 70% of the recovered genistein from bile is in the form of glucuronidated conjugates with smaller amounts reappearing in the distal duodenum and jejunum (Sfakianos et al., 1997). The vast majority of circulating genistein in serum has been found to be in the form of glucuronidated and sulfonated conjugates which represent excretion forms of the molecule (Setchell et al., 2002). Little is known about the bioactivity of conjugated isoflavones. Genistein is composed of two benzene rings (A and B) linked through a heterocyclic pyrane C ring. The hydroxyl group on the A ring confers to genistein a greater activity both *in vitro* and *in vivo*, compared to other isoflavones (Choi et al., 2008).

In addition to competing with endogenous estrogens for binding to the estrogen receptors at high concentrations, genistein may exert anti-estrogenic effects by several potential mechanisms (Tham et al., 1998). Genistein has both ER agonist and antagonist activity in different cell types and works in a promoter specific manner in gene activation *via* ERs. The ER domain structure is typical of nuclear receptors. The amino-terminal region is involved in trans-activation of gene expression and has been termed the activation function domain or AF1 domain. The middle region contains a two-zinc finger structure, which plays an important role in binding to specific DNA response elements and in receptor dimerization. The carboxyl-terminal region contains an activation domain, termed AF2. A ligand binding domain, lying within AF2, is crucial for binding to receptor specific ligands and also corepressors and co-activators. This binding interaction affects receptor dimerization, nuclear translocation and modulation of target gene expression by AF2. The ligand-binding domain is structurally conserved among the nuclear receptor family and consists of between 10 and 12α helices folded in a globular domain. A central hydrophobic pocket accommodates the cognate ligand, which upon binding induces a conformational change in the ligand-binding domain, exposing a coactivator-docking site on ligand binding surface. However the binding of SERMs to ERs results in a different conformation change in which the coactivator-binding site is blocked from interaction with coactivator, thereby blocking AF2 mediated transcriptional activity (Liu et al., 2003). Several classes of nuclear receptor coactivators interact with ERs receptors and are specific for either ERα or ERβ. Using specifying coactivator recruitment and fluorescence resonance energy transfer, the genistein

Genistein Aglycone Demonstrates a Protective and Reversible Effect on the Development

and interleukin-6 gene expression (Rickard et al. 2003).

**2.2 Evidence from** *in vivo* **studies** 

(Arts et al., 1997).

of Steroid-Induced Secondary Osteoporosis and Increases Bone Breaking Strength in Rats 293

interleukin-6 protein. However, actions of genistein at the cellular level depend on the target tissue, receptor status of the tissue, and the level of endogenous estrogen. Both estrogen receptors ERα and ERβ are present in bone tissue, although the expression of these subtypes varies considerably during differentiation. The greatly increased expression of ERβ during bone mineralization is particularly pertinent to the potential hormonal effects of isoflavones because compounds such as genistein show a much higher affinity for ERβ than for ERα

One of the mechanism by which genistein induces apoptosis of mature osteoclasts involves the Ca2+ signaling, the inhibition of protein kinase and the activation of protein tyrosine phosphatase in osteoclasts. In this study the suppressive effect of genistein was completely abolished by the presence of inhibitors of Ca2+-dependent protein kinases (Gao & Yamaguchi, 1999b). The most important cytokine machinery, which is involved in bone metabolism, is osteoprotegerin (OPG)/receptor activator of nuclear factor-κB (RANK)/RANK ligand (RANKL) system. The molecular triad OPG/RANK/RANKL plays important roles in bone remodeling. RANKL is expressed by osteoblasts and is necessary and sufficient for osteoclastogenesis. RANKL binds to its receptor RANK, present at the surface of osteoclast precursors and mature osteoclasts, inducing osteoclast formation and activation. RANKL activity can be blocked by the soluble decoy receptor OPG, resulting in prevention of bone resorption. Studies, *in vitro*, have found that genistein induces apoptosis and inhibits RANKL signaling-related gene expression (Uchiyama & Yamaguchi, 2007). Genistein was also reported to increase osteoblastic differentiation and affect osteoblast activity (Rickard et al., 2003; Pan et al., 2005; Morris et al., 2006; Okumura et al., 2006). In osteoblasts genistein stimulates a concentration-dependent increase in alkaline phosphatase activity. In osteoblastic MC3T3-E1 cells, genistein increases protein content, DNA content and alkaline phosphatase activity (Yamaguchi & Sugimoto, 2000; Sugimoto & Yamaguchi, 2000). This effect has also been demonstrated in tissue cultures. In femoral metaphyseal tissue from elderly female rats genistein increases Ca2+ content and alkaline phosphatase activity. This effect is blocked by tamoxifen, indicating an ER-mediated pathway, and also by cycloheximide, suggesting that this isoflavone influence transcriptional or translational events (Yamaguchi & Gao, 1997, 1998). On the other hand, genistein also causes apoptosis of osteoblasts by activating caspase-3 and cleaving adhesion molecules such as cadherins and catenins (Hunter et al., 2001). In normal fetal osteoblast cells, genistein increases the progesterone receptor and alkaline phosphatase gene expression and inhibits osteopontin

