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Green tea polyphenols suppress nitric oxide–induced apoptosis and acetylcholinesterase activity in human neuroblastoma cells
Nutrition Research 25 (2005) 477 – 483 www.elsevier.com/locate/nutres
Green tea polyphenols suppress nitric oxide–induced apoptosis and acetylcholinesterase activity in human neuroblastoma cells Joo Ho Chunga, Mujo Kimb, Hye Kyung Kimc,T a
Department of Pharmacology, College of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea b Pharma Foods International, Minami-ku, Kyoto 601-8357, Japan c Department of Food and Biotechnology, Hanseo University, Seosan 356-706, Republic of Korea Received 14 December 2003; revised 8 October 2004; accepted 3 February 2005
Abstract Oxidative stress is a main mediator in nitric oxide (NO) –induced neurotoxicity and has been implicated in the pathogenesis of many neurodegenerative disorders. Green tea polyphenols (GTPs) exert a wide range of biochemical and pharmacological effects and have been shown to prevent oxidative stress–related diseases. This paper demonstrated that GTP protected the neurotoxicity of NO, generated by S-nitroso-N-acetylpenicillamine, in human neuroblastoma cells. Green tea polyphenols attenuated the NO-induced apoptotic cell death, assessed by cell viability, Hoechst staining, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling staining. The protective mechanism was via elevated expression of the antiapoptotic bcl2 gene and suppressed expression of the proapoptotic bax gene, thereby arresting NO-induced apoptotic cell death. Furthermore, GTP appeared to be a potent inhibitor of acetylcholinesterase, exhibiting an IC50 of 248 lg/mL. To our knowledge, this is the first report showing the inhibitory effect of GTP on acetylcholinesterase activity, exploiting potential use in neurodegenerative disease. D 2005 Elsevier Inc. All rights reserved. Keywords: Tea polyphenols; Nitric oxide; Human neuroblastoma cells; Apoptosis; Acetylcholinesterase; Neurodegenerative disease
T Corresponding author. Tel.: +82 41 660 1454; fax: +82 41 660 1119. E-mail address: [email protected] (H.K. Kim). 0271-5317/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2005.02.002
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1. Introduction Tea is one of the most widely consumed beverages in the world aside from water. Green tea contains a considerable amount of polyphenolic compounds, and green tea polyphenols (GTPs) have aroused considerable attention in recent years for preventing oxidative stress– related disease [1]. It has been reported that catechins reduce the risk of cardiovascular disease by increasing plasma antioxidative capacity in human beings [2] and that GTPs have a cytoprotective effect against chemical-induced cell injury in cultured cells [3] and oxidative damage in biologic tissues [4]. The generation of oxygen free radicals, reactive oxygen species, and oxidative damage are believed to be involved in the pathogenesis of neurodegenerative disorders [5]. Over the decades, the menagerie of reactive oxygen species has been expanded to include reactive nitrogen species derived from nitric oxide (NO) [6]. Neuronal damage, emerging from oxidative stress, has been reported to involve regulation of cell signaling molecules as well as activation of apoptotic cell death [7,8]. In the central nervous system, apoptosis plays an indispensable role in the development and maintenance of homeostasis within all organisms [9]. However, excessive apoptosis has been suggested to underlie the neuronal loss associated with the exposure to various neurotoxicants [10]. The common mechanism of apoptosis is regulated by several sets of genes, of which the best characterized is the still growing bcl-2 family [9]. Nitric oxide induces apoptosis of a variety of types of cultured cells including neurons and may contribute to several disorders of the nervous system. In recent years, researchers have begun to recognize and explore the putative link between NO and neurodegenerative diseases [11]. Although existing evidence regarding their association are not abundant, emerging data are showing Alzheimer’s disease (AD)–related changes in the NO system and it appears that NO could be related to many AD pathological mechanisms [12]. Alzheimer’s disease is characterized by alterations at the level of various neurotransmitters and related markers and receptors. Of these, the most severely affected is by far the cholinergic system. Loss of cholinergic innervation, demonstrated by reduced choline acetyltransferase and elevated acetylcholinesterase (AChE) activity, is correlated with the degree of dementia and the severity of neuropathological hallmarks of AD [12,13]. Therefore, it is suggested that the elevation of the acetylcholine (ACh) level might be helpful in improving the symptoms of cognitive deficits in AD. In the present study, we examined NO scavenging activity in a noncellular system and the neuroprotective effect of GTP against NO-induced toxicity in human neuroblastoma cells. Furthermore, the inhibitory effect of GTP on AChE was also investigated to evaluate its potential use in neurodegenerative diseases including AD.
