- Cell culture and reagents
The GES-1, AGS and HGC27 cell lines were purchased from the cell bank of the Chinese Academy of Sciences and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). The incubator conditions were 37°C and 5% CO2 (12). FBS and RPMI-1640 medium were purchased from Gibco (New York, USA). Recombinant human TRAIL (#T9701) and 5-FU (#F6627) were purchased from Sigma-Aldrich (Munich, Germany). The small-molecule inhibitors U0126 (#M1977), SP600125 (#M2076) and SB202190 (#M2062) were obtained from AbMole (Houston, USA). Primary antibodies against the following proteins were purchased from Cell Signaling Technology (Beverly, MA, USA): TRAIL (#3219), Caspase-3 (#9662), Caspase-8 (#9746), Caspase-9 (#9502), Cleaved Caspase-3 (#9661), Cleaved Caspase-8 (#9496), Cleaved Caspase-9 (#9505), PARP (#9532), Bid (#2002), DR4 (#42533), DR5 (#8047), c-IAP1 (#7065), c-IAP2 (#3130), Bcl-2 (#4223), Mcl-1 (#94296), Erk (#4695), JNK (#9252), p38 (#8690), phospho-Erk (#4370), phospho-JNK (#4668), phospho-p38 (#4511) and β-actin (#4970). The secondary antibodies used in this study included goat anti-mouse IgG-HRP (abs20001) and goat anti-rabbit IgG-HRP (abs20002), both of which were obtained from Absin (Shanghai, China).
- Determination of cell viability
CCK-8 (Dojindo, Tokyo, Japan) was used to measure cell viability. Briefly, cells were seeded in 96-well plates at a density of 2-4×103 cells/well and allowed to attach for 24 hours. After 24 or 48 hours of treatment with the specified concentrations of TRAIL and 5-FU, a CCK-8 solution (10 µl) was added to each well, and then a microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA) was used to measure the optical density (OD) at 450 nm. The following formula was used to calculate the cell growth inhibition rate: 1-OD experiment / OD control (13). The cells were seeded in a six-well plate and were incubated overnight, after which EdU detection reagent was added, and cell proliferation was observed with a fluorescence inverted microscope (Olympus, Japan).
- Apoptosis analysis by flow cytometry
Flow cytometry was used to analyze cell apoptosis (14). Cells (5x105) were seeded in 35-mm2 petri dishes and treated with TRAIL or/and paclitaxel (PTX) at the specified concentration for 24 hours. The cells were harvested by trypsin digestion at specific time points. Collected cells were washed once with 1× Annexin V binding buffer (eBioscience, San Diego, California, USA) and then incubated in a buffer containing FITC-conjugated Annexin V (eBioscience). After incubating in the dark at room temperature for 30 minutes, the cells were washed once with a binding buffer and then resuspended in 500 μl of binding buffer containing a PI solution (0.5 μg/ml).
- siRNA transfection
C-IAP1-specific siRNA and interfering siRNA were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Cells (2x105) were incubated in a 6-well plate for 24 hours according to the manufacturer's instructions, and then siRNA (100 nmol/l) and Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) in serum-free Opti-MEM were added six hours after seeding. The medium was replaced with fresh medium. Forty-eight hours after transfection, the knockdown effect on C-IAP1 expression was verified by Western blotting. siRNA transfection was performed as described previously (16).
- Western blot assay
GC cells were inoculated into a 6-cm Petri dish, treated for 48 hours, scraped and collected. The cells were completely lysed using RIPA cell lysis reagent (Solarbio, Beijing, China) containing protease and phosphatase inhibitors for 30 minutes. The cell lysate was centrifuged at 12,000 g and 4°C for 20 minutes, and the protein concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China). The supernatant containing the total protein was then mixed with a corresponding volume of 5x SDS loading buffer, and the mixture was heated at 95°C for 5 minutes. Subsequently, 20 μg of total protein from each sample was separated on a 12% premade gel. A constant current of 300 mA was used for wet electrotransfer of the proteins to a 0.22-μm polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk powder in TBST for 2 hours and incubated with an appropriate primary antibody (1:1,000) overnight. The next day, the membrane was washed 3 times with TBST for 10 minutes each time. At room temperature, the membrane was incubated with an HRP-conjugated secondary antibody (1:8,000) for 2 hours and then washed with TBST 3 times for 10 minutes each time. A chemiluminescence kit (Life Technologies, Shanghai, China) was used to observe the bound antibody complexes on a Bio-Rad infrared gel imager system (ChemiDoc XRS+). Western blotting was performed as described previously (15).
- Tumor xenograft study using athymic nude mice
Animal research was conducted strictly in accordance with institutional guidelines. Six-week-old male nude mice were used in the experiment after 7 days of adaptive feeding. At the beginning of the experiment, AGS cells in a healthy logarithmic growth state were inoculated into the right armpit skin of the mice. The tumors were allowed to grow for approximately 1 week (this time was determined to be when the tumor diameter reached 3-5 mm), and then the nude mice were randomly divided into 4 groups, with 3 mice in each group, and drug administration was started. The groups were i) control (vehicle only); ii) TRAIL (100 μg/kg intratumoral injection); iii) 5-FU (10 mg/kg intraperitoneal injection); and iv) TRAIL (100 μg/kg intratumoral injection), followed by 5-FU (10 mg/kg intraperitoneal injection) 24 hours later. All groups were treated every 3 days for a total of 28 days. Tumor size and weight were monitored three times per week. Tumor volume (V) was calculated as V = 0.5×length×width2. To evaluate tumor growth inhibition (TGI), the formula [1-(T-T0)/(C-C0)]×100, where T and C are the average tumor volumes of the treatment group and the control group, respectively, at the end of the experiment and T0 and C0 are the average tumor volumes of the same groups on the 0th day, was used (17).
- TUNEL assay to evaluate apoptosis
The tissue was dehydrated using gradient alcohol. After sectioning, a 2 mg/ml Proteinase K solution was diluted with deionized water at a ratio of 1:100 to a final concentration of 20 μg/ml and incubated at room temperature for 20 min. Dilute 5×Equilibration Buffer with deionized water at a ratio of 1:5. Add 100μl of 1×Equilibration Buffer to each sample to cover the area of the sample to be tested, and incubate at room temperature for 15 minutes. Wash off most of the 100 μl 1×Equilibration Buffer with absorbent paper around the equilibrated area, place the slides in a wet box, and incubate at 37°C for 60 min. Then wash with PBS 3 times, 5min each time. DAPI was added dropwise and incubated for 5 min in the dark to stain the specimen, and the excess DAPI was washed away with PBST 5 min × 4 times. Mount with a mounting solution containing an anti-fluorescence quencher, and then observe and collect the image under a fluorescent microscope. Under the fluorescence microscope, the apoptotic cells on the tissue section showed red fluorescence, and the nucleus showed blue fluorescence.
- Statistical analysis
All statistical tests were performed with SPSS 19.0 (SPSS, Inc., Illinois, USA). Data are expressed as the mean ± standard deviation and were compared by Student's t test or analysis of variance. P <0.05 was considered significant.