FEMS Microbiology Letters 244 (2005) 213–219 www.fems-microbiology.org
Antrodia camphorata prevents rat pheochromocytoma cells from serum deprivation-induced apoptosis Nai-Kuei Huang, Jing-Jy Cheng, Wen-Lin Lai, Mei-Kuang Lu
*
National Research Institute of Chinese Medicine, No. 155-1, Li-Nung St., Sec. 2, Shih-Pai, Peitou, Taipei 112, Taiwan Received 30 September 2004; received in revised form 10 January 2005; accepted 26 January 2005 First published online 3 February 2005 Edited by N. Gunde-Cimerman
Abstract Antrodia camphorata (A. camphorata) is a rare medicinal fungus with antioxidative, vasorelaxtative, anti-inflammatory and anti-hepatitive effects. However, the neuroprotective effect has not been studied. By using serum deprivation-induced apoptosis in neuronal-like PC12 cells as a cell stress model, we found that A. camphorata is effective in preventing serum-deprived apoptosis. Inhibitors of both a serine/threonine kinase and a specific protein kinase A (PKA) inhibited the protective effect of A. camphorata, indicating that A. camphorata prevents serum-deprived PC12 cell apoptosis through a PKA-dependent mechanism. A transcription inhibitor, actinomycin D, and a protein synthesis inhibitor, cyclohexamide, both attenuated the protective effect of A. camphorata, indicating a requirement for gene expression for protection by A. camphorata. On the other hand, A. camphorata also increased phosphorylated CREB, a transcription factor, which is H-89-inhibitable in this study, suggesting the possibility that A. camphorata prevents serum deprivation-induced PC12 cell apoptosis through a PKA/CREB-dependent pathway. 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Antrodia camphorata; Pheochromocytoma cells; Apoptosis; Serum deprivation; Neuroprotection
1. Introduction Antrodia camphorata [1] is a medicinal fungus of the family Polyporaceae that grows slowly on the inner cavity of the camphor tree, Cinnamomum kanehirai. It is an indigenous and rare species in Taiwan. A. camphorata has not only been utilized to treat a wide variety of diseases, but also has drawn the attention of the pharmaceutical industry. Traditionally, it has been used to treat intoxication-caused by food, alcohol, and drugs as well as to treat diarrhea, abdominal pain, *
Corresponding author. Tel.: +886 2 2820 1999x7391; fax: +886 2 2826 4276. E-mail address:
[email protected] (M.-K. Lu).
hypertension, itchy skin, and tumorigenic diseases [2]. Chemical compounds found in A. camphorata include sesquiterpene lactone, steroids, and triterpenoids [3–7]. Its biological effects have rarely been studied. Recently, differential extracts of A. camphorata have been shown to exert antioxidative [8–10], vasorelaxtative [11], anti-inflammatory [12], and anti-hepatitive effects [13]. However, its effect on neuronal protection has never been studied. Besides, since A. camphorata is commercially available and is popularly used in the formulation of neutraceuticals and functional foods in Taiwan, it is worthwhile to fully characterize its expanded activities. On the other hand, since the growth rate of natural A. camphorata in the wild is extremely slow and its cultivation in greenhouses is difficult,
0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.01.048
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obtaining fruiting bodies is expensive. Therefore, using a submerged culture method to obtain useful cellular materials or to produce effective substances from cultured mycelia might be an alternative to overcoming the disadvantages of the retarded growth of the fruiting bodies. Cultured mycelia of A. camphorata were used in this study. Neuronal death induced by apoptosis is a normal aspect of development in which it seems that the death program is triggered by failure of a given neuron to receive limiting supplies of target-derived neurotrophic factors. In the post-development period, neurons also undergo apoptotic death when deprived of appropriate trophic factors or when subjected to a variety of stresses and injuries. The rat pheochromocytoma (PC12) cell line is a commonly used model for studies of neuronal differentiation and cell death. Apoptosis may occur when triggered by deprivation of either serum [14] or trophic factor/nerve growth factor (NGF) [15,16]. Consequently, serum deprivation-induced PC12 cell death was used as an apoptotic model to investigate the therapeutic potential of A. camphorata as a neuroprotectant. The protective mechanism was examined in this study.
