Metformin (1,1-dimethylbiguanide) was purchased from Astellas Pharma (Tokyo, Japan). The Cell Counting Kit (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan) and all other chemicals were obtained from Sigma Chemical (Tokyo, Japan).

In this study, the antibodies used were as follows: anti-β-actin monoclonal antibody (A5441, used at 1:3,000; Sigma-Aldrich), cyclin D1 (RB-9041, used at 1:1,000; Thermo Fisher Scientific, Waltham, MA), cyclin E (used at 1:1,000; BD Biosciences, Franklin Lakes, NJ), Cdk6 (sc-177, used at 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), Cdk4 (no. 2906, used at 1:1,000; Cell Signaling Technology, Danvers, MA), Cdk2 (sc-163, used at 1:2,000; Santa Cruz Biotechnology), and phosphorylated Rb (pRb) (no. 9309, used at 1:1,000; Cell Signaling Technology). Secondary horseradish peroxidase-linked anti-mouse and anti-rabbit IgG antibodies (used at 1:2,000; GE Healthcare, Buckinghamshire, UK) were also used.

The human ESCC cell lines KYSE30, KYSE70 and T.T were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). KYSE30 and KYSE70 cells were grown in Ham’s F12 (Wako, Osaka, Japan)/RPMI-1640 (Gibco Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine serum (533–69545; Wako), penicillin-streptomycin (100 mg/l; Invitrogen) in a humidified atmosphere of 5% CO₂ at 37°C. T.T cells were grown in DMEM/Ham’s F12 medium (Wako) with 10% fetal bovine serum, penicillin-streptomycin (100 mg/l) in a humidified atmosphere of 5% CO₂ at 37°C.

Cell proliferation assays were performed using the CCK-8 according to the manufacturer’s instructions. Each cell type (1×10⁴) was seeded into a well of a 96-well plate and cultured in 100 μl of each culture medium. After 24 h, seeding cells were treated by addition of 1, 5 or 10 mM metformin into the culture medium or left untreated. At the indicated time points, the medium was exchanged for 110 μl of each culture medium with CCK-8 reagent (10 μl CCK-8 and 100 μl each culture medium), and the cells were incubated for 2 h. Absorbance was measured at a wavelength of 450 nm using an auto-microplate reader.

The lysate was performed according to the methods described in our previous reports (10,11). All steps were carried out at 4°C. Protein concentrations were measured using a dye-binding protein assay based on the Bradford method (12).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (13), and western blot analysis was performed as described by Towbin et al(14) using primary antibodies and HRP-conjugated secondary antibodies. Immunoreactive proteins were visualized with an enhanced chemiluminescence detection system (Perkin Elmer Co., Waltham, MA) on X-ray film.

To evaluate the mechanism of growth inhibition by metformin, the cell cycle profile was analyzed after treatment with metformin. KYSE30 cells (1.0×10⁶ cells in a 6-well plate dish) were treated with 10 mM metformin or left untreated for 24–72 h. After treatment, the cells were harvested and fixed in 80% ethanol. The fixed cells were washed with PBS and then stored at −20°C until flow cytometric analysis was performed. On the day of analysis, cells were washed and centrifuged using cold PBS, suspended in 100 μl PBS and 10 μl RNase A solution (250 μg/ml), and incubated for 30 min at 37°C. Then, 110 μl propidium iodide (PI) stain (100 μg/ml) was added to each tube, which was then incubated at 4°C for at least 30 min prior to analysis. Flow cytometric analysis was performed using a Cytomics FC 500 flow cytometer (Beckman Coulter, Brea, CA) equipped with an argon laser (488 nm). The percentages of cells in different phases of the cell cycle were analyzed by using FlowJo software (Tree Star, Ashland, OR). All experiments were performed in triplicate to assess for consistency of response.

