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Division of Life Sciences and Silver Biotechnology Research Center, Hallym University, Chuncheon, Korea
2To whom correspondence should be addressed. E-mail: yhkang{at}hallym.ac.kr.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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10 µmol/L; these inhibitory effects were dose dependent. Western blot data revealed that H2O2 upregulated phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which was rapidly reversed by quercetin within 30 min; H2O2 activation of c-Jun was downregulated. (-)Epigallocatechin gallate inhibited H2O2-induced phosphorylation of JNK and p38 MAPK after 60 min. These results reveal that quercetin blocks JNK- and p38 MAPK-related signaling triggered by the oxidant and may regulate expression of apoptotic downstream genes, preventing apoptosis and promoting cell survival. (-)Epigallocatechin gallate may function as an antiapoptotic agent through other antiapoptotic pathways.
KEY WORDS: • flavonoids • apoptosis • H2O2 • survival signaling
Dysfunction of apoptosis results in neurodegenerative and autoimmune diseases (1). Oxidative injury can induce cardiac and endothelial cell apoptosis and necrosis. The severity of cellular damage from an oxidant determines which mechanism of cell death dominates (2). Accordingly, it was proposed that agents or antioxidants that can inhibit production of reactive oxygen species (ROS)3 such as H2O2 can prevent apoptosis (3–5). The antioxidant N-acetylcysteine, a metabolic precursor of reduced glutathione (GSH), was used as a tool for investigating the role of ROS in numerous biological and pathological processes. However, the underlying molecular mechanisms by which antioxidants protect from apoptosis triggered by diverse stimulators remain to be elucidated.
Redox state–related signaling of ROS may activate the transduction pathways leading to apoptosis (6–8). There is growing evidence that ROS may cause cell death via mediation of mitogen-activated protein kinase (MAPK) under various oxidative conditions (9–13). Hyperoxia generates ROS through activating NADPH oxidase, which mediates lung epithelial cell death via an activation of extracellular signal-regulated kinase (ERK) 1/2 (9). In addition, H2O2 induces neuronal apoptosis via inhibition of ERK 1/2 phosphorylation and activation of p38 MAPK and caspase-3, concomitantly with nuclear factor (NF)-
B transactivation (12). Activation of p38 MAPK primarily mediates ROS-induced apoptosis, whereas concomitant activation of c-Jun N-terminal kinase (JNK) represents a scavenger pathway for inhibiting apoptosis (14). N-Acetylcysteine inhibits the activation of JNK and p38 MAPK and the activity of redox-sensitive activating protein-1 and NF-B regulating expression of apoptotic genes (15,16). Additionally, N-acetylcysteine can also prevent apoptosis and promote cell survival by activating the ERK pathway.
Epidemiologic studies showed that a high consumption of polyphenolic phytochemicals is inversely related to the risk of cardiovascular diseases (17–19), and this phenomenon appears to be associated with their antioxidant capacity. Flavonoids, phytochemicals found in a large number of fruits and vegetables, are natural antioxidants that scavenge various types of radicals in aqueous and organic environments (20–22). Whether such antioxidant effects are apparent in vivo is unclear due to their bioavailability; how flavonoids protect cells and tissues from oxidative damage is clearly undefined. Recently, we showed that flavonoids inhibit endothelial apoptosis induced by H2O2 and that there are differences in the antiapoptotic activity of flavonoids (23), which appeared to stem from their structure. However, actions of antiapoptotic flavonoids are not yet defined. Considering that antioxidant flavonoids inhibit the NAD(P)H oxidase and enhance the intracellular GSH level (24–26), we hypothesized that flavonoid-related redox manipulations block apoptosis by influencing the MAPK signaling pathways.
