(-)-Epigallocatechin Gallate Regulates CD3-mediated T Cell Receptor Signaling in Leukemia through the Inhibition of ZAP-70 Kinase*

The ζ chain-associated 70-kDa protein (ZAP-70) of tyrosine kinase plays a critical role in T cell receptor-mediated signal transduction and the immune response. A high level of ZAP-70 expression is observed in leukemia, which suggests ZAP-70 as a logical target for immunomodulatory therapies. (-)-Epigallocatechin gallate (EGCG) is one of the major green tea catechins that is suggested to have a role as a preventive agent in cancer, obesity, diabetes, and cardiovascular disease. Here we identified ZAP-70 as an important and novel molecular target of EGCG in leukemia cells. ZAP-70 and EGCG displayed high binding affinity (Kd = 0.6207 μmol/liter), and additional results revealed that EGCG effectively suppressed ZAP-70, linker for the activation of T cells, phospholipase Cγ1, extracellular signaling-regulated kinase, and MAPK kinase activities in CD3-activated T cell leukemia. Furthermore, the activation of activator protein-1 and interleukin-2 induced by CD3 was dose-dependently inhibited by EGCG treatment. Notably, EGCG dose-dependently induced caspase-mediated apoptosis in P116.cl39 ZAP-70-expressing leukemia cells, whereas P116 ZAP-70-deficient cells were resistant to EGCG treatment. Molecular docking studies, supported by site-directed mutagenesis experiments, showed that EGCG could form a series of intermolecular hydrogen bonds and hydrophobic interactions within the ATP binding domain, which may contribute to the stability of the ZAP-70-EGCG complex. Overall, these results strongly indicated that ZAP-70 activity was inhibited specifically by EGCG, which contributed to suppressing the CD3-mediated T cell-induced pathways in leukemia cells.

For thousands of years, tea has been the most widely consumed beverage in the world after water. Historically, tea has been credited with various beneficial health effects, including medicinal efficacy in the prevention and treatment of numerous diseases. Thus, longevity and good health have often been associated with the habit of drinking tea (1). Four major polyphenolic catechins are found in green tea and include (Ϫ)-epicatechin (EC), 3 (Ϫ)-epicatechin 3-gallate (ECG), (Ϫ)-epigallocatechin (EGC), and (Ϫ)-epigallocatechin 3-gallate (EGCG). A cup of green tea may contain 100 -200 mg of EGCG (2). Several investigators have reported that green tea exerts cancer preventive activity at a variety of organ sites, including skin, lung, oral cavity, esophagus, stomach, small intestine, colon, pancreas, and mammary gland (1,3,4). However, the mechanisms explaining the cancer preventive activity of tea and tea polyphenols are still not clearly understood.
The -associated 70-kDa protein (ZAP-70) is a Syk (spleen tyrosine kinase) family tyrosine kinase, which is associated with the subunit of the T cell receptor (TCR). The ZAP-70 protein is primarily expressed in T cells and natural killer cells and plays an essential role in signaling through the T cell antigen receptor (5). The TCRs are associated with tyrosine phosphorylation of multiple proteins resulting in activation of various signaling pathways causing alterations in gene expression, increased T cell proliferation, and secretion of cytokines (6). CD3 (cluster of differentiation 3) stimulation of the T cell antigen receptor plays a role in tyrosine phosphorylation of a number of cellular substrates. An important substrate of ZAP-70 is the TCR chain, which can mediate the transduction of extracellular stimuli into cellular effector functions (7,8). ZAP-70 plays a critical role in cell surface expression of T cell antigen receptor-CD3 complex signaling during the early stages of T cell development and differentiation (9 -13). The ZAP-70 tyrosine kinase is reported to play a critical role in T cell activation and the immune response, and therefore might be a logical target for immunomodulatory therapies (5). Crespo et al. (14) observed that among B cell and T cell lymphoproliferative disorders, a high level of ZAP-70 expression is found in T cell proliferative diseases, acute lymphoblastic leukemia, and a subgroup of chronic lymphocytic leukemia (CLL) (15,16). These studies suggested that ZAP-70 could be an excellent prognostic biomarker in CLL.
