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insulin-like signaling pathway
Phytomedicine 17 (2010) 902–909
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Epigallocatechin gallate inhibits beta amyloid oligomerization in Caenorhabditis elegans and affects the daf-2/insulin-like signaling pathway S. Abbas, M. Wink ∗ Institute of Pharmacy and Molecular Biotechnology, Department of Biology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
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Keywords: Caenorhabditis elegans EGCG sHSPs daf-16 Beta amyloid
a b s t r a c t Epidemiological studies have repeatedly demonstrated that green tea protects against oxidative stress involved in many diseases. Health benefits of green tea are attributed to its principal active constituent, epigallocatechin gallate (EGCG). EGCG was shown to increase the stress resistance and lifespan of Caenorhabditis elegans. The mechanism of this action has been investigated in this study. The expression of hsp-16.1 and hsp-16.2 in EGCG-treated worms (N2), as quantified by real-time PCR, was significantly lower under oxidative stress induced by juglone than in controls without EGCG. In the strain TJ356 (DAF-16::GFP) EGCG treatment induced translocation of DAF-16 from the cytoplasm into the nucleus, suggesting that EGCG may affect the daf-2/insulin-like signaling pathway. EGCG decreased the formation of lipofuscin, an aging related pigment. Also, EGCG reduced beta amyloid (A) deposits and inhibited A oligomerization in transgenic C. elegans (CL2006). Thus, the use of green tea and EGCG is apparently rational alternatives for protecting against ROS-mediated and age-related diseases. © 2010 Elsevier GmbH. All rights reserved.
Introduction Green tea (Camellia sinensis) is consumed as a beverage worldwide and famous for its health benefits. The leaves contain several bioactive compounds of which flavonoids are particularly abundant and among these especially the catechins (Weisburger 1997). These compounds possess several phenolic hydroxyl groups which can dissociate into O− ions under physiological conditions; if many such molecules bind to a single protein by forming ionic and hydrogen bonds, it is likely that the conformation and flexibility of the protein will be altered. This explains why polyphenols show several interactions with cellular proteins, including receptors, ion channels, enzymes, regulatory and structural proteins (Wink 2008). Catechins are also known for their antioxidant activity, and monomeric catechin, another constituent of green tea, recently has also been shown to increase the mean lifespan and stress resistance in Caenorhabditis elegans wild type (Saul et al. 2009). It is likely that these polyphenols protect against ROS-mediated and age-related diseases (Hollman et al. 1999). Regular consumption of green tea appears to prevent cardiovascular diseases and even cancer and seems to contribute to a higher life expectancy among Asians (e.g., Japan). EGCG, being the main
∗ Corresponding author. Tel.: +49 6221544880; fax: +49 6221544884. E-mail address: [email protected] (M. Wink). 0944-7113/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2010.03.008
constituent of green tea, has been thoroughly analyzed and shown to exert several beneficial health effects. Several studies have suggested a possible role of EGCG (Fig. 1A) in preventing certain types of cancer including prostate cancer. EGCG inhibits the growth of prostate cancer cells leading to cell cycle arrest and apoptosis without observable toxic effects on normal epithelial cells (Moyers and Kumar 2004; Cooper et al. 2005; Johnson et al. 2010). There is indication that EGCG exerts other medicinal benefits in preventing neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases (Avramovich-Tirosh et al. 2007); EGCG shows antimutagenic effects (Muto et al. 1999) and several studies have provided evidence that green tea consumption reduces the risk of cardiovascular diseases (Kuriyama et al. 2006). These effects of EGCG have been attributed, in part, to its antioxidant action but influences on protein conformation are likely. Clinical pharmacokinetic studies revealed a low bioavailability of EGCG due to a degradation at pH > 6.5 and thus under physiological conditions in the gastrointestinal tract (pH 5–8). Oral administration of decaffeinated green tea polyphenols, marketed as Polyphenon E, on an empty stomach resulted in increased plasma levels of catechins. This indicates that food can delay the gastric emptying which would lead to a raise of stomach pH and thus polyphenol degradation (Chow et al. 2005). Sprague–Dawley rats treated with green tea polyphenols showed highest concentrations of EGCG in the large intestine and significant EGCG concentrations were found in other tissues including kidneys, prostate, and lungs (Kim et al. 2000). The results obtained from [3 H]EGCG-treated mice via gastric tube
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Fig. 1. (A) The chemical structure of EGCG. Expression levels of small heat shock protein genes (hsp-16.1 and hsp-16.2), relative to actin, were determined by quantitative real-time PCR. Graphs represent mean of three independent experiments with SEM; ***P < 0.0001. (B) 20 M juglone-treated worms (applied on third day after hatching and for 24 h) showed high expression levels of hsp-16.1 and -16.2. (C) There is no significant effect in the 220 M EGCG-treated group for 72 h starting 1 day after hatching. (D) The expression levels in 220 M EGCG-treated group (48 h) followed with 20 M juglone (24 h) were reduced, ***P < 0.0001. Cont. = control and set to 1.
administration have demonstrated that frequent administration of EGCG resulted in high plasma levels with a wide distribution in the body (digestive tract, liver, lungs, pancreas, mammary glands, skin, brain, kidneys, ovary, and testes); in these experiments mice were starved for 15 h before gastric intubation (Suganuma et al. 1998). The hepatic first-pass has no significant effect on green tea catechin elimination; gastrointestinal factors seem to largely contribute to the low bioavailability of green tea catechins after oral uptake (Cai et al. 2002). The versatile model nematode Caenorhabditis elegans is useful for understanding aging processes and age-related diseases, due to its rapid life cycle, short lifespan, ease of cultivation, and well-established genetic pathways; in addition, C. elegans contains homologues of nearly two-thirds of the human genome (Sonnhammer and Durbin 1997). The present study has aimed to investigate the mechanisms of action of EGCG, which delays the appearance of several markers of aging and oxidative stress, such as lipofuscin pigments, heat shock proteins, and beta amyloid formation in C. elegans.
Materials and methods Chemicals and reagents (−)-Epigallocatechin gallate (EGCG) (Fig. 1A) 95%, juglone (5hydroxy-1,4-naphthalenedione), TRI reagent, and thioflavin S were obtained from Sigma–Aldrich GmbH (Munich, Germany); sodium azide from Appli-Chem GmbH (Darmstadt, Germany). Caenorhabditis elegans strains and culture conditions The employed C. elegans strains included: N2; BA17, fem1(hc17) (fertile at 20 ◦ C, infertile at 25 ◦ C); TJ356 (DAF-16::GFP) and CL2006, dvIs2 [pCL12 (unc-54/human A beta peptide 1-42) + pRF4]. All strains and Escherichia coli (OP50) were obtained from the Caenorhabditis Genetic Center (CGC) of the NIH National Center for Research Resources. The strains were maintained at 20 ◦ C on nematode growth medium (NGM) (Brenner 1974). The experimental worms were grown in liquid S-medium. E. coli (OP50) as a food
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source was added to media at a concentration of 1 × 109 cells/ml. All worms used in this study were age-synchronized, and the experimental animals were raised from eggs obtained after sodium hypochlorite treatment of hermaphrodites (Sulston and Hodgkin 1988).
