The green tea polyphenol (−)-epigallocatechin gallate attenuates β-amyloid-induced neurotoxicity in cultured hippocampal neurons

Life Sciences 70 (2001) 603–614

The green tea polyphenol (2)-epigallocatechin gallate attenuates b-amyloid-induced neurotoxicity in cultured hippocampal neurons Young-Taeg Choia, Chul-Ho Junga, Seong-Ryong Leeb, Jae-Hoon Baec, Won-Ki Baekd, Min-Ho Suhd, Jonghan Parke, Chan-Woo Parkf, Seong-Il Suhd,* a

Department of Psychiatry, Keimyung University School of Medicine, #194 DongSan Dong, Jung-Gu, Taegu, 700-712, Korea b Department of Pharmacology, Keimyung University School of Medicine, #194 DongSan Dong, Jung-Gu, Taegu, 700-712, Korea c Department of Physiology, Keimyung University School of Medicine, #194 DongSan Dong, Jung-Gu, Taegu, 700-712, Korea d Department of Microbiology, Keimyung University School of Medicine, #194 DongSan Dong, Jung-Gu, Taegu, 700-712, Korea e Department of Psychiatry, Catholic University of Taegu College of Medicine, #3506-6 Daemyung 4 Dong Nam-Gu, Taegu, 705-034, Korea f Jamisung Corporation Kyungju Oriental Hospital, #46-1 Top Dong, Kyungju, 780-170, Korea Received 28 March 2001; accepted 20 July 2001

Abstract Previous evidence has indicated that the neuronal toxicity of amyloid b (bA) protein is mediated through oxygen free radicals and can be attenuated by antioxidants and free radical scavengers. Recent studies have shown that green tea polyphenols reduced free radical-induced lipid peroxidation. The purpose of this study was to investigate whether (2)-epigallocatechin gallate (EGCG) would prevent or reduce the death of cultured hippocampal neuronal cells exposed to bA because EGCG has a potent antioxidant property as a green tea polyphenol. Following exposure of the hippocampal neuronal cells to bA for 48 hours, a marked hippocampal neuronal injuries and increases in malondialdehyde (MDA) level and caspase activity were observed. Co-treatment of cells with EGCG to bA exposure elevated the cell survival and decreased the levels of MDA and caspase activity. Proapoptotic (p53 and Bax), Bcl-XL and cyclooxygenase (COX) proteins have been implicated in bA-induced neuronal death. However, in this study the protective effects of EGCG seem to be independent of the regulation of p53, Bax, Bcl-XL and COX proteins. Taken together, the results suggest that EGCG has protective effects against bA-induced neuronal apoptosis through scavenging reactive oxygen species, which may be beneficial for the prevention of Alzheimer’s disease. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Alzheimer’s disease; (2)-Epigallocatechin gallate; Apoptosis * Corresponding author. *Corresponding author. Tel.: 182-53-250-7442; fax: 182-53-255-1398. E-mail: [email protected]. (S.-I. Suh) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )1 4 3 8 -2

604

Y.-T. Choi et al. / Life Sciences 70 (2001) 603–614

Introduction Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive impairment of cognitive function and loss of memory in association with widespread neuronal death [1–3]. Neuronal loss in AD is accompanied by the deposition of Amyloid beta protein (bA) in senile plaques and the presence of neurofibrillary tangles [4–6]. Generally, bA deposition is considered important in AD. bA is a 40–42 amino acid peptide and is derived from the proteolytic processing of the larger amyloid precursor protein [7, 8]. It has been demonstrated that bA is directly toxic to cultured neuron [9–11], supporting the hypothesis that bA is involved in the neurodegeneration associated with AD, but the mechanisms involved in the bA-mediated neurotoxicity remain unclear. bA has been proposed to cause neuronal cell death by regulation of either inducers or repressors of apoptosis [12–15], to induce oxidative stress [16–18], and to cause death by free radical-mediated pathway [11, 19, 20]. As neurotoxic effects of bA are at least in part mediated by free radicals and some antioxidants such as vitamin E [10], and melatonin [21–23] have been proved to rescue cells from bA-mediated apoptosis, therapeutic efforts aimed at scavenging oxygen free radicals or reducing of their formation may be beneficial in AD. Tea polyphenols are natural plant flavonoids present in the leaves and stem of tea plant. The green tea polyphenols comprise (2)-epigallocatechin-3-gallate (EGCG), (2)-epigallocatechin, (2)-epicatechin, (1)-gallocatechin, and catechin. Many biological functions of tea polyphenols have been reported, including anti-inflammatory [24], anticarcinogenic [25, 26], and antioxidant effects [27–29]. EGCG is the major polyphenol component of green tea and primarily responsible for the green tea effect. EGCG has been demonstrated to display a potent antioxidant property. EGCG possesses two triphenolic groups in its structure, which are reported to be important for its stronger activity [30]. It is important to note that EGCG is thought to act as an antioxidant in biological system. Oxygen free radical-induced lipid peroxidation has been suggested to play an important role in the bA-mediated neurotoxicity. Recent studies showed that frequent consumption of green tea results in high levels of EGCG in various body organs [31], and EGCG has been shown to prevent neuronal damage induced by free radical attack [27, 32, 33]. In this study, we studied whether EGCG prevents or reduces the death of cultured hippocampal neuronal cells exposed to bA. Methods Cell culture Primary culture of hippocampal neuron was prepared from 18 days old embryo of SpragueDawley rats as described previously [34]. Cultures were plated in 6-well plates precoated with poly-D-lysine (Sigma, 50 mg/ml) at a density of 2 3 106 cells per well, and maintained in Neurobasal medium supplemented with B27 components (GibcoBRL). The culture medium was changed to a fresh medium 2 days after plating, and the cells were cultured for a further 4 days. Six days after plating, half of medium was changed again, and then bA (25 – 100 mM) and/or EGCG (10 mM) was added to the culture. Numbers of viable cells per culture were determined by quantifying intact nuclei [35]. Counts were performed on triplicate cultures and expressed as mean 6 SEM.

