This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Navarro-Perán, E. Articles by Rodríguez-López, J. N. Search for Related Content PubMed PubMed Citation Articles by Navarro-Perán, E. Articles by Rodríguez-López, J. N.

The Antifolate Activity of Tea Catechins

¹ Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular A, Facultad de Biología, Universidad de Murcia; ² Servicio de Análisis Clínicos, Hospital Universitario Virgen de la Arrixaca, Murcia, Spain; and ³ Computational Biology Group and ⁴ Department of Biological Chemistry, John Innes Centre, Norwich, United Kingdom

Requests for reprints: José Neptuno Rodríguez-López, Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular A, Facultad de Biología, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain. Phone: 34-968398284; Fax: 34-968364147; E-mail: neptuno{at}um.es.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
A naturally occurring gallated polyphenol isolated from green tea leaves, (–)-epigallocatechin gallate (EGCG), has been shown to be an inhibitor of dihydrofolate reductase (DHFR) activity in vitro at concentrations found in the serum and tissues of green tea drinkers (0.1-1.0 µmol/L). These data provide the first evidence that the prophylactic effect of green tea drinking on certain forms of cancer, suggested by epidemiologic studies, is due to the inhibition of DHFR by EGCG and could also explain why tea extracts have been traditionally used in "alternative medicine" as anticarcinogenic/antibiotic agents or in the treatment of conditions such as psoriasis. EGCG exhibited kinetics characteristic of a slow, tight-binding inhibitor of 7,8-dihydrofolate reduction with bovine liver DHFR (K_I = 0.109 µmol/L), but of a classic, reversible, competitive inhibitor with chicken liver DHFR (K_I = 10.3 µmol/L). Structural modeling showed that EGCG can bind to human DHFR at the same site and in a similar orientation to that observed for some structurally characterized DHFR inhibitor complexes. The responses of lymphoma cells to EGCG and known antifolates were similar, that is, a dose-dependent inhibition of cell growth (IC₅₀ = 20 µmol/L for EGCG), G₀-G₁ phase arrest of the cell cycle, and induction of apoptosis. Folate depletion increased the sensitivity of these cell lines to antifolates and EGCG. These effects were attenuated by growing the cells in a medium containing hypoxanthine-thymidine, consistent with DHFR being the site of action for EGCG.

Key Words: green tea • catechins • dihydrofolate reductase • EGCG • antifolates

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Green tea catechins that include (–)-epigallocatechin gallate (EGCG), (–)-epigallocatechin (EGC), (–)-epicatechin gallate (ECG), and (–)-epicatechin (EC) exhibit a range of biological activities (1–3). EGCG has been the most extensively studied because of its relatively high abundance and strong epidemiologic evidence for cancer prevention (4). EGCG have been shown in vitro to stimulate apoptosis and cell cycle arrest of various cancer cell lines, including prostate, lymphoma, colon, and lung (1). The site of action and mechanism at the molecular level by which EGCG acts as an anticarcinogen is poorly understood. EGCG has been implicated in the modulation of several transcription factors such as activator protein 1 and nuclear factor B, inhibition of gene expression such as tumor necrosis factor , vascular endothelial growth factor, and nitric oxide synthase, and modulation of several cancer-related proteins that include urokinase, ornithine decarboxylase, matrix metalloproteinase, and cyclooxygenase (see ref. 4 and references therein). In addition, ester bond–containing tea polyphenols potently inhibit proteasome activity (5). EGCG binds strongly to many biological molecules and affects a variety of enzyme activities and signal transduction pathways at concentrations from milli- to nanomolar (6). The effective concentration of EGCG in the blood or tissues of tea drinkers is in the range 0.1 to 1.0 µmol/L (6), an important factor in deciding whether an in vitro modulation of biological activity by EGCG is likely to be relevant in vivo.