Quantitative real-time PCR To quantify the expression levels of heat shock protein genes (hsp-16.1 and hsp-16.2), N2 worms were grown for 72 h in the presence or absence of 220 M EGCG on the day after hatching. In case of oxidatively stressed worms the animals were subjected for 48 h to 220 M EGCG on the day after hatching, followed by 20 M juglone for 24 h. To quantify the expression level of the beta amyloid gene, the CL2006 strain was treated with 220 M EGCG on the second day after hatching for 4 days. Then the animals were washed from plates with M9 buffer into Eppendorf tubes, and allowed to settle on ice. The worm pellet was resuspended in 1 ml TRI reagent and total RNA was prepared according to the manufacturer’s instructions. Total RNA was treated with DNase I (Qiagene), followed by chloroform extraction and isopropanol precipitation, and stored frozen at −80 ◦ C. cDNA was prepared with the ImProm-IITM Reverse Transcription System kit (Promega) using 1 g of total RNA per reaction and random primer. Real-time PCR was performed in LightCycler (Roche) using AbsoluteTM QPCR SYPR Green Capillary Mix kit (Abgene), using the following gene-specific primers: Actin
(Forward) 5 -GTGTGACGACGAGGTTGCCGCTCTTGTTGTAGAC-3 (Reverse) 5 -GGTAAGGATCTTCATGAGGTAATCAGTAAGATCAC-3
hsp-16.1
(Forward) 5 -GTCACTTTACCACTATTTCCGTCCAGCTCAACGTTC-3 (Reverse) 5 -CAACGGGCGCTTGCTGAATTGGAATAGATCTTCC-3
hsp-16.2
(Forward) 5 -CTGCAGAATCTCTCCATCTGAGTC-3 (Reverse) 5 -AGATTCGAAGCAACTGCACC-3
Beta amyloid
(Forward) 5 -CAGAATTCCGACATGACTCAGGATATGAAG-3 (Reverse) 5 -CCCACCATGAGTCCAATGATTGC-3
DAF-16 localization via fluorescence microscopy The TJ356 strain was used to examine the intracellular distribution of DAF-16 in the living nematode. In this strain, the gene coding for green fluorescent protein (GFP) was fused to the daf-16 gene (Henderson and Johnson 2001). The effect of 220 M EGCG was studied for 1 h and compared with the effect of heat shock at 37 ◦ C for 15 min and oxidative stress by 20 M juglone for 1 h which were used as positive controls using L2 worms (second larval stage). DAF-16 localization was examined in approximately 20 animals per treatment that were mounted in a drop of 10 mM sodium azide. Fluorescence images were taken at constant exposure times (Nikon-eclipse 90i, Nikon digital sight DS-Qi1Mc; 40× objective; Nikon Imaging Center, University of Heidelberg).
Lipofuscin levels Adult hermaphrodites of the BA17 strain were treated with 220 M EGCG for 16 days on the day after hatching and maintained in S-medium at 25 ◦ C; the worms were transferred to the new medium every second day, then anaesthetized and mounted onto glass slides in M9 medium containing 10 mM sodium azide to visualize intestinal autofluorescence (Nikon-eclipse 90i, Nikon digital sight DS-Qi1Mc; imaging at constant exposure times; Nikon Imaging Center, University of Heidelberg). Lipofuscin levels were measured using ImageJ software by determining average pixel intensity in each animal’s intestines.
Fluorescent staining and quantification of Aˇ deposits CL2006 transgenic nematodes were washed from the plates and transferred to Eppendorf tubes. Thioflavin S-staining was performed as described before (Fay et al. 1998), with some modifications. Briefly, the nematodes were fixed in 4% paraformaldehyde/PBS, pH 7.4, for 24 h at 4 ◦ C. The fixed animals were permeabilized in 5% fresh -mercaptoethanol, 1% Triton X-100, 125 mM Tris pH 7.4, in a 37 ◦ C incubator for 24 h. After washing 2 times with PBS and transferring to poly-l-lysine-coated slides (Sigma) in a drop of PBS, the worms were allowed to settle and the slides completely dried. For staining the slide was immersed in 0.125% thioflavin S (Sigma) in 50% ethanol for 2 min and destained for 2 min in 50% ethanol. Using a drop of PBS, the animals were finally sealed under a coverslip and fluorescence images were acquired (Nikon-eclipse 90i, Nikon digital sight DS-Qi1Mc; 40× objective; Nikon Imaging Center, University of Heidelberg). As cephalic proteins cause a high background with a DAPI filter, we rather chose a FITC filter (excitation 465–495 nm, emission 515–555 nm) (Sun et al. 2002). Quantification of plaques is to be performed instantly, as they gradually bleach under FITC (Wisniewski et al. 1989). Amyloid aggregates were quantified by scoring the thioflavin S-reactive deposits anterior of the pharyngeal bulb in three independent experiments. Western blot of Aˇ A in the CL2006 strain was identified by immunoblotting on a Tris–tricine gel. To detect A monomers and oligomers, total proteins were extracted in PBS containing proteinase inhibitor cocktail. Protein concentration was determined using the Bradford method. Equal amounts of protein (50 g) were loaded and separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). Blots were probed with HRP-labeled beta amyloid 1–16 (6E10) monoclonal antibodies (Signet) at a 1:500 dilution or with HRP-labeled anti-actin polyclonal antibodies (Santa Cruz Biotechnology) at a 1:500 dilution. Proteins were detected using enhanced chemiluminescence (ECL) reagent (WEST-ZOLTM plus Western blot detection system). ImageJ software was used for quantification of band intensities. Statistical analyses Statistical comparison between controls and treatments were done with two-tailed unpaired Student’s t-test, assuming equal variance. All figures indicate means and standard errors of the mean. P < 0.05 was regarded as statistically significant. Results EGCG regulates the mRNA expression of small heat shock protein genes in C. elegans Small heat shock proteins (sHSPs) are a group of low-molecularweight polypeptides found in most organisms (de Jong et al. 1998). The production of HSPs is under the control of the daf-2/insulin-like signaling pathway, which is considered to be a central determinant of the lifespan and stress resistance in C. elegans (Tissenbaum and Ruvkun 1998; Schaffitzel and Hertweck 2006). In the present study, we used C. elegans N2 to examine the effect of EGCG (Fig. 1A) treatment on the expression of the HSP genes hsp-16.1 and hsp16.2. Heat shock and oxidative stress activates these genes (Link et al. 2003). The expression levels of hsp-16.1 and hsp-16.2 were elevated in the worms treated with 20 M juglone, a quinone from Juglans regia for 24 h compared with untreated worms (P < 0.0001) as measured by real-time PCR (Fig. 1B). The expression markedly
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Fig. 2. (A) Effect of different treatments on localization of DAF-16::GFP in C. elegans (TJ356). (B) Heat shock (37 ◦ C, 15 min) or (C) oxidative stress (20 M juglone, 1 h) induce a clear translocation of the DAF-16 transcription factor into the nucleus. (D) EGCG treatment (220 M, 1 h) was also able to induce nuclear localization. Data were obtained from three independent experiments (200 worms each).
decreased in worms pretreated with 220 M EGCG for 48 h followed by oxidative stress of 20 M juglone for 24 h (P < 0.0001) (Fig. 1D). Our results also showed that the EGCG treatment without exposing the worms to any stressors resulted in a no significant decrease of heat shock proteins (P > 0.05) (Fig. 1C).
DAF-16::GFP (Henderson and Johnson 2001). In the nucleus, DAF16 is known to activate transcription of a large number of genes that increase stress resistance and longevity. Our results showed that treatment with 220 M EGCG was apparently able to induce DAF-16 nuclear translocation (Fig. 2).
Localization of DAF-16 in the nucleus
Lipofuscin levels are decreased by EGCG
We previously demonstrated that EGCG is able to increase the mean lifespan and the resistance against oxidative stress in C. elegans (Abbas and Wink 2009). The question remained as to which pathways are involved in the action of EGCG. The DAF-16 transcription factor regulates several biological processes including lifespan and stress resistance (Lin et al. 1997; Partridge and Gems 2002; Lee et al. 2003). Therefore, we used the TJ356 (DAF-16::GFP) strain to investigate whether EGCG is able to affect the cellular localization of DAF-16. DAF-16::GFP does not become localized in the nucleus under normal culturing conditions, but heat treatment or oxidative stress with juglone leads to rapid nuclear localization of
Lipofuscin is the finely yellow-brown pigment in granules composed of lipid-containing residues of lysosomal digestion. It is considered one of the aging pigments, found in the liver, kidney, heart muscle, adrenals, nerve cells, and ganglion cells (Hosokawa et al. 1994; Terman and Brunk 1998). To test the EGCG effect on the lipofuscin levels, the BA17 strain was treated daily with 220 M EGCG from the first day of life for 16 days. Lipofuscin levels were determined by autofluorescence in each animal’s intestines. EGCG treatment was shown to reduce lipofuscin levels (control, mean pixel density 882.74 ± 92.69 vs. EGCG, mean pixel density 636.39 ± 41.82, P < 0.05) (Fig. 3A and B).
