Generation of hydrogen peroxide primarily contributes to the induction of Fe(II)-dependent apoptosis in Jurkat cells by ()-epigallocatechin gallate
Hiroshi Nakagawa1,2,
Keiji Hasumi2,
Je-Tae Woo3,
Kazuo Nagai3 and
Masaaki Wachi1,4
1 Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, 2 Department of Applied Biological Science, Tokyo Noko University, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509 and 3 Department of Biological Chemistry, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan
4 To whom correspondence should be addressed Email: mwachi{at}bio.titech.ac.jp
 |
Abstract
|
---|
Although ()-epigallocatechin gallate (EGCG) has been reported to induce apoptosis in a variety of tumor cells, detailed mechanisms remain to be explored. In the present study, we investigated the antitumor mechanism of EGCG by using human T-cell acute lymphoblastic leukemia Jurkat cells. We focused on the involvement of reactive oxygen species, as we found previously that EGCG caused apoptotic cell death in osteoclastic cells due mainly to promotion of the reduction of Fe(III) to Fe(II) to trigger Fenton reaction, which affords hydroxyl radical from hydrogen peroxide [H2O2 + Fe(II)
OH + OH + Fe(III)]. EGCG (12.550 µM) decreased the viability of Jurkat cells and caused concomitant increase in cellular caspase-3 activity. Catalase and the Fe(II)-chelating reagent o-phenanthroline suppressed the EGCG effects, indicating involvements of both H2O2 and Fe(II) in the mechanism. Unexpectedly, epicatechin gallate (ECG), which has Fe(III)-reducing potency comparable with EGCG, failed to decrease the viability of Jurkat cells, while epigallocatechin (EGC), which has low capacity to reduce Fe(III), showed cytotoxic effects similar to EGCG. These results suggest that, unlike in osteoclastic cells, a mechanism other than Fe(III) reduction plays a role in catechin-mediated Jurkat cell death. We found that EGCG causes an elevation of H2O2 levels in Jurkat cell culture, in cell-free culture medium and sodium phosphate buffer. Catechins with a higher ability to produce H2O2 were more cytotoxic to Jurkat cells. Hydrogen peroxide itself exerted Fe(II)-dependent cytotoxicity. Amongst tumor and normal cell lines tested, cells exhibiting lower H2O2-eliminating activity were more sensitive to EGCG. From these findings, we propose the mechanism that make catechins cytotoxic in certain tumor cells is due to their ability to produce H2O2 and that the resulting increase in H2O2 levels triggers Fe(II)-dependent formation of highly toxic hydroxyl radical, which in turn induces apoptotic cell death.
Abbreviations: EC, ()-epicatechin; ECG, ()-epicatechin gallate; EGC, ()-epigallocatechin; EGCG, ()-epigallocatechin gallate; MTT reagent, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; NHDF, normal human dermal fibroblast; PBS(), phosphate-buffered saline without Ca2+ and Mg2+
 |
Introduction
|
---|
Catechins, contained in some types of foods and plants (especially in green tea), are a subspecies of polyphenols and have been demonstrated in vitro to have a wide range of pharmacological properties such as antioxidative (1,2), antibacterial (3), antimutagenic (4), antiresorptive (5) and antitumor effects (628). Among these effects, the most extensively studied one has been the antitumor effect in which several characteristic phenomena of apoptosis were observed: cell cycle arrest (611), injury of DNA (821) and activation of caspases (1723). The cytotoxicity of catechins has been recognized to be relatively specific to tumor cells as compared with normal cells (8,10,14,23). Phases I and II clinical trials and in vivo study were performed with green tea extract as an anticancer drug, and catechins have been demonstrated to be beneficial substances with little side effects (2931).
The in vitro antitumor mechanism of ()-epigallocatechin gallate (EGCG), a major catechin in green tea, has been suggested to be modulation of the expression of key molecules in cell cycle progression (cyclin kinase inhibitor, cyclin, cyclin-dependent kinase and p27Kip1) (6,7,10,11) and in transcription (inhibitor of nuclear factor-
B) (7), activation of mitogen-activated protein (MAP) kinase cascade (especially apoptosis-regulating kinase-1, MAP kinase kinase, c-Jun N-terminal kinase and p38 MAP kinase) (21,22), inhibition of telomerase (16) and interaction with Fas (19). However, comprehensive mechanisms to explain the diverse effects of EGCG in causing apoptotic cell death remain to be explored.
In the previous study, we found that o-phenanthroline, a Fe(II)-chelating reagent, and catalase, a H2O2-scavenging enzyme, suppressed EGCG-induced apoptotic cell death in cultured osteoclastic cells and demonstrated in a cell-free system that reduction of Fe(III) by EGCG triggered a Fenton reaction to form a highly reactive hydroxyl radical from H2O2 [H2O2 + Fe(II)
OH + OH + Fe(III)] (5). The present study aims to test whether similar mechanisms are involved in the antitumor effect of EGCG in vitro using human T-cell acute lymphoblastic leukemia Jurkat cells. The results demonstrated that EGCG caused Fe(II)- and H2O2-dependent Jurkat cell apoptosis. However, experiments using EGCG analogs with a different potency to reduce Fe(III) demonstrated that, unlike in osteoclastic cells, elevation of H2O2 levels in culture rather than reduction of Fe(III) plays an important role in the cytotoxic mechanism. Exogenously added H2O2 showed Fe(II)-dependent cytotoxicity, and the catechin cytotoxicity changed among cells with a different ability to eliminate H2O2. We propose a novel mechanism that the EGCG-mediated generation of H2O2 primarily triggers Fe(II)-dependent formation of highly toxic molecules (possibly hydroxyl radicals), which in turn induce apoptotic cell death in Jurkat cells.
