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Mechanisms of inhibition of the Ras-MAP kinase signaling pathway in 30.7b Ras 12 cells by tea polyphenols (-)-epigallocatechin-3-gallate and theaflavin-3,3'-digallate ¹

Laboratory for Cancer Research, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA; and
^* Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA

²Correspondence: Laboratory for Cancer Research, College of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Rd., Piscataway, NJ 08854-8020, USA. E-mail: csyang{at}rci.rutgers.edu

SPECIFIC AIMS

Previous work demonstrated the inhibition of tumorigenesis in animal models by tea as well as the growth and transformation of cells in culture by tea polyphenols. Some of the latter activity was closely associated with the inhibition of mitogen-activated protein kinase (MAPK) and activator protein 1 (AP-1) activities. The aim of this study was to investigate the specific targets and biochemical mechanisms for the inhibition of MAPK signal transduction pathway by tea polyphenols. (-)-Epigallocatechin-3-gallate (EGCG) and theaflavin-3,3'-digallate (TFdiG), derived from green tea and black tea, respectively, were selected for the investigation. We used the H-ras transformed mouse epidermal cell line (30.7b Ras 12), which has elevated MAP kinase and AP-1 activities.

PRINCIPAL FINDINGS

1. EGCG and TFdiG treatment of cells decreased the levels of phosphorylated Erk1/2 and MEK1/2
Treatment of cells with EGCG and TFdiG caused a time-dependent decrease in levels of phospho-Erk1/2 and -MEK1/2, as determined by Western blot analysis. Levels of phospho-Erk1/2 and -MEK1/2 decreased by 30% after incubation with EGCG (20 µM) for 30 min. TFdiG (20 µM) treatment caused a more rapid decrease of phospho-Erk1/2 and MEK1/2 levels, by 38–50% at 15 min. The total protein (phosphorylated and nonphosphorylated) levels of Erk1/2 and MEK1/2 were not affected by the treatments.

2. Raf-1 protein levels were rapidly degraded by TFdiG but not with EGCG
The decrease in phospho-MEK1/2 protein levels by EGCG and TFdiG led to the examination of Raf-1, a protein kinase directly upstream of MEK1/2. Treatment of cells with TFdiG (20 µM) caused the degradation of Raf-1 in a time-dependent manner to < 15% of control by 120 min, whereas EGCG had no such effect (Fig. 1 ). The degradation of Raf-1 by TFdiG was blocked by pretreatment of 30.7b Ras 12 cells (for 30 min) by a lysosomal protein degradation inhibitor, chloroquine (100 µM), but not by the proteasome inhibitor N-acetyl-leu-leu-norleu-al (100 µM).

View larger version (35K): [in this window] [in a new window]   Figure 1. Differential effects of EGCG and theaflavin-3,3'-digallate on Raf-1 protein levels. Total cell lysates from 30.7b Ras 12 cells treated with 20 µM EGCG or theaflavin-3,3'-digallate (TFdiG) for different times and lysed using 2% SDS protein loading buffer were used to determine Raf-1 levels with anti-Raf-1 antibodies. A) Western blot analysis of Raf-1. B) Quantification of protein levels using Bio Image Intelligent Quantifier. The values are the mean ± SE of three separate determinations. Values with an asterisk are significantly different (P<0.05) from control and values with (**) are significantly different (P<0.05) from control and (*) as determined by ANOVA, followed by Fisher’s exact test. Con, control. E, EGCG. T, TFdiG. C, D) Effects of N-acetyl-leu-leu-norleu-al and chloroquine on theaflavin-3,3'-digallate-induced Raf-1 degradation. Four plates of 30.7b Ras 12 cells were treated with either 0.1% FBS MEM media for 45 min (Con), media containing 20 µM TFdiG for 15 min, 100 µM N-acetyl-leu-leu-norleu-al/chloroquine (LLnL)/(CQ) for 45 min, or 100 µM N-acetyl-leu-leu-norleu-al/chloroquine for 30 min, followed by 20 µM TFdiG (LLnL+TFdiG)/(CQ+TFdiG) for 15 min per experiment. These cell lysates were used to determine Raf-1 protein levels with anti-Raf-1 antibodies. The values below panel D are the quantification of protein levels using Bio Image Intelligent Quantifier. Values are the mean ± SE of three separate determinations.

