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(−)-Epigallocatechin gallate (EGCG) is the most abundant and biologically active polyphenol in green tea, and many of the therapeutic benefits of the beverage have been attributed to this compound. High concentrations of EGCG are cytotoxic and trigger genotoxic events in mammalian cells. Although this catechin affects a number of cellular systems, the genotoxic effects of several bioflavonoid-based dietary polyphenols are believed to be mediated, at least in part, by their actions on topoisomerase II. Therefore, the effects of green tea extract and EGCG on DNA cleavage mediated by human topoisomerase IIα and β were characterized. The extract and EGCG increased levels of DNA strand breaks generated by both enzyme isoforms. However, EGCG acted by a mechanism that was distinctly different from those of genistein, a dietary polyphenol, and etoposide, a widely prescribed anticancer drug. In contrast to these agents, EGCG exhibited all of the characteristics of a redox-dependent topoisomerase II poison that acts by covalently adducting to the enzyme. First, EGCG stimulated DNA scission mediated by both isoforms primarily at sites that were cleaved in the absence of compounds. Second, exposure of EGCG to the reducing agent dithiothreitol (DTT) prior to its addition to DNA cleavage assays abrogated the effects of the catechin on DNA scission. Third, once EGCG stimulated topoisomerase II-mediated DNA cleavage, exposure to DTT did not effect levels of DNA strand breaks. Finally, EGCG inhibited the DNA cleavage activities of topoisomerase IIα and β when incubated with either enzyme prior to the addition of DNA. Taken together, these results provide strong evidence that EGCG is a redox-dependent topoisomerase II poison, and utilizes a mechanism similar to that of 1,4-benzoquinone.
Green tea, a rich source of polyphenols (i.e., bioflavonoids), is one of the most widely consumed beverages worldwide. Public interest in this popular beverage has grown as a result of the potential health promoting benefits that are attributed to its consumption (1–3). Green tea has been suggested to reduce the incidence of cardiovascular disease and cancers, such as breast, prostate, colorectal, and lung, in humans (3, 4).
(−)-Epigallocatechin gallate (EGCG)1 (Figure 1) is the most abundant and biologically active polyphenol in green tea, and many of the chemopreventative properties of the beverage have been attributed to this compound (1–4). EGCG affects a number of cellular systems, and high concentrations of this catechin are cytotoxic and trigger genotoxic events in cultured mammalian cells (5–9). In addition, EGCG inhibits proliferation, induces apoptosis, inhibits the activity of numerous protein kinases, and blocks the activation of transcription factors such as activator protein-1 and nuclear factor-κB (4, 10). EGCG also enhances DNA cleavage mediated by topoisomerase II (11).
The mechanistic basis for the physiological actions of EGCG is not well defined. Because the compound undergoes redox chemistry, at least some of its cellular functions have been attributed to its antioxidant properties (1, 2, 4, 10). However, the genotoxic effects of several bioflavonoid-based dietary polyphenols are believed to be mediated, at least in part, by their actions on topoisomerase II (12–15).
Type II topoisomerases are ubiquitous enzymes that play essential roles in a number of fundamental DNA processes (16–21). Vertebrates encode two isoforms of the enzyme, topoisomerase IIα and β (16–24). These proteins display ~70% amino acid sequence identity and are enzymologically similar (20–27). However, they have distinct patterns of expression and physiological functions (17, 19–21, 24). Topoisomerase IIα is essential for the survival of actively growing tissues, and its expression rises dramatically during cell proliferation (28–30). This isoform is found at DNA replication forks and remains tightly associated with mitotic chromosomes (17, 31–33). As a result, topoisomerase IIα is thought to be the isoform that functions in growth-dependent processes, such as DNA replication and chromosome segregation (17, 19). In contrast, topoisomerase IIβ is dispensable at the cellular level (34, 35). Protein expression is independent of proliferative status, and the enzyme dissociates from chromosomes during mitosis (17, 24, 30, 36). Topoisomerase IIβ cannot compensate for the loss of the α isoform, and its physiological functions are not well defined (24, 32, 37).