 |
Materials and methods
|
---|
Chemicals
EGCG, o-phenanthroline hydrochloride, human recombinant superoxide dismutase (SOD), hydrogen peroxide (H2O2) and 0.1% (w/v) xylenol orange solution were purchased from Wako Pure Chemical Industries (Osaka, Japan). ()-Epicatechin (EC), ()-epicatechin gallate (ECG) and ()-epigallocatechin (EGC) were from Nagara Science (Gifu, Japan). RPMI-1640 medium (Cat. No. R8758) and Dulbecco's modified Eagle's medium (DMEM, Cat. No. D6046), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT reagent), catalase from bovine liver and D-sorbitol was obtained from Sigma Chemical (St Louis, MO). Protease inhibitor cocktail (CompleteTM), 0.25 M ammonium iron(II) sulfate solution and acetyl-Asp-Glu-Val-Asp-
-(4-methyl-coumaryl-7-amide) (Ac-DEVD-MCA) were obtained from Roche (Mannheim, Germany), Kanto Chemical (Tokyo, Japan) and Peptide Institute (Osaka, Japan), respectively.
Cell culture
HeLa, HL-60, Jurkat and U937 cell lines were maintained in medium A [RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (Valley Biomedical, Winchester, VA), 100 U/ml penicillin and 100 µg/ml streptomycin] at 37°C under 5% CO2 and 95% air. Normal human dermal fibroblast (NHDF) was maintained in DMEM supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C under 5% CO2 and 95% air. HeLa cells and NHDF were transferred to 96-well culture plates (Corning, Corning, NY) at 1 x 105 cells/well or 6-well culture plates (Corning, Corning, NY) at 4 x 105 cells/well 12 h prior to use in experiments. All the cells were cultured in medium A (not containing sodium pyruvate) in experimental use.
MTT assay
HL-60, Jurkat and U937 cells (4 x 105 cells/well) placed on 6-well culture plates and HeLa cells and NHDF pre-cultured for 12 h on 96-well culture plates were treated with catechins (EC, 50 µM; ECG, 50 µM; EGC, 50 µM; EGCG, 050 µM) (dissolved in methanol, the final concentrations of which in culture medium were <0.5%) or H2O2 (50 µM) for 6 h in medium A. After incubation, HL-60, Jurkat and U937 cells were washed with fresh medium A and placed on 96-well culture plates at 1 x 105 cells/well in 100 µl of medium A containing 50 µg of MTT reagent. HeLa cells and NHDF were also incubated with 100 µl of medium A containing 50 µg of MTT reagent after washing with fresh medium A. After incubation for 2 h, 100 µl of 10% (w/v) SDS in phosphate-buffered saline without Ca2+ and Mg2+ [PBS()] was added to solubilize MTT-formazan. After overnight incubation, absorbance at 595 nm was measured. MTT reagent-reducing activity of the cells (MTT-reducing activity) was expressed as percentage of control or percent inhibition by catechins. Data are expressed as the mean ± SD of triplicate cultures in one of two to three experiments. For statistical analysis of the results, groups were compared with Student's t-test.
Assay for caspase-3 activity
Jurkat cells (1 x 106 cells/well of 6-well culture plates) were treated with EGCG (050 µM) for 6 h in medium A. After incubation, cells were collected by centrifugation and washed with ice-cold PBS(). The cells were lysed in lysis buffer [100 mM TrisHCl (pH 7.5), 1 mM DTT, 1% (v/v) Triton X-100 and protease inhibitor cocktail (CompleteTM)], and then passed 10 times through a 27G needle. After centrifugation at 3000 r.p.m. for 10 min, the supernatant [50 µg protein, determined using Bio-Rad Protein Assay kit (Bio-Rad) with bovine serum albumin as a standard] was incubated with 20 µM Ac-DEVD-MCA at 37°C for 1 h. Fluorescence of the liberated aminomethylcoumarin was determined by excitation at 365 nm and emission at 450 nm using a MTP-32 microplate reader (Corona Electric).
Assay for Fe(III)-reduction
To assess Fe(III)-reducing activity of catechins, catechins (1 µM) were added to 1 ml of working solution (1.2 mg/ml o-phenanthroline and 1 mM FeCl3), and the mixture was incubated at room temperature for 20 s. After the incubation, absorbance of Fe(II)o-phenanthroline complex at 510 nm was immediately measured. Fe(III)-reducing activity of catechins was assessed from the amount of Fe(II)o-phenanthroline complex in solutions and expressed as relative to EGCG, of which activity is taken as 100%. Data are expressed as the mean ± SD of triplicate determinations in one of two to three experiments.
Measurement of the concentration of H2O2 in serum-containing medium A in the presence or absence of Jurkat cells
Catechins (50 µM) were added to 6-well culture plates filled with 2 ml of medium A containing Jurkat cells (1 x 106 cells/well) or no cells, and the plates were placed at 37°C under 5% CO2. After 15, 30, 60, 120, 240 and 360 min, part of the medium was collected to measure the concentration of H2O2 by the ferrous ion oxidationxylenol orange method with slight modification (32,33). Briefly, medium (30 µl) was mixed with 0.3 ml of working solution [250 µM ammonium iron(II) sulfate, 25 mM H2SO4, 100 mM sorbitol and 125 µM xylenol orange], followed by vortexing and incubation at room temperature for 20 min. After incubation, absorbance of Fe(III)xylenol orange complex at 595 nm was measured. The concentration of H2O2 was calculated from a standard curve, which was obtained by determining H2O2 concentrations immediately after addition of H2O2 into the culture medium. This assay method was applicable to determine H2O2 concentrations as low as 0.2 µM. Data are expressed as the mean ± SD of three independent experiments. For statistical analysis of the results, groups were compared with Student's t-test.
