Metformin was obtained from Sigma Chemical Co. (St. Louis, MO, USA). RPMI 1640 medium was obtained from Gibco BRL (Gaithersburg, MD, USA). Penicillin and streptomycin were obtained from North China Pharmaceutical Group Corp (Shijiazhuang, China)). TRIzol reagent and crystal violet were obtained from Solarbio (Beijing, China). 0.05% trypsine-EDTA and fetal bovine serum (FBS) were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA).

The B16F10 melanoma cell line was obtained from KeyGen Biotech (Nanjing, China) and cultured in RPMI 1640 medium, containing 10% FBS, 100 g/ml streptomycin and 100 U/ml penicillin at 37°C in a humidified atmosphere of 5% CO₂. The B16F10 cells were cultured in a 6-well plate (1×10⁶ cells/well) for RNA isolation and western blot analysis, and incubated in a 96-well flat-bottom plate (1×10⁴ cells/well) for cell viability analysis. Trypsin/EDTA was used to prepare cells for the experiments and cells were rested for 24 h in FBS-free medium prior to treatment with the indicated concentrations of metformin (specified for each experiment) for 24 h.

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine whether metformin altered cell proliferation. Cells were seeded at a density of 1×10⁴ cells/well in a 96-well plate. The cells were treated with metformin at various concentrations (0, 1, 2.5 or 5 mM) for 24 h. Following incubation, cells were incubated with the MTT solution (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 0.5 mg/ml for 4 h, 37°C in a humidified atmosphere of 5% CO₂. Subsequently, the cells were lysed in dimethyl sulfoxide and the absorbance at 570 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

The wound healing assay was conducted as described previously, by Liang et al (19). B16F10 cells were seeded in a 6-well plate and grown to confluent monolayers. Next, the B16F10 cells were cultured in RPMI-1640 medium without FBS for 12 h. A 200 µl pipette tip was drawn across the center of the well in order to produce a wound area and washed twice with serum-free RPMI 1640 to remove loose cells. The cells were then treated with a medium containing different concentrations of metformin (2.5 or 5 mM) and 1% FBS (1% FBS permits cell survival but not cell proliferation). Subsequently, images of the wound healing process were photographed digitally (x100) at 0, 12 and 24 h. Each value was derived from three randomly selected fields.

B16F10 melanoma cells were incubated in RPMI 1640 with 10% FBS and collected by trypsinization. A cell suspension (200 µl of 5×10⁵ cells/ml with 1% FBS), containing various concentrations of metformin (1, 2.5 or 5 mM) was added into the inner cup of a 24-well Transwell chamber, which had been coated with 50 µl of Matrigel™ (BD Biosciences, Franklin Lakes, NJ, USA; 1:4 dilution in serum-free medium). The medium, supplemented with 10% serum, was added to the outer cup. After 24 h, non-invading cells were removed from the upper surface by gently rubbing with a cotton-tipped swab. Cells that had migrated through the Matrigel and the 8-µm pore membrane, were fixed with 3% paraformaldehyde for 15 min, stained with crystal violet for 30 min and then counted in five random microscopic fields of the lower filter surface. Each experiment was performed in triplicate.

A 24-well Transwell chamber (Corning Life Sciences, Corning, New York, NY, USA) with an 8-µm-pore PET membrane, was used to conduct the migration assay. The lower chamber was filled with 600 µl RPMI 1640, containing 10% FBS. Subsequently, 200 µl B16F10 melanoma cells suspension (5×10⁵ cells/ml with 1% FBS), containing various concentrations of metformin (1, 2.5 or 5 mM) were added into the insert. The cells were allowed to migrate at 37°C with 5% CO₂ over 24 h. Non-migrating cells were removed from the upper surface of the inserts by gently rubbing with a cotton-tipped swab. Cells that had migrated to the lower surface of the inserts were washed, fixed and stained with crystal violet. Quantitative OD at a wavelength of 570 nm of crystal violet staining dissolved in 33% acetic acid, represented migrated cells. Experiments were performed at least in triplicate.

Immunocytochemistry was performed in order to determine the expression of E-cadherin in B16F10 melanoma cells. Briefly, sterilized slides were placed into a 24-well flat-bottomed plate. B16F10 melanoma cells with 1% FBS, containing various concentrations of metformin (0, 1, 2.5 and 5 mM), were added into the 24-well plate (1×10⁵ cells/well); the total volume of each well was 500 ml. Following a 24 h incubation, slides were fixed with 3% paraformaldehyde for 15 min and incubated in 3% hydrogen peroxide to inhibit endogenous peroxidase activity. After blocking with 5% normal goat serum, a rabbit polyclonal primary antibody against E-cadherin (cat. no. ZS-7870; ZSGB-BIO, Beijing, China) was used at a 1:50 dilution and incubated overnight at 4°C. Subsequently, a polyclonal peroxidase-conjugated goat anti-rabbit antibody (cat. no. ZDR-5306, ZSGB-BIO) was added at a 1:500 dilution and incubated for 1 h at room temperature (25°C). Finally, slides were developed in a substrate solution of DAB (Vector Laboratories, Burlingame, CA, USA) and counter-stained with hematoxylin. All Slides were examined and photographed under a light microscope.

