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Division of Life Sciences and Silver Biotechnology Research Center, Hallym University, Chuncheon, Korea and * Food and Nutrition, Cheju University, Cheju, 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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40% (P < 0.05) with distinct nuclear condensation and DNA fragmentation. In the H2O2 apoptosis model, the addition of 50 µmol/L of the flavanol (-)epigallocatechin gallate and the flavonol quercetin, which have in vitro radical scavenging activity, partially (P < 0.05) restored cell viability with a reduction in H2O2-induced apoptotic DNA damage. In contrast, the flavones, luteolin and apigenin, at the nontoxic dose of 50 µmol/L, intensified cell loss (P < 0.05) after exposure to H2O2 and did not protect cells from oxidant-induced apoptosis. The flavanones, hesperidin and naringin, did not have cytoprotective effects. The antioxidants, (-)epigallocatechin gallate and quercetin, inhibited endothelial apoptosis, enhanced the expression of bcl-2 protein and inhibited the expression of bax protein and the cleavage and activation of caspase-3. Therefore, flavanols and flavonols, in particular (-)epigallocatechin gallate and quercetin, qualify as potent antioxidants and are effective in preventing endothelial apoptosis caused by oxidants, suggesting that flavonoids have differential antiapoptotic efficacies. The antiapoptotic activity of flavonoids appears to be mediated at the mitochondrial bcl-2 and bax gene level.
KEY WORDS: • flavonoids • endothelial apoptosis • hydrogen peroxide • bcl-2 • caspase-3
Apoptosis, a morphologically and biochemically distinct form of cell death characterized by cell shrinkage without membrane ruptures, cell membrane blebbing, nuclear chromatin condensation and nonrandom DNA fragmentation, is indispensable for physiologic development and homeostasis of tissues and the elimination of diseased cells in multiorganisms (1 ,2 ). Defects in apoptosis have been implicated in neurodegenerative diseases, cancer and autoimmune diseases (2 ,3 ). Oxidative injury after diverse stimuli including clinical and experimental ischemia/hypoxia, reperfusion and inflammation can induce cardiac and endothelial cell apoptosis (4 –7 ), which is a fundamentally different mode of cell death from necrosis. The severity of cellular damage by an oxidant injury determines which mechanism of cell death dominates (8 ). Accordingly, agents or antioxidants that can inhibit production of reactive oxygen species (ROS) can prevent apoptosis (4 ,9 ,10 ). However, the underlying molecular mechanisms by which antioxidative agents protect cells from stimulator-triggered apoptosis remain to be elucidated.
There is currently intense interest in polyphenolic phytochemicals such as flavonoids, proanthocyanidins and phenolic acids. Epidemiologic studies have shown that a high consumption of these polyphenolics is inversely related to the risk of cardiovascular diseases (11 –13 ), and this phenomenon appears to be associated with their antioxidant capacity (14 ). Flavonoids constitute one of the antioxidant phytochemical groups and are found in a large number of fruits and vegetables. There are several subclasses such as flavonols, flavones, isoflavones, flavonones, flavan-3-ols and anthocyanidins. These flavonoids are natural antioxidants that scavenge various types of radicals in aqueous and organic environments (15 –21 ), and anti-inflammatory agents that inhibit adhesion molecules and matrix proteases (22 –24 ). Whether these flavonoids act in vivo as antioxidants or anti-inflammatory agents appears to depend on their bioavailabilities.
On the basis of the literature evidence that flavonoids are antioxidants and have the ability to scavenge free radicals, we examined the effects of these polyphenolic compounds, when applied in submillimolar doses, on apoptosis in H2O2-exposed human umbilical vein endothelial cells (HUVEC). Cells were exposed to H2O2 for 30 min, killing 40% of cells within 24 h. It has been shown that there are differences in the antioxidant capacity among different groups of flavonoids and within each group of flavonoids (16 ,25 ). By measuring cell viability, nuclear morphology, DNA fragmentation and apoptotic gene protein expression, we assessed the antiapoptotic efficacy of various polyphenolic compounds in the vascular endothelium model. To elucidate the capacity of polyphenolic compounds to inhibit oxidant-induced apoptosis, four different subclasses of polyphenolic flavonoids were used, i.e., flavanols [(-)epigallocatechin gallate and (+)catechin]; flavonols (quercetin and myricetin); flavanones (naringin and hesperidin); and flavones (luteolin and apigenin).
