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Anacardic acid inhibits estrogen receptor alpha-DNA binding and
reduces target gene transcription and breast cancer cell
proliferation
David J. Schultz1,5,7, Nalinie S. Wickramasinghe2,8, Margarita M. Ivanova2,7, Susan M.
Isaacs1,7, Susan M. Dougherty2,8, Yoannis Imbert-Fernandez2,8, Albert R.
Cunningham3,4,6, Chunyuan Chen1,7, and Carolyn M. Klinge2,3,6,8,*
1Department of Biology, Louisville, KY. 40202
2Department of Biochemistry & Molecular Biology, Louisville, KY. 40202
3Department of Medicine, Louisville, KY. 40202
4Department of Pharmacology and Toxicology, Louisville, KY. 40202
5Department of Center for Genetics and Molecular Medicine, Louisville, KY. 40202
6Department of James Graham Brown Cancer Center, Louisville, KY. 40202
7Department of University of Louisville, Louisville, KY. 40202
8Department of University of Louisville School of Medicine, Louisville, KY. 40202
Abstract
Anacardic acid (2-hydroxy-6-alkylbenzoic acid) is a dietary and medicinal phytochemical with
established anticancer activity in cell and animal models. The mechanisms by which anacardic acid
inhibits cancer cell proliferation remain undefined. Anacardic acid 24:1ω5 (AnAc 24:1ω5) was
purified from geranium (Pelargonium × hortorum) and shown to inhibit the proliferation of estrogen
receptor α (ERα)-positive MCF-7 and endocrine-resistant LCC9 and LY2 breast cancer cells with
greater efficacy than ERα-negative primary human breast epithelial cells, MCF-10A normal breast
epithelial cells, and MDA-MB-231 basal-like breast cancer cells. AnAc 24:1ω5 inhibited cell cycle
progression and induced apoptosis in a cell-specific manner. AnAc 24:1ω5 inhibited estradiol (E2)-
induced estrogen response element (ERE) reporter activity and transcription of the endogenous E2-
target genes: pS2, cyclin D1, and cathepsin D in MCF-7 cells. AnAc 24:1ω5 did not compete with
E2 for ERα or ERβ binding, nor did AnAc 24:1ω5 reduce ERα or ERβ steady state protein levels in
MCF-7 cells; rather, AnAc 24:1ω5 inhibited ER-ERE binding in vitro. Virtual Screening with the
molecular docking software Surflex evaluated AnAc 24:1ω5 interaction with ERα ligand binding and
DNA binding domains (LBD and DBD) in conjunction with experimental validation. Molecular
modeling revealed AnAc 24:1ω5 interaction with the ERα DBD but not the LBD. Chromatin
immunoprecipitation (ChIP) experiments revealed that AnAc 24:1ω5 inhibited E2-ERα interaction
with the endogenous pS2 gene promoter region containing an ERE. These data indicate that AnAc
24:1ω5 inhibits cell proliferation, cell cycle progression and apoptosis in an ER-dependent manner
by reducing ER-DNA interaction and inhibiting ER-mediated transcriptional responses.
Request for reprints: Carolyn M. Klinge, Department of Biochemistry & Molecular Biology, University of Louisville School of Medicine,
Louisville, KY 40292. Telephone: 502-852-3668, FAX: 502-852-3659 carolyn.klinge@louisville.edu.
Potential conflicts of interest: none
NIH Public Access
Author Manuscript
Mol Cancer Ther. Author manuscript; available in PMC 2011 March 2.
Published in final edited form as:
Mol Cancer Ther. 2010 March ; 9(3): 594–605. doi:10.1158/1535-7163.MCT-09-0978.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Keywords
anacardic acid; breast cancer; estrogen receptor; computational modeling; endocrine resistance
1. Introduction
Anacardic acid (AnAc) is a mixture of 2-hydroxy-6-alkylbenzoic acid homologs that are
structurally similar to salicylic acid and aspirin (Supplemental Fig. 1A). AnAc is commonly
found in plants of the Anacardiaceae family and is a dietary component found in cashew apple
(Anacardium occidentale) and ginkgo (Ginkgo biloba) leaves and fruits and is found in a
number of medicinal plants that have potential activity against cancer cell lines (1–4). Oral
administration of AnAc to mice had cytotoxic but not genotoxic effects in micronucleus assays
of erythrocytes (5) and AnAc supplied i.p. to mice inhibited the proliferation of implanted
Sarcoma 180 ascites cells (6).
Despite reports demonstrating that AnAc has anti-cancer activity in cell lines (2,4,7) and animal
models (6), its mechanism of action remains largely undefined. AnAc is known to inhibit
histone acetyl transferase (HAT) (8–10); thus, the observed antiproliferative activity may be
associated with chromatin condensation and altered gene transcription. AnAc also induced
apoptosis in chick embryonic neuronal cells, however no direct molecular mechanism was
determined (11). Thus, AnAc has multiple potential molecular targets that are likely cell type
specific as is the case with a variety of natural anti-cancer phytochemicals such as curcumin
(12).
Because AnAc is reported to have higher efficacy in inhibiting the proliferation of breast cancer
cell lines, e.g., MCF-7 and MDA-MB-231, versus cancer cells from other tissues, e.g. lung,
liver, bladder, and melanoma (4,13), we examined the effect of purified AnAc 24:1ω5 on the
proliferation of estrogen- dependent and independent/antiestrogen-resistant breast cancer cells,
primary human mammary epithelial cells (HuMECs), and MCF-10A normal breast epithelial
cells. Our data indicate that AnAc 24:1ω5 inhibits the proliferation of ERα-expressing breast
cancer cell lines, irrespective of endocrine-sensitivity, with greater efficacy than ERα-negative
cells. AnAc 24:1ω5 does not compete with 17β-estradiol (E2) for ER binding. Rather, AnAc
24:1ω5 inhibits ER-estrogen response element (ERE) interaction and inhibits the transcription
of ER target genes.