The manner by which glucocorticoids induce bone loss is complex and incompletely understood (Patschan et al., 2001; Canalis et al., 2007), in part because there are no animal models absolutely comparable to humans. A major effect on the skeleton is a decrease in bone formation and unchanged or enhanced bone resorption (Lane et al., 2001). The main effect of glucocorticoids on bone is inhibition of osteoblastogenesis, augmented continued osteoclastogenesis and osteocyte apoptosis, leading to a decrease in bone formation, a rapid weakening of bone architecture and an increase in fracture risk (Manolagas & Weinstein, 1999). Chronic administration of steroids also causes avascular necrosis *via* an apoptotic mechanism of osteocytes and osteoblasts (Weinstein et al., 2000, Bekler et al., 2007). Once osteonecrosis occurs, glucocorticoids also cause inhibition of bone regeneration (Takano-

interaction with ER receptors was investigated (Kuiper et al., 1998). Compounds with estrogen agonism induce agonist-mediated recruitment and allow fluorescence resonance energy transfer. Compounds with estrogen antagonism block the agonist-mediated recruitment of coactivators and prevents fluorescence resonance energy transfer. Genistein showed full agonism for ER α and only partial agonism for ER β, but higher affinity for ERβ than ERα (Kuiper et al., 1998).

A second action of genistein on membrane associated protein (membrane receptors) has been proposed, and remains still not fully elucidated. In this mechanism, several possible second messengers, including kinase enzymes, are activated and in turn stimulate or inhibit downstream pathways of metabolism and protein products. The duration of this effect *via* a membrane protein is short lasting and of limited efficacy. The positive effects of genistein on primary osteoporosis, due to estrogen fall, have been extensively investigated by our group as well as by others and have been recently summarized in a review article (Bitto et al., 2010); however pre-clinical evidence point out a role for genistein also in secondary osteoporosis induced by corticosteroid use.