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MATERIALS AND METHODS |
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Fetal bovine serum (FBS), penicillin-streptomycin, and all cell growth supplements were purchased from Clonetics (San Diego, CA). Polyphenolic flavonoids [flavanols, (-)epigallocatechin gallate and (+)catechin; flavonols, quercetin and myricetin; flavanones, naringin and hesperidin; and flavones, luteolin and apigenin], M199 chemicals, and 3-(4,5-dimetylthiazol-yl)-diphenyl tetrazolium bromide (MTT) were obtained from Sigma Chemical (St. Louis, MO) as were all other reagents, unless specifically stated elsewhere. All flavonoids were solubilized by dimethyl sulfoxide (DMSO) for culturing with cells (26
); the final culture concentration of DMSO was 
5 g/L.
Measurement of DPPH radical scavenging activity.
The radical scavenging activity of flavonoids was assayed using a stable free radical, 1,1-dipheny-2-picrylhydrazyl (DPPH) by a previously reported method (27 ) with minor modifications. A methanol solution (100 µL) of each flavonoid at different concentrations (10–350 µmol/L) was added to the mixture of 100 mmol/L Tris-HCl buffer (pH 7.4) and 0.2 mmol/L DPPH in methanol. The solution was mixed vigorously and kept for 20 min in the dark. The free radical scavenging activity of each flavonoid was quantified by the decolorization of DPPH at 517 nm. The antioxidant power of flavonoids was expressed as half-maximal scavenging concentration (SC50, their concentration decreasing DPPH 50%).
Primary culture of human vascular endothelial cells.
HUVEC were isolated using collagenase (Worthington Biochemical, Lakewood, NJ), as described elsewhere (28 ). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were incubated in 25 mmol/L HEPES-buffered M199 containing 10% FBS, 2 mmol/L glutamine, 100,000 U/L penicillin, 100 mg/L streptomycin and growth supplements (0.9 g/L bovine brain extract, 0.75 g/L human epidermal growth factor and 75 mg/L hydrocortisone). Cells were passaged at confluence and used within 4–5 passages. Cells were subcultured at 70–80% confluence. Endothelial cells were identified by their cobblestone morphology and uptake of fluorescently labeled acetylated LDL [1.1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; Molecular Probes, Eugene, OR; (28 )].
To determine the dose-viability response of the tested flavonoids, HUVEC were cultured in 25 mmol/L HEPES-buffered M199 containing 10% FBS and 10–100 µmol/L of each tested flavonoid for 24 h.
H2O2-induced oxidant stress.
The cells were preincubated for 30 min with 50 µmol/L of each tested flavonoid, and then incubated for another 30 min with media containing an additional 0.25 mmol/L H2O2. In H2O2 incubations, H2O2 was detoxified by adding catalase (100,000 U/L, Worthington Biochemical) at the end of the 30-min incubation period; this terminated all activity of extracellular H2O2. Non-H2O2–treated control cells were incubated in the same way as those used in the H2O2 protocols. Cells were washed thoroughly with PBS and then resupplied with fresh H2O2-free medium containing 50 µmol/L of the tested flavonoid. Incubation was continued for another 24 h before biochemical and molecular analyses were performed.
Cell viability.
At the end of the 24 h incubation period, the MTT assay was performed to quantitate cellular viability (30
). HUVEC were incubated in fresh medium containing 1 g/L MTT for 3 h at 37°C. After removal of unconverted MTT, the purple formazan product was measured colorimetrically at 
= 570 nm with background subtraction at = 690 nm.
Nuclear morphology.