To test this hypothesis, the current study elucidated the precise sites and mechanisms of action of antiapoptotic flavonoids, (-)epigallocatechin gallate and quercetin, involved in the sequence of events that regulate oxidant-induced cell death. We tested whether flavonoids, when applied in micromolar concentrations, manipulate MAPK-responsive multiple death/survival signaling pathways that converge at the level of transcriptional regulation in human umbilical vein endothelial cells (HUVEC)
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.5% Primary culture of endothelial cells. HUVEC were isolated and cultured using collagenase (Worthington Biochemical), as described elsewhere (23,28). Cells were incubated in 25 mmol/L HEPES-buffered M199 containing 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 100,000 U/L penicillin, and 100 mg/L streptomycin supplemented with growth supplements (0.9 g/L bovine brain extract, 0.75 g/L human epidermal growth factor, and 0.075 g/L hydrocortisone). Endothelial cells were confirmed by their cobblestone morphology and uptake of fluorescently acetylated LDL (29).
To determine the dose response of test flavonoids to caspase-3 activation, HUVEC were cultured in 25 mmol/L HEPES-buffered M199 containing 10% FBS, growth supplements, and 1–50 µmol/L of test flavonoids for 24 h.
H2O2-induced oxidant stress.
We showed that except for apigenin, all other tested flavonoids had little cytotoxicity even at 100 µmol/L when incubated with cells for 24 h, but at concentrations 50 µmol/L, apigenin did not decrease cell viability (23). Accordingly, the maximal nontoxic concentration of the flavonoids used for these mechanistic culture experiments was 50 µmol/L. The HUVEC culture protocols used for inducing the H2O2-induced oxidant stress were described previously (23).
Western blot analysis. Western blot analysis was performed using whole-cell extracts from HUVEC as previously described (23). The membrane was incubated with polyclonal rabbit anti-human cleaved caspase-3 (1:1000 dilution; Cell Signaling Technology), monoclonal mouse anti-human p53 (BD Transduction Laboratory), monoclonal mouse anti-human phospho-p53 (Ser 15, 1:1000 dilution; Cell Signaling Technology), polyclonal rabbit antibody for phospho-JNK (1:1000 dilution; Cell Signaling Technology), polyclonal rabbit for phospho-c-Jun (1:1000 dilution; Cell Signaling Technology) and with polyclonal rabbit antibody for phospho-p38 (1:1000 dilution; Cell Signaling Technology). After being washed in Tris-buffered saline-Tween 20 (TBS-T), the membrane was incubated with a goat anti-rabbit IgG or anti-mouse IgG conjugated to horseradish peroxidase (1:10,000 dilution; Jackson ImmunoResearch Laboratory), followed by washing in TBS-T. Polyclonal rabbit (1:1000 dilution; Santa Cruz Biotechnology) or monoclonal mouse (1:5000 dilution, Sigma) anti-ß-actin was used for comparative control expression.
Caspase-3-like protease activity. The cell extracts were washed with ice-cold PBS and suspended in 100 mmol/L HEPES buffer (pH 7.4) containing 0.5 mmol/L phenylmethanesulfonyl fluoride, 5 mg/L aprotinin and pepstatin, and 10 mg/L leupeptin. The cell suspension was lysed by 3 freeze-thaw cycles, and the cytosolic fraction was obtained by centrifugation at 12,000 x g for 20 min at 4°C. DEVDase activity was determined by measuring proteolytic cleavage of a chromogenic peptide substrate for caspase-3-like protease, Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA) (30).
Intracellular oxidant generation. Oxidant generation of HUVEC was measured according to a previously described method with a minor modification (13,31,32). After the challenge with H2O2, cells were loaded for 30 min with 10 µmol/L 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA, Sigma). The dye solution was freshly prepared in prewarmed M199 (+2% FBS). After dye loading at 37°C, the cells were rinsed twice with PBS, and the cultures were photographed with a fluorescence microscope.
At the end of the 24-h incubation period, the MTT [3-(4,5-dimetylthiazol-yl)-diphenyl tetrazolium bromide] assay was performed to quantify cellular viability (23,33).
Measurement of cellular GSH level. To determine the cellular antioxidant potentials, cellular total GSH was measured using a 5,5'-dithio-bis(2-nitro-benzoic acid) assay with a minor modification (13,34,35). Total GSH in acid-soluble extracts was determined by an enzymatic recycling assay using a commercially available kit (Dojindo Laboratories). Parallel measurements of GSH standards were performed to quantify total GSH expressed as nmol/mg cell protein.
