BACKGROUND
osteosarcoma occurs mainly in the metaphyseal region of the long bones of young people.
the most common sites affected are the distal femur, the proximal tibia and the proximal humerus  <cit> .
osteosarcoma grows aggressively at the primary site and often develops micrometastases in the lung prior to diagnosis.
the primary treatment of osteosarcoma is the complete removal of tumor by wide excision and aggressive adjuvant chemotherapy  <cit> .
despite progress in chemotherapy, the development of metastatic tumors in the lung often has a fatal outcome  <cit> .
therefore, the prevention of pulmonary metastases during the early stage of tumor development is critical for the improvement of the prognosis of osteosarcoma patients.
metastasis is composed of multiple sequential steps, including cell detachment from the primary tumor, cell motility and invasion, angiogenesis, intravasation into blood, and extravasation into distant organs.
a number of factors, such as mmp- <dig> and vascular endothelial growth factor   <cit> , are involved in tumor growth, invasion, and metastasis.
an lm <dig> osteosarcoma cell line with high metastatic potential to the lung was established from murine dunn osteosarcoma cells, which did not develop pulmonary metastasis when implanted subcutaneously  into the backs of c3h mice  <cit> .
lm <dig> cells have been used as an excellent tool for studying inhibitory agents against pulmonary metastasis  <cit> .
previously, we reported that treatment of nude mice implanted s.c. with lm <dig> cells into the backs with troglitazone, a peroxisome proliferator-activated receptor-γ  ligand, decreased the expression of mmp- <dig> and vegf within the primary tumor and inhibited the development of pulmonary metastasis  <cit> .
soy contains phytoestrogen isoflavones .
in japan, soy is widely consumed in a wide variety of forms, such as non-fermented foods  and fermented foods .
an epidemiological study has shown that consumption of miso soup is inversely associated with the risk of breast cancer  <cit> .
several experimental studies have shown that genistein inhibits the growth of tumors  <cit> .
in the present study, we performed in vitro experiments to analyze the effect of genistein on the growth, invasion, and motility of lm <dig> cells.
since β-catenin is associated with tumor cell growth, invasion, motility, and metastasis  <cit> , the effect of genistein on the cellular level and subcellular localization of β-catenin was also examined.
RESULTS
effect of genistein on cell proliferation and dna replication
we first evaluated the anti-proliferative effect of genistein against lm <dig> cells.
for this, lm <dig> cells were treated for  <dig> days with genistein at the indicated concentrations and the dna content of the cultures was measured.
the untreated cultures  contained  <dig>  μg/35-mm plate of dna.
genistein dose-dependently decreased the dna content of the cultures .
genistein at  <dig> μm decreased the dna content by 91%.
figure 1b shows the time course of the genistein-induced changes in the dna content.
in the untreated and genistein-treated cultures, the dna content increased during the 3-day treatment period.
on day  <dig>  there was no difference in the dna content between the two cultures.
on days  <dig> and  <dig>  the dna content of the genistein-treated cultures was significantly lower than that of the untreated cultures.
lm <dig> cells were incubated with 5-bromo-2’-deoxyuridine  during the last  <dig> min of the 3-day treatment period to label dna synthesis .
in the untreated and genistein-treated cultures, positive brdu immunofluorescence staining was observed in the nucleus.
the brdu-labeling index of the genistein-treated cultures  was significantly  lower than that of the untreated cultures .
to examine the effect of genistein on cell viability, the trypan blue exclusion test was performed .
in the untreated and genistein-treated cultures, cells that attached to the bottom of the plates excluded trypan blue.
effect of genistein on subcellular localization and cellular level of β-catenin
the subcellular localization of β-catenin was examined by immunofluorescence.
in the untreated cultures, positive β-catenin immunofluorescence staining was observed in the cytoplasm and/or nucleus and was not observed at the plasma membrane .
in the genistein-treated cultures, positive β-catenin immunofluorescence staining shifted in localization near to the plasma membrane and was not observed in the nucleus .
the cellular level of β-catenin was examined by western blot.
the intensity of the band corresponding to β-catenin was stronger in the genistein-treated cultures than in the untreated cultures , indicating that genistein increased the cellular level of β-catenin.