Despite advances in T cell leukemia therapy, only a minority of patients achieve long term tumor-free survival with conventional chemotherapy but at the cost of significant, irreversible toxic side effects, which often limit effective treatment (17). Therefore, new therapeutic approaches with enhanced tumor selectivity and more favorable toxicity profiles are urgently needed.
EGCG, a major active constituent of green tea, has been shown to stimulate apoptosis and cell cycle arrest in various cancer cell lines, including prostate, colon, lung, leukemias, and lymphomas (3,18). However, the anticancer mechanisms and molecular targets of EGCG are poorly understood, especially in T cell-mediated leukemias and lymphomas. Here we demonstrate that EGCG might be a potential immunomodulator for the management of ZAP-70-dependent T cell activation in human leukemias and lymphomas. The effects of EGCG were extensively investigated in a ZAP-70-deficient Jurkat T cell line (P116 cells) and a cell line in which ZAP-70 activity was recovered (P116.cl39 cells). Results suggested that ZAP-70 antagonists could be useful immunomodulatory therapeutic agents.
Physical Binding and K d Measurement-ZAP-70 binding assays were carried out as described (20) with some modifications. For analyzing concentration-dependent uptake, 1 nM to 10 M concentrations of EGCG were applied. The K d value was determined through nonlinear regression analysis using the Prizm 4.0 software program (Graphpad Inc., San Diego).
Molecular Modeling of the Interactions of EGCG with ZAP-70-Molecular docking studies were carried out using the Maestro software suite (Maestro, version 7.5, Schrödinger, New York). The EGCG molecule was drawn using the builder tool in Maestro and then optimized for docking in Ligprep. The ZAP-70 crystal structure complexed with staurosporine (Protein Data Bank code 1u59) was prepared for docking following the Glide standard procedure (21). Grids defining the protein receptor were generated considering the binding mode of staurosporine. Many protein-binding sites undergo structural rearrangements upon ligand binding, the so-called "induced fit" allows the binding site to follow the shape of the ligand with resulting better interactions. Consequently, molecular docking was performed with the induced fit docking protocol (Schrödinger Suite 2006). The induced fit docking is a part of the Maestro software suite that attempts to reproduce the protein conformational rearrangement upon binding. Initially, EGCG was docked with Glide in extra-precision mode using a softened potential: scaling the receptor (0.70) and the ligand (0.50) van der Waals radius. The best 20 EGCG poses were retained. Then the receptor side chains within 5 Å distance from the ligand were predicted and minimized for each protein-ligand complex, and then another round of minimizations was performed on each protein-ligand complex pose. Finally, Glide redocking in extra-precision mode of each protein-ligand complex within 30.0 kcal/mol of the lowest energy structure was performed. Herein the best-docked representative structure is presented.
Immunoprecipitation and Western Blotting-The P116 and P116.cl39 cells (5 ϫ 10 5 /ml) were grown in 75-cm 3 flasks for 24 h and then serum-starved for 14 h at 37°C. Where indicated, the cells were pretreated with EGCG (4 or 8 M) for 2 h and then washed three times with phosphate-buffered saline. The cells were stimulated for 30 min at 37°C with 2 g/ml mouse IgG 1 monoclonal immunoglobulin isotype control and 2 g/ml mouse anti-human CD3. The reactions were stopped by adding cold phosphate-buffered saline followed by centrifugation for subsequent lysis. Lysates were incubated overnight at 4°C with the indicated antibodies and protein A/G Plus-agarose beads. After centrifugation, the beads were washed three times with washing buffer and were resuspended in SDS sample buffer. Eluted immunoprecipitates or whole-cell lysates were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and then incubated with appropriate antibodies. Immunocomplexes were detected by subsequent incubation with appropriate horseradish peroxidase-conjugated secondary IgG antibodies and visualized by ECL according to the manufacturer's instructions (Amersham Biosciences).
