Adachi Seiji, Shimizu Masahito, Shirakami Yohei, Yamauchi Junichi, Natsume Hideo, Matsushima-Nishiwaki Rie, To Satoshi, Weinstein I Bernard, Moriwaki Hisataka, Kozawa Osamu
Department of Gastroenterology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
Carcinogenesis. 2009 Sep;30(9):1544-52. doi: 10.1093/carcin/bgp166. Epub 2009 Jul 3.
We previously reported that (-)-epigallocatechin gallate (EGCG) in green tea alters plasma membrane organization and causes internalization of epidermal growth factor receptor (EGFR), resulting in the suppression of colon cancer cell growth. In the present study, we investigated the detailed mechanism underlying EGCG-induced downregulation of EGFR in SW480 colon cancer cells. Prolonged exposure to EGCG caused EGFR degradation. However, EGCG required neither an ubiquitin ligase (c-Cbl) binding to EGFR nor a phosphorylation of EGFR at tyrosine residues, both of which are reportedly necessary for EGFR degradation induced by epidermal growth factor. In addition, EGCG induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), a stress-inducible kinase believed to negatively regulate tumorigenesis, and the inhibition of p38 MAPK by SB203580, a specific p38 MAPK inhibitor, or the gene silencing using p38 MAPK-small interfering RNA (siRNA) suppressed the internalization and subsequent degradation of EGFR induced by EGCG. EGFR underwent a gel mobility shift upon treatment with EGCG and this was canceled by SB203580, indicating that EGCG causes EGFR phosphorylation via p38 MAPK. Moreover, EGCG caused phosphorylation of EGFR at Ser1046/1047, a site that is critical for its downregulation and this was also suppressed by SB203580 or siRNA of p38 MAPK. Taken together, our results strongly suggest that phosphorylation of EGFR at serine 1046/1047 via activation of p38 MAPK plays a pivotal role in EGCG-induced downregulation of EGFR in colon cancer cells.
我们之前报道过，绿茶中的（-）-表没食子儿茶素没食子酸酯（EGCG）会改变质膜结构，并导致表皮生长因子受体（EGFR）内化，从而抑制结肠癌细胞的生长。在本研究中，我们调查了EGCG诱导SW480结肠癌细胞中EGFR下调的详细机制。长时间暴露于EGCG会导致EGFR降解。然而，EGCG既不需要泛素连接酶（c-Cbl）与EGFR结合，也不需要EGFR在酪氨酸残基处磷酸化，而据报道这两者对于表皮生长因子诱导的EGFR降解都是必需的。此外，EGCG诱导p38丝裂原活化蛋白激酶（MAPK）磷酸化，p38 MAPK是一种应激诱导激酶，据信对肿瘤发生起负调控作用，使用特异性p38 MAPK抑制剂SB203580抑制p38 MAPK或使用p38 MAPK小干扰RNA（siRNA）进行基因沉默，均可抑制EGCG诱导的EGFR内化及随后的降解。用EGCG处理后，EGFR发生凝胶迁移率改变，而SB203580可消除这种改变，这表明EGCG通过p38 MAPK导致EGFR磷酸化。此外，EGCG导致EGFR在Ser1046/1047位点磷酸化，该位点对其下调至关重要，SB203580或p38 MAPK的siRNA也可抑制这种磷酸化。综上所述，我们的结果强烈表明，通过激活p38 MAPK使EGFR在丝氨酸1046/1047位点磷酸化在EGCG诱导结肠癌细胞中EGFR下调过程中起关键作用。
