Induces Apoptosis in Human Liver Cancer Cells through O - American

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Methyl Antcinate A from Antrodia camphorata Induces Apoptosis in Human Liver Cancer Cells through Oxidant-Mediated Cofilin- and Bax-Triggered Mitochondrial Pathway Yun-Chih Hsieh,† Yerra Koteswara Rao,‡ Chun-Chi Wu,§ Chi-Ying F. Huang,| Madamanchi Geethangili,‡ Shih-Lan Hsu,*,† and Yew-Min Tzeng*,‡ Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan, Republic of China, Institute of Biochemical Sciences and Technology, Chaoyang UniVersity of Technology, Wufeng, Taiwan, Republic of China, Institute of Medical and Molecular Toxicology, Chung Shan Medical UniVersity, Taichung, Taiwan, Republic of China, and Institute of Clinical Medicine, National Yang-Ming UniVersity, Taipei, Taiwan, Republic of China ReceiVed March 15, 2010

We investigated the effects of antcin A, antcin C, and methyl antcinate A (MAA) isolated from Antrodia camphorata on the proliferation of human liver cancer cell lines Huh7, HepG2, and Hep3B and the normal cell rat hepatocytes. The three compounds selectively inhibit the proliferation of tumor cells rather than normal cells, with IC50 values ranging from 30.2 to 286.4 µM. The compound MAA was a more potent cytotoxic agent than antcins A and C with IC50 values of 52.2, 78.0, and 30.2 µM against HepG2, Hep3B, and Huh7 cells, respectively. To elucidate the molecular mechanism, treatment of Huh7 cells with 100 µM MAA induced an apoptotic cell death, which was characterized by the appearance of sub-G1 population, DNA fragmentation, TUNEL positive cells, and caspase activation. MAA triggered the mitochondrial apoptotic pathway, as indicated by an increase in the protein expression of Bax, Bak, and PUMA, as well as a decrease in Bcl-XL and Bcl-2 and disruption of mitochondrial membrane potential and promotion of mitochondrial cytochrome c release, as well as activation of caspases-2, -3, and -9. We also found that pretreatment with inhibitors of caspases-2, -3, and -9 noticeably blocked MAA-triggered apoptosis. Furthermore, intracellular reactive oxygen species (ROS) generation and NADPH oxidase activation were observed in MAA-stimulated Huh7 cells. Mechanistic studies showed that MAA induces mitochondrial translocation of cofilin. When Huh7 cells were treated with cyclosporine A and bongkrekic acid, an inhibitor of the mitochondria permeability transition pore, the levels of cell death induced by MAA were significantly attenuated. Additionally, pretreatment of Huh7 cells with antioxidants ascorbic acid and N-acetyl cysteine markedly attenuated the MAA-induced apoptosis by upregulation of Bax, Bak, and PUMA, mitochondrial translocation of cofilin, activation of caspase-3, and cell death. Taken together, our results provide the first evidence of the activation of the ROS-dependent cofilin- and Baxtriggered mitochondrial pathway as a critical mechanism of MAA-induced cell death in liver cancer cells. Introduction Antrodia camphorata (Niu-Chang-Chih or Zhan-Ku), Polyporaceae, is a medicinal mushroom that has been used for centuries for food intoxication, vomiting, and poisoning and to improve liver and stomach immunity in traditional Chinese medicine (1). The crude extracts of A. camphorata have a broad range of biological activities, and recently, research has focused on elucidating the pharmacological potential including anticancer, immunomodulatory, and anti-inflammatory effects (2). Particularly, the anticancer effects of its crude extracts have been reported in various cancer cell types. For example, it has been reported that A. camphorata ethylacetate extract (EAC) can inhibit the proliferation of superficial and invasive bladder * To whom correspondence should be addressed. (S.-L.H.) Tel: 886-423592525 ext. 4039. Fax: 886-4-23592705. E-mail: [email protected]. (Y.-M.T.) Tel: 886-4-23323000 ext. 4471. Fax: 886-4-23395870. E-mail: [email protected]. † Taichung Veterans General Hospital. ‡ Chaoyang University of Technology. § Chung Shan Medical University. | National Yang-Ming University.

transitional cell carcinoma cell lines (3). The methanol extract can induce HepG2 cell apoptosis possibly via Fas pathway (4). The EAC exhibits the cytotoxic effects on a liver cancer cell line HepG2 via a mitochondrial apoptotic pathway (5). Furthermore, EAC also suppresses another liver cancer cell line Hep3B by the calpain/Bid/Bax and Ca2+/mitochondrial pathway (6). In addition, recent data revealed that A. camphorata extracts induce apoptosis in breast cancer cell lines (7, 8) and in human leukemia HL-60 cells (9). We previously reported that the CHCl3 extract from fruiting bodies of A. camphorata showed cytotoxic activity against human cancer cell lines Jurkat, HepG2, colon 205, and MCF 7 (10). However, all of these previous reports on the pharmacological activities of A. camphorata hitherto utilize the crude extracts, and it is not clear what compounds within the crude extract are responsible for generating the observed anticancer effects. The characteristic constituents of A. camphorata are various terpenoids, benzenoids, lignans, benzoquinones, and maleic/ succinic acid derivatives, in addition to polysaccharides (2). Triterpenoids are the major representative of phytoconstituents and account for ∼60% in the fruiting bodies of A. camphorata

10.1021/tx100116a  2010 American Chemical Society Published on Web 06/17/2010

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Figure 1. (A) Chemical structures of antcin A, antcin C, and MAA. (B) The dose-response curves of antcin A, antcin C, and MAA against Huh7 cell proliferation. (C) HPLC chromatogram of MAA.