2. Materials and methods 2.1. Reagents and cell culture All reagents were purchased from Sigma Chemical (St. Louis, MO, USA) except where specified. H-89 and K-252a were purchased from Biomol (Plymouth Meeting, PA, USA). Actinomycin D (Act D), cycloheximide (CHX), forskolin (FK), LY 294002 and genistein were purchased from Tocris Cookson (Avonmouth, UK). DulbeccoÕs modified EagleÕs medium (DMEM), fetal bovine serum, and horse serum were purchased from HyClone (Logan, UT, USA). First and second antibodies were purchased form Cell Signaling Technology (Beverly, MA, USA). PC12 cells were maintained in DMEM supplemented with 10% (vol/vol) horse serum and 5% (vol/vol) fetal bovine serum and incubated in a CO2 incubator (5%) at 37 C. 2.2. Liquid culture of A. camphorata An A. camphorata isolate, strain B85 from Taitung, Taiwan, was a generous gift from fungi specialist Dr. T.T. Chang (Division of Forest Protection, Taiwan Forest Research Institute, Taipei, Taiwan). A. camphorata was maintained on potato dextrose agar (Sigma) and transferred to fresh medium at 3-week intervals. For liquid culture, 19-day-old seeding mycelium of A. camphorata on the surface of medium was cut into pieces (approximately 0.7 · 0.7 cm) before being transferred to 30 ml of potato dextrose broth (Sigma) in 125-ml
flasks. Flasks were maintained in a stationary condition at 28 C under 90 rpm shaking in the dark for 10 days. Thereafter, 300 ml of the shaking flask culture was inoculated into a 5-L fermentation tank containing 3 L of culture medium (PDB 24 g/l, agar 2 g/l, and glucose 20 g/l, pH 5.6) and then cultured at 28 C for 10 days with an aeration rate of 1 vvm (aeration volume/medium (L)/min) by shaking at 50 rpm to obtain a mucilaginous medium containing mycelia. At the end of the incubation, mycelia were rapidly washed with 1 L of NaCl (250 mM) by an aspirator-suction system to remove the contaminating culture medium. Samples were then lyophilized and resuspended in milli-Q water to achieve a stock concentration of 50 mg/ml. Serially dilution in milli-Q water to adequate concentrations was performed from the stock immediately before use for the following experiments. 2.3. Preparation of mycelial extracts from liquid culture of A. camphorata Lyophilized mycelia were extracted with 80 C water twice in a 1:100 (w/w) ratio for 6 h. Supernatants were collected after centrifugation, whereupon 4 volumes of 95% ethanol was added then precipitated at 4 C overnight. The dilute ethanolic supernatants were then lyophilized following centrifugation.
3. MTT metabolism assay Survival was assessed by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) metabolism assay as described by Mosmann [17]. Cells growing on 150-mm plates were washed 3 times with PBS and resuspended in DMEM. Suspended cells were plated on 96-well plates and treated with the indicated reagent(s) (1 · 104 cells/200 ll/well). These reagents included serine/threonine kinase inhibitor (K-252a: 1–10 lM), tyrosine kinase inhibitor (genistein: 0.1– 100 lM), PI-3 kinase inhibitor (LY 294002: 1–30 lM), PKA inhibitor (H-89: 1–30 lM), RNA synthesis inhibitor (Act D: 0.001–10 mM), and new protein synthesis inhibitor (CHX: 0.001–10 lM). After incubation for 24 h, 20 ll of MTT stock (5 mg/ml) was added to the medium and incubated at 37 C for 3 h. After discarding the medium, DMSO (100 ll) was then applied to the well to dissolve the formazan crystals derived from the mitochondrial cleavage of the tetrazolium ring by live cells. The absorbance at 570/630 nm, which highly correlates with the cell numbers, in each well was measured on a micro-ELISA reader. Cell viability was expressed as a percentage of the results of the MTT metabolism assay (OD 570/630 nm) measured in the serum-containing group.