Fig. 3. Lipofuscin, a marker of aging and oxidative damage, is reduced in EGCG-treated animals. (A, left) Intestinal autofluorescence images from lipofuscin with 0 M or (A, right) 220 M EGCG treatment. (B) The mean fluorescence intensity from intestinal lipofuscin in day-16-adults was measured. Comparisons between treatments and controls were significant, *P < 0.05. Data were obtained from three independent experiments (25 worms in each group).
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Fig. 4. Effects of EGCG on beta amyloid (A). The images represent (A, left) thioflavin S-staining of transgenic C. elegans CL2006 and (A, right) wild type, arrows indicate A deposits. (B) Quantification of A deposits in worms fed 220 M EGCG for 4 days starting on the second day after hatching. Data represent the main of three independent experiments (30 worms in each group). Error bars indicate SEM, *P < 0.05. (C) Representative Western blot of A species in transgenic C. elegans CL2006 fed with or without EGCG. (D) Quantification of A oligomers (∼13 kDa; gray bars) and A monomers (∼4 kDa; black bars) by Western blot analysis of A species demonstrates that EGCG treatment inhibits A oligomerization. Data are expressed as mean intensity of bands from three independent experiments. (E) RT-PCR of A gene expression did not reveal alteration after 4 days treatment with 220 M EGCG.
Aˇ deposits are reduced in transgenic C. elegans treated with EGCG Transgenic worms of the CL2006 strain were used to determine the effect of EGCG treatment on the formation of the A deposits. This strain is able to express human A in muscle cells, accumulating in intracellular cytoplasmic deposits (Link 1995). To quantify the number of A deposits, the worms were stained with thioflavin S, a fluorescent amyloid-specific dye (Fig. 4A). Treatment with 220 M EGCG for 4 days starting from the second day after hatching reduces the mean number of A deposits (4.03 ± 0.18) compared
to the untreated control (6.05 ± 0.61, P < 0.05) (Fig. 4B). In order to determine whether EGCG inhibits the formation of monomers and oligomers, the resulting A was analyzed by Western blotting by measuring the mean densities of A oligomer (band at ∼13 kDa) as well as that of A monomer (band at ∼4 kDa) (Fig. 4C). The amount of A oligomer was significantly lowered (P < 0.05) in worms fed with 220 M EGCG for 4 days starting from the second day after hatching, while no significant effect on A monomers was observed (Fig. 4D). The real-time quantitative PCR analysis of A mRNA did not reveal any significant differences between the control and EGCG-treated worms (Fig. 4E).
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Discussion There is considerable interest in the health benefits derived from green tea and its major catechin, epigallocatechin gallate, which have mainly been attributed to protection against oxidative stress (Abbas and Wink 2009). Therefore, research on the health benefits of epigallocatechin gallate (EGCG) has been expanding greatly in recent years. There is a clear correlation between the enhanced stress resistance and longevity (Larsen 1993; Lithgow et al. 1995). sHSPs are stress response proteins and act as molecular chaperones by preventing accumulation of aggregated proteins (Leroux et al. 1997). The small heat shock proteins (sHSPs) are highly inducible during thermal and oxidative stress (Link et al. 2003). Our previous study had revealed that the lifespan of worms was extended by treatment with 200–400 M EGCG, 220 M being the optimal concentration (Abbas and Wink 2009) – this concentration was thus used in the present study. Consistent with other reports (Link et al. 1999; Strayer et al. 2003), a 24 h treatment with 20 M juglone induces hsp-16.1 and hsp-16.2 expression (Fig. 1B). In the present study we demonstrated that EGCG decreases the expression of hsp-16.1 and hsp-16.2 under oxidative stress (Fig. 1D); this is in agreement with our previous results (Abbas and Wink 2009) showing that EGCG treatment suppresses hsp-16.2::GFP expression (induced by 20 M juglone) in the strain TJ375 (hsp-16.2::GFP). In fact, the presence of EGCG reduces the need to hsp-16.1 and hsp16.2 expression under oxidative stress. Under standard conditions the expression of hsp-16.1 and hsp-16.2 was decreased, but not in a statistically significant manner when the worms were treated with EGCG only (Fig. 1C). mRNA levels of shsps were reduced by blueberry polyphenols treatment, while their effects were separable from antioxidant properties (Wilson et al. 2006), this means that polyphenols possess several ways of mode of actions. The amount of sHSPs decreases in aged worms leading to an increase of unfolded proteins and, as a result, the worms become more sensitive to stress conditions leading to increased mortality (Johnson et al. 2001; Lund et al. 2002). Under oxidative stress, EGCG reduces shsps levels and mortality rates, thus extending the lifespan of worms. The EGCG treatment revealed lifespan prolongation in the mev-1(kn1) strain (Abbas and Wink 2009), which exhibits accelerated aging because of a mutation in the cytochrome b, leading to an elevated level of oxidative damage (Ishii et al. 1998). Similar results were obtained with Ginkgo biloba extract EGb761 (Wu et al. 2002; Strayer et al. 2003), indicating that the benefits of EGCG and EGb761 depend on their antioxidant properties. In one interesting study patients with pulmonary tuberculosis showed increased levels of reactive oxygen and nitrogen species from pulmonary inflammation resulting in high oxidative stress (Reddy et al. 2004); thus, crude green tea catechins may be useful as adjuvant therapy in managing oxidative stress in pulmonary tuberculosis (Agarwal et al. 2010). Approaching the question on the mode of action of EGCG we used transgenic worms TJ356 (DAF-16::GFP) in which a reporter protein (green fluorescent protein, GFP) is fused to the predicted last amino acid of the DAF-16a2 polypeptide chain (Henderson and Johnson 2001). Under normal conditions DAF-16::GFP remains in the cytoplasm (Fig. 2A), but under thermal stress (37 ◦ C, 15 min), oxidative stress (20 M juglone, 1 h), or 220 M EGCG treatment (1 h) a strong nuclear localization of DAF-16::GFP was observed (Fig. 2B–D). These results suggest that EGCG acts through the daf-2/insulin-like signaling pathway which negatively regulates daf-16 (Kimura et al. 1997). This gene encodes the transcription factor DAF-16, a protein that controls the activity of other genes which have a prominent effect on the lifespan of C. elegans (Ogg et al. 1997). The inhibition of daf-2, a homologue of the insulin receptor (Zamore et al. 2000), leads also to nuclear localization of DAF-16::GFP (Henderson and Johnson 2001). The DAF-16 transcription factor is known to up-regulate a large number of genes