In attempting to explain the range of responses of normal and cancer cells to tea phenols observed in our laboratory and those of others, we were intrigued by the structural similarity of EGCG to several inhibitors of dihydrofolate reductase (DHFR), in particular the drugs methotrexate (Fig. 1) and aminopterine. DHFR catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate, which acts as a coenzyme for several one-carbon group transfer reactions that include steps in nucleotide biosynthesis. Consequently, inhibition of DHFR, resulting in the disruption of DNA biosynthesis, is the basis of the chemotherapeutic action of a range of DHFR inhibitors, generically known as "antifolates." Tumor cells that grow rapidly require a higher concentration of dTTP than normal cells, and therefore are more sensitive to antifolates. We have therefore tested our hypothesis that the differential physiologic effects of tea polyphenols on normal and cancer cell lines can be explained if they have pronounced "antifolate activity" by studying in vitro the inhibition of DHFR isolated from two sources, bovine and chicken livers. We have also used the published X-ray structure of human DHFR bound to a tetrahydroquinazoline inhibitor, (R)-6-{[methyl-(3,4,5-trimethoxyphenyl)amino]methyl}-5,6,7,8-tetrahydroquinazoline-2,4-diamine (TQD; Fig. 1) to model the binding of EGCG in a manner that can explain the observed tight binding and competitive inhibition with respect to DHF.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 1. A, structural formulas of (–)-epigallocatechin gallate, TQD, and methotrexate. B, wall-eyed stereo view of EGCG modeled into the folate-binding site of human DHFR. Carbon atoms of the EGCG ligand and surrounding protein are colored green and gray, respectively. Residue Phe31, located behind the EGCG, is unlabeled. Four different ligands from human and chicken DHFR crystal structures were used to define a binding envelope, shown in cyan; these were placed in a common orientation by superimposing backbone atoms from a common set of protein residues located around the ligands. Ligands from the following Protein Data Bank structure files were used: 1DR1 (biopterin), 1S3V (TQD), 1S3W, and 1DLR.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

The action of folate analogues, which act as slow-binding inhibitors (I) on DHFR (E), can be described by the following mechanism:

(A)

Although the DHFR-catalyzed reaction has been shown to occur via a random mechanism (7), it can be simplified to an ordered mechanism whenever [NADPH] >> [DHF]. If the concentration of free inhibitor is not substantially altered by the formation of an enzyme-NADPH inhibitor complex, the progress curve for the inhibition in the presence of saturating NADPH can be described by Eq. B:

(B)

where v_s, v₀, and k' represent the steady-state velocity, initial velocity, and apparent first-order rate constant, respectively. The apparent first-order rate constant is related to the inhibitor concentration by Eq. C, where K_m^DHF is the Michaelis constant of DHFR for DHF:

(C)

(D)

(E)

K_I and K_I* denote the respective dissociation constants for the initial and equilibrium binding of inhibitors to the enzyme-NADPH complex. The slow development of catechin inhibition was determined by continuously monitoring the disappearance of NADPH and DHF after initiation of the reaction by the addition of DHFR (3.3 nmol/L). Reaction mixtures contained buffer mixture, NADPH (100 µmol/L), DHF (20 µmol/L), and various concentrations of catechins from 0 to 20 µmol/L. Experiments to determine the maximum steady-state rate (V) and K_m^DHF at several pH values required the analysis of the curvature evident in the time courses for the disappearance of NADPH and DHF (10 determinations). The initial concentration of saturating NADPH (200 µmol/L) was considered to be constant over the period required for the consumption of 10 µmol/L DHF after the addition of DHFR (6 nmol/L). Data were fitted by nonlinear regression to the integrated form of the Michaelis equation (8). The extent of recovery of enzymatic activity after inhibition induced by preincubation with catechins was determined as follows. DHFR (165 nmol/L) was preincubated for 30 minutes at 25°C in the buffer mixture (pH 8.02) containing catechin (20 to 50 µmol/L) and ascorbic acid (1 mmol/L). An aliquot of the incubation mixture was then diluted 500-fold into a reaction mixture containing buffer mixture (pH 8.02), NADPH (100 µmol/L), and DHF (20 µmol/L) to give a final enzyme concentration of 0.33 nmol/L. Recovery of enzyme activity was followed by continuous monitoring at 340 nm.