Electrophoretic Mobility Shift Assay-Nuclear extracts were prepared as described previously (22). The cells were treated and prepared as described above for Western blotting and then disrupted with a hypotonic buffer. The nuclei pellet was disrupted in a hypertonic buffer, and the nuclear extracts were retained for use in the DNA binding assay. A double-stranded deoxyoligonucleotide corresponding to AP (activator protein)-1 responsive elements (Santa Cruz Biotechnology) was end-labeled with [␥-32 P]ATP using T4 kinase. Nuclear extracts (5 g) were incubated in binding buffer for 15 min with poly(dI⅐dC) and the 32 P-labeled DNA probe. The DNA binding activity was separated from free probe using a TBE Ready Gel Precast Gel (Bio-Rad). Following electrophoresis, the gel was dried and visualized by autoradiography.
Measurement of IL-2 Production-Quantification of interleukin (IL)-2 production from P116 and P116.cI39 cells was performed using a commercial enzyme-linked immunosorbent assay system (Pharmingen). The cells (5 ϫ 10 5 cells/ml) were resuspended in fresh medium in a 48-well plate and incubated for 24 h. The cells were treated with various concentrations of EGCG and 2 g/ml mouse IgG 1 monoclonal immunoglobulin isotype control or 2 g/ml mouse anti-human CD3 for 24 or 48 h at 37°C. The levels of IL-2 in the supernatant fractions were subsequently measured using an enzyme-linked immunosorbent assay kit (Pharmingen) according to the manufacturer's instructions. Annexin V Staining-Apoptosis was performed using the annexin V-FITC apoptosis detection kit as recommended by the manufacturer (MBL International Corp., Watertown, MA). Apoptosis was compared in P116 and P116.cl39 cells that were treated or not treated with 4 or 8 M EGCG for 24, 48, 72, or 96 h. The cells were harvested and washed with phosphatebuffered saline and incubated for 5 min at room temperature with annexin V-FITC plus propidium iodide following the protocol included in the kit. Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences).
Statistical Analysis-Data are presented as means Ϯ S.D. of triplicate samples from at least three independent experiments. Differences between means were assessed by one-way analysis of variance, and the minimum level of significance was set at p Ͻ 0.05.

EGCG Binds with ZAP-70 ex Vivo and in Vitro-Expression
of ZAP-70 was recently reported to be a reliable prognostic marker among patients with CLL (14). In a kinase screening experiment (kinase profiler specificity testing service, Upstate), EGCG (5 M) was found to inhibit ZAP-70 kinase activity. We therefore studied the potential interaction of EGCG and ZAP-70 in wild type Jurkat cells, Jurkat cells (P116) that do not express ZAP-70, and in P116 cells (P116.cl39) in which ZAP-70 protein expression has been restored (Fig. 1A). The interaction of ZAP-70 and EGCG was determined in an EGCG-Sepharose 4B affinity chromatography experiment combined with immunoblotting with anti-ZAP-70. Results indicated that EGCG formed a complex with ZAP-70 in P116.cl39 cells but not in P116 cells (Fig. 1B). To characterize the physical binding between EGCG and ZAP-70, we measured the binding affinity (K d ) of the complex using a GST pulldown assay and 3 H-labeled EGCG. The K d value of ZAP-70 and EGCG binding was determined to be 0.6207 mol/liter (Fig. 1C).