The RayBio™ Human Phospho Array Kit (catalog no. ARY 001) was purchased from RayBiotech Inc. (Norcross, GA). The assay for p-RTK array was performed according to the manufacturer’s instructions. Briefly, p-RTK array membranes were blocked with 5% BSA/TBS (0.01 M Tris-HCl, pH 7.6) for 1 h. Membranes were then incubated with 2 ml of lysate prepared from cell lines or tumorous tissues after normalization with equal amounts of protein. After extensive washing with TBS including 0.1% v/v Tween-20 (3 washings for 10 min each) and with TBS alone (2 washings for 10 min each) to remove unbound materials, the membranes were incubated with anti-phospho-tyrosine-HRP antibody for 2 h at room temperature. The unbound HRP antibody was washed out with TBS including 0.1% Tween-20. Finally, each array membrane was exposed to X-ray film using a chemiluminescence detection system (Perkin Elmer Co.). The density of the immunoreactive band obtained on the p-RTK array was analyzed by densitometric scanning (Tlc scanner; Shimizu Co. Ltd., Kyoto, Japan).

The RayBio™ Human Angiogenesis Antibody Array 1 kit (catalog no. AAH-ANG-1) was purchased from RayBiotech Inc. The assay for array was performed according to the manufacturer’s instructions. Briefly, the angiogenesis antibody membranes were blocked with blocking buffer for 30 min. The membranes were then incubated with 1 ml of lysate prepared from cell lines after normalization with equal amounts of protein. After extensive washing with TBS including 0.1% v/v Tween-20, 3 times for 10 min, and TBS alone, 2 times for 10 min, to remove unbound materials, the membranes were then incubated with anti-phospho-tyrosine-HRP antibody for 2 h at room temperature. The unbound HRP antibody was washed out with TBS including 0.1% Tween-20. Finally, each array membrane was exposed to X-ray film using a chemiluminescence detection system (Perkin Elmer Co.). The density of the immunoreactive band obtained on this array was analyzed by densitometric scanning (Tlc scanner; Shimizu Co. Ltd.).

The samples of cancer cell lines were processed for total-RNA extraction with a miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA samples typically showed A260/280 ratios of between 1.9 and 2.1, as determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

After RNA measurement with an RNA 6000 Nano kit (Agilent Technologies), the samples were labeled using a miRCURY Hy3/Hy5 Power labeling kit and were hybridized on a human miRNA Oligo chip (v.14.0; Toray Industries, Tokyo, Japan). Scanning was performed with a 3D-Gene Scanner 3000 (Toray Industries). 3D-Gene extraction version 1.2 software (Toray Industries) was used to read the raw intensity of the image. To determine the change in miRNA expression between metformin-treated and control samples, the raw data were analyzed via GeneSpringGX v10.0 (Agilent Technologies). Samples were first normalized relative to 28sRNA and baseline-corrected to the median of all samples.

Replicate data were consolidated into two groups: those from metformin-treated cells and those from control cells, and were organized by using the hierarchical clustering and analysis of variance (ANOVA) functions in the GeneSpring software. Hierarchical clustering was done by using the clustering function (condition tree) and Euclidean correlation as a distance metric. Two-way ANOVA analysis and asymptotic p-value computation without any error correction on the samples were performed to search for the miRNAs that varied most prominently across the different groups. The p-value cutoff was set to 0.05. Only changes >50% in at least one of the time points for each sample were considered significant. All the analyzed data were scaled by global normalization. The statistical significance of differentially expressed miRNAs was analyzed by Student’s t-test.

All analyses were performed using the computer-assisted JMP8.0 (SAS Institute, Cary, NC). Paired analysis between the groups was performed using Student’s t-test. A p-value of 0.05 was considered to indicate a significant difference between groups.

To evaluate the effect of the growth activity of metformin on human ESCC cells in vitro, we examined metformin’s effect on the proliferation of three esophageal squamous cell carcinoma cell lines: T.T, KYSE30 and KYSE70. To discern the direct relationship between the decrease in cell viability and the inhibition of cell proliferation, we followed the course of proliferation over 3 days after the addition of metformin. Cells were grown in culture medium and treated with 5 or 10 mM metformin or, as a control, without metformin. As shown in Fig. 1, metformin led to a dose-dependent and strong inhibition of cell proliferation in the ESCC cell lines T.T and KYSE30. In KYSE70 cells, although 5 mM metformin did not effect the proliferation of cancer cells, treatment with 10 mM metformin inhibited the proliferation of cells. These results suggest that metformin inhibits ESCC proliferation.

