Anticancer Effects of Eleven Triterpenoids Derived from Antrodia camphorata

Materials and Methods

Extraction and isolation of compounds from fruiting bodies of A. camphorate. Fruiting bodies of A. camphorata were collected from Yuli, Hualien County, Taiwan, in December 2007. The species was identified by Professor Yew-Min Tzeng at the Natural Products and Bioprocess Laboratory, Chaoyang University of Technology. A voucher specimen (YMT 7002) was deposited in the Herbarium of the Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Taiwan, ROC. Air-dried powder of the fruiting bodies (101.9 g) of A. camphorata was successively extracted (3×1000 ml) with n-hexane, chloroform and methanol under reflux. The resulting extracts were concentrated individually under reduced pressure to yield 3.02 g (2.96% based on dry weight), 46.3 g (45.43%) and 2.7 g (2.64 %) of residues from n-hexane, chloroform and methanol (MeOH) extracts, respectively. Repeated silica-gel column chromatography (5×90 cm) of the chloroform extract residue was carried out with increasing polarity of elutant using mixtures of hexane/ethyl acetate. Following thin-layer chromatographic analysis, eluates with similar profiles were combined to produce six fractions (F1-F6). Fraction F2 was rechromatographed over a silica-gel column eluted with a gradient of hexane/EtOAc to afford methyl antcinate B (MAB, compound 1), dehydroeburicoic acid (DeEA, compound 2) and 15α-acetyl dehydrosulfurenic acid (ADSA, compound 3), respectively. Fraction F3 was purified by silica-gel chromatography eluted with a gradient of hexane/ethyl acetate, resulting in 3β,15α-dihydroxylanosta-7,9,24-triene-21-oic acid (compound 4), and zhankuic acid A (compound 5). Fractions F4 and F5 were purified by column chromatography and eluted with a gradient of n-hexane/ethyl acetate, to afford four subfractions (D1 to D4). Dehydrosulfurenic acid (compound 6) and zhankuic acid C (compound 7), and sulfurenic acid (SA, compound 8) were obtained from subfractions D2, D3 and D4, respectively. Fraction F6 was purified by silica-gel column chromatography using chloroform/methanol from 100% chloroform to 20% methanol to yield antcin A (Ant A, compound 9) and a mixture of compounds that was further separated by column chromatography using chloroform/methanol elution to give antcin C (Ant C, compound 10) and methyl antcinate A (MAA, compound 11). The structures of compounds 1-11 were determined by ¹H- and ¹³C-nuclear magnetic resonance (NMR) spectroscopy and by comparison of the spectral data with published values (13, 14).

Cell culture. Cells used in this study were purchased from Bioresource Collection and Research Center, Food Industry Research and Development Institute (Hsinchu, Taiwan, ROC) or provided by the Cell Bank of Chung Shan Medical University. TsCCA, AsPC, BT474, T47D, HeLa, U2OS and gingival fibroblast (GF) cells were grown in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies). GNM, OC-2, OEC-M1, Panc-1, PC-3 and AGS cells were cultured in RPMI-1640 with 10% FBS. KB cells were maintained in Eagle's minimal essential medium (MEM) with 10% FBS. OVCAE-3 cells were cultured in DMEM with 20% FBS and 0.01 mg/ml bovine insulin. The maintenance growth medium for the MCF-12A cells consisted of a 1:1 mixture of DMEM and Ham's F12 medium, 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 10 mg/ml insulin and 500 ng/ml hydrocortisone. All media included penicillin (100 IU/ml) and streptomycin (100 μg/ml). Briefly, the cells were maintained in the appropriate growth medium at 37°C in a humidified atmosphere of 5% CO₂ and 95% air and were used over a restricted culture period of 10 passages.

Cell viability analysis. The effect of test agents on cell viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in 4 to 6 replicates as previously described (15-19). All tested cell lines were grown in 5% FBS-supplemented culture medium in 96-well plates overnight, and then exposed to the different compounds for 24 hours at different concentrations, including 10, 50 and 200 μM. Controls received 0.1% (V/V) dimethyl sulfoxide (DMSO) vehicle at a concentration equal to the one used for drug-treated cells. At the end of the treatment, the medium was removed, replaced by 200 μl of 0.5 mg/ml of MTT in the same medium, and cells were re-incubated for 2 h. Supernatants were removed from the wells, and the reduced MTT dye was solubilized in 200 μl/well DMSO. Absorbance was determined at 595 nm on a plate reader.

Caspase-3 activity. Caspase-3 activity was determined using the BD ApoAlert Caspase-3 assay according to the manufacturer's instructions (Becton, Dickinson and Company, Palo Alto, CA, USA) as previously described (16, 19, 20). Briefly, 2×10⁶ cells were centrifuged at 400 ×g for 5 min, resuspended in 50 μl of chilled cell lysis buffer and incubated on ice for 10 min, then 50 μl of 2× reaction buffer/ DTT mix and 1 μl of caspase-3 inhibitor (DEVD-fmk, 1 mM) were added to some samples which were then incubated on ice for 30 min as negative controls; 50 μl of 2× reaction buffer/DTT mixture and 1 μl DMSO per 50 μl of 2× reaction buffer were added to the samples without inhibitor to ensure that all samples were tested under similar conditions. Five microliters of 1 mmol/l caspase-3 substrate DEVD-pNA were added and the mixture was incubated at 37°C for 1 h. Cells were analyzed by a FACScan flow cytometer (Becton, Dickinson and Company). All samples were run in triplicate with appropriate controls. Camptothecin (Sigma-Aldrich, St. Louis, MO, USA) served as the positive control.

Western blotting. Western blotting was performed as previously described (12, 19). Cells were lysed in boiled lysis buffer containing 1% SDS and 10 mM/l Tris-HCl (pH 7.4). Following centrifugation at 400 ×g for 10 min, the protein concentration in each lysate was measured (Bradford assay). Equal amounts of total protein from each sample were fractionated by SDS-PAGE, blotted onto a polyvinylidene difluoride membrane, and probed with the primary antibody, mouse monoclonal anti-caspases-3 (1:1000, v/v), or rabbit monoclonal antibodies to poly (ADP-ribose) polymerase (PARP) (1:4000, v/v), Bcl-2-associated × protein (Bax) (1:8000, v/v) and β-actin (1:2000, v/v), followed by 1 h incubation with a 1:10000 (v/v) dilution of the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. Primary antibodies (β-actin, PARP, Bax) were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA), whereas secondary antibodies (anti-rabbit IgG and anti-mouse IgG HRP-labeled) were purchased from PerkinElmer (Boston, MA, USA). The hybridized immunocomplex was detected by Renaissance Chemiluminescence Reagent Plus (NEN Life Science Products, Boston, MA, USA).

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Figure 1.

Chemical structures of triterpenoid compounds 1-11 isolated from the fruiting bodies of Antrodia camphorata.

Statistical analysis. Values are the mean±standard deviation (SD) of experiments carried out in triplicate. Statistical significance was tested by Student's t-test for either paired or unpaired data, where appropriate. Statistical significance was defined as p<0.01.