Cell lines and cell culture
Human pancreatic cancer cell lines SW1990 and BxPC3 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences and the Changhai hospital affiliated to the Second Military Medical University. Mycoplasma testing has been done for the cell lines used. Cells were cultured in 1640 medium (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS), and maintained in a humid atmosphere at 37°C with 5% CO2. The cell medium was changed every 2 to 3 days. Cell passage was performed with trypsin when the confluence of monolayer cells reached 70% to 80%.
Main reagents
ASA was purchased from Sigma (St. Louis, MO, USA) and was dissolved in DMSO (Dimethyl sulfoxide), which was obtained from SRL, India, to make a 5M stock solution. This solution was stored at –20°C. Gemcitabine was obtained from the Lilly Pharmaceuticals (Indianapolis, IN, USA) and dissolved in a sterile saline solution to create a 5 g/L stock solution. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) (St. Louis, MO, USA) was set to 5 mg/ml. The Annexin V-FITC apoptosis detection kit was obtained from R&D (Minneapolis, MN, USA). The rabbit monoclonal antibody against human E-cadherin, Vimentin, Bcl-2, Bax, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR and β-actin was obtained from the Santa Cruz Biotechnology (Santz Cruz, CA, USA). The rabbit monoclonal antibody against human Vimentin was obtained from Cell Signaling Technology (Boston, MA, USA). Horseradish peroxidase-labeled (HRP) goat-anti-rabbit IgG (secondary antibody) antibodies were obtained from CWBIO (Beijing ComWin Biotech, China).
Cell proliferation assay
Cells were cultured in 1640 medium containing 10% FBS, and maintained in a humid atmosphere at 37°C with 5% CO2 for 72 h. Then cells were harvested and inoculated into 96-well plates with 3×103 cells in each well 24 h before treatment. After that, samples were divided into 4 groups. Cells were cultured in: 1)1640 medium containing 10% FBS and 2 mmol/L ASA, 2)1640 medium containing 10% FBS and 0.05 mg/L gemcitabine, 3)1640 medium containing 10% FBS, 2 mmol/L ASA and 0.05 mg/L gemcitabine, 4) negative control (1640 medium containing 10% FBS). 24 hours later, we incubated cells at 37°C with 5% CO2 for next 0 hours, 24 hours, 48 hours and 72 hours. Then, 10% MTT (5 mg/mL) was added into each well. After adding MTT for 4 hours, total of 150 μl DMSO was then added into each well, and the samples were vibrated for 10 minutes. The absorbance (OD) was then measured with a microplate reader set at 570 nm. Each cell proliferation experiment was repeated 3 times and the readings were averaged for the statistical analysis.
Wound Healing Assay
BxPC3 and SW1990 cells were seeded in 6-well plates. When the cell confluence was about 100%, scratches were made on the monolayer cell surface with 200-μl pipette tips. After the cells were washed twice with PBS, the cells were divided into 4 groups, which was mentioned in the cell proliferation assay. Images of cells on both sides of the scratches were captured with an inverted microscope at 0 and 2 hours. Finally, images have analyzed the migration in three fields of view that were randomly selected and analyzed in an independent trial with 400X magnification under an inverted microscope (Olympus Corp). The width (W) of the scratch measured; the percentage of wound area remaining was calculated as W24h/W0h × 100%. All experiments were performed in triplicate
Cell apoptosis
BxPC3 and SW1990 cells were seeded in 6-well plates and cultured with 1640 medium containing 10% FBS. After the cells were attached to the plate for 24 h, they were divided into 4 groups, which was mentioned in the cell proliferation assay, and cultured for 24 h. The cells were then collected, washed with phosphate buffer saline (PBS) solution 2 to 3 times. To count the number of apoptotic cells, they were stained with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). These cells were analyzed by using the BD FACSCalibur in each experiment. All experiments were carried out in triplicate.
Western blot Analysis
The 4 groups cells, which was mentioned in the cell proliferation assay, were harvested after cultured for 24 h and lysed with RIPA lysis buffer. Protein molecules released from the cells were separated and extracted by the SDS-PAGE (Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis), and then transferred to polyvinylidene fluoride (PVDF) membrane for western blotting. Membranes were blocked with 5% non-fat milk and then incubated with primary antibodies against β-actin, Bax, Bcl-2, E-cadherin, Vimentin, PI3K, phospho-PI3K, Akt, phospho-Akt, mTOR and phospho-mTOR at the dilution of 1:1000 at 4°C overnight. The membranes were then rewarmed for 30 minutes, washed with TBST (Tris Buffered saline Tween), and were incubated with the corresponding horse radish peroxidase-labeled secondary antibody at the dilution of 1:5000 at 37°C for 1 hour. Western blots were visualized using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Burlington, MA, USA) followed by film exposure. β-actin was used as the internal reference. The gray level was analyzed, and the results were calculated as the gray level of the target protein divided by the gray level of the internal reference.
Statistical analysis
Statistical analysis was performed with Graphpad Prism software 7.00. Continuous data were calculated as mean ± standard deviation (SD) and statistical significance was defined as p<0.05. Statistically significant differences among experimental groups were determined with student’ t-test.