48
Determination of phenolic compounds
Total phenolic compounds were determined the Folin-Ciocalteau colorimetric method using gallic acid
as a standard by a modified procedure of Singleton
et al
. (1999). For the preparation of calibration curve, 0.5 mL
aliquots of 50-300 ôg/mL ethanolic gallic acid solution (50% ethanol, w/v) were mixed with 2.5 mL of Folin-
Ciocalteau reagent, FCR and 2 mL of 2% (w/v) sodium carbonate solution. Dried pomace was dissolved in 50%
(v/v) ethanol to a final concentration of 20 mg/mL. The sample solutions (0.5 mL) were mixed with the same
reagents as described above. The absorbance of standard and samples were then measured at 760 nm after
standing at room temperature (25
°
C) for 30 min. All the tests were performed in triplicate. Results were expressed
as ôg gallic acid equivalents per g.
Cell Culture
MCF-7 human breast cancer cells obtained from the American Type Culture Collection (Rockville, MD,
USA) were cultured in RPMI 1640 medium supplemented with 10% foetal calf serum (FCS), penicillin (100
IU/ml) and streptomycin (100 ôg/ml). DHT (Sigma-Aldrich, Sydney, Australia) was dissolved in 100% ethanol
and added to media immediately prior to use. For these studies, MCF-7 cultures were used in experiments up to 20
passages following thawing, experiments were repeated two to five times and representative blots are shown.
MTT assay
Antiproliferative assay was determined by MTT assay (McAtee and Davis, 1994). This colorimetric
assay is based on the conversion of the yellow tetrazolium bromide (MTT) to the purple formazan derivatives by
mitochondrial succinate dehydrogenase in viable cells (Hansen
et al
., 1989). Then, the grape samples of various
concentrations (10-1000 ôg/mL) were added to the cells (100 ôL/well) and incubated for 6, 12 and 24 h at 37
°
C
and 5% CO
2.
Fifty microliters of MTT solution (5 mg/mL in phosphate-buffered saline, pH 7.4) was added to each
well and incubated for an additional 4 h. The cultured medium was removed. One hundred microliters of DMSO
were added. The plates were gently agitated until the formazan precipitate was dissolved and the color change was
measured at 570 nm decreasing in absorbance indicated a reduction in cell viability. RPMI-1640 medium was
served as the negative control, and resveratrol was as a positive control. Wells without cells were used as blanks
and were subtracted as blackground from each sample. The results are expressed as the percentage of viable cells
with respect to the control. Experiments for each sample were carried out in triplicate. The absorbance was plotted
against the concentrations of the samples, and
IC
50
was calculated and obtained nonlinear regression.
SDS-PAGE gel electrophoresis and Western blot
Denaturing polyacrylamide gel electrophoresis was performed according to the method of Laemmli
(1970). The blotted membranes were blocked with 0.1% (v/v) Tween-20 in Tris-buffered saline (TBST)
containing 5% (w/v) nonfat dry milk for 20 min at room temperature. The blots were then incubated with the first