Materials and methods
Chemicals
E2 and 4-hydroxytamoxifen (4-OHT) were purchased from Sigma-Aldrich (St. Louis, MO).
AnAc 24:1ω5 was purified to greater than 95% (Supplemental Fig. 1B and C), as previously
reported (14). Multiple preparations of AnAc 24:1ω5 were made throughout the course of these
studies and no difference in bioactivities was detected.
Cell lines
HEK-293, MCF-10A, MCF-7, MDA-MB-231 cell lines were purchased from ATCC
(Manassas, VA) and maintained in the recommended media and supplements. MCF-7-LCC9
(LCC9) and MCF-7-LY2 (LY2) cell lines that express ERα but are estrogen/antiestrogen-
resistant were provided by Dr. Robert Clarke, Georgetown University (15). Primary human
mammary epithelial cells (HuMECs) were purchased from Invitrogen (Carlsbad, CA) and
maintained in HuMEC Ready Medium.
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Cell proliferation assays
Cells were plated in 96 well plates in normal growth media and allowed to attach to the plates
overnight. Media was replaced with phenol red-free IMEM supplemented with 3% dextran
coated charcoal stripped FBS (DCC-FBS) for 24 h. AnAc 24:1ω5 at final concentrations of 1
nM – 100 µM was added for 48 h prior to performing the bromodeoxyudridine (BrdU) ELISA
assay (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions.
Within each experiment, treatments were performed in quadruplicate and values were
averaged. At least 3 separate experiments were performed for each cell line. IC50 values were
calculated using GraphPad Prism (San Diego, CA).
Apoptosis assay
Apoptosis was measured using the Cell Death Detection ELISAPLUS (Roche Diagnostics),
which quantitates cytoplasmic histone-associated DNA fragments (mono- and oligo-
nucleosomes) after induced cell death, according to the manufacturer’s instructions. 4-OHT
and doxorubicin served as positive controls for inducing apoptosis in MCF-7 (16) and MDA-
MB-231 (17) cells, respectively. Cells (10,000) were plated in 24-well plates, in triplicate wells
using normal growth media (IMEM containing 5% FBS and pen-strep) and allowed to attach
for 24 h then replaced with medium containing charcoal-stripped serum for 24 h followed by
treatment with the medium alone (control 1, no treatment), medium containing ethanol (control
2, vehicle control), AnAc 24:1ω5 (0.1–50 µM), 4-OHT (100 nM), or doxorubicin (1 µM).
Whole cell extracts (WCE) were prepared after 2 days of treatment.
RNA Isolation, RT-PCR and Quantitative Real-Time-PCR (QRT-PCR)
Cells were plated in 24 well plates at a density of 5×104 cells/well in phenol red-free OPTI-
MEM I reduced serum medium (Invitrogen) supplemented with 10% DCC-FBS, 1% penicillin/
streptomycin and treated with the indicated concentrations of E2 and AnAc 24:1ω5 alone or in
combination for 6 h. RNA was isolated from the cells using Trizol (Invitrogen). The High
Capacity cDNA archive kit (PE Applied Biosystems, Foster City, CA) was used to reverse
transcribe total RNA from random hexamer primers. Taqman primers and probes for
CCND1 (cyclin D1), TFF1 (pS2), and CATD1 (cathepsin D1), and 18S rRNA were purchased
as Assays-on-Demand™ Gene Expression Products from PE Applied Biosystems. The
expression of each target gene was determined in triplicate in 3 separate experiments and
normalized using 18S. QRT-PCR was performed in the ABI PRISM 7900 SDS 2.1 (PE Applied
Biosystems) using relative quantification. Analysis and fold differences were determined using
the comparative CT method. Fold change was calculated from the ΔΔCT values with the
formula 2−ΔΔCT and data are presented as relative to expression in EtOH-treated cells, i.e.,
vehicle control.
Transient transfection assays
For transient transfection, HEK293 or MCF-7 cells were plated as described above. Transient
transfections were performed using FuGENE 6 (Roche Diagnostics). Each well received 250
ng of a pGL3-pro-luciferase reporter (Promega, Madison, WI) containing 2 tandem copies of
a consensus estrogen response element (ERE, i.e., EREc38 (18)) and 5 ng of a Renilla luciferase
reporter (pRL-TK) from Promega. In addition, HEK293 cells were cotransfected with either
pCMV-rhERα or pSG5-rhERβ (provided by Dr. Benita S. Katzenellenbogen (19) and Dr. Eva
Enmark (20), respectively). Twenty-four h after transfection, triplicate wells were treated with
EtOH (vehicle control), E2, AnAc 24:1ω5 or E2 and AnAc 24:1ω5 simultaneously. The cells
were harvested 30 h post-treatment using Promega’s Passive Lysis buffer. Luciferase and
Renilla luciferase activities were determined using Promega’s Dual Luciferase assay in a Plate
Chameleon luminometer (BioScan, Washington, D.C.). Firefly luciferase was normalized by
Renilla luciferase to correct for transfection efficiency. Values were averaged from multiple
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experiments as indicated in the figure legends and are normalized against vehicle control
(EtOH).
Competition [3H]E2 binding assay
The ability of increasing concentrations of purified AnAc 24:1ω5 to compete with [3H]E2 for
specific binding to baculovirus-expressed recombinant human (rh) ERα and rhERβ (ERβ1)
that was N-terminal FLAG-tagged (21,22) was measured by ER adsorption to hydroxyapatite
(HAP) as previously described (21).
