Melatonin inhibits both ER alpha activation and breast cancer cell proliferation induced by a metalloestrogen, cadmium.
ABSTRACT Cadmium (Cd) is a heavy metal affecting human health both through environmental and occupational exposure. There is evidence that Cd accumulates in several organs and is carcinogenic to humans. In vivo, Cd mimics the effect of estrogens in the uterus and mammary gland. In estrogen-responsive breast cancer cell lines, Cd stimulates proliferation and can also activate the estrogen receptor independent of estradiol. The ability of this metalloestrogen to increase gene expression in MCF7 cells is blocked by anti-estrogens suggesting that the activity of these compounds is mediated by ER alpha. The aims of this work were to test whether melatonin inhibits Cd-induced proliferation in MCF7 cells, and also to study whether melatonin specifically inhibits Cd-induced ER alpha transactivation. We show that melatonin prevents the Cd-induced growth of synchronized MCF7 breast cancer cells. In transient transfection experiments, we prove that both ER alpha- and ER beta-mediated transcription are stimulated by Cd. Melatonin is a specific inhibitor of Cd-induced ER alpha-mediated transcription in both estrogen response elements (ERE)- and AP1-containing promoters, whereas ER beta-mediated transcription is not inhibited by the pineal indole. Moreover, the mutant ER alpha-(K302G, K303G), unable to bind calmodulin, is activated by Cd but becomes insensitive to melatonin treatment. These results proved that melatonin inhibits MCF7 cell growth induced by Cd and abolishes the stimulatory effect of the heavy metal in cells expressing ER alpha at both ERE-luc and AP1-luc sites. We can infer from these experiments that melatonin regulates Cd-induced transcription in both ERE- and AP1 pathways. These results also reinforce the hypothesis of the anti-estrogenic properties of melatonin as a valuable tool in breast cancer therapies.
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ABSTRACT: Cadmium (Cd) is an underground mineral widely used in the steel industry, in plastics, and as a component of batteries. It is an industrial and environmental pollutant released as an air contaminant from fertilizers and, more prominently, in the form of wastewater. Food, drinking water, and, mainly, inhalation of smoke from cigarettes are sources of daily exposure of humans to the heavy metal. Although Cd has no known useful function for humans as well as other organisms, it appears to evoke in cells a number of responses that involve not only death signaling but also protective reactions against the toxicity. This finding prompted a number of experimental studies aimed to elucidate the cellular and molecular aspects of Cd-dependent regulation of gene expression and signal transduction pathways in different model system. Here, the authors briefly review the role of Cd as a transcriptional regulator in diverse cytotypes of human origin, focusing in particular on its effects on two classes of genes, i.e., stress-response genes such as metallothioneins (MTs), heme oxygenase, and heat shock proteins (hsps), and apoptosis-related genes, but giving also an overview of many other examples of genes involved in cell metabolism and both intracellular and extracellular signalization whose expression levels are controlled by Cd.Critical Reviews in Toxicology 11/2010; 41(1):75-82. · 6.25 Impact Factor
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ABSTRACT: Melatonin regulates a variety of biological processes, which are the control of circadian rhythms, regulation of seasonal reproductive function and body temperature, free radical scavenging and so on. Our previous studies have shown that various cells exist in human and mouse tooth germs that express the melatonin 1a receptor (Mel1aR). However, little is known about the effects of melatonin on tooth development and growth. The present study was performed to examine the possibility that melatonin might exert its influence on tooth development. DP-805 cells, a human dental papilla cell line, were shown to express Mel1aR. Expression levels of mRNA for Mel1aR in DP-805 cells increased until 3 days after reaching confluence and decreased thereafter. Real-time reverse transcription-polymerase chain reaction showed that melatonin increased the expression of mRNAs for osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotin (DSPP). Melatonin also enhanced the mineralized matrix formation in DP-805 cell cultures in a dose-dependent manner. These results strongly suggest that melatonin may play a physiological role in tooth development/growth by regulating the cellular function of odontogenic cells in tooth germs.International Journal of Molecular Sciences 01/2014; 15(10):17304-17317. · 2.46 Impact Factor