#### **2.1 Evidence from** *in vitro* **studies**

Genistein has been most investigated for its effect on the proliferation and differentiation of a number of cell types. Through its effect on tyrosine kinase, genistein is able to modulate cell cycle progression in the S phase, to induce G2/M arrest and to induce apoptosis (Matsukawa et al., 1993). Numerous in vitro studies with human or animal osteoclast- or osteoblast-like cell lines have been carried out with consistent observations of direct effects of genistein on both cell types. Effects of genistein on bone metabolism derived from direct and indirect actions on bone cells and can be summarized in stimulation of osteoblastic bone formation and inhibition of osteoclastic bone resorption (Gao & Yamaguchi, 1999a; Sugimoto & Yamaguchi, 2000; Chen et al., 2003; Heim et al., 2004). In vitro studies indicate that genistein is able to stimulate osteoblastic activity and inhibit osteoclast formation and action at a range of concentrations (10−5–10−7 M) consistent with the levels observed in human subjects after ingestion of genistein. With regard to effects on osteoclast genistein inhibits their formation and function (Amano et al., 1998; Gao & Yamaguchi, 2000; Yamagishi et al., 2001; Albertazzi, 2002; Blair et al., 1996; Gao & Yamaguchi, 1999a; Kajiya et al, 2000; Williams et al, 1998). Genistein suppress osteoclast activity by a number of possible mechanisms, including induction of apoptosis, activation of protein tyrosine phosphatase, inhibition of cytokines, changes in intracellular Ca2+, and membrane depolarization. More in details, in mouse marrow cultures genistein has a potent inhibitory effect on osteoclast-like cell formation, and this effect is similar to others anti-bone-resorbing agents, such as calcitonin and 17β-estradiol, and may involve cAMP signaling. Sliwiñski and coworkers (2005) compared the effects of genistein, estradiol and raloxifene, compounds affecting in different ways estrogen receptors, on formation and viability of osteoclast from neonatal rat bone marrow; the results shown that all treatments decrease the number of osteoclasts formed from bone marrow, particularly genistein at the lower concentration decreases the number of osteoclasts by induction cell death, while at higher concentration genistein prevents the formation of osteoclasts. These observations are consistent with the results of another study (Rickard et al. 2003), showing that genistein, acting as agonist of estrogen receptors, mimicked estradiol in affecting gene expression of progesterone receptor, the proteoglycan versican, alkaline phosphatase and osteopontin, and production of interleukin-6 protein. However, actions of genistein at the cellular level depend on the target tissue, receptor status of the tissue, and the level of endogenous estrogen. Both estrogen receptors ERα and ERβ are present in bone tissue, although the expression of these subtypes varies considerably during differentiation. The greatly increased expression of ERβ during bone mineralization is particularly pertinent to the potential hormonal effects of isoflavones because compounds such as genistein show a much higher affinity for ERβ than for ERα (Arts et al., 1997).

One of the mechanism by which genistein induces apoptosis of mature osteoclasts involves the Ca2+ signaling, the inhibition of protein kinase and the activation of protein tyrosine phosphatase in osteoclasts. In this study the suppressive effect of genistein was completely abolished by the presence of inhibitors of Ca2+-dependent protein kinases (Gao & Yamaguchi, 1999b). The most important cytokine machinery, which is involved in bone metabolism, is osteoprotegerin (OPG)/receptor activator of nuclear factor-κB (RANK)/RANK ligand (RANKL) system. The molecular triad OPG/RANK/RANKL plays important roles in bone remodeling. RANKL is expressed by osteoblasts and is necessary and sufficient for osteoclastogenesis. RANKL binds to its receptor RANK, present at the surface of osteoclast precursors and mature osteoclasts, inducing osteoclast formation and activation. RANKL activity can be blocked by the soluble decoy receptor OPG, resulting in prevention of bone resorption. Studies, *in vitro*, have found that genistein induces apoptosis and inhibits RANKL signaling-related gene expression (Uchiyama & Yamaguchi, 2007).

Genistein was also reported to increase osteoblastic differentiation and affect osteoblast activity (Rickard et al., 2003; Pan et al., 2005; Morris et al., 2006; Okumura et al., 2006). In osteoblasts genistein stimulates a concentration-dependent increase in alkaline phosphatase activity. In osteoblastic MC3T3-E1 cells, genistein increases protein content, DNA content and alkaline phosphatase activity (Yamaguchi & Sugimoto, 2000; Sugimoto & Yamaguchi, 2000). This effect has also been demonstrated in tissue cultures. In femoral metaphyseal tissue from elderly female rats genistein increases Ca2+ content and alkaline phosphatase activity. This effect is blocked by tamoxifen, indicating an ER-mediated pathway, and also by cycloheximide, suggesting that this isoflavone influence transcriptional or translational events (Yamaguchi & Gao, 1997, 1998). On the other hand, genistein also causes apoptosis of osteoblasts by activating caspase-3 and cleaving adhesion molecules such as cadherins and catenins (Hunter et al., 2001). In normal fetal osteoblast cells, genistein increases the progesterone receptor and alkaline phosphatase gene expression and inhibits osteopontin and interleukin-6 gene expression (Rickard et al. 2003).