2. Methods and materials 2.1. Nitric oxide scavenging activity S-nitroso-N-acetylpenicillamine (SNAP) was synthesized as described by Yoo et al [14]. Nitric oxide scavenging activity of GTP was determined by incubating 500 lmol/L SNAP, dissolved in 1 mL phosphate-buffered saline (PBS; 50 mmol/L, pH 7.4), with various
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concentrations of GTP. After the desired incubation time, 100 lL of Griess solution was added to 100 lL of aliquot and the absorbance at 550 nm was measured with a spectrophotometer. 2.2. Cell culture The human neuroblastoma cells, SK-N-MC cells, were obtained from the American Type Culture Collection (Rockville, Md). SK-N-MC cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Grand island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) at 378C in a 5% CO2/95% air cell incubator. 2.3. Protective effect of GTP (cell viability and morphological change) For analysis of the protective effects of GTP against NO cytotoxicity, cell viability was determined by an MTT assay kit (Sigma, St. Louis, MO) according to the manufacturer’s protocol. In brief, cells were cultured in each well of a 24-well plate at a density of 2 105 cells per well. The cells were treated with 5 mmol/L SNAP for 4 hours and GTP (60 or 300 lg/mL) was added 1 or 2 hours before treatment with SNAP. 2.4. Hoechst 33258 staining and terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling staining Apoptotic cells were determined by Hoechst staining. Cells were cultured on 4-chamber slides (Nalge Nunc International, Naporvile, Ill) at a density of 5 107 per well. After treatment of SNAP and GTP as described above, the cells were fixed in cold methanol ( 208C) for 10 minutes and washed twice with a PBS buffer before staining with 1 lg/mL Hoechst 33258 for 15 minutes at 378C in the dark. Cells were then washed with PBS, mounted with coverslips using glycergel (Dako, Carpinteria, CA), and observed under an Olympus-BH2 fluorescent microscope at 372 nm. For in situ detection of apoptotic cells, the terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling (TUNEL) assay was performed using an ApoTaq peroxidase in situ apoptosis detection kit (Intergen, Houston, TX). 2.5. Apoptotic gene expression The expression of bcl-2 and bax messenger RNAs (mRNAs) was determined by reverse transcription polymerase chain reaction. Total RNA was isolated from cells with RNAzolB (TEL-TEST, Friendswood, Tex) according to the manufacturer’s instruction. Polymerase chain reaction was performed with the primers for bax (5V-AGA TGA ACT GGA TAG CAA TAT GGA-3V and 5V-CCA CCC TGG TCT TGG ATC CAG ACA-3V) and for bcl-2 (5V-TCC GTG CCT GAC TTT AGC AAG CTG-3Vand 5V-GGA ATC CCA ACC AGA GAT CTC AA3V). Cyclophilin (5V-ACC CCA CCG TGT TCT TCG AC-3Vand 5V-CAT TTG CCA TGG ACA AGA TG-3V) was used as an internal control. The annealing temperatures were 508C for bax and bcl-2 and 568C for cyclophilin. The amplified fragment sizes of bax, bcl-2, and cyclophilin were 260, 333, and 300 base pairs, respectively. The reverse transcription polymerase chain reaction products were electrophoresed on a 1.5% agarose gel and visualized by staining with ethidium bromide.
