The Ishikawa (IK, well-differentiated) and HEC-1B (moderately differentiated) human EC cell lines, provided by Professor LH Wei (Peking University People’s Hospital, Beijing, China), were maintained in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM)/F12 with 10% fetal bovine serum (FBS) at 37°C in an atmosphere containing 5% CO₂. The cell cultures were routinely passaged every 3–5 days. Metformin and PPP (an IGF-1R inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA). IGF-1 and IGF-2 were purchased from Sigma-Aldrich and R&D Systems (Minneapolis, MN), respectively. Compound C (an AMPK inhibitor) was obtained from Calbiochem (Merck Millipore, Billerica, MA, USA). Metformin was diluted in phosphate-buffered saline (PBS) as a stock solution at a concentration of 100 mM.

The IK and HEC-1B cells were plated at a density of 2×10⁵ cells/well in six-well plates for 24 h and were then treated with metformin (1, 10 or 100 μM) in the presence or absence of compound C (1 μM) in phenol red-free DMEM/F12 containing 3% steroid-stripped FBS, produced using dextran-coated charcoal (DCC-FBS) for 72 h. Total RNA was extracted from cells with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was subjected to DNase I digestion to prevent possible genomic DNA contamination and then reverse-transcribed with oligo-dT primers and M-MLV Reverse Transcriptase (Promega Corporation, Madison, WI, USA). qPCR was conducted using SYBR Green sequence detection reagents (Takara Bio, Inc., Shiga, Japan) in a 20 μl reaction volume containing 1 μl cDNA, 10 μl mix, 0.4 μl Rox and 1 μl of each primer (5 μM stock). The primer sequences were as follows: IGFBP-1 forward: 5′-CTATGATGGCTCGAAGGCTC-3′; IGFBP-1 reverse: 5′-TTCTTGTTGCAGTTTGGCAG-3′; IGF-1R forward: 5′-AAGGCTGTGACCCTCACCAT-3′; IGF-1R reverse: 5′-CGATGCTGAAAGAACGTCCAA-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward: 5′-CAGTCAGCCGCATCTTCTTTT-3′, GAPDH reverse: 5′-GTGACCAGGCGCCCAATAC-3′; GAPDH forward: 5′-CTCTCTGCTCCTCCTGTTCG-3′, GAPDH reverse: 5′-TTGATTTTGGAGGGATCTCG-3′. The PCR cycling conditions were as follows: 95°C for 30 sec followed by 40 cycles of two steps at 95°C for 5 sec and 60°C for 31 sec. Fluorescent signals were detected using an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA) and the accumulation of PCR product was measured in real-time as the increase in SYBR green fluorescence. qPCR was performed in triplicate for each sample. The obtained IGF-1R and IGFBP-1 mRNA levels were calculated by normalizing the threshold cycle (Ct) of IGF-1R and IGFBP-1 to the Ct of GAPDH. The relative levels of IGF-1R and IGFBP-1 mRNA were calculated by normalizing the threshold cycle (Ct) to the Ct of GAPDH, which served as a control, using the following formula: 2-^ΔΔCt. The relative levels of mRNA were then expressed as a ratio, compared with that of the control (metformin in the absence or presence of compound C).

The IK and HEC-1B cells were plated at a density of 2×10⁵ cells/well in six-well plates for 24 h and were then treated with metformin (1, 10 or 100 μM) in the presence or absence of compound C (1 μM) in phenol red-free DMEM/F12 containing 3% DCC-FBS for 72 h to observe the changes in IGF-1R and IGFBP-1 protein levels. Cell lysates were prepared using RIPA buffer containing 1% NP40, 0.5 sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS). Subsequently, 20 μg of each protein extract was subjected to SDS-polyacrylamide gel electrophoresis in 10% gels and subsequently electrotransferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk and 0.1% Tween-20 for 1 h at room temperature (RT) with constant agitation, and then incubated with primary monoclonal rabbit anti-human GAPDH (1:1,000; Cell Signaling Technology, Danvers, MA, USA), monoclonal rabbit anti-human IGFBP1 (1:1,1000; Cell Signaling Technology) and monoclonal rabbit anti-human IGF-1R (1:1,1000; Cell Signaling Technology) antibodies overnight at 4°C. Subsequent to washing three times for 5 min each with PBS and Tween-20 (PBST), the membranes were incubated with secondary polyclonal goat anti-rabbit horseradish peroxidase-linked antibody (1:2,000; Cell Signaling Technology) for 2 h. The membranes were then washed again three times for 5 min each with PBST, and bands were visualized by enhanced chemiluminescence using the Pierce ECL Plus Western Blotting Substrates (32132, 32134) according to the manufacturer’s instructions (Pierce Biotechnology, Inc., Rockford, IL, USA). Subsequent to development, the membranes were stripped and reprobed using antibodies against GAPDH (1:1,000; Cell Signaling Technologies) to confirm equal loading. The relative protein expression levels was normalized to the GAPDH expression levels and are expressed as the ratio of treated versus untreated cells. Protein bands, including those of GAPDH, were quantified by densitometry with the Quantity One imaging program (Bio-Rad, Hercules, CA, USA).

