The purpose of this study was to investigate in vivo whether the level of cytoplasmic β-catenin in LM8 cells affected metastatic potential. To this end, we first examined whether untreated and genistein-treated LM8 cells metastasized to the distant organs in nude mice because genistein-treated LM8 cells expressed higher levels of cytoplasmic β-catenin than untreated LM8 cells (Figure 1). In the control group, primary tumor cells formed metastatic lesions in the lung and/or liver of all nude mice (Table 1). This is compatible with the previous reports stating that LM8 cells show an extremely high incidence of pulmonary metastasis in mice [9–12]. In the genistein group, primary tumor cells did not form metastatic lesions in the lung of all nude mice and the liver of 85.7% of nude mice (Table 1). This finding indicates that a majority of primary tumor cells in the genistein group lost metastatic potential.
Next, we performed immunohistochemical staining of β-catenin within the primary tumor. In the control group, 53% of tumor cells within the primary tumor were β-catenin-negative (Figure 4C), and the remaining 47% were β-catenin-positive but the intensity of immunostaining was weak or intermediate (Figure 4A-a). In the genistein/metastasis(-) subgroup, 82% of tumor cells within the primary tumor were β-catenin-positive (Figure 4C) and the intensity of immunostaining was stronger compared with the control group (Figure 4A-c). The results of β-catenin-labeling score showed that primary tumor cells in the genistein/metastasis(-) subgroup contained 1.9-times higher level of cytoplasmic β-catenin than those in the control group (Figure 4D). Based on these findings, we concluded that overexpression of cytoplasmic β-catenin in LM8 cells caused loss of metastatic potential to the lung and liver. Kashima et al. introduced N-cadherin and cadherin-11 cDNAs into LM8 cells, in which there was little endogenous expression of these two cadherins, to investigate the role of the cadherins in osteosarcoma metastasis in vivo. They found that the primary tumor of C3H mice injected with cadherin-transfected LM8 cells contained higher levels of cadherins compared with those injected with control, empty vector-transfected LM8 cells and that a high number of metastatic lesions were present in the lung of the latter mice, whereas there was a marked reduction in pulmonary metastases in the former mice. Based on these findings, they concluded that overexpression of cadherins attenuated the ability of LM8 cells to form pulmonary metastases.
Asai et al.  reported that subcutaneous inoculation of LM8 cells into the backs of C3H mice caused the rapid growth of tumor cells at the inoculation site and the formation of multiple metastatic nodules at the surface of the lung, and both the engraftment rate of tumor cells and metastatic incidence were 100%. The present study confirms this (Table 1). However, genistein-treated LM8 cells inoculated into the backs of C3H mice did not grow at the inoculation site and did not form metastatic nodules at the surface of the lung and liver (Table 1). Even in nude mice, the engraftment rate of the genistein group did not reach 100% (Table 1). Moreover, the metastatic incidence of this group was only 14.3%. These findings indicate that the malignancy of genistein-treated LM8 cells may be low. Since a majority of primary tumor cells in the genistein group was β-catenin-positive (Figure 4C), the present findings suggest that high expression of β-catenin within the primary tumor is associated with low malignancy of tumor cells. In human endometrial carcinoma, positive β-catenin expression has been reported to be associated with decreases in the stage and grade of the tumor [21, 22]. Athanassiadou et al.  reported that loss of β-catenin is a strong and independent predictor of an unfavorable outcome in patients with endometrial carcinoma. In human gastric cancer, decreased expression of E-cadherin and catenins, including β-catenin, correlated with poor differentiation [23, 24].
Invasion of tumor cells into the basement membrane is a critical event for tumor metastasis. Invasive tumors exhibit high levels of MMPs [8, 9, 12, 17]. MMPs are capable of digesting various components of the extracellular matrix (ECM) and play a pivotal role in tumor metastasis by removing physical barriers to invasion [18, 19]. In particular, MMP-2 degrades ECM macromolecules in the basement membranes and other interstitial connective tissues . Asai et al.  reported that LM8 cells secreted higher levels of MMP-2 and exhibited extremely higher invasiveness in vitro compared with Dunn murine osteosarcoma cells with no metastatic potential to the lung. Our previous in vitro study showed that genistein-treated LM8 cells secreted lower levels of MMP-2 and were less invasive compared with untreated LM8 cells . Moreover, our previous study with nude mice inoculated with LM8 cells showed that decreased expression of MMP-2 within the primary tumor was associated with the suppression of the development of metastasis in the lung . Our present study showed that a majority of primary tumor cells of the genistein/metastasis(-) subgroup was MMP-2-negative (Figure 5A-c). The percentage of MMP-2-negative cells to total cells in this subgroup was 80 ± 5% (Figure 5B). This value was similar to that of the β-catenin-labeling index (82 ± 3%) in this subgroup. Taken together, our present findings suggest that decreased expression of MMP-2 in β-catenin-overexpressing LM8 cells may cause the prevention of local invasion, thus resulting in inhibition of the growth of primary tumor and the metastasis to the lung and liver.
In this study, we performed heat-induced antigen retrieval in 10 mM citrate buffer (pH 6.0) for immunohistochemical staining of β-catenin and showed that the primary tumor in the control group expressed lower level of cytoplasmic β-catenin compared with the genistein/metastasis(-) subgroup (Figure 4A). Moreover, we found that the metastatic tumor in the lung and liver also expressed very low level of cytoplasmic β-catenin (Figure 4B). Kashima et al.  also performed antigen retrieval in citrate acid buffer and showed low expression of cytoplasmic β-catenin in human primary osteosarcoma with metastasis and human metastatic osteosarcoma. Thus, osteosarcoma with metastatic potential seems to exhibit low expression of cytoplasmic β-catenin when heat-induced antigen retrieval was performed under acidic pH. Iwaya et al.  performed heat-induced antigen retrieval in 10 mM citrate buffer (pH 8.0) and showed that the expression of cytoplasmic and/or nuclear β-catenin within the primary tumor was higher in C3H mice inoculated with LM8 cells than in those inoculated with Dunn cells. Moreover, they found that in human metastatic osteosarcoma, more than 10% of tumor cells were immunostained for β-catenin in the cytoplasm and/or nucleus . These findings are inconsistent with ours. This inconsistency may be due to the different pH utilized in heat-induced antigen retrieval because the efficiency of heat-induced antigen retrieval is dependent on the pH of the retrieval solutions [26–28].
Preclinical and clinical studies have shown that protein kinases, which are involved in the regulation of a wide variety of cellular processes, are relevant targets for cancer therapy [29, 30]. Bruzzese et al.  reported that treatment of Hep-2 cells with gefitinib, a tyrosine kinase inhibitor , inhibited tyrosine phosphorylation of epidermal growth factor receptor and decreased invasive potential. Genistein also is a specific and potent inhibitor of tyrosine kinase [32, 33]. We previously found that genistein decreased motile and invasive potential of LM8 cells . Whether genistein inhibited tyrosine phosphorylation of proteins in LM8 cells remains unclear. It is unlikely, however, that high expression of cytoplasmic β-catenin in genistein-treated LM8 cells results from inhibition of tyrosine phosphorylation of β-catenin by genistein because phosphorylation of β-catenin by tyrosine kinase leads to an increase in the free pool of cytoplasmic β-catenin during epithelial cell migration . This interpretation may be also supported by reports stating that tyrosine phosphorylation of cell-cell adhesion molecules, including β-catenin, affected their functions, causing unstable cell-cell adhesion and migration of cells [35–37].