(2). However, only few studies have harnessed the pure compounds isolated from the crude extracts and elucidate their underlying anticancer mechanism. Previously, we have reported the cytotoxic effects against various cancer cell types of eight pure triterpenoids from the fruiting bodies, and three of these compounds can induce HT-29 human colon cancer cell apoptosis (11), in addition to their anti-Helicobacter pylori (12) and antiinsecticidal activities (13). A recent study reported that dehydroeburicoic acid (DeA) from fruiting bodies of A. camphorata induced calcium- and calpain-dependent necrosis in human U87MG glioblastomas (14). As part of our study program to evaluate the therapeutic properties of A. camphorata, in this study, three triterpenoid compounds, antcin A (15), antcin C (15), and methyl antcinate A [MAA (16)], were isolated from the fruiting bodies of A. camphorata. It is interesting to note that antcins A and C have potent immunomodulatory effects against reactive oxygen species (ROS) (17, 18); however, the cytotoxic properties of antcins A and C and MAA have not yet been reported. This study is the first to determine the liver cancer and normal cells growth inhibitory activities of these compounds and examine MAA’s effect on apoptosis in human liver cancer cell line Huh7. To further establish MAA’s anticancer mechanism, we assayed the levels of cell cycle control and apoptosisrelated molecules and the involvement of ROS, which are strongly associated with tumor cells death signal transduction pathway.

Materials and Methods Materials. Propidium iodide (PI), 4′,6′-diamindino-2-phenylindole (DAPI), dimethyl sulphoxide (DMSO), ribonuclease (RNase), ascorbic acid (ASC), diphenylamine (DPI), lucigenin and N-acetyl cysteine (NAC) were purchased from Sigma-Aldrich Inc. (St. Louis, MO). 2′,7′-Dichlorofluoroscein diacetate acetyl ester (DCF-DA), and dihydroethidine (HE) were purchased from Molecular Probes (Eugene, OR). Fetal calf serum (FCS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco BRL (Gaithersburg, MD). Antitubulin, -Bcl2, -BclXL, -Bak, and -Bax antibodies were purchased from Santa Cruze Biotechnology (Santa Cruze, CA). Anticytochrome c antibody was purchased from PharMingen (San Diego, CA). Antiactin antibody was purchased from Oncogene Science Inc. (Uniondata, NY). Caspase activity assay kits were purchased from R&D Systems (Minneapolis, MN). Pan-caspase inhibitor (Z-VAD-FMK), caspase-2 inhibitor (ZVDVAD-FMK), caspase-3 inhibitor (Z-DEVD-FMK), caspase-8 inhibitor (Z-IETD-FMK), and caspase-9 inhibitor (Z-LEHD-FMK) were purchased from Kamiya (Seattle, WA). Anticofilin and -COX IV antibodies were purchased from Cell Signaling Technology (Danvers, MA). A TUNEL assay kit was purchased from Roche Diagnostics (Mannheim, Germany). Extraction and Isolation. The compounds antcin A, antcin C, and MAA (Figure 1) were purified and identified from the fruiting bodies of A. camphorata as described previously (13). Briefly, the air-dried powder of the fruiting bodies was successively extracted with n-hexane, chloroform, and methanol under reflux. After exhaustive extraction, the combined extracts were concentrated individually under reduced pressure. The CHCl3 soluble fraction was chromatographed over silica gel using n-hexane/EtOAc gradient

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eluent, and similar fractions were combined to produce six fractions (F1-F6). Fraction F6 was purified by silica gel column chromatography using CHCl3/MeOH to yield antcin A and a mixture of compounds that was further separated by column chromatography using CHCl3/MeOH elution to obtain antcin C and MAA. The structures of antcins A and C (15) and MAA (16) were determined by spectroscopic analysis and by comparison of the spectral data with those of published values. The purity (>95%) of these compounds was confirmed from their sharp melting points, TLC on silica gel (one spot), and NMR studies. HPLC chromatograms of purified MAA (potent compound in this study) are shown in Figure 1C. The identity of the peak of MAA was confirmed by the proportional increase of the chromatographic peak caused by the addition of MAA to the analyzed sample. An analytical HPLC system consisted of a Hitachi (Tokyo, Japan) L-7100 pump; a 20 µL fixed loop and a model L-7400 diode array detector (DAD) were used. Chromatographic analysis was performed with a J’sphere ODS-M80 C18 column (250 mm × 4.6 mm, 4 µm, YMC Sep. Technol., Japan). The mobile phase consisted of acetonitrile (solvent A) and water containing 0.1% formic acid (solvent B). A linear gradient program was used as follows: 60% A in the first 0 min, linear gradient to 90% A over 60 min. The mobile phase flow rate was 1 mL/min, and the detector was monitored at 254 nm. Cell Culture, Cytotoxicity, and Compound Uptake Measurements. Human hepatocellular carcinoma Huh7, HepG2, and Hep3B cell lines were cultured with DMEM, containing 10% FCS, antibiotics (100 U/mL penicillin and 100 U/mL streptomycin), and 2 mM glutamine, at 37 °C in a humidified atmosphere with 5% CO2. The rat hepatocytes were isolated through collagenase perfusion from 250-350 g Sprague-Dawley rats and cultured in 12-well plates in DMEM supplemented with 2% fetal bovine serum (FBS) with some modification (19). The culture medium was changed every 2 days. For the cytotoxicity assay, cells were treated with various concentrations of antcins A and C and MAA for indicated time points. After treatment, the viable cells were evaluated by trypan blue dye exclusion method. For intracellular determination of antcins A and C and MAA, Huh7 cells were harvested and centrifuged at 1400g for 10 min at 25 °C, with two washes using MAA free culture medium. The pellet was ultrasonically homogenized in 50% methanol and then centrifuged. The supernatant was withdrawn, evaporated to dryness under a nitrogen stream, then reconstituted with mobile phase, and placed in autosampler vials for injection. HPLC analysis was carried out as mentioned above for standard MAA. Calibration standard samples were prepared by adding appropriate volumes of stock solution directly to sample from Huh7 cells pellet, and these samples were analyzed identically to collected pellet samples. Peak areas from samples and standards were integrated using chromatography software (Hitachi). The injection volume was 20 µL, and all samples were analyzed in triplicate and averaged. DNA Fragmentation Analysis. Cells were treated with DMSO alone and various concentrations of MAA for 48 h, and then, cells were collected and lysed by DNA extraction buffer (50 mM Tris, pH 7.5, 10 mM EDTA, and 0.3% Triton X-100). The solution was incubated with 0.1 mg/mL proteinase K and 0.2 mg/mL RNase for 1 h at 55 °C. Then, it was extracted with phenol-chloroform (1:1) and separated in 2% agarose gel, and fragmented DNA was visualized with ethidium bromide by UV. Apoptotic Cell Determination. Apoptotic cells were measured by flow cytometry and terminal deoxynucleotidyl transferase dUTP nicked-end labeling (TUNEL) assay. The TUNEL assay was performed according to the manufacturer’s instructions (Boehringer Mannheim). PI staining and flow cytometry were used to determine the cell cycle stage and sub-G1 group. Briefly, 100 µM MAAtreated cells were washed with PBS and fixed with 70% ethanol with gentle vortexing. Fixed cells were spun down and washed with PBS twice, and then, the cells were resuspended in 500 µL of PI (2 µg/mL)/Triton X-100 (0.1% v/v) staining solution with 100 µg/ mL RNase for 30 min in the dark and analyzed by FACScan flow cytometer. The percentage of apoptotic cells (subG1 population) was analyzed using Cell Quest software.

