Cell culture and reagents
The Hep3B hepato-carcinoma cell line was purchased from the American Type Culture Collection (Manassas, VA) and was cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Gibco, Grand Island, NY). Insulin-like growth factor (IGF)-1, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Hoechst 33342 were obtained from Sigma (St Louis, MO). Compound C and 6-bromoindirubin-3′-oxime (BIO) were purchased from Calbiochem (San Diego, CA). Monoclonal antibodies specific for p-AMPK (Thr172), AMPKα1, p-GSK3β (Ser9), GSK3β, β-catenin, Ang-1, VEGF and MMP-9 were purchased from Cell signaling Technology (Beverly, MA, USA). CD31 antibody was purchased from Abcam (Cambridge, UK), and β-actin antibody was obtained from Sigma (St Louis, MO).
Isolation of anthocyanins from Meoru
Anthocyanins were conducted by Won Sup Lee’s group at Gyeongsang National University School of Medicine. The plant with voucher specimen number KNKA200506031111 was deposited in the Korea national arboretum. Fruit of Meoru was collected in the middle of September 2007 at Jiri mountain in Korea, freeze-dried and stored in dark glass containers at −20°C until required for analysis. Anthocyanins pigments were extracted by maceration of the fruits (100 g) in methanol containing 0.1% HCl at 5°C for 24 h. The extraction procedure was repeated three times. After concentration under reduced pressure (Rotavapor R-124, Buchi, Switzerland), the extract was diluted with distilled water (100 ml) and partitioned against ethyl acetate (100 ml). The water layer containing the pigments was concentrated to 50 ml. The concentrate was purified according to established procedures by means of ethyl acetate/water partitioning and adsorption chromatography on a bed of Amberlite XAD-7 (Sigma, Yongin, South Korea).
Cell proliferation measurements
Hep3B cells seeded on 96-well microplates at 4 × 103 cells per well were incubated with the anthocyanins at the indicated concentrations for 48 h. Following incubation with the anthocyanins, the medium was removed, and the cells were then incubated with 100 μl MTT solution (2 mg/ml MTT in phosphate-buffered saline (PBS)) for 4 h. The samples were then solubilized in dimethyl sulfoxide and the purple formazan dye, converted from MTT by viable cells, was quantified by absorbance at 560 nm.
Apoptosis was measured using an FITC-Annexin V apoptosis detection kit (BD Pharmingen™, San Diego, CA) or Hoechst 33342 chromatin staining dye. For Annexin V/propidium iodide staining after treatment with anthocyanins, cells were harvested by trypsinization, washed with ice-cold PBS and suspended in a binding buffer at a density of 1 × 106 cells/ml. Cells were stained with Annexin V-fluorescein isothiocyanate and propidium iodide and analyzed by flow cytometry (Becton-Dickinson Biosciences, Drive Franklin Lakes, NJ). To examine chromatin condensation, cells were stained with 10 μM Hoechst 33342 for 30 min and fixed with 3.7% formaldehyde for 15 min. Changes in chromatin condensation were observed by fluorescence microscopy (Olympus Optical Co., Tokyo, Japan).
Wound healing assay
Hep3B cells were grown on 6-well plate to 100% confluent monolayer and then scratched to form a 100 μm wound by using sterile pipette tips. The cells were then cultured in the presence or absence of AIMs (400 μg/ml) in serum-free media for 24 h. The images were recorded at 0 h and 48 h after scratch using an Olympus photomicroscope (Olympus Optical Co., Tokyo, Japan).
For the cell invasion assays, Hep3B cells were cultured in serum-free media overnight. Cells (5 × 104 cells) were loaded onto pre-coated Matrigel 24-well invasion chambers (BD Biosciences, San Jose, CA) in the presence or absence of anthocyanins. Then 0.5 ml of medium containing 20% FBS was added to the wells of the plate to serve as a chemoattractant. The Matrigel invasion chambers were incubated for 24 h. Invading cells were fixed with 10% formalin, stained with crystal violet, and analyzed according to manufacturer’s instructions.
The gelatinolytic activities of matrix metalloproteinase (MMP)-2 in the conditioning culture medium were assayed by electrophoresis on 8% polyacrylamide gels containing 0.1% gelatin at 4°C. Polyacrylamide gels were run at 120 V, washed in 2.5% Triton X-100 for 1 h, and then incubated for 16 h at 37°C in activation buffer (50 mM Tris–HCl, pH 7.5, 10 mM CaCl2). After staining with Coomassie blue (10% glacial acetic acid, 30% methanol and 0.5% Coomassie brilliant blue) for 2 h, the gel was washed with a solution of 10% glacial acetic acid and 40% methanol without Coomassie blue for 1 h. White lysis zones indicating gelatin degradation were revealed by staining with Coomassie brilliant blue.
After starvation for 12 h in serum-free medium, cells were seeded into six-well plates and treated with test compounds. Total proteins were extracted using a RIPA lysis buffer (50 mM Tris–HCl (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl and 1 mM phenylmethylsulfonyl fluoride) and subjected to western blot analysis with specific antibodies. The proteins were then visualized by enhanced chemiluminescence (Intron, Kyunggi, Korea) and detected using an LAS4000 chemiluminescence detection system (Fuji, Tokyo, Japan).
Transient transfection with small interfering RNA
Specific small interfering RNAs (siRNAs)-targeting AMPKα1 (PRKAA1) and mTOR and non-specific control siRNAs were purchased from Dharmacon (Chicago, IL). For transient transfection, cells were seeded at a density of 5 × 104 cells/ml in antibiotic-free medium, and siRNAs were transfected using the DharmaFECT4 transfection reagent (Dharmacon) according to the manufacturer’s instructions. After incubation for 72 h, the cells were analyzed by MTT assay or western blot.
Five-week-old male Balb/c nu/nu mice were obtained from SLC (Tokyo, Japan) and housed in sterile filter-topped cages. Hep3B hepato-carcinoma cells (1 × 106 cells/150 μl) were subcutaneously injected into the left flank of the mice. One week after the injection of Hep3B cells, anthocyanins were dissolved in PBS and administered intraperitoneally (50 mg/kg/day) for 20 days. The control animals were injected with vehicle (PBS) alone. Tumor size was measured using a caliper at 2 day intervals, and the volume was calculated by the modified formula V = 1/2 (length × width2). After the 20 day treatment, tumors were removed and frozen in liquid nitrogen for western blot analysis or fixed with formalin for immunohistochemistry. All animal experiments were approved by the Ethics Committee for Animal Experimentation, Hannam University.
Tumor specimens from mice were fixed in 10% formaldehyde, embedded in paraffin and sectioned into 5 μm thick slices. Sections were deparaffinized with xylene and dehydrated with 98% ethanol. Serial sections were stained using standard immunoperoxidase techniques with primary antibodies against CD31 (1:100) and p-AMPKα1 (1:50). For epitope retrieval, specimens were microwave treated for 25 min before incubation with primary antibodies. Pre-immune serum was used as a negative control for immunostaining, and positive staining was visualized with diaminobenzidine, followed by a light counter-staining with hematoxylin. All findings were evaluated by a pathologist blinded to the treatment conditions, and samples were evaluated on the basis of stain intensity and percentage of reactive cells. Images of representative results were recorded.
Cell viability and tumor volume data were statistically analyzed using unpaired t-test (SPSS, Chicago, IL). P < 0.05 was considered statistically significant.