Englerin A Inhibits EWS-FLI1 DNA Binding in Ewing Sarcoma Cells

Vittorio Caropreso; Emad Darvishi; Thomas J. Turbyville; Ranjala Ratnayake; Patrick J. Grohar; James B. McMahon; Girma M. Woldemichael

doi:10.1074/jbc.M115.701375

Englerin A Inhibits EWS-FLI1 DNA Binding in Ewing Sarcoma Cells*

From the ^‡Molecular Targets Laboratory, NCI, National Institutes of Health,
^§Optical Microscopy and Analysis Laboratory, Leidos Biomedical Research, Inc., and
^**Basic Science Program, Leidos Biomedical Research, Inc., Molecular Targets Laboratory, Frederick National Laboratory, Frederick, Maryland 21702,
^¶Center for Cancer and Cell Biology, Van Andel Institute, Grand Rapids, Michigan 49503, and
^‖Division of Hematology/Oncology, Helen DeVos Children's Hospital, Grand Rapids, Michigan 49503

↵1 To whom correspondence should be addressed: Molecular Targets Laboratory, Frederick National Lab, Bldg. 538, Rm 131, 1050 Boyles St., Frederick, MD 21702. Tel.: 301-846-5578; E-mail: woldemichaelg{at}mail.nih.gov.

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.

Footnotes

↵* This research was supported in part by the Intramural Research Program of National Institutes of Health, Frederick National Lab, Center for Cancer Research. It has also been funded in part with federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Dept. of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. The authors declare that they have no conflicts of interest with the contents of this article.