Preparation of nuclear protein extract. Nuclear protein extracts were prepared by a detergent lysis procedure from HUVEC (13,36). Cells were washed with PBS, lysed in HEPES buffer, and incubated on ice for 10 min. Nuclei were centrifuged at 8000 x g for 20 min. The cytosolic fraction was collected and proteins of nucleus pellets were extracted with vigorously shaking in a high-salt buffer at 4°C for 20 min. The nuclear debris was pelleted at 8000 x g for 20 min and the supernatant was stored at –70°C.
For the determination of p53 localization, Western blot analysis was carried out with nuclear protein extracts and cytosolic protein fraction using anti-human phospho-p53 (1:1000 dilution; Cell Signaling Technology).
Immunocytochemistry. Cells were rinsed with PBS and permeabilized with a buffer of 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. HUVEC were fixed with 4% ice-cold formaldehyde for 30 min. After fixed cells were washed with PBS containing 0.2% Tween 20 (PBS/T), mouse anti-human phospho-p53 (1:100 dilution; Cell Signaling Technology) in 25% goat serum was added to cells, and incubated overnight at room temperature. Cells were washed with PBS/T and incubated with Cyanine 3-OSu conjugate-goat anti-mouse IgG (1:1000 dilution; Rockland) in TBS as a secondary antibody. Images were obtained with an Olympus BX50 microscope with differential interference contrast and reflected light fluorescence.
Data analysis. The results are presented as means ± SEM. Statistical analyses were conducted using the SAS statistical software package version 6.12 (SAS Institute). One-way ANOVA was used to determine the effects of flavonoids on apoptotic variables. Differences among metabolite treatment groups were analyzed with Duncan’s multiple range test and were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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10 µmol/L (Fig. 2). Thus, micromolar doses of 10–25 µmol/L were required to achieve the full inhibitory effect of flavonoids in endothelial apoptotic models.
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40% cell killing (Table 1). H2O2-exposed cells revealed a substantial yet not full disappearance of DCF staining in the presence of (-)epigallocatechin gallate and quercetin, indicating that these flavonoids can inhibit an accumulation of intracellular oxidants in endothelial cells injured by oxidant stress due to H2O2. In contrast, H2O2-exposed cells treated with apigenin were positive for DCF staining and were damaged (Fig. 3 and Table 1).
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The ability of (-)epigallocatechin gallate and quercetin to block oxidant injury could be due to their antioxidant capacity. The antioxidant N-acetylcysteine reduced the rate of H2O2- and apigenin-induced cell death dose dependently but not completely (data not shown). Thus, it is likely that the antioxidant activity of flavonoids, at least in part, contributed to their blockade of H2O2-induced massive cell death.
Effects on activation of p53. Activation of the nuclear transcription factor p53 is followed by the formation of cellular oxidant (37). There was relatively weak activation of p53 in untreated controls (Fig. 4A). The p53 activation was upregulated in cells injured by H2O2 relative to undamaged cells. However, (-)epigallocatechin gallate-H2O2-, quercetin-H2O2- or flavanone-H2O2-treated cells exhibited marked inhibition of p53 phosphorylation. In contrast, the addition of apigenin did not downregulate the H2O2-induced p53 activation (Fig. 4A). It should be noted that total expression of p53 protein induced by H2O2 was unchanged by incubation with flavonoids. We also explored whether 50 µmol/L hesperetin or naringenin, the corresponding flavanones without the rutinose moiety of hesperidin and naringin, influenced the H2O2-induced p53 activation (Fig. 4A). This implies that naringenin and hesperetin may function as antiapoptotic agents. The flavanone glycosides, naringin and hesperidin at 50 µmol/L did not inhibit the H2O2-induced endothelial apoptosis (23). The presence of the rutinose moiety of flavanones had a substantial effect on mitigating oxidant-induced p53 phosphorylation.
