Esculetin induces antiproliferative and apoptotic response in pancreatic cancer cells by directly binding to KEAP1.
Esculetin induces antiproliferative and apoptotic response in pancreatic cancer cells by directly binding to KEAP1.
复制标题
DOI：
10.1186/s12943-016-0550-2
复制
发表时间：
2016-10-18
期刊：
影响因子：
37.3
通讯作者：
Saluja D
中科院分区：
文献类型：
--
作者：
Arora R;Sawney S;Saini V;Steffi C;Tiwari M;Saluja D
一些研究已经利用了escuoke的抗肿瘤潜力，escuoke是一种二羟基香豆素衍生物;它结合的靶点及其在癌细胞中细胞毒性的可能下游机制仍有待阐明。本研究以胰腺癌细胞系为模型系统，检测七叶壳酮抑制胰腺癌细胞生长的功效，并对其作用机制进行研究，预测其直接结合的靶蛋白。在PANC-1、MIA PaCa-2和AsPC-1细胞系中测定七叶壳的细胞毒性;随后检查细胞内ROS及其相关转录因子p65-NF-κB的水平。在存在和不存在七叶壳的情况下，研究了转录因子Nrf 2及其调节因子KEAP 1之间的相互作用。真实的时间PCR检测Nrf 2对抗氧化反应元件通路基因表达的影响。随后，通过分子对接预测了七叶壳酮的潜在结合靶点，并在体外进行了验证。在所有三种胰腺癌细胞系中，escuoke治疗导致显著的生长抑制，G1期细胞周期停滞，并通过激活半胱天冬酶3、8和9诱导线粒体依赖性凋亡。七叶酮处理后，PANC-1细胞内活性氧和p65-NF-κB蛋白水平明显降低。据报道，抗氧化反应调节剂Nrf 2参与了与NF-κB的串扰。在PANC-1和MIA Paca-2细胞中，发现Nrf 2和KEAP 1之间的相互作用在七叶酮处理后丧失。在PANC-1中观察到的escuoke处理的Nrf 2的核积累和Nrf 2调节基因NQO 1的表达上调进一步支持了Nrf 2的激活。为了解释在七叶酮处理时Nrf 2-KEAP 1相互作用的损失，使用分子对接研究在计算机中描述了七叶酮和KEAP 1之间的直接结合潜力。使用escuoke缀合的琼脂糖珠的下拉测定证实了escuoke和KEAP 1之间的结合。我们建议escuaoke结合KEAP 1，并抑制其与胰腺癌细胞中Nrf 2的相互作用。因此，这促进了PANC-1细胞中Nrf 2的核积累，其可能通过减弱NF-κB诱导抗增殖和凋亡反应。本文的在线版本（doi：10.1186/s12943-016-0550-2）包含补充材料，可供授权用户使用。
A handful of studies have exploited antitumor potential of esculetin, a dihydroxy coumarine derivative; the targets to which it binds and the possible downstream mechanism for its cytotoxicity in cancer cells remain to be elucidated. Using pancreatic cancer cell lines as a model system, herein the study was initiated to check the efficacy of esculetin in inhibiting growth of these cancer cells, to decipher mechanism of its action and to predict its direct binding target protein. The cytotoxicity of esculetin was determined in PANC-1, MIA PaCa-2 and AsPC-1 cell lines; followed by an inspection of intracellular levels of ROS and its associated transcription factor, p65-NF-κB. The interaction between transcription factor, Nrf2 and its regulator KEAP1 was studied in the presence and absence of esculetin. The effect of Nrf2 on gene expression of antioxidant response element pathway was monitored by real time PCR. Thereafter, potential binding target of esculetin was predicted through molecular docking and then confirmed in vitro. Esculetin treatment in all three pancreatic cancer cell lines resulted in significant growth inhibition with G1-phase cell cycle arrest and induction of mitochondrial dependent apoptosis through activation of caspases 3, 8 and 9. A notable decrease was observed in intracellular ROS and protein levels of p65-NF-κB in PANC-1 cells on esculetin treatment. Antioxidant response regulator Nrf2 has been reportedly involved in crosstalk with NF-κB. Interaction between Nrf2 and KEAP1 was found to be lost upon esculetin treatment in PANC-1 and MIA Paca-2 cells. Nuclear accumulation of Nrf2 and an upregulation of expression of Nrf2 regulated gene NQO1, observed on esculetin treatment in PANC-1 further supported the activation of Nrf2. To account for the loss of Nrf2-KEAP1 interaction on esculetin treatment, direct binding potential between esculetin and KEAP1 was depicted in silico using molecular docking studies. Pull down assay using esculetin conjugated sepharose beads confirmed the binding between esculetin and KEAP1. We propose that esculetin binds to KEAP1 and inhibits its interaction with Nrf2 in pancreatic cancer cells. This thereby promotes nuclear accumulation of Nrf2 in PANC-1 cells that induces antiproliferative and apoptotic response possibly by attenuating NF-κB. The online version of this article (doi:10.1186/s12943-016-0550-2) contains supplementary material, which is available to authorized users.