Hsieh et al. Caspase Activity Assay. Cell lysates obtained from MAA-treated or vehicle-treated cells were tested for caspase-2, -3, -8, and -9 activities by addition of caspase-specific peptide substrate conjugated with the fluorescent reporter molecule, according to the manufacturer’s instructions (R&D Systems). An equal amount of protein from each sample was used to determine the level of caspase enzymatic activity and was directly proportional to the fluorescence signal detected with a fluorescent microplate reader (Fluoroskan Ascent; Labsystem, Finland). The relative caspase activity was absorbance at excited by light at 400 nm and emitted fluorescence at 505 nm. Subcellular Fractionation. Cells were washed and harvested and then incubated on ice for 30 min in sucrose buffer (250 mM sucrose, 2 mM EDTA, 2 mM EGTA, and 10 mM DTT). Lysates were centrifuged at 1300g for 10 min at 4 °C, and the soluble cytosolic fraction was collected after further centrifugation of the raw cytosolic fraction at 1300g for 30 min at 4 °C. Mitochondria were purified from a heavy membrane fraction by sucrose density gradient centrifugation; briefly, the heavy membrane pellet was resuspended in 1 mL of sucrose buffer and laid on the top of 1.2 and 1.5 M sucrose buffer gradient before being centrifuged at 1300g for 30 min at 4 °C. Sucrose density gradient-purified mitochondriaenriched fractions were collected at 1.2/1.5 M interphases respectively, washed with PBS, and dissolved in RIPA lysis buffer. All fractions were disrupted by sonication and prepared before Western blot analysis. Protein Preparation and Immunoblotting. Cells were cultured without or with 100 µM MAA at indicated times. After treatment, cells were harvested, washed twice with ice-cold PBS, and lysed in modified RIPA buffer with protease inhibitors. The cell lysates were cleared by centrifugation at 12000g for 30 min at 4 °C, and the total protein content of the supernatant was collected and determined by Bradford method. For Western blot analysis, equal amounts of total protein were loaded onto SDS-polyacrylamide gels, and the proteins electrophoretically transferred onto a PVDF membrane (Millipore, Bedford, MA). The protein expression was detected by each immunoblotting with the corresponding specific primary antibodies (against PARP, Bcl-XL, Bcl-2, Bax, actin, cytochrome c, cofilin, COX IV, and tubulin) at 4 °C for 16 h. After it was washed three times with TBST, the membrane was incubated with horseradish peroxidase-labeled secondary antibody for 1 h. The membrane were washed again, and detection was performed using the enhance chemiluminescence blotting detection system (Amersham, United States). Determination of Intracellular ROS Level and Mitochondrial Membrane Potential (∆Ψm). To assess the intracellular ROS level, the cells were incubated with 100 µM MAA for the indicated periods. Cells were incubated with 10 µM DCF-DA or 10 µM HE for 30 min prior to harvesting. In the presence of ROS, the DCFDA will convert to DCFH, which can be oxidized to the fluorescent compound DCF. The fluorescence intensity of the cells was analyzed by flow cytometry. The ∆Ψm is assessed using lipophilic fluorochrome 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzamidazolyl carbocyanine iodide (JC-1, Molecular Probes). Briefly, cells were treated with 100 µM MAA for the indicated periods. Before they were harvested at each period points, cells were incubated in medium with JC-1 for 30 min and then washed with PBS. Both red and green fluorescence emissions were analyzed by flow cytometry using an excitation wavelength of 488 nm and observation wavelengths of 530 nm for green fluorescence and 585 nm for red fluorescence. Quantitating NADPH Oxidase Activity Assay. This assay was performed following the procedure as described previously (20). The cells were lysed using a Dounce homogenizer for more than 50 strokes per sample, the cell lysates were centrifuged, then supernatant was collected, and the protein concentration was determined by Bradford method. Aliquot proteins were added to the reaction buffer (50 mM phosphate buffer, pH 7.0, 1 mM EGTA, 150 mM sucrose, and 5 µM lucigenin as the electron acceptor and 100 µM NAPDH as the substrate). The reaction was initiated by the addition of 100 µL/tube protein (50 and 25 µg) and 900 µL



Table 1. Growth Inhibition of Antcins A and C and MAA on Human Liver Cancer Cell Lines and Primary Rat Hepatocyte as Calculated from Dose-Response Curvesa compounds IC50b (µM)c cell type

cell line

antcin A

antcin C

MAA

93.6 155.7 132.2

102.5 286.4 152.3

52.2 78.0 30.2

normal rat hepatocytes >500

>500

>500

tumor liver

primary culture

HepG2 Hep3B Huh7

a

Sigmoidal dose-response curves (variable slopes) were generated using GraphPad Prism V. 5.01 (GraphPad Software Inc.). b Tested compound concentration required to inhibit cell proliferation by 50% after 48 h of treatment as compared with vehicle control DMSO. c Values are the mean of triplicate of three independent experiments.

