The inhibitory effect of (-)-epigallocatechin gallate on activation of the epidermal growth factor receptor is associated with altered lipid order in HT29 colon cancer cells
The inhibitory effect of (-)-epigallocatechin gallate on activation of the epidermal growth factor receptor is associated with altered lipid order in HT29 colon cancer cells
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DOI：
10.1158/0008-5472.can-07-0411
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发表时间：
2007-07-01
期刊：
影响因子：
11.2
通讯作者：
Weinstein, I. Bernard
中科院分区：
文献类型：
--
作者：
Adachi, Seiji;Nagao, Tomokazu;Weinstein, I. Bernard
（-）-表没食子儿茶素没食子酸酯（EGCG）是绿茶的主要生物活性成分，可抑制几种人类癌细胞中表皮生长因子受体（EGFR）和下游信号通路的激活，但其确切机制尚不清楚。由于包括FGFR在内的几种质膜相关受体酪氨酸激酶（RTK）定位于洗涤剂不溶性有序膜结构域，即所谓的“脂筏”；我们研究了EGCG对EGFR激活的抑制作用是否与HT29结肠癌细胞膜脂序的改变有关。首先，我们做了冷Triton X-100溶解度测定。磷酸化（活化）的EGFR仅存在于Triton x -100不溶性（脂筏）部分，而细胞总EGFR存在于Triton x -100可溶部分。EGCG预处理可以抑制Alexa Fluor 488标记的EGF与细胞的结合，也可以抑制EGF诱导的EGFR二聚化。为了研究EGCG对膜脂组织的可能影响，我们用荧光脂质类似物1,1 '-二十六进制-3,3,3 ',3 '-四甲基多碳菁高氯酸盐标记细胞，该物质优先结合到细胞的有序膜结构域，并发现随后用EGCG处理导致Triton x -100抗性膜组分显著减少。多酚E，绿茶儿茶素的混合物，有类似的效果，但（类似）表儿茶素（EC），生物活性化合物，没有显著改变Triton X-100膜的溶解度特性。此外，我们发现EGCG而非EC引起双层结合的gramicidin通道功能的剧烈变化。综上所述，这些发现表明EGCG通过改变膜组织抑制EGF与EGFR的结合以及随后的EGFR二聚化和活化。这些作用也可以解释EGCG抑制其他膜相关rtk激活的能力，它们可能在EGCG和相关化合物的抗癌作用中发挥关键作用。
(-)-Epigallocatechin gallate (EGCG), a major biologically active constituent of green tea, inhibits activation of the epidermal growth factor (EGF) receptor (EGFR) and downstream signaling pathways in several types of human cancer cells, but the precise mechanism is not known. Because several plasma membrane-associated receptor tyrosine kinases (RTK) including FGFR are localized in detergent-insoluble ordered membrane domains, so-called "lipid rafts; we examined whether the inhibitory effect of EGCG on activation of the EGFR is associated with changes in membrane lipid order in HT29 colon cancer cells. First, we did cold Triton X-100 solubility assays. Phosphorylated (activated) EGFR was found only in the Triton X-100-insoluble (lipid raft) fraction, whereas total cellular EGFR was present in the Triton X-100-soluble fraction. Pretreatment with EGCG inhibited the binding of Alexa Fluor 488-labeled EGF to the cells and also inhibited EGF-induced dimerization of the EGFR. To examine possible effects of EGCG on membrane lipid organization, we labeled the cells with the fluorescent lipid analogue 1, 1 '-dihexadecyl-3,3,3 ',3 '-tetramethylindocarbocyanine perchlorate, which preferentially incorporates into ordered membrane domains in cells and found that subsequent treatment with EGCG caused a marked reduction in the Triton X-100-resistant membrane fraction. Polyphenon E, a mixture of green tea catechins, had a similar effect but (similar to)-epicatechin (EC), the biologically inactive compound, did not significantly alter the Triton X-100 solubility properties of the membrane. Furthermore, we found that EGCG but not EC caused dramatic changes in the function of bilayer-incorporated gramicidin channels. Taken together, these findings suggest that EGCG inhibits the binding of EGF to the EGFR and the subsequent dimerization and activation of the EGFR by altering membrane organization. These effects may also explain the ability of EGCG to inhibit activation of other membrane-associated RTKs, and they may play a critical role in the anticancer effects of this and related compounds.