Autophagy induced by a sulphamoylated estrone analogue contributes to its cytotoxic effect on breast cancer cells
Verwey et al. Cancer Cell Int
Autophagy induced by a sulphamoylated estrone analogue contributes to its cytotoxic effect on breast cancer cells
Marcel Verwey 0
Elsie M. Nolte 0
Anna M. Joubert 0
Anne E. Theron 0
0 Department of Physiology, Faculty of Health Sciences, University of Pretoria , Private Bag X323, Arcadia, Pretoria 0007, Gauteng , South Africa
Background: Autophagy can either be protective and confer survival to stressed cells, or it can contribute to cell death. The antimitotic drug 2-ethyl-3-O-sulpamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) is an in silico-designed 17-β-estradiol analogue that induces both autophagy and apoptosis in cancer cells. The aim of the study was to determine the role of autophagy in ESE-15-ol-exposed human adenocarcinoma breast cancer cells; knowledge that will contribute to future clinical applications of this novel antimitotic compound. By inhibiting autophagy and determining the cytotoxic effects of ESE-15-ol-exposure, deductions could be made as to whether the process may confer resistance to the drug, or alternatively, contribute to the cell death process. Methods and results: Spectophometrical analysis via crystal violet staining was used to perform cytotoxicity studies. Morphology studies were done using microscopic techniques namely polarization-optical transmitted light differential interference light microscopy, fluorescent microscopy using monodansylcadaverine staining and transmission electron microscopy. Flow cytometry was used to quantify the autophagy inhibition and assess cell viability. Results obtained indicated that 3-methyladenine inhibited autophagy and increased cell survival in both MCF-7 and MDAMB-231 cell lines. Conclusion: This in vitro study inferred that autophagy inhibition with 3-methyladenine does not confer increased effectiveness of ESE-15-ol in inducing cell death. Thus it may be concluded that the autophagic process induced by ESE-15-ol exposure in MCF-7 and MDA-MB-231 cells plays a more significant role in cell death than conferring survival.
Breast cancer; Autophagy; Apoptosis; Cell survival; ESE-15-ol; 3-Methyladenine
2-Methoxyestradiol (2ME), a microtubule
depolymerising agent, is both an anti-cancer and anti-angiogenic drug
that has shown promise in cancer research (Fig. 1a) [1–3].
Although it is formed through the sequential endogenous
metabolism of 17-β-estradiol, the compound exerts its
cytotoxic effect independently of the cellular estrogen receptors
and has no significant systemic hormonal effects [1–4]. 2ME
is able to inhibit cancer cell proliferation, whereas estradiol
promotes proliferation of cancer cells. 2ME inhibits
hypoxiainducible factor-1α (HIF-1α) which, in return causes
inhibition of angiogenesis, as well as disruption of microtubules
[2, 5]. 2ME causes both the intrinsic- and extrinsic apoptotic
pathways to be up-regulated by decreasing B-cell lymphoma
2 (Bcl-2) which has anti-apoptotic properties, or by
increasing the death receptor 5 (DR5) [2, 5]. Actively proliferating
cells, such as cancer cells, are the main target for this
druginduced apoptosis while preferential sparing of normal,
quiescent endothelial cells is observed [3, 6]. 2ME not only
causes a G1 cell cycle arrest, but also a G2/M arrest [3, 7].
However, the molecular mechanism induced by 2ME differs
between cell lines . 2ME has undergone clinical trials for
solid tumors, but shows limited bioavailability and rapid
degradation in vivo [1–3, 8].
2-Ethyl-3-O-sulpamoyl-estra-1,3,5(10),15-tetraen17-ol (ESE-15-ol), a derivative of
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Fig. 1 Structure of 2-methoxyestradiol and ESE-15-ol. a
2-Methoxyestradiol is the parent compound of ESE-15-ol; b ESE-15-ol is a novel
sulphamoylated estradiol analogue
is a novel sulphamoylated estradiol analogue (Fig. 1b)
. ESE-15-ol is an anti-mitotic compound that binds
to the colchicine binding site on microtubules, and is
equally effective in both hormone-receptor
positiveand negative cancer types [9–11]. ESE-15-ol induces
both autophagy and apoptosis in breast cancer cells .
This potential anticancer drug was in silico-designed to
increase the parent compound’s bioavailability via
carbonic anhydrase II (CAII) binding by the addition of
a sulphamate group at the C′3 position . The latter
enzymatic binding occurs in erythrocytes and results
in a slower release of drugs into the circulation, thereby
bypassing the hepatic first-pass metabolism [9, 12, 13].
Tumors have acidic micro-environments in which CAIX
convert carbon dioxide (CO2) to carbonic acid . This
acidic environment enhances metastatic spread due to
proteinase release [9, 15]. Therefore the molecule was
designed to also have an increased binding affinity to
CAIX over CAII. By binding to CAIX, ESE-15-ol should
selectively locate to solid tumours and potentionally
improve chemotherapy by reducing the acidic
surrounding, thereby decreasing metastasis .
Apoptosis is an energy-dependant mode of cell death
and is also known as programmed cell death type I
[16–18]. If apoptosis is down-regulated it permits
tumor growth and multi-drug resistance . Type
IIprogrammed cell death is a degradative process known
as autophagy which is associated with the formation of
autophagic vesicles [19, 20]. Literature points at a
possible dual role associated with autophagy since it can
contribute to either cell survival or cell death, depending on
a myriad of different conditions [19, 20]. It may promote
survival by facilitating an adaptive response to cellular
stress through providing an alternative source of energy
during starvation. Additionally, autophagy can increase
cell survival by protecting them from apoptosis through
the down-regulation of pro-apoptotic proteins .
However, prolonged autophagy can lead to cell death due
to the high protein turnover rate . Autophagy and
apoptosis are interconnected and share common
stimuli for the execution of both pathways. Apoptosis and
autophagy can thus either have synergistic or
antagonistic effects .
3MA, a nucleotide derivative, is an inhibitor of
autophagy . 3MA blocks autophagy through
inhibiting class I and class III phosphatidylinositide-3-kinases
(PI3K) [23, 24]. Class III PI3K is a lipid protein that
phosphorylates the 3rd position on the inositol ring
in phosphatidylinositol to form
phosphatidylinositol3-phosphate (PI3P), which is essential for the initial steps
in autophagy . This leads to the activation of protein
kinase B (Akt) which then phosphorylates the
mechanistic target of rapamycin (mTor) [20, 23]. 3MA can
suppress cell invasion and migration of fibrosarcoma cells
(HT1080) by inhibiting class III PI3K .