reaction buffers, and photon emission was measured every minute for a 15 min time period using a microtiterplate luminometer. The activity was expressed as relative light units (RLU) per minute per milligram of total protein (RLU/minute/mg protein). Immunostaining. Huh7 cells were seeded on coverslips, and after 24 h, cells were treated with 100 µM MAA for 24 h. Cells were washed with PBS and incubated with staining solution containing 50 nM MitoTracker probe for 45 min, and then, cells were fixed with 4% formaldehyde for 2 h. After fixation, cells were washed trice with PBS and incubated with antibody against Bax or cytochrome c for 16 h. Samples were incubated with Alex antirabbit or -mouse secondary antibodies for 2 h, then washed and sealed with cover glass with 90% glycerol, and observed by fluorescence microscope. Statistical Analyses. Each experiment was performed in triplicate and repeated three times (n ) 9). The results were expressed as means ( SDs. Statistical comparisons were made by means of one-way analysis of variance (ANOVA), and the significant difference between the tested groups was found using Student’s t test. The statistical significance was set at *p < 0.05, **p < 0.01, and ***p < 0.001.

Results Growth Inhibition. The chemical structures of antcins A and C and MAA isolated from A. camphorata are illustrated in Figure 1A. To explore whether antcins A and C and MAA have an exclusive beneficial effect in the treatment of liver cancer by blocking cancer cells accumulation, human liver cancer cell lines HepG2, Hep3B, and Huh7 and normal rat liver cells were treated with various concentrations of the aforementioned compounds for the indicated time periods. Control experiments were carried out in the presence of vehicle (DMSO) and served as reference values (100%). The IC50 values as determined by MTT assay from dose-response curves were listed in Table 1. The three compounds showed a wide range of potency with IC50 values ranging from 30.2 to 286.4 µM in liver cancer cell lines (Table 1). The compound MAA is the most potent cytotoxic agent for the liver cancer cell lines than antcins A and C with IC50 values of 52.2, 78.0, and 30.2 µM against HepG2, Hep3B, and Huh7 cells, respectively. The dose-response curves of antcin A, antcin C, and MAA against Huh7 cells proliferation are shown in Figure 1B. Interestingly, the tested compounds were less sensitive (i.e., IC50 values are >500 µM) to the inhibitory activity of rat hepatocyte cells (Table 1). These results pointed to the differential (selective) effect between tumor and normal cells of the aforementioned compounds, and the tested compounds might suppress cancer cell proliferation or induce those undergoing apoptosis that lead to cell growth inhibition. The substituent in positions C-7 and C-26 in the tested

compounds apparently plays a significant role in the regulation of tumor cell growth. The compound MAA differs from antcin C in that there is no hydroxyl group at position 7, while position 26 contains a carboxy methyl group rather than carboxylic acid group. The compounds MAA and antcin A only differ with respect to a methyl group at the position 26, where the former shows a higher inhibitory potency against tested liver cancer cells than the latter. To our knowledge, we have demonstrated here for the first time the cytotoxicity of antcins A and C and MAA against liver cancer cell lines. Among the tested compounds and tumor cell lines, MAA was more sensitive to Huh7 cells and significantly inhibited the cell growth at concentrations higher than 10 µM and was almost completely inhibited at concentrations higher than 110 µM (Figure 1B). To determine whether differences in activity against cellular proliferation were due in part to differences in intracellular compound exposure, the accumulation of antcins A and C and MAA was determined in Huh7 cells. Following treatment with a 100 µM concentration of each compound, Huh7 cells were washed and lysed, and the intracellular compound accumulation was determined by HPLC. MAA exhibited the highest intracellular accumulation followed by antcins A and C. The intracellular concentration of MAA was 63.1 µM/6 × 105 cells 4 h after addition and remained at approximately that level at least to 20 h. In comparison, antcins A and C accumulated to a maximal concentration of 20.8 µM and 13.1 µM/6 × 105 cells only after 20 h of incubation, respectively. The maximal accumulation of MAA as compared with antcins A and C suggests that it displays a particular potent inhibitory effect on Huh7 cell viability. Subsequently, 100 µM MAA was used to examine the changes in cellular and molecular events in Huh7 cells. Effect of MAA on Apoptosis Induction. We studied the mechanisms by which MAA suppresses cell viability. To investigate the effect of MAA on cell cycle progression, Huh7 cells treated with MAA at 100 µM concentration for various time periods was analyzed by flow cytometry. A sub-G1 DNA peak was detected in MAA-treated cells, and the percentages of apoptotic Huh7 cells (sub-G1 population) were gradually increased by 3.0, 12.5, 19.1, 26.7, and 37.9% for times corresponding to 4, 8, 16, 24, and 32 h, respectively (Figure 2A,B). Additional experiments were done on the induction of apoptosis in Huh7 cells by DNA fragmentation assay. The results showed that the typical “DNA laddering” pattern of DNA fragments was first detected after 16 h and reached a maximum in 48 h of MAA treatment, as determined by agarose gel electrophoresis (Figure 2C). A quantitative evaluation was also made using TUNEL to detect DNA strand breaks. As compared with vehicle-treated cells, 100 µM MAA at 48 h induced 90% apoptosis in Huh7 cells, and apoptotic cells were identified according to characteristic cell morphology such as condensation and degradation of the nuclei (Figure 2D). Effect of MAA on Mitochondrial Apoptotic Pathway. To investigate the mitochondrial apoptotic events involved in MAAinduced apoptosis, we analyzed the changes in the caspases-2, -3, -8, and -9, mitochondrial membrane potential (∆Ψm), Bcl-2 family proteins, and cytochrome c activity with 100 µM MAA for various times. The enzymatic activity of caspases was measured using four fluorogenic peptide substrates as follows: VDVAD-AFC, DEVD-AFC, IETD-AFC, and LEHD-AFC, which are specific substrates for caspase-2, -3, -8, and -9, respectively. The results of the caspases assay were almost identical as compared to the cell cycle analysis and DNA fragmentation. As illustrated in Figure 3A, the activities of caspase-2, -3, and -9 were gradually increased with increasing

