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Screening of breast cancer stem cell inhibitors using a protein kinase inhibitor library

Title
Screening of breast cancer stem cell inhibitors using a protein kinase inhibitor library
Authors
Choi, Hack SunKim, Dal-AhChung, HeesungPark, In HoKim, Bo HyeOh, Eok-SooKang, Duk-Hee
Ewha Authors
강덕희오억수최학선김달아정희성
SCOPUS Author ID
강덕희scopus; 오억수scopus; 김달아scopus
Issue Date
2017
Journal Title
CANCER CELL INTERNATIONAL
ISSN
1475-2867JCR Link
Citation
vol. 17
Keywords
Cancer stem cellMammosphereProtein kinase inhibitorBreast cancer
Publisher
BIOMED CENTRAL LTD
Indexed
SCIE; SCOPUS WOS scopus
Abstract
Background: Cancer stem cells (CSCs), a subpopulation in tumors, are known to cause drug resistance, tumor recurrence and metastasis. Based on the characteristic formation of mammospheres in in vitro conditions, the mammosphere formation assay has become an essential tool for quantifying CSC activity in breast cancer research. However, manual counting of mammospheres is a time-consuming process that is not amenable to high-throughput screening, and there are occasional inaccuracies in the process of determining the mammosphere diameter. In this study, we proposed a novel automated counting method of mammosphere using the National Institute of Standards and Technology (NIST)'s Integrated Colony Enumerator (NICE) with a screening of protein kinase library. Methods: Human breast cancer cell line MCF-7 was used for evaluation of tumor sphere efficiency, migration, and phenotype transition. Cell viability was assessed using MTT assay, and CSCs were identified by an analysis of CD44 expression and ALDEFLUOR assay using flow cytometry. Automated counting of mammosphere using NICE program was performed with a comparison to the result of manual counting. After identification of inhibitors to ameliorate CSC formation by screening a library of 79 protein kinase inhibitors using automated counting in primary, secondary and tertiary mammosphere assay, the effect of selected kinase inhibitors on migration, colony formation and epithelial-to-mesenchymal transition (EMT) of MCF-7 cells was investigated. Results: Automated counting of mammosphere using NICE program was an easy and less time-consuming process (< 1 min for reading 6-well plate) which provided a comparable result with manual counting. Inhibition of calcium/ calmodulin-dependent protein kinase II (CaMKII), Janus kinase-3 (JAK-3), and I.B kinase (IKK) were identified to decrease the formation of MCF-7-derived CSCs in primary, secondary and tertiary mammosphere assay. These protein kinase inhibitors alleviated TGF-a1-induced migration, colony formation and EMT of MCF-7 cells. Conclusions: We have developed a novel automated cell-based screening method which provided an easy, accurate and reproducible way for mammosphere quantification. This study is the first to show the efficacy of an automated medium-throughput mammosphere-counting method in CSC-related research with an identification of protein kinase inhibitors to ameliorate CSC formation.
DOI
10.1186/s12935-017-0392-z
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의학전문대학원 > 의학과 > Journal papers
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