In silico Molecular Modeling of the Interaction between EGCG and Dihydrofolate Reductase. Molecular modeling was done using the Discover module of Insight II (release 2000.1, Accelrys Ltd., Cambridge, United Kingdom). Human DHFR X-ray crystal structure 1S3V (9) was retrieved from the protein data bank (10), and its TQD ligand was used as a template for positioning of the EGCG ligand. The composite protein/EGCG model was geometry optimized within Insight II using the consistent valence force field and steepest descent algorithm to a derivative of 1.0. The refined model was validated within InsightII using Prostat.

Cell Culture Experiments. The mouse lymphoma cell line L1210 was maintained at 37°C in a humid 7.5% CO₂/95% air environment for all the experiments. To determine the dose-dependent changes, L1210 cells were plated at a density of 10,000 cells/mL in 96-well plates with a "standard folate" medium [RPMI 1640 supplemented with 10% FCS, 2 mmol/L glutamine, and 100 µg/mL of penicillin and streptomycin (all from Life Technologies, Inc., Barcelona, Spain)] and treated during 4 days with different EGCG concentrations. Cell injury was evaluated by a colorimetric assay for mitochondrial function using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (11). IC₅₀ value was defined as the EGCG concentration that gave a 50% decrease in cellular growth compared with values of untreated control cells. For the time course study, cells were plated as described above and treated with 20 µmol/L EGCG. Reversion experiments were done in a medium containing hypoxanthine-thymidine (HT medium) and/or by adding 50 µmol/L ascorbic acid. With the folate-depleted experiments, IC₅₀ values of EGCG were determined at different folic acid concentrations. The cells were previously adapted over a period of 2 to 3 days to grow in folate-free medium [RPMI 1640 (Sigma) supplemented with 10% dialyzed FCS (Sigma), 2 mmol/L glutamine, and 100 µg/mL of penicillin and streptomycin]. This medium is subsequently referred to as low-folate medium. For the cytotoxicity assay, the cells were grown in 75-cm² NUNC flasks with low-folate medium supplemented with different concentrations of folic acid (Sigma). Different EGCG concentrations were added and the cells were further incubated for various periods. Cell growth was determined with the use of a Z2 Coulter counter (Beckman Coulter, Inc, Fullerton, CA) and a hemocytometer.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Inhibition of Dihydrofolate Reductase by Tea Catechins. Steady-state kinetic data showing the inhibition by EGCG of DHF reduction with chicken liver DHFR are shown in Fig. 2A. A K_I (10.3 µmol/L) for EGCG as a competitive inhibitor of DHF calculated from the secondary plot (Fig. 2B) is compared in Table 1 with values for methotrexate (1.3 nmol/L) and trimethoprim (3.5 µmol/L). Preincubation of the enzyme with EGCG did not produce any measurable inhibition. Thus, the inhibition shown in Fig. 2A and B must involve rapid reversible binding of EGCG to chicken liver DHFR. However, EGCG acted as a slow tight-binding inhibitor of bovine liver DHFR (Fig. 2C). In the absence of EGCG, the steady-state velocity of DHF reduction is rapidly established and only shows a minor deviation from linearity over a 15-minute period due to substrate (DHF) depletion. However, in the presence of EGCG, a time-dependent decrease in the reaction rate, which varies as a function of the inhibitor concentration, is clearly apparent in Fig. 2C and D. Further evidence for slow-binding inhibition was obtained by adding aliquots of preincubation mixtures of EGCG and the bovine liver enzyme to substrate-containing assay mixtures. Such behavior can be described by a mechanism that involves the rapid binding of the inhibitor (EGCG) to the enzyme (DHFR) to form an EI complex which then undergoes a slow isomerization to form an EI* complex (Eq. A). Such a mechanism of inhibition of DHFR has been previously reported for folate analogues such as methotrexate and deazafolates (7, 12). A complete kinetic analysis of the inhibition of bovine liver DFHR yielded the kinetic parameters given in Table 1. Although ECG was also a potent inhibitor, polyphenols lacking the ester bonded gallate moiety (i.e., EGC and EC) did not inhibit bovine DHFR activity. These results indicate that the ester bonded gallate moiety is essential for potent inhibition of bovine liver DHFR. Furthermore, a green tea extract containing significant amounts of EGCG also strongly inhibited the DHFR activity of both the bovine and chicken liver enzymes. Methotrexate is a stronger inhibitor (K_I*, picomolar range) than EGCG (K_I*, nanomolar range) for the two DHFRs studied. EGCG should therefore be assigned to the class of "soft" DHF analogue inhibitors of DHFR (13). Such compounds have several advantages in the treatment and prevention of cancer because they can attenuate the adverse side effects often associated with DHFR inhibitors such as methotrexate that are currently in clinical use.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Inhibition of chicken liver (A and B) and bovine liver (C and D) dihydrofolate reductases by EGCG. A, Lineweaver-Burk plots of the reaction of chicken liver DHFR with DHF and NADPH. EGCG concentrations were () 0, () 25, () 50, and () 100 µmol/L. Points, mean of five separate experiments; bars, SD. B, secondary plots for the apparent Michaelis constant of DHFR for dihydrofolate (KmDHF), obtained from Fig. 1A, versus the concentration of EGCG. C, progress curves for the slow, tight-binding inhibition of bovine liver DHFR by EGCG. D, nonlinear regression analysis of the progress curves presented in Fig. 1C to Eq. A yield the values of k' at different EGCG concentrations. Points, mean of triplicate determinations; bars, SD.