Electrophoretic mobility shift assays (EMSA)
EMSA experiments were performed using baculovirus expressed rhERα and rhER β (ERβ1
that was N-terminal FLAG-tagged) were quantified in a Packard Instruments Instant Imager
and with Packard Imager for Windows v2.04 as previously described (23). Reactions included
1.2 nM ERα or 1.5 nM ERβ, 1.1 nM [32P]EREc38 (5′-
CCAGGTCAGAGTGACCTGAGCTAAAATAACACATTCAG-3′) (24) 10 nM E2, and 1–5
µM AnAc 24:1ω5. The concentrations of free and ER-bound [32P]EREc38 were fit to the one-
site binding model (determination coefficient R2 > 0.93 and 0.98 without ligand and 0.97 and
0.94 with ligand for ERα and ERβ, respectively). Antibodies used in the supershift lanes to
demonstrate the specificity of ER-ERE interaction were G20 (Santa Cruz Biotechnology, Santa
Cruz, CA) and FLAG (Sigma-Aldrich). The IC50 was determined from the Pseudo-Hill plot:
log %/(100−%)=nlog([I] + nlogIC50), where % = percent competition of specific binding,
I=competitor.
ER protein stability and western blot
The effect of AnAc 24:1ω5 and EtOH (vehicle control) on steady state levels of ERα and
ERβ was determined by western blot analysis. MCF-7 cells were seeded into a 10-cm tissue
culture dish in phenol red-free IMEM with 10% DCC-FBS. After 24 h incubation, the same
media containing 10 µM AnAc 24:1ω5 or EtOH was added and cells were harvested after the
indicated times. WCE were prepared and equal amounts of protein, as determined in Bio-Rad
DCC protein assay, were separated by 10% SDS-PAGE. Proteins were transferred to PVDF
membranes for western analysis and data quantified as previously described (25). The
following antibodies were used: HC-20 (ERα) and H150 (ER β) from Santa Cruz
Biotechnology, ERβ from Upstate, AER320 (ERα) from Thermo Scientific, and for
normalization β-actin from Sigma.
Chromatin Immunoprecipitation (ChIP) Assay
MCF-7 cells were transferred to phenol red-free IMEM with 10% DCC-FBS (‘starve medium’)
for 72 h and then treated with 2.5 µM α-amanitin for 2 h. Following three washes in 1X PBS,
the cells were treated with EtOH (vehicle), 10 nM E2, 10 µM AnAc 24:1ω5, or the combination
of E2 and AnAc 24:1ω5, in ‘starve’ media for 20 min. ChIP assays were performed using the
ChIP Assay Kit (USB Corporation, Cleveland, OH) according to the instructions supplied.
Chromatin was cross-linked using 1.5% formaldehyde for 10 min at 37°C and the cells were
collected after 2 washings with PBS. Subsequent chromatin fragmentation and pre-clearing of
chromatin suspensions were completed prior to incubation of the cell extracts with either anti-
ERα antibody (HC-20) or normal rabbit IgG (both from Santa Cruz Biotechnology). After
elution of the antibody-protein complexes using the kit-supplied reagents, the DNA was
purified using the Qiagen PCR Clean-Up Kit (Qiagen, Valencia, CA).
QRT-PCR with ChIP Samples
QRT-PCR was performed using 3 µL of the purified, immunoprecipitated DNA and probed
for pS2 (Trefoil Factor 1; TFF1) using primers flanking the established ERE in the human pS2
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gene promoter (26) and SYBR Green Master Mix (SABiosciences, Frederick, MD). The data
were calculated as described in (27). The average CT values of the input samples (prior to IP)
were subtracted from the average CT values for the ERα antibody immunoprecipitated (IP)
value to obtain the net CT value which was subtracted from the control (IgG) CT value. Relative
promoter enrichment was compared with IgG (28) and expression of the pS2 gene was
expressed relative to EtOH. The pS2 PCR products in representative wells from each treatment
group were separated on a 1.5% agarose gel for visualization of the amplified products. A 1
kb DNA ladder (Promega) was run in parallel with the samples.
Statistical analyses
Student’s t-test or one-way ANOVA followed by Dunn’s multiple comparison or Dunnett’s
post-hoc test were performed with GraphPad Prism (San Diego, CA.).
Molecular modeling
Surflex 2.3 docking module (Surflex-dock) running under Sybyl 8.1 was used to determine the
interaction potential of AnAc 24:1ω5 and several other small reference molecules to the ligand
binding domain (LBD) and DNA binding domains (DBD) of ERα. Surflex-dock GeomX
parameters were selected. Protein Data Bank (PDB) structure 1ERE (29) was used as a
representative ERα LBD structure and 1HCQ (30) was used for the ERα DBD structure. Note
that the crystal structure of the ERα DBD is bound as a homodimer to a consensus estrogen
response element (ERE) palindrome. The ligand binding site for the LBD structure of ERα was
determined by Surflex-dock protomol generation in “ligand mode” with “1ERE A-chain” (the
ERα LBD) and E2. The potential ligand interaction site for the ERα DBD was determined by
Surflex-dock protomol generation in “automatic mode”, i.e. the ERα DBD was not co-
crystallized with a ligand, using ERα DBD (1HCQ) A and B chains with associated Zn atoms.
Sybyl’s Biopolymer Structure Preparation Tool was used to prepare both PDB files for virtual
docking. AnAc 24:1ω5 (2-hydroxy-6-alkylbenzoic acid), aspirin (acetylsalicylic acid), salicylic
acid, E2, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) used for docking were initially
minimized and charged using Sybyl 8.1 MMFF94 force field.