Nuclear morphology was examined by fluorescence microscopy with an Olympus BX50 fluorescent microscope (Olympus Optical Co., Tokyo, Japan) equipped for fluorescence illumination and for photomicroscopy. After the fixation of HUVEC with ice-cold 4% formaldehyde for 1 h, the nuclear stain Hoechst 33258 (Molecular Probes) was added at a final concentration of 10 mg/L for 15 min to allow uptake and equilibration before microscopic observation. The coverslips were mounted while wet in aqueous mounting solution. Cells containing fragmented or condensed nuclei were considered apoptotic, whereas those containing diffuse and irregular nuclei were considered necrotic (31 ).
In situ cell death detection (TdT-mediated dUTP nick end labeling, TUNEL).
DNA strand breaks were detected using the nick-end labeling technique (32 ). To detect in situ DNA fragmentation, the TUNEL assay with HUVEC was performed following a previously published method (34 ). Cultured cells were labeled using a commercially available end-labeling kit (Boehringer Mannheim, Mannheim, Germany). For the detection and visualization, extra-avidin–conjugated alkaline phosphatase was introduced in an alkaline phosphatase substrate buffer (100 mmol/L Tris-HCl, 5 mmol/L MgCl2, pH 9.5) containing nitro blue tetrazolium (NBT; 50 g/L in 70% dimethylformamide) and bromochloroindolyl phosphate toluidine (50 g/L in 70% dimethylformamide). Photomicroscopy took place under a cover slide with a Olympus CH2 light microscope.
Agarose gel electrophoresis.
The calcium-activated endonuclease produces well-defined DNA fragments (33 ), which can be detected as a DNA ladder on agarose gels. Extraction and electrophoresis of genomic DNA were performed according to methods published elsewhere (34 ). DNA from HUVEC was isolated using 5 g/L sodium N-lauroyl sarcosinate, 10 g/L proteinase K and 10 mg/L RNase A. DNA samples were electrophoresed on a 1.8% agarose gel containing ethidium bromide (0.5 mg/L). For apoptotic alterations to DNA integrity, DNA bands were visualized using an UV transilluminator (Hoefer Scientific Instrument, San Francisco, CA), and photographs of gels were obtained using Polaroid Type 667 positive/negative film (Polaroid Co., Wayland, MA).
Immunocytochemistry.
After endothelial cells were thoroughly washed with Tris buffered saline (TBS), cells were incubated for 20 min with 10% normal goat serum in TBS to block any nonspecific binding. After fixed cells were washed twice with TBS, monoclonal mouse bcl-2 antibody (1:100 dilution in TBS; BD Transductional, San Diego, CA) was added to cells and incubated overnight at 4°C. Cells were washed with TBS and incubated with a fluorescein isothiocyanate–conjugated goat anti-mouse immunoglobulin (Ig)G (1:200 dilution in TBS; Sigma) as a secondary antibody. Images were obtained by a fluorescence microscopy with an Olympus BX51 fluorescent microscope.
Western blot analysis.
Whole-cell extracts were prepared from HUVEC in a lysis buffer containing 10 g/L ß-mercaptoenthanol, 1 mol/L ß-glycerophosphate, 0.1 mol/L Na3VO4, 0.5 mol/L NaF and protease inhibitor cocktail. Cell lysates containing equal amounts of total protein were fractionated by electrophoresis on 12 or 15% SDS-PAGE gels and transferred onto a nitrocellulose membrane. Nonspecific binding was blocked by soaking the membrane in a TBS-T buffer [0.5 mol/L Tris-HCl (pH 7.5), 1.5 mol/L NaCl, and 1 g/L Tween 20] containing 50 g/L nonfat dry milk for 3 h. The membrane was incubated for 3 h with a primary antibody [monoclonal mouse anti-human bcl-2 (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal mouse anti-human bax (1:500 dilution; BD Transductional), or polyclonal rabbit anti-human caspase-3 (1:1000 dilution; Cell Signaling Technology, Beverly, MA)]. After five washes with TBS-T, the membrane was then incubated for 1 h with a goat anti-mouse IgG or a goat anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000 dilution, Jackson ImmunoResearch, West Grove, PA). The levels of bcl-2 and bax proteins and caspase-3 protein were determined using Supersignal West Pico chemiluminescence detection reagents (Pierce, Rockford, IL) and Konica X-ray film (Konica, Tokyo, Japan). Incubation with polyclonal rabbit ß-actin antibody (1:1000 dilution, Santa Cruz Biotechnology) was also performed for the comparative control.
