Activation of SAPK/JNK mediated the inhibition and reciprocal interaction of DNA methyltransferase 1 and EZH2 by ursolic acid in human lung cancer cells.
Activation of SAPK/JNK mediated the inhibition and reciprocal interaction of DNA methyltransferase 1 and EZH2 by ursolic acid in human lung cancer cells.
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熊果酸激活 SAPK/JNK 介导人肺癌细胞中 DNA 甲基转移酶 1 和 EZH2 的抑制及其相互作用
DOI：
10.1186/s13046-015-0215-9
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发表时间：
2015-09-11
期刊：
影响因子：
--
通讯作者：
Hann SS
中科院分区：
文献类型：
--
作者：
Wu J;Zhao S;Tang Q;Zheng F;Chen Y;Yang L;Yang X;Li L;Wu W;Hann SS
熊果酸(UA)是一种五环三萜类化合物，对多种癌症具有抗肿瘤活性，其中包括人类非小细胞肺癌(NSCLC)。然而，UA作用的分子机制尚不清楚。方法采用四甲基偶氮唑蓝(MTT)法检测细胞存活率。Annexin V-FITC/PI细胞凋亡检测试剂盒流式细胞仪检测细胞凋亡。Western印迹分析检测应激激活蛋白激酶/c-jun氨基末端激酶(SAPK/JNK)、DNA(胞嘧啶-5)甲基转移酶1[DNA(胞嘧啶-5)-甲基转移酶1]、ZEST 2多梳抑制复合体2亚单位增强子(EZH2)和SP1的磷酸化和蛋白表达。结果UA以剂量和时间依赖的方式抑制NSCLC细胞的生长和诱导细胞凋亡。此外，我们还发现UA诱导SAPK/JNK的磷酸化，并抑制DNMT1和EZH2的蛋白表达。SAPK/JNK抑制剂(SP600125)可阻断UA诱导的DNMT1和EZH2的表达。此外，UA还抑制了SP1蛋白的表达。反之，SP1的过表达逆转了UA对DNMT1和EZH2表达的影响，反馈减弱了UA诱导的SAPK/JNK的磷酸化。结论UA通过SAPK/JNK抑制SP1的表达，进而抑制DNMT1和EZH2的表达，从而抑制NSCLC细胞的生长。过表达DNMT1可减少UA降低的EZH2蛋白表达。SP1和DNMT1对SAPK/JNK信号的负反馈调节以及EZH2和DNMT1的相互作用参与了UA的整体效应。这项研究对UA控制NSCLC细胞生长的机制有了重要的新见解。
BackgroundUrsolic acid (UA), a pentacyclic triterpenoid, is known to have anti-tumor activity in various cancers including human non small cell lung cancer (NSCLC). However, the molecular mechanisms underlying the action of UA remain largely unknown.MethodsCell viability was measured by MTT assays. Apoptosis was analyzed with Annexin V-FITC/PI Apoptosis Detection Kit by Flow cytometry. Western blot analysis was performed to measure the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), DNMT1 [DNA (cytosine-5)-methyltransferase 1], enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and SP1. Exogenous expression of SP1 and DNMT1 was carried out by transient transfection assays.ResultsWe showed that UA inhibited the growth and induced apoptosis of NSCLC cells in the dose- and time-dependent fashion. Furthermore, we found that UA induced phosphorylation of SAPK/JNK and suppressed the protein expression of DNMT1 and EZH2. The inhibitor of SAPK/JNK (SP600125) blocked the UA-reduced expression of DNMT1 and EZH2. In addition, UA suppressed the expression of SP1 protein. Conversely, overexpression of SP1 reversed the effect of UA on DNMT1 and EZH2 expression, and feedback attenuated UA-induced phosphorylation of SAPK/JNK. Moreover, exogenous expression of DNMT1 antagonized the effect of UA on SAPK/JNK signaling, EZH2 protein expression, and NSCLC cell growth.ConclusionOur results show that UA inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of SP1; this in turn results in inhibition the expression of DNMT1 and EZH2. Overexpression of DNMT1 diminishes UA-reduced EZH2 protein expression. The negative feedback regulation of SAPK/JNK signaling by SP1 and DNMT1, and the reciprocal interaction of EZH2 and DNMT1 contribute to the overall effects of UA. This study leads to important new insights into the mechanisms by which UA controls growth of NSCLC cells.