Collection and identification of plant materials
The fruits of P. granatum were field collected from a farm land (22°41'59.3267” N, 120°30'45.1836” E) located in a small township Jiuru, Pingtung county, southern Taiwan from August to September, 2012. The plant specimens were identified by Liao, G.-I. and pressed/dried for voucher specimens (Nan-Kai Lin, STUSTG308-001 to STUSTG308-003) deposited in the herbarium of Taiwan forestry research Institute (TAIF), Taiwan.
Preparation of pomegranate fruit ethanol extract (PEE)
Fresh pomegranate fruit was peeled and the edible portion was squeezed with gauze. The subsequent juice was concentrated by freeze dried with 37.5 ml juice to produce 4.13 g of powder. The powder was first extracted with ethylacetate (EtOAc) at a ratio of 1:3 (w/v) in 50 mL-poly-propylene (PP) centrifugation tube with 360° rotation for 16 hours at room temperature. After extraction, the residue was collected with centrifugation at 10,000 × g and the supernatant was vacuum dried. After centrifugation, the residue was extracted with 70% ethanol as described in EtOAc extraction. After extraction, 17 mg [yield 0.41% (w/w)] and 2.96 g [yield 71.7% (w/w)] of the products were obtained respectively from EtOAc and EtOH extraction of 37.5 ml juice.
Human urinary bladder urothelial carcinoma (UBUC) T24 cell, which is recognized as high grade and invasive, was purchased from Bioresource Collection and Research Center, Hsinchu, Taiwan and cultured at 37°C in McCoy's5A [GIBCO (Life technologies), Grand Island, N.Y., U.S.A.], supplemented with 10% (v/v) fetal bovine serum (FBS). Human papillomavirus E7 immortalized uroepithelial cell was kindly provided by Professor Hsiao-Sheng Liu from department of microbiology and immunology, college of medicine, National Cheng Kung University, Tainan, Taiwan and maintained as described previously. UBUC J82 cells recognized as high grade was offered by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37°C in Dulbecco's Modified Eagle Medium supplemented with 10% (v/v) FBS (GIBCO, Grand Island, N.Y., U.S.A.).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
Appropriate concentrations of PEE were added to a 96-well plate already seeded with 5,000 human T24 cells, 6,000 human J82 cells or 3,000 human E7 cells per well. After exposure for the indicated time duration, 20 μl of MTT solution (Merck, Damstadt, German) (5 mg/ml PBS) was added to each well and the plate was incubated at 37°C for 4 hours. After medium removal, 200 μl of DMSO was added to each well and the plate was gently shaken for 5 minutes. The absorbance was determined at 540 nm. Quadruplicate wells were applied to each concentration for a specific time period. 0.002% (v/v) DMSO (vehicle)-treated bladder cancer cells were employed as the control.
Cell cycle analysis of PEE-treated human T24 cells
Human T24 cells were plated onto a 6-well plate at 8 × 105/well and cultivated overnight. Then 50 or 100 μg/ml of PEE was added onto each well and the cells were harvested with trypsinization at the indicated time period. The harvested cells were incubated with 500 μl of ice-cold 70% ethanol/well at 4°C overnight. After ethanol treatment, the cells were washed with 1 ml ice-cold PBS/well, re-suspended in 100 μl PBS/well and incubated with 300 μl propidium iodide (PI) (Sigma, Saint Louis, Missouri, USA) solution (3 μl RNase and 20 μg PI per ml)/well in the dark at 37°C for 30 minutes. The stained cells are analyzed by FACSCalibur flow cytometer (Becton Dickinson). 0.002% (v/v) DMSO-treated human T24 cells were used as the control and analyzed as described above.
Annexin V/PI staining of PEE-exposed human T24 cells
Human T24 cells were plated onto a 6-well plate at 4 × 105/well and cultivated overnight. Then 50 or 100 μg/ml of PEE was added onto each well and the cells were harvested with trypsinization at the indicated time period. The harvested cells were washed with 1 ml ice-cold PBS/well and re-suspended in 100 μl binding buffer/well. Then 2 μl of annexin V conjugated with FITC (Strong Biotech Corporation, Tainan, Taiwan) plus 2 μl of PI solution (Sigma, Saint Louis, Missouri, USA) were mixed with the re-suspended cells and incubated in the dark on ice for 15 minutes. After staining, the cells were analyzed using FACSCalibur, Becton Dickinson. 0.002% (v/v) DMSO-treated human T24 cells were utilized as the control and analyzed as described above. As to annexin V/PI experiments incorporated with caspase-3 Z-DEVD-FMK inhibitor (BD Bioscience), human T24 cells were incubated with 2 μM inhibitor for 4 hours. Then inhibitor-pre-incubated human T24 cells were exposed in the presence or absence of 100 μg/ml PEE for 48 hours. Apoptosis was detected as described above.
After appropriate treatment with PEE, human T24 or J82 cells were harvested and lysed in lysis buffer [10 mM Tris (pH 8.0), 0.32 M sucrose, 1% (v/v) Triton X-100, 5 mM EDTA, 2 mM DTT, and 1 mM PMSF]. After determining its protein concentration using Bio-Rad DC protein assay kit, equal volume of 2× sample buffer [0.1 M Tris (pH 6.8), 2% SDS, 0.2% β-mercaptoethanol, 10% (v/v) glycerol, and 0.0016% (w/v) bromophenol blue] was combined with the cytoplasmic extract. Appropriate amounts of the lysates were separated by electrophoresis at 100 V with 10% (w/v) sodium dodecyl sulfate (SDS)-polyacrylamide gels, and further transferred on to a PVDF membrane (Strategene, La Jolla, CA, USA). After blocking for 1 hour in 3% (w/v) bovine albumin serum (BSA) at room temperature, membranes were hybridized overnight at 4°C with primary antibodies. The primary antibodies used in this study were listed in supplementary document. The membranes were washed and probed with suitable secondary antibodies for 1 hour at room temperature. Secondary antibodies binding on the membrane were detected by chemiluminescence ECL detection system (Amersham-Pharmacia Biotech Inc., Piscataway, NJ, USA) using Fujifilm LAS-3000 Luminescent Image Analyzer (Fujifilm Corporation, Tokyo, Japan). The intensity of each protein band was quantified by PDQUEST Quantity One software (Bio-Rad Laboratory, Hercules, CA) and normalized with actin protein expression level.
Student’s t-test was used to assess the significance between controls and treatments. Statistical analysis was performed using STATISTICA Advanced V10.0 (Statsofa Holdings, Inc., Tulsa, Oklahoma, USA).