Molecular Modeling of the Interaction between EGCG and Dihydrofolate Reductase. On searching the available ligand-bound human DHFR structures in the Protein Data Bank (10), we identified a 1.8-Å structure (Protein Data Bank accession code 1S3V; ref. 9) containing a tetrahydroquinazoline antifolate ligand, TQD (Fig. 1), as the best available structural match for EGCG. Using the position of TQD as a guide, EGCG was docked into this protein structure and the EGCG-protein composite was then energy minimized (Fig. 1B). Comparison with a range of other DHFR structures containing folate or various inhibitors showed that most of the EGCG lies within the consensual substrate/inhibitor envelope, with the exception of the nonester trihydroxybenzene moiety. To accommodate this ring, the Leu²² side chain is required to adopt a different orientation; a precedent for this movement is provided by the crystal structure of a Tyr²² mutant, which displays a similar geometry at this residue (16). There are specific hydrogen bonding interactions, most notably that involving Glu³⁰. For folate and methotrexate, adjacent heterocyclic and amino nitrogens of the ligand form a pair of hydrogen bonds with the two oxygens of the Glu³⁰ side chain (both O···N distances 2.8 Å). In contrast, EGCG has only a single phenolic OH group available for hydrogen bonding to Glu³⁰ (O···O distance 2.7 Å). This is consistent with the pK_a data discussed above and could explain the soft drug character of this catechin. Other EGCG-protein contacts, shown in Fig. 1B, are similar to those found for TQD.

The difference in the type of inhibition exhibited by EGCG with bovine and chicken DHFR must be due to a difference in primary sequence and hence the three-dimensional structures of these enzymes. The DHFRs from humans, cows, and chickens are quite similar, with sequence identities of 75%, 76%, and 87% for the sequence pairs human/avian, bovine/avian, and human/bovine, respectively. Structural analysis of our model showed only one residue within 4 Å of the EGCG ligand that is variable in the three sequences, namely, residue 31. This residue is located at the active site of DHFR and is Tyr in chicken, but Phe in human and bovine DHFR. Phe³¹ has the same side chain conformation as Tyr³¹ in some, but not all, of the human structures. Because this side chain has quite extensive van der Waals contacts with the EGCG ligand in our model (see Fig. 1), and interacts with folate and inhibitors such as methotrexate and trimethropin, its mutation could have a noticeable effect on ligand binding. Indeed, the crystal structure of chicken liver DHFR complexed with NADP⁺ and biopterin showed two alternative conformations for Tyr³¹, and the implications of this observation in terms of the catalytic mechanism have been discussed (17).