Y.-T. Choi et al. / Life Sciences 70 (2001) 603–614

605

Drugs and materials Anti-p53, anti-Bcl-XL, and anti-Bax antibodies were purchased from Pharmingen Co. (San Diego, CA), and anti-cyclooxygenase (COX)-2 antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). DEVD-pNa, z-VAD-fmk and DEVD-CHO were obtained from Calbiochem (La Jolla, CA). bA (25–35) and EGCG were purchased from Sigma Chemical Co (St Louis, MO). bA (25–35) was dissolved in distilled water at a concentration of 500 mM stock and stored at 2208C until use. Assessment of apoptotic nuclei For propidium iodide (PI) staining, cells were harvested and washed twice with ice-cold phosphate buffered saline and fixed with 4% paraformaldehyde in 0.1% phosphate buffer (pH 7.4) for 10 min at room temperature, followed by ethanol containing 1% HCl for 10 min at 2208C. Fixed cells were placed on slides and stained 1 g/ml PI solution containing 100 mg/ml DNase-free RNase A for 30 min at 378C. Nuclear morphology of cells was examined by a fluorescence microscopy. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling) assay was performed with a kit (Boehringer Mannheim, Indianapolis, IN). Measurement of malondialdehyde (MDA) concentration The amount of MDA was measured by the thiobarbituric acid (TBA) assay which is based on MDA reacts with TBA to give a red species absorbing at 535 nm [36]. The sample was mixed with a TBA reagent consisting of 0.375% TBA and 15% trichloroacetic acid in 0.25 N HCl. The reaction mixtures are placed in a boiling water bath for 15 min and centrifuged at 3,000g for 5 minutes, after which the absorbance of the supernatant was read at 535 nm. The MDA concentration (of the sample) was calculated using an extinction coefficient of 1.56 3 105 M21 cm21. Western blot analysis Cells were lysed on ice for 30 min in NETN buffer (0.5% Nonidet p-40 (NP-40), 1 mM EDTA, 50 mM Tris [pH 7.4], 120 mM NaCl) including 1 mM dithiothreitol, 10 mM NaF, 2 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride. Lysates were cleared by centrifugation at 15, 000 g for 30 min at 48C, and the protein content of the cleared lysates was estimated by the method of Bradford (Bio-Rad, Hercules, CA). Approximately, 50 – 100 mg of protein from each sample was separated on 8 – 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon P membranes (Millipore, Waltham, MA). Immunoblotting was performed by standard procedures, and proteins were detected using alkaline phosphatase-conjugated secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ). Measurement of caspase-3 like protease activity To evaluate caspase-3 like activity, cell lysates were prepared after their respective treatment of bA (25–35) and/or EGCG. Assays were performed in 96-well microtitre plates by incubating 20 mg of cell lysates in 100 ml of reaction buffer (1% NP-40, 20mM Tris-Cl, pH