To study whether or not metformin affects the cell cycle in KYSE30 cells, western blot analysis was used to examine the expression of various cell cycle-related molecules in KYSE30 with and without metformin treatment. Cells were treated with 10 mM metformin or left untreated for 24–72 h. The most remarkable change was the loss of cyclin D1, a key protein implicated in the transition of the G0/G1 phase. In short, the cyclin D1 level was already reduced at 24 h after the addition of metformin and was no longer detectable at 48 and 72 h (Fig. 2). We then studied the expression of other cell cycle-related proteins (Cdk4, Cdk6, cyclin E and Cdk2) implicated in the G0/G1 transition. Cdk4 and Cdk6, the catalytic subunit of cyclin D1, was slightly decreased at 48 and 72 h after the addition of metformin. Cyclin E was decreased at 72 h after the addition of metformin. The catalytic subunit of cyclin E, Cdk2, was also slightly decreased at 48 and 72 h after the addition of metformin. The level of pRb also decreased progressively in metformin-treated cells. These events were also detected in the two other cancer cell lines, i.e., T.T and KYSE70 (data not shown). The amount of β-actin (an internal control for protein loading) was almost the same in each lane in sodium dodecyl sulfate polyacrylamide gel electrophoresis (Fig. 2).

To further investigate the inhibition of KYSE30 cell proliferation in the presence of metformin, the cell cycle progression was examined by flow cytometry. We treated proliferating KYSE30 cells with 10 mM metformin for different durations. After the addition of 10 mM metformin, an increasing number of cells started to accumulate in G0/G1, 55.7% after 48 h and 64.6% by 72 h (Fig. 3A). In parallel, we observed reductions in the percentage of cells in the S phase and G2/M phase (Fig. 3B). These data suggest that metformin inhibits the cell cycle progression from G0/G1 into S phase, resulting in G1 cell cycle arrest.

Having established the antitumor effects of metformin in the ESCC cell lines, we next used a phosphorylated-RTK array system to identify the ‘key’ RTKs in terms of these antitumor effects. By using an antibody array (Fig. 4A), we simultaneously screened the expression of 42 different RTKs in KYSE30 cells with or without metformin. The results showed that the expression of phosphorylated-epidermal growth factor (p-EGFR) (Fig. 4B) was reduced by the treatment of metformin.

The density of the p-EGFR obtained from the membrane array was analyzed by means of a Kodak Image Station (Eastman Kodak, Rochester, NY). The densitometric ratio of the p-EGFR spots of the metformin-treated cell lines to the p-EGFR spots of the cell lines not treated with metformin was reduced to 58.4% (Fig. 4C).

We used an angiogenesis antibody array system to identify the ‘key’ angiogenesis-related protein in terms of the antitumor effect of metformin.

By using the antibody array (Fig. 5A), we simultaneously screened the expression of 20 different angiogenesis antibodies in KYSE30 cells with or without metformin. The expression of interleukin-8 (IL-8), tissue inhibitor of metalloproteinases-1 (TIMP-1) and basic fibroblast growth factor (bFGF) (Fig. 5B) were reduced by the treatment with metformin as detected by the protein array.

The density of the IL-8, TIMP-1 and bFGF obtained from the membrane array was analyzed using the Kodak Image Station (Eastman Kodak). The densitometric ratio of the TIMP-1, IL-8 and bFGF spots from metformin-treated cells to those of cells not treated with metformin were 15.9, 37.4 and 23.3%, respectively (Fig. 5C).

Using a custom microarray platform, we analyzed the expression levels of 985 human miRNA probes in KYSE30 cells with or without metformin treatment in vitro. As shown in Fig. 6 and Table I, when the expression of miRNAs was studied in KYSE30 cells treated with 10 mM metformin and without metformin in vitro, 17 miRNAs were significantly upregulated (Fig. 6 and Table I) in KYSE30 cells after 72 h of metformin treatment, while 45 miRNAs were downregulated (Fig. 6 and Table I) of the 985 miRNAs.

Unsupervised hierarchical clustering analysis, using Pearson’s correlation, showed that KYSE30 cells treated with metformin clustered together and separately from the untreated cells (Fig. 6).