Measurement of the concentration of H2O2 in various solutions
EGCG (50 µM) was added to 6-well culture plates containing serum-free medium A or 100 mM phosphate buffer (pH 5.8, 6.8 and 7.8). Where indicated, 100 U/ml catalase or 500 U/ml SOD was included in the solution. Serum-free medium A and phosphate buffers were incubated at 37°C under 5% CO2 and at 37°C under air, respectively. After incubation for 1 h, measurement of H2O2 concentration was carried out by the ferrous ion oxidationxylenol orange method as described above. The concentration of H2O2 was calculated from a standard curve that had been obtained by determining H2O2 concentrations immediately after addition of H2O2 into each solution. Data are expressed as the mean ± SD of three independent experiments. For statistical analysis of the results, groups were compared with the Student's t-test.
Assay for H2O2-eliminating activity in cells
HL-60, Jurkat and U937 cells (4 x 105 cells/well) were placed on 6-well culture plates and HeLa cells and NHDF were pre-cultured for 12 h on 6-well culture plates in medium A. After addition of 50 µM H2O2, each culture was incubated at 37°C under 5% CO2 for 15, 30, 45 and 60 min. The concentration of H2O2 remaining in the medium was determined by the ferrous ion oxidationxylenol orange method. Data are expressed as the mean ± SD of triplicate determinations in one of two to three experiments.
Absorption spectrum
EGCG (50 µM) was added to 100 mM phosphate buffer at pH 5.8, 6.8 or 7.8. After incubation for 1 h at 25°C, the solutions were subjected to measurement of absorption spectrum on a model 320 spectrophotometer (Hitachi Instruments Service, Tokyo, Japan) at a wavelength range of 200400 nm.
 |
Results
|
---|
Hydrogen peroxide- and Fe(II)-dependent cytotoxicity of EGCG in Jurkat cells
To investigate antitumor mechanism of EGCG, human T-cell acute lymphoblastic leukemia Jurkat cells, widely used in the study of apoptosis, were used in the present study. The cytotoxic effect of EGCG in Jurkat cells was examined by MTT assay (Figure 1A). MTT-reducing activity of Jurkat cells was dose-dependently decreased in the culture with 12.550 µM EGCG for 6 h. The decrease was accompanied by an increase of caspase-3 activity in the cells (Figure 1B). In both experiments, vehicle (methanol) alone did not affect both activities at 0.5% (v/v) (Figure 1A and B). These results indicate that EGCG cause apoptotic cell death in Jurkat cells and that the MTT-reducing activity of Jurkat cells represents the viability of the cells. As shown in Figure 1C and D, catalase (100 U/ml) completely suppressed the cytotoxic inhibitory effect of EGCG. o-Phenanthroline, a Fe(II)-specific chelator, also restored the Jurkat cell viability, whereas its effect was partial (75% restoration, Figure 1C). These results that not all but most of the cytotoxic effects of EGCG in Jurkat cells was mediated both by H2O2 and Fe(II), suggests an involvement of H2O2- and Fe(II)-dependent hydroxyl radical formation in the EGCG-mediated cell death.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1. Effects of EGCG on the viability of Jurkat cells. Jurkat cells were treated in medium A with EGCG (A and B, indicated concentrations; C and D, 50 µM) or vehicle [0.5% (v/v) methanol, open circle and open column] for 6 h in the presence (C and D) or absence (A and B) of 100 U/ml catalase (CL) or 50 µM o-phenanthroline (PNT). (A and C) After incubation, cells were further incubated in fresh medium A containing MTT reagent for 2 h to determine MTT-reducing activity. Data are expressed as percentage of control (mean ± SD of triplicate cultures). *P < 0.005 as compared with control (A) or between the indicated groups (C). (B and D) After incubation, cell lysates were prepared, and then caspase-3 activity in the lysates was determined using fluorogenic substrate. Similar results were obtained in two other experiments.
|
|
EGCG-mediated H2O2 generation causes cell death in Jurkat cells
To elucidate the H2O2- and Fe(II)-dependent cytotoxic mechanism of EGCG in Jurkat cells, three EGCG analogs (EC, ECG and EGC) (Figure 2A) with different activities to reduce Fe(III) were tested for cytotoxicity. Among the three analogs, only EGC decreased the viability of Jurkat cells at 50 µM, and the others showed no effect at 50 µM (Figure 2B). EGC showed a three times lower Fe(III)-reducing activity than the non-cytotoxic analog ECG, which is comparable with EGCG in the activity to reduce Fe(III) (Figure 2B and C). These results partially conflict with the previous observations that osteoclastic cell death is caused by Fenton reaction due mainly to a EGCG-mediated reduction of Fe(III) to Fe(II). Thus, these results suggest that a mechanism other than Fe(III) reduction is involved in the EGCG effects in Jurkat cell death. Although hydrogen peroxide, that ubiquitously exists in cells, can be eliminated by antioxidative systems (3436), an increase of H2O2 levels should cause Fe(II)-dependent radical formation. So we next examined the levels of H2O2 in Jurkat cell culture. Cultures in the presence of cytotoxic catechins (EGC and EGCG) showed higher levels of H2O2 than those in the presence of non-cytotoxic catechins (EC and ECG) (Figure 2B and D). Similar but higher H2O2 accumulations were observed when 50 µM catechins were added