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Figure 2. MAA-induced apoptosis. (A) Cell cycle analysis of MAA-treated cells. Huh7 cells were treated with or without 100 µM MAA for 0, 4, 8, 16, 24, and 32 h and then stained with PI. Cell cycle distribution was determined by flow cytometry. (B) Sub-G1 population refers to apoptotic cells. (C) DNA fragmentation. Huh7 cells were exposed to 100 µM MAA for indicated times, and then, genomic DNA was isolated. DNA samples were loaded into a 2% agarose gel for electrophoresis. (D) TUNEL assay. Huh7 cells were treated with 100 µM MAA for 48 h, TUNEL assay, and DAPI staining was performed. Cells were investigated under a fluorescent microscopy. Magnification, 150×; Scale bar, 20 µm. Data are given as means ( SDs of three independent experiments. **p < 0.01, and ***p < 0.001 as compared to control values.

incubation time in MAA-treated Huh7 cells. In contrast to the significant increases in caspase-2, -3, and -9, a moderate increase in caspase-8 was observed (Figure 3A). To address the activation of caspases required for the induction of apoptosis, Huh7 cells were cotreated with caspase inhibitor and/or MAA. As shown in Figure 3B, incubation with the inhibitor of caspase-2 (ZVDVAD-FMK), caspase-3 (Z-DEVD-FMK), caspase-9 (ZLEHD-FMK), and pan-caspase (Z-VAD-FMK) significantly blocked MAA-triggered apoptosis in Huh7 cells, but the caspase-8 inhibitor (Z-IETD-FMK) could not. In addition, we investigated mitochondrial dysfunction by measuring ∆Ψm by flow cytometry using JC-1 lipophilic fluorochrome for the indicated times. As shown in Figure 4A, which compares Huh7 cells exposed to MAA and control cells, the red shift in fluorescence intensity shifted from 99.4 to 82.3% in MAAinduced apoptotic Huh7 cells at 8 h. These findings show that MAA has an effect on mitochondrial function. Furthermore, to check the change of members of the Bcl-2 family during MAA treatment, the time-course effects of MAA on protein expres-

sion of the Bcl-2 family were evaluated by Western blot. As depicted in Figure 4B, the amounts of pro-apoptotic proteins, including Bax, Bak, and PUMA, were gradually increased in 100 µM MAA-treated cells as compared to vehicle-treated control cells. In contrast, MAA decreases the levels of antiapoptotic proteins Bcl-2 and Bcl-XL, which led to an increase in the pro-apoptotic/antiapoptotic Bcl-2 ratio (Figure 4B). The cytosolic release of cytochrome c is one of the key events in the mitochondria-dependent apoptotic pathway (21). Next, to determine whether the reduction of ∆Ψm and an increase in the Bcl-2 ratio by MAA could lead to cytochrome c release from mitochondria into cytosol, cell lysates were subfractionated, and cytochrome c was determined by Western blot. The cytosolic fraction from untreated Huh7 cells contained no detectable amount of cytochrome c, whereas it did become detectable after 8 h of MAA treatment and increased progressively up to 24 h (Figure 4C), while there was a concomitant decrease of mitochondria cytochrome c levels (Figure 4C). Next, the immunostaining was performed to examine the subcellular


Figure 3. MAA-activated caspases. (A) Huh7 cells were treated with 100 µM MAA for the indicated time periods. The caspase activity was estimated using fluorogenic peptide substrate. (B) Caspase inhibitors attenuated MAA-induced apoptosis. Huh7 cells were pretreated with caspase inhibitor for 1 h and then treated with 100 µM MAA for another 48 h, and apoptosis cells were calculated by TUNEL assay. Data are given as means ( SDs of three independent experiments. ***p < 0.001 as compared to control values.

distribution of Bax and cytochrome c. As expected, treatment with 100 µM MAA for 24 h caused mitochondrial translocation of Bax. Moreover, MAA induced cytochrome c release from mitochondria to cytosol (Figure 4D). Effect of MAA on the Generation of ROS and Mitochondrial Translocation of Cofilin. Dysregulation of cellular redox status can be a potent mechanism of cell death (22). Therefore, we tested the possibility that MAA induces apoptosis allowing for ROS accumulation. The fluorescent dyes DCF-DA and HE were used to detect the levels of hydrogen peroxide and superoxide radical levels. As shown in Figure 5A, the generation of intracellular peroxide increased significantly as early as 0.5 h and peaked at 1 h after 100 µM MAA exposure and then began to decrease afterward, eventually dropping below the untreated control level at 4 h. However, the content of superoxide radical did not change during MAA treatment. Next, the effect of antioxidants NAC and ASC on MAA-induced ROS production was examined. As shown in Figure 5B, NAC and ASC effectively eliminated the MAA-mediated ROS generation. Because one of the major sources of ROS generation in cancer cells is NADPH oxidase activation (23), we then measured the NADPH oxidase activity to address whether it is involved in MAA-induced oxidative stress in Huh7 cells. Figure 5C shows that MAA could enhance NADPH oxidase activity in Huh7 cells, while this event could be inhibited by DPI, a NADPH oxidase inhibitor. DPI also suppressed the MAA-induced ROS production in this cell line (Figure 5D), suggesting that NADPH


