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© 2006 American Society for Nutrition J. Nutr. 136:1166-1170, May 2006

Biochemical, Molecular, and Genetic Mechanisms

The Soybean Isoflavone Genistein Induces Differentiation of MG63 Human Osteosarcoma Osteoblasts1

Christopher Morris*, Julian Thorpe{dagger}, Luigi Ambrosio** and Matteo Santin*,2

* School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ, UK; {dagger} The Sussex Centre for Advanced Microscopy, School of Life Sciences, University of Sussex, Falmer Campus, Brighton BN1 9QG, UK; and ** Institute of Biomedical Composite Materials, CNR, 80130 Naples, Italy

2 To whom correspondence should be addressed. E-mail: m.santin{at}brighton.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
A soybean-rich diet was shown to reduce the incidence of osteoporosis in Eastern countries; its effect on bone metabolism was ascribed to the action of the soybean isoflavones such as genistein. Although many studies have shown isoflavone-induced osteoblast differentiation, its preventative action on bone mass loss has not been clarified. Here, the osteogenetic effects of genistein on human cell line MG63 osteoblasts were elucidated using a variety of approaches. In particular, phalloidin-rhodamine staining revealed that genistein-treated osteoblasts possessed a more organized cytoskeleton, and genistein's inhibitory effect upon cell proliferation was associated with exposure of phosphatidylserines on the external plasmalemma surface. Although this phosphatidylserine exposure is considered a typical apoptotic marker, scanning and transmission electron microscopy revealed that genistein-treated osteoblasts released matrix vesicles and showed no evidence of chromatin condensation. Assays, stainings, and scanning electron microscopy showed that genistein-treated osteoblasts synthesized relatively high levels of collagen and alkaline phosphatase and, even in a nonosteogenic growth medium, formed mineralized bone noduli. A clear pattern of genistein-induced osteoblast activation therefore emerges, in which all of the essential components required for the rapid production of mineralized bone extracellular matrix are stimulated by this soybean isoflavone.

KEY WORDS: • soybean isoflavones • genistein • osteoblast differentiation • bone formation • mineralization

Bone regeneration is regulated by a fine balance of biochemical and cellular events that ultimately stimulate osteoblasts to produce new tissue, in particular, new extracellular matrix composed mainly of collagen (1). The collagen matrix is then mineralized via alkaline phosphatase (ALP)3 activity, which induces formation of calcium phosphate crystal seeds. This process takes place within cell membrane–derived "matrix vesicles" (2–4), whose calcification properties depend upon the presence of annexin V ion channels, through which calcium enters, and of calcium-binding phospholipids, e.g., phosphatidylserine (PS) (5,6). Uptake of calcium ions into the phosphorus-rich vesicles increases the levels of both ions to supersaturating concentrations, whereas their binding to PS generates nucleation loci for hydroxyapatite crystal formation. Although the origin of matrix vesicles is controversial, it was hypothesized that they can be produced as exocytotic bodies following a mechanism different from that of apoptotic membrane blebbing (7).

Recent data highlighted a lower incidence of breast and prostate tumors, as well as of osteoporosis, in human populations that consume a soybean-rich diet (8,9). The antitumor properties of soy were ascribed to the inhibition of cell proliferation by the soybean isoflavones (10–12). Among them, genistein was shown to be one of the most potent inhibitors in vitro, interacting with estrogen receptors on the nuclear envelope to promote G2/M phase arrest (11). Genistein's effects on cellular functions were shown in immunocompetent cells such as monocytes/macrophages and lymphocytes (10). Additionally, it may also interact with plasmalemma tyrosine kinase receptors to reduce free radical secretion by inflammatory cells (10).

Studies at the cellular level have provided insights into the ability of soybeans to prevent osteoporosis (13,14), and others demonstrated the inhibitory action of genistein on osteoclast differentiation and activity (15,16). Genistein was also shown to induce osteoblast differentiation through enhanced expression of ALP, bone morphogenetic protein 2, and osteoprotegerin, and to stimulate stem cell differentiation (15–20). In addition, the treatment of osteoblasts with genistein, and with other soybean isoflavones, induced calcified bone noduli (18). Such findings triggered investigations to elucidate the bioactivational properties of genistein on bone metabolism and, in particular, on processes leading to mineralizaton of the extracellular matrix.