Surflex-dock returns an affinity score reported as −log(Kd) that takes into account
hydrophobic, polar complimentarity, entropic, and solvation terms (31) and a “crash” score
that represents “inappropriate” penetration of a potential ligand into a binding site (32). For
these analyses, crash scores >2 units (indicating an unfavorable protein-ligand interaction),
were used to reject compound interaction even with high estimated affinity scores to limit false
positive predictions of protein-ligand interactions.
Results
AnAc inhibits normal or breast cancer cell proliferation in accordance with ERα status
The effect of AnAc 24:1ω5 on the proliferation of primary human mammary epithelial cells
(HuMECs), one normal breast epithelial and 4 breast cancer cell lines differing in their ER
status and/or sensitivity to the antiestrogens tamoxifen (TAM) and ICI 182,780 (15) was
evaluated by BrdU incorporation (Fig. 1 and Supplemental Figs. 2 and 3). MCF-7 cells
responded proliferatively to E2 while all other cell lines were non E2-responsive, regardless of
ER status, consistent with previously published reports (25,33). As expected, MCF-10A cells
and the TAM-resistant LCC9 and LY2 cells showed no inhibition by 4-OHT whereas E2-
induced proliferation in MCF-7 cells was significantly reduced by 4-OHT. AnAc 24:1ω5 dose-
response curves (Supplemental Fig. 3) indicated ERα positive cell lines are inhibited to a greater
extent with IC50 values ~2- to 6.6-fold lower than cell lines that are ERα negative. In all cell
lines, 50 µM AnAc 24:1ω5 was more effective at inhibiting BrdU incorporation than 100 nM
4-OHT, regardless of TAM-sensitivity (Fig 1), and inhibition by 50 µM AnAc 24:1ω5 was not
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reversed by E2 or 4-OHT. Importantly, AnAc 24:1ω5 did not inhibit the proliferation of ERα-
negative (34) primary HuMECs (Supplemental Fig. 2).
AnAc stimulates apoptosis in breast cancer cell lines
To determine the phase of the cell cycle at which AnAc 24:1ω5 exerts its growth-inhibitory
effect, MCF-7, LY2, and MCF-10A cells were subjected to FACS analysis (Fig. 2A). AnAc
24:1ω5 inhibited cell cycle progression from the G1 phase of the cell cycle in MCF-7 and LY2
cancer cell lines but not in normal MCF-10A cells. Approximately 80% of MCF-7 and LY2
cells were in the G1 phase after 24 h of AnAc 24:1ω5 treatment in comparison to only 60% of
control cells observed to be in G1 after 24 h (Fig. 2A).
Since AnAc 24:1ω5 resulted in more growth inhibition in MCF-7 than MDA-MB-231 cells,
we examined the relative induction of apoptosis in these two cell lines using 4-OHT and
doxorubicin as positive controls, respectively. AnAc 24:1ω5 induced a concentration-
dependent increase in apoptosis in both cell lines with a greater impact on MCF-7 cells (Fig.
2B), results in concordance with the greater inhibition of cell proliferation in MCF-7 cells
(Supplemental Fig. 3).
AnAc inhibits ER-induced gene transcription
Because AnAc 24:1ω5 showed greater efficacy in inhibiting the proliferation of ERα-
expressing breast cancer cells (Fig. 1, Supplemental Figs. 2 and 3), the effect of AnAc
24:1ω5 on the transcription of established endogenous E2-target genes: cyclin D1 (CCND1,
regulates G1 cell cycle progression), Cathepsin D (CTSD, a lysosomal protease involved in
breast cancer metastases), and pS2 (TFF1, a well-established E2-responsive breast cancer
marker gene of unknown function (35)) was evaluated by QRT-PCR (Fig. 3A–C). CCND1
was increased ~3.5-fold in E2-treated MCF-7 cells but was unaffected by E2 in LCC9 or LY2
cells, in agreement with the endocrine-resistant status of these ERα-positive cells (25). In
contrast, 20µM AnAc 24:1ω5 reduced CCND1 to below basal levels in MCF-7 and LCC9 cells,
whereas CCND1 in LY2 cells was slightly, but statistically significantly, increased. Co-
treatment with E2 and 40 µM AnAc 24:1ω5 reduced CCND1 transcript levels to or below basal
in all ERα-positive cell lines. As anticipated, E2 did not increase CCND1 levels in ERα-
negative MCF-10A or MDA-MB-231 cell lines. AnAc 24:1ω5, individually or in combination
with E2, reduced CCND1 to below basal in MCF-10A or MDA-MB-231 cells (Fig. 3A).
E2 increased CTSD expression in MCF-7 and LCC9, while expression in LY2 cells was
unaffected (Fig. 3B). AnAc 24:1ω5 (20 µM) reduced CTSD transcript levels below basal in
MCF-7 but increased CTSD expression in LCC9 and LY2 cells. In combination with E2, all
concentrations of AnAc 24:1ω5 reduced CTSD expression in MCF-7 and 20 and 40 µM AnAc
24:1ω5 reduced CTSD in LY2 cells whereas only 40 µM AnAc 24:1ω5 inhibited CTSD
expression in LCC9 cells (Fig. 3B). As anticipated, E2 did not increase CTSD levels in ERα-
negative MCF-10A or MDA-MB-231 cell lines. AnAc 24:1ω5, alone or in combination with
E2, reduced CTSD below basal levels in MCF-10A and MDA-MB-231 cells (Fig. 3B).