606

Y.-T. Choi et al. / Life Sciences 70 (2001) 603–614

7.5, 137 mM NaCl, 10% glycerol) containing the caspase 3 substrate (DEVD-pNA) at 5 mM. Lysates were incubated at 378C for 4 hr. Photometrical measurements were performed at 405 nm in an ELISA-reader. Reverse transcription polymerase chain reaction (RT-PCR) An alquot of 1 mg of total RNA from each sample was reverse transcribed to cDNA and used for PCR amplification with either rat COX-1, COX-2 or rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific primers by the RNA PCR Core Kit (Perkin-Elmer, Norwalk, CT) according to the manufacture’s instructions. The primer pair for COX-1 amplification was as follows: 59-CCTGCTGACACACGGATACTGG-39 and 59-AGCCGCAGGTGGTACTGTCGTT-39. The predicted fragment size was 465 base pairs. The primer for COX-2 amplification was 59-CTGTACTACGCCGAGATTCCTGA-39 and 59-GTCCTCGCTTCTGATCTGTCTTG-39. The predicted fragment size was 452 base pairs. The primer pair for GAPDH had the following sequence: 59-GGTGAAGGTCGGTGTGAACG-39 and 59-GGTAGGAACACGGAAGGCCA-39. The predicted fragment size was 703 base pairs. Results Previous studies reporting neurotoxicity of bA have used a wide range of concentrations of different forms of bA in a variety of cell types [9, 12, 37]. The active fragment bA, bA (25–35), caused significant toxicity in hippocampal neuronal cells in a dose-dependent manner (Fig 1A). In agreement with previous reports [38, 39], exposure of hippocampal neuronal cells to bA (25–35) for 48 hr resulted in a characteristic cleavage of DNA at intranucleoso-

Fig. 1. Hippocampal neurons were grown in culture for 6 days and then exposed to increasing concentration of bA (25–35). (A) bA(25–35) induces dose-dependent cell death. Survival was assessed after 2 days by counting nuclei in cell lysates (n53). Survival is reported relative to untreated cultures and is given as mean 6 SE. (B) Hippocampal neuron culture treated for 2 days with 50 mM bA(25–35) and stained with propidium iodide (upper panel), and TUNEL staining of apoptotic hippocampal neurons (lower panel).

Y.-T. Choi et al. / Life Sciences 70 (2001) 603–614

607

Fig. 2. EGCG protects against bA(25–35)-induced death in hippocampal neuronal cells. (A) Hippocampal neuronal cells were treated with the indicated concentrations of bA (25–35) for 2 days in the present or absent of EGCG (10 mM). Survival was assessed after 2 days as described in Materials and Methods, is reported relative to untreated cultures, and is given as mean 6 SE. (B) Lipid peroxidation induced by bA (25–35) is prevented by EGCG. Cells were exposed to bA (25–35) for 2 days with or without EGCG (10 mM). The extent of lipid peroxidation was expressed as MDA concentration (nM/mg protein). Values are the means of three determinations, SE in all measurement was ,20% of the mean. Catalase (1000 U) inhibited lipid peroxidation induced by bA (25–35).

mal sites, a biochemical hallmark of apoptosis, as shown in Fig 1B. Because it has been proposed that bA toxicity is caused by the induction of oxidative stress, we evaluated the neuroprotective effects of EGCG which is known as a potent antioxidant against bA-induced neuronal injury. Primary hippocampal neuronal cells were co-treated with bA (25–35) and EGCG. As shown as Fig 2A, EGCG effectively promoted the survival of bA (25–35)-treated neuronal cells. With treatment of bA (25–35) at the concentrations of 25 mM, 50 mM, or 100 mM, the survival rate of hippocampal neuronal cells was 60%, 50%, or 35%, respectively. However, the survival rate was increased with EGCG about 90%, 75%, and 50%, respectively. Lipid peroxidation was induced in cultured neuron after bA (25–35) treatment and was attenuated by catalase or EGCG. These results suggest that the cytoprotective effects of EGCG are related to its antioxidant property. To address the significance of caspase activation in bA (25–35)-induced apoptosis, we used a group II caspase (caspase-2, -3, -7, or –10) inhibitor DEVD-CHO and a potent general caspase inhibitor z-VAD-fmk. Blockage of caspases activities by pretreatment of primary hippocampal neuron with 50 mM of DEVDCHO or 50 mM z-VAD-fmk effectively promoted the survival of bA (25–35)-treated neuronal cells, respectively (Fig 3A). bA (25–35) stimulated caspase protease activity, but

608

Y.-T. Choi et al. / Life Sciences 70 (2001) 603–614

Fig. 3. EGCG and caspase inhibitors, z-VAD-fmk and DEVD-CHO, prevent neuronal apoptosis induced by bA (25–35). (A) Hippocampal neuronal cells were treated with the indicated concentrations of bA (25–35) in the present or absent of EGCG and caspase inhibitors. Survival was assessed after 2 days as described in Materials and Methods, is reported relative to untreated cultures, and is given as mean 6 SE. (B) Inhibitory effect of EGCG and caspase inhibitors on bA (25–35) induced caspase-3 activation. Hippocampal neuronal cells were treated with the indicated concentrations of bA (25–35) for 2 days in the present or absent of EGCG (10 mM) and caspase inhibitors. Data are mean 6 SE of three independent experiments.