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An Annexin V (FITC-conjugated) apoptosis Kit (K101-400; BioVision, Mountain View, CA, USA) was used to analyze the apoptotic cells. The experimental protocol followed the manufactureÕs instructions. In brief, after treatment with serum, serum free, or serum free/ B85 treatments for 24 h, cells growing on 12-well plates at 3–4 · 105 cells/well were loaded with 0.5 ml binding buffer and 5 ll Annexin V-FITC. After incubation for 5 min in the dark, cells were washed once with 1 ml culture medium (without phenol red) for taking fluorescent micrographs or resuspended for flow cytometric analysis (Beckton Dickinson, Franklin Lakes, NJ, USA). The mean values of the fluorescent intensities of FITC were collected using an FL-1 channel (488/530Ex/Em nm). Five thousand live cells were analyzed per sample. 3.2. Western blot analysis Cells were rinsed with ice-cold PBS and lysed in icecold lysis buffer (20 mM HEPES, 1 mM DTT, 20 mM EGTA, 10% glycerol, 50 mM b-glycerophosphate, 10 mM NaF, 1% Triton X-100, 1 mM PMSF, 1 mM Na3VO4, 2 lM aprotinin, 100 lM leupeptin, 2 lM pepstatin, and 0.5 lM OKA). After sonication, cell debris was removed by centrifugation at 14,000 rpm for 10 min, and the supernatant was utilized for Western blot analysis. Equal amounts of sample were separated by 10% polyacrylamide gel electrophoresis. The resolved proteins (50 lg/lane) were then electroblotted onto Immobilon PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% skim milk and then incubated with the first (1:2000) and the second antibodies (1:5000) sequentially for 1 h at room temperature. After washing, blots were processed for visualization using an enhanced chemiluminescence system (Pierce). Blots were then exposed to Kodak XAR-5 film to obtain the fluorographic images. 3.3. Statistical analysis Results were analyzed by one- or two-way analyses of variance according to the suitability. Differences between means were assessed by the Student-Newman– Keuls method and were considered significant at p < 0.05.
4. Results 4.1. A. camphorata prevents serum deprivation-induced apoptosis in PC12 cells The ethanolic extracts of A. camphorata exerted protective effects against serum-deprived PC12 cell death in
a dose-dependent manner as revealed by the MTT assay (Fig. 1). A sub-maximum dosage (300 lg/ml) of the extract (in the following text referred as A. camphonata B85) was adopted and used throughout the following experiments. Annexin V-FITC staining demonstrated that the fluorescent intensities of Annexin V-FITC was significantly increased in PC12 cells after serum deprivation for 24 h as shown by the fluorescent micrograph (Fig. 2(b)) and flow cytometry (Fig. 2(d) and (e)). A. camphorata B85 attenuated the increased fluorescent intensity of Annexin V-FITC after serum deprivation in PC12 cells (Fig. 2(c)–(e)), confirming that A. camphorata B85 prevents serum deprivation-induced apoptosis in PC12 cells. 4.2. A serine/threonine kinase inhibitor blocked the protective effect of A. camphorata B85 The protective effect of A. camphorata B85 was dosedependently inhibited by a serine/threonine kinase inhibitor (K-252a) [18], suggesting the role of serine/ threonine kinase in regulating A. camphorataÕs protection (Fig. 3(a)). On the other hand, the inability of a tyrosine kinase inhibitor (genistein) to attenuate the protective effect of A. camphorata B85 ruled out the tyrosine kinase pathway playing a role in mediating the protection of A. camphorata (Fig. 3(a)). 4.3. A protein kinase A inhibitor blocked the protective effect of A. camphorata B85 The protective effect of A. camphorata B85 was dose-dependently inhibited by a protein kinase A
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Fig. 1. Effect of A. camphorata B85 in preventing serum deprivationinduced PC12 cell death. Serum-deprived PC12 cells were treated with or without different concentrations of A. camphorata B85 ethanolic extract for 24 h. Cell viability was expressed as a percentage of the results of the MTT assay measured in the serum-containing control group. Data points represent means ± SEM of at least three independent experiments (n = 3–6). *p < 0.05 compared to the serum-deprived control group.
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Fig. 2. Effect of A. camphorata B85in preventing serum deprivation-induced PC12 cell apoptosis. The (a) serum containing PC12 cells or serumdeprived PC12 cells that were treated (b) without or (c) with A. camphorata B85 ethanolic extract for 24 h were stained with Annexin V-FITC and analyzed by fluorescent microscopy. (d) Cells stained with different treatments were also analyzed using flow cytometry as described in Section 2. (e) The mean values of FITC fluorescent intensities were collected using an FL-1 channel. Data points represent means ± SEM of at least three independent experiments. *p < 0.05 compared to the serum-containing group (n = 3–6). #p < 0.05 compared to the serum free group.
