Fyn is a novel target of (-)-epigallocatechin gallate in the inhibition of JB6 Cl41 cell transformation

Abstract

The cancer preventive action of (-)-epigallocatechin gallate (EGCG), found in green tea, is strongly supported by epidemiology and laboratory research data. However, the mechanism by which EGCG inhibits carcinogenesis and cell transformation is not clear. In this study, we report that EGCG suppressed epidermal growth factor (EGF)-induced cell transformation in JB6 cells. We also found that EGCG inhibited EGF-induced Fyn kinase activity and phosphorylation in vitro and in vivo. Fyn was implicated in the process because EGF-induced JB6 cell transformation was inhibited by small interfering RNA (siRNA)-Fyn-JB6 cells. With an in vitro protein-binding assay, we found that EGCG directly bound with the GST-Fyn-SH2 domain but not the GST-Fyn-SH3 domain. The K(d) value for EGCG binding to the Fyn SH2 domain was 0.367 +/- 0.122 microM and B(max) was 1.35 +/- 0.128 nmol/mg. Compared with control JB6 Cl41 cells, EGF-induced phosphorylation of p38 MAP kinase (p38 MAPK) (Thr180/Tyr182), ATF-2 (Thr71) and signal transducer and activator of transcription 1 (STAT1) (Thr727) was decreased in siRNA-Fyn-JB6 cells. EGCG could inhibit the phosphorylation of p38 MAPK, ATF-2, and STAT1. The DNA binding ability of AP-1, STAT1, and ATF-2 was also decreased in siRNA-Fyn-JB6 cells. Overall, these results demonstrated that EGCG interacted with Fyn and inhibited Fyn kinase activity and thereby regulated EGF-induced cell transformation. Inhibition of Fyn kinase activity is a novel and important mechanism that may be involved in EGCG-induced inhibition of cell transformation.

Figure 1

EGCG inhibits EGF-induced cell transformation. (A) JB6 Cl41 cells were treated with increasing concentrations of EGCG and viability was assessed with the MTS assay as described in Methods and Materials. (B) For determining the effect of EGCG on proliferation over time, JB6 Cl41 cells were treated with EGCG at 20 µM for different time periods and then proliferation was assessed byMTS assay. For both A and B, data are presented as means ± SD of three independent experiments, each performed in triplicate. The asterisk (*) indicates a significant (*, P<0.05) decrease in viability in EGCG-treated cells relative to untreated control cells. (C) EGCG inhibits JB6 Cl41 anchorage-independent EGF-promoted transformation. Various concentrations of EGCG with or without 10 ng/mL EGF were added into soft agar with JB6 Cl41 cells and colonies were counted automatically after 7 d of incubation at 37°C in a 5% CO₂ incubator. Colony formation in JB6 cells without EGF stimulation (1st plate, upper), with EGF (2nd plate, upper), EGF plus 1 µM EGCG (3rd plate, upper), EGF plus 5 µM EGCG (1st plate, lower), EGF plus 10 µM EGCG (2nd plate, lower) or EGF plus 20 µM EGCG (3rd plate lower). (D) Data are represented as the average number of colonies ± SD as determined from three separate experiments ± SD. The asterisk (*) indicates a significant inhibition compared to EGF only (**, P<0.01 and *, P<0.05).

Figure 2

EGCG inhibits Fyn kinase activity and phosphorylation of Fyn in a dose-dependent manner. (A) For the in vitro kinase assay, phosphorylation of a Fyn substrate peptide was determined with active Fyn (10, 50, or 100 ng alone or with (B) EGCG (1, 5, 10, 20 µM). (C) For the immunoprecipitation kinase assay, cells were treated or not treated with EGCG (5, 10, or 20 µM) 1 h and then treated with EGF (10 ng/mL) and harvested after 30 min. The average ³²P count was determined from three separate experiments. The asterisk (*) indicates a significant EGCG-induced change in kinase activity compared to respective control (**, P<0.01 and *, P<0.05). (D) Cells were treated with various concentrations (0, 5, 10, or 20 µM) of EGCG followed by EGF (10 ng/mL). Phosphorylation of Fyn (Thr12) (upper panel) and total Fyn protein level (lower panel) were then determined by Western blot analysis as described in Materials and Methods with specific antibodies against phosphorylation of Fyn (Thr12) or total Fyn protein.