DEVD-CHO and z-VAD-fmk-pretreated cells reduced bA (25–35)-induced caspase activity (Fig 3B). Interestingly, in co-treatment of bA (25–35) and ECCG, the caspase activity was also reduced compared to that of bA (25–35) alone. These results suggest that EGCG can block the bA (25–35)-induced apoptosis in neuronal cells. We also examined whether bA (25–35) induces neuronal cell death by modulating the expression of p53, Bax, and Bcl-XL, which are ultimately cell responses to apoptotic stimuli. However, the protein expressions of p53, Bax, and Bcl-XL were not changed with treatment of bA (25–35) or co-treatment of bA (25–35) and EGCG (Fig 4A). In AD brains, increased expression of COX-2 protein was reported. To evaluate the effect of bA (25–35) on the treatment in expressions of COX-1 and 2, RT-PCR and Western blot were done. bA (25–35) induced COX-2 mRNA and protein expressions (Fig 4B). However, bA (25–35) failed to change the COX-1 mRNA expression (Fig 4B). EGCG treatment induced the COX-2 mRNA and protein expressions slightly, however catalase induced both of COX-2 mRNA and protein expressions markedly. Concomitant exposure of cells to bA (25–35) and catalase induced greater COX-2 protein expression than catalase alone. However, bA (25–35) and EGCG did not change the COX-1 or -2 mRNA and protein expressions.

Y.-T. Choi et al. / Life Sciences 70 (2001) 603–614

609

Fig. 4. Western blot analysis of apoptosis related proteins and COX-2 protein. Hippocampal neuronal cells were treated with the indicated concentrations of bA (25–35) in the present or absent of EGCG. Expression levels of p53, Bcl-XL, Bax, and COX-2 proteins were assessed by independent immunoblotings using equal amounts of proteins from the same lysates (A and B). Expression of COX-1 and COX-2 mRNAs in bA (25–35) treated cells with or without EGCG. An aliquot of the amplified DNA was separated on an agarose gel and stained with ethidium bromide for qualitative comparison.

Discussion It is now well known that bA is neurotoxic and kills neuronal cells via an apoptotic process. The neurotoxicity of bA has been reported to be mediated with oxygen free radicals and attenuated by antioxidants and free radical scavengers [10, 17, 18, 23]. The present study shows the neuroprotective effect of a potent inhibitor of lipid peroxidation, EGCG, against bA (25–35)-induced neuronal apoptosis. EGCG has been demonstrated to display a potent antioxidant property and recently its antioxidant effects were extensively studied. There are several in vitro and in vivo studies of neuroprotective effect of EGCG against neuronal injury. Kondo et al [40] and Guo et al [32] reported the scavenging and antioxidant effect. EGCG shows protective effects against hippocampal neuronal damage after transient global ischemia [27]. In addition, green tea extract has been reported to protect brain, liver, and kidney from lipid peroxidation injury [33, 41]. However, there is no previous report on the pro-

