The human OS MG63 and U2OS cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 µg/ml penicillin and 100 µg/ml streptomycin (all Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a 5% CO₂ atmosphere at 37°C. Met (1,1-dimethylbiguanide hydrochloride) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The Cell Counting kit-8 (CCK-8) was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Antibodies against β-actin (cat. no. sc-47778), Akt (cat. no. sc-8312), phosphorylated (p)-Akt (cat. no. sc-33437) and phosphatase and tensin homolog (PTEN) (cat. no. sc-6817-R) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and anti-E-cadherin (cat. no. ab1416) from Abcam (Cambridge, UK); vimentin (cat. no. 10366-1-AP) was purchased from ProteinTech Group, Inc. (Chicago, IL, USA); the goat anti-rabbit (cat. no. 32460) and goat anti-mouse (cat. no. 32430) horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Thermo Fisher Scientific, Inc.

PTEN small interfering (si)RNA and negative control (NC) siRNA were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). PTEN-siRNA, 5′-GATCTACTCCTCCAACTCA-3′; NC-siRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′. At 24 h after seeding, the cells were transfected with PTEN-siRNA or NC-siRNA using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After 24 h transfection, cells were collected for the following experiments; 48 h after transfection, cells were collected for western blot analysis.

Cell viability was determined using the CCK-8 assay. MG63 and U2OS cells with or without PTEN- or NC-siRNA (2×10³ per well) were seeded into a 96-well plate. At 24 h, 0, 1, 5 or 10 mM Met was added into the culture medium. Subsequent to incubation at 37°C for a further 24 or 48 h, the medium was exchanged for 100 µl DMEM medium and 10 µl CCK-8 reagent. The cells were incubated for 2 h at 37°C. Finally, the optical density was measured using an EnSpire™ 2300 Multilabel Reader (PerkinElmer, Inc., Waltham, MA, USA) at 450 nm. Three replicates were prepared for each condition.

For the colony formation assay, MG63 and U2OS cells with or without PTEN- or NC-siRNA treated with 0, 1, 5 or 10 mM Met were seeded in triplicate at 1,000 cells/well in a 6-well plate with complete medium. Subsequent to 3 weeks of growth, colonies were fixed with 4% paraformaldehyde for 10 min at room temperature and stained with 1% crystal violet for 15 min at room temperature (0.1% w/v in 20 nM 4-morpholinepropanesulfonic acid). Visible colonies were counted.

A total of 2×10⁶ MG63 and U2OS cells with or without PTEN- or NC-siRNA treated with 0, 1, 5 or 10 mM Met were seeded in 6-well plates, the cell monolayers were wounded by scratching with sterile plastic 20 µl micropipette tips and images were captured using a light microscope at 0 and 24 or 48 h. The migration distance of the cells was measured using Image Pro-Plus v.6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

The invasive capacity of MG63 and U2OS cells with or without PTEN- or NC-siRNA treated with 0, 1, 5 or 10 mM Met was measured with Matrigel-coated (BD Biosciences, Franklin Lakes, NJ, USA) Transwell inserts (Costar, Manassas, VA, USA). The inserts were coated with 50 µl of 1 mg/ml Matrigel according to the manufacturer's protocol. A total of 2×10⁵ cells in 200 µl DMEM serum-free medium were plated in the upper chamber, with 300 µl of medium with 10% FBS added to the lower chamber. Following a 24 h incubation, cells that invaded to the lower surface of the membrane were fixed and stained. For each membrane, five random fields of view were counted.

MG63 and U2OS cells with or without PTEN- or NC-siRNA treated with 0, 1, 5 or 10 mM Met were harvested and lysed in radioimmunoprecipitation assay buffer (Nanjing KeyGen Biotech Co., Ltd.) supplemented with 1 mM phenylmethylsulfonyl fluoride and 1 mM phosphatase inhibitor cocktail. The protein concentration was determined using a bicinchoninic assay kit (Nanjing KeyGen Biotech Co., Ltd.). A total of 30 µg protein samples were mixed with loading buffer, and the proteins were separated using 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequent to blocking in 5% nonfat dry milk for 2 h at room temperature, the blots were incubated at 4°C overnight with the aforementioned primary antibodies (1:1,000) and at 37°C for 2 h with the secondary antibodies (1:20,000). The bands were visualized with WesternBright Sirius HRP substrate (Advansta, Inc., Menlo Park, CA, USA) and were imaged using a Vilber Fusion FX5 (Vilber Lourmat, Marne-la-Valée, France).

SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analysis. Values are presented as the mean ± standard deviation. The statistical analysis of differences between two groups was performed using Student's t-test and the analysis of multiple groups was performed using one-way analysis of variance followed by the Student-Newman-Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of Met on OS cell proliferation, CCK-8 analysis was employed to assess the viability of MG63 and U2OS cells. As presented in Fig. 1A and B, the groups treated with 1, 5 and 10 mM Met exhibited a dose- and time-dependent inhibition of proliferation. Consistent with the results from the CCK-8 assay, the colony formation rates in the cells treated with Met were lower than the control group, particularly in the 5 and 10 mM Met groups (P<0.01; Fig. 1C and D). These data indicated that Met inhibited the growth of OS cells.

To examine the effect of Met on the mobility of MG63 cells, wound-healing and invasion assays were utilized. The speed of wound closure was reduced with increasing concentrations of Met, compared with the control group (Fig. 2A and B). Incubation with Met also suppressed the invasion of MG63 and U2OS cells in a dose-dependent manner (Fig. 2C and D). As EMT is a critical mechanism in the progression of tumor cells to metastasis, whether Met could negatively regulate the EMT of OS cells was analyzed. Following treatment with 10 mM Met, MG63 and U2OS cells exhibited increased levels of the epithelial marker E-cadherin and decreased levels of the mesenchymal marker vimentin (Fig. 2E), which indicated that Met may suppress the metastasis of OS cells by negatively regulating EMT.

The molecular mechanisms responsible for the proliferation, migration and invasion suppressive effect of Met were assessed. Akt serves a key role in regulating the biological functions of tumor cells, including proliferation, apoptosis and metastasis; PTEN is a regulator that inhibits the activation of Akt (31). Following treatment with 10 mM Met for 48 h, the expression level of PTEN, Akt and p-Akt were detected by western blotting. As demonstrated in Fig. 3A, the expression level of PTEN was upregulated following this treatment, whereas the phosphorylation of Akt was downregulated. These data indicated that Met may inhibit the proliferation, migration and invasion of OS cells by suppressing the phosphorylation of Akt through the upregulation of PTEN.

To provide further confirmation that Met exerts its function through the PTEN/Akt pathway, MG63 and U2OS cells were transfected with NC- or PTEN-siRNA prior to Met treatment, and western blotting was performed. The expression of PTEN was confirmed to be lower, and the phosphorylation of Akt, higher, in cells transfected with PTEN-siRNA compared with NC-siRNA-transfected cells (Fig. 3B).

Next, the proliferative, migratory and invasive capacities of cells transfected with PTEN-siRNA in response to Met treatment were assessed. With Met treatment, MG63 and U2OS cells transfected with PTEN-siRNA exhibited an increased rate of proliferation when compared with the corresponding NC-siRNA cells (Fig. 4A and B). The inhibition of migration and invasion in MG63 and U2OS cells were also reversed by PTEN-knockdown (Fig. 4C and D). These data suggest that PTEN/Akt is important for the Met-mediated suppression of proliferation, invasion and metastasis in OS cells.