effect of genistein on invasion, motility, mmp- <dig>  and alkaline phosphatase
lm <dig> cells, which had been treated for  <dig> days without or with  <dig> μm genistein, were harvested by trypsinization, and cell invasion and cell motility assays were performed.
when experiments were performed using matrigel-coated membranes, the absorbance of the dye extracted from genistein-treated cells was 24% of that extracted from untreated cells .
this indicates that genistein-treated cells were less invasive than untreated cells.
when experiments were performed using uncoated membranes, the absorbance of the dye extracted from genistein-treated cells was 40% of that extracted from untreated cells .
this indicates that genistein-treated cells were less motile than untreated cells.
since mmp- <dig> degrades type iv collagen, which is present in vascular and subepithelial basement membranes, and plays a pivotal role in cell invasion  <cit> , the expression of mmp- <dig> was examined by immunohistochemical staining .
in both untreated and genistein-treated cells, positive mmp- <dig> immunostaining was observed in the cytoplasm.
the intensity of immunostaining was weaker in genistein-treated cells than in untreated cells, thus indicating that genistein-treated cells expressed less mmp- <dig> than untreated cells.
the activity of mmp- <dig> secreted into conditioned media during the last  <dig> h of the 3-day treatment period was assayed by gelatin zymography .
mmp- <dig> was secreted from both untreated and genistein-treated cells; however, the activity of mmp- <dig> secreted from genistein-treated cells was lower than that secreted from untreated cells.
low mmp- <dig> activity was detected in conditioned media from untreated cells, but not genistein-treated cells.
the alp activity in lm <dig> cells is higher than that in dunn cells  <cit> , suggesting that the alp activity in osteosarcoma cells may be associated with metastatic potential.
we treated lm <dig> cells without or with  <dig> μm genistein for  <dig> days and measured cellular alp activity .
the alp activity in genistein-treated cells  was significantly lower than that in untreated cells .
does genistein induce the differentiation of lm <dig> cells?
we stained ethanol-fixed lm <dig> cells with hematoxylin-eosin  and observed cell morphology.
in the untreated cultures, the majority of cells  were cuboidal in shape with filopodial and lamellipodial structures surrounding the cell surface .
these cells grew to form numerous multilayer masses even at low cell density .
the genistein-treated cultures contained two morphologically different cell types; one was cuboidal in shape and the other was spindle-shaped, which became more flattened with clear cell-cell margins .
the ratio of the number of spindle-shaped cells to the total number of cells was  <dig>  ±  <dig> %.
multilayered cells were markedly decreased in the genistein-treated cultures.
these findings suggest that genistein may induce the differentiation of lm <dig> cells.
to confirm this, we examined the expression of mrnas of osteocalcin, which is a marker of mature osteoblasts  <cit> , and differentiation-related gene- <dig> , which is associated with the induction of differentiation in tumors  <cit> , by reverse transcription-polymerase chain reaction  .
untreated cells expressed a low level of osteocalcin mrna, while genistein-treated cells expressed a markedly high level of osteocalcin mrna.
untreated cells expressed a significant level of drg- <dig> mrna.
the level of drg- <dig> mrna in genistein-treated cells was similar to that in untreated cells.
the subcellular localization of drg- <dig> was examined by immunohistochemical staining.
in untreated cells, positive drg- <dig> immunostaining was observed in the cytoplasm , while in genistein-treated cells, it was mostly observed in the nucleus .
discussion
since genistein has been reported to inhibit tumor growth  <cit> , we first examined the effect of genistein on the proliferation of lm <dig> cells.
the results of brdu incorporation into dna and dna measurement revealed that genistein inhibited dna replication and cell proliferation in cultures of lm <dig> cells .
of course, there is concern that this inhibition could be due to a cytotoxic effect of genistein on lm <dig> cells.
however, this does not appear to be the case because cells did not detach from the bottom of the plates during the 3-day treatment period.
moreover, the results of the trypan blue exclusion test showed that genistein-treated cells that attached to the bottom of the plates were viable cells .
genistein at a higher concentration  also inhibited the proliferation of lm <dig> cells without affecting cell viability .
