Hiroshi Masuno

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.

Endometrial glandular and stromal breakdown, part 3: cytomorphology of "condensed cluster of stromal cells".

The aim of this study was undertaken to clarify the cytological characteristic of the “condensed clusters of stromal cells,” which may be recognized in endometrial glandular and stromal breakdown (EGBD) cases. The material consists of 60 cases of cytologic smears for which histopathological diagnosis was obtained by endometrial curettage; they comprised 30 cases of EGBD and 30 cases of endometrioid adenocarcinoma grade 1 (G1).

Effect of cortisol on cell proliferation and the expression of lipoprotein lipase and vascular endothelial growth factor in a human osteosarcoma cell line

PurposeThe aim of this study is to investigate whether cortisol inhibited cell proliferation and the expressions of lipoprotein lipase (LPL), a key enzyme involved in the energy metabolism in tumor cells, and vascular endothelial growth factor (VEGF), a potent angiogenic factor in the tumor, in cultures of OST cells, a human osteosarcoma cell line.MethodsOST cells were treated for 48xa0h with or without cortisol. To examine the effect of cortisol on cell proliferation, the expression of proliferating cell nuclear antigen (PCNA) was examined by Western blotting, and the amount of 3H-thymidine incorporated into DNA during the last 30xa0min of the 48-h treatment period was measured. To examine the effect of cortisol on the expression of LPL, the activity and mass of LPL were measured in the extract of acetone/ether powder of cells, and the amount of 35S-methionine incorporated into LPL during the last 2xa0h of the 48-h treatment period was measured by immunoprecipitation. The expression of VEGF was examined by immunohistochemistry and Western blotting.ResultsThe amount of 3H-thymidine incorporated into DNA and the level of PCNA were lower in the cortisol-treated cultures than in the untreated cultures, thus indicating that cortisol inhibited the proliferation of OST cells. The synthetic rate and activity of LPL were lower in the cortisol-treated cultures than in the untreated cultures but no difference in the specific activity of LPL between the two cultures was observed, thus indicating that cortisol inhibited LPL synthesis, thereby resulting in a decreased LPL activity. The expression of VEGF was lower in the cortisol-treated cultures than in the untreated cultures.ConclusionCortisol not only has the ability to inhibit cell proliferation but also the ability to inhibit the expressions of LPL and VEGF in cultures of OST cells.

Genistein inhibits cell invasion and motility by inducing cell differentiation in murine osteosarcoma cell line LM8

BackgroundOne 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.MethodsLM8 cells were treated for 3 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 (BrdU) 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-2 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 (H&E). The expression of osteocalcin mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR).ResultsGenistein dose-dependently inhibits cell proliferation. Genistein-treated cells were less invasive and less motile than untreated cells. The expression and secretion of MMP-2 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 LM8 cells with genistein induced morphological changes, markedly decreased the formation of multilayer masses of cells, and markedly increased the expression of osteocalcin mRNA.ConclusionsGenistein decreased invasive and motile potential by inducing cell differentiation in LM8 cells. Genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects.

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.

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

BackgroundOsteosarcoma 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.MethodsLM8 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.ResultsTGZ 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.ConclusionsInhibition 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.

Prolonged exposure to a low-dose of bisphenol A increases spontaneous motor activity in adult male rats

We investigated the effects of bisphenol A (BPA), an environmental endocrine-disrupting chemical, on spontaneous motor activity in adult male rats. The rats were implanted intraperitoneally with mini-osmotic pumps containing either BPA (50xa0μg/kg body weight per day) in sesame oil (BPA-treated group) or sesame oil only (vehicle-treated group). Spontaneous motor activity during a 24-h period was measured over 5xa0days from day 9 to day 13 after implantation using an animal movement analysis system. Spontaneous motor activity during the last 2 h of the dark phase and during the first 1-h of the light phase was increased in the BPA-treated group. Total spontaneous motor activity during the 12-h light phase, but not the 12-h dark phase, was higher in the BPA-treated group than in the vehicle-treated group. These findings suggest that BPA may induce hyperactivity in adult male rats during the 12-h light phase, especially during the 2 h immediately preceding sleep-onset and 1 h immediately following sleep-onset.

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

BackgroundPreviously, 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.MethodsLM8 cells were treated for 3xa0days with or without 50xa0μM 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 25xa0days 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.ResultsAll mice in the control group (nu2009=u20098) exhibited large primary tumors, while in the genistein group (nu2009=u20098), 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.ConclusionsOverexpression 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.

Efficacy of CytoLyt® hemolytic action on ThinPrep® LBC using cultured osteosarcoma cell line LM8.

Objective: The removal of blood components is necessary to improve the quality of the liquid-based cytology (LBC) preparations. In ThinPrep® (TP) samples a cell suspension in a methanol-based fixative undergoes a vacuum filtration method, whereas in SurePath™ (SP) samples a cell suspension in an ethanol-based fixative is processed through a density gradient centrifugation system prior to gravity deposition of the specimen onto a glass slide. We compared the cyto-architectural features for the cytologic diagnosis of endometrial adenocarcinoma using parallel TP and SP preparations in a previous publication. Study Design: We performed our study on LM8 cells (a cultured osteosarcoma cell line). LM8 cells at a concentration of 1.25 × 103 cell/cm2 were seeded on a 35-mm plate in culture medium, which contained 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μ/ml streptomycin in Dulbeccos modified Eagles medium (DMEM), and aliquots of the cell suspension obtained in this way were compared after the addition of a hemolytic agent, i.e. Cytolyt® (CyL). LBC preparations were then obtained on cell suspensions treated with CyL after different time intervals of hemolysis. Results: Treatment with CyL did not alter the cellularity of the preparation, but reduction of the nuclear area and a tendency towards nuclear chromatin condensation with a subsequent higher brightness were found. Because CyL is a 25% methanol-buffered solution, its alcoholic concentration is low; it was our impression that, while its fixative effect was weak, its hemolytic effect was high. Water influx or efflux through the cell membrane is controlled by osmotic pressure changes induced by the buffer solution in the CyL solution. While CyL was not shown to alter the cell shape, nuclear shrinkage was thought to be probably due to the increasing cell dehydration caused by longer exposure intervals to methanol. Conclusion: This study has allowed us to make significant observations on the hemolytic properties of CyL, and on its combined effects with PreservCyt on the cytomorphology of cells suspensions.