to cell- and serum-free medium A, followed by incubation for 1 h at 37°C (EC, 2.9 ± 0.8 µM; ECG, 7.9 ± 1.6 µM; EGC, 43 ± 2.0 µM; EGCG, 52 ± 2.2 µM). These results indicate that cytotoxic catechins produce H2O2. Since the activity of catechins to generate H2O2 was related to their cytotoxicity (Figure 2B and D), cytotoxicity of exogenously added H2O2 was examined. As shown in Figure 3A, exogenous H2O2 decreased viability of Jurkat cells at H2O2 concentrations comparable with those generated in Jurkat cell culture in the presence of EGCG (3.12550 µM). The cytotoxic effect of H2O2 was suppressed by 100 U/ml catalase and 50 µM o-phenanthroline (Figure 3B). Once again, the effect of o-phenanthroline was partial, and this result suggests that there are Fe(II)-dependent and -independent mechanisms in H2O2 cytotoxicity as well as in EGCG cytotoxicity. Nevertheless, all the results shown here suggest that the production of H2O2 by EGCG primarily contributes to the Fe(II)-dependent cytotoxicity in Jurkat cells.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2. Involvement of H2O2 in cytotoxic effect of catechins. (A) Structures of catechins. (B) Jurkat cells were treated with 50 µM catechins for 6 h in medium A. After incubation, cells were further incubated in fresh medium A containing MTT reagent for 2 h to determine MTT-reducing activity. Data are expressed as inhibition of MTT-reducing activity (%) compared with control (mean ± SD of triplicate cultures). (C) Each catechin (1 µM) was added to a mixture of 1.2 mg/ml o-phenanthroline and 1 mM FeCl3. Twenty seconds after the addition, the absorbance at 510 nm was measured to determine the amount of Fe(II). The amounts of Fe(II) formed after incubation with catechins are expressed as relative to that after incubation with EGCG, which is taken as 100%. Data represent the mean ± SD of triplicate determinations. (D) Each catechin (50 µM) was added to Jurkat cell culture in medium A. Thirty minutes after the addition of catechins, the concentration of H2O2 in the medium was determined by the ferrous ion oxidationxylenol method. Data are expressed as the mean ± SD of three independent experiments.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3. Effects of H2O2 on the viability of Jurkat cells. Hydrogen peroxide (A, indicated concentrations; B, 50 µM) was added to Jurkat cell culture with (B) or without (A) 100 U/ml catalase (CL) or 50 µM o-phenanthroline (PNT). After incubation for 6 h, cells were further incubated in fresh medium A containing MTT reagent for 2 h to determine MTT-reducing activity. Data are expressed as percentage of control (mean ± SD of triplicate cultures). *P < 0.005 as compared with control (A) or between the indicated groups (B).
|
|
Cells may be able to escape from Fenton reaction-mediated cell death by eliminating H2O2 in spite of the ubiquitous existence of H2O2 and Fe(II) in cells. As shown in Figure 4A, H2O2 concentration increased rapidly after addition of EGCG to Jurkat cell culture, and the levels decreased time-dependently, suggesting an existence of H2O2-eliminating ability in Jurkat cells. In the presence of catalase, EGCG-mediated H2O2 generation was not observed (Figure 4A). If cytotoxicity of EGCG is primarily due to its activity to generate H2O2, cells with a higher capacity to eliminate H2O2 might be more resistant to EGCG cytotoxicity. To test this possibility, five different cell lines were examined for their ability to eliminate H2O2 as well as for their sensitivity to EGCG and H2O2. As shown in Figure 4B, all cell lines tested had an evident but distinct capability to eliminate H2O2. The half-life of the added H2O2, as determined from the pseudo-first-order decay curve, was
10 min in HeLa and NHDF,
14 min in U937 and 1820 min in Jurkat and HL-60 cell cultures. As expected, HL-60 cells, as well as Jurkat cells, were sensitive to both EGCG and H2O2 (Figure 4C). On the other hand, HeLa, NHDF and U937 cells, which have a higher ability to eliminate H2O2, were resistant to EGCG and H2O2 at concentrations up to 50 µM.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4. Hydrogen peroxide-eliminating ability and EGCG susceptibility in various cell lines. (A) EGCG (50 µM) was added to Jurkat cell culture in the presence (closed circles in A) or absence (open circles in A) of 100 U/ml catalase. After indicated time, the concentration of H2O2 in the medium was determined by the ferrous ion oxidationxylenol method. Data are expressed as the mean ± SD of three independent experiments. *P < 0.05 as compared with both zero time and catalase-treated cultures at each time point. (B) Hydrogen peroxide (50 µM) was added to the cultures of HeLa (open triangles), HL-60 (open squares), Jurkat (open diamonds), NHDF (cross) and U937 (plus) cells as well as to cell-free medium A (open circles). After indicated times, the concentration of H2O2 in the medium was determined by the ferrous ion oxidationxylenol method. Data are expressed as the mean ± SD of three determinations. (C) Hydrogen peroxide-eliminating ability and sensitivities to EGCG and H2O2 in a variety of cell. aThe concentrations of EGCG and H2O2 to reduce the cell viability by 50% (IC50) as determined by the MTT assay. bHalf life of H2O2 in cell cultures calculated from the pseudo-first-order decay curves in (B).
