Abstract
The Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) system can be used not only to study gene expression at a specific cell cycle stage, but also to monitor cell cycle transitions in real time. In this study, we used a single clone of FUCCI-expressing HeLa cells (FUCCI-HeLa cells) and monitored the cell cycle in individual live cells over time by determining the ratios between red fluorescence (RF) of RFP-Cdt1 and green fluorescence (GF) of GFP-Geminin. Cytotoxic and cytostatic compounds, the latter of which induced G2 or mitotic arrest, were identified based on periodic cycling of the RF/GF and GF/RF ratios in FUCCI-HeLa cells treated with anti-cancer drugs. With this cell cycle monitoring system, ten flavonoids were screened. Of these, apigenin and luteolin, which have a flavone backbone, were cytotoxic, whereas kaempferol, which has a flavonol backbone, was cytostatic and induced G2 arrest. In summary, we developed a system to quantitatively monitor the cell cycle in real time. This system can be used to identify novel compounds that modulate the cell cycle and to investigate structure–activity relationships.
Highlights
Natural compounds are historically important resources to identify novel lead structures for the development of anti-cancer therapeutics
Determination of the cell cycle stage of Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI)-HeLa cells based on the red fluorescence (RF)/green fluorescence (GF) and GF intensity versus the RF intensity (GF/RF) ratios
The intensities of GF of Geminin and RF of Cdt1 in FUCCI-HeLa cells fluctuate according to the cell cycle stage [15]
Summary
Natural compounds are historically important resources to identify novel lead structures for the development of anti-cancer therapeutics. More than 60% of currently used anti-cancer drugs originate from natural sources [1]. A library of natural compounds with unique chemical structures has been established. To develop novel anti-cancer drugs, these phytochemicals are usually screened using cancer cell death as a readout [2,3]. One of the most successful FDA-approved anti-cancer drugs, was identified via this method [4]. Development of a high-throughput screening platform that uses a different readout (other than cell death) will help to identify novel therapeutic phytochemicals
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