Inhibition of Dihydrofolate Reductase by EGCG in Cancer Cells. To determine whether EGCG inhibits DHFR activity in vivo, a mouse lymphoma cell line (L1210) was incubated with various concentrations of EGCG in a standard folate medium. EGCG significantly inhibited L1210 growth in a concentration-dependent manner (IC₅₀ = 20 µmol/L). If this was specifically due to inhibition of DHFR activity by EGCG, cell growth should be restored in HT medium. Antifolates block the de novo biosynthesis of thymine, purines, and pyrimidines by inhibiting the synthesis of 5,6,7,8-tetrahydrofolate, an essential cofactor in these biosynthetic pathways. Cells that express hypoxanthine-guanine phosphoribosyltransferase, an enzyme essential for the recycling of purine nucleotides, can survive in the presence of antifolates in HT medium. Control experiments showed that the inhibition of growth of L1210 cells by methotrexate was greatly attenuated in HT medium (data not shown). Figure 3A shows the time-dependent inhibition of L1210 growth by 20 µmol/L EGCG. Although L1210 grown in HT medium showed a high level of inhibition reversal (Fig. 3A), complete reversal was not obtained after the second day of the experiment. This partial lifting of EGCG inhibition in HT medium is most likely due to secondary effects of EGCG at the concentration used in this assay. EGCG has been reported to have pro-oxidant activity in several cell lines (e.g., hepatoma cells; ref. 18). The production of reactive oxygen species has been associated with the inhibition of cancer cell growth by tea polyphenols (19). The inhibition of L1210 growth by EGCG was partially lifted by the inclusion of the antioxidant ascorbic acid in the reaction medium (Fig. 3A). Similar results were obtained by cotreating the cells with N-acetylcysteine (a glutathione precursor and scavenger of reactive oxygen species) or superoxide dismutase. Growing L1210 in HT medium containing ascorbic acid (Fig. 3A), N-acetylcysteine, or superoxide dismutase completely removed the inhibitory effect of EGCG. These data provide strong evidence that the major site of action of EGCG in vivo is DHFR.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 3. DHFR inhibition by EGCG in cancer cells. A, time-dependent loss of L1210 viability induced by 20 µmol/L EGCG. Columns, mean percentage of untreated control cells determined from three independent experiments; bars, SD. B, effect of different EGCG concentrations on L1210 cell growth after 29 hours of treatment at two different folic acid concentrations, 3 µmol/L (blue line) or 0.3 µmol/L (red line). Treatments were done in triplicate and each experiment was done at least twice. Inset, dependence of the IC50 values on the folic acid concentration added to the medium. C, growth-inhibitory effects of 10 µmol/L EGCG at various folic acid concentrations at two different time intervals. The data were expressed assuming a zero percentage of growth inhibition for untreated control.

We have shown for the first time that gallated tea polyphenols act as DHFR inhibitors in vitro and in vivo, at concentrations usually found in the blood of tea drinkers. The "soft" character of such compounds could be developed for use in the prevention and treatment of cancer with significantly reduced side effects compared with those of the DHFR inhibitors currently in use in chemotherapy, such as methotrexate. An advantage of EGCG is its differential effects on normal and cancer cells. Importantly, at physiologically attainable concentrations, EGCG kills cancer cells through apoptosis, but has little or no effect on normal cells. Inhibition of DHFR by EGCG explains this differential effect because antifolate compounds are more active on cancer cells, which generally have a higher turnover of DNA. Induction of apoptosis can provide highly effective chemotherapeutic and chemopreventative strategies for cancer control. Many chemopreventative agents act through the induction of apoptosis as a mechanism for the suppression of carcinogenesis by eliminating genetically damaged cells, initiated cells, or cells that have progressed to malignancy. Thus, the soft character of EGCG together with its ability to induce apoptosis through DHFR inhibition provides a convincing explanation for the epidemiologic data on the prophylactic effects of diets high in gallated polyphenols for certain forms of cancer.

We conclude that gallated polyphenols and their derivatives have considerable potential for clinical application as anticarcinogenic agents and as antibiotics and for the treatment of psoriasis (1–3). Our data may also explain why neural tube defects such as anencephaly and spina bifida, which are usually associated with folic acid deficiency, have been linked to high levels of maternal green tea consumption during the periconceptional period (20).

