DNA and RNA as New Binding Targets of Green Tea Catechins*

The significance of catechins, the main constituent of green tea, is being increasingly recognized with regard to cancer prevention. Catechins have been studied for interactions with various proteins, but the mechanisms of the various catechins are not yet elucidated. Based on our previous observation that nucleic acids extracted from catechin-treated cells are colored, we studied whether catechins directly interact with nucleic acids using surface plasmon resonance assay (Biacore) and cold spray ionization-mass spectrometry. These two methods clearly showed that (-)-epigallocatechin gallate (EGCG) binds to both DNA and RNA molecules: the Biacore assay indicated that four catechins bound to DNA oligomers, and cold spray ionization-mass spectrometry analysis showed one to three EGCG molecules bound to single strand 18 mers of DNA and RNA. Moreover, one or two molecules of EGCG bound to double-stranded (AG-CT) oligomers of various nucleotide lengths. These results suggest that multiple binding sites of EGCG are present in DNA and RNA oligomers. Double-stranded DNA (dsDNA) oligomers were detected only as EGCG-bound forms at high temperature, whereas at low temperature both the free and bound forms were detected, suggesting that EGCG protects dsDNA oligomers from dsDNA melting to single-stranded DNA. Because both galloyl and catechol groups of EGCG are essential for DNA binding, both groups seem to hold strands of DNA via their branching structure. These findings reveal for the first time the link between catechins and polynucleotides and will intensify our understanding of the effects of catechins on DNA in terms of cancer prevention.

Green tea is an acknowledged cancer preventive in Japan (1)(2)(3)(4)(5)(6)(7). Most of the active principles are assumed to be green tea catechins because they show various cancer-preventive activities in vitro in cell culture and in vivo, including anti-oxidant, anti-cancer, and antimutagenic activities (1)(2)(3)(4)(5)(6)(7). (Ϫ)-Epigallocatechin gallate (EGCG), 2 (Ϫ)-epigallocatechin (EGC), (Ϫ)-epicatechin gallate (ECG), (Ϫ)epicatechin (EC), (Ϫ)-gallocatechin gallate (GCG), and (ϩ)-catechin are the major components of green tea polyphenols (8), and EGCG is the main constituent of green tea (9). Since we first reported in 1987 that topical applications of EGCG inhibited tumor promotion with teleocidin of the 12-O-tetradecanoylphorbol-13-acetate types and okadaic acid on mouse skin in two-stage carcinogenesis experiments (10,11), we have studied the mechanisms of action of green tea catechins in human cancer cell lines (12)(13)(14)(15)(16)(17)(18). During our study of EGCG, we often observed that nucleic acids extracted from EGCGtreated human cancer cells were catechin colored, suggesting that EGCG binds to DNA and RNA molecules in cells. Our speculation was strengthened by results showing that 3 H-EGCG was found in nuclei of human lung cancer cell line PC-9 1 h after treatment (13). Although EGCG apparently protects against DNA damage induced by free radicals, ionization, and ultraviolet radiation, as well as DNA methylation (5,19,20), it was not yet shown whether EGCG and other green tea catechins directly bind to DNA and RNA molecules. Because we had demonstrated that EGCG inhibited expression of the tumor necrosis factor-␣ gene in human cancer cells treated with the tumor promoter okadaic acid (12,16), we looked at the effects of EGCG on the expression of 588 genes in PC-9 cells, using a human cancer cDNA expression array. Results showed up-regulation of four genes (at least 2-fold) and down-regulation of 12 genes (under 0.5-fold) (1,21). It has also been reported that catechins affect DNA replication, DNA repair, and transcription (20 -25) and that all these effects can be explained through the actions of catechin on enzymes (20 -26). Thus, our new research objective was to find out whether EGCG directly interacts with DNA and RNA molecules.
Surface plasmon resonance (Biacore) assay is a real-time analysis that was developed as a methodology for determining molecular interaction under aqueous conditions. Cold spray ionizing (CSI)-mass spectrometry (MS), a variant of electrospray ionization-MS developed by one of the authors, has numerous advantages in determining molecular interaction based on the forming molecular ion in solution (27). Using these two new technologies, we demonstrated for the first time the direct binding of EGCG to DNA and RNA oligomers and investigated the structure-function relationship of this interaction. The results will likely provide new knowledge of the molecular mechanisms of action of green tea catechins and show that green tea catechins have multifunctional targets for the prevention of human cancer.