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2.6. Acetylcholinesterase activity The AChE assay was measured by the slightly modified method of Ellman et al [15] using acetylthiocholine iodide as a substrate. For the enzyme source, SK-N-MC cell cultures were homogenized with a homogenation buffer (10 mmol/L Tris-HCl [pH 7.2] containing 1 mol/L NaCl, 50 mmol/L MgCl2, and 1% Triton X-100) and centrifuged at 10 000g for 30 minutes. The resulting supernatant was used as an enzyme source. Protein concentration was determined by a bicinchoninic acid kit (Sigma) with bovine serum albumin as a standard. The rates of hydrolysis by AChE were monitored spectrophotomatically using a 96-well microtiter plate reader. 2.7. Statistical analysis Data were expressed as mean F SEM of at least 3 separate experiments. Comparison between 2 values was analyzed by one-way analysis of variance followed by Dunnett’s post hoc analysis using SPSS (Chicago, IL). Differences were considered significant at P b .05. 3. Results and discussion
Nitric Oxide Scavenging Effect (%)
It is known that GTP is an excellent scavenger of free radicals [16]. In the present study, it revealed that GTP is also an excellent NO scavenger (Fig. 1). Nitric oxide scavenging effect was time dependent for 8 hours of SNAP treatment and non–time dependent after 8 hours of NO exposure. Nitric oxide scavenging effects after 5 hours were 71.9% F 1.3%, 63.1% F 1.7%, and 55.6% F 2.8% at 1, 3, and 5 mg/mL of GTP treatment, respectively. The effects after 8 hours were 35.7% F 0.6%, 33.8% F 0.7%, and 37.5% F 0.5% at 1, 3, and 5 mg/mL, respectively. Scavenging of the NO radical will contribute to the therapeutic effect of the GTP. The viability of SK-N-MC cells treated with 5 mmol/L SNAP was reduced to 27.8% of the control 120
5 mg/ml 3 mg/ml
100 1 mg/ml 80 60 40 20 0 2
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8
11
Treatment (h) Fig. 1. Nitric oxide scavenging activity of GTP was determined by incubating 500 lmol/L SNAP with various concentrations of GTP.
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value (data not shown). However, a significant degree of protective effect was observed when cells were pretreated with GTP before they were exposed to SNAP. Green tea polyphenols showed a dose- and time-dependent protective effect on NO-induced neurotoxicity. The viabilities of cells pretreated with GTP at 60 lg/mL for 1 or 2 hours were 52.1% F 3.1% and 63.4% F 1.9% and those at 300 lg/mL were 60.8% F 1.8% and 68.9% F 1.9% of the control value, respectively. After treatment with SNAP for 4 hours, there were great changes in cell shape. The cells lost the ability to adhere to the plate surface, with cell rounding, cytoplasmic blebbing, and cell shape irregularity, whereas cells pretreated with GTP before NO exposure appeared to be similar to the control (data not shown). To confirm the effect of GTP against NO-induced cell death, we used Hoechst 33258 staining (Fig. 2A-C) and TUNEL reaction (Fig. 2D-F). Similar results were obtained with these 2 methods. Apoptotic cells exhibiting characteristic figures of apoptosis with condensed and fragmented nuclei were apparent after exposure to NO (Fig. 2B and E). These changes in nuclei characteristic of apoptosis decreased in the cells pretreated with GTP (Fig. 2C and F). It is well known that excessive NO is neurotoxic and kills neuronal cells in various central nervous system disorders. Oxidative stress is believed to play an important role in NOinduced neuronal apoptosis. Some studies reported that an antioxidant attenuated NO-
Fig. 2. Antiapoptotic effect of GTP. Cells were incubated in control medium (A, D), 5 mmol/L SNAP (B, E), or 60 lg/mL GTP pretreated with 5 mmol/L SNAP (C, F). Top panel, cells stained with Hoechst staining. Apoptotic cells exhibit condensed and/or fragmented nuclei (B) and these changes were decreased in GTP-pretreated cells (C). Middle panel, cells stained with TUNEL reaction. Condensed and marginated chromatins were labeled as brown dots in SNAP-exposed cells (E) and GTP protected these changes (F). Lower panel (G), Reverse transcription polymerase chain reaction analysis of mRNA levels of bcl-2 and bax. The expression of the antiapoptotic bcl-2 gene was increased and that of the proapoptotic bax gene was decreased in cells pretreated with GTP compared with those in cells exposed to SNAP. As the internal control, cyclophilin mRNA was also examined. A, Control; B, SNAP-treated group; C, GTP-pretreated group.
