title: Overexpression of cytoplasmic β-catenin inhibits the metastasis of the murine osteosarcoma cell line LM8.
abstract: BACKGROUND
  Previously, we found that treatment of LM8 murine osteosarcoma cells with genistein, an isoflavone found in soy, increased the cellular level of β-catenin and decreased its invasive and motile potential. The purpose of this study is to investigate whether the expression of β-catenin in LM8 cells is associated with metastatic potential in nude mice. To this end, we used untreated and genistein-treated LM8 cells.
  
  
  METHODS
  LM8 cells were treated for 3 days with or without 50 μM genistein and harvested by trypsinization. Untreated (the control group) and genistein-treated (the genistein group) cells were subcutaneously inoculated into the backs of male nude mice. After 25 days of inoculation, the tumors, lungs, and livers were excised, fixed in 10% formalin, and embedded in paraffin. The sections of formalin-fixed, paraffin-embedded lungs and livers were stained with hematoxylin-eosin (H&E) to confirm the absence or presence of metastatic tumors. The expression of β-catenin within the primary tumor was immunohistochemically examined.
  
  
  RESULTS
  All mice in the control group (n = 8) exhibited large primary tumors, while in the genistein group (n = 8), one mouse showed no tumor formation and the remaining seven mice exhibited smaller primary tumors compared with the control group. The tumor mass of the genistein group was 23% of that of the control group. In the control group, multiple metastatic tumors were found in the lung and/or liver and the metastatic incidence was 100% in the lung and 87.5% in the liver. Six of seven tumor-bearing mice in the genistein group developed no metastatic tumors in the lung or liver, and this group was termed the genistein/metastasis(-) subgroup. Positive β-catenin immunostaining was observed in the cytoplasm of tumor cells, and the β-catenin-labeling index was higher in the genistein/metastasis(-) subgroup than in the control group. The intensity of cytoplasmic β-catenin immunostaining was stronger in the genistein/metastasis(-) subgroup compared with the control group, and the β-catenin-labeling score was 1.9-times higher in the former subgroup than in the latter group.
  
  
  CONCLUSIONS
  Overexpression of cytoplasmic β-catenin in LM8 cells causes inhibition of the growth of primary tumors and loss of the metastatic potential to the lung and liver.
authors.forename: Teruki
authors.initials: T
authors.lastname: Kidani
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nakamura
authors.forename: Setsuya
authors.initials: S
authors.lastname: Kamei
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Norimatsu
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Miura
authors.affiliation: Department of Medical Technology, Faculty of Health Sciences, Ehime Prefectural University of Health Sciences, Takooda, Tobe-cho, Iyo-gun, Ehime 791-2101, Japan. hmasuno@epu.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Masuno