Transcript levels of pS2 (TFF1) were increased by E2 in MCF-7 cells while 20µM AnAc
24:1ω5 reduced transcript levels to below basal (0.2-fold) (Fig. 3C). As anticipated, based on
the tamoxifen/endocrine resistance of these cells (25), TFF1 was not detected in LCC9 or LY2
cells data not shown). The TFF1 expression pattern in MCF-7 cells treated simultaneously
with 10 nM E2 and AnAc 24:1ω5 (10, 20 and 40 µM) largely mirrored the pattern observed for
CCND1 expression with a concentration-dependent inhibition of E2-dependent transcription
(Fig. 3C).
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AnAc inhibits ERα- and ERβ-ERE reporter gene transcription
To directly assess the effect of AnAc 24:1ω5 on the transcriptional activity of each ER subtype
(ERα or ERβ), HEK293 cells were transfected with either ERα or ERβ expression plasmids
and an ERE-driven luciferase reporter (Fig. 3D). As expected, E2 increased luciferase activity
for both ERα and ERβ. AnAc 24:1ω5 (alone) did not produce a clear concentration-dependent
response in ER-transfected HEK293 cells; however, a modest, but significant, agonist activity
was apparent at some concentrations, although most concentrations tested were not
significantly different from control. Treatment of HEK293-ERα with E2 in combination with
lower concentrations of AnAc 24:1ω5 (1 and 10 µM) showed no significant difference relative
to E2 alone. However, ≥25 µM AnAc 24:1ω5 inhibited E2-induced reporter activity. Similar
results were seen for HEK293-ERβ except that 50 and 75 µM AnAc 24:1ω5 reduced luciferase
below basal (Fig. 3D). These data indicate that AnAc 24:1ω5 inhibited E2-mediated ERα and
ERβ transcriptional activity. For comparison, the effect of AnAc 24:1ω5 on endogenous ER
activity was examined in MCF-7 cells transiently transfected with the same ERE-luciferase
reporter (Fig. 3D). At the lowest concentrations tested (0.1–10 µM), AnAc 24:1ω5 alone had
no effect on luciferase activity, but at 25 and 50 µM, luciferase activity was completely
inhibited. Co-treatment of MCF-7 cells with 10 nM E2 and AnAc 24:1ω5 resulted in a
concentration-dependent inhibition of E2-mediated reporter activity. These data indicate a
greater sensitivity of MCF-7 cells to AnAc 24:1ω5 inhibition of E2-induced ERE-driven
reporter activity compared to HEK-293 transfected with ERα or ERβ. Further, these data
correlate with the inhibition of endogenous E2-activated gene transcription in MCF-7 cells
(Fig. 3A–C). AnAc 24:1ω5 was more efficacious in inhibiting the ERE-luciferase activity
compared to endogenous gene transcription (Fig. 3), likely reflecting the lack of mature
chromatin structure on the transfected ERE-luciferase plasmid or other factors such as
differences in molar ratio of ER and AnAc 24:1ω5 in each assay.
AnAc 24:1ω5 does not compete with E2 for the ligand binding site of ERα or ERβ, but does
inhibit ER-ERE binding in vitro
Two approaches (ligand binding and DNA binding assays) were used to better define the
interactions between AnAc 24:1ω5 and each ER subtype in vitro. Competition [3H]E2 ligand
binding assays were performed using baculovirus-expressed human ERα or ERβ. Notably,
AnAc 24:1ω5 did not compete with [3H]E2 for binding either ERα or ERβ (Fig. 4A and B),
indicating that AnAc 24:1ω5 does not interact directly with the ligand binding pocket of either
ER subtype.
The effect of AnAc 24:1ω5 on ER binding to a consensus ERE sequence was examined by
EMSA (Fig. 4C and D, Supplemental Fig. 4). The ERE binding of both ER subtypes was
inhibited by AnAc 24:1ω5 in a concentration-dependent manner. Salicylic acid did not inhibit
ER-ERE binding (Supplemental Fig. 4C, and data not shown). Based on IC50 values
(Supplemental Table 1), ERα-ERE binding was more strongly inhibited by AnAc 24:1ω5 than
was ERβ-ERE binding. Together the ligand binding assay and EMSA data indicate that AnAc
24:1ω5 inhibits ER-ERE binding without affecting ligand binding.
AnAc 24:1ω5 inhibits E2- ERα interaction with the pS2 gene promoter in MCF-7 cells
Since AnAc 24:1ω5 inhibited transcription of E2 dependent genes and ER/ERE interactions
(Fig. 3), ChIP assays were used to evaluate whether AnAc 24:1ω5 inhibits E2-induced ERα
interaction with the ERE-containing, E2-regulated, human pS2 (TFF1) (36) gene promoter in
vivo. ERα-specific antibody or IgG (negative control) were used to immunoprecipitate protein
DNA complexes from whole cell extracts of MCF-7 cells treated with EtOH, 10 nM E2, 10
µM AnAc 24:1ω5, or both E2 and AnAc 24:1ω5. QRT-PCR was performed on the ChIP samples
to examine the enrichment of the pS2 promoter by ERα. In agreement with previous reports,
QRT-PCR demonstrated that E2-induced ERα occupancy of the pS2 promoter (Fig. 5A). As
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anticipated based on gene transcription data (Fig. 3C), co-treatment of MCF-7 cells with E2
and AnAc 24:1ω5 blocked E2-induced ERα recruitment (Fig. 5A and 5B). We conclude that
AnAc 24:1ω5 inhibits E2-ERα-DNA interaction on the pS2 promoter in MCF-7 cells.
