Chem. Res. Toxicol. 2009, 22, 1817–1826
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Dehydroeburicoic Acid Induces Calcium- and Calpain-Dependent Necrosis in Human U87MG Glioblastomas Jhu-Yun Deng,†,‡ Sian-Jin Chen,†,‡ Guey-Mei Jow,§ Chao-Wen Hsueh,| and Chung-Jiuan Jeng*,† Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming UniVersity, No. 155, Section 2, Li-Non Street, Taipei 12212, Taiwan, School of Medicine, Fu-Jen Catholic UniVersity, Hsin-Chuang, Taipei County 24205, Taiwan, and Department of Internal Medicine, Kaohsiung Armed Forces General Hospital, No. 2, Chung Cheng First Road, Lingya District, Kaohsiun, 80284, Taiwan ReceiVed July 6, 2009
Dehydroeburicoic acid (DeEA) is a triterpene purified from medicinal fungi such as Antrodia camphorate, the crude extract of which is known to exert cytotoxic effects against several types of cancer cells. We aim to test the hypothesis that DeEA possesses significant cytotoxic effects against glioblastomas, one of the most frequent and malignant brain tumors in adults. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5diphenyltetrazolium bromide and lactate dehydrogenase release assays indicated that DeEA inhibited the proliferation of the human glioblastoma cell U87MG. In addition, Annexin V and propidium iodide staining showed that DeEA treatment led to a rapid increase of glioblastomas in the necrotic/late apoptotic fraction, whereas cell cycle analysis revealed that DeEA failed to significantly enhance the population of U87MG cells in the hypodiploid (sub-G1) fraction. Using electron microscopy, we found that DeEA induced significant cell enlargements, massive cytoplasmic vacuolization, and loss of mitochondrial membrane integrity. DeEA treatment triggered an intracellular Ca2+ increase, and DeEA-induced cell death was significantly attenuated by BAPTA-AM but not ethylenediaminetetraacetic acid or ethylene glycol tetraacetic acid. DeEA instigated a reduction of both mitochondrial transmembrane potential and intracellular ATP level. Moreover, DeEA induced proteolysis of R-spectrin by calpain, and DeEA cytotoxicity in U87MG cells was caspase-independent but was effectively blocked by calpain inhibitor. Interestingly, DeEA also caused autophagic response that was prevented by calpain inhibitor. Taken together, these results suggest that in human glioblastomas, DeEA induces necrotic cell death that involves Ca2+ overload, mitochondrial dysfunction, and calpain activation. Introduction Several types of mushrooms, collectively known as medicinal fungi, have been extensively studied to explore their potential anticancer properties. For example, both Antrodia camphorata (known in Taiwan as Niu-Chang) and Poria cocos (known in Taiwan as Fu-Ling) have been previously reported to display antiproliferation, cytotoxic, or DNA topoisomerase-inhibiting effects against cancer cells (1-4). The majority of those studies involved applying crude extracts of medicinal fungi, which raises the concern that variations in the purity of extract preparation may result in significant discrepancy in tumor-suppressing efficacy. Furthermore, in most cases, it is not clear what compounds within the crude extract are responsible for generating the observed anticancer effects. The major phytochemical constituents of medicinal fungi are polysaccharides and triterpenes (4-8). Polysaccharides such as R- and β-glucans constitute the major component of mushroom fibers and have been shown to display antitumor actions via noncytotoxic immunomodulating mechanisms (5). Triterpenes, on the other hand, are tetra- or pentacyclic lipids that comprise the structural precursors to steroids in both plants and animals * To whom correspondence should be addressed. Tel: 886-2-28267072. Fax: 886-2-28212884. E-mail:
[email protected]. † National Yang-Ming University. ‡ These authors contributed equally to this work. § Fu-Jen Catholic University. | Kaohsiung Armed Forces General Hospital.