Type II topoisomerases alter DNA topology by passing an intact helix through a double-stranded break that they generate in a separate segment of DNA (18, 20, 21, 38–40). To maintain genomic integrity during this process, topoisomerase II forms a covalent bond with the 5′-termini of the cleaved nucleic acid (41, 42, 43). This covalent enzyme-cleaved DNA intermediate is known as the cleavage complex. Despite the essential nature of topoisomerase II, conditions that increase the concentration of cleavage complexes ultimately generate permanent strand breaks in the genetic material that initiate mutagenic recombination/repair pathways (20, 21, 44–46). If these strand breaks overwhelm the cell, they induce death pathways (45).
Agents that increase topoisomerase II-mediated DNA cleavage are called topoisomerase II poisons due to their ability to convert the enzyme to a cellular toxin that fragments the genome (20, 21, 47–50). Many of these compounds are widely prescribed as anticancer drugs and represent some of the most successful chemotherapeutic agents utilized to treat human malignancies (20, 21, 48, 51–54). However, topoisomerase II poisons also have been associated with the development of specific types of leukemia that involve rearrangements of the MLL gene at chromosomal band 11q23 (46, 55–58).
A previous report indicates that EGCG has the potential to poison calf thymus topoisomerase II (11). However, the enzyme preparation employed in this study contained a mixture of topoisomerase II isoforms. Recent studies suggest that the clinical effects of topoisomerase II poisons may be related to individual enzyme isoforms, with the chemotherapeutic properties being attributed primarily to topoisomerase IIα and the leukemogenic properties and off-target toxicity being attributed primarily to topoisomerase IIβ (59, 60). Since chemopreventative and genotoxic properties have both been ascribed to EGCG (1, 3–9), it is important to more fully understand the actions of the catechin on the individual isoforms of topoisomerase II. Therefore, the effects of green tea extract and EGCG on DNA cleavage and ligation mediated by human topoisomerase IIα and β were characterized. The extract and EGCG enhanced DNA cleavage mediated by both enzyme isoforms. In contrast to genistein, a dietary polyphenol, and etoposide, a widely prescribed anticancer drug, EGCG affected enzyme activity in a redox-dependent manner that appears to involve covalent adduction to topoisomerase IIα and β. Thus, EGCG utilizes a mechanism similar to 1,4-benzoquinone and other redox-dependent topoisomerase II poisons.
Recombinant wild-type human topoisomerase IIα and β were expressed in Saccharomyces cerevisiae and purified as described previously (61–63). Negatively supercoiled pBR322 DNA was prepared from Escherichia coli using a Plasmid Mega Kit (Qiagen) as described by the manufacturer. EGCG was purchased from LKT. 1,4-Benzoquinone and etoposide were obtained from Sigma. All compounds were prepared as 20 mM stock solutions in 100% DMSO and stored at −20 °C. Green tea extract was obtained as a retail product (product #44685) manufactured by Sundown, Inc. (Boca Raton, FL). The product was a combination of Camelia Sinensis leaf extract (150 mg per capsule) standardized to 50% total polyphenols and non-standardized C. Sinensis whole leaf (250 mg per capsule). The specific concentration of EGCG in the product was not indicated. Green tea extract was prepared as a 20 mg/mL stock solution in 100% DMSO and stored at −20 °C.
DNA cleavage reactions were performed using the procedure of Fortune and Osheroff (64). Assay mixtures contained 220 nM topoisomerase IIα or β, 5 nM negatively supercoiled pBR322 DNA, 0–100 μg/mL green tea extract or 0–500 μM EGCG in 20 μL of DNA cleavage buffer [10 mM Tris-HCl, pH 7.9, 5 mM MgCl2, 100 mM KCl, 0.1 mM EDTA, and 2.5% (v/v) glycerol]. DNA cleavage mixtures were incubated for 6 min at 37 °C. In some cases, 0–10 min time courses for DNA cleavage were monitored. Enzyme-DNA cleavage intermediates were trapped by adding 2 μL of 5% SDS and 1 μL of 375 mM EDTA, pH 8.0. Proteinase K was added (2 μL of a 0.8 mg/mL solution), and reaction mixtures were incubated for 30 min at 45 °C to digest topoisomerase II. Samples were mixed with 2 μL of 60% sucrose in 10 mM Tris-HCl, pH 7.9, 0.5% bromophenol blue, and 0.5% xylene cyanol FF, heated for 2 min at 45 °C, and subjected to electrophoresis in 1% agarose gels in 40 mM Tris-acetate, pH 8.3, and 2 mM EDTA containing 0.5 μg/mL ethidium bromide. DNA cleavage was monitored by the conversion of negatively supercoiled plasmid DNA to linear molecules. DNA bands were visualized by ultraviolet light and quantified using an Alpha Innotech digital imaging system.
