Chemicals
Cell culture media, Dulbecco's Modified Eagle medium (DMEM)/F-12 1:1 Modified medium (DMEM/F-12) was purchased from Lonza (Walkersville, MD, USA). 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). SB203580, PD98059 were purchased from Calbiochem (San Diego, CA, USA). SB216763 were purchased from Sigma Aldrich. ATF3 antibody and ATF3 siRNA were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Antibodies against β-actin, Poly (ADP-ribose) polymerase (PARP), p38, p-p38, ERK1/2, p-ERK1/2, p-GKS3β and GKS3β were purchased from Cell Signaling (Bervely, MA, USA). ATF3 promoter constructs (-1420/+34, -718/+34, -514/+34, -318/+34, -147/+34 and -84/+34, pATF3-514 del Ftz and pATF3-514 del CRE) were kindly provided by Dr. S-H Lee (University of Maryland College Park, Maryland, USA). All chemicals were purchased from Fisher Scientific, unless otherwise specified.
Sample preparation
The plant parts of A. distichum (voucher number: Park1001(ANH)) was formally identified by Jae Ho Park as the professor of Jungwon University, Korea. Its flower, leaf and branch were cultivated and collected at Goesan-gun, Chungbuk, Korea. Five hundred grams of fresh parts was extracted with 1,000 ml of 80% methanol with shaking for 24 h. The methanol-soluble fraction was filtered and concentrated to approximately 20 ml volume using a vacuum evaporator and a fraction was placed in a separating funnel. The ethyl acetate fractions from the parts of A. distichum was separated from the mixture, evaporated by a vacuum evaporator and prepared aseptically and kept in a refrigerator until use.
Cell culture and treatment
Human colorectal cancer cell lines (HCT116, SW480 and LoVo cells), human breast cancer cell lines (MCF-7 and MDA-MB-231), hepatocellular carcinoma cells (HepG-2) and colon normal cells (CCD-18co) were purchased Korean Cell Line Bank (Seoul, Korea) and grown in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were maintained at 37°C under a humidified atmosphere of 5% CO2. Ethyl acetate fractions from A. distichum were dissolved in dimethyl sulfoxide (DMSO) and treated to cells. DMSO was used as a vehicle and the final DMSO concentration was not exceeded 0.1% (v/v).
Cell viability
Cell viability was measured using MTT assay system. Briefly, cells were plated onto 96-well plated and grown overnight. The cells were treated with 0, 50, 100 and 200 μg/ml of EAFAD-B for 24 and 48 h. Then, the cells were incubated with 50 μl of MTT solution (1 mg/ml) for the additional 2 h. The resulting crystals were dissolved in DMSO. The formation of formazan was measured by reading absorbance at a wavelength of 570 nm.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was prepared using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA (1 μg) was reverse-transcribed using a Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer’s protocol for cDNA synthesis. PCR was carried out using PCR Master Mix Kit (Promega, Madison, WI, USA) with primers for human ATF3 and human GAPDH as follows: human ATF3: 5′-gtttgaggattttgctaacctgac-3′, and reverse 5′-agctgcaatcttatttctttctcgt-3′; huaman GAPDH: forward 5’-acccagaagactgtggatgg-3’ and reverse 5’-ttctagacggcaggtcaggt-3’.
Transient transfections
Transient transfections were performed using the PolyJet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) according to the manufacturers’ instruction. HCT116 and SW480 cells were plated in 12-well plates at a concentration of 2 × 105 cells/well. After growth overnight, plasmid mixtures containing 0.5 μg of ATF3 promoter linked to luciferase and 0.05 μg of pRL-null vector were transfected for 24 h. The transfected cells were cultured in the absence or presence of the ethyl acetate fractions for the indicated times. The cells were then harvested in 1 × luciferase lysis buffer, and luciferase activity was normalized to the pRL-null luciferase activity using a dual-luciferase assay kit (Promega).
Transfection of small interference RNA (siRNA)
The cells were plated in six-well plates and incubated overnight. HCT116 cells were transfected with control siRNA and ATF3 siRNA for 48 h at a concentration of 100 nM using TransIT-TKO transfection reagent (Mirus, Madison, WI) according to the manufacturer's instruction. Then the cells were treated with EAFAD-B (200 μg/ml) for 24 h.
SDS-PAGE and Western blot
After EAFAD-B treatment, cells were washed with 1 × phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma Aldrich) and phosphatase inhibitor cocktail (Sigma Aldrich), and centrifuged at 15,000 × g for 10 min at 4°C. Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). The proteins were separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then incubated with specific primary antibodies in 5% nonfat dry milk at 4°C overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences) and visualized in Polaroid film.
Statistical analysis
Statistical analysis was performed with the Student's unpaired t-test, with statistical significance set at *, P < 0.05.