单宁酸通过抑制TRAF6向细胞质膜的招募抑制Akt信号通路

摘要

目的:表皮生长因子受体(epidermal growth factor receptor,EGFR)/蛋白激酶B(protein kinase B, PKB,也称Akt)信号通路在大多数人类实体肿瘤中过度激活、表达失调,促进肿瘤细胞增殖.植物单宁属于植物多酚类物质,能够抑制肿瘤细胞的增殖.然而,单宁酸通过何种机制调控肿瘤细胞中过度激活的Akt信号通路仍不清楚.方法:本实验选用人胶质瘤U87细胞作为模型,研究单宁酸抑制人胶质瘤U87细胞EGFR/Akt信号通路的分子机制.结果:单宁酸抑制人胶质瘤U87细胞Akt的磷酸化以及受Akt信号通路调控的相关蛋白4EBP-1和核糖体S6蛋白的磷酸化.进一步研究发现,单宁酸抑制肿瘤坏死因子受体相关因子6(tumor necrosis receptor-associated factor 6,TRAF6)向细胞质膜的招募,导致与TRAF6相互作用的Akt蛋白减少,进而抑制人胶质瘤细胞中Akt的磷酸化激活.利用EGFR组成型激活突变体EGFR vIII研究单宁酸对TRAF6向细胞质膜招募的影响发现:一方面单宁酸对EGFR的抑制作用需要EGFR胞外区第2~7外显子区域的存在;另一方面单宁酸对EGFR磷酸化的抑制是导致TRAF6向细胞质膜招募减少的原因.结论:EGFR是单宁酸发挥其抗肿瘤作用的受体蛋白,单宁酸通过抑制EGFR的磷酸化改变TRAF6向细胞质膜的招募进而介导了单宁酸对肿瘤细胞中Akt磷酸化的抑制,从而控制蛋白质的翻译,为食品来源单宁酸的抗肿瘤研究提供理论依据.%Purpose: Epidermal growth factor receptor (EGFR)/protein kinase B (PKB, also named Akt) signaling pathway is over-expressed and abnormally activated in most human solid tumors. EGFR/Akt signaling activation has been known to promote cell proliferation, which is a key factor for tumor survival. Tannins are a class of plant polyphenols, which have been reported for their antitumor activity. However, it remains unclear how tannic acid regulates EGFR/Akt signaling pathway in tumors. Methods: In the present study, human malignant glioma U87 cells with over-activated EGFR/Akt signaling pathway were used as a model to clarify the mechanism underlying the inhibitory effect of tannic acid on cell proliferation and EGFR/Akt signaling pathway. Results: Tannic acid significantly inhibited the phosphorylation of Akt and its two downstream effector proteins, 4EBP-1 and S6, whose phosphorylation are important for protein translation and tumor cells proliferation. Tannic acid also suppressed the proliferation of human glioma U87 cells. Further investigation revealed that tannic acid could block the recruitment of TRAF6 to plasma membrane and decrease the amount of Akt directly interacted with TRAF6, leading to the inhibition of Akt phosphorylation in human glioma U87 cells. In order to validate the role of tannic acid in TRAF6 recruitment, we expressed a constitutively activated EGFR vIII mutant in human glioma U87 cells and found that the inhibition of tannic acid on EGFR phosphorylation, leading to the decrease of TRAF6 plasma membrane recruitment, required the existence of 2-7 exons of EGFR extracellular domain; and the inhibition of tannic acid in EGFR phosphorylation. Conclusion: EGFR is the receptor of tannic acid in human glioma U87 cells. Tannic acid manipulates TRAF6 recruitment and inhibits Akt activation, thereby executing the signal transduction of tannic acid across the cell plasma membrane into tumor cells. These results provide a possible mechanism for the potential anti-tumor therapy with food-derived tannin.