Human colon carcinoma RKO cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (Serana Europe GmBH, Pessin, Germany) containing 10% fetal bovine serum (Biotechnics Research, Inc., Lake Forest, CA, USA; catalog no. 7101). Metformin was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany; catalog no. D150959). Compound C (catalog no. 171260) and MG132 (catalog no. 474790) were purchased from EMD Millipore (Billerica, MA, USA). Wnt3a was purchased from R&D Systems Inc. (Minneapolis, MN, USA; catalog no. 5036-WN).

Cells were seeded into 96-well microplates at a density of 1×10⁴ cells/well and incubated with 0, 0.05, 0.1, 0.5, 1, 2.5, 5, 10 or 20 mM metformin for 24 h at 37°C in an atmosphere containing 5% CO₂. Following incubation with the test compound, the medium was removed and the cells were incubated with 5 µl MTT solution (5 mg/ml MTT in PBS) for 1 h followed by solubilization in dimethyl sulfoxide. The purple formazan dye converted from MTT by viable cells was quantified by measuring the absorbance at a wavelength of 595 nm.

The 5-bromodeoxyuridine (BrdU) incorporation assay was performed using a BrdU Cell Proliferation ELISA kit (Roche Applied Science, Rotkreuz, Switzerland), according to the manufacturer's protocol. Absorbance at 370 nm was measured using an ELISA reader (SpectraMax plus 384; Molecular Devices, LLC, Sunnyvale, CA, USA).

Intracellular ATP levels were measured using a Luminescence ATP Detection assay system (ATPlite; PerkinElmer, Inc., Waltham, MA, USA, catalog no. 6016941), according to the manufacturer's protocol. Cells were cultured into 96-well microplates at a density of 1×10⁴ cells/well for 16 h at 37°C. The cells were then treated with 100 µl culture medium containing 0, 5, 10 or 20 mM metformin for 24 h at 37°C. After 24 h of incubation, 50 µl mammalian cell lysis solution from the Luminescence ATP Detection assay system was added to each well and the plate was placed in an orbital shaker at 55 × g for 5 min. Subsequently, 50 µl substrate solution from the Luminescence ATP Detection assay system was added and the plate was placed in an orbital shaker at 55 × g for 5 min. ATP amount was normalized by the cell number, which was assessed by BrdU assay.

RKO cells were washed with ice-cold PBS. Total protein was then extracted using protein lysis buffer (20 mM HEPES-KOH (pH 7.4), 1 mM EGTA, 50 mM KCl and 2 mM MgCl₂) with protease inhibitor cocktail and 1 mM dithiothreitol. The protein concentration was determined with a DC protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA; catalog no. 500-0113). Proteins (10 µg) were loaded on 9% SDS-PAGE gels and then electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were blocked with 10% skim milk at room temperature for 1 h. The proteins were then subjected to immunoblot analysis using specific antibodies. Primary antibodies were added at 4°C for 16 h, followed by horseradish peroxidase-conjugated secondary antibodies, including goat anti-rabbit IgG (Bio-Rad Laboratories, Inc.; 1:3,000; catalog no. 170-6515) or goat-anti-mouse IgG (Bio-Rad Laboratories, Inc.; 1:3,000; catalog no. 172-1011) at room temperature for 1 h. Proteins were visualized using enhanced chemiluminescent reagent (PerkinElmer, Inc; catalog no. NEL120001EA). The following primary antibodies were used: phosphorylated (p)-AMPKα1 (Thr172) (1:1,000; catalog no. 2535s; Cell Signaling Technology, Inc., Danvers, MA, USA), p-β-catenin (Ser33/37) (1:500; catalog no. 9561s; Cell Signaling Technology, Inc.), p-β-catenin (Ser552) (1:1,000; catalog no. 9566s; Cell Signaling Technology, Inc.), β-catenin (1:1,000; catalog no. 610154; BD Biosciences, San Jose, CA, USA), lamin A/C (1:1,000; catalog no. 612163; BD Biosciences), AMPKα1/2 (1:1,000; catalog no. sc-74461; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); β-actin (1:1,000; catalog no. sc-sc-47778; Santa Cruz Biotechnology, Inc.) and GAPDH (1:1,000; catalog no. sc-32233; Santa Cruz Biotechnology, Inc.).