Structural Analysis of the Interaction between EGCG and ZAP-70-To better characterize the possible interaction between EGCG and the kinase/ATP binding domain of ZAP-70, we used the x-ray co-crystal structure of ZAP-70 complexed with staurosporine (Protein Data Bank code 1u59) as a starting point for a docking experiment. The accuracy of small molecule/protein docking increases when considering holo-proteins (23), and staurosporine is a rigid and flat molecule that creates an open conformation to the ZAP-70 ATP binding pocket. An open conformation is usually adopted by kinases when bound to small ligands, such as EGCG. In our docking model, EGCG presented a calculated binding affinity of Ϫ13.07 kcal/mol. One of the hydroxyl groups of the EGCG A ring, similar to other known kinase inhibitors, appears to form two hydrogen bonds with the kinase hinge region and, in particular, with the backbone carbonyl group of Glu 415 and the amide group of Ala 417 (Fig. 2A). The A-C ring system acts as an adenine mimic and likewise interacts with the front cleft hydrophobic pocket (Fig. 2B). The gallate moiety (D ring) occupies mainly the hydrophilic and solvent-exposed pocket covered by the G-loop, which is believed to be less important for ligand affinity (24). The side chains of lysine (Lys 369 ) and aspartate (Asp 479 ) may form a network of hydrogen bonds with the hydroxyl groups of the B ring. In addition, the side chain of Glu 386 is expected to form a hydrogen bond with one of the hydroxyl groups of this ring (Fig. 2, A and B). This docking experiment suggested that disrupting specific hydrogen bonding networks by point mutation should affect the ability of EGCG to bind with ZAP-70 as modeled.  OCTOBER 17, 2008 • VOLUME 283 • NUMBER 42

JOURNAL OF BIOLOGICAL CHEMISTRY 28373
Therefore, to continue to assess the importance of the interaction between EGCG and the ZAP-70 kinase/ATP-binding site, we generated a variety of ZAP-70 point mutants, including ZAP-70 E415Q, ZAP-70 E415Q/K369R, ZAP-70 E415Q/ D479N, ZAP-70 E415Q/K369R/D479N, and ZAP-70 E415Q/ K369R/D479N/E386Q/R465K. The various ZAP-70 mutants were each transfected into HEK 293 cells to determine the effect on ZAP-70 binding with EGCG using EGCG-Sepharose 4B or Sepharose 4B combined with Western blot analysis (Fig.  2C). As predicted, the E415Q substitution did not affect the binding because EGCG forms hydrogen bonds with the main chain atoms in the hinge region. The binding seems to be dependent on the interactions of EGCG with Lys 369 and Asp 479 , which we predicted would form a network of hydrogen bonds with the ligand B ring. The bulkier arginine in the K369R mutant very likely affects potential hydrogen bonding with EGCG but also may alter the shape of the cavity, preventing the ligand from assuming the correct orientation for satisfying all the necessary connections with the binding site. Replacing aspartate 479 with asparagine (D479N) also affects the ability of EGCG to bind. We predicted a hydrogen bond between the B ring and the aspartate side chain. The mutant, in which both Lys 369 and Asp 479 were substituted, consistently showed inhibition of EGCG binding. Thus, these data suggested that ZAP-70 Asp 479 and Lys 369 are required for the essential interactions of ZAP-70 with EGCG within the catalytic site.
The information from the docking experiment suggested that EGCG might effectively inhibit ATP binding to active ZAP-70. Thus, we examined the effect of EGCG on ATP binding by pulldown assay using ATP-agarose 4B and the active ZAP-70 protein. Results confirmed that the binding of ATP with ZAP-70 decreased with increasing amounts of EGCG (Fig. 3A). This suggested that EGCG might inhibit ZAP-70 kinase activity by competing with ATP binding.
We next determined whether EGCG could prevent ZAP-70 phosphorylation and/or suppress kinase activity. Kinase activity was analyzed using an in vitro kinase assay with active ZAP-70 and the preferred kinase substrate, poly(Glu 4 -Tyr) peptide. Results indicated that increasing concentrations of EGCG markedly suppressed ZAP-70 kinase activity, and 3 M EGCG significantly inhibited ZAP-70 kinase activity by 50% (Fig. 3B). ZAP-70 is recruited to the phosphorylated CD3 and subunits after TCR stimulation (25). The immunoreceptor tyrosine-based activation motifs (ITAM) of the signal-transducing antigen receptor subunit (CD3 and ) are phosphorylated by Src PTK thus allowing the Syk family PTK ZAP-70 to bind to the ITAM (26). EGCG had no effect on CD3-induced phosphorylation of CD3 (Fig. 3C). TCR-mediated Lck activity leads to phosphorylation of ZAP-70 on Tyr 493 in the regulatory loop of the PTK domain resulting in the up-regulation of ZAP-70 kinase activity (27). Our data showed that EGCG had no effect on phosphorylation of ZAP-70 (Tyr 493 ) (Fig. 3C). Tyrosine 319 is a key phosphorylation site of ZAP-70 and is phosphorylated upon recruitment of ZAP-70 to T cell antigen receptor signaling or by ZAP-70 itself (28 -31). Experiments were performed to compare total tyrosine phosphorylation of ZAP-70 and autophosphorylation of Tyr 319 . EGCG inhibited autophosphorylation of ZAP-70 at Tyr 319 in a dose-dependent manner (Fig. 3C). Notably, EGCG at 4 or 8 M inhibited Tyr 319 autophosphorylation by 76 or 93%, respectively, compared with cells treated with only CD3 (Fig.  3C). On the other hand, total tyrosine phosphorylation was inhibited at about 55% by EGCG (Fig. 3D). The percent inhibition was determined by densitometric analysis of EGCGtreated cells compared with CD3-treated cells after normalization to ZAP-70 total levels (Fig. 3D). Overall, these results indicated that EGCG does not affect ZAP-70 recruitment to the TCR activation complex, but specifically inhibits ZAP-70.

