Tea extract from black tea, polyphenols from green tea, (-)-epicatechin, (-)-epicatechin gallate (ECG); (-)-epigallocatechin (EGC); (-)-epigallocatechin gallate (EGCG), ellagic acid, and gallic acid were obtained from Sigma Chemical Co. (St. Louis, MO) and were dissolved in sterile water at 1 mg/ml; epicatechin was dissolved in a 1:1 mixture of actone:water, and ellagic acid was dissolved in 1 M sodium hydroxide. All were used fresh or stored at -20°C. Lipoprotein-free calf serum, acadesine (AICAR), 1,1-dimethylbiguanide hydrochloride (Metformin), and protease inhibitor cocktail were also obtained from Sigma. HALT phosphatase inhibitor and the BCA protein assay kit were purchased from Pierce Biotechnology, Inc. (Rockford, IL). Dulbecco's modified media (DMEM), penicillin-streptomycin-glutamine (PSG), and trypsin were obtained from GIBCO (Invitrogen, Carlsbad, CA). Fetal bovine serum was obtained from BioWhittaker, Inc. (Walkersville, MD) or GIBCO. Radiochemicals (14C-acetate at 56 mCi/mmol, 14C-mevalonate at 65 mCi/mmol, and 14C-Hydroxymethylglutaryl-CoA at 55 mCi/mmol) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). McARH7777 rat hepatoma cells were obtained from American Type Culture Collection and used between passages 1-40.
2.2 Cytotoxicity assays
McARH7777 rat hepatoma cells were grown in DMEM supplemented with 10% FBS and 1.0% PSG in 6-well plates at 37°C under a humidified atmosphere of 5% CO2. The cells were used between passages 1 and 40. After 48 h the media was replaced with fresh media with the addition of the selected tea compound or extract and incubation was continued for 3 h. Control cells received an equal volume of vehicle. The cells were detached with trypsin, suspended in DMEM containing 0.2% trypan blue, and counted in a hemocytometer. Leakage of lactate dehydrogenase from cells was determined by measuring NADH oxidation from added pyruvate spectrophotometrically at 340 nm and compared to total lactate dehydrogenase activity from cells lysed with 0.1% Triton X100.
2.3 Determination of cholesterol synthesis
Hepatoma cells were cultured for 48 h in 6-well plates, at which time the media was replaced and appropriate concentrations of the tea extracts or compounds were added along with 1 μCi of 14C-acetate or 14C-mevalonate. Incubations were carried out for 3 h, after which time the cells were washed twice with phosphate-buffered saline, harvested by trypsinization or scraping, resuspended in 20 mM Tris buffer, pH 7.4, containing 0.1% Triton X100, and lysed by sonication (Sonic Dismembrator, Fisher Scientific, Pittsburgh, PA) at medium setting on ice with ten 8-sec pulses, separated by 30 sec each. Lipids were extracted into 5 ml of chloroform:methanol (2:1), the solvent was removed by evaporative centrifugation, and the lipids were resuspended in 50 μl of chloroform/methanol and spotted onto silica thin layer plates (Whatman). Chromatography was carried out in petroleum ether:ethyl ether:acetic acid (60:40:1). Cholesterol was identified by co-chromatography of an authentic standard visualized by iodine vapor staining and quantified by electronic autoradiography (Packard Instant Imager).
2.4 Determination of 14C-acetate uptake
Hepatoma cells treated with tea extract were incubated for 3 h with 1 μCi of 14C-acetate or 14C-mevalonate, after which time the media was removed, the cells were washed twice with phosphate-buffered saline, harvested by trypsinization or scraping, resuspended in 20 mM Tris buffer, pH 7.4, containing 0.1% Triton X100, and lysed by sonication (Sonic Dismembrator, Fisher Scientific) at medium setting on ice with ten 8-sec pulses, separated by 30 sec each. Radioactivity was counted by scintillation spectrophotometry and normalized to protein concentration, as determined by BCA method (Pierce Chemical, Inc., Rockford, IL).