(6). Several types of purified triterpenoid compounds have been previously demonstrated to possess significant cytotoxic as well as noncytotoxic effects against cancer cells (9, 10). Overall, the cell death mechanisms mediated by those characterized triterpenoids involve both apoptosis and necrosis. Dehydroeburicoic acid (DeEA) is a lanostane triterpene (see Figure 2A) that is found in both Antrodia (8) and Poria (7). It remains unknown, however, whether DeEA can display any cytotoxic anticancer effect as did the forgoing triterpenoid compounds. The purpose of this study is therefore to test the hypothesis that DeEA may exert substantial cytotoxic effects against glioblastomas, one of the most frequent and malignant brain tumors in adults (11). We applied cell viability tests to confirm that DeEA caused significant cell death of the glioblastoma cell U87MG. Furthermore, we presented several lines of evidence showing that the mechanism of DeEA-induced cytotoxicity involves Ca2+- and calpain-mediated necrosis.
Experimental Procedures Cell Culture. Human glioblastoma U87MG (American Type Culture Collection, Manassas, VA) and GBM8401 (Bioresource Collection and Research Center, Taiwan) cells were cultured in Dulbecco’s modified Eagle’s medium and RPMI1640 (Invitrogen, Carlsbad, CA), respectively, containing 10% bovine calf serum (Hyclone, Logan, UT), 100 units/mL penicillin, and 50 µg/mL streptomycin. Cells were maintained at 37 °C in a 95% air and 5% CO2 humidified incubator. DeEA was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM as a stock solution.
10.1021/tx9002275 CCC: $40.75 2009 American Chemical Society Published on Web 10/22/2009
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Chem. Res. Toxicol., Vol. 22, No. 11, 2009
The stock was diluted to the required concentration immediately before use. Cells grown in media containing an equivalent amount of DMSO without DeEA were used as the negative control. MTT Assay. Cells were plated in 96-well culture plates for 24 h and then treated with various compounds for an additional 24 h. In control experiments, cells were grown in the same media containing drug-free vehicle (DMSO). The 3-(4,5-Dimethyl-thiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was performed by incubating cells with 0.5 mg/mL MTT for 2 h at 37 °C. Cells were then washed twice with phosphate-buffered saline (PBS) and lysed with DMSO. The assay for the mitochondria-dependent reduction of MTT into formazan was measured at 550 nm in a scanning multiwell spectrophotometer. The absolute optical density was normalized and expressed as the percentage of cell viability. Lactate Dehydrogenase (LDH) Release Assay. Cells were plated into 96-well plates the day before the experiment. After treatment with various compounds, both the total cellular LDH content (LDH in lysed cell pellet plus medium) and the LDH leakage from injured cells into the medium were determined using the CytoTox96 Non-Radioactive cytotoxicity assay kit (Promega, Madison, WI) according to the manufacturer’s instructions. Measurement of Annexin V-FITC and Propidium Iodide (PI) Staining. Cells were stained according to the manufacturer’s instruction (BD Bioscience Pharmingen, San Diego, CA). Briefly, following treatment with DeEA, cells were harvested with trypsin/ ethylenediaminetetraacetic acid (EDTA) and washed twice with icecold PBS. Cells were resuspended in 100 µL of Annexin V binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2) and incubated with 1 µg/mL FITCconjugated Annexin V plus 5 µg/mL PI (Sigma Chemical Co., St. Louis, MO) for 15 min in the dark. After 400 µL of Annexin V binding buffer was added, cells were analyzed by flow cytometry (the FACSCalibur flow cytometer system, BD Biosciences, San Jose, CA) using the CellQuest software (BD Biosciences). At least 10000 cells were analyzed in each sample. Cell Cycle Analysis. For the measurement of the sub-G1 fraction, both adherent and floating cells were collected, washed with PBS, fixed in ice-cold absolute ethanol, treated with RNase, stained with PI, and then analyzed by flow cytometry. The CellQuest software was then utilized to calculate the percentage of the sub-G1 cell population. Analysis of Morphological Changes. Morphological changes were examined by either phase-contrast or