2.1 Reagents and Cell culture
Reagents: Human bladder cancer cell lines T24 and 5637 were provided by the cell bank of the Chinese Academy of Sciences in Shanghai. The present study used metformin (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China), Roswell Park Memorial Institute (RPMI)-1640 medium (Cellmax Company, Groningen, Netherlands), fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA), phosphate-buffered saline (PBS) (Beijing Solarbio Company, Beijing, China), Cell counting Kit-8 (CCK-8) (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China), and an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Assay Kit (BD Biosciences, San Jose, CA, USA). Antibodies recognizing PI3K/Akt/mTOR signaling pathway associated proteins (phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), phosphorylated (p)-PI3K, protein kinase B (Akt), p-Akt, mechanistic target of rapamycin (mTOR), p-mTOR), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Apoptosis-related protein (cleaved-caspase 3 and cleaved‑poly(ADP-ribose) polymerase (PARP)) were all purchased from Affinity Biosciences (Cincinnati, OH, USA). We also used a microplate analyzer (Thermo Scientific, Waltham, MA, USA), a flow cytometer (Bio-Rad, Hercules, CA, USA), and a western blotting imaging system (Bio-Rad).
Cell culture: Bladder cancer T24 and 5637 cells were taken out of -80°C liquid nitrogen storage and thawed in a beaker for 1 min. The cells were suspended in RPMI-1640 medium containing 10% FBS and placed into an incubator at 37 °C and 5% CO2 for culture. After the cell density reached about 90%, the cells were passaged. T24 and 5637 cells in the logarithmic growth phase were used for the following experiments.
2.2 Wound healing assay
We made horizontal lines on the back of the 6-well plate, about every 0.5–1 cm, across the holes. A single cell suspension was prepared by adding 0.25% trypsin to the cells for digestion. The cell plate count method was used to adjust the cell concentration to about 5.0 ×105/mL and the cells were inoculated into a 6-well plate. The control group (A) and Group B (metformin 5, 10, 15, and 20 mmol/L) were placed in an incubator for 24 h until the cells were attached to the wall and a confluent cell layer had developed. Scratches were made in the cell layer using a micropipette tip. The cells were washed with PBS three times to remove the scratched cells. Serum-free RPMI-1640 medium was added vertically and gently at the apex of the plate. Photographs were taken at time zero and after 48 hours of culture using an Olympus camera (Tokyo, Japan).
2.3. Transwell assay
A single cell suspension was prepared by adding 0.25% trypsin to the cells for digestion. 500 μL of medium containing 10 %FBS was added to the lower chamber of the Transwell apparatus (8 μm in diameter), and 200 μL of cell suspension without serum was added to the upper chamber. After incubation for 48 h, the medium in the upper and lower chambers was removed, and 500 μL of 4% paraformaldehyde was added to the lower chamber to fix the cells for half an hour, after which the paraformaldehyde was removed. The cells that did not penetrate the membrane were wiped off using a cotton swab. Then, 500 μL of 0.1 % crystal violet were added to the lower chamber to stain the cells for 15 minutes. The chamber was washed with PBS until it was completely clear, and the cells were observed and counted under a microscope (Olympus).
2.4 Assay for cell growth
A Cell Counting Kit-8 (CCK-8) assay was used to determine cell proliferation. Logarithmic growth phase cells were digested with 0.25% trypsin, and a single cell suspension was prepared. The cell density was adjusted to about 1× 103/mL, and 100 μL cells of the suspension were inoculated in each well of a 96‑well plate, and cultured in an incubator for 24 h. After the cells adhered to the wall, the medium was discarded and 5, 10, 15, and 20 mmol\L metformin were added and incubated for 48 h, respectively. Five replicates were set for each concentration, and a blank control group was set with the same volume of medium. After the treatment, each well received 10 μL of CCK-8 solution, the 96-well plate was gently knocked to mix, and then incubated for 1.5 hours. The absorbance value at 450 nm was measured using an enzyme plate analyzer (Thermo Scientific)
2.5 Assay for colony formation
The proliferation of T24 and 5637 cells was determined using a colony formation assay. T24 and 5637 cells were plated in 6-well plates at a density of 100 cells per well and cultured for 12 h. Thereafter, the cells were treated with metformin at 5, 10, 15, and 20 mmol/L, or with medium only (RPMI-1640 medium + 10% FBS). The culture medium was changed every three days and observed continuously for at least 10–12 days. Thereafter, 4% paraformaldehyde was used to fix the cells, followed by staining with using 0.1% Crystal violet solution. The stained cells were counted under an inverted microscope (Olympus).
