Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
Z. Bai and R. Gust
133
Review
Breast Cancer, Estrogen Receptor and Ligands
Zhenlin Bai and Ronald Gust
Institute of Pharmacy, Freie Universitt Berlin, Berlin, Germany
This review emphasizes the relationship of breast cancer, estrogen receptor and ligands, especially the centrality of the estrogen receptor, which mediates on one hand the hormone-induced
gene transcription and on the other hand the anti-estrogen action against breast cancer. The
characterization of the estrogen receptor ligand-binding domain co-crystallized with agonists or
antagonists provided a molecular basis to gain an insight into the regulation of estrogen receptor and, thereby, to describe the mechanism of the hormone therapy in treating breast cancer.
Keywords: Anti-estrogen action / Breast cancer / Estrogen receptor / Ligand / Estrogen action /
Received: June 6, 2008; accepted: September 22, 2008
DOI 10.1002/ardp.200800174
Introduction
The discovery of the estrogen receptor b (ER)b and the
determination of the crystal structures of ligand-ER
ligand-binding domain (LBD) complexes belonged to the
most valuable developments in the area of ERs in the last
decade and facilitated greatly the investigation of hormone action through the ER. Therefore, plenty of new
information was achieved and some of the previous
knowledge must be expounded in a new way; there is
clearly inspiring information dealing with this topic. The
aim of this review is to systematically summarize the
most important results in a paper as short as possible.
Yet, reading the review saves a lot of time and one can
easily study these fascinating research achievements
Correspondence: Ronald Gust, Institute of Pharmacy, Freie Universitt
Berlin, Knigin-Luise-Strasse 2 + 4, D-14195 Berlin, Germany.
E-mail: rgust@zedat.fu-berlin.de
Fax: +49 30 838-56906
Abbreviations: (trans)activation function (AF); diethylstilbestrol (DES);
DNA-binding domain (DBD); 17b-estradiol (E2); estrogen receptor (a/b)
(ER(a/b)); estrogen response elements (ERE); genistein (GEN); G-protein-coupled receptor (GPCR); heat shock protein 90 (Hsp90); histone
acetyltransferase (HAT); human estrogen receptor (a/b) (hER(a/b)); ICI
164,384 (ICI); immunophilin-FK-binding protein 52 (FKBP52); insulin-like
growth factor (IGF); ligand-binding domain (LBD); mitogen-activated protein kinase (MAPK); 4-hydroxytamoxifen (OHT); nuclear receptor corepressor (NcoR); phosphoinositide 3-kinase (PI3K); protein kinase A
(PKA); steroid receptor co-activator (Src); selective estrogen receptor
modulators (SERMs); transforming growth factor-b (TGF-b)
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without perusing all relevant articles. This review structures and orders the literatur and advances step by step.
Breast cancer and estrogen
Breast cancer is the most frequently diagnosed cancer in
women. In Germany and other industrialized countries,
breast cancer is ascertained with approximately every
tenth woman in the course of her life. Breast cancer is
there responsible for 27.8% of all new cancer cases among
women. It is estimated that there are 57 230 new breast
cancer cases each year [1]. Though very rarely, also men
can be suffer from breast cancer. Breast cancer is frequently hormone-dependent. That is: hormones stimulate the cancer cells to grow. Vice versa this means that
the growth of the cancer cells can be down regulated by
the oppositely active hormones or so-called antihormones. Therefore, a hormone therapy is possible as an
adjuvant treatment on breast cancer as well as with metastases. Today surgery, radio-, chemo-, and hormonal therapy form a common combination which has to be coordinated for each individual treatment.
The “dependent” natural female sex hormone means
primarily estrogen, 17b-estradiol (E2), which plays a
prominent role in mediating the maturation, proliferation, differentiation, apoptosis, inflammation, metabolism, homeostasis, and brain function and influences the
growth and development of breast cancer [2, 3, 4]. The
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Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
Scheme 1. Interconversion between E2 and the less active hormone, estrone, by 17b-hydroxysteroid dehydrogenase.
biological activity of E2 in hormone-sensitive tissues is
actively regulated by the interconversion by 17b-hydroxysteroid dehydrogenase between E2 and the less active
hormone, estrone [5] (Scheme 1).
Because of its numerous involvement and particular
importance, the deficiency of E2 in healthy women may
be associated with an increased risk of various diseases.
On the contrary, the normal existence of E2 in women
with hormone-positive breast cancer may worsen the disease. In all these cases, hormone therapy may be sensible,
effective, and practical. For the deficiency of E2, the hormone-replacement therapy means an extra supplementation with exogenous natural estrogens which include
estradiol, estrone, and estriol as well as synthetic estrogens that have been used for years imitating the natural
estrogens with similar characteristics, but they are not
the same biological substances like the ones that exist in
our bodies from birth on. In theory, treating hormonal
deficiencies with natural estrogens should have many
benefits over synthetic estrogens and, in fact, currently
many patients do use natural estrogens for hormonal
supplementation. For instance, E2 is used especially in
postmenopausal women with reduced ovarian hormonal
concentrations to prevent and treat cardiovascular diseases, to reduce lipoprotein cholesterol levels and lower
blood pressure, to prevent spinal bone loss, to inhibit
skin aging, and improve glycemic control in patients
with noninsulin-dependent diabetes mellitus [6]. For
breast cancer, however, the hormone therapy means that
synthetic estrogens and, in particular, anti-estrogens are
used to inhibit the physiological activities of E2, that
would otherwise stimulate the growth and development
of breast cancer, so as to control and treat the disease.
Estrogen agonists and antagonists
In clinical settings, exogenous estrogens and anti-estrogens are used for hormon-replacement therapy and as
anticancer agents [7]. They can be categorized into three
pharmacological classes: agonists, mixed agonist-antagonists, and pure antagonists. The mixed agonist-antagonists are also referred to as selective estrogen receptor
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Figure 1. Selected structures of estrogens, anti-estrogens, and
SERMs.
modulators (SERMs), because of their tissue-selective agonist or antagonist activities.
Agonists
Aside from the above-mentioned natural estrogens E2
and estrone, there are many other estrogens, e. g. diethylstilbestrol (DES, see Fig. 1). DES, a synthetic estrogen, was
once thought to revolutionize estrogen therapy and was
used to treat breast and prostate cancer with high-doses
of the drug as standard endocrine therapy before the discovery of the anti-estrogens [8].
Selective estrogen receptor modulators
Since estrogens are known to play a role in the growth
and development of many breast cancers, a logical
approach for the treatment of estrogen-sensitive breast
cancer is the use of anti-estrogens that inhibit the estrogen function in breast cancer cells. The first “classic antiestrogen” tamoxifen (TAM) (see Fig. 1) was therefore
developed. However, tamoxifen is now reclassified as a
typical selective estrogen receptor modulator (SERM).