Studies by Xie et al.  and Li et al.  have shown
that autophagy inhibition by 3-methyladenine (3MA)
increased apoptotic cell death in human colon cancer cell
lines and human hepatoma cells (HepG2). By inhibitiong
the protective mechanism of autophagy, chemoresistance
was overcome. However, Bonet-Ponce et al. 
demonstrated that inhibition of autophagy by 3MA inhanced
cell survival through reducing the oxidative
stressinduced cell death. This indicates that the mechanisms of
autophagy are cell and drug dependant.
In this study autophagy inhibition with 3MA
during ESE-15-ol exposure was conducted to allow insight
into whether autophagy will confer resistance to
drugexposed cells, or whether it will contribute to
programmed cell death.
Dulbecco’s minimum essential medium Eagle (DMEM),
trypsin–EDTA, 3-methyladenine (3MA) and all other
reagents not specifically mentioned were of
analytical grade and purchased from Sigma-Aldrich (St. Louis,
USA). Streptomycin, fungizone and penicillin were
manufactured by Thermo Fisher Scientific (Massachusette
(MA), USA). Crystal violet, gluteraldehyde and triton
X-100 were purchased from Merck (Darmstadt,
Germany). Anti-LC3B/MAP1LC3B-antibody was supplied
by Novus Biological [Littleton, Colorado (CO), USA].
Chemical compounds and appropriate controls
The novel estradiol analogue, ESE-15-ol, was synthesized
by Ithemba Pharmaceuticals (PTY) Ltd. (Modderfontein,
Gauteng, South Africa) as it is not commercially
available. A working 1 mM stock solution of ESE-15-ol in
dimethyl sulfoxide (DMSO) was prepared at the
Department of Physiology, University of Pretoria and stored
at −20 °C. DMSO was used as a vehicle control, never
exceeding a 0.05% concentration in the final dilution.
Actinomycin D (Sigma-Aldrich, St Louis, USA) was used
as a positive control for apoptosis at a final concentration
of 0.1 μg/ml. A final concentration of 20 μM tamoxifen
(Sigma-Aldrich, St Louis, USA) was used as a positive
control for autophagy. Tamoxifen (20 μM) combined with
5 mM 3MA was used as a positive control for autophagy
inhibition. All chemicals were of analytical grade and
purchased from Sigma-Aldrich (St Louis, USA), unless
Cell lines and general cell culture protocols
For this in vitro study, the human adenocarcinoma breast
cancer MCF-7 cell line and the metastatic human
adenocarcinoma breast cancer MDA-MB-231 cell line were
used (Cellonex, Johannesburg, South Africa).
Penicillin G (100 U/ml), fungizone (250 μg/l), streptomycin
(100 μg/l) and 10% heat activated fetal calf serum (FCS)
(Gibco® Invitrogen, California, USA) were added to
DMEM. For the 96 well plates, 5000 cells were seeded in
200 μl growth medium in each individual well. For the
6 well plates, 375,000 cells were seeded in 3 ml growth
medium in each individual well. For the 25 cm2 culture
flasks, 1 × 106 cells/5 ml growth medium were seeded.
Cells were incubated in a Forma Scientific water-jacketed
incubator (Ohio, USA) in a humidified atmosphere at 5%
CO2 (37 °C) for 24 h before exposure to ESE-15-ol, with
or without 3MA. A final concentration of 5 mM 3MA
was used to inhibit autophagy during exposure with
ESE15-ol or tamoxifen. Cells were pre-exposed to 3MA for
an hour prior to addition of ESE-15-ol or tamoxifen.
Spectrophotometry: crystal violet
Crystal violet is a triarylmethane dye (purple) that stains
the deoxyribonucleic acid (DNA) within cells in a
monolayer culture. Cytotoxicity of a drug may be determined
by measuring the absorbance of the crystal violet stained
cells via spectrophotometry. The half maximal growth
inhibitory concentration (GI50) may thus be determined
[29, 30]. After a 24 h-exposure to an ESE-15-ol dose
concentration series, 100 μl 1% gluteraldehyde was added to
the cells and samples were incubated for 15 min at room
temperature. Gluteraldehyde was discarded and 100 μl
0.1% crystal violet was added for 30 min for staining to
take place. Crystal violet was discarded and the plate
was washed under running tap water (±10 min). Plates
were left to dry for 24 h. Triton X-100 (0.2%) (200 μl) was
added to the wells and samples were incubated for 40 min
at room temperature for solubilisation. After incubation,
100 μl of the solution was pipetted to a clean 96 well
plate. Absorbance was read using the ELx800 Universal
Microplate Reader (Bio-tek Instruments Inc. Vermont,
USA) at 570 nm.
Polarization‑optical transmitted light differential
Viable cells were seeded at a density of 375,000 cells/3 ml
growth medium in 6 well plates. After attachment, cells
were exposed to ESE-15-ol in the presence or absence of
3MA for 24 h (37 °C) along with appropriate controls.
PlasDIC images were viewed at a 40× magnification with
a Zeiss Axiovert-40 microscope (Göttingen, Germany)
and captured with the Zeiss Axiovert MRm monochrome
camera (Göttingen, Germany).
Cells were exposed to ESE-15-ol in the presence or
absence of 3MA for 24 h (37°) along with the
appropriate controls. Cells were stained with 0.05 mM
monodansylcadaverine (MDC) in PBS for 10 min (37 °C) and
protected against light after which cells were washed four
times with PBS. Using the Zeiss inverted Axiovert CFL 40
microscope and the Zeiss Axiovert MRm monochrome
camera (Göttingen, Germany) fluorescent images were
visualized with a UV filter with excitation of 380 nm and
emission of 480 nm at a 40× magnification.
Transmission electron microscopy
After 24 h of exposure to ESE-15-ol with or without
3MA alongside all the appropriate controls, cells were
trypsinized and fixed in 2.5% gluteraldehyde in 0.075 M
phosphate buffer for 1 h (room temperature). Cells were
washed three times for 10 min with 0.075 M phosphate
buffer followed by fixation in 0.5% aqueous osmium
tetroxide for 1 h (room temperature). Cells were washed
3 times in distilled water. Increasing ethanol
concentrations (10, 30, 50, 70 and 100%) were used to dehydrate
the fixed cells for 10 min in each concentration. Quetol
(30%) in ethanol was used to infiltrate the cells (1 h)
followed by 60% quetol (1 h) and lastly in 100% quetol (4 h).
Specimen were embedded and polymerized (60 °C, 39 h).