In assays that determined the reversibility of topoisomerase II-mediated DNA cleavage induced by EGCG, EDTA (final concentration of 18 mM) was added to reaction mixtures prior to treatment with SDS. To determine whether cleaved DNA was protein-linked, proteinase K treatment was omitted. To examine the effects of a reducing agent on the actions of EGCG against human topoisomerase IIα or β, 500 μM dithiothreitol (DTT) was incubated with 500 μM EGCG, 25 μM 1,4-benzoquinone, 50 μM genistein, or 50 μM etoposide for 5 min prior to initiating DNA cleavage reactions. Alternatively, DTT was added to reaction mixtures for 5 min following a 6 min DNA cleavage reaction.
To examine the effects of EGCG on human topoisomerase IIα or β in the absence of DNA, 500 μM EGCG, 25 μM 1,4-benzoquinone, 50 μM genistein, or 50 μM etoposide was incubated with 220 nM enzyme for 0–10 min at 37 °C in 15 μL of DNA cleavage buffer. Cleavage was initiated by adding 5 nM negatively supercoiled pBR322 DNA (in 5 μL of cleavage buffer) to the reaction mixture.
Finally, to determine whether EGCG undergoes a molecular alteration that enhances its reactivity toward human topoisomerase IIα or β, 500 μM EGCG was incubated alone in DNA cleavage buffer for 3 min at 37 °C prior to the initiation of reactions.
DNA ligation mediated by human topoisomerase IIα or β was monitored according to the procedure of Byl et al. (65). DNA cleavage/ligation equilibria were established for 6 min at 37 °C as described above in the absence of compound or in the presence of 500 μM EGCG or 50 μM genistein. Ligation was initiated by shifting samples from 37 to 0 °C. Reactions were stopped at time points up to 15 s by the addition of 2 μL of 5% SDS followed by 1 μL of 375 mM EDTA, pH 8.0. Samples were processed and analyzed as above. Ligation was monitored by the loss of linear DNA.
DNA cleavage sites were mapped using a modification (66) of the procedure of O’Reilly and Kreuzer (67). A linear 4330 bp fragment (HindIII/EcoRI) of pBR322 plasmid DNA singly labeled with 32P on the 5′-terminus of the HindIII site was used as the cleavage substrate. The pBR322 DNA substrate was linearized by treatment with HindIII. Terminal 5′-phosphates were removed by treatment with calf intestinal alkaline phosphatase and replaced with [32P]phosphate using T4 polynucleotide kinase and [γ-32P]ATP. The DNA was cut with EcoRI, and the 4330 bp singly end labeled fragment was purified from the small EcoRI-HindIII fragment by passage through a CHROMA SPIN+TE-100 column (Clontech). DNA cleavage reaction mixtures contained 0.7 nM labeled pBR322 DNA and 90 nM human topoisomerase II in 50 μL of DNA cleavage buffer. Assays were performed in the absence of compound or in the presence of 12.5 μM etoposide, 50 μM genistein, 50 μM 1,4-benzoquinone, or 0–500 μM EGCG. Reactions were incubated for 6 min (topoisomerase IIα) or 0.5 min (topoisomerase IIβ) at 37 °C. Cleavage intermediates were trapped by adding 5 μL of 5% SDS followed by 4 μL of 250 mM EDTA, pH 8.0. Topoisomerase II was digested with proteinase K (5 μL of a 0.8 mg/mL solution) for 30 min at 45 °C. DNA products were precipitated twice in 100% ethanol, washed in 70% ethanol, dried, and resuspended in 6 μL of 40% formamide, 10 mM NaOH, 0.02% xylene cyanol FF, and 0.02% bromophenol blue. Samples were subjected to electrophoresis in a denaturing 6% polyacrylamide sequencing gel in 100 mM Tris-borate, pH 8.3, and 2 mM EDTA. The gel was fixed in a 10% methanol/10% acetic acid mixture for 2 min and dried. DNA cleavage products were analyzed on a Bio-Rad Molecular Imager FX.