|
|
Mechanism of EGCG-mediated generation of H2O2
As mentioned above, catechins promote H2O2 generation in Jurkat cell culture. We next examined dose dependency of EGCG and the effect of catalase on H2O2 generation. As shown in Figure 5A, the generation of H2O2 gradually increased as the concentration of EGCG elevated (12.550 µM). The increase in H2O2 levels was completely inhibited by 100 U/ml catalase. The generation of H2O2 was also observed in the absence of Jurkat cells and serum (Figure 5B). These results indicate that the EGCG-mediated H2O2 generation is a cell- and serum-independent process. On the other hand, EGCG failed to generate H2O2 in pure water, ethanol or methanol (data not shown). So we explored the essential constituents of the medium for EGCG-induced H2O2 generation. As shown in Figure 6A, EGCG-mediated H2O2 generation was detected in 100 mM sodium phosphate buffer at pH 7.8, but H2O2 generation was very small at pH 6.8 and not detected at pH 5.8. This result suggests that EGCG-mediated H2O2 generation is dependent on pH levels. The absorption spectrum of EGCG in 100 mM sodium phosphate buffer at pH 5.8 was distinct from that at pH 7.8: the absorption at 300370 nm increased with elevation of the pH levels (Figure 6B). This indicates a pH-dependent change in aromatic resonance structure in the EGCG molecule. The pKa1 value for EGCG is reported to be at 7.597.75 (37,38), and the change in absorption spectrum may represent the deprotonation of phenolic hydroxyl group(s).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5. Effects of EGCG on H2O2 generation under various conditions. EGCG (indicated concentrations) was added to Jurkat cell culture (A), cell-free medium A (B) or cell- and serum-free medium A (B) in the presence (closed symbols) or absence (open symbols) of 100 U/ml catalase. After 1 h, the concentration of H2O2 in the medium was determined by the ferrous ion oxidationxylenol method. Data are expressed as the mean ± SD of three independent experiments. *P < 0.05; **P < 0.005, as compared with control.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6. Generation of H2O2 in sodium phosphate buffer by EGCG. EGCG (50 µM) was added to 100 mM sodium phosphate buffer at pH 5.8 (A and solid line a in B), 6.8 (A and broken line b in B) or 7.8 (A, bold solid line c in B and C). (A and C) One hour after the addition of EGCG, the concentration of H2O2 in the buffers was determined by the ferrous ion oxidationxylenol method. Data are expressed as the mean ± SD of three independent experiments. *P < 0.005, as compared between the indicated groups. (B) One hour after the addition of EGCG to 100 mM phosphate buffers, absorption spectra were measured at a wavelength range of 200400 nm. Similar results were obtained in two other experiments.
|
|
As H2O2 can be generated from superoxide anion (
) under physiological conditions (
), we next examined the involvement of
in the EGCG-mediated H2O2 generation by using SOD, which catalyses H2O2 formation from
. SOD inhibited EGCG-mediated H2O2 generation in 100 mM sodium phosphate buffer at pH 7.8 (Figure 6C). SOD also inhibited EGCG-mediated H2O2 generation in Jurkat cell culture and suppressed cytotoxicity of EGCG to Jurkat cells in a dose-dependent manner (20500 U/ml) (Figure 7AC), while bovine serum albumin, tested as a control, did not exert such activities at the same concentration (2 mg protein/ml) as those of SOD (data not shown). On the other hand, the cytotoxic effect of H2O2 was not suppressed by SOD (Figure 7D), excluding the possibility that
plays a role after H2O2 is formed. These results suggest that
participates in the process of H2O2 generation by EGCG.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 7. Suppression by SOD of EGCG-mediated H2O2 generation and Jurkat cell death. Jurkat cells were cultured in the presence or absence of either SOD (A and B, indicated concentrations; C and D, 500 U/ml) or catalase (C, 100 U/ml) in medium A. (A, B and C) After incubation with 50 µM EGCG or 50 µM H2O2 (D) for 6 h, cells were further incubated in fresh medium A containing MTT reagent for 2 h to determine MTT-reducing activity (A and D) or directly used for lysate preparation to determine caspase-3 activity (C). (A and D) Data are expressed as percentage of control (mean ± SD of triplicate cultures). (B) Similar results were obtained in two other experiments. (C) Thirty minutes after the addition of EGCG (50 µM), the concentration of H2O2 in the medium was determined by the ferrous ion oxidationxylenol method. Data are expressed as the mean ± SD of triplicate determinations. *P < 0.005, as compared between the indicated groups.
|
|
 |
Discussion
|
---|
In the present study, we investigated the antitumor mechanism of EGCG by using human T-cell acute lymphoblastic leukemia Jurkat cells. EGCG was found to reduce viability of the cells and activate cellular caspase-3. These results were consistent with the previous findings that the antitumor effect of EGCG resulted from apoptosis (1723). Yang et al. reported that catalase suppressed the antitumor effect of EGCG (12,24), indicating a major role of H2O2 in the cytotoxic effect of EGCG. The present study demonstrated that the Fe(II)-specific chelator o-phenanthroline, as well as catalase, suppressed the cytotoxic effect of EGCG in Jurkat cells. Although the suppression by catalase was complete, the effect of o-phenanthroline was partial (
75% restoration of viability). In addition, o-phenanthroline partially blocked the H2O2 cytotoxicity itself (
60% restoration of viability). These results suggest that cytotoxicity of EGCG is due to its ability to generate H2O2 and that resulting H2O2 exerts cytotoxicity through two distinct mechanisms, Fe(II)-dependent and -independent ones. In Jurkat cells, the Fe(II)-dependent mechanism may play a major role because o-phenanthroline restores
75% of EGCG cytotoxicity. Our results also suggest that EGCG itself is not directly involved in Jurkat cell death, but a substance formed in the presence of H2O2 and Fe(II) may act as a direct effector in the cytotoxic effect. One possible candidate is hydroxyl radical, a highly reactive oxygen species that can be formed from H2O2 in the presence of Fe(II) through a Fenton reaction.
Our previous findings have suggested that EGCG acted mainly in reducing Fe(III) to Fe(II), which in turn promoted radical formation via Fenton chemistry, leading to apoptotic cell death in osteoclastic cells (5). However, experiments using EGCG analogs with a different activity to reduce Fe(III) suggested that the Fe(III)-reducing activity of catechins was not related to their cytotoxic effects in the Jurkat cell system. On the other hand, we have found that catechins with a pyrogallol moiety have H2O2-producing activity and this activity is responsible for the cytotoxic effect in Jurkat cells. The amount of H2O2 generated by EGCG was demonstrated to be sufficient to exert Fe(II)-dependent cytotoxicity in Jurkat cells. Antunes and Cadenas have shown that, following the exposure to H2O2, the cytosolic concentration of H2O2 in Jurkat cells reaches a steady-state level at the few seconds, with a ratio of intracellular to extracellular concentration of 1:7 (39). They also have shown that H2O2 induces apoptosis in Jurkat cells at intracellular concentrations of 13 µM (40). Thus, the intracellular concentration of H2O2 in EGCG-treated Jurkat cells could be assumed to be
1 µM (1 h after treatment with 50 µM EGCG).