Ultra thin sections were prepared using a microtome and
each section was mounted on a copper grid. Samples
were contrasted in 4% aqueous uranyl acetate (10 min)
and then in Reynolds lead citrate (2 min). To view the
TEM images the JEM-2100F field emission transmission
electron microscope (JEOL, Tokyo, Japan) was used. All
Chemicals provided by the Electron Microscopy unit,
University of Pretoria.
Cell cycle progression
Propidium iodide (PI) is a fluorescent dye used to
quantify DNA content at 488 nm excitation . Flow
cytometry employing PI was used to distinguish cells in the
different cell cycle phases. After exposure, cells were
trypsinized and washed in 1 ml ice-cold PBS and
resuspended in 200 μl ice-cold PBS. Cells were fixed in 4 ml
ice-cold 70% ethanol, added drop wise while vortexing
and incubated overnight at 4 °C. Cells were centrifuged
(5 min at 100×g) and the pellet resuspended in PBS
containing 0.01% triton X-100, PI (40 μg/ml) and RNase A
(100 μg/ml) and incubated for 40 min at 37 °C. PI
fluorescence was measured using flow channel 3 (FL3) on the
FC500 system flow cytometer (Beckman Coulter,
California, USA) excited at 488 nm. A minimum of 10,000 cells
were analized using three biological repeats. Cell cycle
distributions were analized with Cyflogic flow cytometry
analysis software (Beckman Coulter, California, USA)
using the DNA content per cell which illustrates the
subG1, G1, S, G2/M fractions.
Apoptosis detection: annexinV‑ FITC
Viable cells contain phosphatidylserine (PS), located on
the inside of cell membrane. When apoptosis occurs, PS
flip will occur. PS moves to the outside surface of cells
to which annexin-V binds . Washed cells were
centrifuged and the supernatant discarded. The pellet was
resuspended in annexin-V FITC binding buffer (0.25–
107 cells/ml). Cell suspension (100 μl) was pipetted into a
5 ml test tube. Annexin-V FITC (5 μl) and 10 μl of PI was
added and samples were incubated at room temperature
(15 min) in the dark. After incubation 400 μl
annexinV binding buffer was added to each test tube. PI (FL3)
and annexin-V (FL1) fluorescence was analyzed with the
FC500 system flow cytometer (Beckman Coulter,
California, USA) equipped with an air-cooled argon laser
excited at 488 nm. Three biological repeats with a
minimum of 10,000 cells were analized. Data was analyzed
with Cyflogic flow cytometry analysis software (Beckman
Coulter, California, USA) with PI plotted on the x-axis
and annexin V-FITC on the y-axis.
Autophagy detection: microtubule‑associated protein
1A/1B‑light chain 3
To detect autophagy, flow cytometry was used
employing microtubule-associated protein 1A/1B-light chain
3 (LC3) using a conjugated rabbit polyclonal
antiLC3B antibody which can be detected in FL1
(excitation = 488 nm; emission = 525 nm) . After 24 h of
exposure to ESE-15-ol with or without 3MA, washed
trypsinized cells were resuspended in ice-cold PBS
containing 0.01% formaldehyde and samples were
incubated for 10 min at 4 °C. Cells were centrifuged and
resuspended in 200 μl PBS to which 1 ml ice-cold 100%
methanol was added and incubated for 15 min at 4 °C.
Cells were washed twice with PBS. Cell pellet was
resuspended in 200 μl PBS containing 0.05% triton X-100, 1%
BSA, 40 μg/ml PI and anti-LC3B/MAP1LC3B-antibody
(1:200) (Novus Biological, Littleton, CO, USA) and
incubated for 2 h at 4 °C. Cells were washed twice with 1 ml
PBS containing 0.05% triton X-100 and 1% BSA. LC3
fluorescence was measured in flow channel 1 (FL1) on the
FC500 system flow cytometer (Beckman Coulter,
California, USA). Three biological repeats with a minimum of
10,000 cells were used and data was analyzed with
Cyflogic flow cytometry analysis software (Beckman Coulter,
California, USA). Any cell debris and cell clumps were
removed from analysis.
Western blot analysis: LC3II
After 24 h exposure, DMEM was aspirated and cells were
washed with ice-cold PBS. 200 μl RIPA cell lysis buffer
(150 mM NaCl, 0.1% SDS, 10 mM Tris–HCl 0.5% sodium
deoxcylate, 1 mM EGTA and 1 mM EDTA in ddH20,
adjusted to a pH of 7.4) was added to cells and incubated
for 5 min on ice. Lysed cells were scraped and centrifuged
for 30 min at 4 °C (1000×g). Protein concentration was
determined by use of the Pierce® BCA protein assay kit
(Thermo Fisher Scientific Inc., Rockford, Illinois, USA).
In a 96 well plate, 20 μl of the cytosolic extract was added
to 100 μl of the protein assay working solution. The
absorbance was read at 570 nm by use of ELx800
Universal Microplate Reader (Bio-tek Instruments Inc.
Vermont, USA). The protein concentration was calculated
by using a standard curve. After protein quantification
25 μg protein of each sample, containing 5%
b-mercaptoethanol and NuPAGE LDS buffer (1:4) (Sigma-Aldrich,
St. Louis, USA) was denaturated at 96 °C (10 min).
Samples (20 μl) were loaded into NuPAGE 4–12% Bis–Tris
gel wells alongside a relevant protein band size ladder.
Samples were run in 1× MOPS buffer (190 mM Glycine,
25 mM Tris and 0.1% SDS in ddH20, adjusted to a pH of
8.3) at 120 V for 90 min for protein separation via
electrophoresis. Separated proteins were transferred to a PVDF
0.2 μm membrane (Amersham Hybond, GE Healthcare
Life Sciences) in 1× transfer buffer (48 mM Tris, 39 mM
glycine, 20% methanol and 0.0375% SDS), which was
activated with 100% methanol. Wet transfer was achieved
overnight at 40 V. After transfer, the membrane was
blocked in 5 ml blocking buffer (2% BSA in 0.2%
PBSTWEEN) for 30 min at room temperature. Membranes
were incubated overnight at 4 °C in the primary antibody
cocktail (1:1000 monoclonal LC3B/MAP1LC3B antibody
produced in rabbit in 0.2% PBS-Tween, 2% BSA)
purchased from Novus Biological [Littleton, Colorado (CO),
USA]. Membranes were washed three times in washing
buffer for 15 min each. This was followed by 1 h
incubation with the secondary antibody (anti-rabbit IgG
antibody raised in goat labelled with horse-radish peroxidase
(HRP) (1:10,000) (KPL, Mayland, USA) in 2.5% BSA in
PBS. Membranes were washed three times in washing
buffer for 15 min each. Pierce® ECL western blotting
reagent (Thermo Fisher Scientific, MA, USA) was used to
activate HRP activity to visualize proteins using
ChemiDoc MP (Bio-Rad, CA, USA). Monoclonal anti-actin
antibody produced in mouse (1:5000, Sigma-Aldrich, St.
