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

292 Basic and Clinical Endocrinology Up-to-Date

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

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

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

than ERα (Kuiper et al., 1998).

osteoporosis induced by corticosteroid use.

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

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-

Genistein Aglycone Demonstrates a Protective and Reversible Effect on the Development

**<sup>C</sup> Femur breaking strength**

**A B**

of Steroid-Induced Secondary Osteoporosis and Increases Bone Breaking Strength in Rats 295

**methylprednisolone methylprednisolone**

Fig. 3. A and B: Light microscopy of the bone structure of the femur head obtained from animals treated with methylprednisolone (A) or methylprednisolone plus genistein aglycone (B). (H&E original magnification X5). C: Femur breaking strength: \**p*<0.001 vs. methylprednisolone. Data are shown as % of variation from vehicle treated animals.

In addition, genistein caused a significant increase in b-ALP and OPG over methylprednisolone and vehicle confirming its role as an anabolic bone-forming agent (Figure 4A and B). Genistein administration also significantly reduced CTX circulating

Corticosteroid therapy has been shown to cause apoptosis in osteoblasts and inhibit the production of OPG required for bone formation, while RANKL accumulates resulting in further bone resorption (Bejar et al. 2005). Genistein has been shown to stimulate the production of osteoblasts *via* inhibition of a RANKL-resorptive mechanism by producing OPG (Viereck et al. 2002). This was also confirmed in our study where genistein increased OPG levels in glucocorticoid-treated animals. Osteoprotegrin is a glycoprotein secreted by osteoblasts in a differentiation-dependent manner and acts as a 'decoy receptor' (a soluble receptor that acts as antagonist) for RANKL regulating osteoclast functions and lifespan. In postmenopausal women with bone loss, genistein stimulated the production of OPG, downregulated RANKL and decreased the RANKL/OPG ratio suggesting a direct effect on the RANK cytokine system (Marini et al., 2008). This work on genistein also suggests that increases in OPG in humans modulates the maturation process of osteoclast formation, thus

**% of change vs vehicle**

levels suggesting an anti-resorptive effect (Figure 4C).

 **+ genistein**

Murakami et al., 2009). In a rat model, we studied genistein preservative effects on methylprednisolone-induced bone loss and osteonecrosis of the femoral head (Bitto et al., 2009). In our study (Figure 1), genistein succeeded in preventing osteoporosis and osteonecrosis of the femoral head when co-administered with the glucocorticoid. The isoflavone statistically maintained bone mineral density (Figure 2) and content (Bitto et al., 2009) over the methylprednisolone-treated group and showed comparable efficacy with the vehicle group. Genistein co-administered with methylprednisolone also statistically maintained femoral bone's resistance to rupture compared with the methylprednisolone group (Figure 3C) and preserved the normal architecture of cartilage as well as both cortical and trabecular bones with a well-organized matrix in femoral head (Figure 3 A and B).

Fig. 1. Flow-chart of the experimental protocol.

Fig. 2. Effects of aglycone genistein on femoral bone mineral density (BMD) in methylprednisolone-treated rats. Data are shown as % of variation from basal values in the same group of animals. *\* p*<0.005 vs. methylprednisolone.

Murakami et al., 2009). In a rat model, we studied genistein preservative effects on methylprednisolone-induced bone loss and osteonecrosis of the femoral head (Bitto et al., 2009). In our study (Figure 1), genistein succeeded in preventing osteoporosis and osteonecrosis of the femoral head when co-administered with the glucocorticoid. The isoflavone statistically maintained bone mineral density (Figure 2) and content (Bitto et al., 2009) over the methylprednisolone-treated group and showed comparable efficacy with the vehicle group. Genistein co-administered with methylprednisolone also statistically maintained femoral bone's resistance to rupture compared with the methylprednisolone group (Figure 3C) and preserved the normal architecture of cartilage as well as both cortical and trabecular bones with a well-organized matrix in femoral head

> Methylprednisolone + Genistein (n=7)

Assessment of Basal BMD and BMC

**Time of treatment 60 days**

Methylprednisolone (n=7)

Sacrifice, assessment of final BMD and BMC. Evaluation of breaking strength, biochemical and histological assays.

Fig. 2. Effects of aglycone genistein on femoral bone mineral density (BMD) in

same group of animals. *\* p*<0.005 vs. methylprednisolone.

methylprednisolone-treated rats. Data are shown as % of variation from basal values in the

Fig. 1. Flow-chart of the experimental protocol.

(Figure 3 A and B).

Genistein (n=7)

Vehicle (n=7)

Fig. 3. A and B: Light microscopy of the bone structure of the femur head obtained from animals treated with methylprednisolone (A) or methylprednisolone plus genistein aglycone (B). (H&E original magnification X5). C: Femur breaking strength: \**p*<0.001 vs. methylprednisolone. Data are shown as % of variation from vehicle treated animals.

In addition, genistein caused a significant increase in b-ALP and OPG over methylprednisolone and vehicle confirming its role as an anabolic bone-forming agent (Figure 4A and B). Genistein administration also significantly reduced CTX circulating levels suggesting an anti-resorptive effect (Figure 4C).