Tamoxifen is largely inhibitory and functions as estrogen
antagonist in breast cancer cells, but it also functions as
an agonist in some tissues including the bone, uterus,
liver, and the cardiovascular system. These estrogen-like
activities of tamoxifen are significant for woman taking
anti-estrogen against breast cancer. Its stimulatory
effects on uterus and liver may underlie the increased
incidence of endometrial hyperplasia that may lead to
cancer, as well as alterations in liver function. The agonistic effects of tamoxifen in bone cells and in the cardiovascular system enhance bone maintenance, preserve a
favorable blood-lipid profile, and reduce the risk of corowww.archpharm.com
Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
nary problems ([9] and refs. therein). Because of this selectivity, up to now, tamoxifen has been used as standard
therapy in adjuvant hormone treatment on breast cancer.
However, the latest results of several major international trials showed that aromatase inhibitors work better than tamoxifen in post-menopausal women with
early-stage breast cancer that is ER positive, progesterone
receptor positive, or both. So, in the near future, it may
be possible that aromatase inhibitors will be the new
standard of care for post-menopausal women with invasive hormone-receptor-positive breast cancer, both early
and advanced-stage [10].
Another typical SERM is raloxifene (RAL), which has
been shown to function as an antagonist in the breast
and uterus, while functioning as an estrogen in the bone
and cardiovascular system. RAL was developed initially
as an anti-estrogen for breast cancer in the late 1980s,
but since it was found to maintain bone density, to prevent rodent breast cancer, and to inhibit tamoxifenstimulated endometrial cancer growth, it was developed
for osteoporosis, for which it is now an approved drug
[11]. RAL is an inhibitor of cultured breast cancer cells
and, in vivo, it possesses antitumor activity. Like tamoxifen, RAL reduces total cholesterol but does not increase
high-density lipoprotein cholesterol, a feature that may
lessen any cardioprotective effects [11, 12].
Furthermore, it was demonstrated that RAL has a
potential in the treatment of myo-cardial ischemia. It is
able to relax porcine coronary arteries in vitro due to activation of the mitogen-activated protein kinase (MAPK)
pathway [13]. P38 MAPK activation has been shown to be
responsible for cardioprotection during ischemic preconditioning [14].
Antagonists
Several classes of pure anti-estrogens, which possess no
known estrogen agonist effects, have been developed for
the treatment of breast cancer. Pure anti-estrogens, such
as ICI 164 384, ICI 182 780 (fulvestrant, Fig. 1), and RU 54
876, may perhaps prove to be more effective than tamoxifen in treating hormone-responsive breast cancer, but
are not effective in preventing bone loss and may have
detrimental effects on the cardiovascular system [9 and
refs. cited therein]. Therefore, a pure anti-estrogen, e. g.
fulvestrant, is recommended for treatment of breast cancer after failure of a first-line therapy with tamoxifen
[15].
The biological effects of estrogens and anti-estrogens
are mostly mediated through the ER, which acts as hormone-activated transcription factor.
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Estrogen Receptor and Ligands
135
Figure 2. Overall distribution of ERa and ERb in different tissues
from [23].
The estrogen receptor
Discovery of the estrogen receptor
The ER is a ligand-inducible transcription factor that
belongs to the nuclear receptor super family and acts as a
dimeric species. In the early 1960s, Jensen and Jacobsen
first demonstrated that a specific protein was responsible
for the concentration of physiological levels of E2 in target tissues [16]. This protein is now known as the ER. Jensen and colleagues translated the basic science into clinical utility by proposing a predictive test, the ER assay, to
determine which patients would respond to endocrine
ablation. It was then established that patients with ERrich tumors respond to endocrine therapy, whereas
patients with ER-negative tumors are unlikely to respond
[17, 18].
For a long tim, it was assumed that only one human ER
(hERa), cloned and sequenced in 1986 from MCF-7
human breast cancer cells [19, 20], exists. But ten years
later, a second receptor was cloned from a rat prostate
cDNA library [21] at first, and then, the human ERb
(hERb) was identified and characterized [22].
Both receptors are expressed next to one another in
many tissues, including the central nervous system, the
cardiovascular system, the urogenital tract, the breast,
and the bone (Fig. 2). In the uterus and mammary gland,
ERa is an important estrogen receptor and much more
frequently expressed than ERb. In addition, ERa is also
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Z. Bai and R. Gust
Figure 3. Domain-structure representation of human ERa and
ERb isoforms [26].
Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
Figure 4. The DBD of hERa comprises two zinc finger motifs
according to Ruff [29], Pettersson [31], Chen [32], and Tsai [33].
present in liver, while in the gastro-intestinal tract it is
only ERb [23].
Both ERa and ERb are mainly regulated by the endogenous estrogen E2. ER modulation is involved in the development and regulation of reproductive, cardiovascular,
and bone health, in addition to controlling various
aspects of cognitive function [24]. Besides, an excessive
activity of the ER has been correlated with the development and proliferation of certain breast and uterine carcinomas [25].
Structure of the estrogen receptors
ERa and ERb represent two separate gene products. The
hERa protein consists of 596 amino acids with a molecular weight of 66 kDa [26, 19] and is located on chromosome 6 [27], while the hERb sequence encodes a protein
of 530 amino acid residues with a molecular weight of
59 kDa [28] and is positioned on chromosome 14 [22].
Like other nuclear receptors, the ER has a multidomain
structure consisting of six functional regions, from the
N-terminal A/B domain to the C-terminal F domain,
which show various degrees of sequence conservation
(Fig. 3) [26].
The poorly conserved combined A/B region contains
the autonomous transactivation function AF-1. In this
region, no clear secondary structure can be identified
and no structural data have been obtained until now [29].
The better characterized parts, for which functional and
structural data are available, are the highly conserved C
region harboring the DNA-binding domain (DBD) and the
conserved E region containing the ligand-binding
domain (LBD) as well as the transactivation function AF2. The D domain can be considered as a linker peptide
between the DBD and the LBD, whereas the F domain, a
C-terminal extension region of the LBD, is not conserved
[29]. Both ERa and ERb share a modest overall sequence
identity (47%) [30]. The DBDs of ERa and ERb show a high
degree of homology (97%; only three amino acids differ),
but the LBD possesses only 47% homology [23, 26].
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Figure 5. Three-dimensional structure of the ERa (A, ligand E2;
B, ligand genistein) and ERb (C, ligand genistein) LBD monomer
[37, 29, 38].
The DBDs of the two ER isoforms share the same
response elements. DBD structures are available only for
ERa [29]. The topology of ER DBDs is characterized by two
zinc finger motifs with eight cysteines that constitute
the tetrahedral coordination of two zinc ions [29, 31,
32, 33] (Fig. 4). These zinc fingers are essential components of the ER due to their non-fungible DNA-binding
function [34, 35]. The first zinc finger sequence is neutral
to slightly acidic, which determines the binding specificity to the so called estrogen response elements (ERE),
whereas the second zinc finger structure harbors a positive net charge and governs unspecific DNA contacts as
well as dimerization of the two DBD molecules [36]. The
helical structure of the “P-box” (E, G, A) and downstream
amino acids provides important deoxynucleotide contacts and fits into the major groove of the DNA helix. The
amino acids in the “P-box” are responsible for base recognition and discrimination, whereas the residues participating in the “D-box” (P, A, T, N, and Q) have been shown
to be involved in the dimerization interface [29, 33].