Louis, USA) was used to standardise the membranes and
developed using the anti-mouse IgG secondary antibody
raised in goat labelled with HRP (KPL, Mayland, USA).
Image Lab 5.2.1 (Bio-Rad, CA, USA) was used to
determine band size. Three biological repeats were done.
Morphology studies supplied qualitative data and
crystal violet staining and flow cytometry analysis supplied
quantitative data. Flow cytometry was repeated 3 times
and involved the analysis of at least 10,000 cells per
run. Three independent experiments were performed
and data were shown as the mean ± standard
deviation. Analysis of variance (ANOVA) single factor model
of significance was used to statistically analyze data and
followed by a two-tailed Student’s t-test. A statistical
significant P value of <0.05 was used and indicated with an
asterisk (*) and (#) was used to indicate a statistically
significant difference between ESE-15-ol-exposed cells and
those exposed in the presence of 3MA. Means are
presented with bar-graphs with T-bars referring to standard
Inhibition of autophagy decreases the cytotoxicity
of ESE‑15‑ol as determined by spectrophotometry
Spectrophotometry was used to determine the effect
of autophagy inhibition on the cytotoxicity of
ESE-15ol-exposed human adenocarcinoma breast cancer cells
(MCF-7) and metastatic human adenocarcinoma breast
cancer cells (MDA-MB-231). The half maximum growth
inhibitory concentrations (IG50) of ESE-15-ol with and
without 3MA were determined by dose-dependent
studies over a 24 h period. The IG50 of ESE-15-ol was
calculated at 0.05 ± 0.018 μM and at 0.15 ± 0.014 μM for
ESE-15-ol with 3MA in MCF-7 cells (Fig. 2a). The IG50
of ESE-15-ol was calculated at 0.065 ± 0.005 μM, and at
0.13 ± 0.06 μM for ESE-15-ol with 3MA-exposed
MDAMB-231 cells (Fig. 2b). Autophagy inhibition was thus
seen to have caused a statistically significant decrease in
ESE-15-ol cytotoxicity, with a P value of 0.007 in MCF-7
cells and 0.0195 in MDA-MB-231 cells.
Morphological features of cell death induced by ESE‑15‑ol
were atteniated by addition of 3MA
Polarization-optical transmitted light differential
interference light microscopy (PlasDIC) was used to evaluate
the morphological response of cells to ESE-15-ol with
or without 3MA. MCF-7 (Fig. 3ai) and MDA-MB-231
(Fig. 3aii) cells exposed to DMSO showed no signs of
cell distress. Confluent cell growth was seen with
visible nucleoli as for the 3MA-exposed cells (Fig. 3bi, bii).
Cells were mostly present in interphase.
Actinomycin D-treated cells showed a decrease in cell density for
both MCF-7 (Fig. 3ci) and MDA-MB-231 (Fig. 3cii) cells.
Apoptotic body formation, cell debris and shrunken cells
were visible, which are characteristic of apoptotic cell
death. ESE-15-ol-treated MCF-7 (Fig. 3di) and
MDAMB-231 (Fig. 3dii) cells demonstrated an increased
proportion of rounded cells as well as the presence of
apoptotic bodies. ESE-15-ol-treated cells together with
3MA showed apoptotic body formation and rounded
cells in both MCF-7 (Fig. 3ei) and MDA-MB-231 cells
(Fig. 3eii), but to a lesser extend when compared to cells
treated with ESE-15-ol without 3MA.
Acidic vacuoles were decreased in cells treated
with ESE‑15‑ol in combination with 3MA
Monodansylcadaverine (MDC) is a weak base
fluorescent stain which stains acidic vacuoles that suggest
occurence of autophagy . MCF-7 and MDA-MB-231
cells exposed to DMSO (Fig. 4ai, aii) and 3MA (Fig. 4bi,
bii) showed non-specific MDC staining. Both controls
displayed confluent cell growth. Tamoxifen-treated cells
(Fig. 4ci, cii) showed an increase in MDC-stained
vacuoles in both cell lines. ESE-15-ol-treated cells
demonstrated an increase in MDC-stained vacuoles, as well as
decreased cell density (Fig. 4di, dii). ESE-15-ol treated
cells in the presence of 3MA showed a decrease in MDC
staining, with less acidic vacuole formation apparent
when compared to the drug treated sample (Fig. 4ei, eii).
Vacuole formation in response to ESE‑15‑ol exposure was
evident but diminshed when co‑incubated with 3MA
Transmission electron microscopy was used to
analyze the ultrastructure of ESE-15-ol-treated MCF-7 and
MDA-MB-231 cells, with and without autophagy
inhibition by 3MA. Cells propagated in DMSO (Figs. 5a, 6a)
showed a smooth cell membrane with minimal cellular
protrusions, together with an intact nuclear envelope.
No morphological differences were seen between cells
exposed to DMSO and 3MA (Figs. 5b, 6b). Cells treated
with actinomycin D (Figs. 5c, 6c) increased membrane
blebbing and apoptotic body formation representative of
apoptotic cell death. Tamoxifen-treated cells showed an
increase in vesicle formation (Figs. 5d, 6d). Less vacuole
formation was observed in tamoxifen-treated cells with
3MA (Figs. 5e, 6e). This suggests partial autophagy
inhibition by 3MA. Intact cell membranes were observed in
tamoxifen-treated cells, with and without 3MA.
ESE15-ol-treated cells (Figs. 5f, 6f ) displayed an increase in
vacuolar structures indicative of autophagy. Apoptotic
bodies, hypercondensed chromatin and increased cell
Fig. 2 Cytotoxicity study for ESE-15-ol with/without 3MA over a 24 h exposure period in MCF-7 and MDA-MB-231 breast cancer cells. a The dose
dependent curve for MCF-7 cells showed an IG50 of 0.15 μM for ESE-15-ol with 3MA and 0.05 μM for ESE-15-ol only (P-value = 0.007); b
MDAMB-231 cells showed an IG50 of 0.13 μM for ESE-15-ol with 3MA and 0.065 μM for ESE-15-ol only (P-value = 0.0195). Bars indicate averages of three
independent biological repeats, each with n = 3. Error bars represent standard deviation
protrusions were also observed in cells exposed to
ESE15-ol. ESE-15-ol-treated cells with 3MA inhibition of
autophagy (Figs. 5g, 6g) revealed cells with intact
nuclearand cell-membranes. A decrease in cellular distress with
fewer vesicles was observed in these cells.