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that increase stress resistance and extend the lifespan of worms. EGCG has been shown to increase the lifespan and stress resistance of C. elegans (Abbas and Wink 2009), and these effects may be due to inhibition of daf-2 resulting in DAF-16 migration into the nucleus and to induction of gene expression which promotes longevity and stress resistance (Murphy et al. 2003). In addition, the nuclear localization of DAF-16::GFP is essential for activation a several of genes including antioxidative enzymes like the superoxide dismutase and the catalases (Oh et al. 2006). Studies on aging have revealed that longevity is influenced by environmental, nutritional, and genetic factors. Telomerase activity is also assumed to be involved in longevity. Telomerase, which is active in embryonic but not in adult cells acts by adding DNA sequence repeats to the 3 end of DNA strands in telomeric regions of chromosomes. Shortening of telomeres during life could thus be affecting aging and survival. However, one study has revealed that telomerase overexpression does not alter aging in mice (Artandi et al. 2002). An inactivation of telomerase leads to sterile worms after few generations (Ahmed and Hodgkin 2000). All somatic cells in adult worms are postmitotic and they do not proliferate; since telomere shortening is related to cell division, telomere shortening should not be thus important in worms not relevant for the aging process. The role of telomerase in aging of C. elegans remains uncertain, because one study has demonstrated that long telomeres promote increased lifespans in C. elegans (Joeng et al. 2004). Telomerase inhibition and apoptosis activation have been observed in several cancer cells treated with EGCG such as human breast carcinoma MCF-7 cells (Mittal et al. 2004), drug-resistance lung cancer cells (Sadava et al. 2007), as well as oral cavity, thyroid and liver carcinoma cell lines (Lin et al. 2006). Another aging marker investigated in this study is lipofuscin, also called age pigment. It is a brown-yellowish autofluorescent material which accumulates in postmitotic cells such as neurons, cardiac myocytes, and skeletal muscle fibers (Terman and Brunk 1998). The current study shows that EGCG treatment is able to reduce the amount of lipofuscin in the worms’ intestines (Fig. 3). Blueberry polyphenols, which are also potent antioxidants, have been shown to exert a similar effect (Wilson et al. 2006). Oxidative stress promotes lipofuscin formation and antioxidant treatment prevents lipofuscin accumulation (Terman and Brunk 1998), in agreement with the free radical theory of aging and supporting the correlation between oxidative stress and aging (Harman 1956). Vitamins E and C, which are known antioxidant compounds, have also been tested on C. elegans. Vitamin E, but not vitamin C, increased the lifespan by slowing down the rate of reproduction and of development (Harrington and Harley 1988). Vitamin C is also able to protect the worms against oxidative stress induced by juglone (Link et al. 1999; Hartwig et al. 2009). The polyphenols quercetin and resveratrol induced a lifespan extension in C. elegans. Although these polyphenols exert antioxidant activity, they apparently act through different mechanisms. Antioxidant properties and nuclear translocation of DAF-16 may contribute to the mode of action of quercetin (Kampkotter et al. 2008). Resveratrol promotes longevity by delaying mortality onset and also activating sir-2.1 (silencing information regulator 2 or sirtuins) which is essential for longevity related to dietary restriction (Wood et al. 2004; Gruber et al. 2007). In order to determine additional health benefits of EGCG we employed transgenic C. elegans CL2006, which expresses human A in muscle cells (Link 1995). Alzheimer’s disease (AD) has been confirmed to be associated with A accumulation in senile plaques (Masters et al. 1985) playing a central role in the pathology of this disease (Selkoe 2001). CL2006 worms produce A deposits which can be stained by thioflavin S, an amyloid-specific dye (Fay et al. 1998). Thioflavin S-staining reveals that EGCG reduces the number of A deposits (Fig. 4A and B), A oligomers being inhibited, and
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A monomers remaining unaltered (Fig. 4C and D). EGCG treatment does not alter A mRNA levels (Fig. 4E). Other study also revealed that EGCG treatment of human SH-SY5Y neuroblastoma cells does not change the levels of amyloid precursor protein (APP) mRNA, while at the same time reducing the amount of APP. One may interpret that the decrease in A peptide is attributed to the suppression of translation of APP (Reznichenko et al. 2006) or A in our transgenic worms. Oxidative stress may induce amyloid formation and lead to neuronal cell death in AD (Butterfield 2003). Thus, the antioxidant properties of EGCG may play a central role in preventing A oligomerization but also non-covalent interactions might be possible. Ginkgo biloba extract EGb761 has decreased A oligomers while increasing A monomers in transgenic C. elegans; in contrast, vitamin C did not show any alteration in these A species in worms treated accordingly (Wu et al. 2006). The present study demonstrates that EGCG down-regulates hsp-16.1 and -16.2 under oxidative stress, protecting the macromolecules from oxidation damage. EGCG inhibits the formation of lipofuscin and A deposits. We conclude that EGCG plays an important role in protection against age-related diseases mediated by reactive oxygen species. Based on the present data, the mechanism of action of EGCG seems to be linked to the daf-2/insulin-like signaling pathway and to the antioxidant properties of EGCG. Acknowledgements Dr. U. Engel and Dr. C. Ackermann (Nikon Imaging Centre, University of Heidelberg) are gratefully acknowledged for help and support. The Caenorhabditis Genetics Center (University of Minnesota) kindly supplied the worm strains. Prof. Dr. S. Galas (Montpellier) and Dr. Christopher D. Link (University of Colorado) provided valuable advice in the early phase of our experiments. We thank Dr. Y. Ibrahim for discussion and Theodor C. H. Cole for valuable suggestions and for improving the English of the manuscript. References Abbas, S., Wink, M., 2009. Epigallocatechin gallate from green tea (Camellia sinensis) increases lifespan and stress resistance in Caenorhabditis elegans. Planta Med. 75, 216–221. Agarwal, A., Prasad, R., Jain, A., 2010. Effect of green tea extract (catechins) in reducing oxidative stress seen in patients of pulmonary tuberculosis on DOTS Cat I regimen. Phytomedicine 17, 23–27. Ahmed, S., Hodgkin, J., 2000. MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature 403, 159–164. Artandi, S.E., Alson, S., Tietze, M.K., Sharpless, N.E., Ye, S., Greenberg, R.A., Castrillon, D.H., Horner, J.W., Weiler, S.R., Carrasco, R.D., DePinho, R.A., 2002. Constitutive telomerase expression promotes mammary carcinomas in aging mice. Proc. Natl. Acad. Sci. U.S.A. 99, 8191–8196. Avramovich-Tirosh, Y., Reznichenko, L., Mit, T., Zheng, H., Fridkin, M., Weinreb, O., Mandel, S., Youdim, M.B., 2007. Neurorescue activity, APP regulation and amyloid-beta peptide reduction by novel multi-functional brain permeable iron-chelating-antioxidants, M-30 and green tea polyphenol, EGCG. Curr. Alzheimer Res. 4, 403–411. Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Butterfield, D.A., 2003. Amyloid beta-peptide [1-42]-associated free radical-induced oxidative stress and neurodegeneration in Alzheimer’s disease brain: mechanisms and consequences. Curr. Med. Chem. 10, 2651–2659. Cai, Y., Anavy, N.D., Chow, H.H., 2002. Contribution of presystemic hepatic extraction to the low oral bioavailability of green tea catechins in rats. Drug Metab. Dispos. 30, 1246–1249. Chow, H.H., Hakim, I.A., Vining, D.R., Crowell, J.A., Ranger-Moore, J., Chew, W.M., Celaya, C.A., Rodney, S.R., Hara, Y., Alberts, D.S., 2005. Effects of dosing condition on the oral bioavailability of green tea catechins after single-dose administration of Polyphenon E in healthy individuals. Clin. Cancer Res. 11, 4627–4633. Cooper, R., Morre, D.J., Morre, D.M., 2005. Medicinal benefits of green tea: part II. Review of anticancer properties. J. Altern. Complement Med. 11, 639–652. de Jong, W.W., Caspers, G.J., Leunissen, J.A., 1998. Genealogy of the alpha-crystallinsmall heat-shock protein superfamily. Int. J. Biol. Macromol. 22, 151–162. Fay, D.S., Fluet, A., Johnson, C.J., Link, C.D., 1998. In vivo aggregation of beta-amyloid peptide variants. J. Neurochem. 71, 1616–1625. Gruber, J., Tang, S.Y., Halliwell, B., 2007. Evidence for a trade-off between survival and fitness caused by resveratrol treatment of Caenorhabditis elegans. Ann. N. Y. Acad. Sci. 1100, 530–542.
