Methods

2.3.1 Cell culture

Human aortic smooth muscle cells (HASMCs) were purchased from Food Industry Research and Development Institute, 新竹, Taiwan (CCRC 60293). They were maintained in Ham’s F12K containing 10 % fetal bovine serum, 2 mmol/l L-glutamine, 1.5 g/l sodium bicarbonate, 10 mmol/l HEPES, 10 mmol/l TES, 0.05 mg/ml ascorbic acid, 0.01 mg/ml transferrin, 0.01 mg/ml insulin, 10 ng/ml sodium selenite, 0.03 mg/ml ECGs. All experiments were performed with HASMCs from passages 21 to 31, which were grown to 80-90 % confluence and made quiescent by serum starvation (0.1 % FBS) for at least 24 h.

2.3.2 Cell viability assay (MTT assay)

The cytotoxic effect of curcumin on HASMCs was investigated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay (Chen et al, 2002). The principle of this assay is that mitochondria dehydrogenase in viable cells reduces MTT to a blue formazan. Briefly, the cells were grown in 96-well culture plates at a density of 1×10⁴ cells per well in F-12K culture medium and incubated with various concentrations of curcumin for 24 hours. 10 μl MTT (5 mg/ml) were then added to each well and incubation continued at 37 °C for an additional 4 hours. The medium was then carefully removed, so as not to disturb the formazan crystals which had formed. Dimethyl sulphoxide (100 μl), which solubilizes formazan crystals, was

28

added to each well and absorbance of the solubilized blue formazan was measure the optical density at 590 nm using µQuant Microplate Spectrophotometer (Bio-Tek, VT, USA). All determinations were performed according to three individual experiments.

The data were shown mean±SD as percentage of control.

2.3.3 Gelatin zymography for MMP-9

MMP-9 activity in conditioned medium of cultured HASMCs was analyzed by substrate-gel electrophoresis (zymography) using SDS-PAGE (10 %) containing 0.1 % gelatin. Substrate gel zymography of the activity of MMP-9 was performed with a Mini-Protein II apparatus from Bio-Rad, according to a method described previously (Demeule et al, 2000). Cells were grown to sub-confluence and were rinsed with phosphate-buffered saline (PBS) and then incubated in serum-free medium for 24 h.

Equal volumes of samples of conditioned cell culture medium were mixed with sample buffer containing 62.5 mmol/l Tris-HCl (pH 6.8), 10 % glycerol, 2 % SDS, and 0.00625 % (w/v) bromophenol blue, loaded onto the gel and separated by electrophoresis. Thereafter, gels were washed 3 times for 30 minutes at room temperature in buffer (50 mmol/l Tris-HCl, pH 8.0, 5 mmol/l CaCl2, 0.02 % NaN3, and 2.5 % Triton X-100) and incubated for 18 h at 37 °C with the same buffer except Triton X-100. Gels were stained with Coomasssie Brillant Blue R-2500 (0.1 %) and destained in 5 % methanol and 7 % acetic acid. Gelatinolytic activity appeared as a clear band on a blue background.

2.3.4 Bradford protein assay

The Bradford assay (Bradford, 1976), a colorimetric protein assay, is based on an absorbance shift in the dye Coomassie when bound to arginine and hydrophobic amino acid residues present in protein. The anionic (bound) form of the dye is blue and has an absorption spectrum maximum historically held to be at 595 nm. The cationic (unbound) forms are green and red. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample. Standard solutions contain a range of 0 to 25 micrograms protein (BSA) in 800 μl H2O, followed by adding 200 μl dye reagent and incubate 5 min. l μl of sample solution add into 799 μl H2O, followed by adding 200 μl dye reagent and incubated for 5 min. The absorbance was read at 595 nm. The results made a standard curve and the protein concentration of sample was determined by standard curve.

2.3.5 Western blot analysis

HASMCs were treated with various concentrations of curcumin in the presence of 100 ng/ml TNF-α. Cellular lysates were prepared in a lysis buffer containing 10 mmol/l Tris/HCl (pH 8), 0.32 mol/l sucrose, 5 mmol/l Ethyienediamine Teraacetate Disodium Salt (EDTA), 1 % Triton X-100, 2 mmol/l 1, 4-Dithio-D,L-thereitol (DTT), 1 mmol/l PMSF. The cells were disrupted and extracted at 4 °C for 30 min. After centrifugation at 13,000 rpm for 15 min, the supernatant was obtained as the cell lysate.