610

Y.-T. Choi et al. / Life Sciences 70 (2001) 603–614

tective effects of EGCG against bA (25–35)-induced neuronal injury. This study is the first to report the protective effects of EGCG against neuronal apoptosis induced by bA (25–35). In the present study, EGCG significantly reduced hippocampal neuronal cell death induced by bA (25–35) in comparison with its vehicle (Fig 2A). It has been well known that reactive oxygen species-induced lipid peroxidation has been strongly suggested to play a role in bAinduced neurotoxicity [11, 18, 22]. And the exposure of hippocampal neuronal cells to bA (25–35) resulted in increased lipid peroxidation (Fig 2B), and this effect was significantly prevented by addition of EGCG to the culture medium. For comparison, the effect of the hydrogen peroxide degrading enzyme catalase was checked. bA (25–35)-induced hippocampal neuronal cell death and MDA levels were attenuated by catalase treatment. These results support that the neuroprotective effects of EGCG are related to its antioxidant property. Green tea is one of the most common beverages consumed worldwide, especially in China, Japan and Korea. Polyphenols are the major effective compounds of tea for which numerous biological activities have been reported [24–26, 33]. EGCG is the major polyphenol component of green tea and primarily responsible for the green tea effects. Apoptosis, programmed cell death, is known as a discrete form of cell death different from necrosis and is regarded as an ideal way of cell elimination. Imbalance in apoptosis can lead to many pathological states including cancers and neurodegenerative disorders. As to the intracellular death effectors, the most important family in process of apoptosis is caspases [42]. Caspases activation plays a critical role in the apoptosis of neurons [43, 44]. According with other studies [11, 16, 17, 20], catalase protected neuronal cells against bA-induced toxicity in this study. It supports that hydrogen peroxide mediates bA-induced toxicity and is known to induce caspase-mediated apoptosis [45–47]. In this study, we found that caspase activity was activated in hippocampal neurons by bA (25–35) treatment, and was attenuated by caspase inhibitors (Fig 3A). Therefore, caspases might be one of main effector proteins in bA-induced neuronal cell death. Interestingly, EGCG inhibited the increase of caspase activity-induced by bA (25–35). These results suggest that EGCG can attenuate bA (25–35)induced apoptosis in neuronal cells through scavenging reactive oxygen species. p53 activation was shown to play a significant role in several models of neuronal apoptosis [48–50]. In particular, activation of p53 in response to various stressful stimuli leads to increased expression of several proapoptotic genes, such as Bax and IGFBP. However, Blasko et al [51] reported that p53 is not essential for bA (25–35)-induced apoptosis. Bax protein is well known to homodimerize to form heterodimers with Bcl-2 and Bcl-XL anti-apoptotic proteins [52, 53]. The ratio of Bax to Bcl-2 and Bcl-XL was suggested to be a critical determinant of cell sensitivity to stimuli that trigger apoptosis. As shown in Fig 4A, in contrast to other studies, the expression levels of p53, Bax, and Bcl-2 proteins were insignificantly affected by bA (25–35). The data reported here suggest that apoptosis induced by bA (25–35) indeed account for the neuronal loss in AD, indicating that these proteins do not seem to play a key role in the mechanisms of cell death in this disorder. Recent studies suggested that inflammatory events are associated with plaque formation in brains of AD patients [54–56]. Treatment with non-steroidal anti-inflammatory drug of AD patients appears to slow the progression of disease [57–59]. Pasinetti [60], Ho et al [61], and Yasojima et al [62] reported that COX-2 expression was up-regulated in the brain of patients with AD, whereas COX-1 expression was unchanged in AD hippocampal neurons [63]. In the present study, bA (25–35)