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% inhibition of AchE activity
induced neurotoxicity [17,18]. Recently, it has been demonstrated that epigallocatechin gallates scavenge NO reagents [19] as well as inhibit oxidative damage to DNA induced by NO [9]. The present study showed that SNAP-induced apoptosis of human neuroblastoma cells and NO-induced cytotoxicity were significantly protected by GTP pretreatment. Changes in nuclear morphology revealed that treatment with SNAP exhibits condensed and/or fragmented nuclei, which are characteristic of apoptosis (Fig. 2B and E), whereas GTPpretreated cells showed a noticeable decrease in these features (Fig. 2C and F). Because the bcl-2 family controls apoptosis triggered by a variety of signals, the mRNA levels of bcl-2 and bax were examined. Bcl-2 usually inhibits apoptosis whereas another member of this family, bax, can promote cell death. It has been suggested that the bcl-2–to-bax ratio determines survival or cell death by modulating the release of proapoptotic factors [10]. In agreement with mitochondria-dependent activation of caspases during the induction of apoptosis, in the present study, SNAP treatment decreased the mRNA level of bcl-2 and increased that of bax in human neuroblastoma cells, whereas GTP pretreatment increased the mRNA level of bcl-2 mRNA and decreased that of bax (Fig. 2G). This suggests that the increased bcl-2–to-bax ratio by GTP treatment may contribute to the inhibition of NOtriggered apoptosis. Green tea polyphenols inhibited the AChE activity in a dose-dependent manner (Fig. 3). The concentration that was required for the 50% enzyme inhibition (IC50) was 248 lg/mL. The reduction of choline acetyltransferase and AChE activity is correlated with the degree of dementia and the severity of neuropathological hallmarks of AD. Therefore, it is suggested that the elevation of the ACh level might be helpful in improving the symptoms of cognitive deficits in neurodegenerative diseases such as AD. Several groups have tried to supplement the ACh level in synaptic sites by the administration ACh precursors, cholinergic agonists, or AChE inhibitors such as tacrine and physotigmine that prevent ACh hydrolysis. Acetylcholinesterase inhibitors are the only source of compound currently approved for the treatment of AD yet. Therefore, seeking a new active constituent that has a potent inhibitory effect of AChE and antiamnesic activity from natural resource is very promising. To our knowledge, this is the first report showing the inhibitory effect of GTP on AChE activity. On the basis of these in vitro studies, it would be of considerable interest to test GTP in 150
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Fig. 3. Dose-dependent inhibitory effect of GTP on AChE activity. Each value represents mean F SEM. The inhibition efficacy was expressed as a percentage of enzyme activity inhibited compared with the control value (100%).
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comparison to in vivo animal NO toxicity or dementia models to evaluate its potential use in therapy. Acknowledgment This research was supported by Pharma Foods International (Kyoto, Japan). References [1] Hollman PC, Feskens EJ, Katan MB. Tea flavonols in cardiovascular diseases and cancer epidemiology. Proc Soc Exp Biol Med 2001;220:198 - 202. [2] Nakagawa K, Ninomiya M, Okubo T, Aoi N, Juneja LR, Kim M, et al. Tea catechin supplementation increases antioxidant capacity and prevents phospholipid hydroperoxidation in plasma of humans. J Agric Food Chem 1999;47:3967 - 73. [3] Miyagawa C, Wu C, Kenedy DO, Nakatani T, Ohtan K, Sakanaka S, et al. Protective effect of green tea extract and tea polyphenols against the cytotoxicity of 1,4-naphtoquinone in isolated rat hepatocytes. Biosci Biotechnol Biochem 1997;61:1901 - 5. [4] Chen H, Tappel A. Protection by multiple antioxidants against lipid peroxidation in rat liver homogenate. Lipids 1996;31:47 - 50. [5] Mattson MP, Liu D. Energetics and oxidative stress in synaptic plasticity and neurodegenerative disorders. Neuromolecular Med 2002;2:215 - 31. [6] Squadrito GL, Pryor WA. Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide. Free Radic Biol Med 1998;25:392 - 403. [7] Chun HS, Gibson GE, DeGiorgio LA, Zhang H, Kidd VJ, Son JH. Dopaminergic cell death induced by MPP(+), oxidant and specific neurotoxicants shares the common molecular mechanism. J Neurochem 2001;76:1010 - 21. [8] Hu S, Peterson PK, Chon CC. Cytokine mediated neuronal apoptosis. Neurochem Int 1997;30:427 - 31. [9] Tsujimoto Y, Schimizu S. Bcl-2 family: life-or-death switch. FEBS Lett 2000;466:6 - 10. [10] Kelly MR, Geigerman CM, Loo G. Epigallocatechin gallate protects U937 cells against nitric oxide– induced cell cycle arrest and apoptosis. J Cell Biochem 2001;81:647 - 58. [11] Norris PJ, Faull RL, Emson PC. Neuronal nitric oxide synthase (nNOS) mRNA expression and NADPHdiaphorase staining in the frontal cortex, visual cortex and hippocampus of control and Alzheimer’s disease brains. Mol Brain Res 1996;41:36 - 49. [12] Zubenko GS, Moosy J, Martinez AJ, Rao GR, Kopp U, Hanin I. A brain regional analysis of morphologic and cholinergic abnormalities in Alzheimer’s disease. Arch Neurol 1989;46:634 - 8. [13] Law A, Gauthier S, Qurion L. Say no to Alzheimer’s disease: the putative links between nitric oxide and dementia of the Alzheimer’s type. Brain Res Rev 2001;35:73 - 96. [14] Yoo YM, Yim S, Kim S, Jang HY, Lea HZ, Hwang G, et al. Melatonin suppresses NO-induced apoptosis via induction of Bcl-2 expression in PGT-b immortalized pineal cells. J Pineal Res 2002;33:146 - 50. [15] Ellman GL, Courtney D, Valentino A, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88 - 95. [16] Zhao BL, Guo Q, Xin WJ. Free radical scavenging by green tea polyphenols. Methods Enzymol 2001;335:217 - 31. [17] Imam SZ, el-Yazai J, Newport GD, Itzhak Y, Cadet JL, Slikker Jr W, et al. Methamphetamine-induced dopaminergic neurotoxicity: role of peroxynitrite and neuroprotective role of antioxidants and peroxynitrite decomposition catalysts. Ann N Y Acad Sci 2001;939:366 - 80. [18] Bredt DS, Snyder SH. Nitric oxide, a novel neuronal messenger. Neuron 1992;8:3 - 11. [19] van Acker SABE, Tromp MNJL, Haenen GRMM, van der Vijgh WJF, Bast A. Flavonoids as scavengers of nitric oxide radical. Biochem Biophys Res Commun 1995;214:755 - 9.
Green tea polyphenols suppress nitric oxide–induced apoptosis and acetylcholinesterase activity in human neuroblastoma cells Joo Ho Chunga, Mujo Kimb, Hye Kyung Kimc,T a
Department of Pharmacology, College of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea b Pharma Foods International, Minami-ku, Kyoto 601-8357, Japan c Department of Food and Biotechnology, Hanseo University, Seosan 356-706, Republic of Korea Received 14 December 2003; revised 8 October 2004; accepted 3 February 2005
Abstract Oxidative stress is a main mediator in nitric oxide (NO) –induced neurotoxicity and has been implicated in the pathogenesis of many neurodegenerative disorders. Green tea polyphenols (GTPs) exert a wide range of biochemical and pharmacological effects and have been shown to prevent oxidative stress–related diseases. This paper demonstrated that GTP protected the neurotoxicity of NO, generated by S-nitroso-N-acetylpenicillamine, in human neuroblastoma cells. Green tea polyphenols attenuated the NO-induced apoptotic cell death, assessed by cell viability, Hoechst staining, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling staining. The protective mechanism was via elevated expression of the antiapoptotic bcl2 gene and suppressed expression of the proapoptotic bax gene, thereby arresting NO-induced apoptotic cell death. Furthermore, GTP appeared to be a potent inhibitor of acetylcholinesterase, exhibiting an IC50 of 248 lg/mL. To our knowledge, this is the first report showing the inhibitory effect of GTP on acetylcholinesterase activity, exploiting potential use in neurodegenerative disease. D 2005 Elsevier Inc. All rights reserved. Keywords: Tea polyphenols; Nitric oxide; Human neuroblastoma cells; Apoptosis; Acetylcholinesterase; Neurodegenerative disease
T Corresponding author. Tel.: +82 41 660 1454; fax: +82 41 660 1119. E-mail address: [email protected] (H.K. Kim). 0271-5317/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2005.02.002