Figure 3

EGCG specifically binds with the Fyn SH2 domain and not the SH3 domain. (A) For the in vitro EGCG pull-down assay, lane 1 indicates the Fyn protein detected in the recombinant Fyn sample as a positive control and in lane 2 the cell lysates incubated with EGCG-Sepharose 4B beads. Lanes 3 and 4 show no Fyn protein detected in cell lysates incubated with only Sepharose 4B beads. (B) Purified GST-Fyn (SH2) and GST-Fyn (SH3) are shown. (C) Lanes 1 and 2 show no Fyn protein detected in the mixture of GST-Fyn (SH3) and EGCG-Sepharose 4B beads or elution buffer, respectively. Lanes 3 and 4 show Fyn protein detected in the mixture of GST-Fyn (SH2) with EGCG-Sepharose 4B beads or in the elution solution from this mixture, respectively. (D) GST-Fyn (SH2) binding with EGCG affinity and K_d value (0.367 ± 0.122 µM) are shown.

Figure 4

siRNA-Fyn inhibits phosphorylation of STAT1 (Ser727), ATF-2 (Thr71), and p38MAPK (Thr180/Tyr182). Cells were treated as described in Methods and Materials and phosphorylation of selected proteins was determined by Western blot. (A) Phosphorylation of ERKs (Ser42/Ser 44) was detected with a specific phospho-ERKs (Ser^42/44) antibody and total ERKs protein levels were detected by a non-phospho-ERKs antibody (A, left). Fyn expression was confirmed in SiRNA-Fyn JB6 cells and mock cells by Western blot (A, right). (B) Phosphorylation of STAT1 (Ser727), ATF-2 (Thr71), and p38 MAPK (Thr180/Tyr182) were separately detected with specific phospho-STAT1, ATF-2, and p38MAP kinase antibodies. Total protein levels of STAT1, ATF-2, and p38 MAP kinase were detected by a non-phospho-STAT1, ATF-2, or p38 MAP kinase antibody, respectively. (C) SiRNA-Fyn JB6 cells and mock cells were treated with EGCG as described in Methods and Materials. Phosphorylation of STAT1 (Ser727), ATF-2 (Thr71), and p38 MAPK (Thr180/Tyr182) was also detected by Western blot. Respective total protein levels served as controls.

Figure 5

siRNA-Fyn inhibits EGF-induced JB6 Cl4l anchorage-independent cell transformation. (A) Colony formation promoted by various concentrations of EGF in mock-transfected JB6 Cl41 cells (upper row) or siRNA-Fyn-JB6 Cl41 cells (lower row) was determined automatically as described in Methods and Materials. (B) Bars indicate the average colony number calculated from three separate experiments ± SD. The asterisk (*) indicates a significant inhibition by siRNA-Fyn-JB6 compared to mock-JB6 cells (*, P<0.05).

Figure 6

siRNA-Fyn inhibits EGF-induced AP-1, STAT1, and CREB/ATF-2 DNA binding. Mock-transfected JB6 Cl41 cells and siRNA-Fyn-JB6 Cl41 cells were treated with EGF and then nuclear proteins were extracted as described in Materials and Methods. siRNA-Fyn cells effectively blocked EGF-induced AP-1 DNA binding (A, lanes 8–10), STAT1 DNA binding (B, lanes 8–10), and CREB/ATF-2 DNA binding (C, lanes 8–10) compared with EGF-induced AP-1 DNA binding in mock-transfected JB6 Cl41 cells (A, lanes 3–5), STAT1 DNA binding (B, lanes 3–5), and CREB/ATF-2 DNA binding (C, lanes 3–5). Lane 1 in each figure shows only ³²P as the “cold” probe to confirm specificity and lane 2 indicates that the corresponding antibody was added in the reaction mixture. (D) Proposed signal transduction pathways of EGCG inhibition of JB6 Cl41 cell transformation promoted by EGF. EGCG effectively inhibited EGF-induced Fyn kinase activity and phosphorylation. siRNA-Fyn kinase inhibited EGF-induced phosphorylation of STAT1, ATF-2, and p38 MAP kinase and AP-1, STAT1, and CREB/ATF-2 DNA binding abilities. Thus Fyn plays an important role in EGF-promoted JB6 Cl41 cell transformation through inhibition of its downstream target kinase and transcription factors.