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Epigallocatechin gallate inhibits beta amyloid oligomerization in Caenorhabditis elegans and affects the daf-2/insulin-like signaling pathway S. Abbas, M. Wink ∗ Institute of Pharmacy and Molecular Biotechnology, Department of Biology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
a r t i c l e
i n f o
Keywords: Caenorhabditis elegans EGCG sHSPs daf-16 Beta amyloid
a b s t r a c t Epidemiological studies have repeatedly demonstrated that green tea protects against oxidative stress involved in many diseases. Health benefits of green tea are attributed to its principal active constituent, epigallocatechin gallate (EGCG). EGCG was shown to increase the stress resistance and lifespan of Caenorhabditis elegans. The mechanism of this action has been investigated in this study. The expression of hsp-16.1 and hsp-16.2 in EGCG-treated worms (N2), as quantified by real-time PCR, was significantly lower under oxidative stress induced by juglone than in controls without EGCG. In the strain TJ356 (DAF-16::GFP) EGCG treatment induced translocation of DAF-16 from the cytoplasm into the nucleus, suggesting that EGCG may affect the daf-2/insulin-like signaling pathway. EGCG decreased the formation of lipofuscin, an aging related pigment. Also, EGCG reduced beta amyloid (A) deposits and inhibited A oligomerization in transgenic C. elegans (CL2006). Thus, the use of green tea and EGCG is apparently rational alternatives for protecting against ROS-mediated and age-related diseases. © 2010 Elsevier GmbH. All rights reserved.
Introduction Green tea (Camellia sinensis) is consumed as a beverage worldwide and famous for its health benefits. The leaves contain several bioactive compounds of which flavonoids are particularly abundant and among these especially the catechins (Weisburger 1997). These compounds possess several phenolic hydroxyl groups which can dissociate into O− ions under physiological conditions; if many such molecules bind to a single protein by forming ionic and hydrogen bonds, it is likely that the conformation and flexibility of the protein will be altered. This explains why polyphenols show several interactions with cellular proteins, including receptors, ion channels, enzymes, regulatory and structural proteins (Wink 2008). Catechins are also known for their antioxidant activity, and monomeric catechin, another constituent of green tea, recently has also been shown to increase the mean lifespan and stress resistance in Caenorhabditis elegans wild type (Saul et al. 2009). It is likely that these polyphenols protect against ROS-mediated and age-related diseases (Hollman et al. 1999). Regular consumption of green tea appears to prevent cardiovascular diseases and even cancer and seems to contribute to a higher life expectancy among Asians (e.g., Japan). EGCG, being the main
∗ Corresponding author. Tel.: +49 6221544880; fax: +49 6221544884. E-mail address: [email protected] (M. Wink). 0944-7113/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2010.03.008
constituent of green tea, has been thoroughly analyzed and shown to exert several beneficial health effects. Several studies have suggested a possible role of EGCG (Fig. 1A) in preventing certain types of cancer including prostate cancer. EGCG inhibits the growth of prostate cancer cells leading to cell cycle arrest and apoptosis without observable toxic effects on normal epithelial cells (Moyers and Kumar 2004; Cooper et al. 2005; Johnson et al. 2010). There is indication that EGCG exerts other medicinal benefits in preventing neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases (Avramovich-Tirosh et al. 2007); EGCG shows antimutagenic effects (Muto et al. 1999) and several studies have provided evidence that green tea consumption reduces the risk of cardiovascular diseases (Kuriyama et al. 2006). These effects of EGCG have been attributed, in part, to its antioxidant action but influences on protein conformation are likely. Clinical pharmacokinetic studies revealed a low bioavailability of EGCG due to a degradation at pH > 6.5 and thus under physiological conditions in the gastrointestinal tract (pH 5–8). Oral administration of decaffeinated green tea polyphenols, marketed as Polyphenon E, on an empty stomach resulted in increased plasma levels of catechins. This indicates that food can delay the gastric emptying which would lead to a raise of stomach pH and thus polyphenol degradation (Chow et al. 2005). Sprague–Dawley rats treated with green tea polyphenols showed highest concentrations of EGCG in the large intestine and significant EGCG concentrations were found in other tissues including kidneys, prostate, and lungs (Kim et al. 2000). The results obtained from [3 H]EGCG-treated mice via gastric tube
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Fig. 1. (A) The chemical structure of EGCG. Expression levels of small heat shock protein genes (hsp-16.1 and hsp-16.2), relative to actin, were determined by quantitative real-time PCR. Graphs represent mean of three independent experiments with SEM; ***P < 0.0001. (B) 20 M juglone-treated worms (applied on third day after hatching and for 24 h) showed high expression levels of hsp-16.1 and -16.2. (C) There is no significant effect in the 220 M EGCG-treated group for 72 h starting 1 day after hatching. (D) The expression levels in 220 M EGCG-treated group (48 h) followed with 20 M juglone (24 h) were reduced, ***P < 0.0001. Cont. = control and set to 1.
administration have demonstrated that frequent administration of EGCG resulted in high plasma levels with a wide distribution in the body (digestive tract, liver, lungs, pancreas, mammary glands, skin, brain, kidneys, ovary, and testes); in these experiments mice were starved for 15 h before gastric intubation (Suganuma et al. 1998). The hepatic first-pass has no significant effect on green tea catechin elimination; gastrointestinal factors seem to largely contribute to the low bioavailability of green tea catechins after oral uptake (Cai et al. 2002). The versatile model nematode Caenorhabditis elegans is useful for understanding aging processes and age-related diseases, due to its rapid life cycle, short lifespan, ease of cultivation, and well-established genetic pathways; in addition, C. elegans contains homologues of nearly two-thirds of the human genome (Sonnhammer and Durbin 1997). The present study has aimed to investigate the mechanisms of action of EGCG, which delays the appearance of several markers of aging and oxidative stress, such as lipofuscin pigments, heat shock proteins, and beta amyloid formation in C. elegans.
Materials and methods Chemicals and reagents (−)-Epigallocatechin gallate (EGCG) (Fig. 1A) 95%, juglone (5hydroxy-1,4-naphthalenedione), TRI reagent, and thioflavin S were obtained from Sigma–Aldrich GmbH (Munich, Germany); sodium azide from Appli-Chem GmbH (Darmstadt, Germany). Caenorhabditis elegans strains and culture conditions The employed C. elegans strains included: N2; BA17, fem1(hc17) (fertile at 20 ◦ C, infertile at 25 ◦ C); TJ356 (DAF-16::GFP) and CL2006, dvIs2 [pCL12 (unc-54/human A beta peptide 1-42) + pRF4]. All strains and Escherichia coli (OP50) were obtained from the Caenorhabditis Genetic Center (CGC) of the NIH National Center for Research Resources. The strains were maintained at 20 ◦ C on nematode growth medium (NGM) (Brenner 1974). The experimental worms were grown in liquid S-medium. E. coli (OP50) as a food
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source was added to media at a concentration of 1 × 109 cells/ml. All worms used in this study were age-synchronized, and the experimental animals were raised from eggs obtained after sodium hypochlorite treatment of hermaphrodites (Sulston and Hodgkin 1988).