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1. Introduction Tea is one of the most widely consumed beverages in the world aside from water. Green tea contains a considerable amount of polyphenolic compounds, and green tea polyphenols (GTPs) have aroused considerable attention in recent years for preventing oxidative stress– related disease [1]. It has been reported that catechins reduce the risk of cardiovascular disease by increasing plasma antioxidative capacity in human beings [2] and that GTPs have a cytoprotective effect against chemical-induced cell injury in cultured cells [3] and oxidative damage in biologic tissues [4]. The generation of oxygen free radicals, reactive oxygen species, and oxidative damage are believed to be involved in the pathogenesis of neurodegenerative disorders [5]. Over the decades, the menagerie of reactive oxygen species has been expanded to include reactive nitrogen species derived from nitric oxide (NO) [6]. Neuronal damage, emerging from oxidative stress, has been reported to involve regulation of cell signaling molecules as well as activation of apoptotic cell death [7,8]. In the central nervous system, apoptosis plays an indispensable role in the development and maintenance of homeostasis within all organisms [9]. However, excessive apoptosis has been suggested to underlie the neuronal loss associated with the exposure to various neurotoxicants [10]. The common mechanism of apoptosis is regulated by several sets of genes, of which the best characterized is the still growing bcl-2 family [9]. Nitric oxide induces apoptosis of a variety of types of cultured cells including neurons and may contribute to several disorders of the nervous system. In recent years, researchers have begun to recognize and explore the putative link between NO and neurodegenerative diseases [11]. Although existing evidence regarding their association are not abundant, emerging data are showing Alzheimer’s disease (AD)–related changes in the NO system and it appears that NO could be related to many AD pathological mechanisms [12]. Alzheimer’s disease is characterized by alterations at the level of various neurotransmitters and related markers and receptors. Of these, the most severely affected is by far the cholinergic system. Loss of cholinergic innervation, demonstrated by reduced choline acetyltransferase and elevated acetylcholinesterase (AChE) activity, is correlated with the degree of dementia and the severity of neuropathological hallmarks of AD [12,13]. Therefore, it is suggested that the elevation of the acetylcholine (ACh) level might be helpful in improving the symptoms of cognitive deficits in AD. In the present study, we examined NO scavenging activity in a noncellular system and the neuroprotective effect of GTP against NO-induced toxicity in human neuroblastoma cells. Furthermore, the inhibitory effect of GTP on AChE was also investigated to evaluate its potential use in neurodegenerative diseases including AD.
2. Methods and materials 2.1. Nitric oxide scavenging activity S-nitroso-N-acetylpenicillamine (SNAP) was synthesized as described by Yoo et al [14]. Nitric oxide scavenging activity of GTP was determined by incubating 500 lmol/L SNAP, dissolved in 1 mL phosphate-buffered saline (PBS; 50 mmol/L, pH 7.4), with various
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concentrations of GTP. After the desired incubation time, 100 lL of Griess solution was added to 100 lL of aliquot and the absorbance at 550 nm was measured with a spectrophotometer. 2.2. Cell culture The human neuroblastoma cells, SK-N-MC cells, were obtained from the American Type Culture Collection (Rockville, Md). SK-N-MC cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Grand island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) at 378C in a 5% CO2/95% air cell incubator. 