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Effects on MAPK activation. We attempted to determine whether (-)epigallocatechin gallate and quercetin might inhibit apoptosis in H2O2-exposed cells through blocking MAPK signaling cascades. The effects of cytoprotective flavonoids on the phosphorylation of JNK, c-Jun, and p38 MAPK were examined in HUVEC. The 30-min pulse treatment with 0.25 mmol/L H2O2 induced phosphorylation of JNK, c-Jun, and p38 MAPK (Fig. 5). Quercetin blocked the phosphorylation of JNK and c-Jun, which occurred rapidly within 30 min (Fig. 5A). In addition, the phosphorylation of p38 MAPK by H2O2 was nearly completely and immediately attenuated by quercetin (Fig. 5B). The phosphorylation of JNK, c-Jun, and p38 MAPK was modestly inhibited in HUVEC treated with (-)epigallocatechin gallate (Fig. 6). On the other hand, in apigenin-treated cells, the phosphorylation of ROS-activated JNK and p38 MAPK followed by caspase activation in HUVEC, remained high up to 120 min (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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GSH constitutes a key cellular defense mechanism against oxidative injury, and GSH depletion results in mitochondrial damage as a major consequence (7,32). H2O2 produced by mitochondria can cause extensive damage to this organelle when GSH levels are greatly reduced. Cell survival correlates with enhanced total GSH level in cytoprotective flavonoid-treated HUVEC exposed to H2O2. There is accumulating evidence that flavonoids enhance cellular antioxidant defenses and in turn protect against the damaging effects induced by oxygen radicals (25,26,34). Accordingly, the observed beneficial effects of antiapoptotic flavonoids may reflect their ability to protect endothelial cells from the deleterious consequences following H2O2 injury-induced GSH depletion. In addition, treatment with N-acetylcysteine before H2O2 exposure improved cell viability reduced by H2O2 injury. However, the quantitative importance of such antioxidant effects of these flavonoids on the H2O2 injury is far from clear.
Antioxidant effects of (-)epigallocatechin gallate and quercetin may function on the turn-off phase of death signal transduction triggered by ROS and determine the outcome in terms of survival by downregulating activation of ROS-sensitive caspases. Unlike apigenin, which has weak DPPH radical-scavenging activity (23), (-)epigallocatechin gallate and quercetin in H2O2-induced endothelial cells inhibited the activation of caspase-3 (Fig. 1), a key effecter in apoptotic signaling pathways. The activation of caspases in the presence of H2O2 could be due to direct oxidative stress or could be mediated by mitochondria or by Fas; any of these mechanisms might be inhibited by (-)epigallocatechin gallate and quercetin. It was demonstrated that the death signaling pathway of the Fas ligand is activated by H2O2 through a mediation of tyrosine kinase (38). The present study did not determine activation of caspase-8 by the Fas ligand in the presence of H2O2. A recent report showed that 2-methoxyestradiol effectively sensitizes a human prostate cancer cell line to Fas-mediated apoptosis (39). Generation of intracellular ROS elicits the translocation of cytosolic Bax to mitochondria, which in turn activates Bax to induce the dissociation of cytochrome c from the inner mitochondrial membrane resulting in increased activity of cytosolic caspases (9). H2O2 downregulated antiapoptotic Bcl-2 and upregulated proapoptotic Bax in HUVEC, thereby rendering caspase-3 cleaved (23,40). These H2O2 effects were inhibited by preincubation with (-)epigallocatechin gallate and quercetin (23).
Experimental manipulations of cellular antioxidant systems can influence induction of transcription factors and the expression of related gene proteins (7,15). The transcription factor p53 may affect the mitochondrial membrane potential through ROS generation (41), and may induce apoptosis as a consequence of mitochondrial depolarization (42,43). It was shown that p53, activated by NF-B, plays a role in H2O2-induced apoptosis in glioma cells (43). Functional p53 may play an important role in Bax translocation and mitochondrial fragmentation in Helicobacter pylori-induced apoptosis (44). Our previous report (23) and the present data propose that Bcl-2 and Bax may be controlled by downstream target genes of p53 in the H2O2 toxicity. It is tempting to speculate that flavonoid-related cellular manipulation could stabilize or activate expression of enzymes and transcription factors that are involved in DNA repair and modulation of apoptotic pathways. Indeed, we observed functional activation of p53 after the H2O2 insult, suggesting that p53 could play a pivotal role in H2O2-induced endothelial apoptosis (Fig. 4). The blockade of functional activation of p53 by (-)epigallocatechin gallate and quercetin supports this idea.