Quantitative real-time PCR To quantify the expression levels of heat shock protein genes (hsp-16.1 and hsp-16.2), N2 worms were grown for 72 h in the presence or absence of 220 M EGCG on the day after hatching. In case of oxidatively stressed worms the animals were subjected for 48 h to 220 M EGCG on the day after hatching, followed by 20 M juglone for 24 h. To quantify the expression level of the beta amyloid gene, the CL2006 strain was treated with 220 M EGCG on the second day after hatching for 4 days. Then the animals were washed from plates with M9 buffer into Eppendorf tubes, and allowed to settle on ice. The worm pellet was resuspended in 1 ml TRI reagent and total RNA was prepared according to the manufacturer’s instructions. Total RNA was treated with DNase I (Qiagene), followed by chloroform extraction and isopropanol precipitation, and stored frozen at −80 ◦ C. cDNA was prepared with the ImProm-IITM Reverse Transcription System kit (Promega) using 1 g of total RNA per reaction and random primer. Real-time PCR was performed in LightCycler (Roche) using AbsoluteTM QPCR SYPR Green Capillary Mix kit (Abgene), using the following gene-specific primers: Actin
(Forward) 5 -GTGTGACGACGAGGTTGCCGCTCTTGTTGTAGAC-3 (Reverse) 5 -GGTAAGGATCTTCATGAGGTAATCAGTAAGATCAC-3
hsp-16.1
(Forward) 5 -GTCACTTTACCACTATTTCCGTCCAGCTCAACGTTC-3 (Reverse) 5 -CAACGGGCGCTTGCTGAATTGGAATAGATCTTCC-3
hsp-16.2
(Forward) 5 -CTGCAGAATCTCTCCATCTGAGTC-3 (Reverse) 5 -AGATTCGAAGCAACTGCACC-3
Beta amyloid
(Forward) 5 -CAGAATTCCGACATGACTCAGGATATGAAG-3 (Reverse) 5 -CCCACCATGAGTCCAATGATTGC-3
DAF-16 localization via fluorescence microscopy The TJ356 strain was used to examine the intracellular distribution of DAF-16 in the living nematode. In this strain, the gene coding for green fluorescent protein (GFP) was fused to the daf-16 gene (Henderson and Johnson 2001). The effect of 220 M EGCG was studied for 1 h and compared with the effect of heat shock at 37 ◦ C for 15 min and oxidative stress by 20 M juglone for 1 h which were used as positive controls using L2 worms (second larval stage). DAF-16 localization was examined in approximately 20 animals per treatment that were mounted in a drop of 10 mM sodium azide. Fluorescence images were taken at constant exposure times (Nikon-eclipse 90i, Nikon digital sight DS-Qi1Mc; 40× objective; Nikon Imaging Center, University of Heidelberg).
Lipofuscin levels Adult hermaphrodites of the BA17 strain were treated with 220 M EGCG for 16 days on the day after hatching and maintained in S-medium at 25 ◦ C; the worms were transferred to the new medium every second day, then anaesthetized and mounted onto glass slides in M9 medium containing 10 mM sodium azide to visualize intestinal autofluorescence (Nikon-eclipse 90i, Nikon digital sight DS-Qi1Mc; imaging at constant exposure times; Nikon Imaging Center, University of Heidelberg). Lipofuscin levels were measured using ImageJ software by determining average pixel intensity in each animal’s intestines.
Fluorescent staining and quantification of Aˇ deposits CL2006 transgenic nematodes were washed from the plates and transferred to Eppendorf tubes. Thioflavin S-staining was performed as described before (Fay et al. 1998), with some modifications. Briefly, the nematodes were fixed in 4% paraformaldehyde/PBS, pH 7.4, for 24 h at 4 ◦ C. The fixed animals were permeabilized in 5% fresh -mercaptoethanol, 1% Triton X-100, 125 mM Tris pH 7.4, in a 37 ◦ C incubator for 24 h. After washing 2 times with PBS and transferring to poly-l-lysine-coated slides (Sigma) in a drop of PBS, the worms were allowed to settle and the slides completely dried. For staining the slide was immersed in 0.125% thioflavin S (Sigma) in 50% ethanol for 2 min and destained for 2 min in 50% ethanol. Using a drop of PBS, the animals were finally sealed under a coverslip and fluorescence images were acquired (Nikon-eclipse 90i, Nikon digital sight DS-Qi1Mc; 40× objective; Nikon Imaging Center, University of Heidelberg). As cephalic proteins cause a high background with a DAPI filter, we rather chose a FITC filter (excitation 465–495 nm, emission 515–555 nm) (Sun et al. 2002). Quantification of plaques is to be performed instantly, as they gradually bleach under FITC (Wisniewski et al. 1989). Amyloid aggregates were quantified by scoring the thioflavin S-reactive deposits anterior of the pharyngeal bulb in three independent experiments. Western blot of Aˇ A in the CL2006 strain was identified by immunoblotting on a Tris–tricine gel. To detect A monomers and oligomers, total proteins were extracted in PBS containing proteinase inhibitor cocktail. Protein concentration was determined using the Bradford method. Equal amounts of protein (50 g) were loaded and separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). Blots were probed with HRP-labeled beta amyloid 1–16 (6E10) monoclonal antibodies (Signet) at a 1:500 dilution or with HRP-labeled anti-actin polyclonal antibodies (Santa Cruz Biotechnology) at a 1:500 dilution. Proteins were detected using enhanced chemiluminescence (ECL) reagent (WEST-ZOLTM plus Western blot detection system). ImageJ software was used for quantification of band intensities. Statistical analyses Statistical comparison between controls and treatments were done with two-tailed unpaired Student’s t-test, assuming equal variance. All figures indicate means and standard errors of the mean. P < 0.05 was regarded as statistically significant. Results EGCG regulates the mRNA expression of small heat shock protein genes in C. elegans Small heat shock proteins (sHSPs) are a group of low-molecularweight polypeptides found in most organisms (de Jong et al. 1998). The production of HSPs is under the control of the daf-2/insulin-like signaling pathway, which is considered to be a central determinant of the lifespan and stress resistance in C. elegans (Tissenbaum and Ruvkun 1998; Schaffitzel and Hertweck 2006). In the present study, we used C. elegans N2 to examine the effect of EGCG (Fig. 1A) treatment on the expression of the HSP genes hsp-16.1 and hsp16.2. Heat shock and oxidative stress activates these genes (Link et al. 2003). The expression levels of hsp-16.1 and hsp-16.2 were elevated in the worms treated with 20 M juglone, a quinone from Juglans regia for 24 h compared with untreated worms (P < 0.0001) as measured by real-time PCR (Fig. 1B). The expression markedly
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Fig. 2. (A) Effect of different treatments on localization of DAF-16::GFP in C. elegans (TJ356). (B) Heat shock (37 ◦ C, 15 min) or (C) oxidative stress (20 M juglone, 1 h) induce a clear translocation of the DAF-16 transcription factor into the nucleus. (D) EGCG treatment (220 M, 1 h) was also able to induce nuclear localization. Data were obtained from three independent experiments (200 worms each).
decreased in worms pretreated with 220 M EGCG for 48 h followed by oxidative stress of 20 M juglone for 24 h (P < 0.0001) (Fig. 1D). Our results also showed that the EGCG treatment without exposing the worms to any stressors resulted in a no significant decrease of heat shock proteins (P > 0.05) (Fig. 1C).
DAF-16::GFP (Henderson and Johnson 2001). In the nucleus, DAF16 is known to activate transcription of a large number of genes that increase stress resistance and longevity. Our results showed that treatment with 220 M EGCG was apparently able to induce DAF-16 nuclear translocation (Fig. 2).
Localization of DAF-16 in the nucleus
Lipofuscin levels are decreased by EGCG
We previously demonstrated that EGCG is able to increase the mean lifespan and the resistance against oxidative stress in C. elegans (Abbas and Wink 2009). The question remained as to which pathways are involved in the action of EGCG. The DAF-16 transcription factor regulates several biological processes including lifespan and stress resistance (Lin et al. 1997; Partridge and Gems 2002; Lee et al. 2003). Therefore, we used the TJ356 (DAF-16::GFP) strain to investigate whether EGCG is able to affect the cellular localization of DAF-16. DAF-16::GFP does not become localized in the nucleus under normal culturing conditions, but heat treatment or oxidative stress with juglone leads to rapid nuclear localization of
Lipofuscin is the finely yellow-brown pigment in granules composed of lipid-containing residues of lysosomal digestion. It is considered one of the aging pigments, found in the liver, kidney, heart muscle, adrenals, nerve cells, and ganglion cells (Hosokawa et al. 1994; Terman and Brunk 1998). To test the EGCG effect on the lipofuscin levels, the BA17 strain was treated daily with 220 M EGCG from the first day of life for 16 days. Lipofuscin levels were determined by autofluorescence in each animal’s intestines. EGCG treatment was shown to reduce lipofuscin levels (control, mean pixel density 882.74 ± 92.69 vs. EGCG, mean pixel density 636.39 ± 41.82, P < 0.05) (Fig. 3A and B).