	Acknowledgments

 
Grant support: EU INTAS Program project INTAS00-0727 (F. Garc;ía-Cánovas, R.N.F. Thorneley, and J.N. Rodríguez-López), Fondo de Investigación Sanitaria projects 01/3025 and 02/1567 (J. Cabezas-Herrera), and Biotechnology and Biological Sciences Research Council, United Kingdom (R.N.F. Thorneley and M.C. Durrant). E. Navarro-Perán has a fellowship from the Ministerio de Educación, Cultura y Deporte.

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.

Received 9/27/04. Revised 11/18/04. Accepted 1/12/05.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

1. Mukhtar H, Ahmad N. Tea polyphenols: prevention of cancer and optimizing health. Am J Clin Nutr 2000;71:1698–702S.
2. Mabe K, Yamada M, Oguni I, et al. In vitro and in vivo activities of tea catechins against Helicobacter pylori. Antimicrob Agents Chemother 1999;43:1788–91.[Abstract/Free Full Text]
3. Heyendael VMR, Spuls PI, Opmeer BC, et al. Methotrexate versus cyclosporine in moderate-to-severe chronic plaque psoriasis. N Engl J Med 2003;349:658–65.[Abstract/Free Full Text]
4. Jung YD, Ellis LM. Inhibition of tumour invasion and angiogenesis by epigallocatechin gallate (EGCG), a major component of green tea. Int J Exp Pathol 2001;82:309–16.[CrossRef][Medline]
5. Nam S, Smith DM, Dou QP. Ester bond-containing tea polyphenols potently inhibit proteasome activity in vitro and in vivo. J Biol Chem 2001;276:13322–30.[Abstract/Free Full Text]
6. Yang CS. Inhibition of carcinogenesis by tea. Nature 1997;389:134–5.[CrossRef][Medline]
7. Stone SR, Morrison JF. Mechanism of inhibition of dihydrofolate reductases from bacterial and vertebrate sources by various classes of folate analogues. Biochim Biophys Acta 1986;869:275–85.[CrossRef][Medline]
8. Cornish-Bowden A. Fundamentals of enzyme kinetics. London: Butterworth and Co; 1979. p. 34–7.
9. Cody V, Luft JR, Pangborn W, et al. Structure determination of tetrahydroquinazoline antifolates in complex with human and Pneumocystis carinii dihydrofolate reductase: correlations between enzyme selectivity and stereochemistry. Acta Crystallogr D Biol Crystallogr 2004;60:646–55.[CrossRef][Medline]
10. Berman HM, Westbrook J, Feng Z, et al. The protein data bank. Nucleic Acids Res 2000;28:235–42.[Abstract/Free Full Text]
11. Carmichael J, Degraff WG, Gazdar AF, et al. Evaluation of a tretazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 1987;47:936–42.[Abstract/Free Full Text]
12. Williams EA, Morrison JF. Human dihydrofolate reductase: reduction of alternative substrates, pH effects, and inhibition by deazafolates. Biochemistry 1992;31:6801–11.[Medline]
13. Graffner-Nordberg M. Approaches to soft drug analogues of dihydrofolate reductase inhibitors. Design and synthesis. Acta Universitatis Upsaliensis. Comprehensive summaries of Uppsala dissertations from the Faculty of Pharmacy 252. Uppsala: Tryck and Medier, Uppsala; 2001.
14. Baumann H, Wilson KJ. Dihydrofolate reductase from bovine liver. Enzymatic and structural properties. Eur J Biochem 1975;60:9–15.[Medline]
15. Cody V, Luft JR, Ciszak E, et al. Crystal structure determination at 2.3 Å of recombinant human dihydrofolate reductase ternary complex with NADPH and methotrexate--tetrazole. Anticancer Drug Des 1992;7:483–91.[Medline]
16. Lewis WS, Cody V, Galitsky N, et al. Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. J Biol Chem 1995;270:5057–64.[Abstract/Free Full Text]
17. Mc Tigue MA, Davies JF, Kaufman BT, et al. Crystal structure of chicken liver dihydrofolate reductase complexed with NADP+ and biopterin. Biochemistry 1992;31:7264–73.[CrossRef][Medline]
18. Waltner-Law ME, Wang XL, Law BK, et al. Epigallocatechin gallate, a constituent of green tea, represses hepatic glucose production. J Biol Chem 2002;277:34933–40.[Abstract/Free Full Text]
19. Backus HH, Pinedo HM, Wouters D, et al. Folate depletion increases sensitivity of solid tumor cell lines to 5-fluorouracil and antifolates. Int J Cancer 2000;15:771–8.
20. Correa A, Stolley A, Liu Y. Prenatal tea consumption and risk of anencephaly and spina bifida. Ann Epidemiol 2000;10:476–7.