EXPERIMENTAL PROCEDURES
Materials-EGCG was purified from Japanese green tea leaves with 99.7% purity. EGCG, EGC, ECG, EC, gallic acid (GA), GCG, (Ϫ)-catechin gallate (CG), (Ϫ)-gallocatechin (GC), (ϩ)-catechin (C), and theaflavin were purchased from Funakoshi Co. Ltd. (Tokyo, Japan). DNA and RNA oligomers were synthesized and further purified by high performance liquid chromatography. Their purities were examined using polyacrylamide gel electrophoresis and TOF-MASS by Hokkaido System Science Co., Ltd. Double-stranded DNAs (dsDNAs) were made * This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan and the Smoking Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed. by annealing complementary single strand DNAs (ssDNAs) in 100 mM ammonium acetate. The DNA and RNA oligomers used were as follows: oligo(dA) 18 , oligo(dT) 18 , oligo(dC) 18 , oligo(dG) 18 , oligo(A) 18 , oligo(U) 18 , oligo(G) 18 , oligo(C) 18 , oligo(dA⅐dT) 9 , and oligo(dG⅐dC) 9 . In addition, dsDNA oligomers of dAdC⅐dTdG from 6 -14 nucleotides were also used (28). For Biacore assay, 20 mers of oligo DNA biotinylated at the 5Ј-end were immobilized on sensor chip SA (streptavidine). Surface Plasmon Resonance Assay (Biacore Assay)-Biacore 3000 (Biacore AB, Uppsala, Sweden) was used for surface plasmon resonance analysis according to the manufacturer's instructions. The methods with buffer can be described briefly as follows. We used sensor chip SA and HBS-N buffer (10 mM HEPES, pH 7.4, 150 mM NaCl) as running buffer. 10 l of 2 M biotinylated DNA oligomers were immobilized to sensor chip SA. EGCG and other catechins were originally dissolved in methanol at 10 mM as stock solution. At various concentrations (1.6, 3.3, 6.5, 13, 25, and 50 M) in HBS-N buffer, they were applied to the immobilized ssDNA and dsDNA at 20 l/min as flow rate (Figs. 1 and 2). Blank was used as reference and was subtracted from all the raw data. For the affinity analysis, we used BIAevaluation steady state affinity software. The results with 10 M EGCG and other catechins are presented in Fig. 2.

Interaction between DNA Oligomers and EGCG Determined by Biacore Assay and Their Structure-Function
Relationship-We tested the interaction between catechins and DNA using two different methods: Biacore analysis, which indicates the real-time binding, and CSI-MS, which shows the end point binding. The Biacore assay method makes it possible to observe the association and subsequent dissociation of DNA FIGURE 1. Interaction between DNA oligomer and EGCG determined by surface plasmon analysis (Biscore assay). Biotinized oligomer DNA was immobilized on sensor chip SA. Various concentrations of EGCG were applied to a DNA-immobilized sensor chip. Resonance unit reflects the number of attached molecules. A, red, purple, green, blue, dark blue, and brown lines indicate the binding curves of 1.6, 3.3, 6.5, 13, 25, and 50 M EGCG with DNA, respectively. The data show the resonance unit after subtracting the resonance unit of DNA (Ϫ) as background from the experimental unit. The resonance units of EGCG to DNA increased dose dependently. Association and dissociation represent the buffer in the presence or absence of the input molecules. B, the affinity analysis of EGCG with DNA, which was conducted based on the data of panel A. with EGCG in real time. We analyzed the interaction between EGCG and poly(dT) 20-mer oligo ssDNA at various concentrations (1.6, 3.3, 6.5, 13, 25, and 50 M) of EGCG (Fig. 1). Fig. 1 shows both the association, represented by an increase in resonance unit, and the dissociation, represented by a decrease in resonance unit. Poly(dT) ssDNA oligomers immediately associated with EGCG and rapidly dissociated from it. The resonance units of EGCG to DNA increased dose dependently (red for 1.6 M, purple for 3.3 M, green for 6.5 M, blue for 13 M, dark blue for 25 M, and brown for 50 M EGCG) (Fig. 1A). This is the first demonstration that EGCG directly binds to DNA. For the binding parameters, we conducted steady state affinity analysis using BIAevaluation software. The K d value was estimated to be 5.4 ϫ 10 Ϫ5 M (Fig. 1B). The effective concentrations of EGCG for growth inhibition of a human lung cancer cell line are almost the same as this K d value (3), and EGCG subsequently accumulated in the cells. Thus we think that the K d value 5.4 ϫ 10 Ϫ5 M is reasonable (for more details, see "Discussion").
(The results with poly(dC) are not shown). Poly(dT) oligomer bound to EGCG, and poly(dA) and poly(dG) oligomers showed weak binding to EGCG, indicating the presence of base preference for EGCG-DNA binding. Because ethidium derivatives are known to bind to AT-rich sequences via a minor groove, EGCG may recognize a higher order structure of DNA.