The LBD is a globular domain that harbors a hormone(ligand-) binding site, a dimerization interface, and a coactivator and corepressor interaction function. Despite
low sequence identity in LBDs of the nuclear receptor
superfamily, the three-dimensional structures of the
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Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
LBDs are similar [29]. The first reported crystal structure
for a steroid receptor was that of the ERa LBD in complex
with E2 (Fig. 5) and RAL (cf. Fig. 10) [37]. In addition, the
crystal structure of the ERb LBD was characterized in
complex with genistein (Fig. 5C) and RAL [38]. ER LBDs
are arranged in an antiparallel a-helical ”sandwich” fold
that was first described for the human RXRa apolipoprotein LBD [40]. The liganded ER LBD (Fig. 5A and cf. Fig. 9)
contains 11 a-helices (H1, H3-H12) organized in a threelayered sandwich structure with H4, H5, H6, H8, and H9
flanked on one side by H1 and H3, and on the other side
by H7, H10, and H11 [29]. The ligand pocket is closed after
hormone binding on one side by an antiparallel b-sheet
and on the other side by H12, which is known to be
directly involved in the transactivation function AF-2 by
mutagenesis studies [41], for which several “agonist” or
“antagonist” conformations have been evidenced [42].
AF-1 located in the A/B regions mediates a constitutive
activation potential and is responsible for the promoterspecific transcriptional activation independent of the
presence of a ligand. In addition, the AF-1 is thought to
be responsible for the partial agonist activity of tamoxifen in cells that express ERa [43]. AF-2 enclosed in the E
domain provides ligand-specific activation. AF-1 and AF-2
are independently autonomous in their regions and also
synergistic with each other in most cases [44, 45].
Estrogen receptor transcription
The ER is a ligand-inducible transcription factor. Both
ERa and ERb stimulate transcription of an ER-responsive
gene containing an ERE in an E2-dependent manner [46].
Ligand-binding experiments revealed high affinity and
specific binding of E2 to both ER isotypes, and no obvious
differences between the two isotypes alone or combined
were observed in ERE transcriptional assays in the presence of E2 [46]. ER-mediated transcription is a highly
complex process involving a multitude of coregulatory
factors and “cross-talk” between distinct signaling pathways [47, 48], which could be depicted in a mode including ligand-dependent and ligand-independent ER transcriptional activations (Figs. 6 and 7) [9, 47, 2].
The basic pathway follows an E2-regulated ER transcription line (Fig. 6): Upon binding E2, ER becomes activated through a process that involves dissociation from
protein chaperones, conformational change, dimerization, and binding to EREs of target genes. ERE-bound ER
recruits coregulators that stimulate gene transcription.
In the non-active state, the ER exists as a heterocomplex consisting of the heat-shock protein 90 (HSP90) and
immunophilin-FK-binding protein 52 (FKBP52). HSP90
binds directly to the ER LBD to form a less stable complex,
which is stabilized by FKBP52 through a direct binding to
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Estrogen Receptor and Ligands
137
Figure 6. A simple mode of estrogen action through estrogen
receptor [15].
HSP90 and an electrostatical interaction with the nuclear
localization signal (NLS) contained at the C-terminal end
of the ER DBD [49]. The role of HSP90 and other chaperons may be to maintain the receptors folded in an appropriate conformation to respond rapidly to hormonal signals [49, 50]. This inactive ER complex continually shuttles between the nucleus and cytoplasm with nuclear
localization and nuclear export sequences.
E2 diffuses through the plasma membrane and cytoplasm of cells into the nucleus where it binds to the ER
LBD. Once E2 binds to the ER, HSP90 and FKBP52 dissociate and the receptor undergoes a conformational change
transforming the receptors to the active form [49].
It has been demonstrated that different ligands induce
different changes in the receptor (ERa) conformation (see
Chapter on The X-ray crystal structure of ER LBD-ligand
complexes) and target cells can distinguish between
these ERa-ligand complexes. These conformational
changes have been shown to influence ERa cofactor binding and, therefore, have a profound impact on ERa pharmacology [51, 52, 53, 54]. In addition, the nature of the
bound ligand also influences the stability of ERa, and the
rate of ERa degradation in the presence of E2 directly correlates with transcriptional activity [54]. It was even concluded that acute degradation of ERa followed by an E2dependent transcriptional activation of ERa mRNA is a
general E2 response [55]. With these conformational
changes, the receptors dimerize as homodimers (ERa/
ERa and ERb/ERb) or heterodimers (ERa/ERb) [56, 57]. The
dimer complex is translocated to the nucleus of the target cells by nuclear localization sites.
As mentioned above (Structure of the estrogen receptors), the ER contains two dimerization domains, one in
the DBD and one in the LBD. Dimerization by the LBD is
ligand-dependent, whereas dimerization by the DBD is
ligand-independent and mediated by sequences in the
DNA, therein Ser236 located in the second zinc finger of
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Z. Bai and R. Gust
ERa DBD plays an important role [32]. However, hERa is
phosphorylated by protein kinase A (PKA) on Ser236 and
phosphorylation at this site can inhibit dimerization in
the absence of estrogen and, therefore, inhibit DNA binding. Binding of estrogen to the ER can overcome this
inhibition [32].
Even though this inhibition of dimerization was found
in ERa, phosphorylation on multiple sites of ERa as a
phosphoprotein, by ligand binding and other events,
increases transcriptional activation of the receptor [32].
The dimer complex either directly binds to EREs in target
genes or indirectly interacts with DNA through tethering
to other DNA-bound transcription factors, e. g. AP-1
[58, 59] or Sp1 [60], in a way that stabilizes the DNA binding of that transcription factor in the absence of direct
ER-DNA binding, to alter the rate of transcription.
These EREs may be consensus or no consensus and may
exist as single or multiple full or half sites; they may also
be composite sites, consisting of EREs flanked by
response elements for other transcription factors (such
as Sp1, Sp1 may play two roles, either in direct binding as
“half site” or in indirect interaction as “tethering” [60]),
which themselves may or may not be occupied by their
respective transactivating factors [61]. The ERE sequence
is an allosteric effector of ER action. Binding of the ER to
natural and synthetic EREs with different nucleotide
sequences alters ER-binding affinity, conformation, and
transcriptional activity and, therefore, impacts physical
and functional interaction of ERa and ERb with coregulators [62]. Both direct and indirect interaction between
the ER and EREs result in recruitment of coregulators
and components of the RNA polymerase II transcription
initiation complex that enhances target gene transcription (Fig. 7) [63].