electron microscopy. For electron microscopy experiments, U87MG cells were plated in 10 cm Petri dishes the day before the experiment. After treatment with either vehicle or 30 µM DeEA for 12 h, cells were harvested and fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), postfixed in 1% buffered osmium tetraoxide for 1 h, and dehydrated in an ascending series of ethanol concentrations. Specimens for transmission electron microscopy were embedded in resin EMBed 812. Ultrathin sections were cut with an ultramicrotome (Reichert Ultracut E, Leica, Wetzlar, Germany) and double stained with uranyl acetate and lead citrate. Images were viewed and captured using a Hitachi H-800 transmission electron microscope (TEM, JEM-2000EXII, JEOL, Japan) with 100 kV accelerating voltage. Immunofluorescence. U87MG cells were grown on glass coverslips coated with poly-D-lysine. At the end of treatment, cells were rinsed with cold PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, permeabilized with cold methanol at -20 °C for 15 min, and blocked with a blocking buffer [5% normal goat serum and 0.1% (v/v) Triton X-100 in PBS, pH 7.4] for 60 min at 4 °C. Cells were then incubated with rabbit antiLC3B antibody (Cell Signaling, Danvers, MA) at 1:200 dilution in the blocking buffer overnight at 4 °C. After three washes with PBS, the coverslips were incubated with goat-anti-mouse antibodies conjugated to Alexa488 (Invitrogen Molecular Probes) for 1 h at room temperature. Nuclei were labeled with DAPI. Finally, the coverslips were rinsed once in blocking buffer, twice in PBS, and twice in 0.1 M carbonate buffer, pH 9.2, before they were mounted on glass slides in a mounting medium (4% n-propyl gallate, 90%
Deng et al. glycerol, and 0.1 M carbonate, pH 9.2). The fluorescence images of the fixed cultures were viewed and acquired with a Leica TCS SP2 laser-scanning confocal microscope (Leica, Mannheim, Germany). Fluo3-AM Staining. U87MG cells were preloaded with the Ca2+ indicator Fluo3-AM (4 µM) (Invitrogen Molecular Probe) in the presence of 0.02% pluronic acid (Invitrogen Molecular Probe) in Krebs-Ringer buffer (KRB: 120 mM NaCl, 0.5 mM MgCl2, 0.5 mM KCl, 10 mM glucose, 0.7 mM Na2HPO4, and 1.5 mM NaH2PO4, pH 7.4) for 60 min at 37 °C. After they were briefly rinsed with KRB, cells were treated with DMSO, DeEA, or the Ca2+ ionophore A23187 (Sigma). At indicated time points after drug treatments, fluorescence images were taken under an inverted fluorescence microscope (Nikon, Tokyo, Japan). Fluorescence images were stored as gray level images and analyzed with the Simple PCI 5.3.1 software (Compix Inc., Cranberry Township, PA). Measurement of Mitochondrial Membrane Potential. The reduction of mitochondrial transmembrane potential (4Ψm) was analyzed with the cationic fluorochrome DiOC6(3) (Invitrogen Molecular Probe) that stains the intact mitochondria. Briefly, U87MG cells were treated with DeEA for 12 h, followed by incubation with 20 nM DiOC6 at 37 °C for 30 min. After the cells were washed with PBS, the fluorescence intensity was analyzed using flow cytometry. ATP Content. Cells were seeded into 96-well plate for 24 h and then treated with various concentrations of DeEA for 6 h. The cellular ATP content was determined by using the ATP determination kit (BioVision, Mountain View, CA). Immunoblotting. At the end of treatments, cells were lysed in ice-cold lysis buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM Na2HPO4, 1% Triton X-100, 1% Na-deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride] containing protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After they were blocked with 5% nonfat milk, the membranes were probed with appropriate primary antibodies, including mouse anti-spectrin (1: 1000) (Millipore Chemicon, Billerica, MA), rabbit anti-LC3B (Cell Signaling, 1:000), and mouse anti-β-actin (Sigma, 1:5000). Blots were exposed to peroxidase-conjugated goat-anti-rabbit or goatanti-mouse secondary antibodies (Pierce) and revealed by an enhanced chemiluminescence detection system. Statistical Analysis. All experiments were repeated at least three times and performed in duplicates or triplicates of samples. Data were expressed as means ( SEMs. Student’s t test was used to compare differences between the test and the control group. A P value
