Cells culture
A total of 4 human colon adenocarcinoma cell lines were used in the current study. SW480 and SW620 cells were obtained from the Cell Resource Center of the institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College (PUMC) (Beijing, China), respectively, and cultured in Iscove’s Modified Dulbecco’s Medium and Eibovitz’s L-15 Medium (ThermoFisher Scientific, Waltham, MA, USA). HCT-116 cells were kept in our laboratory in RPMI-1640 medium (ThermoFisher Scientific). DiFi cell line was a gift from Professor Wang Zhen at our institute and cultured in Dulbecco’s Modified Eagle Medium: nutrient mixture F-12 (1:1) medium (ThermoFisher Scientific). For all cell lines, the culture medium was supplemented with 10% fetal bovine serum (Gibco, Carlsbad, California, USA), 100-U/mL penicillin and 100-µg/mL streptomycin (North China Pharmaceutical Inc, Beijing, China) at 37 °C in a 5% CO2 incubator.
Reagents and antibodies
Azithromycin and N-acetylcysteine (NAC) were obtained from National Institutes for Food and Drug Control (Beijing, China). Azithromycin was dissolved in anhydrous ethanol; NAC was dissolved in distilled water. Anti-caspase-3, anti-Akt (pan), anti-p44/42 MAPK (Erk 1/2), anti-p38 MAPK, anti-PARP and p62 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3B antibody was purchased from Sigma (St. Louis, MO, USA). Azide-free anti-human CD261 (DR4) antibody was obtained from Diaclone (Besancon, France). Anti-DR5 antibody was obtained from ProSci (San Diego, California, USA). β-Actin (6G3) and GAPDH (1C4) monoclonal antibodies were purchased from AmeriBiopharma (Wilmington, Delaware, USA). Secondary antibodies included peroxidase-conjugated affiniPure goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) (ZSGB-BIO, Beijing, China). Caspase inhibitor zVAD-fmk and RIP1 inhibitor necrostatin-1 were purchased from Selleck.cn (Houston, TX, USA). Chloroquine (CQ), acridine orange hemi (zinc chloride) salt (AO) and sulforhodamine B (SRB) were from Sigma.
Expression and purification of TRAIL protein
Recombinant TRAIL was constructed in this laboratory and expressed in P. pastoris. TRAIL expressing strains were inoculated in 100-mL BMGY medium (100 mmol/L potassium phosphate buffer, pH 6.0, 1% yeast extract, 2% peptone, 1% glycerol, 1.34% YNB, 0.00004% biotin) in a shaking incubator at 30 °C for 36 h. Yeast cells were precipitated and re-suspended in 100-mL BMMY with added 1.5% methanol every 24 h, at 26 °C for 72 h. Supernatant was collected by centrifugation at 7800 × g, 4 °C for 15 min. TRAIL protein was purified with Ni2+ affinity chromatography (His Trap HP, GE Healthcare, Pittsburgh, Pennsylvania, USA). Protein concentration was examined using a BCA method.
Cell survival assay
Cells were seeded in 96-well plates at 3 × 103 cells/well in 100-µL culture medium. Twenty-four hour later, cells were exposed to test drugs (azithromycin and TRAIL of varying concentrations, and combination) for 24, 48, or 72 h prior to survival assay using a sulforhodamine B (SRB) method [17]. Cell survival was calculated relative to the control group.
Western blot analysis
Cells were lysed with a lysis buffer (50 mmol/L Tris–HCl pH 8.0; 2% NP-40; 150 mmol/L NaCl; 0.2% SDS; 0.5% sodium deoxycholate) containing 1% protease inhibitor (Beyotime, Jiangsu, China) and 100-µmol/L phenylmethylsulfonyl fluoride (PMSF). The supernatant was collected after centrifugation at 13,000 × g for 15 min at 4 °C, prior to Western blotting analyses, as described previously [18].