RKO cells were plated into XF24 cell culture microplates (Seahorse Bioscience, North Billerica, MA, USA) at a density of 20,000 cells/well. The cells were then treated with 10 mM at 37°C in an atmosphere containing 5% CO₂. Following 24 h of treatment, the cell culture growth medium in the microplate was replaced with warmed Seahorse XF base medium (catalog no. 102353-100; Seahorse Bioscience) using a multichannel pipette. Oligomycin (2 µM), FCCP (0.1 µM), antimycin A (1 µM) and rotenone (1 µM), from the Agilent Seahorse XF Cell Mito Stress Test kit (Agilent Technologies, Inc., Santa Clara, CA, USA; catalog no. 103015-100) were then consecutively added to the each well. The cells were then incubated at 37°C in an incubator with 0% CO₂ for 45 min to 1 h prior to the assay. OCR was measured using an XF24 Analyzer (Seahorse Bioscience). Measured values were normalized by cell number, which was assessed by BrdU assay.

Cells were seeded onto cover glasses in a 12-well plate. After 16 h the cells were treated with 10 mM metformin at 37°C in an atmosphere containing 5% CO₂. After 24 h, the cells were fixed with 4% paraformaldehyde for 10 min and permeabilized in 0.1% Triton X-100 for 20 min at room temperature. Cells were then blocked with 2% bovine serum albumin for 1 h at room temperature and incubated at 4°C overnight with primary antibodies against AMPK and β-catenin. Following washing, cells were incubated with fluorochrome-conjugated anti-mouse (1:200; catalog no. TI-2000) and anti-rabbit IgG (1:100; catalog no. FI-1000; both from Vector Laboratories, Inc., Burlingame, CA, USA) at room temperature for 1 h. Subsequently, nuclei were stained with 10 µM Hoechst 33342 (Invitrogen; Thermo Fisher Scientific, Inc.; catalog no. H1399) for 10 min at room temperature, and visualized using a fluorescence microscope (magnification, ×400) and a digital camera.

Cells were cultured in a 100 mm culture dish for 24 h and then treated with 10 mM metformin for 24 h at 37°C in an atmosphere containing 5% CO₂. Cells were harvested by centrifugation at 1,000 × g at 4°C for 10 min, washed with PBS, and lysed with protein lysis buffer. Cell extracts were then incubated with Protein G agarose beads at 4°C for 3 h. Following washing with lysis buffer three times, extracts were incubated with an anti-AMPKα1/2 antibody at 4°C overnight. Following washing with lysis buffer, SDS-loading buffer containing β-mercaptoethanol was added and the samples were boiled for 5 min. The proteins [whole cell lysate (WCL), 10 µg; and immunoprecipitation (IP), 10 µl] were loaded on 9% SDS-PAGE gels and then electrophoretically transferred to polyvinylidene fluoride membranes. Subsequently, the membranes were blocked with 10% skim milk at room temperature for 1 h. The proteins were then subjected to immunoblot analysis using AMPK1/2α, β-catenin and β-actin antibodies. Primary antibodies were added for 16 h at 4°C followed by horseradish peroxidase-conjugated goat-anti-mouse IgG secondary antibodies (Bio-Rad Laboratories, Inc.; 1:3,000; catalog no. 172-1011) at room temperature for 1 h. Proteins were visualized using enhanced chemiluminescent reagent (PerkinElmer, Inc; catalog no. NEL120001EA).

Data are presented as the mean ± standard deviation. All data were analyzed using SPSS 20.0 software (IBM Corp., Armonk, NY, USA). Comparisons between groups were performed using Student's t-test or one-way analysis of variance followed by Duncan's multiple-range test. P<0.05 was considered to indicate a statistically significant difference. All experiments were conducted in triplicate.

