Overexpression of cytoplasmic ß-catenin inhibits the metastasis of the murine osteosarcoma cell line LM8.

BACKGROUND: Previously, we found that treatment of LM8 murine osteosarcoma cells with genistein, an isoflavone found in soy, increased the cellular level of ß-catenin and decreased its invasive and motile potential. The purpose of this study is to investigate whether the expression of ß-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 ß-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 ß-catenin immunostaining was observed in the cytoplasm of tumor cells, and the ß-catenin-labeling index was higher in the genistein/metastasis(-) subgroup than in the control group. The intensity of cytoplasmic ß-catenin immunostaining was stronger in the genistein/metastasis(-) subgroup compared with the control group, and the ß-catenin-labeling score was 1.9-times higher in the former subgroup than in the latter group. CONCLUSIONS: Overexpression of cytoplasmic ß-catenin in LM8 cells causes inhibition of the growth of primary tumors and loss of the metastatic potential to the lung and liver.

Bisphenol A downregulates Akt signaling and inhibits adiponectin production and secretion in 3T3-L1 adipocytes.

AIM: The aim of this study was to investigate whether environmental endocrine-disrupting chemicals, bisphenol A (BPA) and BPA-related chemicals, affect adiponectin production and secretion in 3T3-L1 adipocytes and whether BPA acts through Akt signaling. METHODS: 3T3-L1 adipocytes were treated for 24 h with BPA at various concentrations (20-80 microM) in serum-deprived medium. The medium was filtered through a 0.2 microm filter. Adiponectin in the infranatants of cell homogenates and in the media was measured using an adiponectin ELISA kit. The levels of Akt and p-Akt in cultures treated for 24 h with or without 80 microM BPA were analyzed by Western blot. RESULTS: The control cultures (i.e., BPA was absent during a 24-h treatment period) contained 49.4 microg/mg DNA of adiponectin in the cells and secreted 35.5 microg/mg DNA of adiponectin into the medium. BPA at 80 microM dose-dependently decreased the amounts of intracellular and medium adiponectin by 60% (p<0.01) and 56% (p<0.01), respectively, and decreased the levels of Akt and p-Akt by 46% (p<0.01) and 29% (p<0.01), respectively, compared with the control cultures. Like BPA, bisphenol F (BPF), bisphenol E (BPE), and bisphenol B (BPB) decreased the amounts of intracellular and medium adiponectin. The order of the potential to decrease the amount of intracellular adiponectin was BPB>BPA>BPE>BPF. CONCLUSIONS: BPA downregulates Akt signaling and inhibits adiponectin production and secretion in 3T3-L1 adipocytes.

Effect of troglitazone on tumor growth and pulmonary metastasis development of the mouse osteosarcoma cell line LM8.

BACKGROUND: Osteosarcoma often develops micrometastases in the lung prior to diagnosis, causing a fatal outcome. Therefore, the prevention of pulmonary metastases is critical for the improvement of the prognosis of patients with osteosarcoma. The purpose of this study was to investigate whether troglitazone (TGZ) is considered as possible therapeutics in the treatment of growth and metastasis of osteosarcoma. METHODS: LM8 cells were treated for 3 days with various concentrations of TGZ. The effect of TGZ on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2'-deoxyuridine incorporation study. The assay of cell invasion and motility was performed using either the Matrigel-coated cell culture inserts or the uncoated cell culture inserts in the invasion chambers. The effect of TGZ on Akt signaling was assessed by Western blot analysis of Akt and p-Akt. The effects of oral administration of either TGZ (TGZ group) or ethanol (control group) on the growth of primary tumor and the development of pulmonary metastasis were examined in nude mice implanted with LM8 cells on their backs. The expression and activity of matrix metalloproteinase 2 (MMP-2) within the tumor were determined by immunohistochemistry and zymography. The microvessel density (MVD) within the tumor was determined by immunohistochemistry for CD34. RESULTS: TGZ dose-dependently inhibits cell proliferation. TGZ-treated cells were less invasive and less motile than untreated cells. The activity of MMP-2 secreted by TGZ-treated cells was lower than that secreted by untreated cells. TGZ decreased the level of p-Akt. The primary tumor mass was smaller in the TGZ group than in the control group. The TGZ group had less metastatic tumors in the lung compared with the control group. The expression and activity of MMP-2 within the tumor of the TGZ group were lower than those of the control group. The MVD within the tumor of the TGZ group was lower than that of the control group. CONCLUSIONS: Inhibition of Akt signaling by TGZ may decrease the secretion of MMP-2, resulting in the decrease of invasiveness and motility in LM8 cells. Treatment of tumor-bearing mice with TGZ decreases the expression and activity of MMP-2 within the tumor, and inhibits primary tumor growth and pulmonary metastasis development. TGZ may offer a new approach in chemotherapy for osteosarcoma.

Ketoprofen in topical formulation decreases the matrix metalloproteinase-2 expression and pulmonary metastatic incidence in nude mice with osteosarcoma.