The results that some catechins produce H2O2 in culture medium are consistent with observations by other investigators (33,41), while its relationship with cell death remains to be elucidated in those studies. Hydrogen peroxide has been reported to act as a second messenger that is involved in chemokine expression in macrophages (42), activation of lymphocytes (43) and activation of transcription factors (44). Hydrogen peroxide is potentially harmful when it oxidizes proteins, lipids and nucleic acids (45,46). Furthermore, it becomes more harmful when it reacts with transition metal ions [such as Fe(II) and Cu(II)] to afford hydroxyl radicals via a Fenton reaction (47,48). As mentioned above, we have shown that cytotoxicity of EGCG was due to its activity to produce H2O2 and that the effect of H2O2, as well as the effect of EGCG, was dependent mainly on Fe(II). These results suggest an important role of the Fe(II)-dependent mechanism, rather than the role as a second messenger or direct modifier of biological molecules in the H2O2 cytotoxicity in Jurkat cells. Taken together, we propose a mechanism that EGCG primarily acts to generate H2O2 and that the resulting H2O2 triggers an Fe(II)-dependent reaction to form highly toxic radicals, which in turn induce apoptotic cell death in Jurkat cells. The idea that cytotoxic potency of EGCG is due to its activity to generate H2O2 is further supported by the observation that cells with lower activity to eliminate H2O2 are more sensitive to EGCG. Higher activity to eliminate H2O2 in normal cells, as demonstrated with NHDF, may explain relative specificity of EGCG to certain tumor cells as compared with normal cells.
Although detailed mechanism of EGCG to generate H2O2 in cell culture medium has not been investigated fully, the observation that EGCG caused H2O2 generation even in sodium phosphate buffer indicates that most of the medium components, including amino acids, vitamins and inorganic salts, were not essential to EGCG-mediated H2O2 generation. Rather, pH levels may be an important factor, as EGCG-mediated H2O2 generation prefers alkaline pH and is not observed at pH 5.8. The experiments using some EGCG analogs indicated that a pyrogallol moiety in the catechin molecule played an essential role in H2O2 generation and cytotoxic effects. As the pKa1 value of EGCG is reported to be at 7.597.75 (37,38), the process for deprotonation in the pyrogallol moiety and/or deprotonated form of EGCG may play a crucial role in H2O2 generation. It was reported previously that varying amount of H2O2 was generated when EGCG was added to several cell culture media (33). Our preliminary experiments suggested that pyruvate, which is included in some types of medium, suppressed EGCG-mediated H2O2 generation. Pyruvate non-enzymatically reacts with H2O2 to afford acetate, carbon dioxide and water (CH3COCOO + H2O2
CH3COO + CO2 + H2O) (49). It is probable that such variation is due to the difference in the amount of antioxidative components such as pyruvate in the medium. We have found that SOD inhibited EGCG-mediated H2O2 generation as well as the cytotoxic effect of EGCG, but not of H2O2. These observations raise the possibility that
plays a key role in EGCG-mediated H2O2 generation, and further investigation on the role of
may provide us with important information to understand the entire mechanism.
In conclusion, a novel antitumor mechanism of EGCG was proposed here, and this mechanism may be a potential candidate that explains the diverse effects of EGCG.
 |
Acknowledgments
|
---|
This work was supported partly by the Grant of the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
 |
References
|
---|
- Anderson,R.F., Fisher,L.J., Hara,Y., Harris,T., Mak,W.B., Melton,L.D. and Packer,J.E. (2001) Green tea catechins partially protect DNA from OH radical-induced strand breaks and base damage through fast chemical repair of DNA radicals. Carcinogenesis, 22, 11891193.[Abstract/Free Full Text]
- Higdon,J.V. and Frei,B. (2003) Tea catechins and polyphenols: health effects, metabolism and antioxidant functions. Crit. Rev. Food Sci. Nutr., 43, 89143.[ISI][Medline]
- Ikigai,H., Nakae,T., Hara,Y. and Shimamura,T. (1993) Bactericidal catechins damage the lipid bilayer. Biochim. Biophys. Acta, 1147, 132136.[ISI][Medline]
- Toering,S.J., Gentile,G.J. and Gentile,J.M. (1996) Mechanism of antimutagenic action of (+)-catechin against the plant-activated aromatic amine 4-nitro-o-phenylenediamine. Mutat. Res., 361, 8187.[ISI][Medline]
- Nakagawa,H., Wachi,M., Woo,J.T., Kato,M., Kasai,S., Takahashi,F., Lee,I.S. and Nagai,K. (2002) Fenton reaction is primarily involved in a mechanism of ()-epigallocatechin-3-gallate to induce osteoclastic cell death. Biochem. Biophys. Res. Commun., 292, 94101.[CrossRef][ISI][Medline]