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ABSTRACT: In vitro and animal data suggest cadmium, a heavy metal that contaminates some foods and tobacco plants, is an estrogenic endocrine disruptor. Elevated estrogen exposure is associated with breast, endometrial, and ovarian cancer risk. Examine the association between dietary cadmium intake and risk of these cancers in the large, well-characterized Women's Health Initiative (WHI). 155,069 postmenopausal women, age 50-79, enrolled in the WHI clinical trials or observational study participated in this study. We estimated dietary cadmium consumption by combining baseline food frequency questionnaire responses with US Food and Drug Administration data on food cadmium content. Participants reported incident invasive breast, endometrial, or ovarian cancer and WHI centrally adjudicated all cases through August 2009. We applied Cox regression to estimate adjusted hazard ratios (HRs) and 95% confidence intervals (95% CIs) for each cancer, comparing quintiles of energy-adjusted dietary cadmium intake. Over an average of 10.5 years, 6,658 invasive breast cancers, 1,198 endometrial cancers, and 735 ovarian cancers were reported. We observed no statistically significant associations between dietary cadmium and risk of any of these cancers after adjustment for potential confounders including total dietary energy intake. Results did not differ in any sub-group of women examined. We found little evidence that dietary cadmium is a risk factor for breast, endometrial, or ovarian cancers in postmenopausal women. Misclassification in dietary cadmium assessment may have attenuated observed associations.Environmental Health Perspectives 03/2014; · 7.26 Impact Factor
Melatonin inhibits both ERa activation and breast cancer cell
proliferation induced by a metalloestrogen, cadmium
Cadmium (Cd) is an environmental pollutant widely used in
industry and which is also present in tobacco smoke.
Activities such as smelting, fuel combustion, smoking, or
Ni-Cd battery manufacturing are the main sources of
human exposure to this heavy metal which, because of its
long biological half-life, estimated to be between 15 and
20 yr , accumulates in the body.
Cadmium has been classified as a human carcinogen by
the International Agency for Research on Cancer .
Epidemiological studies have identified lung, prostate,
kidney and stomach as targets for Cd-induced tumorigen-
esis . In rats, Cd can induce prostate and testicular
tumors . The mechanisms of Cd carcinogenesis are not
well understood. Cd is cytotoxic and induces radical-
dependent DNA-damage at concentrations ranging from
0.1 to 10 mm , whereas at lower doses (1–100 lm) it
decreases DNA replication and repair , and up-regulates
the expression of proto-oncogenes such as c-fos, c-jun and
c-myc . It has also been described that Cd perturbs the
folding of p53 and impairs p53 induction by DNA-
damaging agents inactivating the wild-type protein . Cd
also damages the E-Cadherin-dependent junctions between
cells, a fact that could represent an important step in tumor
initiation and promotion [8, 9].
There is increasing evidence relating Cd and breast
cancer. It has been reported that Cd has a potent estrogen-
like activity in vivo. In rats, the exposure to this heavy
metal increases uterine weight, induces the expression of
progesterone receptor (PgR), increases the proliferation of
the endometrium, and promotes growth and development
of the mammary glands increasing the formation of side
branches and alveolar buds and well as the production of
casein and whey acidic protein .
In breast cancer cells, Cd acts like steroidal estrogens
which induce MCF7 proliferation, probably by forming a
high-affinity complex with the hormone binding domain of
the estrogen receptor alpha (ERa) [11–12]. MCF7 cells
treated with Cd showed decreased levels of ER protein and
ER mRNA . On the contrary, Cd treatment increases
PgR receptor and cathepsin D mRNA levels. The heavy
Abstract: Cadmium (Cd) is a heavy metal affecting human health both
through environmental and occupational exposure. There is evidence that Cd
accumulates in several organs and is carcinogenic to humans. In vivo, Cd
mimics the effect of estrogens in the uterus and mammary gland. In estrogen-
responsive breast cancer cell lines, Cd stimulates proliferation and can also
activate the estrogen receptor independent of estradiol. The ability of this
metalloestrogen to increase gene expression in MCF7 cells is blocked by anti-
estrogens suggesting that the activity of these compounds is mediated by
ERa. The aims of this work were to test whether melatonin inhibits
Cd-induced proliferation in MCF7 cells, and also to study whether melatonin
specifically inhibits Cd-induced ERa transactivation. We show that
melatonin prevents the Cd-induced growth of synchronized MCF7 breast
cancer cells. In transient transfection experiments, we prove that both
ERa- and ERb-mediated transcription are stimulated by Cd. Melatonin is a
specific inhibitor of Cd-induced ERa-mediated transcription in both estrogen
response elements (ERE)- and AP1-containing promoters, whereas
ERb-mediated transcription is not inhibited by the pineal indole. Moreover,
the mutant ERa-(K302G, K303G), unable to bind calmodulin, is activated
by Cd but becomes insensitive to melatonin treatment. These results proved
that melatonin inhibits MCF7 cell growth induced by Cd and abolishes the
stimulatory effect of the heavy metal in cells expressing ERa at both ERE-luc
and AP1-luc sites. We can infer from these experiments that melatonin
regulates Cd-induced transcription in both ERE- and AP1 pathways. These
results also reinforce the hypothesis of the anti-estrogenic properties of
melatonin as a valuable tool in breast cancer therapies.