Coregulators can be broadly divided into co-activators,
which augment the activity of receptors, and corepressors, which mediate the repressive effects of receptors
[64]. In recent years, at least 28 different ERa co-activator
proteins have been identified [62]. Many co-activators
required for the ER activity are histone acetyltransferases
(HATs), e. g. CBP/p300 [65].
Transcriptional activation involves alterations in chromatin structure mediated by ATP-dependent chromatinremodeling enzymes in conjunction with factors that
contain HAT activity [66]. Transcriptional competence
correlates with the acetylation of chromosomal histone
proteins at their N-termini, which results in destabilization of protein-DNA contacts and chromatin decompaction [67].
Briefly, co-activators facilitate ER transcription
through their functions of (i) acetylating the N-terminal
tails of lysine residues in histones H3 and H4 leading to
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Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
The abbreviations used are: E, estrogen; R, receptor; ERE, estrogen-response element; GF, growth factor; TBP, TATA binding protein; TAFs, TBP-associated factors;
RNA polymerase II (pol II).
Figure 7. A model for estrogen-receptor transcription from [9].
“relaxed” chromatin structure, (ii) acetylating other transcription factors and co-activators, (iii) recruiting secondary co-activators including co-activator associated arginine methyltransferase 1 (CARM 1) and protein arginine
methyltransferase 1 (PRMT 1) that methylate histones,
(iv) interacting with components of various ATP-dependent chromatin-remodeling complexes, and (v) directly
interacting with and stabilizing basal transcription factor binding [62 and refs. cited therein].
Most of the co-activators, e. g. p160 family proteins,
interact with the AF-2 domain of agonist-bound ERs
through multiple LxxLL (L = leucine, x = any amino acid)
amino-acid motifs [68], whereas some co-activators, such
as the steroid receptor RNA activator SRA and the RNA
helicases p68/p72, interact with and regulate the AF-1
domain of ER [69, 70, 71].
Opposing the co-activators, corepressors negatively
regulate transcription, namely promote transcriptional
repression, via their recruitment of histone deacetylases
(HDACs). The best characterized corepressors are the
structurally related proteins NcoR (nuclear receptor corepressor) and SMRT, which are recruited by ER to the promoter of target genes in the presence of antagonists such
as tamoxifen [72, 73]. But NcoR and SMRT differentially
impact E2-induced transcriptional activity in an ER subtype- and ERE sequence-dependent manner [62]. Other
proteins act to repress ER-mediated transcription by distinct mechanisms. For instance, the ER-specific corepressor REA, as well as the orphan receptors SHP and DAX-1
act by competing with the p160 coactivators for binding
agonist-bound ER [74 and refs. therein].
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Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
Upon binding to ERE and in cooperation with coregulators, ER binds to a promoter and forms a transcription
pre-initiation complex. Upon further interaction with
coregulators, components of the core transcriptional
machinery “TATA-box” and RNA polymerase II (pol II), a
transcription-initiation complex is complete. RNA polymerase II is recruited to the transcription start site and
begins transcription [75, 33].
Besides the E2-dependent basic pathway (i), there are
other E2-dependent or -independent pathways that play
an important role for gene transcription of the ER. These
include signaling pathway (ii) regulated by membrane
ER (mER), and modulation of ER activity by growth factors (including epidermal growth factor, insulin-like
growth factor-1, insulin, and transforming growth factor-b), neurotransmitters such as dopamine, and second
messengers such as cAMP and others that affect protein
kinase cascades including the MAP kinase signaling pathway (iii), and (iv) (Fig. 7) [2, 47, 9].
Pathway (ii) is about mER action. ER is one of the
nuclear receptor super families. The majority of them in
the cell reside in the nucleus in the presence of estrogen.
But a small fraction of total receptors are also localized at
or near the cell membrane in either the presence or
absence of estrogen. Several studies suggest that ER localization to the cell membrane is facilitated by association
with other proteins that themselves translocate to the
cell membrane. Candidate proteins reported to fulfill
this role are caveolin-1, the adaptor molecule Shc, and
insulin-like growth factor (IGF)1 receptor [2 and refs.
therein]. The adaptor molecule Shc, through a coupling
of ER with IGF1 receptor, has also been suggested to
mediate ER translocation to the cell membrane in an
estrogen dependent manner.
ERa and ERb have all been demonstrated to associate
with Src and activate downstream mitogen-associated
protein kinases that confer proliferative and differentiating effects [76, 77].
The signaling pathway (iii) is also mediated by the
interaction between growth-factor receptor and Shc as
well as the membrane ER. The ER LBD alone is sufficient
for estrogen-dependent translocation to the cell membrane and also sufficient for mediating many of the
described effects of estrogen on signal-transduction pathways in different cell types [2 and refs. therein]. These
extranuclear signaling pathways converge upon and activate nuclear transcription factors by phosphorylation
and thus may ultimately affect gene-expression patterns
by integration with nuclear signaling by the ER in the
cell.
In several papers, it was speculated about an involvement of classical steroid receptors localized at the cyto-
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Estrogen Receptor and Ligands
139
plasmic membrane in the rapid effects of steroids [76]. In
addition, there are reports that support the existence of a
distinct receptor, associated to the plasma membrane,
being different from the classical, intracellular one.
Moreover the non-genomic effect of steroids could be
attributed to their binding with the sex-hormon-binding
globulin (SHBG) receptor, that is a G-protein-coupled
receptor (GPCR), located at the membrane and has been
functionally identified in a number of tissues such as
prostate, testis, liver, and breast [78, 79]. Finally, the
rapid steroid action could potentially be induced in the
absence of a receptor, by influencing membrane fluidity
[80, 81].
In recent years, several rapid estrogen-mediated effects
could be traced back to membrane-associated estrogenresponsive receptors [82]. These are unrelated to their
intracellular counterparts, but, instead, exert characteristics of GPCRs. In the course of these discoveries, attention was directed to an orphan member of GPCRs,
GPR30, which revealed facilities of an estrogen-responsive receptor [83]. Regarding its structural sequence
homology to receptors for angiotensins, interleukins,
and a variety of chemokines, it had been suggested to be
a receptor for peptide ligands [84]. However, this assumption has been disproved [85]. Characteristically for members of the large superfamily of GPCRs, it consists of seven
membrane-spanning helices and transduces extracellular stimuli into intracellular signals through interaction
of its cytosolic domain with heteromeric G-proteins.
