Cannabinoid Receptor Agonist as an Alternative Drug in 5-Fluorouracil-resistant Gastric Cancer Cells

Materials and Methods

Drugs. The mixed CB₁/CB₂ agonist R-(+)- WIN-55,212-2 (2,3 dihydro-5-methyl-3 [(morpholinyl)-methyl]pyrollo (1,2,3 de)-1,4-benzoxazinyl]-[1-naphthaleny]methanone, C₂₇H₂₆N₂O₃·CH₃SO₃H) was purchased from Tocris Bioscience (Bristol, UK). 5-FU and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA).

Cell culture. The 5-FU-resistant cell line of the SNU-620 human gastric cancer cell line, SNU-620-5FU/1000, was obtained from the Korean Cell Line Bank (Seoul, Korea). RPMI-1640 medium and fetal bovine serum (FBS) were purchased from WelGENE (Daegu, Korea), and penicillin–streptomycin was purchased from Sigma. SNU-620-5FU/1000 cells were cultured in RPMI-1640 medium, and supplemented with 10% heat-inactivated FBS, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. The cells were maintained under standard cell culture conditions at 37°C and 5% CO₂ in a humid environment.

CCK-8 assay. The Cell Counting Kit-8 (CCK-8) assay was performed to determine cell viability. CCK-8 assay detection kit from Dojindo Molecular Technologies (Rockville, MA, USA) was used according to the manufacturer's instructions. Briefly, cells were seeded in 96-multi-well plates and the cells were treated with WIN 55,212-2 (1, 2.5, 3, 3.5, 4, 4.5, 5, 10, 30 μM), 5-FU (0.1, 0.5, 1, 2.5, 5, 7.5, 10, 100 μM) or an equivalent amount of DMSO for 24 or 48 h. After incubation for the specified time, 10 μl of CCK-8 solution was added to each well in an assay plate. The plate was then placed in a CO₂ incubator for 4 h. After incubation, absorbance was recorded on a microplate reader (Spectra Max 250, Molecular Devices, Sunnyvale, CA, USA) at 450 nm. Six wells were used for each treatment condition.

Apoptosis analysis by flow cytometry. Phycoerythrin (PE)-annexin V apoptosis detection kit from BD Biosciences (Bedford, MA, USA) was used according to the manufacturer's instructions. Briefly, the cells were treated with WIN 55,212-2 (1, 3.4, or 5 μM) or an equivalent amount of DMSO for 48 h. The cells were then washed twice with cold phosphate-buffered saline (PBS) and resuspended in 1× binding buffer [0.01 M HEPES (pH 7.4), 0.14 M NaCl, 2.5 mM CaCl₂] at a density of 1 × 10⁶ cells/ml. One hundred microliters of the cell suspension was transferred to a 5-ml culture tube, and 5 μl of PE-annexin V and 5 μl of 7-amino-actinomycin D (7-AAD) were added. The cells were incubated at room temperature for 15 min in the dark, and 400 μl of 1× binding buffer was then added. Apoptosis was analyzed by flow cytometry using a FACSCalibur apparatus (Becton Dickinson, San Jose, CA, USA).

Cell-cycle analysis by flow cytometry. Aliquots of 1×10⁶ cells were plated in 60-mm culture dishes. The cells were treated with WIN 55,212-2 (1, 3.4, or 5 μM) or an equivalent amount of DMSO for 48 h. After incubation for the specified time, the cells were then washed twice with cold PBS and pelleted. The pellet was suspended in cold PBS and the cells were fixed in a final concentration of 70% ethanol for 1 h at 4°C. The cells were washed with cold PBS and incubated with 100 μg/ml RNase A for 15 min at 37°C. Nuclei were stained with 50 μg/ml propidium iodide (PI) (Sigma-Aldrich) for 30 min at 37°C in the dark. Samples were then analyzed by flow cytometry using a FACSCalibur apparatus (Becton Dickinson). For flow cytometric evaluation of the cell cycle, 10,000 events corrected for debris and aggregate were analyzed for each sample. The assay was carried out in three replicates.

Preparation of cell extracts. The cells were plated in 100-mm culture dishes and then treated with WIN 55,212-2 (1, 3.4, or 5 μM) or an equivalent amount of DMSO for 48 h. The cells were then washed with cold PBS, harvested, disrupted on ice for 5 min using lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin; Cell Signaling Technology, Beverly, MA, USA), supplemented with protease inhibitors (Sigma-Aldrich) and centrifuged at 14,000 ×g for 15 min at 4°C. The supernatants were removed, flash-frozen in clean 1.5 ml tubes and stored at −70°C for subsequent western blot analysis.

Western blot analysis. The cell protein concentration was determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA, USA). For western blotting, equal amounts of protein samples were loaded onto 10% sodium dodecyl sulfate polyacrylamide electrophoresis gels and subjected to electrophoresis. Proteins were then transferred onto nitrocellulose membranes at 350 mA for 1 h at room temperature. The membranes were subsequently blocked with 5% non-fat dry milk/Tris-buffered saline containing 0.1% Triton X-100 (TBST) for 1 h at room temperature and washed thrice for 5 min in TBST. The membranes were incubated overnight at 4°C with specific primary antibodies then washed for 5 min in TBST and hybridized with the secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. Then, the membranes were washed thrice for 5 min in TBST. Reactions were detected using West-Q Chemiluminescent Substrate Plus Kits (GenDEPOT, Barker, TX, USA). Antibody binding detection and intensities of the different proteins were quantified using a western blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ, USA). All experiments were repeated at least three times and yielded similar results. Antibodies to phospho- extracellular-signal-regulated kinases (ERK) 1/2, ERK1/2, phospho-Akt (Ser473), B-cell lymphoma-2 (BCL2), BCL2-associated X (BAX), and cleaved poly (ADP-ribose) polymerase (PARP) were from Cell Signaling Technology. Cleaved caspase-3 antibody was from Abcam (Cambridge, UK). β-Actin antibody (loading control) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Statistical analysis. Statistical analysis was performed with SAS software (SAS Institute, Cary, NC, USA). Data were analyzed using the two-tailed Student's t-test. A value of p<0.05 was considered significant. Data are expressed as the mean±standard error of the mean (SEM).