2.3. Protective effect of GTP (cell viability and morphological change) For analysis of the protective effects of GTP against NO cytotoxicity, cell viability was determined by an MTT assay kit (Sigma, St. Louis, MO) according to the manufacturer’s protocol. In brief, cells were cultured in each well of a 24-well plate at a density of 2 105 cells per well. The cells were treated with 5 mmol/L SNAP for 4 hours and GTP (60 or 300 lg/mL) was added 1 or 2 hours before treatment with SNAP. 2.4. Hoechst 33258 staining and terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling staining Apoptotic cells were determined by Hoechst staining. Cells were cultured on 4-chamber slides (Nalge Nunc International, Naporvile, Ill) at a density of 5 107 per well. After treatment of SNAP and GTP as described above, the cells were fixed in cold methanol ( 208C) for 10 minutes and washed twice with a PBS buffer before staining with 1 lg/mL Hoechst 33258 for 15 minutes at 378C in the dark. Cells were then washed with PBS, mounted with coverslips using glycergel (Dako, Carpinteria, CA), and observed under an Olympus-BH2 fluorescent microscope at 372 nm. For in situ detection of apoptotic cells, the terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling (TUNEL) assay was performed using an ApoTaq peroxidase in situ apoptosis detection kit (Intergen, Houston, TX). 2.5. Apoptotic gene expression The expression of bcl-2 and bax messenger RNAs (mRNAs) was determined by reverse transcription polymerase chain reaction. Total RNA was isolated from cells with RNAzolB (TEL-TEST, Friendswood, Tex) according to the manufacturer’s instruction. Polymerase chain reaction was performed with the primers for bax (5V-AGA TGA ACT GGA TAG CAA TAT GGA-3V and 5V-CCA CCC TGG TCT TGG ATC CAG ACA-3V) and for bcl-2 (5V-TCC GTG CCT GAC TTT AGC AAG CTG-3Vand 5V-GGA ATC CCA ACC AGA GAT CTC AA3V). Cyclophilin (5V-ACC CCA CCG TGT TCT TCG AC-3Vand 5V-CAT TTG CCA TGG ACA AGA TG-3V) was used as an internal control. The annealing temperatures were 508C for bax and bcl-2 and 568C for cyclophilin. The amplified fragment sizes of bax, bcl-2, and cyclophilin were 260, 333, and 300 base pairs, respectively. The reverse transcription polymerase chain reaction products were electrophoresed on a 1.5% agarose gel and visualized by staining with ethidium bromide.
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2.6. Acetylcholinesterase activity The AChE assay was measured by the slightly modified method of Ellman et al [15] using acetylthiocholine iodide as a substrate. For the enzyme source, SK-N-MC cell cultures were homogenized with a homogenation buffer (10 mmol/L Tris-HCl [pH 7.2] containing 1 mol/L NaCl, 50 mmol/L MgCl2, and 1% Triton X-100) and centrifuged at 10 000g for 30 minutes. The resulting supernatant was used as an enzyme source. Protein concentration was determined by a bicinchoninic acid kit (Sigma) with bovine serum albumin as a standard. The rates of hydrolysis by AChE were monitored spectrophotomatically using a 96-well microtiter plate reader. 2.7. Statistical analysis Data were expressed as mean F SEM of at least 3 separate experiments. Comparison between 2 values was analyzed by one-way analysis of variance followed by Dunnett’s post hoc analysis using SPSS (Chicago, IL). Differences were considered significant at P b .05. 3. Results and discussion
Nitric Oxide Scavenging Effect (%)
It is known that GTP is an excellent scavenger of free radicals [16]. In the present study, it revealed that GTP is also an excellent NO scavenger (Fig. 1). Nitric oxide scavenging effect was time dependent for 8 hours of SNAP treatment and non–time dependent after 8 hours of NO exposure. Nitric oxide scavenging effects after 5 hours were 71.9% F 1.3%, 63.1% F 1.7%, and 55.6% F 2.8% at 1, 3, and 5 mg/mL of GTP treatment, respectively. The effects after 8 hours were 35.7% F 0.6%, 33.8% F 0.7%, and 37.5% F 0.5% at 1, 3, and 5 mg/mL, respectively. Scavenging of the NO radical will contribute to the therapeutic effect of the GTP. The viability of SK-N-MC cells treated with 5 mmol/L SNAP was reduced to 27.8% of the control 120
5 mg/ml 3 mg/ml
100 1 mg/ml 80 60 40 20 0 2
5
8
11
Treatment (h) Fig. 1. Nitric oxide scavenging activity of GTP was determined by incubating 500 lmol/L SNAP with various concentrations of GTP.