This article has been cited by other articles:

				T. Naganuma, S. Kuriyama, M. Kakizaki, T. Sone, N. Nakaya, K. Ohmori-Matsuda, A. Hozawa, Y. Nishino, and I. Tsuji Green Tea Consumption and Hematologic Malignancies in Japan: The Ohsaki Study Am. J. Epidemiol., September 15, 2009; 170(6): 730 - 738. [Abstract] [Full Text] [PDF]

				T. D. Shanafelt, T. G. Call, C. S. Zent, B. LaPlant, D. A. Bowen, M. Roos, C. R. Secreto, A. K. Ghosh, B. F. Kabat, M.-J. Lee, et al. Phase I Trial of Daily Oral Polyphenon E in Patients With Asymptomatic Rai Stage 0 to II Chronic Lymphocytic Leukemia J. Clin. Oncol., August 10, 2009; 27(23): 3808 - 3814. [Abstract] [Full Text] [PDF]

				M. Inoue, K. Robien, R. Wang, D. J. Van Den Berg, W.-P. Koh, and M. C. Yu Green tea intake, MTHFR/TYMS genotype and breast cancer risk: the Singapore Chinese Health Study Carcinogenesis, October 1, 2008; 29(10): 1967 - 1972. [Abstract] [Full Text] [PDF]

				B. D. Zeitlin, I. J. Zeitlin, and J. E. Nor Expanding Circle of Inhibition: Small-Molecule Inhibitors of Bcl-2 as Anticancer Cell and Antiangiogenic Agents J. Clin. Oncol., September 1, 2008; 26(25): 4180 - 4188. [Abstract] [Full Text] [PDF]

				D. J. Castro, Z. Yu, C. V. Lohr, C. B. Pereira, J. N. Giovanini, K. A. Fischer, G. A. Orner, R. H. Dashwood, and D. E. Williams Chemoprevention of dibenzo[a,l]pyrene transplacental carcinogenesis in mice born to mothers administered green tea: primary role of caffeine Carcinogenesis, August 1, 2008; 29(8): 1581 - 1586. [Abstract] [Full Text] [PDF]

				T.-T. Kao, K.-C. Wang, W.-N. Chang, C.-Y. Lin, B.-H. Chen, H.-L. Wu, G.-Y. Shi, J.-N. Tsai, and T.-F. Fu Characterization and Comparative Studies of Zebrafish and Human Recombinant Dihydrofolate Reductases--Inhibition by Folic Acid and Polyphenols Drug Metab. Dispos., March 1, 2008; 36(3): 508 - 516. [Abstract] [Full Text] [PDF]

				S. B. van Waalwijk van Doorn-Khosrovani, J. Janssen, L. M. Maas, R. W.L. Godschalk, J. G. Nijhuis, and F. J. van Schooten Dietary flavonoids induce MLL translocations in primary human CD34+ cells Carcinogenesis, August 1, 2007; 28(8): 1703 - 1709. [Abstract] [Full Text] [PDF]

				M. D. Navarro-Martinez, F. Garcia-Canovas, and J. N. Rodriguez-Lopez Tea polyphenol epigallocatechin-3-gallate inhibits ergosterol synthesis by disturbing folic acid metabolism in Candida albicans J. Antimicrob. Chemother., June 1, 2006; 57(6): 1083 - 1092. [Abstract] [Full Text] [PDF]

				M. D. Lucock and P. D. Roach The Antifolate Activity of Tea Catechins Cancer Res., September 15, 2005; 65(18): 8573 - 8573. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Navarro-Perán, E. Articles by Rodríguez-López, J. N. Search for Related Content PubMed PubMed Citation Articles by Navarro-Perán, E. Articles by Rodríguez-López, J. N.