Y.-T. Choi et al. / Life Sciences 70 (2001) 603–614

611

increased the COX-2 mRNA and protein expression but not COX-1. However, EGCG did not change the COX-1 and -2 mRNAs and proteins expressions in bA (25–35) treated neuronal cells. These results suggest that the protective effects of EGCG are independent of the regulation of COX mRNAs and proteins. Taken together, these data suggest that a complicated interaction between bA, reactive oxygen species, and apoptosis might exist and contribute to the neuronal cell death in AD, and that EGCG prevents bA-induced hippocampal neuronal cell death through its antioxidant property. After drinking a single cup of tea, the calculated maximum blood concentration of EGCG may reach 60 mM in adults weighing 60 Kg [64, 65]. EGCG has been demonstrated to pass the blood-brain barrier and reach brain parenchyma in animal study [31]. Therefore, daily intakes of green tea may be reduced the risk of AD by helping rid reactive oxygen species induced by bA. Acknowledgments This work supported in part by Ministry of Science & Technology (MOST) and the Korean Science and Engineering Foundation (KOSEF) through the Center for Traditional Microorganism Resources (TMR) at Keimyung University. References 1. Barinaga M. Missing Alzheimer’s gene found. Science 1995;269(5226):917–8. 2. Berg L, McKeel DW, Miller JP, Baty J, Morris JC. Neuropathological indexes of Alzheimer’s disease in demented and nondemented persons aged 80 years and older. Arch Neurol 1993;50(4):349–58. 3. Yamazaki T, Koo EH, Selkoe DJ. Trafficking of cell-surface amyloid beta-protein precursor. II. Endocytosis, recycling and lysosomal targeting detected by immunolocalization. J Cell Sci 1996;109(Pt 5):999–1008. 4. Braak H, de Vos RA, Jansen EN, Bratzke H, Braak E. Neuropathological hallmarks of Alzheimer’s and Parkinson’s diseases. Prog Brain Res 1998;117:267–85. 5. Masters CL, Multhaup G, Simms G, Pottgiesser J, Martins RN, Beyreuther K. Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer’s disease contain the same protein as the amyloid of plaque cores and blood vessels. EMBO J 1985;4(11):2757–63. 6. Selkoe DJ. Deciphering Alzheimer’s disease: the amyloid precursor protein yields new clues. Science 1990; 248(4959):1058–60. 7. Glenner GG, Wong CW. Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 1984;120(3):885–90. 8. Robakis NK, Ramakrishna N, Wolfe G, Wisniewski HM. Molecular cloning and characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides. Proc Natl Acad Sci U S A 1987; 84(12):4190–4. 9. Pike CJ, Walencewicz AJ, Glabe CG, Cotman CW. In vitro aging of beta-amyloid protein causes peptide aggregation and neurotoxicity. Brain Res 1991;563(1–2):311–4. 10. Behl C, Davis J, Cole GM, Schubert D. Vitamin E protects nerve cells from amyloid beta protein toxicity. Biochem Biophys Res Commun 1992;186(2):944–50. 11. Behl C, Davis JB, Lesley R, Schubert D. Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 1994;77(6):817–27. 12. Estus S, Tucker HM, van Rooyen C, Wright S, Brigham EF, Wogulis M, Rydel RE. Aggregated amyloid-beta protein induces cortical neuronal apoptosis and concomitant “apoptotic” pattern of gene induction. J Neurosci 1997;17(20):7736–45.