3MA co‑incubation increased cell viability and reduced
G2/M block as well as the sub‑G 1 population in response
to ESE‑15‑ol exposure
The quantification of cells at various stages within the
cell cycle was determined with flow cytometry. Cell cycle
distribution of MCF-7 cells exposed to DMSO (Fig. 7ai)
showed an average of 3.24 ± 0.54% in the sub-G1 phase,
56.2 ± 4.89% in the G1 phase and 18.79 ± 0.89% in the
G2/M phase. Cell cycle distribution of MDA-MB-231
cells grown in DMSO (Fig. 7aii) showed an average of
2.53 ± 1.62% in the sub-G1 phase, 57.8 ± 0.61% in the
G1 phase and 29.5 ± 1.83% in the G2/M phase. No
statistical significance was found between cells grown in
medium only and treated with DMSO or 3MA,
indicating that the vehicle was non-toxic to the cells. MCF-7
and MDA-MB-231 cells exposed to actinomycin D
and ESE-15-ol had statistically significant changes in
their cell cycle distributions when compared to the
DMSO vehicle control. Actinomycin D-treated MCF-7
cells (Fig. 7bi) had a significant increase in the
subG1 phase (9.47 ± 0.31%; P = 0.00015) and the G2/M
(22.98 ± 2.32%; P = 0.04) phase. ESE-15-ol-treated
MCF-7 cells (Fig. 7ci) had a significant increase in the
sub-G1 phase (18.14 ± 2.81%; P = 0.001) and the G2/M
phase (60.28 ± 1.34%; P = 0.0009) when compared to the
DMSO control. ESE-15-ol-treated MCF-7 cells together
with 3MA autophagy inhibition (Fig. 7di) had a
statistically significant increase in the G1 phase (35.70 ± 7.45%;
P = 0.02) when compared to ESE-15-ol without 3MA
treatment (13.59 ± 0.36%). Actinomycin D treated
MDAMB-231 cells (Fig. 7bii) had a significant decrease in the
G1 phase (31.94 ± 2.75%; P = 0.0004) and concomitant
increase in the G2/M phase (48.52 ± 2.63%; P = 0.02)
when compared to DMSO-exposed cells.
ESE-15-oltreated MDA-MB-231 cells (Fig. 7cii) had a significant
decrease in the G1 phase (44.45 ± 1.59%; P = 0.008) with
a significant increase in the G2/M phase (43.23 ± 2.13%;
P = 0.009) when compared to DMSO vehicle control.
ESE-15-ol-treated MDA-MB-231 cells with a
concurrent treatment with 3MA (Fig. 7dii) had a statistically
significant increase in the G1 phase (63.64 ± 4.46%; P 0.03)
when compared to cells exposed to ESE-15-ol without
(See figure on next page.)
Fig. 3 PlasDIC images of MCF-7 and MDA-MB-231 cells exposed to the compound with/or without 3MA for 24 h. i MCF-7 cells and ii MDA-MB-231
cells grown in a DMSO and b 3MA served as negative controls. Confluent cell growth with no signs of cell distress was demonstrated. c
Actinomycin D (0.1 μg/ml) served as a positive control for apoptosis, resulting in apoptotic body formation and compromised cell density. d
ESE-15-oltreated cells revealed the presence of rounded cells, formation of apoptotic bodies and decreased cell density. e ESE-15-ol exposure to cells in
which autophagy had been inhibited with 3MA showed an increase in cell viability. (Arrow colour key: Yellow = interphase cells; orange = rounded
cells in metaphase; white = apoptotic bodies.)
(See figure on previous page.)
Fig. 4 Fluorescent microscopy with monodansylcadaverine staining of MCF-7 and MDA-MB-231 cells. i MCF-7 and ii MDA-MB-231 cells treated
with a DMSO and b 3MA served as negative controls and displayed non-specific MDC staining. c Tamoxifen was used as a positive control for
acidic vacuoles and displayed clear MDC stained vacuoles. d ESE-15-ol treated cells showed increased MDC staining, while e ESE-15-ol treated cells
together with 3MA showed less distinctive MDC staining, indicating partial autophagy inhibition (40× magnification)
3MA. These results indicate that autophagy inhibition
decreases the cytotoxic effect of ESE-15-ol exposure in
MCF-7 and MDA-MB-231 breast cancer cells.
Apoptosis induction in response to ESE‑15‑ol was
diminshed with addition of 3MA
Flow cytometry employing annexin V-FITC and PI was
used to distinguish between viable-, apoptotic- and
necrotic cells. Statistical analysis of dot plots data
indicated a statistically significant increase in apoptosis
in actinomycin D-treated (Fig. 8bi) (40.06 ± 5.55%;
P = 0.001) and ESE-15-ol-treated (Fig. 8ci)
(21.21 ± 0.13%; P = 0.002) MCF-7 cells when compared
to the DMSO-vehicle control (Fig. 8ai). A statistically
significant increase in apoptosis in actinomycin D-treated
(Fig. 8bii) (25.39 ± 3.44%; P = 0.0006) and
ESE-15ol-treated (Fig. 8cii) MDA-MB-231 cells (38 ± 7.02%;
P = 0.004) was detected when compared to the
DMSOvehicle control (Fig. 8aii). No statistically significant
difference in viability was detected between cells grown
in medium only, DMSO-exposure and 3MA-exposure
in both MCF-7 and MDA-MB-231 cells. A statistically
significant increase in viability in ESE-15-ol-treated
MCF-7 and MDA-MB-231 cells together with 3MA
(81.31 ± 2.05%; P = 0.01 and 83.97 ± 5.6%; P = 0.01,
respectively) was observed when compared to
ESE-15-oltreated cells (Fig. 8ei, eii).
LC3 expression was increased in cells exposed to ESE‑15‑ol
Flow cytometry employing a microtubule-associated
protein 1A/1B-light chain 3 (LC3) conjugated rabbit
polyclonal anti-LC3B antibody (Novus Biologicals,
Littleton, CO) was used to quantify and confirm
induction and inhibition of autophagy. Figure 9a illustrates an
overlay histogram of MCF-7 (1) and MDA-MB-231(2)
cells exposed to ESE-15-ol with and without 3MA.
