Phenformin (Aladdin Chemistry, Shanghai, China) was prepared in a range of concentrations in culture medium. Gefitinib (Selleck-Biotool, Shanghai, China) was prepared as a stock solution of 5 mmol/L in DMSO. Antibodies against the following target proteins were obtained from Cell Signaling (Beverly, MA, USA): total p70 S6 kinase, phospho-p70 S6 kinase (Thr389), total AMPKα, phospho-AMPKα (Thr172), total mTOR, phospho-mTOR (Ser2448), total 4E-BP1, phospho-4EBP1, total EGFR, phospho-EGFR and β-actin.
Cell lines and culture conditions
The mouse bladder cancer cell line MB49 and the human bladder cancer cell lines T24 and UMUC3 were generously provided by Dr. P. Guo of the Institute of Urology at Xi’an Jiaotong University (Xi’an, Shaanxi, China) . All cell lines were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% penicillin–streptomycin. Cultures were incubated at 37 °C in humidified air containing 5% CO2.
Cell viability assay
Cell viability was assessed using a tetrazolium-based assay. Briefly, cells were seeded at 8 × 103 per well in 96-well culture plates and incubated in medium containing 10% FBS. At 24 h later, cells were treated for 48 h with different concentrations of phenformin alone or in combination with gefitinib. The tetrazolium salt of MTT (50 μL; Sigma) was dissolved in Hank’s balanced salt solution to a concentration of 2 mg/mL, and added to each well. The plates were incubated another 5 h. The medium was aspirated from each well, DMSO (150 μL; Sigma) was added to dissolve formazan crystals, and absorbance was measured using a microplate reader (Biotek, SYNERGY HTX, VT, USA) at 490 nm (against reference absorbance at 630 nm). Dose–response curves were generated and used to calculate the half-maximal inhibitory concentration (IC50) using SPSS 16.0 (IBM, Chicago, IL, USA).
Briefly, 8 × 103 cells were seeded into 24-well dishes in 0.5 mL of medium. At 24 h, cells were treated with different concentrations of phenformin alone or combined with gefitinib for a further 6–8 day period in medium containing 10% FBS. Cells were fixed with 10% formaldehyde, stained with 0.1% crystal violet. Absorbance was measured using a microplate reader (Biotek) at 550 nm wavelength. Colony formation images were captured under a microscope (DFC450C; Leica, Wetzlar, Germany).
Cells (5 × 103) were seeded into 6-well plates and allowed to reach confluence. The monolayer was scratched using a cocktail stick. Cells were incubated with serum-free DMEM medium for different time periods before capturing the digital images with a DFC450C microscope (Leica). Wound closure was determined by measuring the migrated distance of cells from the 0 h using Image J (US National Institutes of Health, Bethesda, MD, USA). Experiments were repeated three times.
In one set of experiments, apoptosis was assessed using fluorescence microscopy. Cells (1.2 × 104) were seeded into 96-well plates. After 24 h, cells were treated with phenformin alone or with gefitinib for 24 h. Then the cells were incubated at room temperature in the dark for 15 min with 100 μL binding buffer, 1 μL FITC-conjugated Annexin V (MultiSciences Biotech, Hangzhou, China) and 1 μL of propidium iodide (MultiSciences Biotech). Cells were observed under a DFC450C fluorescence microscope (Leica).
Apoptosis was assessed using flow cytometry in a separate experiment. Briefly, cells treated with the different drug combinations were harvested with trypsinization, washed twice with phosphate-buffered saline (PBS), and resuspended in binding buffer to 1 × 106 cells/mL. Then 5 μL of Annexin V-FITC and 10 μL of propidium iodide were added to 100 μL of cell suspension, incubated for 30 min at room temperature in the dark, and then mixed with 400 μL of binding buffer. Within 30 min, labeled cells were counted by flow cytometry on a FACS Calibur flow cytometer [excitation wavelength, 488 nm; emission wavelengths, 530 nm (FL-1 channel, FITC) and 670 nm (FL-3 c3 channel, propidium iodide)]. Data were analyzed using Cell Quest software (Becton–Dickinson). Non-apoptotic cells were defined as those negative for Annexin-V and propidium iodide; necrotic/late apoptotic cells as those positive for both labels; and early apoptotic cells as those positive for Annexin V but negative for propidium iodide.
Proteins were fractionated by SDS-PAGE, transferred to membranes, and then incubated overnight at 4 °C with different primary antibodies described in Reagents section above (Cell Signaling, Beverly, MA, USA) in buffer containing bovine serum albumin (BSA). Membranes were washed with TBS containing 0.05% Tween-20, blotted with secondary antibody for 1 h at room temperature, then washed again three times. Pierce Super Signal chemiluminescent substrate (Rockford, IL, USA) was added, and the blot was imaged immediately on a Chemi Doc system (Bio-Rad, Hercules, CA, USA) and a Perfection V500 camera (Epson). Band intensities were quantified using Image J.
All data are presented as mean ± SD. Statistical analysis was performed using SPSS 16.0. Differences between groups were assessed for significance using Student’s t test for experiments involving only two groups and using ANOVA and the least significant difference (LSD) test for experiments involving more than two groups. Graphs were generated using GraphPad Prism 6.0. Two levels of statistical significance were considered: *P < 0.05 and #P < 0.01.