(PKA) inhibitor (H-89), suggesting a role of PKA in regulating A. camphorataÕs protection (Fig. 3(b)). Consequently, FK, a known PKA activator, as a positive control was used and found that the protective effect of FK was also abolished by H-89 (Fig. 3(b)), confirming the role of PKA pathway in preventing serumdeprived apoptosis. In addition, the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway was not involved in the protection of A. camphorata B85, since a PI3-K inhibitor (LY 294006) did not block the effect of
A. camphorata B85 in antagonizing serum-deprived PC12 cell apoptosis (Fig. 3(b)). 4.4. Both Act D and CHX blocked the protective effect of A. camphorata B85 Pretreatment of PC12 cells with Act D, an RNA synthesis inhibitor, or CHX, a protein synthesis inhibitor, blocked the antiapoptotic effect of A. camphorata B85 (Fig. 4(a)), suggesting that gene expression is required
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Fig. 3. Effects of serine/threonine kinase, tyrosine kinase, PKA, and PI3-K inhibitors on the protection provided by A. camphorata B85 during serum deprivation. (a) Cells were pretreated with K-252a or genistein for 30 min before the addition of A. camphorata during serum deprivation. (b) Cells were pretreated with LY 294002 or H-89 for 30 min before the addition of A. camphorata B85 or FK (10 lM) during serum deprivation. Cell viability was expressed as a percentage of the results of the MTT assay measured in the serum-containing group. Data points represent means ± SEM of at least three independent experiments (n = 3–6). *p < 0.05 compared to the serum-deprived control group.
for A. camphorata B85 to prevent serum deprivationinduced apoptosis in PC12 cells.
Fig. 4. Effects of RNA transcription and protein translation inhibitor on the protection provided by A. camphorata B85 and the effects of A. camphorata B85 on CREB and Akt activity. (a) Cells were pretreated with Act D or CHX for 30 min before the addition of A. camphorata B85 during serum deprivation. Cell viability is expressed as a percentage of the results of the MTT assay measured in the serumcontaining control group. Data points represent means ± SEM of at least three independent experiments (n = 3–6). *p < 0.05 compared to the serum-deprived control group. (b) Cell lysates with or without A. camphorata B85 treatment for the indicated times during serum deprivation were harvested and analyzed by Western blotting. (c) After pretreatment with H-89 (20 lM) for 30 min during serum deprivation, cell lysates in the presence or absence of A. camphorata B85 or FK (10 lM) for another 30 min were harvested and analyzed by Western blotting.
5. Discussion 4.5. A. camphorata B85 induced cyclic-AMP response element binding protein phosphorylation during serum deprivation During serum deprivation, the addition of A. camphorata B85 significantly increased the phosphorylation of cyclic-AMP response element binding protein (CREB) (Fig. 4(b)). Pretreatment with H-89 blocked A. camphorata-induced CREB phosphorylation, indicating that CREB is the downstream target of PKA (Fig. 4(c)). On the other hand, A. camphorata B85 did not induce Akt phosphorylation (Fig. 4(b)), further confirming the lack of involvement of PI3-K/ Akt in regulating the protective effect of A. camphorata.
Apoptosis plays an important role during neuronal development, toxicity, and stress, and may underlie various neurodegenerative disorders. We utilized serum deprivation-induced apoptosis in neuronal-like PC12 cells as an apoptotic model to screen potential therapeutic neuroprotectants from Chinese herbal extracts (Fig. 1). The maximum effect is ranging from 300 to 500 lg/ ml. The drop of MTT metabolism only occurred at the last highest dose (1000 lg/ml). The less protective effect at the highest dose is not clear, however, it could be owing to the toxic effect by other components in A. camphorata B85. Primarily, we found that the extracts of A. camphorata exerted significant protection against serumdeprived apoptosis (Fig. 2). This protection seemed to
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be serine/threonine kinase-dependent (Fig. 3(a)). Further investigations indicated that A. camphorata B85 might prevent serum-deprived apoptosis through PKA-dependent pathway and possibly through downstream CREB activation (Figs. 3 and 4). The requirement of gene expression for the protective effect of A. camphorata B85 (Fig. 4) strengthened the possibility that activated transcription factor(s), such as CREB, turn on gene expression and the subsequent protection. 