Protein concentrations were measured using the bradford assay. Total protein (20 μg) were subjected to SDS-PAGE (10 %) and blotted on PVDF membranes (Shishodia et al, 2003). Nonspecific binding was blocked by soaking the membrane in PBS-Tween

30

20 (PBST) buffer containing 50 g/l nonfat milk. The membrane was incubated with monoclonal mouse anti-human β-actin (1:1000) and polyclonal rabbit anti-human MMP-9 (1:1000). Subsequently, the membrane was incubated with sheep anti-mouse IgG antibody (1:5000) and goat anti-rabbit IgG antibody (1:5000). The protein levels were determined using the enhanced chemiluminescence detection reagents (Upstate, CA, USA) and high performance chemiluminescence film (Amersham, IL, USA).

Incubation with mouse anti-human β-actin antibody was also performed as an internal control. Results were quantified with scanning densitometer using an image analysis system with software.

2.3.6 Preparation of nuclear extract

Nuclear protein extracts of HASMCs were prepared using a nuclear extract kit (TransAM nuclear extract kit, CA, USA). Cells were lysed in hypotonic buffer and centrifuge suspension for 30 seconds at 14,000×g in a microcentrifuge pre-cooled at 4

°C (Dschietzig et al, 2001). Then resuspend nuclear pellet in 50 μl complete lysis buffer containing 10 mmol/l DTT, lysis buffer AM2, and protease inhibitor cocktail by pipetting up and down. The suspension was incubated for 30 min on ice, and centrifuged for 10 min at 14,000×g in a microcentrifuge pre-cooled at 4 °C. Transfer supernatant and stored at -80 °C. Protein concentrations were measured using the bradford protein assay.

2.3.7 ELISA-Based Nuclear Factor-κB Assay

Additionally to gel-shift assays, an ELISA-based kit was used for quantitative detection of NF-κB activity (TransAM NF-κB kit, CA, USA). For each sample, 20 μl of nuclear extracts (5 μg protein) were used according to the instructions of the manufacturer (Yu et al, 2007). Nuclear extracts were incubated with the oligonucleotide-coated wells for 60 min. Where indicated a competitor for NF-κB binding (NF-κB wild-type consensus oligonucleotide) was added in molar excess prior to the probe. The wells were then washed and incubated with the primary antibodies for p50 and p65 for 60 min. After incubation with a horseradish peroxidase-conjugated secondary antibody, a substrate was added to produce blue colour and then for quantitation by µQuant Microplate Spectrophotometer (Bio-tek, VT, USA). The absorbance was read at 590 nm and the blank was subtracted from all measurements.

2.3.8 Cell migration assay

VSMCs invasion through the extracellular matrix was determined by using a commercial cell invasion assay kit (Chemicon, CA, USA). HASMCs (1.5×10⁵ cells/300 μl) were resuspended in conditioned medium collected from pretreatment with curcumin and TNF-α-treated cells for 23 hours, and added to the upper components of migration chamber (Bedoui et al, 2005). Five hundred microliters of same conditioned medium were added to the lower compartment of migration chamber.

Cells without TNF-α-treated conditioned medium served as control. The migration chambers were incubated at 37 °C for 24 hours in 5 % CO2. After incubation, the inserts were removed from the wells, and the cells on the upper side of the filter were removed using cotton swabs. The filters were fixed, and stained according to the

32

manufacturer’s instructions. The cells that invaded and were located on the underside of the inserts. Then transfer 100 μl of the dye mixture to a 96-well plate, and measure the optical density at 560 nm.

2.3.9 Measurement of intracellular ROS

HASMCs were pretreated with 10 and 20 μmol/l curcumin for 1 hour and induced by TNF-α (100 ng/ml) for 23 hours. Then were incubated with 10 μmol/l 2,7-dichlorofluorescein (DCF) diacetate (DCFH-DA) for 30 minutes, which is converted to DCF by intracellular esterase (Kim et al, 2006). The latter was then oxidized by ROS to the highly fluorescent DCF. The fluorescence of each dish was immediately analyzed at excitation wavelength of 485 nm and emission wavelength of 528 nm by FLx800 microplate fluorescence reader (Bio-tek, VT, USA). All measurements were at least triplicated.

2.3.10 Statistical analysis

Results are shown as mean±SD. Statistical analyses of MTT were performed using One-way ANOVA followed by Dunnett’s test and others were performed using One-way ANOVA followed by Duncan’s Multiple Range Test. A value of p<0.05 was considered statistically significant.

34