MAPK signal transduction pathways differentially relay numerous extracellular signals within cells and are involved in diverse cellular functions including stress responses and apoptosis (11,14,45,46). In particular, many protein kinases and transcription factors are activated due to ROS under the conditions of oxidative stress (6,10,12,14,16,45,46). It is not known whether flavonoid manipulation could stabilize or activate expression of these kinases and transcription factors. Western blot analysis revealed that the rapid phosphorylation of JNK and p38 MAPK in H2O2-exposed HUVEC was substantially downregulated by the flavanol (-)epigallocatechin gallate and the flavonol quercetin. The antioxidant (-)epigallocatechin gallate and quercetin inhibited endothelial apoptosis by selectively acting on JNK and p38 MAPK, which are believed to be components of death pathways triggered by oxidative stress and which converge at the level of transcription regulation of c-Jun. In addition, the cytoprotective effect of hesperetin appeared to be mediated via other survival pathways. On the other hand, apigenin caused endothelial cell death with elevating intracellular ROS via JNK- and p38 MAPK-responsive activation, an effect upstream of caspase activation. Indeed, H2O2-induced apoptosis, if mediated at least in part by ROS-activated JNK and p38 MAPK, was clearly inhibited by quercetin and (-)epigallocatechin gallate, but not by apigenin. Nevertheless, MAPK phosphatase–responsive mechanisms of cytoprotection of (-)epigallocatechin gallate and quercetin against oxidative damage cannot be ruled out. It was reported recently that ERK activation and MAPK phosphatase-1, which downregulate stress-activated protein kinase or JNK, play a role in sphingosine-1-phosphate–mediated inhibition of apoptosis in mouse fibroblast C3H10T 1/2 cells (11). Thus, we propose that inhibition of H2O2 toxicity by (-)epigallocatechin gallate or quercetin could be mediated at least in part via an activation of MAPK phosphatases, and that the quantitative balancing between proapoptotic MAPK and MAPK phosphatase may be responsible for blunting apoptosis induced by oxidative stress and leading to improved cell survival.
In summary, our results provide new insights into the relative contributions of JNK, p38 MAPK, and transcription factors of p53 and c-Jun responsible for effects of (-)epigallocatechin gallate and quercetin on cell survival after oxidant injury (Fig. 7). H2O2-induced endothelial apoptosis was abolished by quercetin, but not by apigenin through blunting ROS-triggered activation of JNK and p38 MAPK. The cytoprotective effect of (-)epigallocatechin gallate may be partially mediated via JNK- and p38 MAPK-dependent pathways and possibly via survival pathways entailing other kinases such as ERK 1/2. (-)Epigallocatechin gallate and quercetin appeared to switch off apoptotic death cascades via inhibiting activation of caspase-3 and most likely via enhancing the intrinsic cellular tolerance against apoptotic triggers. The death-survival signaling pathways might be causally linked to cellular antioxidant systems readily responsive to flavanol-type or flavonol-type flavonoids. Consequently, interventions with antioxidants could be promising in the design and development of new treatment strategies aimed at limiting cellular oxidative damage.
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FOOTNOTES |
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3 Abbreviations used: Ac-DEVD-pNA, DEVD-pNA, Asp-Glu-Val-Asp-p-nitroanilide; DCF-DA, 2',7'-dichlorodihydrofluorescein diacetate; DMSO, dimethyl sulfoxide; DPPH, 1,1-dipheny-2-picrylhydrazyl; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GSH, glutathione; HUVEC, human umbilical vein endothelial cells; JNK, c-Jun N-terminal protein kinase; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimetylthiazol-yl)-diphenyl tetrazolium bromide; NF, nuclear factor; ROS, reactive oxygen species; TBS-T, Tris buffered saline-Tween 20.
Manuscript received 5 October 2004. Initial review completed 24 October 2004. Revision accepted 13 January 2005.
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indicates activation or induction, and 
indicates inhibition or blockade.