AnAc does not reduce steady-state protein levels of ERα or ERβ
ERα ligands impact ERα protein stability (37,38). An alternative explanation for the observed
reduction in E2-activated gene transcription by AnAc 24:1ω5 could be reduced ER protein
levels. The effect of 10 µM AnAc 24:1ω5 steady state protein levels of ERα and ERβ was
evaluated by western blotting with two different antibodies for each ER subtype (Fig. 5C and
D). There was no statistical difference in ERα or ERβ protein expression over the 12 h time
course that was selected to parallel gene transcription (Fig. 3) and ChIP (Fig. 5A and B) studies.
These data indicate that AnAc 24:1ω5 does not cause a rapid reduction in ER protein.
Molecular modeling of AnAc 24:1ω5 interaction with ERα
Molecular modeling approaches were used to assess the potential interactions of ERα with
AnAc 24:1ω5 and structurally similar molecules (aspirin and salicylic acid, Supplemental Fig.
1), as well as with known positive and negative controls for the ligand binding domain (LBD)
and/or the DNA binding domain (DBD) of ERα. To validate that Surflex will detect high
affinity E2-ERα LBD interaction, Surflex-docking experiments were performed and the natural
ligand E2 was successfully docked to the ERα LBD with an affinity score of 7.17 and a crash
score of only −0.79. Aspirin and Salicylic acid were estimated to bind to the ERα LBD with
apparent lower affinities of 4.45 and 3.59 respectively (with crash scores of −1.20 and −0.32).
TCCD (2,3,7,8-tetrachlorodibenzo-p-dioxin) functioned as our negative control since it is not
known to bind ERα (39). TCCD was estimated to have a score of −0.03 and a crash score of
−1.86 (i.e., no affinity) for the ERα LBD. AnAc 24:1ω5 was estimated to have a 9.05 affinity
score for the ERα LBD. However, the accompanying crash score of −5.36 indicates a high
degree of inappropriate ligand-protein interactions and thus allows the conclusion that
AnAc24:1ω5 is not an ERα ligand, consistent with HAP assay results showing no competition
of AnAc 24:1ω5 with [3H]E2 for ERα or ERβ in vitro (Fig. 4A).
When modeling the ERα DBD as a potential target for small molecule interactions, aspirin and
salicylic acid had low affinity (3.91 and 4.05, with crash scores of −0.48 and −0.34 respectively)
for the ERα DBD and TCDD again had almost no affinity (value of only 1.31 with crash score
of −0.33). Remarkably, AnAc 24:1ω5 was found to have an affinity value of 8.01 units and a
crash score or only −1.28 for the ERα DBD, indicating that AnAc 24:1ω5 may interact directly
with the DBD and thus interfere with the ER’s ability to interact with an ERE. Visualization
of AnAc 24:1ω5 docked to the ERα DBD reveals that the compound lies between the Zn fingers
(Fig. 6A) and traverses from one side of the protein to the other (Fig. 6B). Since the structurally
similar aspirin and salicylic acid did not yield comparable modeling results, it appears the alkyl
chain of AnAc 24:1ω5 may be an important factor, in combination with the salicylic ring
structure, for ERα DBD interaction. More complete structure-activity relationship studies are
needed to fully address this suggestion.
Discussion
Tamoxifen/endocrine resistance is a major problem in the treatment of breast cancer patients
(40). Here we demonstrate that AnAc 24:1ω5 displayed greater efficacy in inhibiting the
proliferation of ERα-expressing breast cancer cells, regardless of endocrine/tamoxifen-
sensitivity, compared to ERα-negative primary HuMECs, normal MCF-10A breast epithelial
cells, or MDA-MB-231 breast cancer cells. AnAc 24:1ω5 inhibited cell cycle progression and
induced apoptosis in an ERα-dependent manner, consistent with inhibition of cell proliferation
in these cell lines. Furthermore, AnAc 24:1ω5 inhibits ER-ERE binding without affecting
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ligand binding and AnAc 24:1ω5 displays selectivity in inhibiting ERα- over ERβ- ERE binding
in vitro. This result is further supported by in vivo ChIP assays demonstrating that AnAc
24:1ω5 inhibits E2-induced ERα occupancy of the endogenous, ERE-containing promoter of
the TFF1 (pS2) gene in MCF-7 breast cancer cells. Since E2-induced breast cell proliferation
is mediated by ERα activation (41), these data provide a possible mechanism, i.e., inhibition
of DNA binding, to explain the greater inhibition of ERα-expressing breast cancer cell
proliferation by AnAc 24:1ω5 compared to HuMECs, MCF-10A, and MDA-MB-231 cells.
Our data showing greater AnAc 24:1ω5 inhibition of ERα positive breast cancer cell
proliferation combined with an absence of AnAc 24:1ω5 -ER ligand domain binding, inhibition
of ERE binding in vitro, inhibition of ERα-interaction with an endogenous target gene
promoter, and inhibition of both ERE-reporter and endogenous E2-regulated gene transcription
cumulatively indicate a potential interaction of AnAc 24:1ω5 with another site on ER that
modulates E2-activation, e.g., the DBD. This assertion is supported by the virtual molecular
docking experiments wherein AnAc 24:1ω5 was estimated to have a relatively high affinity for
the ERα DBD and no affinity for the ERα LBD. The computational modeling is supported by
in vitro EMSA data confirming DBD interference (Fig. 4 C and D), the lack of LBD interaction
detected in the E2 binding competition assays (Fig. 4 A and B), and the ChIP data showing
that treatment of MCF-7 cells with AnAc 24:1ω5 blocked E2-induced ERα occupancy of the
endogenous pS2 gene promoter in MCF-7 cells. Together, based on our in vitro, ChIP, and in
silico experimental data, we suggest that the molecular mechanism by which AnAc 24:1ω5
preferentially inhibits the cell proliferation of ERα positive breast cancer cell lines is by
interfering with ER-DNA interactions.