oxidase plays a crucial role in MAA-mediated ROS production. Driven by the evidence that mitochondrial translocation of cofilin is an early step in oxidant-triggered apoptosis induction (24), we subsequently attempted to correlate the effect of MAA on the cellular distribution of cofilin in Huh7 cells and apoptosis induction. As shown in Figure 5E,F, Western blot analyses confirmed that, indeed, cytosolic cofilin was down-regulated by MAA, while increase the same in mitochondrial fraction. No changes in cellular cofilin distribution were detected when total cell lysates were used. Furthermore, exposure of Huh7 cells to the inhibitors of mitochondria permeability transition pore, cyclosporine A (CsA) and bongkrekic acid (BA), dose dependently protected cells against MAA-induced apoptosis (Figure 5G). These results imply that MAA-elicited apoptosis is partly through a mitochondrial signaling pathway. Effect of Antioxidants on the ROS Generation and MAAInduced Apoptosis. To investigate whether ROS generation is directly associated with the MAA-induced mitochondrial apoptotic pathway, we assessed apoptotic events in Huh7 cells pretreated with antioxidants NAC and ASC for 1 h followed by treatment with 100 µM MAA. As shown in Figure 6A, pretreatment of Huh7 cells with NAC and ASC could decrease the levels of pro-apoptotic proteins Bax, Bak, and PUMA caused by MAA. We further assessed the effect of antioxidant agents NAC and ASC in MAA-mediated mitochondrial apoptotic events and caspase-3 activity. As shown in Figure 6B, the majority of Bax and Bak were detected in the cytosol of control cells, following incubation with MAA translocated into the mitochondrial fraction. Importantly, Bax and Bak translocation during MAA treatment were suppressed by NAC and ASC. The cytochrome c was in mitochondria in control cells and was released into the cytosol following MAA treatment, which was attenuated by NAC and ASC (Figure 6B). To confirm the morphological observations, we also measured caspase-3 activity. As shown in Figure 6C, MAA stimulated caspase-3 activity, which was suppressed by NAC and ASC. Furthermore, the percent of apoptotic cells in MAA-treated Huh7 cells was also attenuated by NAC and ASC (Figure 6D). Collectively, the results suggest that NAC and ASC, by scavenging ROS, may attenuate the MAA-induced mitochondrial apoptotic pathway in Huh7 cells. To characterize the role of Bax in MAA-triggered cell death, Bax specific small interfering RNA (siRNA) was transfected into Huh7 cells for 16 h and then treated without or with MAA, and the levels of Bax protein (24 h MAA treatment) and cell survival (48 h MAA treatment) were determined. As shown in Figure 6E, transfection of Huh7 cells with Bax siRNA effectively reduced the expression of Bax protein at a concentration range of 75-100 nM. In contrast, at 100 nM, control-siRNA did not cause an alteration in Bax protein expression. Moreover, reduction of Bax expression by transfection with Bax siRNA resulted in a significant increase in cell viability. From the MTT reduction assay, viable cells were increased from 22.4 to 43.9 and 64.2% in 75 and 100 nM BAX siRNA-transfected groups, respectively. Control-siRNA had no significant effect on cell viability as compared with MAA-treated control (22.4 ( 5.3 vs 27.8 ( 7.6%).

Discussion Liver cancer is the most leading cause of death in both developing and developed countries including Taiwan (25). In this context, many dietary natural substances were found to induce apoptosis in various tumor cell lines and suggest that it might be useful for the treatment of cancer complications (26). Many investigators have focused on the manipulation of the

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Figure 4. MAA-induced apoptosis through the initiation of the mitochondrial pathway. (A) Evaluation of mitochondrial membrane potential in Huh7 cells by flow cytometry. JC-1 was used to trace the alteration of mitochondrial membrane potential. The fluorescence intensity change indicated MAA-induced depolarization. Distinct populations were detected following apoptosis-inducing treatment with 100 µM MAA for 0, 0.5, 2, 4, 6, and 8 h. (B) Regulation of Bcl-2 family molecules by MAA. Huh7 cells were treated with 100 µM MAA for 2, 8, 16, and 24 h, and total protein was isolated. An equal amount of cell lysates was analyzed by Western Blot with corresponding antibodies. (C) The release of cytochrome c was detected after treatment with 100 µM MAA for indicated time points. (D) Subcellular distribution of Bax and cytochrome c. Cells were treated without or with 100 µM MAA for 8 h, and subcellular distribution of Bax and cytochrome c was examined by immunostaining. Mitotracker was used as a mitochondria marker.

apoptotic process for the treatment and prevention of cancer, and they have also searched for compounds that influence apoptosis to understand their mechanism of action (27, 28). A. camphorata is a folk medicinal mushroom that has been used

for liver complications (2). The beneficial anticancer effects of A. camphorata extracts have been reported in rats and cell culture studies (3-10). However, the characteristic chemical compounds of A. camphorata against liver cancer are not well-



Figure 5. MAA provoked ROS generation and cofilin translocation. (A) Cells were treated without or with 100 µM of MAA for 0, 0.5, 1, 2, and 4 h, and the ROS levels were measured by FACS analysis after incubation with DCF-DA or HE fluorescent probe. (B) Antioxidants eliminated MAA-induced ROS generation. Cells were pretreated with NAC (1 mM) or ASC (0.4 mM) for 1 h and then treated with 100 µM MAA for another 1 h, and the ROS level was measured using DCF-DA fluorescent probe by FACS analysis. (C) NADPH oxidase activity assay. Cells were treated with 100 µM MAA for 0.5, 1, and 2 h in the absence or presence of 0.5 µM DPI. The reaction velocity (V) was calculated as the change in relative light unit (RLU) per minute per microgram of protein. The statistic difference level was shown as *p < 0.05 as compared with untreated control. (D) DPI blocked MAA-provoked ROS production. Huh7 cells were pretreated with 0.5 µM DPI for 1 h and then treated with 100 µM MAA for another 1 h, and the ROS level was detected. (E) Cellular distribution of cofilin. Subcellular fractionation was carried out before analysis by a Western blot assay using anticofilin, -Cox IV (as a mitochondrial marker), or R-tubulin (as a cytosolic marker) antibodies after treated with 100 µM MAA for 16 h. (F) Densitometric analysis of cofilin expression in different subcellular compartments was shown in untreated and treated cells. (G) Cells were pretreated with the inhibitors of mitochondria permeability transition pore, cyclosporine A (CsA; 1, 2, and 4 µM), or bongkrekic acid (BA; 25, 50, and 100 µM) for 1 h and then treated without or with 100 µM MAA for another 48 h. After treatment, apoptotic cells were measured by TUNEL assay. Data are given as means ( SDs of three independent experiments. **p < 0.01, and ***p < 0.001 as compared to control values.