2.6 Assay of cell apoptosis
Cell apoptosis was detected using flow cytometry. Logarithmic growth phase cells were digested with 0.25% trypsin, and a single cell suspension was prepared. The density of the cells was adjusted to 1.0 × 105/mL, and the cells were inoculated in a 6-well plate at 1 mL/ well. The cells were cultured in an incubator for 24 h. After cell adherence, we discarded the medium and the cells were divided into the control group and the metformin group, which was treated with 5, 10, 15, and 20 mmol/L metformin for 48 h, respectively, and the control group was treated with the same volume of medium. Cells in each group were collected and rinsed with precooled PBS three times. After centrifugation at 1500 × g for 5 min at 4 °C, the supernatant was discarded, the cells were resuspend in AnnexinⅤ-FITC binding solution, mixed with PI staining solution, and incubated in the dark for 15 min. The apoptosis rate was detected by flow cytometry, carried out according to the manufacturers' protocol (Cell Sorter BD FACSAria II, BD Biosciences)
2.7 Western blotting analysis
Logarithmic phase cells were and digested using 0.25% trypsin to prepare a single cell suspension, and the cell density was adjusted to 1.0 × 106/mL. The cells were inoculated in a 6-well plate at 1 mL/well, and cultured in an incubator for 24 h. After the cells had attached to the wall, we discarded the medium and the cells were divided into control group and metformin group. The metformin group was treated with 5, 10, 15, and 20 mmol/L metformin for 48 h, and the control group was treated with the same volume of medium containing 10 % FBS. The cells were collected, and cell lysis buffer containing protease and phosphatase inhibitors was added and the cells were lysed on ice for 20 min. After centrifugation at 1500 × g for 5 min at 4 °C, the supernatant was retained and its protein concentration was determined using the bicinchoninic acid (BCA) method. Then, 40 mg of protein was subjected to SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to 0.45 μm or 0.22 mm polyvinylidene fluoride (PVDF) membranes, and blocked using 5% skimmed milk at room temperature for 40 min. Primary antibodies were added (the dilution ratio of cleaved-caspase 3 and cleaved-PARP was 1:500, the dilution ratio of PI3K, p-PI3K, Akt, p-Akt, mTOR and p-mTOR was 1:1000, and the dilution ratio of GAPDH was 1:10,000), and placed in a refrigerator at 4 °C for overnight incubation. The membranes were washed with 1´ Tris-buffered saline-Tween 20 (TBST) three times for 5 min each time and incubated with labeled secondary antibody at room temperature for 40 min. After washing with 1 × TBST three times (5 min each time), ECL exposure luminescence solution was added at a 1:1 ratio onto the PVDF membrane for luminescence development. The immunoreactive protein bands were analyzed using Image-J software. The relative level of each protein was represented by the ratio of the gray level of target protein band to the gray level of internal reference, GAPDH. For the phosphorylated proteins, the relative expression level was represented by the ratio of the gray level of the phosphorylated target protein to that of the total target protein. Finally, the ChemiDocxRS imaging system was used to obtain images of the protein bands, which were analyzed using Quantity One software (Bio-Rad).
2.8 Statistical analysis
SPSS 22.0 statistical software (IBM Corp., Armonk, NY, USA) was used for the statistical analyses. Measurement data were expressed as the mean ± SD. One-way analysis of variance was used for comparisons between multiple groups. GraphPad Prism 8.0 (GraphPad inc., La Jolla, CA, USA) was used to draw the graphs and Image-J software (NIH, Bethesda, MD, USA) was used to calculate the western blotting gray values, cell counts and wound areas. Differences were judged to be statistically significant at a P value < 0.05.