The effects of metformin on cell proliferation and energy production in colon cancer RKO cells were examined. RKO cells were treated with metformin at low to high concentrations for 24 h. AMPK activation was observed in the groups treated with metformin at 100 µM to 20 mM and cell growth inhibition was observed in groups treated with 5–20 mM metformin (Fig. 1). Therefore, the concentrations of metformin that effectively activated AMPK and inhibited cell growth were selected for further experiments. Treatment with metformin at different concentrations (5–20 mM) for 24 h significantly inhibited RKO cell viability (Fig. 2A and B) and reduced ATP production (Fig. 2C). Subsequent analysis demonstrated that metformin increased the expression level of pAMPK, but decreased the expression level of β-catenin (Fig. 2D). AMPK is a master regulator of cellular energy homeostasis (6); therefore, AMPK may be activated by the reduction of cellular ATP. Several types of cancer cell have demonstrated increased energy metabolism through glycolysis and/or oxidative phosphorylation with increased production of ATP (14). In metformin-treated RKO cells in the present study, mitochondrial oxidative phosphorylation and glycolysis were reduced compared with that in the controls (Fig. 2E and F).

β-catenin is a major effector of Wnt signaling as it transcriptionally regulates the expression of c-myc, c-Jun and cyclin D1 to increase cell proliferation and oncogenesis (15). Upon activation of Wnt signaling, β-catenin binds to Tcf and translocates into the nucleus (16). The suppression of β-catenin translocation by metformin was evaluated according to the status of AMPK activity (Fig. 3). Treatment with metformin decreased nuclear β-catenin expression, according to immunoblotting and immunofluorescence assays (Fig. 3A and C), which suggests that a decrease of cytosolic β-catenin resulted in β-catenin nuclear translocation. A subsequent immunoprecipitation assay demonstrated the association between AMPK and β-catenin following metformin treatment. Co-localization of AMPK with β-catenin was confirmed (Fig. 3B and C). These results suggest that AMPK can bind to β-catenin in the cytosol and such binding may suppress translocation of β-catenin into the nucleus.

Cytoplasmic β-catenin is complexed with adenomatous polyposis coli, axin and glycogen synthase kinase (GSK), and subjected to proteasomal degradation following GSK-mediated phosphorylation of β-catenin (17). Serine 33/37 phosphorylation of β-catenin is mediated by GSK3β. Phosphorylation at this site leads to ubiquitination of β-catenin, which acts as a bridge for proteasomal degradation (18). In addition, it has been reported that serine 552 phosphorylation can facilitate β-catenin translocation from the cytoplasm to the nucleus (18). The present study examined whether the AMPK-mediated decrease in β-catenin expression may be attributable to proteasomal degradation. In the presence of MG132, metformin still activated AMPK; however, it failed to reduce β-catenin expression. Instead, a ubiquitination laddering pattern was produced (Fig. 4). Since β-catenin phosphorylation on residues serine 33/37 was observed in metformin-treated cells, GSK-mediated β-catenin phosphorylation and subsequent proteosomal degradation may have adequately occurred. In addition, β-catenin phosphorylation on serine 552 was observed in metformin-treated cells (Fig. 4). These results suggest that an association of β-catenin with AMPK may sequester β-catenin in the cytoplasm, even when serine 552 is phosphorylated. This cytoplasmic sequestering of β-catenin may subject β-catenin to degradation.

Metformin-induced suppression of cell proliferation was partially restored by inhibition of AMPK using compound C (Fig. 5A). Subsequently, the current study examined whether metformin could suppress RKO cell proliferation and inhibit β-catenin expression when Wnt signaling was hyperactivated. It was identified that Wnt-treatment increased cell proliferation. This increase in cell proliferation was significantly inhibited by co-treatment with metformin (Fig. 5B). β-catenin expression was markedly lower in cells co-treated with Wnt and metformin compared with that in cells treated with Wnt alone (Fig. 5C). Wnt treatment did not affect the phosphorylation status of AMPK (Fig. 5C). To determine whether these effects were attributable to AMPK, cells were treated with compound C, an AMPK inhibitor. The results revealed that the metformin-induced decrease in β-catenin expression level was restored following treatment with compound C (Fig. 5D). These results suggest that metformin-induced AMPK activation can regulate β-catenin expression.