defined. An anticancer drug should ideally selectively kill the tumor and spare normal tissues. However, they are generally also toxic to normal cells, and this limits their useful dose range. In this study, we have provided the first evidence showing that antcins A and C and MAA selectively inhibit tumor cell growth of three liver cancer cell lines HepG2, Hep3B, and Huh7 at

lower IC50 values, as compared with values for normal rat hepatocytes (Table 1). These results suggest that the tested compounds mediated growth inhibition and were probably specific for tumor cells and were in agreement with results as we reported previously (11). The compound MAA is the most potent among tested, and the extent of the growth inhibitory

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Figure 6. ROS production contributed to MAA-induced mitochondrial apoptosis. Huh7 cells were pretreated with various concentrations of ASC (0.2 and 0.4 mM) and NAC (1 and 2 mM) for 1 h and then treated without or with 100 µM MAA for another 24 h. (A) Total amounts of Bax, Bak, and PUMA and (B) the subcellular distribution of cofilin and cytochrome c were examined by Western blot. (C) The caspase-3 activity was examined using fluorogenic peptide substrate after 24 h of 100 µM MAA treatment. (D) Antioxidants abolished MAA-induced apoptosis. Huh7 cells were pretreated with ASC (0.1, 0.2, and 0.4 mM) and NAC (0.5, 1, and 2 mM) for 1 h and then incubated without or with 100 µM MAA for another 48 h. Apoptotic cells were measured by TUNEL assay. Huh7 cells were transfected without or with Bax specific siRNA (75 nM and 100 nM) or control siRNA (100 nM) for 16 h and then treated with 100 µM MAA for 24 h to detect Bax protein expression (E), and for 48 h to measure the cell viability by MTT assay (F). Data are given as means ( SDs from three independent experiments. **p < 0.01, and ***p < 0.001 as compared to control values.

effect appears to be cell line-dependent, being clearly more pronounced in the liver cancer cell line Huh7 cells (Table 1). There was also a higher intracellular accumulation of MAA than antcins A and C in Huh7 cells. Further studies are currently in progress to elucidate the effect of MAA on a broad spectrum of different tumor cell types. Apoptosis is modulated by complex pathways that involve a series of biochemical regulators and molecular interactions (29).

The distinct sub-G1 peaks in the flow cytometry analysis of Huh7 cells (Figure 2A) argue for the induction of apoptotic cell death. In addition to the flow cytometry, DNA fragmentation was determined by agarose gel electrophoresis (Figure 2C). Because caspases play a critical role in the morphology of apoptosis and in some cases are essential for its induction (30), we decided to study the effect of MAA on the activation of caspases-2, -3, -8, and -9 in Huh7 cells. Both the DNA


fragmentation and the caspase test systems gave nearly identical results (Figures 2 and 3). In accordance with the potent growth inhibitory properties, MAA showed by far the highest rate of DNA fragmentation and activation of caspases-2, -3, and -9. To clarify the relationship underlying the cascade activation of caspases during MAA-induced apoptosis, we utilized a caspase inhibitor system, which resulted in the almost complete inhibition of apoptotic cell death induced by MAA. Taken together, the results of the flow cytometry, DNA fragmentation analysis, and caspases activity assay, it can be concluded that the compound MAA induces apoptosis in the liver cancer cell line Huh7 cells. To further elucidate the molecular mechanism responsible for the MAA-induced apoptosis in Huh7 cells, the expression of mitochondria-dependent members of the Bcl-2 family proteins was analyzed. Many reports have indicated that mitochondria may play a critical role in the commitment of cells to apoptosis (31), and this prompted us to investigate whether mitochondria were the target organelles in agent-induced apoptosis. Mitochondria play a central role in cellular homeostasis, and their homeostatic center is the mitochondrial membrane potential (∆Ψm). Thus, the assessment of ∆Ψm in cells is worthy of investigation. In this study, MAA exhibited significant ∆Ψm disruption. Bcl-2 family proteins, including the antiapoptotic and pro-apoptotic proteins, have been reported to regulate cytochrome c release from mitochondria (32). Pro-apoptotic Bcl2-family molecules permeabilize the mitochondrial membrane (thereby promoting cytochrome c release), whereas antiapoptotic Bcl-2-family molecules prevent cytochrome c release (32). Our data demonstrated that treatment of Huh7 cells with MAA resulted in an increase in the expression levels of pro-apoptotic proteins Bax, Bak, and PUMA while decreasing the amounts of antiapoptotic Bcl-XL and Bcl-2 proteins (Figure 4B). It is known that mitochondrial cytochrome c release from the inner membrane into the cytosol is a common early event in the induction of apoptosis by multiple agents and that cytochrome c release is linked to caspase activation and subsequent DNA fragmentation (33). The present data showed that MAA induced cytochrome c release from mitochondria to cytosol (Figure 4C,D). According to our data, the increased DNA damage in MAA-treated Huh7 cells was in line with activation of caspases 2, -3, and -9, increase in pro-apoptotic/antiapoptotic Bcl-2 ratio, and cytochrome c release. This finding suggested that the effect of the Bax gene product via the mitochondria might be responsible for the modulation of MAA-induced apoptosis in the Huh7 cells. These results are consistent with previous reports that treatment with the extract of A. camphorata initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 in human hepatoma HepG2 and PLC/ PRF/5 cells (6) and in human breast cancer cell lines MCF-7 and MDAMB-231 (7, 8). One proposed model indicated that Bax translocation to the mitochondrial membrane leads to the formation of a protein complex with the mitochondrial voltagedependent anion channel (VDAC) (34). The Bax-VDAC complex induces opening of the mitochondrial permeability transition pore, which results in mitochondrial membrane potential (∆Ψ) collapse and subsequent release of apoptosis promoting factors into the cytoplasm (35). This finding is in line with our results in MAA-treated cells subsequent to reduced ∆Ψ, increased pro-apoptotic (Bax, Bak, and PUMA), and decreased antiapoptotic (Bcl-2 and Bcl-XL) protein levels and cytochrome c release.


