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
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
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DOI：
10.1096/fj.01-0031fje
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发表时间：
2001-07-01
期刊：
影响因子：
4.8
通讯作者：
Yang, CS
中科院分区：
文献类型：
--
作者：
Chung, JY;Park, JO;Yang, CS
我们的前期研究表明，茶多酚抑制小鼠表皮JB 6细胞和相应的H-ras转化细胞系30.7b Ras 12的MAP激酶和AP-1活性。本研究探讨了这种抑制的机制。将细胞与（-）-表没食子儿茶素-3-没食子酸酯（EGCG）或茶黄素-3，3 ′-二没食子酸酯（TFdiG）（20 μ M）孵育不同的时间，并通过免疫印迹分析细胞裂解物。EGCG处理时间依赖性地降低磷酸化-Erk 1/2和-MEK 1/2的水平（在60分钟时降低60%）。TFdiG在15分钟时使它们的水平降低38%-50% JFdiG有效地降低总Raf-1蛋白水平，最可能是通过溶酶体降解。EGCG并没有影响蛋白质水平或活性的Raf-1显着，但减少其与MEK 1的关联，如通过免疫共沉淀法测定。此外，EGCG和TFdiG（10 μ M）在体外通过分离的磷酸化Erk 1/2抑制Elk-1的磷酸化。Erk 1/2活性的这种抑制是Elk-1浓度依赖性和ATP浓度无关性的，这表明EGCG和TFdiG干扰蛋白质底物与激酶的结合。目前证明的EGCG和TFdiG抑制MAP激酶的具体机制可能有助于我们了解茶叶消费对癌症，炎症性疾病和心血管疾病的影响。
Our previous study showed that tea polyphenols inhibited MAP kinase and AP-1 activities in mouse epidermal JB6 cells and the corresponding H-ras-transformed cell line 30.7b Ras 12. The present study investigated the mechanisms of this inhibition. The cells were incubated with (-)-epigallocatechin-3-gallate (EGCG) or theaflavin-3,3'-digallate (TFdiG) (20 muM) for different times, and the cell lysate was analyzed by immunoblotting. EGCG treatment decreased the levels of phospho-Erk1/2 and -MEK1/2 time-dependently (by 60% at 60 min). TFdiG lowered their levels by 38%-50% at 15 min. TFdiG effectively decreased total Raf-1 protein levels, most likely through lysosomal degradation. EGCG did not affect protein levels or the activity of Raf-1 significantly but decreased its association with MEK1 as determined by co-immunoprecipitation. In addition, EGCG and TFdiG (10 muM) inhibited the phosphorylation of Elk-1 by isolated phospho-Erk1/2 in vitro. This inhibition of Erk1/2 activity is Elk-1 concentration-dependent and ATP concentration-independent, which suggests that EGCG and TFdiG interfere with the binding of the protein substrate to the kinase. The presently demonstrated specific mechanisms of inhibition of MAP kinases by EGCG and TFdiG may help us to understand the effects of tea consumption on cancer, inflammatory diseases, and cardiovascular diseases.