Fig. 3. Lipofuscin, a marker of aging and oxidative damage, is reduced in EGCG-treated animals. (A, left) Intestinal autofluorescence images from lipofuscin with 0 M or (A, right) 220 M EGCG treatment. (B) The mean fluorescence intensity from intestinal lipofuscin in day-16-adults was measured. Comparisons between treatments and controls were significant, *P < 0.05. Data were obtained from three independent experiments (25 worms in each group).
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Fig. 4. Effects of EGCG on beta amyloid (A). The images represent (A, left) thioflavin S-staining of transgenic C. elegans CL2006 and (A, right) wild type, arrows indicate A deposits. (B) Quantification of A deposits in worms fed 220 M EGCG for 4 days starting on the second day after hatching. Data represent the main of three independent experiments (30 worms in each group). Error bars indicate SEM, *P < 0.05. (C) Representative Western blot of A species in transgenic C. elegans CL2006 fed with or without EGCG. (D) Quantification of A oligomers (∼13 kDa; gray bars) and A monomers (∼4 kDa; black bars) by Western blot analysis of A species demonstrates that EGCG treatment inhibits A oligomerization. Data are expressed as mean intensity of bands from three independent experiments. (E) RT-PCR of A gene expression did not reveal alteration after 4 days treatment with 220 M EGCG.
Aˇ deposits are reduced in transgenic C. elegans treated with EGCG Transgenic worms of the CL2006 strain were used to determine the effect of EGCG treatment on the formation of the A deposits. This strain is able to express human A in muscle cells, accumulating in intracellular cytoplasmic deposits (Link 1995). To quantify the number of A deposits, the worms were stained with thioflavin S, a fluorescent amyloid-specific dye (Fig. 4A). Treatment with 220 M EGCG for 4 days starting from the second day after hatching reduces the mean number of A deposits (4.03 ± 0.18) compared
to the untreated control (6.05 ± 0.61, P < 0.05) (Fig. 4B). In order to determine whether EGCG inhibits the formation of monomers and oligomers, the resulting A was analyzed by Western blotting by measuring the mean densities of A oligomer (band at ∼13 kDa) as well as that of A monomer (band at ∼4 kDa) (Fig. 4C). The amount of A oligomer was significantly lowered (P < 0.05) in worms fed with 220 M EGCG for 4 days starting from the second day after hatching, while no significant effect on A monomers was observed (Fig. 4D). The real-time quantitative PCR analysis of A mRNA did not reveal any significant differences between the control and EGCG-treated worms (Fig. 4E).
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Discussion There is considerable interest in the health benefits derived from green tea and its major catechin, epigallocatechin gallate, which have mainly been attributed to protection against oxidative stress (Abbas and Wink 2009). Therefore, research on the health benefits of epigallocatechin gallate (EGCG) has been expanding greatly in recent years. There is a clear correlation between the enhanced stress resistance and longevity (Larsen 1993; Lithgow et al. 1995). sHSPs are stress response proteins and act as molecular chaperones by preventing accumulation of aggregated proteins (Leroux et al. 1997). The small heat shock proteins (sHSPs) are highly inducible during thermal and oxidative stress (Link et al. 2003). Our previous study had revealed that the lifespan of worms was extended by treatment with 200–400 M EGCG, 220 M being the optimal concentration (Abbas and Wink 2009) – this concentration was thus used in the present study. Consistent with other reports (Link et al. 1999; Strayer et al. 2003), a 24 h treatment with 20 M juglone induces hsp-16.1 and hsp-16.2 expression (Fig. 1B). In the present study we demonstrated that EGCG decreases the expression of hsp-16.1 and hsp-16.2 under oxidative stress (Fig. 1D); this is in agreement with our previous results (Abbas and Wink 2009) showing that EGCG treatment suppresses hsp-16.2::GFP expression (induced by 20 M juglone) in the strain TJ375 (hsp-16.2::GFP). In fact, the presence of EGCG reduces the need to hsp-16.1 and hsp16.2 expression under oxidative stress. Under standard conditions the expression of hsp-16.1 and hsp-16.2 was decreased, but not in a statistically significant manner when the worms were treated with EGCG only (Fig. 1C). mRNA levels of shsps were reduced by blueberry polyphenols treatment, while their effects were separable from antioxidant properties (Wilson et al. 2006), this means that polyphenols possess several ways of mode of actions. The amount of sHSPs decreases in aged worms leading to an increase of unfolded proteins and, as a result, the worms become more sensitive to stress conditions leading to increased mortality (Johnson et al. 2001; Lund et al. 2002). Under oxidative stress, EGCG reduces shsps levels and mortality rates, thus extending the lifespan of worms. The EGCG treatment revealed lifespan prolongation in the mev-1(kn1) strain (Abbas and Wink 2009), which exhibits accelerated aging because of a mutation in the cytochrome b, leading to an elevated level of oxidative damage (Ishii et al. 1998). Similar results were obtained with Ginkgo biloba extract EGb761 (Wu et al. 2002; Strayer et al. 2003), indicating that the benefits of EGCG and EGb761 depend on their antioxidant properties. In one interesting study patients with pulmonary tuberculosis showed increased levels of reactive oxygen and nitrogen species from pulmonary inflammation resulting in high oxidative stress (Reddy et al. 2004); thus, crude green tea catechins may be useful as adjuvant therapy in managing oxidative stress in pulmonary tuberculosis (Agarwal et al. 2010). Approaching the question on the mode of action of EGCG we used transgenic worms TJ356 (DAF-16::GFP) in which a reporter protein (green fluorescent protein, GFP) is fused to the predicted last amino acid of the DAF-16a2 polypeptide chain (Henderson and Johnson 2001). Under normal conditions DAF-16::GFP remains in the cytoplasm (Fig. 2A), but under thermal stress (37 ◦ C, 15 min), oxidative stress (20 M juglone, 1 h), or 220 M EGCG treatment (1 h) a strong nuclear localization of DAF-16::GFP was observed (Fig. 2B–D). These results suggest that EGCG acts through the daf-2/insulin-like signaling pathway which negatively regulates daf-16 (Kimura et al. 1997). This gene encodes the transcription factor DAF-16, a protein that controls the activity of other genes which have a prominent effect on the lifespan of C. elegans (Ogg et al. 1997). The inhibition of daf-2, a homologue of the insulin receptor (Zamore et al. 2000), leads also to nuclear localization of DAF-16::GFP (Henderson and Johnson 2001). The DAF-16 transcription factor is known to up-regulate a large number of genes