C. Martı ´nez-Campa1, C. Alonso-
Gonza ´lez1, M. D. Mediavilla1,
S. Cos1, A. Gonza ´lez1, S. Ramos2
and E. J. Sa ´nchez-Barcelo ´1
1Department of Physiology and Pharmacology,
School of Medicine, University of Cantabria,
Santander;2Department of Biochemistry and
Molecular Biology, IUOPA, University of
Oviedo, Oviedo, Spain
Key words: cadmium, estrogen receptors,
MCF-7 cells, melatonin
Address reprint requests to Emilio J. Sa ´nchez-
Barcelo ´, Departamento de Fisiologı ´a y Farm-
acologı ´a, Facultad de Medicina, Universidad
de Cantabria, Cardenal Herrera Oria s/n
39011 Santander, Spain.
Received October 24, 2005;
accepted December 7, 2005.
J. Pineal Res. 2006; 40:291–296
? 2006 The Authors
Journal compilation ? 2006 Blackwell Munksgaard
Journal of Pineal Research
metal also stimulates estrogen response elements (ERE) in
transient transfection experiments .
Melatonin, the major secretory product of the pineal
gland, has oncostatic and anti-proliferative effects on
endocrine-responsive neoplasms, especially in those con-
cerning the mammary gland [13, 14]. The most common
conclusion in animal models of tumorigenesis is that
either experimental manipulations that activate the pineal
gland or the administration of melatonin is what reduces
the incidence and development of chemically induced
mammary tumors, whereas pinealectomy usually stimu-
lates breast cancer growth [15–17]. Different mechanisms
have been proposed to explain how melatonin could
reduce the development of mammary tumors. Studies
using MCF7 human breast cancer cells demonstrate that
physiological concentrations of melatonin (1 nm to 1 pm)
exert a direct anti-proliferative effect on estradiol (E2)-
induced proliferation of these cells [18, 19] and reduce
their invasiveness, causing a decrease in cell attachment
and cell motility, probably by interacting with estrogen-
mediated mechanisms . It has been demonstrated that
melatonin inhibits MCF7 cell proliferation by inducing
an arrest of cell cycle, dependent on an increased
expression of p21WAF1 protein, which is mediated by
the p53 pathway . Melatonin acts as an anti-estrogen
by preventing the E2-dependent transcriptional activation
in MCF7 cells through destabilization of the E2-ER
complex from binding to DNA . Calmodulin (CaM)
was proposed as a potential candidate for mediating the
anti-estrogenic effects of melatonin . Indeed, it has
been recently reported that melatonin is a specific
inhibitor of E2-induced ERa-mediated transcription in
both ERE- and AP1-containing promoters, whereas ERb-
mediated transactivation is not affected. Moreover, the
mutant ERa (K302G, K303G), unable to bind CaM,
normally transactivates in response to E2treatment, but
becomes insensitiveto melatonin
melatonin does not affect the binding of coactivators to
ERa, indicating that melatonin action is different from
that of current therapeutic anti-estrogens used in breast
cancer therapy .
The aims of the present work were to test whether
melatonin inhibits Cd-induced proliferation in MCF7
cells, and also to study whether melatonin specifically
inhibits Cd-induced ERa transactivation. The results
obtained proved that, indeed, melatonin inhibits MCF7
cell growth induced by Cd and the stimulatory effect of
the heavy metal in cells expressing ERa at both ERE-luc
and AP1-luc sites was abolished by treatment with
melatonin. We can infer from these experiments that
melatonin regulates Cd-induced transcription in both
ERE- and AP1 pathways.