- Ahmad,N., Cheng,P. and Mukhtar,H. (2000) Cell cycle dysregulation by green tea polyphenol epigallocatechin-3-gallate. Biochem. Biophys. Res. Commun., 275, 328334.[CrossRef][ISI][Medline]
- Nam,S., Smith,D.M. and Dou,Q.P. (2001) Ester bond-containing tea polyphenols potently inhibit proteasome activity in vitro and in vivo. J. Biol. Chem., 276, 1332213330.[Abstract/Free Full Text]
- Ahmad,N., Feyes,D.K., Nieminen,A.L., Agarwal,R. and Mukhtar,H. (1997) Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells. J. Natl Cancer Inst., 89, 18811886.[Abstract/Free Full Text]
- Tan,X., Hu,D., Li,S., Han,Y., Zhang,Y. and Zhou,D. (2000) Differences of four catechins in cell cycle arrest and induction of apoptosis in LoVo cells. Cancer Lett., 158, 16.[CrossRef][ISI][Medline]
- Ahmad,N., Gupta,S. and Mukhtar,H. (2000) Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappaB in cancer cells versus normal cells. Arch. Biochem. Biophys., 376, 338346.[CrossRef][ISI][Medline]
- Gupta,S., Hussain,T. and Mukhtar,H. (2003) Molecular pathway for ()-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells. Arch. Biochem. Biophys., 410, 177185.[CrossRef][ISI][Medline]
- Yang,G.Y., Liao,J., Kim,K., Yurkow,E.J. and Yang,C.S. (1998) Inhibition of growth and induction of apoptosis in human cancer cell lines by tea polyphenols. Carcinogenesis, 19, 611616.[Abstract]
- Otsuka,T., Ogo,T., Eto,T., Asano,Y., Suganuma,M. and Niho,Y. (1998) Growth inhibition of leukemic cells by ()-epigallocatechin gallate, the main constituent of green tea. Life Sci., 63, 13971403.[CrossRef][ISI][Medline]
- Chen,Z.P., Schell,J.B., Ho,C.T. and Chen,K.Y. (1998) Green tea epigallocatechin gallate shows a pronounced growth inhibitory effect on cancerous cells but not on their normal counterparts. Cancer Lett., 129, 173179.[CrossRef][ISI][Medline]
- Saeki,K., Sano,M., Miyase,T., Nakamura,Y., Hara,Y., Aoyagi,Y. and Isemura,M. (1999) Apoptosis-inducing activity of polyphenol compounds derived from tea catechins in human histiolytic lymphoma U937 cells. Biosci. Biotechnol. Biochem., 63, 585587.[ISI][Medline]
- Naasani,I., Oh-Hashi,F., Oh-Hara,T., Feng,W.Y., Johnston,J., Chan,K. and Tsuruo,T. (2003) Blocking telomerase by dietary polyphenols is a major mechanism for limiting the growth of human cancer cells in vitro and in vivo. Cancer Res., 63, 824830.[Abstract/Free Full Text]
- Islam,S., Islam,N., Kermode,T., Johnstone,B., Mukhtar,H., Moskowitz,R.W., Goldberg,V.M., Malemud,C.J. and Haqqi,T.M. (2000) Involvement of caspase-3 in epigallocatechin-3-gallate-mediated apoptosis of human chondrosarcoma cells. Biochem. Biophys. Res. Commun., 270, 793797.[CrossRef][ISI][Medline]
- Pan,M.H., Liang,Y.C., Lin-Shiau,S.Y., Zhu,N.Q., Ho,C.T. and Lin,J.K. (2000) Induction of apoptosis by the oolong tea polyphenol theasinensin A through cytochrome c release and activation of caspase-9 and caspase-3 in human U937 cells. J. Agric. Food Chem., 48, 63376346.[CrossRef][ISI][Medline]
- Hayakawa,S., Saeki,K., Sazuka,M., Suzuki,Y., Shoji,Y., Ohta,T., Kaji,K., Yuo,A. and Isemura,M. (2001) Apoptosis induction by epigallocatechin gallate involves its binding to Fas. Biochem. Biophys. Res. Commun., 285, 11021106.[CrossRef][ISI][Medline]
- Sakagami,H., Arakawa,H., Maeda,M., Satoh,K., Kadofuku,T., Fukuchi,K. and Gomi,K. (2001) Production of hydrogen peroxide and methionine sulfoxide by epigallocatechin gallate and antioxidants. Anticancer Res., 21, 26332641.[ISI][Medline]
- Chen,C., Shen,G., Hebbar,V., Hu,R., Owuor,E.D. and Kong,A.N. (2003) Epigallocatechin-3-gallate-induced stress signals in HT-29 human colon adenocarcinoma cells. Carcinogenesis, 24, 13691378.[Abstract/Free Full Text]
- Saeki,K., Kobayashi,N., Inazawa,Y., Zhang,H., Nishitoh,H., Ichijo,H., Isemura,M. and Yuo,A. (2002) Oxidation-triggered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways for apoptosis in human leukaemic cells stimulated by epigallocatechin-3-gallate (EGCG): a distinct pathway from those of chemically induced and receptor-mediated apoptosis. Biochem. J., 368, 705720.[CrossRef][ISI][Medline]
- Vergote,D., Cren-Olive,C., Chopin,V., Toillon,R.A., Rolando,C., Hondermarck,H. and Le Bourhis,X. (2002) ()-Epigallocatechin (EGC) of green tea induces apoptosis of human breast cancer cells but not of their normal counterparts. Breast Cancer Res. Treat., 76, 195201.[CrossRef][ISI][Medline]
- Yang,G.Y., Liao,J., Li,C., Chung,J., Yurkow,E.J., Ho,C.T. and Yang,C.S. (2000) Effect of black and green tea polyphenols on c-jun phosphorylation and H2O2 production in transformed and non-transformed human bronchial cell lines: possible mechanisms of cell growth inhibition and apoptosis induction. Carcinogenesis, 21, 20352039.[Abstract/Free Full Text]
- Morre,D.J., Bridge,A., Wu,L.Y. and Morre,D.M. (2000) Preferential inhibition by ()-epigallocatechin-3-gallate of the cell surface NADH oxidase and growth of transformed cells in culture. Biochem. Pharmacol., 60, 937946.[CrossRef][ISI][Medline]