Effects of EGCG on the CD3-induced Phosphorylation of Downstream Kinases-Activation of T cells can be initiated through cell
surface molecules in addition to the TCR-CD3 complex (7,32,33). Expression of the TCR-CD3 complex is associated with acute-type adult T cell leukemia and is observed in lymphoma-type adult T cell leukemia patients (34,35). TCR activates signaling by recruiting and activating PTK of the Src, Syk, and Tec families (8, 36 -39). Activated ZAP-70 phosphorylates adapter LAT at multiple conserved tyrosine residues (Tyr 171 and Tyr 191 ) within the SH2-binding motifs, exposing these motifs as the docking sites for PLC␥1 (40,41). Precisely how the formation of the LAT-associated signaling complex leads to ERK1/2 activation is unclear. ERK1/2 and MEK both need to be activated in order for TCR engagement to result in T cell activation (42). We examined the effect of EGCG on the phosphorylation of several of these downstream kinases in ZAP-70 expressing (P116.cl39) or deficient (P116) cells. The cell lines were treated with increasing concentrations of EGCG (0 -8 M) for 2 h followed by stimulation with CD3 (2 g/ml) for 30 min. An EGCG-induced dose-dependent decrease in phosphorylation of LAT (Tyr 171 , Tyr 191 ), PLC␥1, MEK, and ERK1/2 (Tyr 202 /Tyr 204 ) was observed in P116cl.39 cells compared with untreated control cells (Fig. 4). On the other hand, EGCG had no effect on phosphorylation of these various kinases in ZAP-70-deficient P116 cells.
Effect of EGCG on CD3-induced IL-2 Production and DNA Binding Activity of AP-1-Activated ERK1/2 translocates to the nucleus and directly regulates various transcription factors. Multiple transcription factors regulate the IL-2 gene, including AP-1, nuclear factor-B (NF-B), Oct-1, and nuclear factor of activated T cells. We hypothesized that the inhibition of ZAP-70 by EGCG might decrease CD3-induced IL-2 secretion and DNA binding activity of AP-1. We therefore examined the effect of EGCG on IL-2 secretion and DNA binding activity of AP-1 in supernatant fractions from P116 and P116.cl39 cells. Results indicated that EGCG suppressed CD3-induced AP-1 The asterisk (*, p Ͻ 0.05; **, p Ͻ 0.005; ***, p Ͻ 0.001) indicates a significant decrease in kinase activity compared with untreated control cells. C, phosphorylation of CD3, pZAP-70 (Tyr 319 ), and pZAP-70 (Tyr 493 ) was detected by Western blot as described under "Experimental Procedures." Equal protein loading and protein transfer were confirmed by stripping and incubating the same membrane with antibodies against ␣-tubulin, total CD3, or ZAP-70. D, effect of EGCG on CD3-induced ZAP-70 total tyrosine phosphorylation. P116.cl39 cells were starved for 14 h followed by incubation for 1 h with EGCG at different concentrations (0, 1, 2, 4, or 8 M). Cells were washed and then stimulated with 2 g/ml CD3 for 30 min. Immunoprecipitation with anti-ZAP-70 was followed by anti-phosphotyrosine immunoblot analysis. Membranes were stripped and blotted with anti-ZAP-70. OCTOBER 17, 2008 • VOLUME 283 • NUMBER 42 DNA binding (Fig. 5A) and IL-2 production at 24 h (Fig. 5B) or 48 h (Fig. 5C) in P116.cl39 (ZAP-70 expressing) cells but had no effect on P116 ZAP-70-deficient cells.