612

Y.-T. Choi et al. / Life Sciences 70 (2001) 603–614

13. Forloni G, Chiesa R, Smiroldo S, Verga L, Salmona M, Tagliavini F, Angeretti N. Apoptosis mediated neurotoxicity induced by chronic application of beta amyloid fragment 25–35. Neuroreport 1993;4(5):523–6. 14. Loo DT, Copani A, Pike CJ, Whittemore ER, Walencewicz AJ, Cotman CW. Apoptosis is induced by betaamyloid in cultured central nervous system neurons. Proc Natl Acad Sci U S A 1993;90(17):7951–5. 15. Paradis E, Douillard H, Koutroumanis M, Goodyer C, LeBlanc A. Amyloid beta peptide of Alzheimer’s disease downregulates Bcl-2 and upregulates bax expression in human neurons. J Neurosci 1996;16(23):7533–9. 16. Huang X, Atwood CS, Hartshorn MA, Multhaup G, Goldstein LE, Scarpa RC, Cuajungco MP, Gray DN, Lim J, Moir RD, Tanzi RE, Bush AI. The A beta peptide of Alzheimer’s disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry 1999;38(24):7609–16. 17. Miranda S, Opazo C, Larrondo LF, Munoz FJ, Ruiz F, Leighton F, Inestrosa NC. The role of oxidative stress in the toxicity induced by amyloid beta-peptide in Alzheimer’s disease. Prog Neurobiol 2000;62(6):633–48. 18. Pike CJ, Ramezan-Arab N, Cotman CW. Beta-amyloid neurotoxicity in vitro: evidence of oxidative stress but not protection by antioxidants. J Neurochem 1997;69(4):1601–11. 19. Guo Q, Sebastian L, Sopher BL, Miller MW, Ware CB, Martin GM, Mattson MP. Increased vulnerability of hippocampal neurons from presenilin-1 mutant knock-in mice to amyloid beta-peptide toxicity: central roles of superoxide production and caspase activation. J Neurochem 1999;72(3):1019–29. 20. Zhang L, Zhao B, Yew DT, Kusiak JW, Roth GS. Processing of Alzheimer’s amyloid precursor protein during H2O2-induced apoptosis in human neuronal cells. Biochem Biophys Res Commun 1997;235(3):845–8. 21. Bachurin S, Oxenkrug G, Lermontova N, Afanasiev A, Beznosko B, Vankin G, Shevtzova E, Mukhina T, Serkova T. N-acetylserotonin, melatonin and their derivatives improve cognition and protect against betaamyloid-induced neurotoxicity. Ann N Y Acad Sci 1999;890:155–66. 22. Chyan YJ, Poeggeler B, Omar RA, Chain DG, Frangione B, Ghiso J, Pappolla MA. Potent neuroprotective properties against the Alzheimer beta-amyloid by an endogenous melatonin-related indole structure, indole3-propionic acid. J Biol Chem 1999;274(31):21937–42. 23. Daniels WM, van Rensburg SJ, van Zyl JM, Taljaard JJ. Melatonin prevents beta-amyloid-induced lipid peroxidation. J Pineal Res 1998;24(2):78–82. 24. Stoner GD, Mukhtar H. Polyphenols as cancer chemopreventive agents. J Cell Biochem Suppl 1995;22:169–80. 25. Gensler HL, Timmermann BN, Valcic S, Wachter GA, Dorr R, Dvorakova K, Alberts DS. Prevention of photocarcinogenesis by topical administration of pure epigallocatechin gallate isolated from green tea. Nutr Cancer 1996;26(3):325–35. 26. Kato T, Harashima T, Moriya N, Kikugawa K, Hiramoto K. Formation of the mutagenic/carcinogenic imidazoquinoxaline-type heterocyclic amines through the unstable free radical Maillard intermediates and its inhibition by phenolic antioxidants. Carcinogenesis 1996;17(11):2469–76. 27. Lee S, Suh S, Kim S. Protective effects of the green tea polyphenol (2)-epigallocatechin gallate against hippocampal neuronal damage after transient global ischemia in gerbils. Neurosci Lett 2000;287(3):191–4. 28. Lin JK, Chen PC, Ho CT, Lin-Shiau SY. Inhibition of xanthine oxidase and suppression of intracellular reactive oxygen species in HL-60 cells by theaflavin-3,39-digallate, (2)-epigallocatechin-3-gallate, and propyl gallate. J Agric Food Chem 2000;48(7):2736–43. 29. Shi X, Ye J, Leonard SS, Ding M, Vallyathan V, Castranova V, Rojanasakul Y, Dong Z. Antioxidant properties of (2)-epicatechin-3-gallate and its inhibition of Cr(VI)-induced DNA damage and Cr(IV)- or TPA-stimulated NF-kappaB activation. Mol Cell Biochem 2000;206(1–2):125–32. 30. Matsuo N, Yamada K, Shoji K, Mori M, Sugano M. Effect of tea polyphenols on histamine release from rat basophilic leukemia (RBL-2H3) cells: the structure-inhibitory activity relationship. Allergy 1997;52(1):58–64. 31. Suganuma M, Okabe S, Oniyama M, Tada Y, Ito H, Fujiki H. Wide distribution of [3H](2)-epigallocatechin gallate, a cancer preventive tea polyphenol, in mouse tissue. Carcinogenesis 1998;19(10):1771–6. 32. Guo Q, Zhao B, Li M, Shen S, Xin W. Studies on protective mechanisms of four components of green tea polyphenols against lipid peroxidation in synaptosomes. Biochim Biophys Acta 1996;1304(3):210–22. 33. Lin AM, Chyi BY, Wu LY, Hwang LS, Ho LT. The antioxidative property of green tea against iron-induced oxidative stress in rat brain. Chin J Physiol 1998;41(4):189–94. 34. Farinelli SE, Greene LA, Friedman WJ. Neuroprotective actions of dipyridamole on cultured CNS neurons. J Neurosci 1998;18(14):5112–23. 35. Troy CM, Stefanis L, Greene LA, Shelanski ML. Nedd2 is required for apoptosis after trophic factor with-

