Human CML K562 cells were purchased from the Korea Cell Line Bank (Seoul, Korea), and KU812 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and antibiotic-antimycotic solution (Life Technologies, Carlsbad, CA, USA). The cells were cultured in a humidified incubator at 37°C with 5% CO₂.

Nilotinib was purchased from Cayman (Ann Arbor, MI, USA) and was dissolved in dimethyl sulfoxide. Metformin hydrochloride was purchased from Wako (Richmond, VA, USA) and was dissolved in distilled water. Rabbit antibodies against c-Jun N-terminal kinase (JNK), phosphor-JNK, ERK, phosphor-ERK, p38, phosphor-p38, BCR-ABL, phosphor-BCR-ABL, caspase 3, caspase 8, PARP, and Bid, as well as mouse antibody against caspase 9 were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Mouse antibody against Bcl-2 and rabbit antibody against Bcl-xL were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-actin was obtained from Sigma-Aldrich (St. Louis, MO, USA).

For the cell proliferation assay, we performed an EZ-Cytox assay. Aliquots of cells (8 × 10³) were plated into 96-well plates (200 μL growth medium per well) and then treated with metformin or nilotinib at various concentrations. After 48 hours of incubation, 20 μL of the Kit reagent (EZ-Cytox Kit, DoGEN, Seoul, Korea) was added to each well. Next, the cells were incubated at 37°C for 3 hours, and the absorbance was measured at 450 nm by a microplate reader.

For the trypan blue exclusion assay, centrifuged cells were resuspended in 1 mL of phosphate-buffered saline solution (PBS) and then mixed with a 0.4% trypan blue solution at a 1:1 ratio. The samples were incubated at 25°C for 5 minutes and visualized by light microscopy. At least 200 cells were counted for each survival determination.

Cells were harvested and resuspended in 100 μL of binding buffer provided in the Annexin V-FITC apoptosis detection kit (BioBud, Seoul, Korea). Cells were mixed with 1.25 μL Annexin V-FITC reagent and 10 μL propidium iodide (PI) and incubated for 15 minutes at room temperature in the dark. The staining was terminated by addition of binding buffer and the cells were immediately analyzed by flow cytometry (Beckman Coulter, Atlanta, GA, USA).

After various treatments, the cells were washed with PBS before RIPA buffer (Sigma-Aldrich) was added to the cells. A protease and phosphatase inhibitor cocktail (Sigma-Aldrich) was added to the RIPA buffer before lysing the cells. Protein concentrations were measured with a BCA protein assay reagent (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein were separated by 10% to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then electro-transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL, USA). The membrane was blocked with 5% skim milk in Tris buffered saline (TBS)-Tween20 for 2 hours and incubated with primary antibodies overnight at 4°C. The membrane was then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G as a secondary antibody. Immunoreactive proteins were visualized using a chemiluminescence reagent (DoGen).

Cells were seeded on poly-L-lysine (Sigma-Aldrich) pre-coated cover slips on 12-well plates. After incubation for 24 hours, the cells were treated with 10 nM nilotinib for 48 hours. Next, the cells were harvested, washed with PBS, and fixed with 3.7% formaldehyde at room temperature for 15 minutes. The cells were then stained with DAPI (Invitrogen, Carlsbad, CA, USA) solution at 37°C for 10 minutes in the dark. The cover slips were mounted onto glass microscope slides and examined by fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).

Cells were maintained in suspension cultures and gradually exposed to increasing concentrations of nilotinib, starting with a concentration of 1 nM and ending with a final dose of 10 nM nilotinib. The parental, sensitive cell lines were maintained without nilotinib to be used as controls.

All experiments were repeated at least three times. Statistical analysis was carried out using the GraphPad Prism version 5 software (GraphPad Software, San Diego, CA, USA). The results were expressed as mean with standard error of the mean and evaluated using a paired Student’s t test. The results were considered statistically significant at p < 0.05.

To investigate the anti-proliferative efficacy of metformin and nilotinib, cells were treated as described in the Methods section. As shown in Fig. 1A, both metformin and nilotinib induced cytotoxicity in a dose-dependent manner. However, metformin alone did not significantly affect cell proliferation (Fig. 1A). Next, we investigated the effect of combined treatment with metformin and nilotinib in CML cell lines. First, we observed the effect of 5 mM metformin in combination with 5 nM nilotinib on CML cell morphology under a light microscope. After the combined treatment, cell adhesion to each other significantly decreased in comparison to control or nilotinib alone-treated cells (Fig. 1B). We further examined whether combined treatment with metformin and nilotinib enhances cell death/apoptosis compared to metformin or nilotinib alone in CML cell lines. After combined treatment for 48 hours, the cells were stained with trypan blue. The results showed that the combined treatment induced 40% to 50% cell death (Fig. 1C). To clarify the apoptosis mechanism, we performed a western blotting assay with anti-PARP; anti-caspase 3, 8, and 9; anti-Bcl-xL; anti-Bcl2; and anti-Bid. The results showed that combination treatment induced activation of caspase 3, 8, and 9, which led to an increase in PARP cleavage in K562 cells. Additionally, decreased expression of Bcl-xL (anti-apoptotic protein) was majorly responsible for induction of apoptosis after the treatment (Fig. 1D). Furthermore, when the cells were stained with annexin V and PI, the early apoptotic cell percentage after combination treatment was almost eight-fold higher than that after control treatment (Fig. 1E). Collectively, combination therapy with metformin and nilotinib enhanced apoptosis mediated by an increase in caspase activation by decreasing Bcl-xL expression in nilotinib-sensitive CML cells.