these findings indicate that genistein-induced inhibition of cell proliferation was not due to a cytotoxic effect.
there are reports on the effect of genistein on the proliferation of normal cells.
in cultures of 3t3-l <dig> fibroblasts, genistein at  <dig> and  <dig> μm inhibits cell proliferation  <cit> .
in cultures of endothelial cells, genistein at lower concentrations  stimulates cell proliferation, while at a higher concentration  this chemical inhibits it  <cit> .
when lm <dig> cells are implanted s.c. into the backs of mice, a 100% pulmonary metastatic rate is observed within  <dig> weeks  <cit> .
the initial step of metastasis is cell detachment from the primary tumor.
since β-catenin is essential for cadherin-mediated cell-cell adhesion, reduced expression of β-catenin and cadherins at the cell surface is associated with tumor metastasis  <cit> .
loss of membranous β-catenin occurs commonly in primary colorectal cancers with metastatic potential and in the corresponding colorectal liver metastases  <cit> .
our results of immunofluorescence showed loss of membranous β-catenin in lm <dig> cells , indicating that cell-cell adhesiveness may be reduced in lm <dig> cells.
genistein affects the subcellular localization and expression level of β-catenin in normal cells.
for example, treatment of mouse hc <dig> mammary epithelial cells with genistein increases membranous β-catenin  <cit> .
in mammary glands of young female rats fed a genistein-containing diet, β-catenin is predominantly localized to ductal epithelial cell membranes  <cit>  and the level of this protein is higher compared with those fed a control diet  <cit> .
therefore, we examined whether genistein affects the subcellular localization and cellular level of β-catenin in lm <dig> cells.
in genistein-treated cells, immunofluorescence staining of β-catenin was observed near the plasma membrane .
the results of western blot showed that genistein-treated cells contained higher levels of β-catenin than untreated cells .
these findings suggest that genistein may promote intracellular adhesion of lm <dig> cells.
this is the first report to focus on the subcellular localization and cellular level of β-catenin following genistein treatment in osteosarcoma cells.
the next step of metastasis is invasion of tumor cells into basement membranes, which involves distinct events, such as cell motility and mmp expression.
invasive tumors exhibit active migration and high levels of mmps  <cit> .
lm <dig> cells have been reported to exhibit higher invasive potential, higher mmp- <dig> activity, and higher alp activity than dunn cells with no metastatic potential  <cit> .
in patients with osteosarcoma, high levels of alp activity in serum are associated with a poor clinical outcome  <cit> .
in the present study, we found that genistein-treated cells were less invasive and less motile , exhibited lower expression and secretion of mmp- <dig> , and exhibited lower alp activity  compared with untreated cells.
taken together, our findings suggest that genistein-treated cells might lose metastatic potential.
when β-catenin is translocated to the nucleus, it activates target genes, such as cyclin d <dig> and c-myc, and promotes the growth of tumor cells  <cit> .
in intestinal-type gastric cancer, β-catenin in the nucleus not only plays a role in early tumor growth, but it also functions in initiation of invasive processes  <cit> .
in osteosarcoma that has metastasized to the lung, β-catenin is detected in the cytoplasm and/or nucleus, whereas in primary osteosarcoma that has not metastasized for more than five years, it is not detected in the cytoplasm and/or nucleus  <cit> , indicating that the presence of β-catenin in the cytoplasm and/or nucleus is a marker of metastatic potential of osteosarcoma to the lung.
our results of immunofluorescence showed that β-catenin was present in the cytoplasm and/or nucleus of lm <dig> cells  and that after treatment of lm <dig> cells with genistein, β-catenin was translocated near to the plasma membrane and was not found in the nucleus .
this translocation of β-catenin may result in decreases in proliferative rate and invasive potential of genistein-treated cells.
a similar translocation of β-catenin from the nucleus to the plasma membrane has been reported in human ht- <dig> colon cancer cells treated with thiazolidinedione, a pparγ ligand  <cit> .
treatment of lm <dig> cells with genistein induced morphological changes  and markedly increased the level of osteocalcin mrna .
