Bisphenol A Inhibits Cell Proliferation and Reduces the Motile Potential of Murine LM8 Osteosarcoma Cells.

AIM: The aim of this study was to examine the effect of bisphenol A (BPA) on the proliferation and motility potential of murine LM8 osteosarcoma cells. MATERIALS AND METHODS: LM8 cells were treated for 3 days with or without 80 µM BPA. The effect of BPA on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2'-deoxyuridine (BrdU) incorporation study. Ethanol-fixed cells were stained with hematoxylin-eosin (H&E) to visualize cell morphology. Cell motility was assayed using inserts with uncoated membranes in invasion chambers. Expression of cell division cycle 42 (CDC42) was determined by immunofluorescence staining and western blotting. RESULTS: BPA reduced the DNA content of cultures and the number of BrdU-positive cells. BPA induced a change in morphology from cuboidal with multiple filopodia on the cell surface to spindle-shaped with a smooth cell surface. BPA-treated cells expressed less CDC42 and were less motile than untreated cells. CONCLUSION: BPA inhibited DNA replication and cell proliferation. BPA inhibited filopodia formation and motile potential by inhibiting CDC42 expression in LM8 cells.

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.

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 × 10(3) cell/cm(2) 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 Dulbecco's modified Eagle's 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.

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 (50 µg/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 5 days 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.

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

BACKGROUND: 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: LM8 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 ß-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). RESULTS: Genistein 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. ß-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 ß-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. CONCLUSIONS: Genistein 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.

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.

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). The following parameters were examined for "condensed clusters of stromal cells" in EGBD and for "clumps of cancer cells" in G1, respectively: (1) the occurrence of "condensed clusters of stromal cells"; (2) the nuclear shape; (3) a longer/shorter axis ratio in cell nuclei; (4) the area of cell nuclei; (5) the presence of overlapping nuclei; (6) nuclear crowding; (7) immunostaining. (1)"Condensed clusters of stromal cells" were only observed in EGBD. (2) A reniform nuclear shape was observed in 100% EGBD (P < 0.0001) in comparison to G1 (3%). (3) The longer/shorter axis ratio in cell nuclei, G1 (1.37 +/- 0.2) was significantly lower in comparison to EGBD (1.53 +/- 0.12, P = 0.0005). (4) Nuclear area in G1 (51.6 +/- 11.9, P < 0.0001) was significantly higher in comparison to EGBD (24.3 +/- 3.9 microm(2)). (5) The score of overlapping nuclei in EGBD (2.5 +/- 0.49) was significantly higher in comparison to G1 (1.8 +/- 0.44, P < 0.0001). (6) The nuclear crowding score was the same both in EGBD (2) and G1 (2) and these findings were not statistically significant. (7) Both CD10 and Wilms' tumor protein 1 were positive in the "condensed clusters of stromal cells" in the EGBD. The anti-cytokeratin staining was positive in "clumps of cancer cells" in the G1. The evaluation of the immunocytochemical findings by combining the Wilms' tumor 1 protein, CD10, and the anti-cytokeratin with the considered cytomorphologic features (reniform nucleus) may be useful for a correct diagnosis of EGBD in endometrial cytology.

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.

The synthesis and activity of lipoprotein lipase in the subcutaneous adipose tissue of patients with musculoskeletal sarcomas.

The purpose of this study was to explore the triacylglycerol (TG) deposition and lipoprotein lipase (LPL) activity in the adipose tissue of patients with muculoskeletal sarcoma. Subcutaneous adipose tissue was obtained from the thighs of 19 patients with musculoskeletal sarcomas (sarcoma group) and 20 patients with osteoarthritis of the hip joint (control group) at surgery. The adipose tissue was homogenized and aliquots of the homogenate were used to measure the TG content and to prepare an acetone/ether powder to measure the LPL activity. The TG content was higher, but not significantly, in the sarcoma group than in the control group. The LPL activity of the sarcoma group was significantly higher than that of the control group. The TG content of the sarcoma group correlated positively with the LPL activity. [35S]Methionine incorporation investigation showed that the rate of LPL synthesis was significantly higher in the sarcoma group than in the control group. These results indicated that LPL was up-regulated at the transcriptional/translational level, thus resulting in an increased TG deposition in the adipose tissue of patients with muculoskeletal sarcoma.

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.

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

PURPOSE: The 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. METHODS: OST cells were treated for 48 h 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 (3)H-thymidine incorporated into DNA during the last 30 min 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 (35)S-methionine incorporated into LPL during the last 2 h of the 48-h treatment period was measured by immunoprecipitation. The expression of VEGF was examined by immunohistochemistry and Western blotting. RESULTS: The amount of (3)H-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. CONCLUSION: Cortisol 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.

Perinatal and postnatal exposure to bisphenol a increases adipose tissue mass and serum cholesterol level in mice.

