A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516.
A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516.
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DOI：
10.1371/journal.pone.0162394
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Itoh M
中科院分区：
文献类型：
--
作者：
Arai K;Eguchi T;Rahman MM;Sakamoto R;Masuda N;Nakatsura T;Calderwood SK;Kozaki K;Itoh M
上皮-间质转化（EMT）是肿瘤的重要病理过程，尤其是肿瘤细胞的出芽和转移。因此，控制EMT可以代表癌症的新治疗策略。在这里，我们介绍了一种创新的三维（3D）高通量筛选（HTS）系统，导致EMT抑制剂的识别。为了建立新的3D-HTS系统，我们选择了纳米培养板（NCP），它为细胞提供了无凝胶的微图案支架，并且独立于使用软琼脂的其他球状体形成系统。在基于NCP的3D细胞培养系统中，A549肺癌细胞在7天内迁移、聚集，然后形成多个球状体。活细胞成像实验表明，已建立的EMT诱导剂TGF-β促进球状体核心周围的外周细胞获得间充质纺锤体形状，丧失细胞间粘附，并从球状体迁移。沿着这种形态学变化，EMT相关基因表达特征发生改变，特别是ECAD/CDH 1、NCAD/CDH 2、Vim和ZEB 1/TCF 8的mRNA水平发生改变。这些EMT相关的表型变化可被TGF-β受体I（TGFβR1）抑制剂SB 431542阻断。球体内部高度缺氧;相比之下，球体衍生的外周迁移细胞含氧量正常，通过使用缺氧探针的可视化和定量显示。因此，TGF-β触发的EMT引起球状体发育不全和缺氧丧失。从低氧探针的荧光强度计算SB 431542的球形EMT抑制（SEMTIN）活性，然后用于EMT抑制性小分子化合物的药物筛选。在初步筛选中，1，330种化合物中有9种高于SEMTIN活性和细胞活力的阈值。最后，两个化合物SB-525334和SU 9516以剂量依赖性方式显示SEMTIN活性。SB-525334是一种已知的TGFβR1抑制剂。SU 9516是一种细胞周期蛋白依赖性激酶2（CDK 2）抑制剂，我们发现它也具有EMT抑制活性。SB-525334和SU 9516的半数抑制浓度（IC 50）分别为0.31 μM和1.21 μM，而SB 431542的IC 50为2.38 μM。综上所述，结果表明，这种基于3D NCP的HTS系统可用于筛选EMT调节药物。
Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. The half maximal inhibitory concentration (IC50) of SB-525334 and SU9516 were 0.31 μM and 1.21 μM, respectively, while IC50 of SB431542 was 2.38 μM. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.
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影响因子：
4
作者：
Mizushima, Hiroto;Wang, Xiaobiao;Mekada, Eisuke
通讯作者：
Mekada, Eisuke
影响因子：
3.7
作者：
Kumar M;Allison DF;Baranova NN;Wamsley JJ;Katz AJ;Bekiranov S;Jones DR;Mayo MW
通讯作者：
Mayo MW
影响因子：
3.7
作者：
Moen I;Øyan AM;Kalland KH;Tronstad KJ;Akslen LA;Chekenya M;Sakariassen PØ;Reed RK;Stuhr LE
通讯作者：
Stuhr LE
DOI：
10.1038/nrm3758
发表时间：
2014-03
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
通讯作者：
--
DOI：
10.1186/bcr762
发表时间：
2004
期刊：
Breast cancer research : BCR
影响因子：
--
作者：
Brown KA;Roberts RL;Arteaga CL;Law BK
通讯作者：
Law BK