5.1. Serine/threonine kinases and apoptosis Since serine/threonine kinases including tyrosine kinases have been suggested to play a role in apoptosis [19], a general serine/threonine kinase and tyrosine kinase inhibitor were used to screen their involvement in the control of A. camphorataÕs protection. To our surprise, only K-252a blocked the protective effect of A. camphorata B85 but not genistein (Fig. 3(a)), confirming that the protection may involve a serine/threonine kinase-dependent pathway. However, since serine/threonine kinase comprises a large family that contains diverse kinases, such as the mitogen-activated protein kinase (MAPK) family, PKA, Akt, protein kinase C (PKC), etc., we selected two kinases (PKA and Akt), which are known for their antiapoptotic effects [20,21] in this system, as the primary targets. Basically, H-89 but not LY294002 blocked the protective effect of A. camphorata B85 (Fig. 3(b)), suggesting that A. camphorata B85 prevents serum-deprived PC12 cell death through a PKA-dependent pathway consistent with results from a previous article [20]. 5.2. PKA and transcription factor(s) Since the protective effect of A. camphorata B85 is PKA-dependent, it is possible that A. camphorata B85 may contain a cyclic AMP analogue, an andenlyl cyclase activator, or a receptor agonist, which indirectly activates andenlyl cyclase. However, further investigations are required to resolve these questions. On the other hand, although an extranuclear locus of PKAÕs action has been shown to be necessary and sufficient for promotion of spiral ganglion neuronal survival by cyclic AMP [22], our data demonstrate that the protective mechanism of A. camphorata B85 might be transcription- and translation-dependent (Fig. 4(a)), suggesting a requirement of gene expression for the protective effect of A. camphorata B85 in this system. Thus, activation of downstream transcription factor(s) is likely to be necessary. Currently, PKA-mediated transcription factors may include CREB, activating transcription factor-1, AP-2, NF-jB, etc. [23]. Since the signaling pathway of PKA/CREB is well known for its antiapoptotic effect [24], we thus characterized whether A. camphorata B85 can activate CREB.
5.3. CREB and apoptosis The transcriptional activation of CREB is crucially dependent on phosphorylation of Ser133 by PKA [25] or by other kinases, such as Ca2+-activated calmodulin kinases, ribosomal S6 kinase 2, or mitogen-activated protein kinase-activated protein kinase 2 [26]. In its active form, CREB has been shown to regulate many aspects of neuronal functioning, including neuronal excitation [27], development [28] and long-term synaptic plasticity [29]. Recent evidence suggests that CREB might also be involved in an active process of neuroprotection [30] or that its disruption in the brain might lead to neurodegeneration [31], suggesting a pivotal role of CREB in preventing cell death [32]. Since the protection of A. camphorata B85 was mediated through a PKA-dependent pathway, the downstream target was subsequently assayed and revealed that CREB phosphory lation increased after A. camphorata treatment (Fig. 4(b)). Pretreatment with H-89 blocked A. camphoratainduced CREB activation, indicating that A. camphorata B85 may have the potential to prevent serum-deprived PC12 cell death through the PKA/CREB signaling pathway. However, the expression of a dominant negative or positive mutant of CREB in this system might be helpful to verify if PKA-mediated CREB activation is responsible for the protective effect of A. camphorata. We determined the composition of A. camphorata B85 to elucidate which active components are associated with anti-apoptotic effect. In this study, three nucleoside-like components, cytidine, adenosine, and thymidine were identified. The amount of cytidine, adenosine and thymidine were determined to be 7.58 ± 0.09, 3.36 ± 0.06, and 7.83 ± 0.22 mg/g ethanolic extract, respectively, and may play a role in the observed anti-apoptotic activity in the A. camphorata B85 (supplementary data). Further study should be performed to validate its specific components. Taken together, in this study we found for the first time that the extract of A. camphorata prevented PC12 cells from serum deprivation-induced apoptosis through a PKA-dependent pathway, suggesting the therapeutic potential for treating neurotoxicity. The requirement of gene expression for the protective effect of A. camphorata suggests that activation of transcription factor(s), such as CREB, might be responsible for the PKAtargeted protective mechanism.
Acknowledgements We thank Dr. Tun-Tschu Chang for kindly supplying the strain of A. camphorata and Mr. D.P. Chamberlin for critically reading the manuscript. This work was supported in part by Grant NSC93-2320-B-077012 to NKH and NSC92-2313-B-077-001 to MKL
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from the National Science Council, Taiwan, and in part by Grant NRICM-94-DBCMR and NRICM-94DHM from National Research Institute of Chinese Medicine, Taiwan.
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