The present in vitro study is a coordinated assessment of the effect of genistein on osteoblast osteogenic potential. Morphology and ultrastructure, proliferation, collagen and matrix vesicle synthesis, ALP activity, and calcification potential in cells cultured with genistein were analyzed in both nonosteogenic and osteogenic media.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Cell viability and proliferation. Human MG63 osteosarcoma osteoblast cells (ATCC catalog CRL-1427) were incubated in osteogenic medium as previously described (21). The growth medium was then replaced with fresh medium containing increasing concentrations of soybean genistein [from soy (Glycine max), 98% pure, Sigma). The range of genistein concentrations tested was 2.5–30 µmol/L. Cells were allowed to proliferate for 48 h, washed with calcium-free PBS, pH 7.4 (Oxoid), and their nuclei stained with Hoerscht-propidium iodide dye (21). Six different fields per sample were counted. The early induction of cell apoptosis was studied by detecting the exposure of PS on the outer plasmalemma surface by an Annexin V kit (BD Biosciences, catalog PF032–1ES) following the manufacturer's instructions. The number of live and apoptotic cells was scored by fluorescence microscopy at X40 magnification.

    Cell morphology. After 48 h of incubation with increasing concentrations of genistein, MG63 osteoblasts were fixed in formalin for 1 h at ambient temperature, washed 3 times with PBS, and the cytoskeleton stained with 1:100-diluted rhodamine-phalloidin dye. The cell cytoskeleton was analyzed by fluorescence microscopy at X40 magnification. Additionally, osteoblasts were prepared for scanning electron microscopy (SEM) as described elsewhere (21). The experiments were performed in triplicate on different days.

    Cell ultrastructure. Cells were fixed in situ on thermanox coverslips (Nunc) with 2.5% (wt:v) glutaraldehyde in PBS for 3 h at ambient temperature, followed by postfixation in 0.5% (wt:v) OsO4 in PBS for 4 h at ambient temperature. After being rinsing with distilled water, the samples were dehydrated in an ethanol series and embedded in Spurr resin. Thin sections were cut onto formvar-coated grids and poststained for 1 h in 0.5% (wt:v) aqueous uranyl acetate and then 10 min in lead staining with citrate. Sections were examined in a Hitachi-7100 transmission electron microscope (TEM) at 100 kV and images acquired with a Gatan Ultrascan 1000 CCD camera (Gatan UK). The experiments were performed in duplicate on different days.

    Collagen synthesis. The synthesis of collagen by the MG63 osteoblasts was evaluated in both nonosteogenic and osteogenic medium. After 48 h of incubation at different concentrations of genistein, the cells were trypsinized, lysed by the addition of 0.1% (wt:v) Triton, centrifuged for 5 min at 500 x g and the supernatants (50 µL) tested for their collagen content by a quantitative Sirius Red method as previously described (21). Protein concentration was assessed by the Bradford's method (21).

    ALP activity. Cells were incubated for 24 h in either nonosteogenic medium or ascorbic acid–depleted osteogenic medium (21). The cells were detached from the substrate by trypsin and lysed. The samples were centrifuged at 1000 x g and the supernatants stored at –70°C until use. An ALP microplate assay was performed as described elsewhere (21).

    Calcification potential. The effect of genistein on the formation of calcification foci in osteoblast nonosteogenic and osteogenic cultures was evaluated by Alizerin Red S staining as described elsewhere (22). After rapid rinsing in toluene, the cells were analyzed by visible light microscopy at X40 magnification. Experiments were performed in duplicate on different days.