A right shift is seen in ESE-15-ol-treated cells
without 3MA. A decrease in LC3 detection is seen in cells
Fig. 5 Transmission electron micrographs of MCF-7 cells. MCF-7 cells treated with a DMSO and b 3MA showed intact nuclear envelope and
cytoplasmic membranes with minimal cell protrusions. c Actinomycin D-exposed cells showed membrane blebbing and apoptotic body formation. d
Tamoxifen-treated cells showed an increase in vesicle formation when compared to e tamoxifen with 3MA. f ESE-15-ol-treated cells showed
hypercondensed chromatin with an increase in vesicle- and apoptotic body formation. g Cells exposed to ESE-15-ol concurrent to autophagy inhibition
with 3MA showed less signs of cell distress, fewer vesicles and intact nuclear membranes (Scale bar 5 μM) (Arrow colour keys: white = intact nuclear
membrane; red = apoptotic bodies; yellow = vesicles)
Fig. 6 Transmission electron micrographs of MDA-MB-231 cells. MDA-MB-231 cells treated with a DMSO and b 3MA showed an intact nuclear
membrane and well-defined cell membranes with minimal cell protrusions. c Actinomycin D-exposed cells showed membrane blebbing and
apoptotic body formation. d Tamoxifen-treated cells showed more vesicle formation when compared to cells treated with e tamoxifen with 3MA.
f ESE-15-ol-treated cells showed hyper condensed chromatin with increased vesicles and apoptotic body formation. g Cells exposed to
ESE15-ol with 3MA showed fewer vesicles with an intact nuclear membrane (Scale bar 5 μM) (Arrow colour keys: white = intact nuclear membrane;
red = apoptotic bodies; yellow = vesicles)
exposed to ESE-15-ol together with 3MA indicating
partial autophagy inhibition. No difference was detected
between cells grown in medium only and cells exposed
to the DMSO vehicle control and 3MA (not shown in
overlay). Graphical representations in Fig. 9b show a
statistically significant fold increase of 2.075 ± 0.05 LC3
after tamoxifen exposure and 1.67 ± 0.17 LC3 in
ESE15-ol when compared to the DMSO vehicle control. A
statistically significant fold decrease of LC3 is seen after
tamoxifen with 3MA exposure (1.40 ± 0.25; P = 0.022)
when compared to tamoxifen exposure alone, as well as
between ESE-15-ol with 3MA (1.13 ± 0.11; P = 0.004)
when compared to ESE-15-ol alone.
LC3I conversion to LC3II decreased in cells treated
with ESE‑15‑ol together with 3MA
LC3II has a role in cytoplasmic cargo selection,
autophagosome membrane tethering and fusion .
Increased levels of LC3-II may be associated with an
upregulation in autophagy or a decrease in turnover (altered
autophagic flux). Western blots were used to quantify
the formation of LC3II in the presence of the analogue
with- and without 3MA (Fig. 10). Protein quantification
revealed a statistically significant fold-increase in LC3II
expression when MCF-7 cells were exposed to
ESE15-ol (1.23 ± 0.11, P = 0.02) and tamoxifen (2.55 ± 0.35,
P = 0.003, as did the MDA-MB-231 cells (2.75 ± 0.35,
P = 0.002 and 2.4 ± 0.42, P = 0.004 respectively), when
compared to the DMSO control. Addition of 3MA
reduced LC3II expression to 0.73 ± 0.09-fold (MCF-7
cells) and to 1.4 ± 0.56 (MDA-MB-231 cells), statistically
significant when compared to ESE-15-ol only-exposed
MCF-7 (P = 0.015) and MDA-MB-231 cells (P = 0.04)
respectively. Similar trends were seen in the
tamoxifentreated positive control samples.
2ME is an anti-proliferative agent which shows
promise in cancer treatment, including breast cancer . It
induces both the intrinsic- and extrinsic apoptotic
pathways through inactivation of Bcl-2 and increased DR5
expression . Several promising analogues of 2ME,
such as 2-methoxyestradiol-bis-sulfamate
(2MEOE2bisMATE) have been developed for improved potency and
bioavailability of the parent drug [9, 36, 37]. Our
laboratory has in silico-designed a range of sulphamoylated
estrone analogues with the added intention to localise
them to solid tumour micromilieus through an increased
CAIX binding affinity [9, 38].
Of these compounds, ESE-15-ol,
2-ethyl-3-O-sulphamoyl-etsra-1,3,5 (10) 15-tetraene-3-ol-17-one
(ESE15-one) and 2-ethyl-3-O-sulphamoyl-etsra-1,3,5 (10)
16-tetraene (ESE-16) have been investigated for modes
of cell death induction (apoptosis and autophagy) on
Fig. 7 Cell cycle analysis of MCF-7 and MDA-MB231 cells exposed to ESE-15-ol with- and without 3MA. Cells were exposed to DMSO as a negative
vehicle control (ai, aii) which showed a prominent G1 phase. Actinomycin D (bi, bii) was used as a positive control for apoptosis which resulted
in an increase in the sub-G1 phase. An increase in the G2/M phase was seen in ESE-15-ol-treated cells (ci, cii) with a concurrent decrease in the
G1 phase. ESE-15-ol treated cells together with 3MA (di, dii) showed a decrease in the G2/M phase with an increase in the G1 phase. Graphical
representation of ei MCF-7 and eii MDA-MB-231 cell cycle analysis. ESE-15-ol-treated cells together with 3MA demonstrated an increase in the G1
phase when compared to ESE-15-ol-treated cells (P value <0.05; standard deviation represented by T-bars; *indicates statistical difference between
compounds and DMSO vehicle control; #indicates statistical difference between ESE-15-ol and ESE-15-ol with 3MA)
Fig. 8 Annexin-V FITC flow cytometric analysis of cell viability. Dot blots of i MCF-7 and ii MDA-MB-231 cells exposed to a DMSO as a vehicle
control and demonstrated a viable cell population. Dot blots of b actinomycin D (positive apoptosis control) and c ESE-15-ol-treated cells showed
increased cell death via apoptosis. d ESE-15-ol-treated cells with 3MA showed increased cell viability when compared to cells exposed to ESE-15-ol
only. Graphical representation of (ei) MCF-7 and (eii) MDA-MB-231 cells showed a decrease in cell viability in cells treated with ESE-15-ol when
compared to DMSO. An increase in viable cells is observed in ESE-15-ol treated cells with 3MA when compared to ESE-15-ol treated cells. Bars represent
averages of three biological repeats (P value <0.05; standard deviations represented by T-bars; *indicates statistical difference between compounds
and DMSO vehicle control; #indicates statistical difference between ESE-15-ol and ESE-15-ol with 3MA) (Dot plot keys: i = viable cells; ii = necrosis;
iii = late apoptosis; iv early apoptosis)
Fig. 9 LC3 fluorescence determination within MCF-7 and MDA-MB-231 cells after a 24 h exposure. Overlay histogram of ai MCF-7 and aii
MDAMB-231 exposed cells. Cells treated with ESE-15-ol showed a right shift which was greater than ESE-15-ol with 3MA. b Graphical representation
shows an increase in LC3 detection within ESE-15-ol-treated cells. Bars represent averages of three biological repeats (P value <0.05; standard
deviations represented by T-bars; *indicates statistical differences between compounds and DMSO vehicle control; #indicates statistical difference
between ESE-15-ol and ESE-15-ol with 3MA; †indicates statistical difference between tamoxifen and tamoxifen with 3MA)
various cancer cell lines [9, 36, 39–41]. Authors have
indicated the increased presence of autophagic vesicles
at certain time points after drug exposure .