GPR30 exhibits a widespread expression pattern with
abundant levels not only in breast cancer, but also in placental, bone, brain, ovarian, prostate, vascular epithelial,
and hepatic tissues. A large number of investigators
proved that GPR30 accounts for a substantial set of rapid
cellular responses to estrogens. Since most of GPR30expressing tissues are considered to be ER positive, it
seems likely to assume that their estrogenic responses
are partially mediated by GPR30. GPR30 demonstrably
regulates the phosphorylation state of ERK1/2
[86, 87, 88], induces mobilisation of intracellular calcium
[89, 90], cAMP (cyclic adenosine monophosphate) release
[87], and synthesis of phosphoinositide 3-kinase (PI3K)
[90]. In the course of mER signalling, a trans-activation of
epithelial growth factor receptor (EGFR), IGF-1 receptor,
and human epithelial growth factor receptor type 2/neu
(Her-2/neu) occurs, leading to the activation of their
downstream mediators ERK1/2 and PI3K/Akt [87]. By this
means, estrogens may induce cell proliferation without
involvement of gene transcription. Moreover, ERK1/2
activation by E2 in hormone-dependent MCF-7 breast
cancer cells also partially occurs by secretion of heregulin, which activates the HER-2/neu receptor, PKC d, and
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Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
less, it may be a great help to understand more about ERtranscriptional action.
As mentioned above, both ER subtypes recognize similar target-DNA sequences and bind and respond similarly
to E2, but there are differences in DNA-binding affinity
and specificity for pharmacological ligands. In addition,
ERa is a more potent transcriptional activator than ERb,
and in tissues, where both ERs are expressed, ERb has
been suggested to have a role as an attenuator of ERa [2
and refs. cited therein).
Estrogen and anti-estrogen action through
estrogen receptor
Membrane ER and / or growth-factor receptors, can interact directly with components
of the cytosolic signaling molecules, including the regulatory subunit of PI3K, leading
to the activation of the serine / threonine kinase Akt pathway (ii); growth factors such
as epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), insulin, and
transforming growth factor-b (TGF-b) bind to and activate their receptors, which, and /
or membrane ER, in turn activate the Src-RAS – RAF – MEK-MAPK and the phosphoinositide 3-kinase (PI3K) pathways (iii); other extracellular stimuli such as dopamine
and cyclic AMP bind G-protein-coupled receptors and activate adenylyl cyclase (AC)
and protein kinase A (PKA) pathway (iv). The activated kinases subsequently phosphorylate and activate ER and other transcription factors in the nucleus [47, 2].
Figure 8. Extranuclear-signaling pathways in ER transcription.
the Ras pathway [91]. As depicted in Fig. 8, activated
MAPKs and PI3K may phosphorylate certain residues of
nuclear ERs promoting their transcriptional activity or
stability.
Malek [92, 93, 94] showed that comparable to E2, the
pure anti-estrogen faslodex, which has been proved to
bind to GPR30 in an agonistic manner, impaired migration and Smad phosphorylation in response to transforming growth factor (TGF)-b through a pertussis toxin(PTX-) sensitive mechanism. Concrete evidence for GPR30
as a mediator of E2-induced interruption of TGF-b signalling could successfully be provided by transfection
experiments. Thus, migration and Smad2 phosphorylation of E2-sensitized MCF-7 cells could be restored after
down-regulation of GPR30 expression by siRNA technique. These findings could be corroborated, as the inhibitory properties of the hormone on TGF-b signalling
could be established by transfection of hormone-independent MDA-MB-231 breast cancer cells with a GPR30
expression vector.
Currently, there is better understanding of molecular
mechanisms of steroid receptors in terms of transcriptional signaling in the nucleus, whereas the clear mechanisms by which conventional steroid receptors interact
with and modulate the activities of extranuclear cell signaling pathways still remain to be uncovered [2]. The
mechanisms with those pathways depicted in Fig. 7 are
only a model established on present studies. Neverthe-
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The ER transcription described above is based on ER
action regulated by the natural ligand E2 and the ability
of cells to distinguish and response. In clinical settings,
the bound ligands to ER are not only estrogens but also
anti-estrogens including SERMs, and the ER can adopt
multiple conformations upon binding different ligands
[95, 52]. The impact of such conformational changes was
further revealed when steroid receptor co-activator-l
(SRC-l), and subsequently other cofactor proteins, co-activators and corepressors, were isolated [96, 97]. Furthermore, analysis of the crystal structure of the ligand-ER
LBD complexes provided the molecular basis of the interaction of the ER with its ligands [37, 98, 99, 100, 101] and
so that a better understanding of estrogen and anti-estrogen actions through the ER was established.
The X-ray crystal structure of ER LBD-ligand
complexes
The first crystal structure of an ERa LBD was reported in
complexes with E2 and the nonsteroidal selective estrogen antagonist RAL [37].
In the E2-ERa LBD complex (Fig, 9), the E2 cavity is completely shielded from the external environment involving parts of H3, H6, H8, H11, and H12 as well as a small
two-strand antiparallel b-sheet (Fig. 5). E2 binds diagonally across the cavity between H11, H3, and H6 and
adopts a low-energy conformation. H12 sits over the
ligand-binding cavity, without direct contact with E2
and is packed against H3, H5/6, and H11, with its inner
hydrophobic surface toward the bound hormone [37].
Hormone recognition is achieved through a combination of specific hydrogen bonds and van-der-Waals contacts of the binding cavity to E2's non-polar character
(Fig. 9). They are involved in the anchoring of the E2
hydroxyl moiety at positions 3 and 17. The phenolic
hydroxyl group of the A-ring (3-OH) is hydrogen bonded
to Glu353 from H3, and to Arg394 from H6 and a water
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Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
Figure 9. Left: The three-dimensional protein structure of the
E2-ERa LBD complex including H12 (blue cylinder) and hydrophobic residues (yellow); dotted lines indicate unmodelled
regions of the structures. Right: The interaction of E2 with critical amino acids in the ERa LBD [37].
molecule. The hydroxyl group of the D-ring (17b-OH)
forms a single hydrogen bond with His524 in H11. The
remainder of the molecule participates in van-der-Waals
contacts that are concentrated over the A, A/B interface,
and D-rings [37].
In the RAL-ERa LBD complex (Fig. 10), RAL binds at the
same site as E2 within the LBD. The side chain of RAL
makes extensive hydrophobic contacts with H3 and H5/6,
H11 and the loop between H11 and H12. In addition, the
long side chain displaces H12 and protrudes from the
pocket between H3 and H11. Instead of the alignment of
H12 over the cavity in E2-ERa LBD complex, the helix H12
lies in a groove formed by H5 and the carboxy-terminal
end of H3, with a rotation of 1308 combined with a 10 rigid-body shift toward the amino terminus of the LBD
compared with the E2-induced conformation [37].
Hydrogen bonds between the hydroxyl group of the
benzothiophene moiety and H3 (Glu353), H6 (Arg394)
and a water molecule (Fig. 10) are similar to those of the
A-ring phenolic OH of E2. In the binding mode of RAL at
the “D-ring end” of the cavity, the phenolic hydrogen
bonds with His524 whose imidazole ring makes a rotation. The remainder of the core is involved in non-polar
contacts similar to those seen for E2. The side chain of
RAL is anchored to the protein by a direct hydrogen bond
between Asp351 and the piperazine ring nitrogen [37].