    Statistical analysis. Unless specified differently, the experiments were performed in duplicate on 6 different days and the data are means ± SD, n = 6. The data were tested by ANOVA and Dunnett's test. Differences from the control were considered significant at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Cell viability and proliferation. MG63 proliferation was significantly reduced by genistein in the range of concentrations used; however, the numbers of early apoptotic cells (cells exposing PS on their surface) (23) were constant (Fig. 1). Apoptotic cells with clear DNA damage appeared only after incubation with genistein concentrations >10 µmol/L (Fig. 1, late apoptosis bar).


Figure 1
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  		FIGURE 1  Effect of genistein osteoblast proliferation and apoptosis in MG63 cells cultured with genistein in both nonosteogenic and osteogenic media. Early apoptotic cells (Early) are those with exposed phosphatidylserine on their external cell membrane surface. Late apoptotic cells (Late) exhibit DNA fragmentation. Values are means ± SD, n = 6. *Different from the control, P ≤ 0.05.

 
    Cell morphology. Differences in proliferation and viability were accompanied by changes in cell morphology (Fig. 2A–C). When incubated with genistein, the osteoblast cytoskeleton became more organized than in control cells (Fig. 2A, B). Condensed areas of fluorescence were also visible (Fig. 2B, arrowheads). At high genistein concentrations, the formation of a material of a woolly appearance and of needle-shaped crystals deposited on the cells was also highlighted by the fluorescent staining (Fig. 2C, arrow). EM revealed vesicles emerging from the cell membrane of genistein-treated osteoblasts (Fig. 3A, B, arrowheads). Interestingly, osteoblasts treated with genistein also had a tendency to form cell clusters (Fig. 3A) and secrete collagen-like fibrillar material (Fig. 3B, arrows). The crystals observed by fluorescence microscopy were easily washed off during sample preparation for SEM and therefore were not detectable by this technique.


Figure 2
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  		FIGURE 2  Genistein's effect on MG63 osteoblast cytoskeletal organization in control osteoblasts (A), osteoblasts treated with a relatively low concentration of genistein (2.5 µmol/L) (B), and osteoblasts treated with a relatively high concentration of genistein (30 µmol/L) (C). Magnification bars = 20 µm. Arrowheads indicate area of fluorescent dye concentration. Arrow indicates a typical needle-shaped hydroxyapatite crystal.

 

Figure 3
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  		FIGURE 3  SEM analysis of the effect of 2.5 µmol/L genistein on MG63 osteoblast cell membrane morphology in cells cultured with genistein in both nonosteogenic and osteogenic media. Cell nodules (A), cell detail (B). Arrowheads indicate vesicles emerging from the cell membrane. Arrow indicates collagen-like fibril deposition.

 
    Cell ultrastructure. Ultrastructural studies by TEM showed that both control and genistein-treated osteoblasts had a normal ultrastructural appearance (Fig. 4A, B). In particular, the genistein-treated cells did not show any sign of chromatin condensation typical of apoptotic cells (Fig. 4B). However, genistein-treated osteoblasts often exhibited marked accumulations of vesicles near the plasmalemma (Fig. 4B, insert, 4C, arrowheads).


Figure 4
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  		FIGURE 4  TEM analysis of the effect of genistein on MG63 osteoblast ultrastructure in control cells (A) and genistein-treated (30 µmol/L) cells (B); cell plasmalemma detail (C) from (B). Arrowheads indicate the formation of matrix vesicles.

 
    ALP activity. ALP activity (IU/L) was evaluated in cells incubated with increasing concentrations of genistein, under nonosteogenic and osteogenic conditions. Cells incubated in both nonosteogenic and osteogenic medium were not affected by genistein (data not shown). However, when the data were expressed as ALP specific activity (IU/mg of protein), activity of cells cultured in osteogenic medium under a genistein stimulus was lower than that of controls at a concentration ≥7.5 µmol/L (Fig. 5). Similarly, in nonosteogenic medium, a significant ALP activity reduction occurred at 20 and 30 µmol/L. The ALP-specific activities, however, were significantly affected by the high cell protein synthesis induced by genistein (Fig. 6A). In both nonosteogenic and osteogenic medium, there was an isoflavone-induced increase in osteoblast protein synthesis.