Green tea is a rich source of biologically active polyphenols that are believed to impact a number of important physiological processes (4, 10). Since several classes of polyphenols, including flavones, flavonols, isoflavones, and catechins have been reported to affect the activity of mammalian type II topoisomerases (11, 12, 68), the effects of green tea extract on the DNA cleavage activity of human topoisomerase IIα and β were assessed.
Green tea extract increased levels of DNA cleavage mediated by both enzyme isoforms (Figure 2). Scission rose ~3-fold for topoisomerase IIα and ~5-fold for topoisomerase IIβ. In both cases, optimal cleavage enhancement was observed at ~25 μg/mL green tea extract, with cleavage decreasing at higher concentrations. The basis for the decline in activity of the extract above 25 μg/mL is not known. One possibility is that compounds in the extract intercalate into the plasmid substrate and inhibit the enzymes by diminishing topoisomerase II-DNA binding or by altering the apparent supercoiled state of the double helix. However, no DNA intercalation was observed at concentrations of extract up to 200 μg/mL (data not shown). As a second possibility, green tea leaves contain tannins, and some of these compounds have been shown to inhibit the activity of type II topoisomerases (69, 70).
EGCG, the major polyphenol in green tea, represents ~40–60% of the bioflavonoids in green tea extract (6, 71). Since ~25 μM/mL green tea extract contains ~20–30 μM EGCG, the effects of the isolated catechin on the DNA cleavage activities of topoisomerase IIα and β were determined (Figure 3). EGCG increased levels of scission with both isoforms (~5- and 8-fold, respectively) over a wide range of concentrations. It is notable that the concentration of EGCG in plasma and salivary samples is estimated to be as high as 4 and 48 μM, respectively, following consumption of ~3 cups of green tea (5, 72). As seen in Figure 3, significant topoisomerase II-DNA cleavage enhancement was observed in this range.
No decline in cleavage enhancement was observed at concentrations as high as 500 μM EGCG. This indicates that the decrease in activity seen at high concentrations of green tea extract was not due to an effect of EGCG.
A series of control reactions was performed to ensure that the DNA cleavage enhancement observed with EGCG was topoisomerase II-mediated (Figure 4). No EGCG-induced DNA scission was seen in the absence of enzyme. Furthermore, with both topoisomerase IIα and β, EGCG-induced cleavage was reversed when reactions were incubated with EDTA prior to trapping cleavage complexes with SDS. This reversibility is inconsistent with an enzyme-independent reaction. Finally, cleaved plasmid was covalently linked to topoisomerase II. In the absence of proteinase K, the linear DNA band disappeared and was replaced by a band that remained at the origin of the gel or by a diffuse smear with reduced electrophoretic mobility (data not shown).
The DNA cleavage and ligation reactions of topoisomerase II exist in a tightly coupled equilibrium (18, 20, 21, 38–40). Some topoisomerase II poisons, such as etoposide or genistein, increase levels of cleavage complexes by inhibiting the DNA ligation activity of the enzyme (68, 73). In contrast, abasic sites and other lesions that act as topoisomerase II poisons have little effect on ligation and act primarily by stimulating the forward rate of scission (74–78). Finally, some bioflavonoid-based topoisomerase II poisons have a modest effect on ligation and presumably affect both the cleavage and ligation reactions (68). EGCG appears to fall into this last category (Figure 5). The effects of the catechin on DNA ligation mediated by topoisomerase IIα or β were intermediate to those of no drug reactions and those that contain genistein.
With the exception of DNA lesions, topoisomerase II poisons can be categorized into two broad classes. Members of the first group act by a redox-independent mechanism. These compounds interact with topoisomerase II at the protein-DNA interface (in the vicinity of the active site tyrosine) in a non-covalent manner (20, 21, 48–50). Redox-independent topoisomerase II poisons include several anticancer drugs (e.g., etoposide) and dietary bioflavonoids (e.g., genistein), and display a number of characteristic properties. They generally alter the DNA cleavage specificity (site utilization) of the enzyme due to their interactions with the double helix in the tertiary enzyme-DNA-drug complex (79). Furthermore, because their actions against topoisomerase II do not depend on redox chemistry, they are unaffected by the presence of reducing agents (80). Finally, these compounds induce similar levels of enzyme-mediated DNA scission whether they are added to the binary topoisomerase II-DNA complex or are incubated with the enzyme prior to the addition of the double helix (80).