The crystal structures of the synthetic agonist DES and
the selective antagonist 4-hydroxytamoxifen (OHT),
respectively bound to the ERa LBD [98] are similar to
those of E2 and RAL, namely, the conformation and interactions of ERa LBD with DES are similar to those of ERa
LBD with E2, and the conformation and interactions of
ERa LBD with OHT resemble that of ERa LBD with RAL
(Figs. 11 and 12). It is remarkable that the DES-LBD com-
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Estrogen Receptor and Ligands
141
Figure 10. Left: The three-dimensional protein structure of the
RAL-ERa LBD complex including H12 (green cylinder) and
hydrophobic residues (yellow). Right: the interaction of RAL with
critical amino acids in the ERa LBD [37].
Figure 11. Above: The interaction of DES with critical amino
acids in the ERa LBD [98]. Below: The three-dimensional protein structure of the DES-ERa LBD-GRIP1 NR box II peptide
(gold) complex; two orthogonal views of the complex including
the co-activator peptide (gold), helix 12 (red), H3, H4, and H5
(blue). DES (green) shown in space-filling representation.
plex binds to the NR box II peptide, while the OHT-LBD
cannot [98].
In the DES-LBD-NR box II peptide complex, the ligand is
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Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
Figure 13. Superposition of the three-dimensional structure of
ERa LBD complexed with estrogens (green conformation), antiestrogens (red and blue conformations) [29].
Figure 12. Above: The interaction of OHT with critical amino
acids in the ERa LBD [98]. Below: The three-dimensional protein structure of the OHT-ERa LBD complex; two orthogonal
views of the complex including helix 12 (red), H3, H4, and H5
(blue) are presented; OHT (blue) shown in space-filling representation.
ity with two of the phenolic hydrogens binding to the
corresponding amino acid residues and a water molecule. Besides, DES contacts two regions of the ligandbinding pocket not occupied by E2, located at the 7a and
11b positions of E2, and filled by the two ethyl groups of
DES. H12 makes a similar conformation as that in E2-LBD
complex. In OHT-LBD complex, OHT is bound within the
same pocket that recognizes DES, E2, and RAL. Next to
the hydrogen bonds of its hydroxyl group with Glu353,
Arg394 and water, OHT stretches its side chain between
H3 and H6, and the positioning of the flexible dimethylaminoethyl region of the side chain is stabilized by vander-Waals contacts with Thr347, Ala350, and Trp383 and
by a salt bridge between the dimethylamino group of the
side chain and the b-carboxylate of Asp351. The orientation of H12 mimics that in RAL-LBD complex. The remainder of the molecule of DES as well as OHT participates in
van-der-Waals contacts with the corresponding residues
[98].
Based on these analyses of the crystal structures of the
ligand-ERa LBD complexes, the relative positioning of
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
H12 is compared in Fig. 13 [29]. These analyses revealed
that the activation function 2 (AF-2) pocket, when bound
by an agonist, undergoes a conformational change that
forms a hydrophobic groove on the surface of an agonistbound LBD formed by residues from H3, H4, H5, and H12
and allows the docking of a conserved leucine-rich NR
box LxxLL motif present in all p160 and most of other coactivator proteins. Conversely, binding of an antagonist
alters the AF-2 structure, especially H12 with an NR boxlike sequence (LxxML versus LxxLL) functions as an intramolecular mimic of the co-activator helix interacts with
the hydrophobic groove so that it is incompatible with
co-activator docking [98].
These different conformational changes of LBD with
binding to agonist and antagonist also were revealed by
analyses of the crystal structure of ERb LBD complexed
with genistein (GEN) [38], RAL [100], and ICI 164,384 (ICI)
[101]. In the GEN-ERb LBD complex (Fig. 5), H12 is bound
over the ligand-binding pocket in a position such that it
occludes the co-activator recognition surface only partially, being consistent with that genistein acts as an ERb
partial agonist [100]. The position of H12 in the RAL-ERb
complex is similar to that in RAL-ERa LBD, also with a
tethering interaction between H12 and the hydrophobic
groove [100]. In the ICI-ERb LBD complex, the pure antagonist ICI side chain binds directly to the co-activator-binding site of ERb LBD, causing H12 to be completely disordered [101].
Nevertheless, the estrogenic properties of some ligands
are selective for both subtypes ERa and ERb. For instance,
5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol
(THC) (Fig. 14) acts as an ERa agonist and as an ERb antagonist, which can be explained by analysis of the crystal
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Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
Figure 14. Left: The three-dimensional protein structure of the
THC-ERa LBD-GRIP1 NR box II peptide complex including the
co-activator peptide (gold), helix 12 (red), and THC (green)
shown in space-filling. Middle: The three-dimensional protein
structure of the THC-ERb LBD complex including helix 12 (red),
and THC (green) shown in space-filling [99]. Left: Graphical
drawing of THC.
structure of their complexes (Fig. 14), and therein a novel
antagonistic mechanism was proposed [99].
The ERa LBD when bound to THC adopts the same conformation (Fig. 14) as it does when bound to the full agonists E2 and DES, whereas the THC-ERb complex shows a
H12 orientation (Fig. 14) similar to that observed in the
GEN-ERb complex (Fig. 5C), without H12 binding to the
region of the co-activator-recognition groove. In contrast
to that antagonism (OHT, RAL, or ICI) with a bulky side
chain that directly or “actively” precludes the agonistbound conformation of H12 by steric hindrance, termed
“active antagonism”, the antagonism by THC-ERb, without H12 precluding from adopting the agonist-bound
conformation, was termed “passive antagonism”. This
“passive antagonism” lies in the difference of ligandbinding pocket residues of ERb from that of ERa [99].
These analyses of crystal structures of ligand-ER LBD
complexes reveal that the position and orientation of
H12 are important indicators for understanding the conformation changes by ER binding to agonists and antagonists including SERMs, but not determining factors. The
determining conformation changes lie in overall structure of ligand-ER complex, including AF2 and AF1 as well
as degradation of ER.
Comparison of estrogen and anti-estrogen actions
Due to the fact that ligand actions are mostly mediated
by the transcription factor ligand receptor, the ligand
receptor actions, on the other hand, are mostly regulated
by ligands, estrogen, and anti-estrogen actions, in fact,
are interconnected with ER transcriptions to form united
physiological events. The ER transcription described
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Figure 15. A simple modal of anti-estrogen action through the
estrogen receptor (ER) [18].
above relates to the normal transcriptional process
including estrogen action, but there is rarely a distinction from anti-estrogen action. This section focuses on a
brief comparison between the normal estrogen action
and clinical anti-estrogen action.
Estrogen binding to the ER facilitates such a conformational change as to be favorable for ER binding to co-activators. This change brings AF-2 and AF-1 of the ER in
direct association with one another leading to synergy.