Extensive analysis of the drug’s effect on induction or flux of
autophagy have not been completed, although
up-regulation of autophagic genes have been reported on
microarrays . Descriptions of autophagy conceptualize a
dual role within the context of cellular survival, either
inducing programmed cell death type II or activating a
pro-survival phenotype . Autophagy can be activated
by tumor microenvironments to protect hypoxic and
nutrient deprived cells, thus inducing resistance to drug
treatment . The current study aimed to determine
the role of autophagy associated demonstrated on cancer
cell exposure to ESE-15-ol by inhibiting autophagy with
Fig. 10 Western blot analysis of LC3II protein expression levels. Western blots of ai MCF-7 and aii MDA-MB-231 cells treated with ESE-15-ol showed
a more prominent LC3II band than cells treated with ES-15-ol with 3MA. b Graphical representation demonstrates a statistically significant decrease
in LC3II protein levels within cells exposed to ESE-15-ol concurrently with 3MA as well as tamoxifen with 3MA. Bars represent averages of three
biological repeats (P value <0.05; standard deviations represented by T-bars; *indicates statistical differences between compounds and DMSO
vehicle control; #indicates statistical difference between ESE-15-ol and ESE-15-ol with 3MA; †indicates statistical difference between tamoxifen and
tamoxifen with 3MA)
Cytotoxicity studies on MCF-7 and MDA-MB-231
breast adenocarcinoma cells were conducted over a 24 h
period by spectrophotometric quantification of
crystal violet. IG50 values were determined for
ESE-15-oltreated cells, and compared to the value obtained when
5 mM 3MA, an inhibitor of autophagy, was added for the
duration of the drug exposure. This was done to
determine the effect which autophagy inhibition would have
on the cytotoxicity of ESE-15-ol on MCF-7 and
MDAMB-231 cells. IG50 values of 0.05 and 0.065 μM were
calculated for MCF-7 and MDA-MB-231 cells respectively
when exposed to ESE-15-ol without 3MA. IG50 values
of 0.15 and 0.1 μM in MCF-7 and MDA-MB-231 cells
respectively were determined for ESE-15-ol together
with 3MA. The increase in IG50 values with the addition
of 3MA were statistically significant in both cell lines
(P = 0.007; P = 0.0195). ESE-15-ol without 3MA had an
increased cytotoxicity than with 3MA. The results
indicate the possibility that autophagy contributes to the cell
death process in cells exposed to ESE-15-ol.
Various microscopic techniques were used to
determine the effect of autophagy inhibition on the
morphology of MCF-7 and MDA-MB-231 cells after
ESE-15-ol-exposure. PlasDIC microscopy revealed that
ESE-15-ol-exposure resulted in numerous rounded
cells, most likely in mitotic arrest, supporting its action
as a spindle poison. Cell cycle analysis corroborated this
induction due to an increase in cells present in the G2/M
phase. Previous studies have reported that a mitotic
block was caused in MCF-7 cells exposed to 1 μM 2ME,
the parent compound of ESE-15-ol . Apoptotic bodies
and shrunken cells were visualized on the micrographs
of ESE-15-ol-exposed cells. This suggests that
ESE15-ol induced cell death via apoptosis over a 24 h
exposure period due to spindle disruption. Theron et al. 
showed that ESE-16-ol and ESE-15-one induced
metaphase block due to abrogation of microtubule dynamics
and non-satisfaction of the spindle assembly checkpoint
(SAC), thereby inducing apoptosis in MDA-MB-231 and
HeLa cells. In this study, only a small proportion of cells
demonstrated a mitotic arrest, although apoptotic bodies
were still visible when cells were treated with ESE-15-ol
combined with 3MA (an effect which was more subtle
when compared to cells exposed to the compound-only).
Thus signs of apoptotic cell death were still present when
autophagy was inhibited. For optimal cell death by
ESE15-ol both apoptosis and autophagy are hypothesized to
Fluorescent microscopy employing MDC was used
to detect acidic vacuoles that may be indicative of
autophagy. MCF-7 and MDA-MB-231 cells treated with
ESE-15-ol over a 24 h period showed positive
staining for MDC, with more discrete vacuolar formation.
Cells treated with ESE-15-ol in combination with 3MA
showed a decrease in MDC staining when compared to
cells treated with ESE-15-ol alone. This indicates that
3MA blocked autophagy induced by ESE-15-ol, albeit
incompletely. TEM was conducted to investigate the
effects of ESE-15-ol as well as, autophagy inhibition on
the ultrastructure of MCF-7 and MDA-MB-231 cells.
Cells exposed to ESE-15-ol and tamoxifen (positive
control) showed an increased number of vacuoles which are
indicative autophagic vacuoles. Apoptotic bodies were
observed in ESE-15-ol-treated cells, together with
cellular distress, chromatin hypercondensation and
membrane blebbing, indicating cell death via apoptosis. Cells
exposed to ESE-15-ol together with 3MA demonstrated
significantly less distress and a decrease in vacuole
Apoptotic characteristics observed in the morphology
studies of the drug-exposed cells were quantified using
flow cytometry. Analysis of cell cycle progression showed
a statistically significant decrease in mitotic arrest when
MCF-7 and MDA-MB-231 cells were treated with
ESE15-ol with 3MA when compared to ESE-15-ol without
3MA. A statistically significant increase in the G1 phase
was also demonstrated when cells were treated with
ESE15-ol with 3MA when compared to ESE-15-ol without
3MA. Quantification of the translocation of PS to the
outer membrane of the cell during apoptosis was also
done by flow cytometry. ESE-15-ol-exposed cells showed
an increase in apoptosis to 21.21% for MCF-7 cells and
38% for MDA-MB-231 cells with a decrease in cell
viability. Autophagy inhibition demonstrated a decrease in
apoptosis (16.13% for MCF-7 cells and 6.74% for
MDAMB-231 cells). This suggests that 3MA increased cell
viability when combined with ESE-15-ol.