EGCG Inhibits Cell Viability and Induces Apoptosis in ZAP-70-expressing Cells but Has No Effect on ZAP-70-deficient Cells-Results
thus far suggested that ZAP-70 induces phosphorylation of pLAT, pPLC␥1, pMEK, and pERK and DNA binding activity of AP-1. These signaling molecules are critical for cell survival (43,44), and the blocking of these signals by EGCG may at least partially explain the ability of EGCG to induce apoptosis. ZAP-70-deficient P116 cells will be more resistant to the effect of EGCG on viability or apoptosis compared with the ZAP-70-expressing P116.cl39 cells. P116 and P116.cl39 cells were cultured for 72 h in a 96-well culture plate with increasing concentrations of EGCG, and then EGCG cytotoxicity was measured using the MTS assay. Treatment with EGCG (2 M) resulted in about a 25% decrease in the viability of P116.cl39 cells (Fig. 6A). Only about 40% of P116.cl39 cells survived treatment with 4 M EGCG, whereas more than 95% of the P116 cells remained viable after treatment with 4 M EGCG. Treatment with the EGCG analogues, EC, ECG, or EGC, had no effect on P116 or P116.cl39 cells (Fig. 6B), suggesting a specific effect for EGCG. To determine whether the effect of EGCG on cell viability was because of apoptosis, flow cytometry was used in combination with Western blot analysis of caspase-3 and cleaved caspase-3. Caspase-3 is used as a marker for apoptosis in these experiments. Results indicated that the percentage of annexinV-stained positive cells (early apoptotic and late apoptotic/necrotic cells) increased substantially in P116.cl39 cells treated for 72 or 96 h with 4 or 8 M EGCG (Fig.  6C). Very little apoptosis was observed in P116 ZAP-70-deficient cells. The key components of the biochemical pathways of caspase activation play a central role in the execution of apoptosis (45,46). We therefore examined the effect of EGCG on caspase-3 and cleaved caspase-3 in P116 and P116.cl39 cells by Western blot analysis. Caspase-3 was detected in P116 cells but did not change with EGCG treatment, whereas capase-3 was substantially decreased in P116.cl39 cells following EGCG treatment (Fig. 6D). Cleaved caspase-3 was detected in the EGCG-treated P116.cl39 cells but not in the EGCG-treated P116 ZAP-70-deficient cells (Fig. 6D). These results suggested that EGCG treatment induces apoptosis only in ZAP-70-expressing leukemia cells through a caspase-3-dependent pathway. In addition, results confirmed that ZAP-70-deficient P116 cells are more resistant to the effects of EGCG on viability or apoptosis than the ZAP-70-expressing P116.cl39 cells. Overall, these results suggested that EGCG suppressed cell viability and induced cell apoptosis through a ZAP-70-mediated mechanism.

DISCUSSION
The ability of tea to prevent tumorigenesis in various in vitro and in vivo systems has been reported. However, the mechanisms of action to explain the effects are not clearly understood. In particular, although EGCG inhibits cellular viability and  . The effect of EGCG on CD3-mediated AP-1 DNA binding and IL-2 cytokine release. A, gel-shift assay. Nuclear extracts were obtained from cells pretreated with different concentrations of EGCG (0, 4, or 8 M) for 1 h and then exposed to mouse IgG 1 monoclonal immunoglobulin isotype control (2 g/ml) or mouse anti-human CD3 (2 g/ml) for 24 h. The nuclear extracts were loaded onto a 5% polyacrymide gel and probed with ␥-32 Plabeled AP-1. Specific binding of nuclear proteins to the AP-1-responsive element was analyzed by the electrophoretic mobility shift assay. B and C, IL-2 secretion was measured from cells pretreated for 1 h with various concentrations of EGCG and then exposed to mouse IgG 1 monoclonal immunoglobulin isotype control (2 g/ml) or mouse anti-human CD3 (2 g/ml) for 24 (B) or 48 h (C). IL-2 production was measured in supernatant fractions by an enzyme-linked immunosorbent assay according to the manufacturer's instructions. Data are represented as the average of triplicate samples from three independent experiments. The asterisks indicate a significant change relative to P116.cl39 cell stimulated with CD3 (*, p Ͻ 0.005; **, p Ͻ 0.001).