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value (data not shown). However, a significant degree of protective effect was observed when cells were pretreated with GTP before they were exposed to SNAP. Green tea polyphenols showed a dose- and time-dependent protective effect on NO-induced neurotoxicity. The viabilities of cells pretreated with GTP at 60 lg/mL for 1 or 2 hours were 52.1% F 3.1% and 63.4% F 1.9% and those at 300 lg/mL were 60.8% F 1.8% and 68.9% F 1.9% of the control value, respectively. After treatment with SNAP for 4 hours, there were great changes in cell shape. The cells lost the ability to adhere to the plate surface, with cell rounding, cytoplasmic blebbing, and cell shape irregularity, whereas cells pretreated with GTP before NO exposure appeared to be similar to the control (data not shown). To confirm the effect of GTP against NO-induced cell death, we used Hoechst 33258 staining (Fig. 2A-C) and TUNEL reaction (Fig. 2D-F). Similar results were obtained with these 2 methods. Apoptotic cells exhibiting characteristic figures of apoptosis with condensed and fragmented nuclei were apparent after exposure to NO (Fig. 2B and E). These changes in nuclei characteristic of apoptosis decreased in the cells pretreated with GTP (Fig. 2C and F). It is well known that excessive NO is neurotoxic and kills neuronal cells in various central nervous system disorders. Oxidative stress is believed to play an important role in NOinduced neuronal apoptosis. Some studies reported that an antioxidant attenuated NO-
Fig. 2. Antiapoptotic effect of GTP. Cells were incubated in control medium (A, D), 5 mmol/L SNAP (B, E), or 60 lg/mL GTP pretreated with 5 mmol/L SNAP (C, F). Top panel, cells stained with Hoechst staining. Apoptotic cells exhibit condensed and/or fragmented nuclei (B) and these changes were decreased in GTP-pretreated cells (C). Middle panel, cells stained with TUNEL reaction. Condensed and marginated chromatins were labeled as brown dots in SNAP-exposed cells (E) and GTP protected these changes (F). Lower panel (G), Reverse transcription polymerase chain reaction analysis of mRNA levels of bcl-2 and bax. The expression of the antiapoptotic bcl-2 gene was increased and that of the proapoptotic bax gene was decreased in cells pretreated with GTP compared with those in cells exposed to SNAP. As the internal control, cyclophilin mRNA was also examined. A, Control; B, SNAP-treated group; C, GTP-pretreated group.
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% inhibition of AchE activity
induced neurotoxicity [17,18]. Recently, it has been demonstrated that epigallocatechin gallates scavenge NO reagents [19] as well as inhibit oxidative damage to DNA induced by NO [9]. The present study showed that SNAP-induced apoptosis of human neuroblastoma cells and NO-induced cytotoxicity were significantly protected by GTP pretreatment. Changes in nuclear morphology revealed that treatment with SNAP exhibits condensed and/or fragmented nuclei, which are characteristic of apoptosis (Fig. 2B and E), whereas GTPpretreated cells showed a noticeable decrease in these features (Fig. 2C and F). Because the bcl-2 family controls apoptosis triggered by a variety of signals, the mRNA levels of bcl-2 and bax were examined. Bcl-2 usually inhibits apoptosis whereas another member of this family, bax, can promote cell death. It has been suggested that the bcl-2–to-bax ratio determines survival or cell death by modulating the release of proapoptotic factors [10]. In agreement with mitochondria-dependent activation of caspases during the induction of apoptosis, in the present study, SNAP treatment decreased the mRNA level of bcl-2 and increased that of bax in human neuroblastoma cells, whereas GTP pretreatment increased the mRNA level of bcl-2 mRNA and decreased that of bax (Fig. 2G). This suggests that the increased bcl-2–to-bax ratio by GTP treatment may contribute to the inhibition of NOtriggered apoptosis. Green tea polyphenols inhibited the AChE activity in a dose-dependent manner (Fig. 3). The concentration that was required for the 50% enzyme inhibition (IC50) was 248 lg/mL. The reduction of choline acetyltransferase and AChE activity is correlated with the degree of dementia and the severity of neuropathological hallmarks of AD. Therefore, it is suggested that the elevation of the ACh level might be helpful in improving the symptoms of cognitive deficits in neurodegenerative diseases such as AD. Several groups have tried to supplement the ACh level in synaptic sites by the administration ACh precursors, cholinergic agonists, or AChE inhibitors such as tacrine and physotigmine that prevent ACh hydrolysis. Acetylcholinesterase inhibitors are the only source of compound currently approved for the treatment of AD yet. Therefore, seeking a new active constituent that has a potent inhibitory effect of AChE and antiamnesic activity from natural resource is very promising. To our knowledge, this is the first report showing the inhibitory effect of GTP on AChE activity. On the basis of these in vitro studies, it would be of considerable interest to test GTP in 150
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Fig. 3. Dose-dependent inhibitory effect of GTP on AChE activity. Each value represents mean F SEM. The inhibition efficacy was expressed as a percentage of enzyme activity inhibited compared with the control value (100%).
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