Flow cytometry studies employing LC3 detection
were used to quantify the presence of autophagic
vacuoles. MCF-7 and MDA-MB-231 cells treated with
ESE15-ol showed a significant fold increase in LC3 detection
when compared to the DMSO vehicle control.
Additionally, 3MA inhibition of autophagy were quantified and
verified in tamoxifen-treated cells. The decrease of acidic
vacuoles seen with MDC staining of ESE-15-ol treated
cells with 3MA as well as the TEM findings were
corroborated by the decrease in LC3 detection via flow
cytometry. These results verified that 3MA incompletely blocked
autophagy. LC3 is a marker for autophagic induction or
to detect a decrease in autophagosome formation, but
does however, have limitations. There are multiple
isoforms of LC3 (LC3A, LC3B, LC3B2 and LC3C), as well
as variation in the antibody specificity (LC3I vs LC3II).
ATG8-PE/LC3II is a protein marker which detects
completed autophagosome formation but does not measure
or quantify autophagy flux (flow cytometry indicative of
total LC3 levels and does not differentiate between LC3I
and LC3II). Autophagic flux is often quantified via
LC3II turnover as detected by Western blotting . When
co-incubated with the autophagy inhibitor 3MA, the
decreased LC3II expression in comparison to the
elevated expression induced by ESE-15-ol, alludes to a
disruption in autophagic flux, although these results must
be interpreted with cauation due to the analytic
constraints of autophagy quantification. This study indicated
that the drug treatment did increase the LC3II
expression, a response which was muted by co-incubation with
Autophagosomes are transported to the
microtubuleorganising centre (MTOC) of cells along the microtubules
where lysosome cluster are found . In the MTOC,
lysosome fusion and content exchange occurs after which
autophagosomal degradation by lysosomal hydrolases
take place . It is hypothesised that ESE-15-ol increases
the induction of autophagy, possibly by the increased
formation of reactive oxygen species . Microarray
analysis of autophagy gene expression indicated up-regulation
of autophagy-related genes after exposure to 17-beta
estradiol analogues . However, ESE-15-ol disrupts
microtubule dynamics which are essential for autophagy,
potentially retracting autophagosome fusion and
degradation. A possible explanation for the induction of
autophagy by ESE-15-ol could be the inhibition of
beclin-1. 3MA can possibly increase beclin-1 expression
causing autophagy inhibition. These results support
previous studies done with estradiol analogues and 2ME that
indicate a molecular crosstalk between apoptosis and
autophagy . In addition, the exact effect that 3MA
may have on the cells in the combination treatment would
need to be determined (such as induction of p70S6K
activity ). As inhibitors of PI3K, the loss of this
signalling cascade may have further reaching consequences
than just autophagy inhibition itself. Further investigation
needs to be done to determine whether the increase in
autophagosomes observed during ESE-15-ol exposure is
due to an increase in autophagy induction or due to
abrogated flux (fusion or degradation).
Data from this study supports the concept that the novel
17-β-estradiol analogue, ESE-15-ol, induces both
apoptosis and autophagy in MCF-7 and MDA-MB-231 cells.
Results obtained from the morphological studies
indicated that 3MA incompletely blocked autophagy, and
thereby increased cell survival. Flow cytometry was used
to quantify the autophagy inhibition and increased cell
viability. This in vitro study revealed that
3MA-mediated inhibition of autophagy increased cell viability of
MCF-7 and MDA-MB-231 cells when exposed to
ESE15-ol. Thus it is proposed that the ESE-15-ol-induced
autophagic process in MCF-7 and MDA-MB-231 cells
contributes to the novel compounds’ cytotoxic effect by
inducing programmed cell death type II, as opposed to
conferring resistance to the neoplastic cells to drug
exposure as a mechanism to compensate for cellular distress.
Further quantification of the contribution of autophagy
in cell death induction in response to the novel
compound, as well as its role in possible tumor resistance will
be carried out in future in vitro and in vivo studies.
2-ME: 2-methoxyestradiol; 2MEOE2bisMATE:
2-methoxyestradiol-bissulfamate; 3MA: 3-methyladenine; Akt: protein kinase B; ANOVA: analysis of
variance; Bcl-2: B cell lymphoma 2; CAII: carbonic anhydrase II; CO: colorado;
CO2: carbon dioxide; DMEM: dulbecco’s Minimum Essential Medium Eagle;
DMSO: dimethyl sulfoxide; DNA: deoxyribonucleic acid; DR5: death receptor
5; E2: estradiol; ESE-15-ol:
2-ethyl-3-O-sulpamoyl-estra-1,3,5(10),15-tetraen17-ol; ESE-15-one: 2-ethyl-3-O-sulpamoyl-estra-1,3,5(10),15-tetraen-17-one;
ESE-16: 2-ethyl-3-O-sulphamoyl-etsra-1,3,5 (10) 16-tetraene; FCS: fetal calf
serum; FL1: flow channel 1; FL3: flow channel 3; HepG2: human heptoma
cells; HIF-1α: hypoxia-inducible factor -1α; HT1080: fibrosarcoma cell line;
IG50: half maximum growth inhibitory concentrations; LC3:
microtubuleassociated protein 1A/1B-light chain 3; MA: Massachusetts; MCF-7: human
adenocarcinoma breast cancer cell line; MDA-MB-231: metastatic human
adenocarcinoma breast cancer cell line; MDC: monodansylcadaverine; MTOC:
microtubule-organising centre; mTor: mechanistic target of rapamycin; PI:
propidium iodide; PI3 K: phosphatidylinositide-3-kinase; PI3P:
phosphatidylinositol-3-phosphate; PlasDIC: polarization-optical transmitted light differential
interference light microscopy; PS: phosphatidylserine; SAC: spindle assembly
checkpoint; USA: United States of America.
MV, EN, AMJ and AET conceived and designed the experiments; MV and EN
performed the experiments; MV analyzed the data; AMJ and AET contributed
reagents/materials/analysis tools and supervised the project; MV wrote the
paper. All authors read and approved the final manuscript.
Prof AM Joubert and Dr. AE Theron for general support. This work was
supported by Grants from the Medical Research Council of South Africa, the
Cancer Association of South Africa, National Research Foundation and the
Struwig-Germeshuysen Cancer Research Trust of South Africa.
Availability of data and materials
Please contact author for data requests.
Prof. AM Joubert and Dr. AE Theron for general support. This work was
supported by Grants from the Medical Research Council of South Africa, the
Cancer Association of South Africa, National Research Foundation and the
Struwig-Germeshuysen Cancer Research Trust of South Africa.
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