Statistical analysis.
The results are presented as mean ± SEM. Statistical analyses were conducted using SAS statistical software package version 6.12 (SAS Institute, Cary, NC). One-way ANOVA was used to determine effects of both H2O2 and individual flavonoids. The differences among 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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Among the tested flavonoids in the cell-free system, the flavanols, (-)epigallocatechin gallate and (+)catechin, and the flavonols, quercetin and myricetin, had high DPPH scavenging activities with half-maximal scavenging concentrations, SC50, lower than 10 µmol/L (Table 1 ). These compounds were more potent than L-ascorbic acid, the classic reference antioxidant, with 14.0 µmol/L SC50. In contrast, the flavanones, naringin and hesperidin, and the flavones, luteolin and apigenin, were not active even at concentrations up to 100 µmol/L.
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Except for apigenin, all other flavonoids tested showed little cytotoxicity even at 100 µmol/L when incubated with cells for 24 h, but at concentrations 50 µmol/L, apigenin did not significantly decrease cell viability (P > 0.05, data not shown). Accordingly, the maximal nontoxic concentration of all the flavonoids used for culture experiments was 50 µmol/L. During 24-h incubations, the cytotoxicity of H2O2 increased dose dependently at concentrations ranging from 0.05 to 0.25 mmol/L with a decrease in cell viability by 40% at 0.25 mmol/L. Data in tables and figures were obtained when HUVEC were treated with 0.25 mmol/L H2O2 for 30 min (Fig. 1
,upper panel).
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Nuclear condensation and DNA fragmentation.
The H2O2 produced cells that exhibited fragmented and/or condensed nuclei within 24 h and there were nonnucleated cell fragment apoptotic bodies (Fig. 2 , arrows in microphotographs with Hoechst 33258 nuclear stain). In the H2O2-untreated control cells, no signs of morphological nuclear damage or chromatin condensation were observed (Fig. 2) . The nuclear morphology of cells exposed to H2O2 with (-)epigallocatechin gallate, (+)catechin or quercetin was comparable to, if not indistinguishable from that of the H2O2-untreated control cells. In marked contrast, the morphology of cells treated with H2O2 in the presence of luteolin or apigenin compared poorly with that of the H2O2-untreated cells but well with that of cells treated with H2O2 alone. In cells exposed to H2O2 and incubated with luteolin and apigenin, cell density was markedly reduced and nuclear condensations and appearance of apoptotic body–like structures became increasingly frequent in response to H2O2 (Fig. 2) .
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Immunostaining assays were used to compare the effects of the selected flavonoids on expression of bcl-2 in the presence of H2O2. As expected, there was substantial cytoplasmic staining in the H2O2-free control cells, whereas this staining did not occur in cells exposed to H2O2 alone, suggesting the down-regulation of bcl-2 expression at the single cell level (Fig. 5 ). However, addition of (-)epigallocatechin gallate or quercetin to H2O2-exposed cells increased the staining of bcl-2. In marked contrast, the flavone apigenin did not increase the expression of bcl-2 reduced by H2O2, suggesting that not all of the flavonoids can attenuate H2O2-induced apoptotic gene expression in the vascular endothelium.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Oxidative stress contributes to cellular injury after experimental ischemic reperfusion (5 ) and appears to be the common apoptotic mediator, most likely via lipid peroxidation (39 ). The literature has supported the role of ROS in apoptotic cell death. This study demonstrated that treatment of HUVEC with H2O2, a precursor of other ROS such as highly reactive hydroxyl radicals, leads to cell death via apoptotic processes. These findings are consistent with previous reports showing apoptotic death processes in various types of cells induced by H2O2 (34 ,40 ). Agents that inhibit production of ROS or enhance cellular antioxidant defenses can prevent apoptosis and protect cells from the damaging effects of oxygen radicals (4 ,9 ,10 ). Consistent with these reports, the flavanols and flavonols, in particular (-)epigallocatechin gallate and quercetin, had high antiapoptotic activities in the H2O2-treated vascular endothelial cells. In contrast, at nontoxic doses, the flavone-type flavonoids, luteolin and apigenin, had no antiapoptotic effects and intensified the apoptosis-like alterations, including nuclear condensation and DNA fragmentation. These results were in agreement with those obtained for DPPH scavenging activity, indicating that there is a major structural feature responsible for the antiapoptotic activity against reactive radicals (Fig. 7) . In addition, the antiapoptotic activities of polyphenolic flavonoids proved to be diverse.