The fact that AnAc 24:1ω5 inhibited ERα-negative MDA-MB-231 and MCF-10A cell
proliferation, although with reduced efficacy, also indicates that AnAc24:1ω5 acts through
ERα-independent mechanisms. Both MDA-MB-231 and MCF-10A express ERβ (42), and
given the high homology between the DBDs of ERα and ERβ (43), it is possible that AnAc
would also interact with the ERβ DBD. Since the crystal structure of the ERβ DBD has not
been examined, this possibility cannot be tested in Surflex. Alternatively, AnAc has been
reported to inhibit HAT activity in vitro (8–10). Many transcription factors, including ER,
recruit coactivators with HAT activity to initiate gene transcription (44). Thus, inhibition of
HAT activity by AnAc may reduce the expression of genes required for cell proliferation.
Interestingly, a series of substituted phenoxyacetic acid ethyl esters, structurally related to
AnAc, were shown to inhibit MCF-7 cell proliferation and this was correlated with HAT
inhibition in vitro (45). Thus, the potential inhibition of HAT activity by AnAc 24:1ω5 would
fit the inhibition of basal cyclin D1 expression that we observed (Fig 3 A).
AnAc 24:1ω5 inhibited E2-induced endogenous cyclin D1 (CCND1) transcription in MCF-7
cells. Cyclin D1 is a well established ERα genomic target (46) involved in cell cycle progression
(47). Based on our EMSA and molecular modeling data, we suggest that the inhibition of E2-
induced CCND1 transcription by AnAc 24:1ω5 may result from the direct interaction of AnAc
24:1ω5 with the DBD of ERα that could prevent ERα interaction with a 3′ flanking region
(46). However, because E2-ERα regulates cyclin D1 transcription via multiple mechanisms
including tethering of ERα with AP-1 (48), the precise mechanism of inhibition remains to be
established. Likewise, the inhibition of E2-induced endogenous CTSD (cathepsin D1)
transcription in MCF-7 and LCC9 cells may not be due to blocking direct ER-DNA interaction
since transcription is mediated by ERα-Sp1 interaction at GC-boxes in the CTSD promoter
(49). It is possible that AnAc 24:1ω5 interaction with the DBD could impact ERα-Sp1
interaction since deletion studies indicated the importance of the ERα DBD for ligand-activated
Sp1 interaction (50). Notably, AnAc 24:1ω5 suppressed basal cathepsin D transcription in LY2
cells, a promising result given the greater endocrine-resistance in LY2 cells compared to LCC9
cells (25). The apparent biphasic effect of AnAc 24:1ω5 in the ERE-luciferase assay in ER-
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transfected HEK-293 cells is similar to that for other natural ER inhibitors, e.g., apigenin,
although apigenin acts by a different mechanism than AnAc 24:1ω5, i.e., apigenin induces
ERα degradation (37), which AnAc 24:1ω5 does not.
In conclusion, our data provide a mechanism to account for the observation that breast cancer
cells expressing ERα are more than twice as sensitive to inhibition by AnAc 24:1ω5 regardless
of their endocrine/tamoxifen-sensitivity. AnAc 24:1ω5 may preferentially inhibit ERα positive
breast cancer cell proliferation by direct ER DBD interaction. The fact that AnAc 24:1ω5
inhibits the proliferation of estrogen-dependent and -independent breast cancer cells, but not
primary HuMECs, is an indication that the distinct mode(s) of AnAc 24:1ω5–mediated
inhibition might be further therapeutically exploited.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Abbreviations
AnAc anacardic acid
ER estrogen receptor
E2estradiol
EMSA electrophoretic mobility shift assays
ERE estrogen response element
4-OHT 4-hydroxytamoxifen
ChIP Chromatin Immunoprecipitation
DBD DNA binding domain
LBD ligand binding domain
HAT histone acetyl transferase
PDB Protein Data Bank
TAM tamoxifen
Acknowledgments
We thank Dr. Kathleen A. Mattingly and Emily Darling for performing some of the experiments included here and
Christopher A. Worth of JG Brown Cancer Center Core Sorting Facility for FACS.
Financial support: This work was supported by NIH RO1 DK 53220 and Susan G. Komen For the Cure grants
BCTR0201438 and KG072365 to CMK; and by Intramural Research Incentive Grants from the Office of the Senior
Vice President for Research to CMK and DJS. KAR was supported pre-doctoral fellowships from NIH/NIEHS T32
ES011564. SMI was supported by a summer fellowship from NIH R25CA044789. ARC was supported by NIH P20
RR018733 and the Congressionally Directed Medical Research Program for Breast Cancer Idea Award
W81XWH-05-1-0236.
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Fig. 1.
AnAc 24:1ω5 inhibits the proliferation of human breast cancer cells. MCF-10A normal,
immortalized, breast cells and MCF-7, LCC9, LY2, and MDA-MB-231 breast cancer cells
were grown in the presence of 10 nM E2, 100 nM 4-OHT, or 50 µM AnAc 24:1ω5, alone or in
combination, as indicated, for 48 h prior to examining BrdU incorporation as described in
Materials and Methods. Values are the mean ± SEM of 3–5 independent experiments in which
each treatment within that experiment was performed in quadruplicate. Treatments that were
significantly different (P<0.05) from EtOH control are designated (a) and treatments in
combination with E2 that were significantly different (P<0.05) from E2 alone are designated
(b).
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Fig. 2.