Figure 5
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  		FIGURE 5  Effect of genistein on MG63 osteoblast ALP-specific activity (IU/mg of protein) in nonosteogenic (non-osteo) and osteogenic (osteo) medium. Values are means ± SD, n = 6. *Different from the control, P ≤ 0.05.

 

Figure 6
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  		FIGURE 6  Effect of genistein on MG63 osteoblast protein synthesis in nonosteogenic (non-osteo) and osteogenic (osteo) medium for protein concentration (A) and collagen concentration (B). Values are means ± SD, n = 6. *Different from the control, P ≤ 0.05.

 
    Collagen synthesis. The induction of protein synthesis, as well as the visible secretion of collagen-like fibrils detected by SEM, prompted a quantitative determination of collagen synthesis and secretion. Collagen synthesis was significantly higher than in controls when cells were incubated with concentrations of genistein ≥5.0 µmol/L in a nonosteogenic medium (Fig. 6B), whereas genistein had no effect in cells incubated in an osteogenic environment.

    Calcification potential. Alizerin staining showed that genistein stimulated the formation of calcification foci on osteoblasts in both nonosteogenic and osteogenic medium (data not shown). There was no calcification when control osteoblasts were grown under the same conditions (data not shown). In particular, Alizerin staining showed the needle-like crystals already observed with phalloidin-rhodamine (Fig. 2C, arrows). Unlike SEM (Fig. 3A), light microscopy showed the tendency of the cell noduli to calcify in the presence of genistein, a feature not observed in the control (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Biomineralization in living tissues is associated with proteins and biological membranes as a consequence of the calcium-binding potential of acidic phospholipids such as the PS (4,24). Matrix vesicles appear to be formed from exocytotic bodies of the osteoblast plasmalemma and released into the surrounding medium to facilitate formation of apatite crystal nuclei (4,24).

Soybean-rich diets reduce the incidence of osteoporosis (5,6). Recent research showed the potential of genistein and daidzein to induce the expression of bone morphogenetic protein 2 by osteoblasts as well as the synthesis of osteoprotegerin and other markers of osteoblast differentiation (15–20). However, the mechanisms of genistein-induced bone formation at the biochemical and cellular level have not yet been elucidated.

Previous work highlighted the ability of genistein to inhibit cell proliferation in vitro and to stimulate dermal collagen synthesis in vivo (9,25). However, specific studies on osteoblasts with 1 nmol/L soybean isoflavones did not show any effect on cell proliferation or collagen synthesis (15). The established effect of genistein on osteoblast differentiation and calcified noduli formation (15,18) would suggest that it can induce the formation of new mineralized extracellular matrix.

For these reasons, correlation analyses were employed to determine the sequence of cellular events initiated by genistein to shed light on the mechanisms of new mineralized tissue formation. In particular, genistein's ability to reduce cell proliferation was related to the formation of an apoptotic-like plasmalemma and its ability to induce the nucleation of hydroxyapatite crystals by MG63 human osteoblasts, a cell line widely used because of its ability to retain a differentiated phenotype under culturing conditions (22). During apoptosis, cells flip their plasmalemma PS from the cytoplasmic to the external surface. PS is, in fact, a recognition molecule for macrophages whose role is to clear apoptotic cells (23). However, PS is also a potent calcium-binding phospholipid, able to induce the formation of calcium phosphate crystal nuclei in mineralizing tissues (4). The proliferation studies confirmed the inhibitory effect of genistein and clearly showed the exposure of the calcium-binding phospholipid PS on the plasmalemma external surface. Therefore, these combined data suggested that genistein could induce cell apoptosis. However, a complete apoptotic process, with DNA fragmentation, could be observed only with relatively high concentrations of genistein. Indeed, TEM studies revealed no DNA condensation below these concentrations. Furthermore, electron microscopy clearly showed the formation of matrix vesicles stemming from cell surfaces. These vesicles were not detected on control osteoblasts, suggesting that genistein is able to stimulate the formation of these structures, which are key for bone mineralization. We speculated that the exposure of PS detected was not caused by apoptosis, but rather by the exocytosis of membrane vesicles (4). These vesicles acted as potent calcification centers in both nonosteogenic and osteogenic media. This shows that the biochemical pathway induced by genistein to produce membrane vesicles is independent of any effect of the addition of ß-glycerophosphate, an inducer of calcification, to the medium. Unpublished studies of genistein localization in osteoblasts seem to indicate that, when present at relatively low concentrations, genistein can bind plasmalemma, whereas it condenses in the proximity of the nucleus at higher concentrations, while being tightly associated with the matrix vesicle exocytosis process.