moreover, this treatment markedly decreased the formation of multilayer masses , suggesting that genistein-treated cells grew with contact inhibition.
contact inhibition is a characteristic of normal cells grown on plastic culture plates, which is lost in cancerous cells  <cit> .
on the basis of these findings, we concluded that genistein induced the differentiation of lm <dig> cells.
there are a few reports on the association of genistein with cell differentiation.
treatment of mouse b <dig> melanoma cells with genistein induces morphological changes characteristic of differentiation, such as enlargement of the soma and nuclei together with the formation of dendritic-like structures  <cit> .
treatment of human mg  <dig> osteosarcoma osteoblasts with genistein increases the level of collagen and stimulates the formation of calcification foci  <cit> .
drg- <dig> is expressed at lower levels in tumors than in normal tissue  <cit> .
overexpression of drg- <dig> gene in human sw <dig> metastatic colon cancer cells induces morphological changes that are similar to differentiation-specific changes induced by known differentiation reagents such as tributyrin and a ligand of retinoid x receptor lg <dig>  <cit> , thus indicating that drg- <dig> plays a role in differentiation of tumor cells.
however, there is a contradictory report on the involvement of drg- <dig> in cell differentiation.
human hepatocellular carcinomas express a higher level of drg- <dig> than non-tumor liver or cirrhotic and benign liver lesions  <cit> .
moreover, hepatocellular carcinomas that are moderately and poorly differentiated express a higher level of drg- <dig> than those that are well differentiated  <cit> .
the positive or negative association of drg- <dig> with cell differentiation depends on tumor types.
therefore, we examined the effect of genistein on the expression of drg- <dig> mrna by rt-pcr.
lm <dig> cells significantly expressed drg- <dig> mrna.
genistein did not increase the expression of drg- <dig> mrna in lm <dig> cells .
thus, the expression of drg- <dig> in lm <dig> cells appears not to be associated with the induction of cell differentiation.
drg- <dig> is located in the cytoplasm of human ej bladder carcinoma cells  <cit> , human hepatocellular carcinoma cells  <cit> , and human prostate cancer cells  <cit> .
our results of immunohistochemical staining also reveal that drg- <dig> in lm <dig> cells was located in the cytoplasm .
after treatment of lm <dig> cells with genistein, drg- <dig> mostly shifted in localization from the cytoplasm to the nucleus .
a study with human u2os osteosarcoma cells expressing drg- <dig> small interfering rna showed that drg- <dig> is associated with the production of osteocalcin  <cit> .
taken together, our findings suggest that drg- <dig> in the nucleus of genistein-treated cells may play a role in the expression of osteocalcin mrna.
in postmenopausal japanese women  <cit>  and asian-american women  <cit> , the risk of breast cancer is inversely associated with isoflavone intake during adult life.
asian-american women who are high consumers of isoflavone  show a significantly reduced risk of breast cancer compared with those who are low consumers   <cit> .
however, isoflavones at the levels commonly consumed by non-asian us women  have little effect on breast cancer risk  <cit> .
in mice implanted with human 253j b-v bladder cancer cells, dietary genistin , the natural form of genistein in soy, inhibits tumor growth at the primary site and pulmonary metastasis  <cit> .
in mice implanted with human pc3-m prostate cancer cells, dietary genistein  inhibits pulmonary metastasis without affecting tumor growth at the primary site  <cit> .
CONCLUSIONS
genistein induced cell differentiation and translocated β-catenin near to the plasma membrane, resulting in lowering of the proliferative rate and invasive potential of lm <dig> cells.
genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects.