AIM: To investigate whether the perinatal and postnatal exposure of mice to bisphenol A (BPA) caused the development of obesity and/or hyperlipidemia. METHODS: Pregnant mice were exposed to BPA in drinking water at concentrations of either 1 microg/mL (LD group) or 10 microg/mL (HD group) from gestation day 10 and throughout the lactating period. After weaning, the pups were allowed free access to drinking water containing the appropriate concentrations of BPA. The body weight, adipose tissue weight, and serum lipid levels were measured in the offspring at postnatal day 31. RESULTS: In females, the mean body weight increased by 13% in the LD group (p<0.05) and 11% in the HD group (p<0.05) compared with the control group. The mean adipose tissue weight increased by 132% in the LD group (p<0.01). The mean total cholesterol level increased by 33% in the LD group (p<0.01) and 17% in the HD group (p<0.05). In males, the mean body weight and mean adipose tissue weight increased by 22% (p<0.01) and 59% (p<0.01), respectively, in the HD group compared with the control group. The mean triacylglycerol level increased by 34% in the LD group (p<0.05). CONCLUSIONS: The continuous exposure of mice to BPA during the perinatal and postnatal periods caused the development of obesity and hyperlipidemia.

Elevated concentration of hyaluronan in the cerebrospinal fluid is a secondary marker of spinal disorders: hyaluronan in the cerebrospinal fluid in patients with spinal disorders.

BACKGROUND: Hyaluronan (HA) was measured in cerebrospinal fluid (CSF) to ascertain the clinical significance of this substance in patients with spinal disorders, a topic that, to the best of our knowledge, has not previously been studied. METHODS: We examined correlations of CSF HA concentration with age, sex, height, body weight, and spinal disorders. By using a sandwich-binding protein assay, HA was measured in CSF samples obtained from 500 patients aged 12 to 104 years who underwent lumbar spinal anesthesia for surgery, myelography, or CSF examination. These patients were classified into 3 groups: (1) a control group (306 patients with injury or benign tumor of the lower limbs); (2) a cervical disorders group (84 patients with cervical disc herniation, cervical spondylotic myelopathy, or ossification of the posterior longitudinal ligament); and (3) a lumbar disorders group (110 patients with lumbar disc herniation, lumbar spinal canal stenosis tethered cord syndrome, lumbar fracture, or spondylolytic spondylolisthesis). RESULTS: CSF HA concentration was found to be positively correlated with age, and was significantly higher in patients with cervical spondylotic myelopathy, ossification of the posterior longitudinal ligament, or lumbar spinal canal stenosis tumor than in the control group. CONCLUSIONS: CSF HA concentration might be a secondary marker for inflammation in patients with spinal disease.

Bisphenol a accelerates terminal differentiation of 3T3-L1 cells into adipocytes through the phosphatidylinositol 3-kinase pathway.

In order to identify whether bisphenol A (BPA) acts as an adipogenic agent, following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for six days with BPA alone. Treatment with BPA increased the triacylglycerol (TG) content of the cultures, increased the percentage of Oil Red O-staining cells in the cultures, and increased the levels of lipoprotein lipase (LPL) and adipocyte-specific fatty acid binding protein (aP2) mRNAs. These findings indicate that BPA was able to accelerate terminal differentiation of 3T3-L1 cells into adipocytes. LY294002, a chemical inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), blocked completely the increasing effect of BPA on TG accumulation and expression of LPL and aP2 mRNAs. Western blot analysis revealed that BPA increased the level of phosphorylated Akt kinase. Based on these findings, we concluded that BPA acted through the PI 3-kinase and Akt kinase pathway, resulting in increased TG accumulation and expression of adipocyte genes. The structure-activity relationship for BPA-related chemicals was examined. Eight derivatives of BPA (three diphenylalkanes with different substituents at the central carbon atom, three diphenylalkanes with ester bonds on hydroxyl groups in the phenolic rings, one bisphenol consisting of a sulphur atom at the central position, one chemical with cyanic groups, instead of hydroxyl groups, in the phenolic rings) accelerated terminal adipocyte differentiation and their potencies to increase TG accumulation were 73-97% of that of BPA. Two diphenylalkanes with ether bonds on hydroxyl groups and two alkylphenols (4-nonylphenol and 4-tert-octylphenol) did not have the ability to accelerate terminal adipocyte differentiation.

Effect of ketoprofen in topical formulation on vascular endothelial growth factor expression and tumor growth in nude mice with osteosarcoma.

