Abstract
High-throughput screening of extracts from plants, marine, and micro-organisms led to the identification of the extract from the plant Phyllanthus engleri as the most potent inhibitor of EWS-FLI1 induced luciferase reporter expression. Testing of compounds isolated from this extract in turn led to the identification of Englerin A (EA) as the active constituent of the extract. EA induced both necrosis and apoptosis in Ewing cells subsequent to a G2M accumulation of cells in the cell cycle. It also impacted clonogenic survival and anchorage-independent proliferation while also decreasing the proportion of chemotherapy-resistant cells identified by high ALDH activity. EA also caused a sustained increase in cytosolic calcium levels. EA appears to exert its effect on Ewing cells through a decrease in phosphorylation of EWS-FLI1 and its ability to bind DNA. This effect is mediated, at least in part, through a decrease in the levels of the calcium-dependent protein kinase PKC-βI after a transient up-regulation.
Highlights
High-throughput screening of extracts from plants, marine, and micro-organisms led to the identification of the extract from the plant Phyllanthus engleri as the most potent inhibitor of Ewing sarcoma breakpoint region gene (EWS)-FLI1 induced luciferase reporter expression
The hallmark that defines the Ewing sarcoma family of tumors is the presence of non-random chromosomal rearrangements between the Ewing sarcoma breakpoint region 1 gene (EWS)2 located on chromosome 22q12 and genes of the E26 transformation-specific sequence (ETS) family of transcription factors
High Throughput Screening Identifies Englerin A (EA) as an Inhibitor of EWS-FLI1 Activity—A high throughput screen (HTS) developed using the Ewing sarcoma cell line TC32 stably transduced with a luciferase reporter construct that reports on EWS-FLI1 activity was used to screen over 60,000 natural products extracts [11]
Summary
High-throughput screening of extracts from plants, marine, and micro-organisms led to the identification of the extract from the plant Phyllanthus engleri as the most potent inhibitor of EWS-FLI1 induced luciferase reporter expression. EA Treatment Increases Cytosolic Calcium Levels—To get a clearer picture of signaling pathways that are involved in mediating EA effects, we undertook transcription factor (TF) activity profiling of treated cells transiently transfected with luciferase reporters for various TFs. Because some impact on cell viability only became detectable 24 h after treatment with EA, a TF activity profile was generated at this time point.
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