 osteosarcoma occurs mainly in the metaphyseal region of the long bones of young people. the most common sites affected are the distal femur, the proximal tibia and the proximal humerus. osteosarcoma grows aggressively at the primary site and often develops micrometastases in the lung prior to diagnosis. the primary treatment of osteosarcoma is the complete removal of tumor by wide excision and aggressive adjuvant chemotherapy. despite progress in chemotherapy, the development of metastatic tumors in the lung often has a fatal outcome. therefore, the prevention of pulmonary metastases during the early stage of tumor development is critical for the improvement of the prognosis of osteosarcoma patients. metastasis is composed of multiple sequential steps, including cell detachment from the primary tumor, cell motility and invasion, angiogenesis, intravasation into blood, and extravasation into distant organs. a number of factors, such as mmp- and vascular endothelial growth factor, are involved in tumor growth, invasion, and metastasis. an lm osteosarcoma cell line with high metastatic potential to the lung was established from murine dunn osteosarcoma cells, which did not develop pulmonary metastasis when implanted subcutaneously into the backs of c3h mice. lm cells have been used as an excellent tool for studying inhibitory agents against pulmonary metastasis. previously, we reported that treatment of nude mice implanted s.c. with lm cells into the backs with troglitazone, a peroxisome proliferator-activated receptor-γ ligand, decreased the expression of mmp- and vegf within the primary tumor and inhibited the development of pulmonary metastasis. soy contains phytoestrogen isoflavones. in japan, soy is widely consumed in a wide variety of forms, such as non-fermented foods and fermented foods. an epidemiological study has shown that consumption of miso soup is inversely associated with the risk of breast cancer. several experimental studies have shown that genistein inhibits the growth of tumors. in the present study, we performed in vitro experiments to analyze the effect of genistein on the growth, invasion, and motility of lm cells. since β-catenin is associated with tumor cell growth, invasion, motility, and metastasis, the effect of genistein on the cellular level and subcellular localization of β-catenin was also examined.  effect of genistein on cell proliferation and dna replication we first evaluated the anti-proliferative effect of genistein against lm cells. for this, lm cells were treated for days with genistein at the indicated concentrations and the dna content of the cultures was measured. the untreated cultures contained μg/35-mm plate of dna. genistein dose-dependently decreased the dna content of the cultures. genistein at μm decreased the dna content by 91%. figure 1b shows the time course of the genistein-induced changes in the dna content. in the untreated and genistein-treated cultures, the dna content increased during the 3-day treatment period. on day there was no difference in the dna content between the two cultures. on days and the dna content of the genistein-treated cultures was significantly lower than that of the untreated cultures. lm cells were incubated with 5-bromo-2’-deoxyuridine during the last min of the 3-day treatment period to label dna synthesis. in the untreated and genistein-treated cultures, positive brdu immunofluorescence staining was observed in the nucleus. the brdu-labeling index of the genistein-treated cultures was significantly lower than that of the untreated cultures. to examine the effect of genistein on cell viability, the trypan blue exclusion test was performed. in the untreated and genistein-treated cultures, cells that attached to the bottom of the plates excluded trypan blue. effect of genistein on subcellular localization and cellular level of β-catenin the subcellular localization of β-catenin was examined by immunofluorescence. in the untreated cultures, positive β-catenin immunofluorescence staining was observed in the cytoplasm and/or nucleus and was not observed at the plasma membrane. in the genistein-treated cultures, positive β-catenin immunofluorescence staining shifted in localization near to the plasma membrane and was not observed in the nucleus. the cellular level of β-catenin was examined by western blot. the intensity of the band corresponding to β-catenin was stronger in the genistein-treated cultures than in the untreated cultures, indicating that genistein increased the cellular level of β-catenin. effect of genistein on invasion, motility, mmp- and alkaline phosphatase lm cells, which had been treated for days without or with μm genistein, were harvested by trypsinization, and cell invasion and cell motility assays were performed. when experiments were performed using matrigel-coated membranes, the absorbance of the dye extracted from genistein-treated cells was 24% of that extracted from untreated cells. this indicates that genistein-treated cells were less invasive than untreated cells. when experiments were performed using uncoated membranes, the absorbance of the dye extracted from genistein-treated cells was 40% of that extracted from untreated cells. this indicates that genistein-treated cells were less motile than untreated cells. since mmp- degrades type iv collagen, which is present in vascular and subepithelial basement membranes, and plays a pivotal role in cell invasion, the expression of mmp- was examined by immunohistochemical staining. in both untreated and genistein-treated cells, positive mmp- immunostaining was observed in the cytoplasm. the intensity of