- Wang,Y.C. and Bachrach,U. (2002) The specific anti-cancer activity of green tea ()-epigallocatechin-3-gallate (EGCG). Amino Acids, 22, 131143.[CrossRef][ISI][Medline]
- Morre,D.J., Morre,D.M., Sun,H., Cooper,R., Chang,J. and Janle,E.M. (2003) Tea catechin synergies in inhibition of cancer cell proliferation and of a cancer specific cell surface oxidase (ECTO-NOX). Pharmacol. Toxicol., 92, 234241.[ISI][Medline]
- Naasani,I., Seimiya,H. and Tsuruo,T. (1998) Telomerase inhibition, telomere shortening and senescence of cancer cells by tea catechins. Biochem. Biophys. Res. Commun., 249, 391396.[CrossRef][ISI][Medline]
- Pisters,K.M., Newman,R.A., Coldman,B., Shin,D.M., Khuri,F.R., Hong,W.K., Glisson,B.S. and Lee,J.S. (2001) Phase I trial of oral green tea extract in adult patients with solid tumors. J. Clin. Oncol., 19, 18301838.[Abstract/Free Full Text]
- Jatoi,A., Ellison,N., Burch,P.A. et al. (2003) A phase II trial of green tea in the treatment of patients with androgen independent metastatic prostate carcinoma. Cancer, 97, 14421446.[CrossRef][ISI][Medline]
- Gupta,S., Hastak,K., Ahmad,N., Lewin,J.S. and Mukhtar,H. (2001) Inhibition of prostate carcinogenesis in TRAMP mice by oral infusion of green tea polyphenols. Proc. Natl Acad. Sci. USA, 98, 1035010355.[Abstract/Free Full Text]
- Nourooz-Zadeh,J., Tajaddini-Sarmadi,J. and Wolff,S.P. (1994) Measurement of plasma hydroperoxide concentrations by the ferrous oxidation-xylenol orange assay in conjunction with triphenylphosphine. Anal. Biochem., 220, 403409.[CrossRef][ISI][Medline]
- Long,L.H., Clement,M.V. and Halliwell,B. (2000) Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of ()-epigallocatechin, ()-epigallocatechin gallate, (+)-catechin and quercetin to commonly used cell culture media. Biochem. Biophys. Res. Commun., 273, 5053.[CrossRef][ISI][Medline]
- Chance,B., Sies,H. and Boveris,A. (1979) Hydroperoxide metabolism in mammalian organs. Physiol. Rev., 59, 527605.[Free Full Text]
- Takagi,Y., Mitsui,A., Nishiyama,A., Nozaki,K., Sono,H., Gon,Y., Hashimoto,N. and Yodoi,J. (1999) Overexpression of thioredoxin in transgenic mice attenuates focal ischemic brain damage. Proc. Natl Acad. Sci. USA, 96, 41314136.[Abstract/Free Full Text]
- Bai,J., Rodriguez,A.M., Melendez,J.A. and Cederbaum,A.I. (1999) Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury. J. Biol. Chem., 274, 2621726224.[Abstract/Free Full Text]
- Jovanovic,S.V., Hara,Y., Steenken,S. and Simic,M.G. (1995) Antioxidant potential of gallocatechins. A pulse radiolysis and laser photolysis study. J. Am. Chem. Soc., 117, 98819888.[ISI]
- Kumamoto,M., Sonda,T., Nagayama,K. and Tabata,M. (2001) Effects of pH and metal ions on antioxidative activities of catechins. Biosci. Biotechnol. Biochem., 65, 126132.[CrossRef][ISI][Medline]
- Antunes,F. and Cadenas,E. (2000) Estimation of H2O2 gradients across biomembranes. FEBS Lett., 475, 121126.[CrossRef][ISI][Medline]
- Antunes,F. and Cadenas,E. (2001) Cellular titration of apoptosis with steady state concentrations of H2O2: submicromolar levels of H2O2 induce apoptosis through Fenton chemistry independent of the cellular thiol state. Free Radic. Biol. Med., 30, 10081018.[CrossRef][ISI][Medline]
- Dashwood,W.M., Orner,G.A. and Dashwood,R.H. (2002) Inhibition of beta-catenin/Tcf activity by white tea, green tea and epigallocatechin-3-gallate (EGCG): minor contribution of H2O2 at physiologically relevant EGCG concentrations. Biochem. Biophys. Res. Commun., 296, 584588.[CrossRef][ISI][Medline]
- Jaramillo,M. and Olivier,M. (2002) Hydrogen peroxide induces murine macrophage chemokine gene transcription via extracellular signal-regulated kinase- and cyclic adenosine 5'-monophosphate (cAMP)-dependent pathways: involvement of NF-kappa B, activator protein 1 and cAMP response element binding protein. J. Immunol., 169, 70267038.[Abstract/Free Full Text]
- Reth,M. (2002) Hydrogen peroxide as second messenger in lymphocyte activation. Nat. Immunol., 3, 11291134.[CrossRef][ISI][Medline]
- Rhee,S.G. (1999) Redox signaling: hydrogen peroxide as intracellular messenger. Exp. Mol. Med., 31, 5359.[ISI][Medline]
- Stadtman,E.R. (1992) Protein oxidation and aging. Science, 257, 12201224.[ISI][Medline]
- Stadtman,E.R. and Berlett,B.S. (1998) Reactive oxygen-mediated protein oxidation in aging and disease. Drug Metab. Rev., 30, 225243.[ISI][Medline]
- Halliwell,B. and Gutteridge,J.M. (1990) Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol., 186, 185.[Medline]
- Stohs,S.J. and Bagchi,D. (1995) Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med., 18, 321336.[CrossRef][ISI][Medline]
- Hinoi,E., Fujimori,S., Takemori,A. and Yoneda,Y. (2002) Cell death by pyruvate deficiency in proliferative cultured calvarial osteoblasts. Biochem. Biophys. Res. Commun., 294, 11771183.[CrossRef][ISI][Medline]
Received October 29, 2003;
revised March 14, 2004;
accepted April 9, 2004.