The osteogenetic potential of genistein was shown not to be limited to the formation of matrix vesicles. Previous studies demonstrated that genistein and daidzein can induce osteoblast ALP activity and, in general, their differentiation (15–19). In particular, a stimulatory effect of daidzein and genistein on osteoblastic MC3T3-E1 cells in a range of concentration from 10–6 to 10–5 mol/L was demonstrated (19). Conversely, the present study was performed on MG63 osteoblast-like cells at a partially overlapping (but relatively higher) range of concentration (2.5 x 10–6 to 3 x 10–5 mol/L) and shows a reduction of the ALP-specific activity (mU/g of protein). Therefore, the effect of genistein on osteoblast ALP activity in vitro seems to depend on the cell type and culturing conditions (proliferative or confluent) (19), on the concentration range considered, as well as on the way data are expressed. Indeed, in this study, although the expression of ALP data as an enzyme-specific activity suggested an inhibition of osteoblast differentiation by genistein, it was clear that the lower values were due to the relatively high protein content synthesized by the genistein-treated cells rather than a lower enzyme synthesis. However, an induction of ALP activity by genistein becomes apparent when data are considered on a per cell basis because this isoflavone reduces cell proliferation.

Elevated protein synthesis in osteoblasts incubated in genistein, combined with SEM showing secretion of collagen-like fibrils, prompted a specific study on collagen synthesis by osteoblasts under stimulus with genistein. The protein synthesis data confirmed previous studies carried out under nonosteogenic conditions (19) and offered a better understanding of genistein's potential to induce collagen synthesis by osteoblasts. Indeed, these studies showed that the high protein concentrations corresponded to significantly higher levels of collagen, but only in cells grown under nonosteogenic conditions. Because SEM revealed the formation of collagen fibrils around genistein-stimulated osteoblasts, these data may suggest that the in vivo release of the matrix vesicles induced by genistein takes place in a collagen-rich extracellular matrix, thus creating the ideal extracellular environment for rapid tissue mineralization. This hypothesis was corroborated by alizarin red (data not shown) and fluorescent staining, which showed the formation of needle-like apatite crystals in genistein-treated osteoblasts.

The data herein confirm the findings of others on another important soybean isoflavone, daidzein (19). More importantly, rationalized study of the different parameters leading to bone formation seems to demonstrate that the soybean isoflavone genistein can induce bone formation through a very specific and coordinated cell activation pathway. Genistein switches the osteoblasts toward a more differentiated phenotype that is able to synthesize all of the main biochemical components required for the production of a new mineralized extracellular matrix. These effects, combined with the ability of genistein to inhibit macrophage (7) and osteoclast activity (15,16), would shift the metabolic balance toward the deposition of new tissue, thus preventing loss of bone mass and osteoporosis.


   ACKNOWLEDGMENTS
 
The authors are grateful to Mr. Michael Helias and Dr. Gary Phillips for their scanning electron microscopy technical support.


   FOOTNOTES
 
1 Supported by an EC grant contract n. NMP3-CT-2005-013912. Back

3 Abbreviations used: ALP, alkaline phosphatase; PS, phosphatidylserine; SEM, scanning electron microscopy; TEM, transmission electron microscopy. Back

Manuscript received 20 October 2005. Initial review completed 1 December 2005. Revision accepted 14 February 2006.


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