immunostaining was weaker in genistein-treated cells than in untreated cells, thus indicating that genistein-treated cells expressed less mmp- than untreated cells. the activity of mmp- secreted into conditioned media during the last h of the 3-day treatment period was assayed by gelatin zymography. mmp- was secreted from both untreated and genistein-treated cells; however, the activity of mmp- secreted from genistein-treated cells was lower than that secreted from untreated cells. low mmp- activity was detected in conditioned media from untreated cells, but not genistein-treated cells. the alp activity in lm cells is higher than that in dunn cells, suggesting that the alp activity in osteosarcoma cells may be associated with metastatic potential. we treated lm cells without or with μm genistein for days and measured cellular alp activity. the alp activity in genistein-treated cells was significantly lower than that in untreated cells. does genistein induce the differentiation of lm cells? we stained ethanol-fixed lm cells with hematoxylin-eosin and observed cell morphology. in the untreated cultures, the majority of cells were cuboidal in shape with filopodial and lamellipodial structures surrounding the cell surface. these cells grew to form numerous multilayer masses even at low cell density. the genistein-treated cultures contained two morphologically different cell types; one was cuboidal in shape and the other was spindle-shaped, which became more flattened with clear cell-cell margins. the ratio of the number of spindle-shaped cells to the total number of cells was  ±  %. multilayered cells were markedly decreased in the genistein-treated cultures. these findings suggest that genistein may induce the differentiation of lm cells. to confirm this, we examined the expression of mrnas of osteocalcin, which is a marker of mature osteoblasts, and differentiation-related gene-, which is associated with the induction of differentiation in tumors, by reverse transcription-polymerase chain reaction. untreated cells expressed a low level of osteocalcin mrna, while genistein-treated cells expressed a markedly high level of osteocalcin mrna. untreated cells expressed a significant level of drg- mrna. the level of drg- mrna in genistein-treated cells was similar to that in untreated cells. the subcellular localization of drg- was examined by immunohistochemical staining. in untreated cells, positive drg- immunostaining was observed in the cytoplasm, while in genistein-treated cells, it was mostly observed in the nucleus. discussion since genistein has been reported to inhibit tumor growth, we first examined the effect of genistein on the proliferation of lm cells. the results of brdu incorporation into dna and dna measurement revealed that genistein inhibited dna replication and cell proliferation in cultures of lm cells. of course, there is concern that this inhibition could be due to a cytotoxic effect of genistein on lm cells. however, this does not appear to be the case because cells did not detach from the bottom of the plates during the 3-day treatment period. moreover, the results of the trypan blue exclusion test showed that genistein-treated cells that attached to the bottom of the plates were viable cells. genistein at a higher concentration also inhibited the proliferation of lm cells without affecting cell viability. these findings indicate that genistein-induced inhibition of cell proliferation was not due to a cytotoxic effect. there are reports on the effect of genistein on the proliferation of normal cells. in cultures of 3t3-l fibroblasts, genistein at and μm inhibits cell proliferation. in cultures of endothelial cells, genistein at lower concentrations stimulates cell proliferation, while at a higher concentration this chemical inhibits it. when lm cells are implanted s.c. into the backs of mice, a 100% pulmonary metastatic rate is observed within weeks. the initial step of metastasis is cell detachment from the primary tumor. since β-catenin is essential for cadherin-mediated cell-cell adhesion, reduced expression of β-catenin and cadherins at the cell surface is associated with tumor metastasis. loss of membranous β-catenin occurs commonly in primary colorectal cancers with metastatic potential and in the corresponding colorectal liver metastases. our results of immunofluorescence showed loss of membranous β-catenin in lm cells, indicating that cell-cell adhesiveness may be reduced in lm cells. genistein affects the subcellular localization and expression level of β-catenin in normal cells. for example, treatment of mouse hc mammary epithelial cells with genistein increases membranous β-catenin. in mammary glands of young female rats fed a genistein-containing diet, β-catenin is predominantly localized to ductal epithelial cell membranes and the level of this protein is higher compared with those fed a control diet. therefore, we examined whether genistein affects the subcellular localization and cellular level of β-catenin in lm cells. in genistein-treated cells, immunofluorescence staining of β-catenin was observed near the plasma membrane. the results of western blot showed that genistein-treated cells contained higher levels of β-catenin than untreated cells. these findings suggest that genistein may promote intracellular adhesion of lm cells. this is the first report to focus on the subcellular localization and cellular level of β-catenin following genistein treatment in osteosarcoma cells. the next step of metastasis is invasion of tumor cells into basement membranes, which involves distinct events, such as cell motility and mmp expression. invasive