Topoisomerase II poisons in the second class act in a redox-dependent fashion (21, 80–87) and form covalent adducts with the enzyme at amino acid residues distal to the active site (84). The best-characterized members of this group are quinones, such as 1,4-benzoquinone and polychlorinated biphenyl (PCB) metabolites (80–86). In contrast to redox-independent topoisomerase II poisons, this latter class generally enhances DNA cleavage at sites normally cut by the enzyme (80, 84). Moreover, because of the dependence on redox chemistry, their ability to form covalent adducts with topoisomerase II (and hence their ability to enhance topoisomerase II-mediated DNA cleavage) is blocked by the presence of reducing agents (80, 84, 88, 89). Finally, while redox-dependent poisons enhance topoisomerase II-mediated DNA cleavage when added to the enzyme-DNA complex, these compounds display the distinguishing feature of inhibiting topoisomerase II function when incubated with the protein prior to the addition of DNA (80, 84, 88, 89).
A previous study demonstrated that the bioflavonoid genistein (which contains a single hydroxyl group on its B-ring) poisons topoisomerase IIα and β in a redox-independent manner (68). EGCG contains a similar three-ring bioflavonoid core, but undergoes a complex set of redox-dependent reactions, presumably due to the three hydroxyl groups on its B-ring (see Figure 1) (4, 90–94). Therefore, it is not obvious whether EGCG acts in a manner that requires redox chemistry.
As a first step in elucidating the basis for the actions of EGCG as a topoisomerase II poison, the effects of the compound on the DNA cleavage site specificity of human topoisomerase IIα and β were assessed. As seen in Figure 6, EGCG stimulated DNA scission mediated by both isoforms primarily at sites that were cleaved by the enzymes in the absence of compounds. A similar result was seen with 1,4-benzoquinone. In contrast, etoposide and genistein enhanced cleavage at a number of sites that were not intrinsic to either topoisomerase II isoform. These findings suggest that EGCG may be a redox-dependent topoisomerase II poison.
To test this hypothesis, the effects of the reducing agent, DTT, on the activity of EGCG were determined (Figure 7). Prior to the addition of the catechin or other agents to the topoisomerase II (α or β)-DNA complex, compounds were incubated with 500 μM DTT for 5 min. While this treatment had no significant effect on the enzyme alone or on the actions of the redox-independent poisons, genistein and etoposide, it markedly decreased the ability of EGCG to enhance DNA cleavage. Levels of scission dropped ~90% as compared to reactions in the absence of DTT. A similar result was observed for 1,4-benzoquinone. This finding provides strong evidence that EGCG is a redox-dependent topoisomerase II poison.
A common feature of redox-dependent topoisomerase II poisons is that they form covalent adducts with the enzyme (80–86). Since the maintenance of this covalent interaction is independent of the redox state of the poison, DTT has no effect on DNA cleavage after adducts are formed. Similar to results seen with 1,4-benzoquinone, once cleavage complexes were formed in the presence of EGCG, DTT did not diminish the efficacy of the catechin (Figure 8). Together with the data shown in Figure 7, this finding suggests that EGCG acts by forming covalent adducts with topoisomerase IIα and β.
A final distinguishing feature of redox-dependent topoisomerase II poisons is the fact that they inactivate enzyme function when they are incubated with the protein prior to the addition of DNA (80, 82, 84, 88, 89, 95). To determine whether this was the case for EGCG, the catechin was added to reaction mixtures that contained topoisomerase IIα or β for times up to 10 min before the addition of plasmid. Levels of cleavage were compared to those observed when EGCG was added to a mixture that contained the enzyme-DNA complex (set to 100%). As seen for other redox-dependent topoisomerase II poisons, when topoisomerase IIα or β was incubated with EGCG before the addition of DNA, there was a time-dependent decrease in cleavage activity (Figure 9).
It is notable that the t1/2 of inactivation was considerably longer for the catechin than for 1,4-benzoquinone. Moreover, there was a lag of ~2 and ~1 min, respectively, before EGCG-induced inactivation of topoisomerase IIα or β was observed. However, when the catechin was incubated alone in reaction buffer for 3 min prior to its addition to the enzyme, its reactivity rose considerably. T1/2 values for inactivation were comparable to those seen for 1,4-benzoquine, and the previously observed delay disappeared. These results suggest that EGCG undergoes redox chemistry that enhances its reactivity toward topoisomerase IIα and β. However, the nature of the redox-induced change(s) is unknown at the present time.