Y.-T. Choi et al. / Life Sciences 70 (2001) 603–614

36. 37. 38. 39. 40.

41.

42. 43. 44.

45.

46. 47. 48.

49. 50.

51. 52.

53. 54. 55.

56.

613

drawal, but not superoxide dismutase (SOD1) downregulation, in sympathetic neurons and PC12 cells. J Neurosci 1997;17(6):1911–8. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52(302–10. Ii M, Sunamoto M, Ohnishi K, Ichimori Y. beta-Amyloid protein-dependent nitric oxide production from microglial cells and neurotoxicity. Brain Res 1996;720(1–2):93–100. Mattson MP, Partin J, Begley JG. Amyloid beta-peptide induces apoptosis-related events in synapses and dendrites. Brain Res 1998;807(1–2):167–76. Watt JA, Pike CJ, Walencewicz-Wasserman AJ, Cotman CW. Ultrastructural analysis of beta-amyloidinduced apoptosis in cultured hippocampal neurons. Brain Res 1994;661(1–2):147–56. Kondo K, Kurihara M, Miyata N, Suzuki T, Toyoda M. Scavenging mechanisms of (2)-epigallocatechin gallate and (2)- epicatechin gallate on peroxyl radicals and formation of superoxide during the inhibitory action. Free Radic Biol Med 1999;27(7–8):855–63. Sano M, Takahashi Y, Yoshino K, Shimoi K, Nakamura Y, Tomita I, Oguni I, Konomoto H. Effect of tea (Camellia sinensis L.) on lipid peroxidation in rat liver and kidney: a comparison of green and black tea feeding. Biol Pharm Bull 1995;18(7):1006–8. Pettmann B, Henderson CE. Neuronal cell death. Neuron 1998;20(4):633–47. Marin N, Romero B, Bosch-Morell F, Llansola M, Felipo V, Roma J, Romero FJ. Beta-amyloid-induced activation of caspase-3 in primary cultures of rat neurons. Mech Ageing Dev 2000;119(1–2):63–7. Masumura M, Hata R, Nishimura I, Uetsuki T, Sawada T, Yoshikawa K. Caspase-3 activation and inflammatory responses in rat hippocampus inoculated with a recombinant adenovirus expressing the alzheimer amyloid precursor protein. Brain Res Mol Brain Res 2000;80(2):219–27. Dumont A, Hehner SP, Hofmann TG, Ueffing M, Droge W, Schmitz ML. Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB. Oncogene 1999;18(3):747–57. Matsura T, Kai M, Fujii Y, Ito H, Yamada K. Hydrogen peroxide-induced apoptosis in HL-60 cells requires caspase-3 activation. Free Radic Res 1999;30(1):73–83. Simizu S, Takada M, Umezawa K, Imoto M. Requirement of caspase-3(-like) protease-mediated hydrogen peroxide production for apoptosis induced by various anticancer drugs. J Biol Chem 1998;273(41):26900–7. Daily D, Barzilai A, Offen D, Kamsler A, Melamed E, Ziv I. The involvement of p53 in dopamine-induced apoptosis of cerebellar granule neurons and leukemic cells overexpressing p53. Cell Mol Neurobiol 1999; 19(2):261–76. de la Monte SM, Sohn YK, Wands JR. Correlates of p53- and Fas (CD95)-mediated apoptosis in Alzheimer’s disease. J Neurol Sci 1997;152(1):73–83. Yamaguchi A, Tamatani M, Matsuzaki H, Namikawa K, Kiyama H, Vitek MP, Mitsuda N, Tohyama M. Akt activation protects hippocampal neurons from apoptosis by inhibiting transcriptional activity of p53. J Biol Chem 2001;276(7):5256–64. Blasko I, Wagner M, Whitaker N, Grubeck-Loebenstein B, Jansen-Durr P. The amyloid beta peptide abeta (25–35) induces apoptosis independent of p53. FEBS Lett 2000;470(2):221–5. Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X, Nunez G,Thompson CB. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 1993; 74(4):597–608. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993;74(4):609–19. Aisen PS. Anti-inflammatory therapy for Alzheimer’s disease. Neurobiol Aging 2000;21(3):447–8; discussion 51–3. Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, Cooper NR, Eikelenboom P, Emmerling M, Fiebich BL, Finch CE, Frautschy S, Griffin WS, Hampel H, Hull M, Landreth G, Lue L, Mrak R, Mackenzie IR, McGeer PL, O’Banion MK, Pachter J, Pasinetti G, Plata-Salaman C, Rogers J, Rydel R, Shen Y, Streit W, Strohmeyer R, Tooyoma I, Van Muiswinkel FL, Veerhuis R, Walker D, Webster S, Wegrzyniak B, Wenk G,Wyss-Coray T. Inflammation and Alzheimer’s disease. Neurobiol Aging 2000;21(3):383–421. Sugaya K, Uz T, Kumar V, Manev H. New anti-inflammatory treatment strategy in Alzheimer’s disease. Jpn J Pharmacol 2000;82(2):85–94.

614

Y.-T. Choi et al. / Life Sciences 70 (2001) 603–614

57. Dzenko KA, Weltzien RB, Pachter JS. Suppression of A beta-induced monocyte neurotoxicity by antiinflammatory compounds. J Neuroimmunol 1997;80(1–2):6–12. 58. Netland EE, Newton JL, Majocha RE, Tate BA. Indomethacin reverses the microglial response to amyloid beta-protein. Neurobiol Aging 1998;19(3):201–4. 59. Nourhashemi F, Ousset PJ, Reyes G, Micas M, Adoue D, Vellas BJ, Albarede JL. [NSAID in Alzheimer disease]. Presse Med 1998;27(1):25–8. 60. Pasinetti GM. Cyclooxygenase and inflammation in Alzheimer’s disease: experimental approaches and clinical interventions. J Neurosci Res 1998;54(1):1–6. 61. Ho L, Pieroni C, Winger D, Purohit DP, Aisen PS, Pasinetti GM. Regional distribution of cyclooxygenase-2 in the hippocampal formation in Alzheimer’s disease. J Neurosci Res 1999;57(3):295–303. 62. Yasojima K, Schwab C, McGeer EG, McGeer PL. Distribution of cyclooxygenase-1 and cyclooxygenase-2 mRNAs and proteins in human brain and peripheral organs. Brain Res 1999;830(2):226–36. 63. Yermakova AV, Rollins J, Callahan LM, Rogers J, O’Banion MK. Cyclooxygenase-1 in human Alzheimer and control brain: quantitative analysis of expression by microglia and CA3 hippocampal neurons. J Neuropathol Exp Neurol 1999;58(11):1135–46. 64. Lin JK, Liang YC, Lin-Shiau SY. Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade. Biochem Pharmacol 1999;58(6):911–5. 65. Yang CS, Wang ZY. Tea and cancer. J Natl Cancer Inst 1993;85(13):1038–49.