tumors exhibit active migration and high levels of mmps. lm cells have been reported to exhibit higher invasive potential, higher mmp- activity, and higher alp activity than dunn cells with no metastatic potential. in patients with osteosarcoma, high levels of alp activity in serum are associated with a poor clinical outcome. in the present study, we found that genistein-treated cells were less invasive and less motile, exhibited lower expression and secretion of mmp-, and exhibited lower alp activity compared with untreated cells. taken together, our findings suggest that genistein-treated cells might lose metastatic potential. when β-catenin is translocated to the nucleus, it activates target genes, such as cyclin d and c-myc, and promotes the growth of tumor cells. in intestinal-type gastric cancer, β-catenin in the nucleus not only plays a role in early tumor growth, but it also functions in initiation of invasive processes. in osteosarcoma that has metastasized to the lung, β-catenin is detected in the cytoplasm and/or nucleus, whereas in primary osteosarcoma that has not metastasized for more than five years, it is not detected in the cytoplasm and/or nucleus, indicating that the presence of β-catenin in the cytoplasm and/or nucleus is a marker of metastatic potential of osteosarcoma to the lung. our results of immunofluorescence showed that β-catenin was present in the cytoplasm and/or nucleus of lm cells and that after treatment of lm cells with genistein, β-catenin was translocated near to the plasma membrane and was not found in the nucleus. this translocation of β-catenin may result in decreases in proliferative rate and invasive potential of genistein-treated cells. a similar translocation of β-catenin from the nucleus to the plasma membrane has been reported in human ht- colon cancer cells treated with thiazolidinedione, a pparγ ligand. treatment of lm cells with genistein induced morphological changes and markedly increased the level of osteocalcin mrna. moreover, this treatment markedly decreased the formation of multilayer masses, suggesting that genistein-treated cells grew with contact inhibition. contact inhibition is a characteristic of normal cells grown on plastic culture plates, which is lost in cancerous cells. on the basis of these findings, we concluded that genistein induced the differentiation of lm cells. there are a few reports on the association of genistein with cell differentiation. treatment of mouse b melanoma cells with genistein induces morphological changes characteristic of differentiation, such as enlargement of the soma and nuclei together with the formation of dendritic-like structures. treatment of human mg osteosarcoma osteoblasts with genistein increases the level of collagen and stimulates the formation of calcification foci. drg- is expressed at lower levels in tumors than in normal tissue. overexpression of drg- gene in human sw metastatic colon cancer cells induces morphological changes that are similar to differentiation-specific changes induced by known differentiation reagents such as tributyrin and a ligand of retinoid x receptor lg, thus indicating that drg- plays a role in differentiation of tumor cells. however, there is a contradictory report on the involvement of drg- in cell differentiation. human hepatocellular carcinomas express a higher level of drg- than non-tumor liver or cirrhotic and benign liver lesions. moreover, hepatocellular carcinomas that are moderately and poorly differentiated express a higher level of drg- than those that are well differentiated. the positive or negative association of drg- with cell differentiation depends on tumor types. therefore, we examined the effect of genistein on the expression of drg- mrna by rt-pcr. lm cells significantly expressed drg- mrna. genistein did not increase the expression of drg- mrna in lm cells. thus, the expression of drg- in lm cells appears not to be associated with the induction of cell differentiation. drg- is located in the cytoplasm of human ej bladder carcinoma cells, human hepatocellular carcinoma cells, and human prostate cancer cells. our results of immunohistochemical staining also reveal that drg- in lm cells was located in the cytoplasm. after treatment of lm cells with genistein, drg- mostly shifted in localization from the cytoplasm to the nucleus. a study with human u2os osteosarcoma cells expressing drg- small interfering rna showed that drg- is associated with the production of osteocalcin. taken together, our findings suggest that drg- in the nucleus of genistein-treated cells may play a role in the expression of osteocalcin mrna. in postmenopausal japanese women and asian-american women, the risk of breast cancer is inversely associated with isoflavone intake during adult life. asian-american women who are high consumers of isoflavone show a significantly reduced risk of breast cancer compared with those who are low consumers. however, isoflavones at the levels commonly consumed by non-asian us women have little effect on breast cancer risk. in mice implanted with human 253j b-v bladder cancer cells, dietary genistin, the natural form of genistein in soy, inhibits tumor growth at the primary site and pulmonary metastasis. in mice implanted with human pc3-m prostate cancer cells, dietary genistein inhibits pulmonary metastasis without affecting tumor growth at the primary site.  genistein induced cell differentiation and translocated β-catenin near to the plasma membrane, resulting in lowering of the proliferative rate and invasive potential of lm cells. genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects. 