Exploring the possible oxidant-triggered molecular mechanisms underlying Huh7 cell apoptosis by MAA, we found that the pro-apoptotic activity of MAA was accompanied by accumulation of ROS such as hydrogen peroxide (H2O2) (Figure 5A), suggesting that Huh7 cell death by MAA is a ROSdependent process. ROS are a family of active molecules containing free radicals and involved in the modulation of biological cell functions. However, excessive ROS bring about oxidative stresses that cause injury to various cellular constituents such as lipid, protein, and DNA, finally resulting in growth arrest, senescence, or apoptosis (36). Intracellular ROS can be generated from aberrant mitochondria, which are well-known as sites of ROS generation and targets for ROS action (36). NADPH oxidase is another important source of ROS, which accounts, at least partially, for increased levels of ROS in several types of cancer (37). Enhancement of ROS production has long been associated with the apoptotic response induced by anticancer agents (36). Here, we demonstrated that MAA induced the loss of ∆Ψ accompanied with increased ROS generation in Huh7 cells (Figures 4A and 5A). Furthermore, an increased activity of NADPH oxidase was observed in MAA-treated Huh7 cells as compared with untreated cultures. Incubation with a NADPH oxidase specific inhibitor DPI significantly suppressed MAA-induced NADPH oxidase activation and ROS production (Figure 5C,D). These results provide a possible fundamental explanation for why MAA brings about excessive ROS generation in Huh7 cells. Growing evidence indicates that in response to oxidative stress, movement of key proteins in or out of mitochondria during apoptosis is essential for the regulation of apoptosis. Cofilin has been recently identified as an interesting key protein that participates in the initiation phase of mitochondrial apoptosis in response to oxidative stress (38, 39). Several studies showed cofilin-mediated apoptosis in response to staurosporine, VP16, and transforming growth factor-β through its translocation to the mitochondria and that it regulates apoptotic morphology (40, 41). A recent report demonstrates that H2O2 has the ability to oxidize cofilin, and oxidized cofilin loses its affinity for actin, translocates to the mitochondria, and induces apoptosis-related mitochondrial damage (24). In this study, we found that treatment of Huh7 cells with MAA caused an increase of intracellular H2O2 production and induced translocation of cofilin and Bax from the cytosol to the mitochondria (Figures 4D and 5E,F), and this change in the subcellular localization of cofilin and Bax was concurrent with a measurable decrease in mitochondrial membrane potential (Figure 4A). Moreover, MAAinduced apoptosis was drastically inhibited by CsA and BA, which blocks mitochondrial pore opening (Figure 5G). To the best of our knowledge, this is the first evidence on cofilinmediated apoptosis of A. camphorata extracts or its isolated compounds in human liver cancer cells. These results suggest that mitochondrial translocation of cofilin and Bax contributed to MAA-induced mitochondrial dysfunction and apoptosis. Mitochondria appear to be the most powerful intracellular source of ROS; mitochondria might also be a primary target for the damaging effects of ROS. A previous study showed that treatment with the extract of A. camphorata induced dosedependent ROS generation in the breast cancer cell line MCF-7 cells (7). In this study, the time-course experiments showed that the elevation of ROS generation occurred as early as 1 h post-MAA exposure, indicating that this event was earlier than mitochondrial membrane potential disruption and apoptotic execution (Figures 2, 4A, and 5A). Interestingly, the MAAtriggered mitochondrial disruption, Bax, and mitochondrial

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might contribute its overall chemotherapy effects in the fight against liver cancer and its possible future therapeutic applications. Acknowledgment. This study was supported by a research grants from the National Science Council of Taiwan, ROC (NSC 98-2811-M-324-001, NSC 98-2811-B-038-013, and NSC-983112-B-075A-001), and Taichung Veterans General Hospital (TCVGH-977327D).

References

Figure 7. Schematic representation of MAA-induced apoptotic pathway in Huh7 cells.

translocation of cofilin, cytochrome c release, caspases activation, and apoptosis in Huh7 cells is apparently dependent on ROS generation, because these events can be abolished or attenuated following treatment with antioxidants NAC and ASC (Figures 6A to 6D). Besides, knockdown of Bax expression by Bax specific siRNA effectively blocked MAA-induced Bax upregulation and cell death, suggesting that Bax is also an important pro-apoptotic molecule in MAA-triggered cell death. Taken together, our results indicate that MAA-induced ROS production is a major upstream event in the cofilin and Baxmediated mitochondrial apoptotic pathway (Figure 7). In conclusion, A. camphorata is now believed to be a potential chemotherapeutic agent for cancer in Taiwan, especially liver cancer. This study shows for the first time that MAA from A. camphorata has antiproliferation activity in human liver cancer cell line Huh7 cells through a ROS-provoked mitochondrial apoptotic pathway. This event is associated with a significant increase in the levels of Bcl-2 family proteins Bax, Bak, and PUMA and a decrease in Bcl-2 and Bcl-XL, as well as induction of Bax and cofilin translocation, which target the mitochondria to induce cytochrome c release and subsequently activate the caspase cascade, finally leading to apoptotic death. These data provide a basic mechanism for the chemotherapeutic properties of MAA in liver cancer cells. Future in vivo studies may ascertain whether this cell growth inhibition effect of MAA

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