Dietary flavonoid supplementation reduces the incidence of myocardial infarction (12 ) and plasma LDL cholesterol concentration (11 ), and alleviates lipid peroxidation (41 ). Rodent feeding studies have supported the possibility that certain polyphenolics may have antioxidant functions in vivo (42 ,43 ). However, the underlying mechanisms for their cardio- and cytoprotective actions are still unknown. Their antioxidant actions in oxidant-induced endothelial apoptosis have been shown to be mediated through their H+-donating properties (38 ). However, not all flavonoids tested in this study exhibited antiapoptotic activity in the H2O2-treated cells, suggesting that other mechanisms for cytoprotection against oxidant insults must be involved.
(-)Epigallocatechin gallate and quercetin, with potent antiapoptotic actions, and apigenin, which was not antiapoptotic, were further tested for their effects on cascade events of the apoptotic pathway. It has been proposed that ROS down-regulate the antiapoptotic bcl-2 gene and up-regulate the pro-apoptotic bax gene, as shown in the present study. Bcl-2–expressing cells have been reported to have the enhanced antioxidant capacity that suppresses oxidative stress signals (44 ,45 ). In addition, enhancement of ROS has been reported to elicit translocation of cytosolic bax to mitochondria, and to activate bax to induce the release of cytochrome c from mitochondria, stimulating caspases (46 ). The H2O2-induced decrease in bcl-2 protein expression and the increase in bax expression were blocked in (-)epigallocatechin gallate- and quercetin-treated cells, providing compelling evidence in support of their potent antiapoptotic actions.
H2O2 strongly activated caspase-3, and the activation was partially blocked by (-)epigallocatechin gallate and almost completely by quercetin. The substantial difference between these flavonoids in inhibiting the activated caspase-3 appeared to be responsible for the difference in their antiapoptotic activities. (-)Epigallocatechin gallate and quercetin appeared to switch off the apoptotic death cascade by inhibiting the activation of caspase-3 and likely by enhancing the intrinsic cellular tolerance against apoptotic triggers. Conversely, the H2O2-activated caspase-3 was sustained in apigenin-treated cells that had no antiapoptotic ability. Thus, our findings suggest that the antiapoptotic flavonoids may function by acting selectively through various endothelial death signaling cascades. In addition, phytochemicals affect multiple signaling pathways that converge at the level of transcriptional regulation, i.e., mitogen-activated protein kinase–responsive pathways (47 ).
In summary, this endothelial cell model demonstrates that there are differences in antiapoptotic capacity among flavonoids, which appears to stem from their disparate chemical structures. Unlike flavones, the flavanol (-)epigallocatechin gallate and the flavonol quercetin protected the endothelium from oxidant-induced apoptosis. This antiapoptotic protection was possibly mediated at least in part by a mechanism linked to proapoptotic bax blockade and antiapoptotic bcl-2 activation. It is crucial to elucidate the precise sites of action of antiapoptotic flavonoids in the sequence of events that regulate oxidant-induced cell death and to further evaluate the potential of dietary flavonoids as cardio- and cytoprotective agents.
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FOOTNOTES |
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3 Abbreviations used: DMSO, dimethyl sulfoxide; DPPH, 1,1-dipheny-2-picrylhydrazyl; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cells; Ig, immunoglobulin; MTT, 3-(4,5-dimetylthiazol-yl)-diphenyl tetrazolium bromide; NBT, nitro blue tetrazolium; ROS, reactive oxygen species; SC50, half-maximal scavenging concentration; TBS-T, Tris buffered saline-Tween 20; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
Manuscript received 16 September 2002. Initial review completed 23 October 2002. Revision accepted 10 December 2002.
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