To simulate nilotinib resistance, Philadelphia chromosome-positive cell lines (K562 and KU812) were cultured in the presence of increasing concentrations of nilotinib. After approximately 6 months, the cell lines continued to survive in the presence of 10 nM nilotinib. Almost completely nilotinib-resistant cells were able to grow in the presence of nilotinib at concentrations of up to 10 nM; however, parental cell lines could not grow at this concentration (Fig. 2A). The IC₅₀ of the nilotinib-resistant K562R and KU812R cells to nilotinib was 120.7 and 151.5 nM, respectively, which was six times higher than that of the corresponding parental cell lines (22.86 and 25.85 nM, respectively). Next, the cells were stained with DAPI after being exposed to nilotinib for 48 hours. The result indicated that nilotinib-resistant cells were spherical and had homogeneous nuclei, whereas nilotinib-sensitive cells showed apoptotic bodies (Fig. 2B). The survival of sensitive and resistant cells was measured using trypan blue exclusion assay. The survival of nilotinib-sensitive cells, but not nilotinib-resistant cells, was decreased in a dose-dependent manner by treatment with up to 10 nM nilotinib (Fig. 2C). Lastly, the effect of increasing concentrations of nilotinib on Bcr/Abl phosphorylation and apoptosis was evaluated by western blotting. Phosphorylated Bcr/Abl level was maintained in nilotinib-resistant cells, but not in nilotinib-sensitive cells, after treatment with 10 nM nilotinib. Similar results were observed for PARP, caspase 3, and Bcl-xL (Fig. 2D).

To investigate whether nilotinib-resistant cell lines were associated with MAP kinase expression, western blotting was performed using anti-JNK, anti-ERK, and anti-p38 antibodies. The results indicated an overexpression of phosphor-JNK expression, but no significant differences in ERK and p38 expression were detected between nilotinib-sensitive and nilotinib-resistant cells (Fig. 3A). To confirm whether nilotinib resistance can be reversed by targeting the JNK gene, nilotinib-resistant cells were treated concomitantly with a JNK inhibitor (SP600125) and nilotinib. The JNK inhibitor (20 μM) together with increasing concentrations of nilotinib decreased the proliferation of nilotinib-resistant cells in a dose-dependent manner (Fig. 3B). Next, we observed whether metformin decreases JNK phosphorylation. The result indicated that metformin downregulated JNK phosphorylation in a dose-dependent manner in both nilotinib-sensitive and nilotinib-resistant cell lines (Fig. 3C). However, the treatment resulted in a significant reduction of JNK expression in nilotinib-resistant cells, compared to that in nilotinib-sensitive cells. To confirm whether combined treatment with metformin and nilotinib can restore nilotinib sensitivity by targeting the JNK protein, the EZ-Cytox assay was performed. Combined treatment with metformin (10 mM) and increasing concentrations of nilotinib decreased the proliferation of nilotinib-resistant cells in a dose-dependent manner (Fig. 3D). The cell growth inhibition effect of combined treatment with nilotinib and metformin was more potent than that of combined treatment with nilotinib and JNK inhibitor, as shown in Fig. 3B.

After treating the cells with 10 mM metformin and 10 nM nilotinib for 48 hours, we measured apoptosis induction by measuring the levels of PARP, caspase 3, caspase 9, and Bcl-xL, which are hallmark proteins of apoptosis. Neither metformin nor nilotinib alone induced PARP, caspase 3, or caspase 9 cleavage in nilotinib-resistant cells. However, the combination therapy induced PARP, caspase 3, and caspase 9 cleavage. Furthermore, the combination treatment notably decreased the expression of phosphorylated JNK in nilotinib-resistant cells compared to control. These data indicated that JNK downregulation by metformin reversed nilotinib resistance (Fig. 4A). Similar results were observed by trypan blue exclusion assay (Fig. 4B). The early apoptotic cell percentage increased by seven-or eight-fold compared to control (Fig. 4C). Finally, we confirmed whether the apoptosis-inducing effect of the combination treatment was dependent on caspase. Combined treatment with metformin and nilotinib markedly enhanced apoptosis signaling, whereas combined treatment with metformin, nilotinib, and z-VAD-fmk (25 μM), a pan-caspase inhibitor, significantly attenuated apoptosis signaling (PARP, caspase 3 and caspase 9 cleavage). These data implied that the apoptosis-enhancing effect of combination treatment with metformin and nilotinib was mediated via increased caspase activation (Fig. 4D).