To study the structure-function relationship of EGCG for DNA binding, we next determined which chemical group of EGCG is important for the interaction with DNA. We used several catechins and related chemicals: EGCG, ECG, EGC, EC, GA, GCG, CG, GC, C and theaflavin. In addition to EGCG, ECG, GCG, and CG also showed the association with poly(dT) 18 mer (Fig. 2, a, b, g, and h).
Other catechins, such as EGC, EC, GC, C, and theaflavin, did not interact with DNA oligomers (Fig. 2, c, d, i-k). Based on our results, we think that the binding to DNA oligomers is not influenced by the (Ϫ)-epi-or non (Ϫ)-epi forms of catechins. Next, we designed a putative tertiary structure of various catechins and used them for binding analysis (Fig. 2). The structure-function relationship of catechins and DNA oligomers indicated the significance of the branching structure, consisting of galloyl and catechol groups (see the putative molecular models of the tertiary structures in Fig. 2, right column). To confirm the usefulness of the branching structure, a mixture of EC and GA was tested, but no association was observed (Fig. 2, f and l ). This suggests an insufficient interaction when the two chemical groups are separated but strengthened the importance of these two groups when linked by covalent bond. Although the exact binding site and the mode of interaction between catechins and DNA have not been resolved yet, we assume that the branching structure of EGCG is attached to some parts of DNA molecule. All the results strongly encouraged us to pursue further experiments with DNA oligomers and EGCG using CSI-MS analysis.
Multiple EGCG-binding Sites in DNA Oligomers Determined by CSI-MS Analysis-Mass spectrometry can show the exact molecular ratio of binding molecules. CSI-MS is a direct analysis method that promotes electrolytic dissociation forming the molecular ion in solution at low temperature (27,28). This method has the advantage of detecting extremely labile complexes of biological molecules without causing decomposition. For the experiments, 50 M DNA oligomers, such as single-stranded 18 mers of poly(dA), poly(dT), poly(dG), poly(dC), poly(dA⅐dT), and poly(dG⅐dC) were mixed with 0.1 mM EGCG. For example, the molecular weights of poly(dT) 18 mer and EGCG are 5,414 and 458, respectively. In the CSI-MS analysis, the molecular weights are presented as numbers divided by the charge numbers. Various peaks of poly(dT) 18 mer and the bound form, DNA ϩ EGCG, appear in Fig. 3. As an example, peaks of [DNA] 4ϩ and [DNA ϩ 2EGCG] 4ϩ were output in the m/z ranges of 1350.3 and 1579.0, respectively. The number 4 represents the charge number of molecules in the ionization in this case; when the difference between the two values is multiplied by four, resulting in 915, the value corresponds to the molecular weight of two molecules of EGCG, which is 916. These results indicate that two molecules of EGCG directly associated with DNA. Other peaks were also calculated and identified as shown in Fig. 3. Specifically, three kinds of bound forms, (DNA ϩ EGCG), (DNA ϩ 2EGCG), and (DNA ϩ 3EGCG), were observed, showing that at least three molecules of EGCG bind to poly(dT) 18 mer and suggesting that multiple binding sites for EGCG are present in DNA oligomer (Table 1).
To study the nucleotide specificity of DNA oligomer for EGCG binding, the next experiments were conducted with 18 mers of poly(dA), poly(dG), and poly(dC) and 9 mers of poly(dA⅐dT) and poly(dG⅐dC). The results are summarized in Table 1. Whereas poly(dT) oligomers bound to three molecules of EGCG, poly(dA), poly(dC), and poly(dA⅐dT) showed weak binding to at least one molecule of EGCG, which corresponds to the results of the Biacore assay. No interaction between poly(dG) 18 mer and EGCG was detected, probably due to difficulty of ionization in the assay conditions. All the results in CSI-MS analysis showed that EGCG binds to ssDNA oligomers. The question of why three molecules of catechin, but not more, bind to the 18 mers will be discussed in the next part.
