The human osteosarcoma (OS) cell lines MG 63 and U2OS (Korean Cell Line Bank, Seoul, Korea) were cultured. All experiments were carried out in Eagle's modified minimal essential medium containing 10% inactivated fetal bovine serum (GIBCO, Carlsbad, CA). In all the experiments, the OS cells were cultured in 10% serum and harvested with trypsin which was blocked by complete media as soon as the cells had become detached. Trypan blue exclusion was used to test for the cell viability in order to exclude the possibility that the cells had been damaged by trypsin. In all the experiments, the cells were left to recover from trypsinization for 24h before being treated. The media were replaced after the third day, only in the experiments requiring longer treatment times. In all experiments, the cells were plated at the same density, i,e., at densities equivalent to 3,000 cells/well of a 96 well plate.

AM (1-52) was purchased from Sigma (St. Louis, MO) and was used at concentrations of 10 nM, 50 nM, and 100 nM in both cell lines.

The level of cell proliferation was determined by counting the cells, as described elsewhere1). Briefly, the MG63 and U2OS cells (10,000 cells/well) were plated in a 12-well tissue culture plate containing the minimum essential medium (MEM) with 10% of fetal bovine serum (FBS). Immediately after attachment to the plates, cells were switched to serum-free MEM for 24h. Subsequently, the cells were either left untreated or treated with AM (1, 10, or 100 ng/ml) in the presence or absence of PD98059 (10 uM) and incubated at 37℃ for 3 days. Subsequently, they were trypsinized and the living cells (trypan blue exclusion) were counted using a hemocytometer.

The viabilities of the osteosarcoma cells was determined using a WST-1 quantitative colorimetric assay. The assay detects living cells only and the results are thus dependant on the degree of cellular viability. In this study, the sulfonated tetrazolium salt,4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate (WST-1) assay, was used because this compound produces a highly water-soluble formazan dye, which makes the assay procedure easier to perform. Subconfluent osteosarcoma cells grown in 96-well plates were treated with the agents for 48h. WST-1 (16 mg) and 0.2 mM 1-methyl-phenazium methylsulfate were dissolved in a 20 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) buffer (pH 7.4). Ten ul and 10 ul of the reaction solution was added immediately to 100 ul of the culture medium per well. The cells were incubated for an additional 2 h at 37℃ in a humidified atmosphere of 5 % CO₂ and 95 % air, and the cell viability was determined using a WST-1 assay. The absorbance of the lysate was measured at 405 nm for the test samples and 650 nm as a reference wavelength using an Immuno Mini NJ-2300 microplate reader (Japan Intermed, Tokyo, Japan).

The antibodies (Promega, Madison, WI, USA) specific to the phosphorylated, active forms of extracellular signal-regulated kinase-1 and -2 (ERK1/2) were used for the Western blotting. After culturing in 60 mm dishes, the cells were collected and added to 600 ul extraction buffer (25 mM HEPES, 0.5% Triton X-100, 5 mM ethylene glycol-bis (β-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA), 5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethysulfonyl fluoride, 50 nM beta-glycerol phosphate, and 10 mM sodium orthovanadate). The samples were then heated to 95℃ for 5 min, sonicated and centrifuged for 10 min at 15,000×g. Aliquots of 20 ul were mixed with 5 ul of a 5x sample buffer and subjected to SDS-polyacrylamide gel electrophoresis using 10% gels. The proteins were blotted onto Immobilin-P transfer membranes (Millipore, San Jose, CA, USA) and the nonspecific binding was blocked using Tris-buffered saline (TBS) containing 5% skim milk for 90 min at 37℃. The membranes were then incubated with the primary antibody for 3 h at room temperature and the protein bands were revealed using horseradish peroxidase-conjugated goat antirabbit immunoglobulins and visualized using an enhanced chemiluminescence detection system. The activated kinase bands were quantitated by densitometry using NIH Image software (National Institutes of Health, Bethesda, MD) soft ware analysis.

The MG63 and U2OS cells were incubated for 30 min in the presence of 10 uM of 2'-amino-3'-methoxyflavone (PD 98059; a specific MEK1 inhibitor) (Calbiochem, CA, USA). The cells were also treated with the MAPK inhibitors and cultured in the absence of AM for comparison purposes. The number of viable cells was determined by Western blotting, as described above.

All the data is expressed as the means±SD of experiments performed in triplicate. The analysis was performed using SPSS Ver.12.0 program. The mean values were compared using a Student's t test or ANOVA where appropriate. p values <0.05 were considered significant.

The proliferative effects of AM on the two osteosarcoma cell lines (MG63, U2OS) were assessed by cell counting. Treatment with AM for 24 h, in cultures grown in medium containing 10% inactivation. FBS induced dose-dependant increases in the numbers of cells in both cell lines (Fig. 1) and significant increases were observed at AM concentrations of 50 nM or greater. The stimulations were maintained for 48 h (Fig. 2).

The cell viability was determined using WST-1 assays. Fig. 3 shows that AM increased the cell viability in both cell lines in a dose-dependant manner up to 50 nM. AM at 50 nM increased the cell viability of MG63 and U2OS cells to approximately 285±20.5% and of U2OS cells 262± 15.3% of the control level, respectively. Treatment with AM for 24 h produced a dose-dependant increase in WST-1 assay (Fig. 4). The stimulation were maintained for 48 h.

As shown in Fig. 1, 100 nM of AM caused the maximum proliferation of both cell lines in the serum-deprived media. PD98059, selective inhibitor of MEK, was used to determine the role of ERK activation in mitogenesis. As shown in Fig. 5, treatment with 10 uM PD98059 completely inhibited AM-induced proliferation in both cell lines.

Polyclonal antibodies against the conserved, non-phosphorylated regions in mammalian ERK1 and ERK2 MAP kinase were used to probe the immunoblots of the osteosarcoma cell lines (MG63, U2OS). Phospho-specific antibodies, which detect the active forms of the MAP kinase, were used. ERK1 and 2, MAPK were found to be activated within 3 hours after the AM treatment. A strongly immunoreactive protein of molecular weight of about 42 kDa was obtained. Treatment with AM at 100 nM caused a two-fold increase in the phosphor-MAP kinase levels without affecting the total MAPK protein levels (Fig. 6). Furthermore, this increase in the phosphor-MAP kinase level was prevented by a co-treatment with 100 nM AM and 10 uM (Fig. 7).