2.5 Determination of squalene and lanosterol synthesis
For the determination of squalene and lanosterol synthesis, cells were incubated as described above for cholesterol synthesis with the inclusion of 60 μM terbinafine (100× in ethanol), an inhibitor of squalene monooxygenase (for the determination of squalene) or 10 μM ketoconazole (100× in methanol), an inhibitor of lanosterol demethylase (for the determination of lanosterol), along with the selected tea extract or component. Lipids were saponified by addition of 0.5 ml of 10% methanolic potassium hydroxide and incubated at 80°C for 1 h. For the determination of squalene the neutral lipids were extracted into 5 ml of petroleum ether, the solvent was removed by centrifugal evaporation, and the samples were resuspended in 50 μl of petroleum ether and resolved by silica thin-layer chromatography in 5% ethyl acetate in hexane. Lanosterol was determined as described for cholesterol. Authentic standards for squalene and lanosterol were visualized by iodine-vapor staining.
2.6 Determination of HMG-CoA reductase activity in microsomes
The microsomal fraction (100,000×g pellet resuspended at ~15 mg protein/ml) was prepared by standard procedures from the livers of untreated male Harlan Sprague-Dawley rats (~200 g). HMG-CoA reductase activity was determined by the procedure of Brown and Goldstein [18
] as follows: Microsomes (100 μg) were incubated at 37°C in a final volume of 200 μl of 0.1 M potassium phosphate buffer, pH 7.5, containing the indicated tea extract or component, 20 mM glucose 6-phosphate, 2.5 mM NADP+
, 1 unit of glucose-6-phosphate dehydrogenase, 5 mM dithiothreitol, and 0.2 μCi 14
The reaction was stopped after 3 h by the addition of 25 μl of 6M HCl. Mevalonate was converted to the lactone by standing at 37°C for 30 min, extracted into 5 ml of ethyl acetate, and brought to dryness by evaporative centrifugation. The sample was redissolved in 50 μl of ethyl acetate and fractionated by silica thin-layer chromatography with benzene:acetone (1:1). Mevalonolactone was identified by co-migration with authentic mevalonolactone visualized by iodine vapor staining and quantified by electronic autoradiography.
2.7 Immunoquantitation of AMP-kinase and HMG-CoA reductase
Hepatoma cells between passages 12-15 were cultured in 6 cm plates for 2-3 days, until reaching confluence, with daily changes of media. Cells were treated with the indicated tea extract or AMP-kinase activator (AICAR or metformin, 1 mM final concentration) for 3 hr in fresh media, after which cells were washed once with phosphate-buffered saline, scraped from the plates, pelleted by low-speed centrifugation, and lysed by two cycles of freeze-thawing in 0.25 M Tris buffer, pH 7.5, containing protease and phosphatase inhibitor cocktails at 2× standard concentration. The lysates were cleared by centrifugation (18,300 × g, 10 min, at 4°C) and the supernatant was stored as aliquots at -80°C. Thirty μg of protein (determined by BCA analysis) was fractionated by sodium dodecyl sulfate-polyacrylamide gel (8%) electrophoresis and electroblotted to nitrocellulose. The membrane was blocked with 0.05% Tween-20 and 5% defatted milk, and then incubated in this same buffer with rabbit antibody to total AMP-kinase (Anti-AMPK α-pan, 1:2000; Upstate USA, Inc., Charlottesville, VA) or to phosphorylated AMP-kinase (Anti-phospho-AMPKα, 1:500; Upstate USA, Inc.) overnight at 4°C. The immunoblot was developed with a secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature and the chemiluminescent image (Supersignal West Pico Chemiluminescent Substrate, Pierce Biotechnology, Inc.) captured by autoradiography. Band intensity was measured with Image J software on the scanned image with background subtraction. For estimation of HMG-CoA reductase phosphorylation, lysates were incubated for 1 hr at 4° with 20 μl of mouse monoclonal antibody to phosphoserine/phosphothreonine/phosphotyrosine (Abcam Inc., Cambridge, MA) and antibody conjugates were precipitated with 35 μl of protein G Plus-Agarose from Calbiochem (EMD Chemicals, La Jolla, CA) by following an Amersham Biosciences immunoprecipitation protocol. Immunoprecipitated protein was released by boiling in gel loading buffer and fractionated by SDS-gel electrophoresis, followed by electroblotting to nitrocellulose. HMG-CoA reductase was detected and quantified with an antibody to the enzyme (Upstate USA, Inc.) followed by a secondary antibody conjugated with horseradish peroxidase as described above.