The aim of this study was to investigate whether ketoprofen (KP) in topical formulation affected the tumor growth and pulmonary metastasis of LM8 cells, which were inoculated subcutaneously into the back space of male nude mice. At 7 days after inoculation, the tumor was treated topically for 3 weeks with either a KP-containing patch (KP group) or a placebo-containing patch (placebo group). The pulmonary metastatic incidence was 100% in the placebo group and 60% in the KP group. The tumor mass of the KP group without pulmonary metastasis, termed the KP/metastasis(-) group, was smaller than that of the placebo group. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor (VEGF) was performed. The tumors of the KP/metastasis(-) group contained fewer PCNA-positive cells and many more TUNEL-positive cells in comparison to the placebo group. In the placebo group, MMP-2 and VEGF were extensively expressed within the tumor, whereas in the KP/metastasis(-) group the expression of these two proteins was very low. In conclusion, the topical treatment of osteosarcoma with KP decreased the expression of MMP-2 and VEGF, thus resulting in the suppression of tumor growth and pulmonary metastasis.

Perinatal and postnatal exposure to 4-tert-octylphenol inhibits cortical bone growth in width at the diaphysis in female mice.

The aim of this study was to investigate the effects of 4-tert-octylphenol (OP) on bone growth in vivo. Pregnant mice were exposed to drinking water containing either 1microg/ml OP (LD group) or 10microg/ml OP (HD group) from gestational day 10 and throughout the lactation period. After weaning, the pups were allowed free access to drinking water containing the appropriate OP concentrations. The serum osteocalcin level of the females, but not the males, was significantly lower in the LD and HD groups than in the control group on postnatal day 31. The femurs of the females were analyzed by peripheral quantitative computed tomography and immunohistochemistry for alkaline phosphatase (ALP) was performed in the sections of the formalin-fixed femurs. The periosteal and endosteal circumferences of the cortical bone at the diaphysis were significantly smaller in the LD group, but not the HD group, than in the control group (4% and 6% smaller, respectively), while there were no differences in the cortical bone density, cortical bone area, or cortical thickness among the three groups. There were fewer ALP-positive cells on the periosteal surfaces at the diaphysis in the LD group than in the control group. The values of the strength strain index (xSSI, ySSI, and pSSI) decreased with decreasing the periosteal circumference. In conclusion, the exposure of female mice to OP during the perinatal and postnatal periods inhibited the periosteal bone formation in the cortical bone at the diaphysis of the femur, thereby causing a reduction in bone growth in width.

4-tert-octylphenol regulates the differentiation of C3H10T1/2 cells into osteoblast and adipocyte lineages.

The aim of this study was to investigate whether 4-tert-octylphenol (OP) affects the differentiation of multipotent C3H10T1/2 cells, a cell line established from mouse embryonic connective tissue, into osteoblast and adipocyte lineages. Confluent C3H10T1/2 cells were incubated for 7 days with (OP-treated cultures) or without (control cultures) 15 microg/ml of OP. The 7-day treatment of confluent cells with OP decreased alkaline phosphatase activity by 81%, inhibited the expression of transforming growth factor beta2, and inhibited the morphological changes in cells to an osteoblastic appearance. These results indicate that the 7-day treatment of confluent C3H10T1/2 cells with OP inhibited their differentiation into osteoblasts. Since this treatment strongly induced the expression of peroxisome proliferator-activated receptor r (PPARr) but did not stimulate triacylglycerol (TG) accumulation in cells, C3H10T1/2 cells in the control and OP-treated cultures were incubated for 2 days with a hormone mixture (insulin [INS], dexamethasone, and 1-methyl-3-isobutylxanthine) and incubated for an additional 5 days with INS alone. The TG and adiponectin contents of the OP-treated cultures were 4.2 and 4.1 times higher, respectively, than those of the control cultures. There were many more Oil Red O-staining cells in the OP-treated cultures than in the control cultures. The expression of PPARr in the OP-treated cultures was higher than that in the control cultures. These results indicate that the OP-treated cultures contained a larger number of adipocytes than the control cultures. In conclusion, treatment of C3H10T1/2 cells with OP inhibited osteoblast differentiation, causing a lineage shift toward adipocytes.

Diagnostic and therapeutic problems of giant cell tumor in the proximal femur.

Primaly giant cell tumor of bone (GCT) in the proximal femur is relatively rare, and can prove difficult to diagnose, and can require therapeutic methods. Subjects comprised 10 patients (8 males, 2 females). Mean patient age was 27.5 years, and mean follow-up was 89.9 months. Tumors in the present study were limited to H1 and H2 according to the International Society of Limb Salvage (ISOLS) system. All patients received surgical treatment only. Second surgery after preoperative open biopsy was performed for two patients, while the remaining eight patients received excisional biopsy to determine treatment methods using rapid intraoperative pathological examination of frozen sections. The mean functional score was 28.2 out of 30 (93.9%). Local recurrence was observed in two patients. The long-term follow-up reveals that one of the important problem is pre-operative diagnosis. Excisional biopsy is effective for surgery of GCT in the proximal femur.