Interaction Between ssRNA Oligomers and EGCG Determined by CSI-MS Analysis-Based on evidence that ssDNA oligomers directly bind to EGCG, we next tested the binding of ssRNA oligomers by CSI-MS analysis. For this experiment, 0.5 mM 18 mers of poly(A), poly(U), poly(G), and poly(C) and 18 mers of poly(AU) and poly(GC) and 0.1 mM EGCG were mixed. Fig. 4 shows three bound forms of EGCG, (RNA ϩ EGCG), (RNA ϩ 2EGCG), and (RNA ϩ 3EGCG), suggesting that poly(A) 18 mer binds to at least three molecules of EGCG (Table 1). In addition, the 18 mers of poly(AU) bound to one molecule of EGCG, indicating that poly(A), poly(U), and poly(AU) also bind to EGCG. However, the interaction between poly(G) and EGCG was not clearly observed, similar to the interaction between poly(dG) and EGCG. This suggests that oligomers of poly(G) and poly(dG) do not readily ionize under these assay conditions. Because EGCG binds to both ssDNA and ssRNA oligomers, we assume that the 2Ј-hydroxyl group of ribose does not influence the interaction with EGCG.     Interaction between dsDNA Oligomers and EGCG, Determined by CSI-MS Analysis and Biacore Assay-It is necessary to study the interaction between dsDNA and EGCG more precisely, because DNA exists as a double strand in the genome. As in the case of ssDNA, we conducted two different assays for this subject. Initially, various lengths of double-stranded AG⅐CT oligomers (from 6 to 14 base pairs) were mixed with EGCG under identical conditions. Fig. 5 shows that one or two molecules of EGCG bound to all double-stranded AG⅐CT oligomers without any relation to nucleotide length.
To study the binding mode of EGCG and dsDNA, the assay was conducted at high (48°C) and low temperatures (28°C), using six-base oligomer dsDNAs (Fig. 6). As Fig. 6A shows, dsDNA oligomers were detected only as EGCG-bound forms at high temperature, although at low temperature both the free and EGCG-bound forms were present. At the high temperature this length of oligomer dsDNA normally melts into ssDNA. These data suggest that EGCG protects dsDNA oligomers from dsDNA melting to ssDNA.
In the Biacore assay, we detected the association of EGCG with the annealed 20-mer DNA (Fig. 7a), but no association with other catechins was found (Fig. 7, b-d). Judging from these different experiments, we conclude that EGCG also binds to dsDNA. However, the other catechins did not, which indicates that one hydroxyl group of the trihydroxyphenyl group in EGCG is essential for binding to dsDNA. The galloyl group is also essential for binding to both ssDNA and dsDNA. The association-dissociation curve is different in the experiments between EGCG-ssDNA and EGCG-dsDNA, and the difference probably reflects the binding mode of EGCG to each DNA.

DISCUSSION
Based on numerous reports, the interaction of EGCG with various proteins and lipids is now widely accepted. Catechins affect reactions associated with DNA, and in those cases EGCG is thought to interact with enzymes on DNA molecules (20 -25). This is the first experiment demonstrating a direct interaction between catechin and polynucleotides, and so, this report shows a new molecular mechanism of action for green tea catechins.
We previously reported that duplicate 3 H-EGCG administrations at 6-h intervals enhanced incorporation of 3 H-EGCG 4 to 9 times in most organs compared with a single administration. Because this enhancement showed that EGCG accumulates in cells, we named it the "Fujiki-Suganuma Effect" (29,30). Although it is not clear yet which molecules promote EGCG accumulation in cells, the results of the current experiment allow us to assume that both DNA and RNA molecules can act as biological reservoirs for EGCG. In most experiments in cell lines treated with EGCG, the concentrations of EGCG are relatively higher than those of the usual active compounds, but we think such concentrations are significant in vivo: EGCG and green tea catechins accumulate in the whole body by consumption of green tea throughout the day.
It is of interest to note why more than three molecules of catechin were not found on the 18 mers, and there are at least two possible explanations. One is the limitation in the sensitivity of mass spectrometry; the complex of ssDNA with more than three molecules of EGCG would be broken upon ionization of CSI, and a peak of the complex of ssDNA with more than three molecules of EGCG would be covered by the background peaks. Another possibility is that the binding surface is limited because of the tertiary structure of ssDNA, which influences the interaction. This problem can be resolved by NMR or x-ray crystallography.
Recently, administration of green tea was reported to alter the mutation profile of p53 (31), and our findings here on the binding of catechins to DNA support this change in the mutation profile of DNA. Catechin inhibits the activities of various proteins attached to DNA, such as DNA polymerase, cAMP-response element-binding protein, DNA methyltransferase, and DNA topoisomerase (21)(22)(23)(24)(25)(26). These reactions are likely affected by EGCG binding to DNA and RNA.
Catechins induce apoptosis in cells and sometimes cause DNA damage (21)(22)(23)(24)(25)(26), phenomena that are due to oxidative stress. However, this might be triggered by the direct binding of catechins to DNA. Judging from our findings, the mechanisms of anti-cancer and anti-aging activities of catechins are intensified through the binding of catechins to DNA and RNA.