Apoptosis assay
Apoptosis was determined using an annexin V-FITC/PI apoptosis detection kit from DOJINDO (Shanghai, China). A schematic plot was used to display the results: the lower left quadrant represents live cells; the lower right and upper right quadrants represent early and late apoptotic cells, respectively; the upper left quadrant represents necrotic cells. Cell death refers to the sum of early and late apoptotic and necrotic cells.
Acridine orange (AO) staining
HCT-116 and SW480 cells were plated into 6-well plates and treated with drugs for 24 h. Later, cells were washed by PBS twice and stained with 700 µL/well AO (1 µg/mL) for 15 min at 37 °C in the dark. Then, the cells were washed by PBS twice. Watching the images under a fluorescence microscope through a 490 nm band-pass excitation filter and a 515 nm long-pass barrier filter. The green color represented the nucleus, while the red represented the acidic vesicles.
siRNA transfection
DR4 siRNA (sense: 5′-AACGAGATTCTGAGCAACGCA-3′, anti-sense: 3′-TTGCTCTAAGACTCGTTGCGT-5′), DR5 siRNA (sense: 5′-AAGACCCTTGTGCTCGTTGTC-3′, anti-sense: 3′-TTCTGGGAACACGAGCAACAG-5′), LC-3B siRNA (sense: 5′-GGTGTATGAGAGTGAGAAA-3′, anti-sense: 3′-CCACATACTCTCACACTTT-5′) and negative siRNA were purchased from Ruibo Biotechnology (Guangzhou, China) and dissolved in RNase-free water as a 20 µmol/L stock. Negative siRNA was designed by Ruibo biotechnology and belonged to scrambled control. Cells were transfected with siRNAs using the Ruibo FECT™ CP transfection kit, plated in 96-well or 6-well plates and incubated at 37 °C for 24 h. siRNAs were diluted in transfection reagent and incubated for 15 min at room temperature to allow the formation of transfection complexes prior to addition to the cells (final concentration: 30 nmol/L). Experiments with test drugs started 24 h after the transfection. Efficiency of transfection was verified with Western blotting.
Colon cancer xenograft
All animal experiments were performed in accordance with relevant guidelines and regulations. Briefly, HCT-116 cells (1 × 107 cells in 200-µL PBS) were injected into the right armpits of 6-week-old female BALB/c nude mice (SPF Biotechnology Co., Ltd., Beijing, China). At 21 days after the inoculation, tumors were removed and cut into 2 m × 2 m × 2 m prisms, and transplanted into the right flanks of other mice through a trocar. Seven days later, mice were randomized to receive azithromycin (50 mg/kg/day, via oral administration, for 3 consecutive days in a week) or TRAIL (10 mg/kg, via the tail vein, once a week). Tumor volumes and body weights were monitored once every 2 days. The tumor volume was calculated by the following formula: V = ab2/2 (a represents the length of the tumor and b represents the width). The animal experiment lasted for a total of 32 days. At the end of experiment, the tumors were removed and fixed with formalin to detect cell proliferation by immunohistochemistry.
Ki-67
Tissue sections (5 µm) were quenched with 3% H2O2 for 10 min at room temperature after dewaxing and antigen retrieval in hot citrate buffer. After blocking with 5% BSA for 30 min at 37 °C, tissue sections were incubated with a monoclonal anti-Ki-67 nuclear antigen antibody (ZSGB-BIO) at 4 °C overnight. Immunostaining was assessed in 3 randomly selected fields under a microscope with a 200 × objective lens and photographed. Images were analyzed using a microscope-matched analytical software (Leica QWin Standard).
Assessment of drug interaction
The mode of drug interaction was evaluated based on the coefficient of drug interaction (CDI), calculated as: CDI = AB/(A × B), where AB is the survival rate of the cells exposed to both agents and A or B is the survival rate of cells exposed to either agent alone. A CDI at < 1.0 indicates synergistic effect; a CDI at < 0.7 indicates strongly synergistic effect [17].
Statistical analysis
All experiments were repeated for at least three times. Data are expressed as the mean ± SD, and analyzed with Student’s t test for independent samples. Statistical significance was set at P < 0.05.