Total RNA was extracted from B16F10 cells that had been treated with various concentrations of metformin (0,1, 2.5 or 5 mM) for 24 h. An RNA extraction kit (Solarbio,) was used for the extraction of total RNA. The concentration of total RNA was quantified by measuring the optical density (OD) at 260 nm and RNA integrity was confirmed using denaturing agarose gel electrophoresis. Reverse transcription was conducted using a PrimeScript™ RT Master Mix kit (Takara, Dalian, China), according to the manufacturer's instructions. RT-qPCR was conducted in the Stratagene Mx3000P (Stratagene, La Jolla, CA, USA) using a SYBR® Premix DimerEraser™ kit (Takara). By detecting the increase in the level of the reporter dye (SYBR), the PCR product was monitored continuously during the reaction using MxProTMQPCR software (Stratagene, La Jolla, CA, USA). The cycling conditions were in two stages. An initial denaturation step at 95°C for 30 sec for one cycle. The PCR amplification step was 40 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec. The expression of E-cadherin mRNA in the treated cells was compared to that in the control cells at each timepoint, using the comparative cycle threshold (Ct) method. The following primers were used: Forward, 5′-ATTGCAAGTTCCTGCCATCCTC-3′ and reverse, 5′-CACATTGTCCCGGGTATCATCA-3′ for E-cadherin and forward, 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ and reverse, 5′-ATGGAGCCACCGATCCACA-3′ for β-actin. According to the manufacturer's instructions, the quantity of each transcript was calculated and normalized to the quantity of β-actin, as an internal standard.

B16F10 melanoma cells (1×10⁶) in the exponential phase of growth, were plated in a 6-well plate and treated with metformin (0, 1, 2.5 or 5 mM). Following treatment, cells were collected and washed with phosphate-buffered saline. The total protein was extracted using a RIPA lysis buffer kit (BestBio, shanghai, China), on ice for 30 min. The lysates were collected and centrifuged at 12,000 × g for 30 min at 4°C. Protein concentrations were detected using a bicinchoninic acid protein assay kit (Multisciences, Hangzhou, China). Aliquots of the lysates were boiled for 5 min, electrophoresed on 10% SDS-PAGE gels and transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany). The membrane was blocked with 1% BSA at room temperature for 1 h and then probed with primary polyclonal antibodies to E-cadherin at a 1:50 dilution (cat. no.ZS-7870; ZSGB-BIO) and β-actin at a 1:1,000 dilution (cat. no. sc-7210; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C, followed by incubation with the HRP-labeled goat-anti-rabbit secondary antibodies at a 1:5,000 dilution (KPL, Gaithersburg, MD, USA) for 1 h at 25°C. Finally, protein bands were detected using an enhanced chemiluminescence Western blotting detection kit (Santa Cruz Biotechnology, Inc.). The bands were quantified with a Gel Doc 2000 system and Quantity One software, version 4.6 (Bio-Rad Laboratories), and expressed as a ratio of the quantity of E-cadherin to β-actin, followed by standardization, with the ratio of the normal control set as 1.

All experiments were repeated at least three times. The mean ± standard deviation was calculated for each group, and data were analyzed using one-way analysis of variance followed by a post hoc least significant differences multiple comparison test. Statistical analysis was performed using SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA). P≤0.05 was considered to indicate a statistically significant difference.

The cytotoxicity of metformin in the B16F10 murine melanoma cell line was evaluated by treating the cells with metformin, at concentrations of 0, 1, 2.5 or 5 mM for 24 h. As shown in Fig. 1, no significant cytotoxicity was observed. Since metformin was not cytotoxic in B16F10 cells at 5 mM, metformin were used at concentrations of 5 mM or lower in subsequent experiments.

Increased cell motility is a characteristic that is associated with malignancy. The ‘wound’ repair model was used to evaluate the effect of metformin on the motility of B16F10 cells. As shown by the wound healing assay (Fig. 2), in the group treated with metformin the rate of cell migration into the wounded area was significantly reduced compared with that in the control group (control group, 51.2 and 33%; 2.5 mM group, 80.8 and 56.6%; and 5 mM group, 94.7 and 81.6% at 12 and 24 h, respectively). Furthermore, Transwell migration and Matrigel invasion assays were used to investigate the inhibitory effects of metformin on the invasive potency of cells. The Matrigel invasion assay, a three dimensional model, showed that metformin significantly suppressed B16F10 cell invasion, while 1 mM metformin inhibited the invasion of cells by 17%, 2.5 mM inhibited invasion by 36% and 5 mM by 47% (Fig. 3A and B). In the Transwell migration assay, a significantly reduced number of migrating cells was observed when cells were treated with metformin for 24 h, compared with that in the control group (Fig. 3C). These results suggested that non-toxic concentrations of metformin inhibit the motility, invasion and migration of B16F10 melanoma cells in a dose-dependent manner.

E-cadherin is involved in the progression of cancer. The results of the immunocytochemistry experiment demonstrated that the level E-cadherin was markedly reduced in B16F10 cells following 24 h treatment with metformin, compared with that in the control group (Fig. 4A). Western blot analysis showed that E-cadherin expression was significantly suppressed by metformin at concentrations of 1, 2.5 and 5 mM, compared with the control group (all P<0.05; Fig. 4B). RT-qPCR demonstrated that the level of E-cadherin mRNA in B16F10 cells was significantly reduced in the groups treated with metformin at concentrations of 1, 2.5 and 5 mM, compared with that in the control group (all P<0.05; Fig. 4C).