A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516.
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.
SCIVAX Life Sciences, Inc., Kawasaki, Kanagawa, Japan.|SCIVAX USA, Inc., Woburn, Massachusetts, United States of America.
Kazuya
http://orcid.org/0000-0003-2420-4596
K
Arai
Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.|Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.|Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School / Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8525, Japan.
Takanori
T
Eguchi
SCIVAX USA, Inc., Woburn, Massachusetts, United States of America.
M Mamunur
MM
Rahman
SCIVAX Life Sciences, Inc., Kawasaki, Kanagawa, Japan.
Ruriko
R
Sakamoto
SCIVAX Life Sciences, Inc., Kawasaki, Kanagawa, Japan.
Norio
N
Masuda
Division of Cancer Immunotherapy, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Kashiwa, Chiba, Japan.
Tetsuya
T
Nakatsura
Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.
Stuart K
SK
Calderwood
Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
Ken-Ichi
K
Kozaki
SCIVAX Life Sciences, Inc., Kawasaki, Kanagawa, Japan.
Manabu
M
Itoh
D001549:Benzamides / Q000494:pharmacology; D015687:Cell Hypoxia / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors*; D004149:Dioxoles / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor / Q000295:instrumentation / Q000379:methods*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology*; D010865:Pilot Projects; D018874:Spheroids, Cellular; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology
C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D004149:Dioxoles; D007093:Imidazoles; D007211:Indoles; C433985:SU 9516; C509041:TGFB2 protein, human; D053781:Transforming Growth Factor beta2; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2