To determine whether the redox-dependent changes in EGCG also enhance its actions as a topoisomerase II poison, the ability of the catechin to stimulate DNA cleavage mediated by topoisomerase IIα and β was examined over time (Figure 10). The time course for stimulation of DNA scission (when EGCG was added to the enzyme-DNA complex) was similar to that for inactivation of the enzyme isoforms. EGCG was less reactive than 1,4-benzoquinone, and a lag was observed prior to cleavage enhancement. Once again, reactivity increased, and the lag disappeared when EGCG was incubated in reaction buffer prior to its addition to the enzyme-DNA complex. Therefore, it is likely that redox chemistry induces a change in EGCG that makes it a more potent topoisomerase II poison.
EGCG is the most abundant polyphenol in green tea and is believed to be responsible for many of the beneficial effects of this popular beverage on human health, including chemoprevention (1–4). High concentrations of the catechin trigger genotoxic events in mammalian cells (5–9). Despite the potential therapeutic benefits of polyphenols in adults, the consumption of bioflavonoid-rich diets during pregnancy increases the risk of infant leukemias with MLL rearrangements ~3-fold (14). Type II topoisomerases are believed to initiate the chromosomal rearrangements in these leukemias, (12, 14, 15, 54, 96–98) and a number of polyphenols have been found to enhance DNA cleavage mediated by these enzymes (11, 68). Therefore, the effects of EGCG on the DNA cleavage activities of human topoisomerase IIα and β were assessed.
Results indicate that EGCG is a strong topoisomerase II poison against both isoforms. In contrast to genistein, a bioflavonoid that acts in a redox-independent, non-covalent manner (68), EGCG is a redox-dependent topoisomerase II poison that appears to act by covalently adducting the enzyme. Thus, the mechanistic basis for the actions of EGCG against topoisomerase II is similar to those of quinone-based compounds, such as 1,4-benzoquinone (80–86).
Green tea extract increases levels of DNA cleavage mediated by human topoisomerase IIα and β, and the concentration of EGCG (~20–30 μM) at maximal extract activity is consistent with this catechin being an important (if not the most important) topoisomerase II-active compound in green tea. However, comparing the cleavage titrations of green tea extract and EGCG, it is clear that other topoisomerase II-active compounds (either stimulatory or inhibitory) are present in the extract. Although the specific nature of these compounds has not been elucidated, myricetin, quercetin, kaempferol, and other bioflavonoids that are topoisomerase II poisons are present in green tea (3, 99).
When EGCG is added to initiate reactions, there is a 1–2 min delay before alterations in topoisomerase II activity are observed. This time delay disappears when the compound is incubated in reaction buffer for 3 min prior to its addition to topoisomerase II or the enzyme-DNA complex. EGCG is known to undergo a complex set of redox-dependent chemical changes that have been ascribed primarily to its B-ring (4, 10, 90–94). Thus, it is likely that an oxidized derivative of EGCG is more reactive toward topoisomerase II than the parent catechin. On the basis of previous studies with redox-dependent topoisomerase II poisons (80–86), it is predicted that this reactive species contains a quinone on the B-ring. However, given the complexity of the redox chemistry of EGCG, other structural rearrangements cannot be eliminated.
In summary, EGCG stimulates DNA cleavage mediated by human topoisomerase IIα and β. Given the high concentration of this catechin in green tea, its potency against the type II enzymes, and its plasma concentration range after consumption of the beverage, it is possible that topoisomerase II mediates some of the physiological actions of EGCG.
We are grateful to Jo Ann Byl, Joseph Deweese, and Amanda Gentry for critical reading of the manuscript.
1Abbreviation: EGCG, (−)-epigallocatechin gallate.
†This work was supported by National Institutes of Health research grant GM33944. OJB was a trainee under grant 5 T32 CA09582 from the National Institutes of Health and was supported in part by Ruth L. Kirschstein National Research Service Award Predoctoral Fellowship F31 GM78744 from the National Institutes of Health.