Cell proliferation assays were performed using a 5-bromodeoxyuridine (BrdU)-enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics GmbH, Mannheim, Germany). The IK and HEC-1B cells were plated into 96-well plates at 8×10³ or 1×10⁴ cells/well, respectively. At 24 h after plating, the cells were serum-starved for an additional 24 h and were then treated with increasing concentrations of metformin (0.1, 1, 10 or 100 μM) in the absence or presence of PPP (0.5 or 1 μM) for 72 h. The effects of metformin and PPP treatment were calculated as the percentage of control cell growth obtained in PBS- or DMSO-treated cells grown in the same 96-well plates. Assays were performed under serum-free conditions. DNA synthesis was monitored as determined by the incorporation of BrdU into DNA, which was detected by immunoassay according to the manufacturer’s instructions (Roche Diagnostics GmbH). Briefly, following incubation, the cells were incubated again with 10 μl/well BrdU labeling solution for an additional 2 h at 37°C. The labeling medium was removed, 200 μl/well FixDenat was added and the cells were incubated for 30 min at 20°C. Subsequently, the FixDenat solution was removed completely and the cells were incubated with 100 μl/well anti-BrdU POD working solution for 90 min at 20°C. The antibody conjugate was removed and the cells were rinsed three times with washing solution. Following removal of the washing solution, 100 μl/well substrate solution was added and the cells were incubated at 20°C for 20 min, followed by incubation with 25 μl 1 M H₂SO₄ for 1 min on a shaker at 100 × g. The absorbance of the samples was measured using the Fluostar Optima ELISA reader (BMG Labtech GmbH, Ortenberg, Germany) at 450 nm (reference wavelength, 690 nm). Each experiment was performed in triplicate and repeated three times to assess the consistency of the results. The BrdU assay results were compared using MTT assays and the validity of the findings was confirmed (data not shown).

The apoptotic cells were detected in situ using a Roche TUNEL kit (Roche Diagnostics GmbH). TUNEL was conducted according to the manufacturer’s instructions to visualize the 3’-OH ends of DNA fragments in apoptotic cells. Subsequent to xylene dewaxing, the sections were rinsed three times in distilled water for 5 min and then dipped in methanol containing 0.3% H₂O₂ at RT for 30 min to inhibit endogenous peroxidase activity. Following rinsing in PBS three times at RT for 5 min, the sections were treated with proteinase K (Sigma-Aldrich Chemie GmbH, Mannheim, Germany) at 37°C for 8 min. The sections were then rinsed again in PBS three times at RT for 5 min, soaked in TdT buffer for 10 min and then incubated at 37°C for 60 min in a moist chamber with 50 μl TdT buffer. Subsequent to rinsing in PBS three times at RT for 5 min, the sections were placed in 50 μl fluorescein isothiocyanate (Roche Diagnostics GmbH) and then incubated at 37°C for 40 min. Following a further three 5-min rinses in PBS, the sections were dipped in 3,3′-diaminobenzidine (Roche Diagnostics GmbH) at RT for 3 min and the reaction was observed under a microscope (Olympus IMT-2; Olympus Corporation, Tokyo, Japan). The reactions were terminated with distilled water and the nuclei were counterstained with hematoxylin buffer. Normal nuclei were stained blue by DAPI, and apoptotic nuclei were stained green using TUNEL. The number of apoptotic cells was then calculated as a percentage of the total cells.

All data are presented as the mean ± standard error of the mean. The data were analyzed by one-way analysis of variance using SPSS software (version 13.0; SPSS, Inc., Chicago, IL, USA) and P<0.05 was considered to indicate a statistically significant difference.

The expression levels of IGF-1R mRNA and protein in IK and HEC-1B cells following treatment with metformin and/or compound C were analyzed. Metformin markedly reduced IGF-1R mRNA and protein expression levels in a concentration-dependent manner in the two cell lines. The most evident effect was observed following 100 μM metformin treatment. This inhibitory effect was partially reversed by compound C treatment (Figs. 1 and 2).

The effects of metformin and compound C on IGFBP-1 expression levels in IK and HEC-1B cells were analyzed. Metformin markedly increased IGFBP-1 mRNA and protein expression levels in a concentration-dependent manner in the two cell lines. Similar to IGF-1R, the most marked effect was detected following 100 μM metformin treatment. This increase was partially reversed by compound C treatment (Figs. 1 and 2).

The effects of metformin with or without PPP on the proliferation of IK and HEC-1B EC cells were examined. As shown in Fig. 3, 0.5 and 1 μM PPP significantly inhibited the proliferation of EC cells (P<0.01). In addition, using BrdU incorporation assays, PPP was found to enhance the inhibitory effects of metformin on cell proliferation. The greatest effect was observed when using 1 μM PPP combined with 10 μM metformin.

The effects of high-concentration metformin on apoptosis in EC cells were examined using TUNEL assays. The data demonstrated that incubation of IK and HEC-1B cells with 1 or 2 mM metformin significantly increased the rate of apoptosis, compared with that of the control (P<0.01; Fig. 4).