After dimerization, the ligand-ER dimer binds to ERE and
promotor, with the help of coregulators and the transcriptional machinery as well as growth factors; this
results in transcription (Fig. 7) [18].
Anti-estrogens, including SERMs, can be used to inhibit
or prevent this estrogen action in the breast, so as to treat
estrogen-dependent breast cancer. Such an anti-estrogen
action mediated also by ER is depicted in a simple model
in Fig. 15 [18].
Anti-estrogen competitively binds to the ER and induces an ER conformational change, which blocks ER binding to co-activators and / or is favorable for ER binding to
corepressors [73, 102] as well as it affects ER dimerization
and interaction with ERE. Thus, the genetic estrogen
response is inhibited and the growth of breast cancer
cells is stopped. In addition, the pure anti-estrogens can
destroy the ER [18]. The ER is synthesized in the cytoplasm and transported to the nucleus where it functions
as a transcription factor. A pure anti-estrogen such as fulvestrand binds to the newly synthesized receptor in the
cytoplasm and prevents transport to the nucleus. Then,
the paralyzed receptor complex is destroyed rapidly
[103]. The complete destruction of available ER will prevent the occurence of any estrogen-regulated events.
Besides these antagonistic effects in breast tissue,
SERMs such as tamoxifen and RAL also act as agonists in
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some tissues, e. g. bone and the cardiovascular system.
These biological selectivities in different tissues may be
explained, by the nature of the ligands, by both the multitude of transcription factors and cofactors in the
modes, i. e., how they take part in the mode of action, and
the differences and particularities of various tissues
[47, 104, 18]. For instance, (i) different transcription factors (or subtypes) such as ERa and ERb can regulate different, even opposing biological events with a same ligand;
(ii) different cofactors such as co-activators or corepressors possess different recognition features for the ER; (iii)
specific EREs or a particular promoter that interacts with
the altered ligand-ER complex result in a corresponding
response; (iv) different intracellular environments influence or even determine the ligands' biocharacter
through different signaling pathways. However, an exact
mechanism of SERM action in tissues is not presented up
to now.
Despite an increasing understanding of the hormone
action and the success of hormone therapy in preventing
and treating breast cancer, there are still many clinical
problems and theoretical questions to solve and answer.
For example, prolonged treatment with the most widely
used SERM tamoxifen may develop tamoxifen resistance
[9] and increases the risk for endometrial cancer [7, 105].
Therefore, much current research focuses on identifying
alternative estrogen-receptor modulators that will minimize harmful side effects while preserving the ability to
block cancer growth. Besides further investigation of the
analogues of the clinically used SERMs and the pure antiestrogen fulvestrant, a new series of potential ER ligands
were developed.
Estrogen-receptor ligands
ER ligands can be categorized into three pharmacological classes: estrogens, SERMs, and pure anti-estrogens.
SERMs such as tamoxifen and RAL, pure anti-estrogens
such as fulvestrant and other steroid hormones have
been largely investigated and reported [11, 15,
106, 107, 108], and got interpreted above. Hence, we will
limit this section to some new potential estrogens or lead
structures, which may be also developed into SERMs or
pure anti-estrogens by introduction of an appropriate
active group onto a suitable position [109].
Classification of estrogens
Based on the binding mode to the ER, estrogens are classified in two types: Type-I estrogens are linear or planar
molecules similar to E2 or DES and bind analogously to
the ER (Figs. 9 and 11).
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Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
Figure 16. Interaction model of the Type-II estrogens (Left:
(2R,3S)/(2S,3R)-2-(2-Chloro-4-hydroxyphenyl)-3-(2,6-dichloro4-hydroxyphenyl)piperazine; Right: (4R,5S)/(4S,5R)-N-Ethyl4,5-bis(2-chloro-4-hydroxyphenyl)-2-imidazoline) located in the
LBD of ERa. Contacting the amino acids Glu353 and Arg394 as
well as the water molecule like other agonists or antagonists, the
Type-II estrogens are suggested to be attached also at the
amino acid Asp351 [110] or Thr347 [112].
Type-II estrogens are the second typ with angular spatial structures and are anchored in part to other amino
acids within the ER LBD [110, 111]. The amino acids
Asp351 and Thr347 are the alternative hydrogen-bond
anchor points at ERa (Fig. 16) [110, 112]. Being suitable in
size to the pocket of the ER LBD, numerous compounds,
above all hydroxylated aryl-substituted heterocycles
were synthesized and biologically evaluated.
Novel estrogens or lead structures
A series of aryl-substituted five-membered heterocycles
containing a single heteroatom, such as furans, pyrroles,
and thiophenes were investigated and, of them, several
furan derivatives (Fig. 17) were found in biological studies to possess a very interesting character [113]. Furan 1
proved to be an agonist with high selectivity for ERa versus ERb in both ER-binding affinity and transcriptionalactivation activity in human endometrial cancer HEC-1
cells. This selectivity benefits allegedly by the third phenolic hydroxyl on the C(5) phenyl group, which is possibly H-bonded to the amino acid Thr347 within the ERa
LBD according to the molecular modeling investigation
on the binding orientation of a furan ligand in ERa.
Furan 2, derived by grafting an N-piperidinylethyl side
chain on the C(4) phenol of furan 1, was an ERa-selective
antagonist with high binding affinity [114].
More studies focused on the ring system bearing two
heteroatoms, above all pyrazoles, imidazoles, 2-imidazolines, and piperazines. Pyrazole 3 (PPT) (Fig. 18), having
high ERa selectivity in terms of affinity and gene-transcriptional activity [115]. The different interactions of the
ligand with ERa and ERb were postulated to account for
this specificity. Appending an N-piperidinylethyl side
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Estrogen Receptor and Ligands
145
Figure 17. Furan derivatives with interesting character in biological studies.
Figure 20. Structures of imidazoles 6 and 7.
Figure 18. Structures of pyrazole 3 (PPT) and pyrazole 4
(MPP).
Figure 19. Model of the interaction of imidazole 5 with the ERa
LBD (Type-I binding mode) [121].
selective full antagonist similar to furan 2 [116, 117].
Such an ERa-selective antagonist as pyrazole 4 (MPP)
(Fig. 18) was suggested to be used as a new tool for investigating the biological functions of ERa and ERb [116].
A number of imidazoles were investigated as ER
ligands and cytotoxic inhibitors of the cyclooxygenase
(COX) [118, 119, 120, 121]. Imidazole 5 (Fig. 19) showed
full agonist activity in ERa-positive MCF-7-2a breast cancer cells stably transfected with the plasmid EREwtcluc,
even though its relative binding affinity (RBA) was very
low. Only in light of the molecular structure, imidazole 5
(Fig. 19) may dock into the ER LBD by taking both binding
modes of Type-I and Type-II estrogens. Yet from the data
of the luciferase assay – its analogues with angular pharmacophores were completely inactive – a Type-II estrogen-like orientation was excluded. Therefore, the interac-
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tion of imidazole 5 with the ERa LBD was proposed as
Type-I binding mode [121]. A further binding to Met522 is
very likely and must be taken into consideration when
discussing the attachment of hormonal active ligands in
the LBD (Fig. 19).