3. EGCG inhibited the association between Raf-1 and MEK1
To further investigate the mechanism for the decrease in phospho-MEK1/2 protein levels by EGCG, kinase activity of Raf-1 was examined. Raf-1 was immunoprecipitated from cells treated with EGCG (20 µM) for 1 h and kinase activity was measured. The treatment did not significantly lower Raf-1 activity. In cells treated with EGCG (20 µM), however, the association of Raf-1 with MEK1 decreased, as determined by immunoprecipitation. The effect of TFdiG on the association was not measured accurately because of the degradation of Raf-1 during the experiment.

4. EGCG and TFdiG inhibited Erk1/2-catalyzed phosphorylation of Elk-1 in a substrate-dependent and ATP-independent manner
We examined the ability to directly inhibit Erk1/2 activity by EGCG and TFdiG using different ATP and substrate concentrations. Active phospho-Erk1/2 was immunoprecipitated from 30.7b Ras 12 cells using phospho-specific Erk1/2 antibodies and kinase activity was assayed by the addition of substrate Elk-1 and ATP. Both EGCG and TFdiG (10 µM) strongly inhibited the phosphorylation of Elk-1 by Erk1/2 (>60% inhibition). The ability of EGCG and TFdiG to inhibit Erk1/2 activity was decreased as the Elk-1 concentration increased from 1 µg to 6 µg, but was not affected by varying the ATP concentration from 50 µM to 600 µM (Fig. 2 ).

View larger version (47K): [in this window] [in a new window]   Figure 2. Effects of EGCG and theaflavin-3,3'-digallate on Erk-catalyzed phosphorylation of Elk-1. 30.7b Ras 12 cell lysate was used to immunoprecipitate active Erk1/2 using a phospho-specific monoclonal antibody against Erk1/2. These immunoprecipitates were washed and kinase activity was performed using the substrate GST-Elk (307–428). A) ATP (200 µM) with or without EGCG, TFdiG, or EC at 20 µM. B) Different ATP (50, 200, 600 µM) concentrations with or without EGCG or TFdiG, at 10 µM. C) Different substrate GST-Elk-1 (307–428) concentrations and ATP (200 µM) with or without EGCG or TFdiG, at 10 µM. The values below the figures are the mean ± SE of three separate determinations. Con, control. E, EGCG. T, TFdiG. D, DMSO control. EC, (-)-epicatechin.

CONCLUSIONS

The present study demonstrates potent inhibitory activities of EGCG and TFdiG on specific protein kinases in the Ras-activated MAP kinase signaling pathway. Both EGCG and TFdiG potently and rapidly inhibited phospho-Erk1/2 and -MEK1/2 protein levels, but with different mechanisms. EGCG inhibits the association of Raf-1 with MEK1 as well as the phosphorylation of Elk-1 by Erk1/2. Proline-rich regions are involved both in the interactions between Raf-1 and MEK1 and between Erk1/2 and Elk-1; the results are consistent with earlier reports on the affinity of polyphenolic compounds for proteins rich in proline residues (Fig. 3 ). The rapid decrease in Raf-1 protein levels by TFdiG is a novel mechanism that contributes to the decrease in phospho-MEK1/2 levels, but raises another question as to how TFdiG stimulates lysosome-mediated Raf-1 degradation. TFdiG may possibly alter the plasma membrane, which leads to vacuole formation and entrapment of Raf-1 protein, followed by fusion with the lysosome, since we have observed drastic morphological changes, namely, cell contraction by TFdiG treatment.

View larger version (33K): [in this window] [in a new window]   Figure 3. Inhibitory mechanisms of EGCG and TFdiG on the Ras-MAP kinase pathway. The inhibitory effects of EGCG and TFdiG are shown at two sites on Ras-activated signal transduction pathway. EGCG disrupts the association of MEK1 with Raf-1 possibly by binding to the proline-rich sequences on MEK1 (shown as a dark line on MEK1), whereas TFdiG promotes lysosome-mediated degradation of Raf-1 protein. These mechanisms lead to decreased phospho-MEK1 and phospho-Erk1/2 levels within the cell and thus inhibit the signal transduction to the nucleus. The in vitro studies suggest that EGCG and TFdiG both may also affect Erk1/2 activity in a substrate-dependent manner through association with critical proline residues near the phosphorylation site on Elk-1.

Further research is needed to determine whether the mechanism we observed occurs in other experimental systems. Elevated MAP kinase and AP-1 activities are involved in many disease processes such as inflammation, neoplastic transformation, cancer cell invasion, metastasis, and angiogenesis. The presently observed inhibitory actions of EGCG and TFdiG may help us to understand the effects of tea consumption on cancer, inflammatory diseases, and cardiovascular diseases.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0031fje ; to cite this article, use FASEB J. (July 9, 2001) 10.1096/fj.01-0031fje

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