OST cells, a low metastatic cell line established from human osteosarcoma, were inoculated under the periosteum of the ossa cranii of nude mice. Four weeks later, tumors were percutaneously treated for an additional 4 weeks with a patch containing either placebo or ketoprofen (KP). In the placebo group, OST cells formed osteoid and invaded the cranial bone. Tumor mass weighed 3.54 g. Approximately 85% of cells within the tumor expressed proliferating cell nuclear antigen (PCNA), indicating that they were proliferating with a high mitotic activity. Many feeder vessels were located within the tumor. The majority of tumor cells expressed intensely vascular endothelial growth factor (VEGF). In the KP group, invasion of OST cells into the cranial bone was suppressed and the tumor mass was 47% of that of the placebo group. Approximately 65% of cells within the tumor were PCNA-negative, indicating that their growth was arrested. There were considerably fewer feeder vessels within the tumor in the KP group than in the placebo group. Only a small number of cells expressed VEGF. Based on these findings, we concluded that topical administration of KP to nude mice with osteosarcoma inhibited VEGF expression, reduced the development of feeder vessels for supply of nutrients and oxygen, and suppressed tumor growth.

Effect of 4-nonylphenol on cell proliferation and adipocyte formation in cultures of fully differentiated 3T3-L1 cells.

The effect of 4-nonylphenol (NP) on cell proliferation and adipocyte formation was examined in cultures of fully differentiated 3T3-L1 cells. Following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for 8 days with or without NP. NP at 5 and 10 microg/ml increased the DNA content by 32% and 68%, respectively, compared with that of the untreated cultures, in which NP was absent during the treatment period. There were many more bromodeoxyuridine (BrdU)-positive cells in the NP-treated cultures, in which NP was present at a concentration of 10 microg/ml during the treatment period, compared to the untreated cultures. These results indicate that NP had the ability to stimulate the proliferation of fully differentiated 3T3-L1 cells. NP at 5 and 10 microg/ml decreased the triacylglycerol (TG) content by 26% and 58%, respectively, and decreased the lipoprotein lipase (LPL) activity by 51% and 71%, respectively. The lipid droplets in individual cells of the NP-treated cultures were smaller than those of the untreated cultures. The mRNA levels of LPL and adipocyte-specific fatty acid binding protein (aP2) were considerably lower in the NP-treated cultures than in the untreated cultures. Thus, NP also had the ability to inhibit adipocyte formation in cultures of fully differentiated 3T3-L1 cells. A study using an antiestrogen ICI 182,780 showed that the NP-stimulated cell proliferation was mediated partly by the estrogen receptor, while the NP-induced inhibition of adipocyte formation was mediated by a mechanism other than the estrogen receptor.

Active-dimeric form of lipoprotein lipase increases in the adipose tissue of patients with rheumatoid arthritis treated with prednisolone.

The synthesis, activity and mass of LPL in adipose tissue were studied in patients with rheumatoid arthritis (RA) treated with prednisolone (PSL) (PSL-treated group) and untreated patients with osteoarthritis (untreated group). LPL activity and mass in the extracts of acetone/ether powder of adipose tissue were 2.4 and 1.6 times, respectively, higher in the PSL-treated group than in the untreated group. There were no differences in the amount of 35S incorporated into LPL during the 2-h incubation of adipose tissue with [35S]methionine between PSL-treated and untreated groups. These results indicate that degradation of LPL was inhibited in the adipose tissue of the PSL-treated group. In the adipose tissue of the untreated group, 72% of the LPL was the inactive-monomeric form, which was eluted with 0.4-0.75 M NaCl from the heparin-Sepharose column, and 28% was the active-dimeric form, which was eluted with 0.8-1.2 M NaCl. In the adipose tissue of the PSL-treated group, 40% was inactive-monomeric, and 60% was active-dimeric. Thus, the relative amount of the active-dimeric form of LPL was increased in the adipose tissue of the PSL-treated group. Taken together, our present results indicate that the higher level of LPL activity in the PSL-treated group was a result of the inhibition of the degradation of the active-dimeric form.

Bisphenol A in combination with insulin can accelerate the conversion of 3T3-L1 fibroblasts to adipocytes.

The confluent cultures of 3T3-L1 fibroblasts were treated with or without bisphenol A (BPA) for 2 days and subsequently treated with insulin (INS) alone for 9 days. When BPA was absent during the first 2-day treatment period, the cultures contained 1.6 microg/microg DNA of triacylglycerol (TG), 202 mU/mg DNA of lipoprotein lipase (LPL) activity, and 462 nmol/min/mg DNA of glycerol-3-phosphate dehydrogenase (GPDH) activity. The presence of BPA during the same period caused a 150% increase in the TG content, a 60% increase in the LPL activity, and a 500% increase in the GPDH activity. Thus, BPA by itself can trigger 3T3-L1 fibroblasts to differentiate into adipocytes. Next, the confluent cultures were treated with BPA for 2 days and subsequently treated with a combination of INS and BPA for 9 days. The simultaneous presence of BPA with INS caused a 370% increase in the TG content, a 200% increase in the LPL activity, and a 225% increase in the GPDH activity compared with the cultures treated with INS alone. The amount of [(3)H]thymidine incorporated into DNA was lower in the cultures treated with INS in the presence of BPA than in those treated with INS alone, indicating that BPA has an anti-proliferative activity on 3T3-L1 cells. Taken together, our results indicate that BPA in combination with INS can accelerate the adipocyte conversion.