AnAc 24:1ω5 inhibits cell cycle progression and stimulates apoptosis. FACS analysis (A) was
used to determine the distribution of cells in G1-, S-, and G2/M- phases of the cell cycle in
MCF-7, LY2 and MCF-10A breast cell lines. Cells were treated with EtOH, 10 nM E2, 100
nM 4-OHT or 20 µM AnAc 24:1ω5 alone or in combination as indicated and described in
Materials and Methods. For apoptosis assays (B), MDA-MB-231 and MCF-7 cells were
incubated with the indicated concentrations of AnAc 24:1ω5 or, as positive controls, 100 nM
4-OHT for MCF-7 and 1 µM doxorubicin for MDA-MB-231 for 48 h. Apoptosis was evaluated
by an Elisa kit that measures histone-associated DNA fragments in mono- and
oligonucleosomes as an index of relative apoptosis as described in Materials and Methods.
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Values are the mean of quadruplicate determinations ± SEM. * Significantly different from
the EtOH control, p < 0.05.
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Fig. 3.
AnAc 24:1ω5 inhibits E2-induced target gene transcription. To measure endogenous gene
transcription, MCF-7, LCC9, LY2, MCF-10A, and MDA-MB-231 cells were serum-starved
for 48 h and then treated for 6 h with EtOH, 10nM E2, and 10, 20, or 40 µM AnAc 24:1ω5
alone or in combination as indicated and as described in Materials and Methods. RNA levels
of the target genes CCND1 (cyclin D1, A), CATD (cathepsin D, B) and TFF1 (pS2, C) were
analyzed by real-time QRT-PCR as described in Materials and Methods. For MCF-10A and
MDA-MB-231, CCND1 basal expression was ~25- and 114- fold higher than MCF-7 cells,
respectively. For LY2, MCF-10A, and MDA-MB-231, CTSD basal expression was ~2-, 172-,
and 138- fold higher than MCF-7 cells, respectively. No TFF1 expression was detected in cell
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lines other than MCF-7 (C). The effect of AnAc 24:1ω5 on each ER subtype (D) was examined
in HEK-293 cells that were co-transfected with ERα (top) or ERβ (middle) in addition to an
ERE-luciferase reporter and pRL-TK as described in Materials and Methods. MCF-7 cells
(bottom) were transfected with the same ERE-luciferase reporter and pRL-TK as described in
Materials and Methods. Twenty-four hours after transfection, the cells were treated with
ethanol (EtOH), 10 nM E2 or the indicated concentrations of AnAc 24:1ω5 alone (solid lines,
open squares) or in combination with 10 nM E2 (dashed lines, filled circles). Dual luciferase
activity was assayed as described in Materials and Methods. Data are displayed as relative
luciferase activity (fold difference) in which the EtOH activity was set to 1. For all panels, data
are the mean ± SEM from 3 separate experiments. Values that were significantly different
(P<0.05) from EtOH control are designated (a) and values from combined treatments that were
significantly different (P<0.05) compared to E2 alone are designated (b).
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Fig. 4.
AnAc 24:1ω5 does not compete with [3H]E2 for binding ERα or ERβ, but inhibits ERE binding.
Ligand binding assays (A and B) utilized baculovirus expressed human ERα or ERβ incubated
with [3H] E2 and the indicated concentrations of E2 or AnAc 24:1ω5. [3H]E2 specific binding
was determined by HAP assay as described in Materials and Methods. Values are the average
± SEM of triplicates. EMSA assays utilized baculovirus-expressed ERα (C) and ERβ (D)
incubated with [32P]-labeled EREc38 in the absence (no ligand) or presence of E2 (with ligand),
plus increasing concentrations of AnAc 24:1ω5 as indicated. EMSA was performed as
described in Materials and Methods. An antibody against either ERα (G20 in C) or FLAG (used
to detect FLAG-ERβ in D) was added to the indicated reaction mixtures to confirm the
specificity of the retarded ER-ERE complex. SS = supershift of the ER-ERE with the indicated
ERα or FLAG antibodies.
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Fig. 5.
AnAc 24:1ω5 inhibits E2-ERα occupancy of the pS2 (TFF1) gene promoter in MCF-7 cells
and does not accelerate ERα or ERβ protein degradation. MCF-7 cells were treated with EtOH,
10 nM E2, 10 µM AnAc 24:1ω5, or 10 nM E2 plus 10 µM AnAc 24:1ω5 for 20 min and ChIP
assays were performed as described in Materials and Methods. QRT-PCR (A) was performed
for ERα occupancy on the pS2 ERE in ChIP samples and calculation of relative promoter
enrichment was described in Materials and Methods. Values are the average ± std of two
separate experiments. PCR products of pS2 reactions (B) from the input or indicated ChIP
assay samples were separated on a 1.5% agarose gel and visualized by EtBr staining. To
examine ERα (C) and ERβ (D) protein stability, MCF-7 cells were treated with 10 µM AnAc
24:1ω5 or EtOH for the indicated times. WCE were separated by SDS PAGE and
immunoblotted for ERα or ERβ using two different subtype-specific antibodies for each ER
subtype. Blots were stripped and reprobed with β-actin for normalization and the bar graphs
show the average of two replicate experiments ± std. No statistical differences were determined.
Arrows (D) indicate the expected 50 kDa band of ERβ.
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Fig. 6.
Surflex-dock depiction of PDB structure of AnAc 24:1ω5 docking with the ERα DBD. In each
panel, the structure on left shows original PDB structure and the one on the right illustrates
AnAc 24:1ω5 bound to the DNA binding domain (DBD) as side view (A) and top view (B),
i.e., looking through ERα DBD protein to the DNA.
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