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that increase stress resistance and extend the lifespan of worms. EGCG has been shown to increase the lifespan and stress resistance of C. elegans (Abbas and Wink 2009), and these effects may be due to inhibition of daf-2 resulting in DAF-16 migration into the nucleus and to induction of gene expression which promotes longevity and stress resistance (Murphy et al. 2003). In addition, the nuclear localization of DAF-16::GFP is essential for activation a several of genes including antioxidative enzymes like the superoxide dismutase and the catalases (Oh et al. 2006). Studies on aging have revealed that longevity is influenced by environmental, nutritional, and genetic factors. Telomerase activity is also assumed to be involved in longevity. Telomerase, which is active in embryonic but not in adult cells acts by adding DNA sequence repeats to the 3 end of DNA strands in telomeric regions of chromosomes. Shortening of telomeres during life could thus be affecting aging and survival. However, one study has revealed that telomerase overexpression does not alter aging in mice (Artandi et al. 2002). An inactivation of telomerase leads to sterile worms after few generations (Ahmed and Hodgkin 2000). All somatic cells in adult worms are postmitotic and they do not proliferate; since telomere shortening is related to cell division, telomere shortening should not be thus important in worms not relevant for the aging process. The role of telomerase in aging of C. elegans remains uncertain, because one study has demonstrated that long telomeres promote increased lifespans in C. elegans (Joeng et al. 2004). Telomerase inhibition and apoptosis activation have been observed in several cancer cells treated with EGCG such as human breast carcinoma MCF-7 cells (Mittal et al. 2004), drug-resistance lung cancer cells (Sadava et al. 2007), as well as oral cavity, thyroid and liver carcinoma cell lines (Lin et al. 2006). Another aging marker investigated in this study is lipofuscin, also called age pigment. It is a brown-yellowish autofluorescent material which accumulates in postmitotic cells such as neurons, cardiac myocytes, and skeletal muscle fibers (Terman and Brunk 1998). The current study shows that EGCG treatment is able to reduce the amount of lipofuscin in the worms’ intestines (Fig. 3). Blueberry polyphenols, which are also potent antioxidants, have been shown to exert a similar effect (Wilson et al. 2006). Oxidative stress promotes lipofuscin formation and antioxidant treatment prevents lipofuscin accumulation (Terman and Brunk 1998), in agreement with the free radical theory of aging and supporting the correlation between oxidative stress and aging (Harman 1956). Vitamins E and C, which are known antioxidant compounds, have also been tested on C. elegans. Vitamin E, but not vitamin C, increased the lifespan by slowing down the rate of reproduction and of development (Harrington and Harley 1988). Vitamin C is also able to protect the worms against oxidative stress induced by juglone (Link et al. 1999; Hartwig et al. 2009). The polyphenols quercetin and resveratrol induced a lifespan extension in C. elegans. Although these polyphenols exert antioxidant activity, they apparently act through different mechanisms. Antioxidant properties and nuclear translocation of DAF-16 may contribute to the mode of action of quercetin (Kampkotter et al. 2008). Resveratrol promotes longevity by delaying mortality onset and also activating sir-2.1 (silencing information regulator 2 or sirtuins) which is essential for longevity related to dietary restriction (Wood et al. 2004; Gruber et al. 2007). In order to determine additional health benefits of EGCG we employed transgenic C. elegans CL2006, which expresses human A in muscle cells (Link 1995). Alzheimer’s disease (AD) has been confirmed to be associated with A accumulation in senile plaques (Masters et al. 1985) playing a central role in the pathology of this disease (Selkoe 2001). CL2006 worms produce A deposits which can be stained by thioflavin S, an amyloid-specific dye (Fay et al. 1998). Thioflavin S-staining reveals that EGCG reduces the number of A deposits (Fig. 4A and B), A oligomers being inhibited, and
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A monomers remaining unaltered (Fig. 4C and D). EGCG treatment does not alter A mRNA levels (Fig. 4E). Other study also revealed that EGCG treatment of human SH-SY5Y neuroblastoma cells does not change the levels of amyloid precursor protein (APP) mRNA, while at the same time reducing the amount of APP. One may interpret that the decrease in A peptide is attributed to the suppression of translation of APP (Reznichenko et al. 2006) or A in our transgenic worms. Oxidative stress may induce amyloid formation and lead to neuronal cell death in AD (Butterfield 2003). Thus, the antioxidant properties of EGCG may play a central role in preventing A oligomerization but also non-covalent interactions might be possible. Ginkgo biloba extract EGb761 has decreased A oligomers while increasing A monomers in transgenic C. elegans; in contrast, vitamin C did not show any alteration in these A species in worms treated accordingly (Wu et al. 2006). The present study demonstrates that EGCG down-regulates hsp-16.1 and -16.2 under oxidative stress, protecting the macromolecules from oxidation damage. EGCG inhibits the formation of lipofuscin and A deposits. We conclude that EGCG plays an important role in protection against age-related diseases mediated by reactive oxygen species. Based on the present data, the mechanism of action of EGCG seems to be linked to the daf-2/insulin-like signaling pathway and to the antioxidant properties of EGCG. Acknowledgements Dr. U. Engel and Dr. C. Ackermann (Nikon Imaging Centre, University of Heidelberg) are gratefully acknowledged for help and support. The Caenorhabditis Genetics Center (University of Minnesota) kindly supplied the worm strains. Prof. Dr. S. Galas (Montpellier) and Dr. Christopher D. Link (University of Colorado) provided valuable advice in the early phase of our experiments. We thank Dr. Y. Ibrahim for discussion and Theodor C. H. Cole for valuable suggestions and for improving the English of the manuscript. References Abbas, S., Wink, M., 2009. Epigallocatechin gallate from green tea (Camellia sinensis) increases lifespan and stress resistance in Caenorhabditis elegans. Planta Med. 75, 216–221. Agarwal, A., Prasad, R., Jain, A., 2010. Effect of green tea extract (catechins) in reducing oxidative stress seen in patients of pulmonary tuberculosis on DOTS Cat I regimen. Phytomedicine 17, 23–27. Ahmed, S., Hodgkin, J., 2000. MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature 403, 159–164. Artandi, S.E., Alson, S., Tietze, M.K., Sharpless, N.E., Ye, S., Greenberg, R.A., Castrillon, D.H., Horner, J.W., Weiler, S.R., Carrasco, R.D., DePinho, R.A., 2002. Constitutive telomerase expression promotes mammary carcinomas in aging mice. Proc. Natl. Acad. Sci. U.S.A. 99, 8191–8196. Avramovich-Tirosh, Y., Reznichenko, L., Mit, T., Zheng, H., Fridkin, M., Weinreb, O., Mandel, S., Youdim, M.B., 2007. Neurorescue activity, APP regulation and amyloid-beta peptide reduction by novel multi-functional brain permeable iron-chelating-antioxidants, M-30 and green tea polyphenol, EGCG. Curr. Alzheimer Res. 4, 403–411. Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Butterfield, D.A., 2003. Amyloid beta-peptide [1-42]-associated free radical-induced oxidative stress and neurodegeneration in Alzheimer’s disease brain: mechanisms and consequences. Curr. Med. Chem. 10, 2651–2659. Cai, Y., Anavy, N.D., Chow, H.H., 2002. Contribution of presystemic hepatic extraction to the low oral bioavailability of green tea catechins in rats. Drug Metab. Dispos. 30, 1246–1249. Chow, H.H., Hakim, I.A., Vining, D.R., Crowell, J.A., Ranger-Moore, J., Chew, W.M., Celaya, C.A., Rodney, S.R., Hara, Y., Alberts, D.S., 2005. Effects of dosing condition on the oral bioavailability of green tea catechins after single-dose administration of Polyphenon E in healthy individuals. Clin. Cancer Res. 11, 4627–4633. Cooper, R., Morre, D.J., Morre, D.M., 2005. Medicinal benefits of green tea: part II. Review of anticancer properties. J. Altern. Complement Med. 11, 639–652. de Jong, W.W., Caspers, G.J., Leunissen, J.A., 1998. Genealogy of the alpha-crystallinsmall heat-shock protein superfamily. Int. J. Biol. Macromol. 22, 151–162. Fay, D.S., Fluet, A., Johnson, C.J., Link, C.D., 1998. In vivo aggregation of beta-amyloid peptide variants. J. Neurochem. 71, 1616–1625. Gruber, J., Tang, S.Y., Halliwell, B., 2007. Evidence for a trade-off between survival and fitness caused by resveratrol treatment of Caenorhabditis elegans. Ann. N. Y. Acad. Sci. 1100, 530–542.
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