the expression of osteocalcin mrna was determined by reverse transcription-polymerase chain reaction. treatment of lm cells with genistein induced morphological changes, markedly decreased the formation of multilayer masses of cells, and markedly increased the expression of osteocalcin mrna. β-catenin in untreated cells was located in the cytoplasm and/or nucleus, while in genistein-treated cells it was translocated near to the plasma membrane. the purpose of this study is to investigate the effect of genistein, an isoflavone found in soy, on the invasive and motile potential of osteosarcoma cells. in japan, soy is consumed in a wide variety of forms, such as miso soup and soy sauce. the subcellular localization and cellular level of β-catenin were determined by immunofluorescence and western blot. therefore, the prevention of pulmonary metastases during the early stage of tumor development is critical for the improvement of the prognosis of osteosarcoma patients. the effect of genistein on cell proliferation was determined by dna measurement in the cultures and 5-bromo-2’-deoxyuridine incorporation study. genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects. the assays of cell invasion and motility were performed using the cell culture inserts with either matrigel-coated membranes or uncoated membranes in the invasion chambers. genistein decreased invasive and motile potential by inducing cell differentiation in lm cells. for examining cell morphology, the ethanol-fixed cells were stained with hematoxylin-eosin. the expression and secretion of mmp- were determined by immunohistochemistry and gelatin zymography.
 one of the problems associated with osteosarcoma is the frequent formation of micrometastases in the lung prior to diagnosis because the development of metastatic lesions often causes a fatal outcome. therefore, the prevention of pulmonary metastases during the early stage of tumor development is critical for the improvement of the prognosis of osteosarcoma patients. in japan, soy is consumed in a wide variety of forms, such as miso soup and soy sauce. the purpose of this study is to investigate the effect of genistein, an isoflavone found in soy, on the invasive and motile potential of osteosarcoma cells. methods lm cells were treated for days with various concentrations of genistein. the effect of genistein on cell proliferation was determined by dna measurement in the cultures and 5-bromo-2’-deoxyuridine incorporation study. the assays of cell invasion and motility were performed using the cell culture inserts with either matrigel-coated membranes or uncoated membranes in the invasion chambers. the expression and secretion of mmp- were determined by immunohistochemistry and gelatin zymography. the subcellular localization and cellular level of β-catenin were determined by immunofluorescence and western blot. for examining cell morphology, the ethanol-fixed cells were stained with hematoxylin-eosin. the expression of osteocalcin mrna was determined by reverse transcription-polymerase chain reaction.  genistein dose-dependently inhibits cell proliferation. genistein-treated cells were less invasive and less motile than untreated cells. the expression and secretion of mmp- were lower in the genistein-treated cultures than in the untreated cultures. β-catenin in untreated cells was located in the cytoplasm and/or nucleus, while in genistein-treated cells it was translocated near to the plasma membrane. the level of β-catenin was higher in genistein-treated cells than in untreated cells. treatment of lm cells with genistein induced morphological changes, markedly decreased the formation of multilayer masses of cells, and markedly increased the expression of osteocalcin mrna.  genistein decreased invasive and motile potential by inducing cell differentiation in lm cells. genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects. genisteinlm8cell invasionmatrix metalloproteinase-2β-catenincell differentiation 