induces apoptosis in various leukemias, the molecular mechanism(s) by which this occurs in T cell-mediated leukemia is not clear. The biological activities of EGCG and other components need to be characterized more fully to enhance our understanding of the biological effects of tea consumption. Cancer is a multistep process, with accumulation of mutations in tumor-suppressor genes and the dominant expression of oncogenes. Tyrosine kinases include the largest group of oncoproteins (47). The recent development of a series of relatively specific protein-tyrosine kinase (PTK) inhibitors with the ability to suppress the proliferation of tumor cells expressing the target PTK in vivo shows that inhibition of deregulated, dominant oncogenic PTKs is often enough to slow tumor progression (48). Research results suggest that targeted molecular cancer therapies can potentially deliver treatment directly to a specific protein or gene target and optimize efficacy and thus reduce adverse side effects often associated with traditional chemotherapy. Many research groups have reported that the ZAP-70 family of tyrosine kinases are selectively retained and expressed in T cell proliferative diseases (49). A subgroup of CLL was reported to overexpress ZAP-70 thus enabling T cells to develop and differentiate through CD3-TCRmediated signaling pathways (16). Therefore, ZAP-70 has been suggested to be an excellent biomarker for prognosis in CLL (9 -11) and might be a logical target for the development of potent immunosuppressive agents for T cell-mediated diseases (5,50). We showed that the green tea polyphenol, EGCG, directly interacts with and suppresses ZAP-70 activity. Molecular modeling and biochemical experiments confirmed that EGCG interacted with the ATP-binding pocket and effectively competed with ATP for binding.
Activation of the TCR induces numerous downstream signaling events, including the activation of various transcription factors, IL-2 promoter-driven transcription, protein tyrosine phosphorylation, intracellular calcium mobilization, MAPK activation, and phosphorylation of multiple important signaling molecules (51). To understand the effect of tea consumption on leukemias with ZAP-70 overexpression, we used ZAP-70-deficient Jurkat (P116) mutant cells and ZAP-70-recovered (P116.c139) cells as a model system to investigate the involvement of EGCG in T cell activation. Our results showed that EGCG impaired CD3-in- duced activation of TCR signaling through the inhibition of ZAP-70 kinase activity and autophosphorylation of ZAP-70 at Tyr 319 for T cell antigen receptor-dependent signaling. The mutation of ZAP-70 Tyr 319 site dramatically impaired TCR signaling (28). EGCG had no effect on phosphorylation of ZAP-70 on Tyr 493 . ZAP-70 is a protein-tyrosine kinase thought to play a critical role in T cell receptor (TCR) signal transduction. During T cell activation, ZAP-70 binds to a conserved signaling motif known as the ITAM and becomes phosphorylated at Tyr 493 (27,52). This result indicated that EGCG did not affect ZAP-70 recruitment to the TCR activation complex and that EGCG specifically inhibited ZAP-70. EGCG inhibited CD3-induced phosphorylation of several ZAP-70 downstream signaling molecules, including LAT (Tyr 171/191 ), PLC␥1 (Tyr 783 ), MEK (Ser 217/221 ), and ERK (Thr 202 /Tyr 204 ). EGCG also suppressed CD3-induced IL-2 production and activation of transcription factor AP-1. ZAP-70 was required for inhibition of T cell leukemia proliferation and induction of apoptosis by EGCG because EGCG had no effect on ZAP-70-deficient P116 cells, which also display severe defects in TCR signaling. These results indicated that EGCG specifically targets ZAP-70. Others have reported that EGCG induces apoptosis in human T cell acute lymphoblastic leukemia Jurkat cells (53), peripheral blood T lymphocytes, or leukemic blast cells of adult T cell leukemia patients (54,55). However, how the formation of this signaling complex leads to apoptosis is unclear. Our results provide strong evidence that ZAP-70 is a direct mediator of EGCGinduced apoptosis in leukemia cells and suggest that EGCG suppresses CD3-TCR-mediated signaling (pLAT, pPLC␥1, pMEK, and pERK, and DNA binding activity of AP-1 and IL-2) in leukemia through its inhibition of ZAP-70.