Corticosteroid therapy has been shown to cause apoptosis in osteoblasts and inhibit the production of OPG required for bone formation, while RANKL accumulates resulting in further bone resorption (Bejar et al. 2005). Genistein has been shown to stimulate the production of osteoblasts *via* inhibition of a RANKL-resorptive mechanism by producing OPG (Viereck et al. 2002). This was also confirmed in our study where genistein increased OPG levels in glucocorticoid-treated animals. Osteoprotegrin is a glycoprotein secreted by osteoblasts in a differentiation-dependent manner and acts as a 'decoy receptor' (a soluble receptor that acts as antagonist) for RANKL regulating osteoclast functions and lifespan. In postmenopausal women with bone loss, genistein stimulated the production of OPG, downregulated RANKL and decreased the RANKL/OPG ratio suggesting a direct effect on the RANK cytokine system (Marini et al., 2008). This work on genistein also suggests that increases in OPG in humans modulates the maturation process of osteoclast formation, thus

Genistein Aglycone Demonstrates a Protective and Reversible Effect on the Development

**4. References** 

Intern Med. 2003; 163:2619-24.

genistein? Climacteric 2002; 5: 190–196

osteoclasts. J Bone Miner Res, 1998; 13: 846–853.

regression. J Bone Miner Res. 2002; 17: 1512-1526.

outpatient study. Bone 2006; 39: 253–259

of Steroid-Induced Secondary Osteoporosis and Increases Bone Breaking Strength in Rats 297

be attributed to chronic, high-dose glucocorticoid administration in humans (van Staa et al., 2001). Prior and current exposure to glucocorticoids increases the risk of fractures beyond that explained by values of BMD (Civitelli & Ziambaras, 2008). Pharmacological intervention for prevention of glucocorticoid-induced osteoporosis is needed depending on dose, expected duration of treatment, age and gender of the patient, and sometimes BMD at the start of the glucocorticoid therapy. At present, calcium and vitamin D3 supplementation are considered as important support for the prevention of glucocorticoid-induced osteoporosis (Williams et al., 2004). Bisphosphonates are largely used to avoid bone loss and are cost effective in certain subgroups of patients depending on age, gender, glucocorticoid dose, and previous fracture history (Williams et al., 2004; Prinsloo & Hosking, 2006). Unfortunately, calcium and vitamin D3 supplementation may not be enough to stave off bone deterioration and bisphosphonates have safety risks associated with long-term use, such as esophageal burns, bone and muscular pain (Ettinger et al., 1998; Wysowski & Chang, 2005). Osteonecrosis of the jaw, although rare and found primarily in cancer patients undergoing dental surgery, is a very serious and debilitating side effect of bisphosphonate use (Durie et al., 2005). Therefore, a safe and effective treatment for the prevention of bone loss and osteonecrosis of the femoral head in glucocorticoid-treated subjects is still needed. Collectively, our results strongly suggest that genistein might be a new potential therapy for the prevention of glucocorticoid-induced osteoporosis, the most important secondary cause of osteoporosis in humans. And in the minority of cases, genistein may prevent necrotic deterioration of the femoral head. Usually, drugs used in management of osteoporosis have been classified as predominantly 'antiresorptive agents' or as 'bone-forming agents', but, on the basis of the present results, genistein might represent the first therapy to overcome this classification combining a powerful bone-forming as well as an anti-resorptive activity.

Adler, R.A. & Hochberg, M.C. Suggested guidelines for evaluation and treatment of

Albertazzi, P. Purified phytoestrogens in postmenopausal bone health: is there a role for

Amano, H., Yamada, S. & Felix, R. Colony-stimulating factor-1 stimulates the fusion process in

American college of Rheumatology Ad Hoc Committee on glucocorticod-induced

induced osteoporosis. 2001 Update. Arthritis and Rheumat 2001; 44:1496-503. Amin, S., Lavalley, M.P., Simms, R.W. & Felson, D.T. The comparative efficacy of drug

Angeli, A., Guglielmi, G., Dovio, A., Capelli, G., de Feo, D., Giannini, S., Giorgino, R., Moro, L.

Arts, J., Kuiper, G.G., Janssen, J.M., Gustafsson, J.A., Löwik, C.W., Pols, H.A. & van Leeuwen,

glucocorticod-induced osteoporosis for the Department of Veterans Affairs. Arch

osteoporosis. Recommendation for the prevention and treatment of glucocorticod-

therapies used for the management of corticosteroid induced osteoporosis: a meta-

& Giustina, A. High prevalence of asymptomatic vertebral fractures in postmenopausal women receiving chronic glucocorticoid therapy: a cross-sectional

J.P. Differential expression of estrogen receptors alpha and beta mRNA during differentiation of human osteoblast SV-HFO cells. Endocrinology. 1997; 138: 5067-70

clarifying the putative antiresorptive effect being a non-apoptotic process, rather than an apoptotic process induced in antiresorptive therapy with bisphosphonates. Other mechanisms of genistein binding to glucocorticoid receptors have also been implicated.

Fig. 4. A-C: Effects of aglycone genistein on serum bone-alkaline phosphatase (b-ALP), osteoprotegerin (OPG) and collagen C-telopeptides (CTX). Data are shown as % of variation from vehicle treated animals. b-ALP: \* *p*<0.005 vs methylprednisolone. OPG: \**p*<0.001 vs. methylprednisolone. CTX: \**p*<0.001 vs. methylprednisolone.

It has been demonstrated that genistein inhibits glucocorticoid receptor transactivation and may also induce a proteosomal degradation of the glucocorticoid receptor complex *via* the p53 and ubiquitin pathways (Kinyamu & Archer 2003). Another mechanism might involve genistein activity as a tyrosine kinase inhibitor *via* the limitation of the subcellular nuclear transport and the recycling of the glucocorticoid receptors, blunting in turn the effects of glucocorticoids on bone (Yang et al., 1997).
