BACKGROUND : 2-Methoxyestradiol (2ME2) is an estradiol metabolite with well documented antiproliferative properties
in many cancer cell lines. However, it is rapidly metabolised in vivo which limits its clinical application. Therefore, more
stable derivatives with potentially improved clinical features have been designed by our group. Here we describe an
estrone-like derivative of 2ME2, namely EE-15-one, that unlike other derivatives which induce cell cycle arrest, induces
a rapid loss of cell–substrate adhesion through the inactivation and disassembly of focal adhesions.
METHODS : To assess the effect of 2-ethyl-estra-1,3,5 (10),15-tetraen-3-ol-17-one (EE-15-one) on breast cancer cell
lines, cell survival was quantified. The effect of EE-15-one on cell attachment was assessed by measuring cell adhesion
and cell rounding via light microscopy. Effects on focal adhesion dynamics and actin cytoskeleton organisation were
visualised by immunofluorescence while focal adhesion signalling was assessed by western blot. Cell death was quantified
using a lactate dehydrogenase activity (LDH) assay. To investigate specificity towards cell–substrate over cell–cell
contact inhibition, EE-15-one effects on 3D cell cultures were assessed.
RESULTS : Cell survival assays show an almost complete loss of cells within 24 h of EE-15-one exposure in contrast to
published sulphamoylated 2ME2 derivatives. Cell loss is linked to rapid detachment and adhesion inhibition. Focal
adhesion size and number are rapidly diminished while actin fibres became severed and disappeared within 2 h post
exposure. These changes were not due to cell necrosis as LDH activity only slightly increased after 24 h. Cells grown in
cell–cell adhesion dependent spheroids did not respond to EE-15-one exposure suggesting that EE-15-one specifically
inhibits cell–substrate adhesions but not cell–cell adhesions and does not directly impact the actin cytoskeleton.
CONCLUSION : We show that a novel 2ME2 derivative, EE-15-one, induces rapid loss of focal adhesion function leading
to cell–substrate detachment through interference with integrin-based cell–substrate adhesions, but not cadherin
dependent cell–cell adhesions. Therefore, EE-15-one is the first 2ME2 derivative that has an alternative mode of action
to the antimitotic activity of 2ME2. As such EE-15-one shows potential as a lead compound for further development
as an inhibitor of cell–substrate adhesion which is essential for metastatic dissemination.
Additional file 1: Figure S1. Chemical structure of EE-15-one. The chemical
structure of 2-Ethyl-estra-1,3,5(10),15-tetraen-3-ol-17-one (EE-15-one)
showing the ketone group at position C17 which makes EE-15-one an
estrone derivative rather than an estradiol derivative along with an alkene
group at C15. Additionally, the chemical structures of ESE-15-one, EE-one,
EE-15-ol are depicted.
Additional file 2: Figure S2. EE-15-one related cell detachment in MDAMB-
231 cells is not due to induced cell death. Medium from MDA-MB-231
cells exposed to 5 μM EE-15-one was collected at the indicated times and
analysed for LDH activity. The graph shows the percentage LDH activity as
compared to the positive control. The average result of three independent
experiments was plotted with error bars representing s.e.m. *Indicates significant
differences between negative control medium and medium from
EE-15-one treated cells as calculated by student’s t-test at a P < 0.05.
Additional file 3: Video S1. EE-15-one exposure results in the disassembly
of the focal adhesions, severance of actin stress fibres connected
to focal adhesions. For timelapse imaging, MCF-7 cells were transfected
with LifeAct-mRFP (actin, red) and paxillin-GFP (focal adhesions, green).
The following day cells were prepared for imaging every 30 s on a Zeiss
LSM800 confocal microscope using a 63× magnification objective with
environmental control. Cells were incubated with 5 μM EE-15-one. Scale
bar, 20 μm.