Imidazole 6 and 7 (Fig. 20) were estrogenically inactive
in MCF-7-2a cells. They exhibited, however, antiproliferative effects against MCF-7 and MDA-MB 231 cells and
showed strong inhibitory effects on COX enzymes [120].
Upregulation of the inducible isoform COX-2 has been
identified in many human cancers and precancerous
lesions. Initially recognized in the context of colorectal
cancer, COX-2 over-expression has also been detected in
approximately 40% of cases of human breast carcinoma.
Furthermore, epidemiologic analyses suggest a protective effect of COX inhibitory drugs with respect to both
colon and breast cancer. Together, these observations
have stimulated widespread enthusiasm for COX-2 as a
molecular target for cancer prevention [122].
Elevated COX-2 protein levels have been detected
immunohistochemically in approximately 40% of invasive breast carcinomas, with individual studies reporting
frequencies ranging from 17% to 84% [123]. COX-2 protein is predominantly confined to the tumor epithelium,
with negligible expression in normal epithelium. In contrast, COX-1 appears to be ubiquitously expressed in
mammary tissues [124]. Since COX-2 is over-expressed in
mammary tumors from rodent breast-cancer models,
these animals provide useful experimental systems in
which to evaluate the role of COX enzymes. Numerous
studies have shown that experimental breast cancer can
be suppressed by inhibiting COX activity with either conventional NSAIDs or COXibs [125].
Interestingly, correlations between COX and aromatase expression have been observed in human breast carcinomas [126]. These correlations are thought to reflect a
causal link, because prostaglandine signaling can stimulate transcription of the CYP19 gene [127].
In our studies, we demonstrated a clear synergism
between the selective COX-2 inhibitor celecoxib and the
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Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
Figure 22. Linear chemical structure of the active compounds
investigated usually as ER ligands and for chemopreventing and
treating breast cancer.
Figure 21. Structures of hydroxylated aryl-substituted 2-imidazolines.
aromatase inhibitor formestane regarding their cytotoxic effects against MCF-7 cells [128].
Hydroxylated aryl-substituted 2-imidazolines (Fig. 21)
belong to the effective Type-II estrogens [129, 130]. Their
relative binding affinities were very low, but many of
them exhibited full agonist activity in the luciferase
assay with MCF-7-2a cells. Of them, 2-imidazoline 8 was
the most active compound. Other vast variations of the
substituents and the substitution sites led to a reduction
or even a complete loss of agonist activity. Several 2-imidazolines grafted separately by a basic side chain on one
of the phenol group showed distinct hormonal effects
[131]. The majority of them exhibited agonistic effects.
Imidazoline 9 was evaluated as a stronger agonist at ERa
versus a weaker one at ERb, whereas two C2 ethyl-substituted compounds possessed high antagonistic properties. For instance, imidazoline 10 antagonized the E2
effect at ERa strongly, while its antagonistic properties at
ERb were distinctly lower, mixing with partial agonistic
effects [131].
Aryl-substituted six-membered heterocycles, 2,3-diarylpiperazines such as 11, can also activate gene expression
but to a lesser degree than 2-imidazolines [132]. This is
the consequence of different contacts in the LBD. While
one hydroxyl group of the 2,3-diarylpiperazines comes
near to Asp351, the spatial structure of the 2-imidazolines allows the phenolic ring a close contact to Thr347
in the LBD of ERa as depicted in Fig. 16.
Numerous diazenes (pyrazines, pyrimidines, and pyridazines) were also investigated and several of them were
found to be considerably more agonistic on ERa than on
ERb [133].
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All these results described above indicate that the spatial structure of the ligands determines their hormonal
activity [110]. This, in particular, signals the significance
to develop new lead structures. Despite investigating the
novel synthetic ligands, those safe natural phytoestrogens were not out of our sight.
Phytoestrogens
Phytoestrogens are plant-derived chemicals that have
estrogenic activity, combining with ERs and initiating
estrogen-dependent transcription [134, 135]. They are
classified as several different groups according to their
chemical structure: isoflavones, flavones, flavanones,
coumestans, stilbenes, and lignans. The most widely
studied are the isoflavones, present in high concentrations in soy products and red clover [136], followed by the
flavones and then coumestans [134]. The active compounds investigated usually as ER ligands and for chemopreventing and treating breast cancer have a linear
chemical structure similar to that of E2 (Fig. 22)
[134, 137, 138], so that they can bind to ERa with the
Type-I mode and to ERb.
The increasing research interest in phytoestrogens
probably stemmes from following five arguments: (1)
there is a possibility that the traditionally low breast cancer incidence rates in Asia are associated with the high
dietary intake of phytoestrogens; (2) widely diverse beneficial biological effects, such as anti-inflammatory, antioxidant, and anticancer effects; (3) safety in use as natural products; (4) partial selectivity for ERb; (5) derivatization i. e., novel biologically active derivatives can be
derived from the core structure of phytoestrogens. For
the investigation of phytoestrogens, the key questions
are whether or not and how they act as anticancer drugs
or chemopreventing agents; should they be consumed
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Arch. Pharm. Chem. Life Sci. 2009, 342, 133 – 149
(or not) at very high levels in the diet, or should they
rather (or not) be taken as medicine, because they are
already more or less present in the daily food. There is
still no conclusive evidence that a high dietary intake of
phytoestrogens and the reduced incidence of breast cancer are directly related [134]. But as ER ligands, varieties
of phytoestrogens remain in binding to the ER and breast
cancer.
Conclusion
The estrogen receptor plays a central role in the hormone
action. Its conformational changes upon binding different ligands initiate an intra- and /or intercellular biological response and, therefore, offset a corresponding physiological action. The considerably increased understanding of the molecular mechanism of the ER transcription,
the advance in uncovering the ER response pathways, the
exactly illustrative interaction between the estrogen
receptor and ligands and the more clinical research
achievements provide a better basis for the development
of novel and more effective anticancer agents.
It should be noted that there is an indirect hormone
therapy approach beyond the topic of this article, that is,
aromatase inhibitor therapy. The aromatase is a key
enzyme in the conversion of androgens to estrogens. The
aromatase inhibitor, such as formestane, anastrozole, or
letrozole, inhibit the activity of the aromatase enzyme as
to block the synthesis of estrogens. This method becomes
more and more important in adjuvant hormone therapy
for treating breast cancer [139, 140].
This work was supported by grants Gu-285/3-1, Gu-285/3-2 and
Gu-285/5-1 to Gu-285/5-4 as well as the SFB 765 of the Deutsche
Forschungsgemeinschaft.
The authors have declared no conflict of interest.
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