Materials and methods
Melatonin, 17b-estradiol (E2), Epidermal Growth Factor
(EGF), cadmium chloride, 4-hydroxytamoxifen, and other
chemicals were purchased from Sigma-Aldrich (Madrid,
The expression vector pcDNA-ERa has been previously
described . pCMX-mERb were kindly provided by
Dr V. Gigue ´ re from the R.W. Johnson Pharmaceutical
Research Institute, Don Mills, Ontario, Canada. The
plasmid 3x-ERE-TATA-Luc was kindly provided by
Dr S. Safe from the Department of Veterinary Physiology
and Pharmacology, Texas A& M University, College
Station, TX, USA. DColl-73 was kindly provided by
Dr A. Aranda from Instituto de Investigaciones Biome ´ dicas
?Alberto Sols’CSIC Madrid, Spain. pRL-TK (Promega
Corp., Madison, WI, USA) was also used in this work.
Cell proliferation assays
MCF7 cells were seeded in 96-multiwell plates at a density of
6000 per well and incubated for 48 hr at 37?C in Dulbecco’s
Modified Eagle’s Medium (DMEM) with 5% of charcoal/
dextran-treated fetal calf serum (sFCS). At 60–80% conflu-
ence, the media were replaced by fresh ones containing 5%
sFCS as well as CdCl2 (1 lm), 17b-estradiol (10 nm),
melatonin (1 nm) or Cd plus melatonin at the same concen-
trations as above. Cells were cultured for 5 days. Medium
was renewed 72 hr after the beginning of treatment. Cell
proliferation was measured by the MTT [3(4,5dimethylthi-
azol-2-yl)-2,5-diphenyl tetrazolium bromide (Molecular
Probes Inc. Eugene, OR, USA) method reading absorbance
at 570 nm in a microplate reader.
Transient transfection assays
HeLa cells were propagated as previously described .
Before transfection, HeLa cells were seeded in 12-well
plates and incubated 12–18 hr at 37?C. Then, cells were
transferred to phenol-red free DMEM containing 0.5%
sFCS and maintained for 3 days. At 60–80% confluency,
cells were transfected with 0.75 lg of ERE-driven or AP1-
driven reporter plasmids, 0.1 lg of ER expression vector
and 75 ng of an internal control Renilla luciferase plasmid,
pRL-TK (Promega Corp., Fitchburg, WI, USA) using
FuGENE 6 Transfection Reagent from Roche Molecular
Biochemicals (Mannheim, Germany) following the manu-
facturer’s protocols. After 18–24 hr, medium was renewed
and cells were stimulated during 24 hr with different
chemicals, as indicated.
Luciferase was assayed with the Dual Luciferase System
(Promega Corp.). Luciferase activities were normalized to
Renilla luciferase activity, in order to correct for differences
in transfection efficiency.
mean ± S.D. of three independent experiments performed
at least in duplicates.
MCF-7 cells were propagated in RPMI 1640 medium
containing 25 mm HEPES/NaOH pH 7.3 and synchronized
cells were transfected as above.
The data on luciferase activity and cell proliferation are
expressed as the mean ± standard errors of the mean
(S.E.M.). Statistical differences between groups were
Martı´nez-Campa et al.
processed by one way analysis of variance (ANOVA)
followed by the Student–Newman–Keuls test. Results were
considered as statistically significant at P < 0.05.
To investigate whether or not melatonin inhibits the growth
of MCF-7 cells induced by Cd, cells were cultured in
DMEM with 5% of estrogen-depleted fetal bovine serum
(sFCS). The addition of 1 lm CdCl2into media stimulates
cell proliferation (Fig. 1, bar 2). The stimulation levels on
MCF7 proliferation reached with this concentration of Cd
was similar to that obtained with 10 nm E2(Fig. 1, bar 3).
This proliferative effect of Cd was significantly inhibited by
1 nm melatonin (Fig. 1, bar 5).
To assess whether Cd stimulates both ERa- and
ERb-mediated transcription, and whether melatonin is able
to specifically inhibit ERa transactivation mediated by the
heavy metal (as has already been described for E2), HeLa
cells were transiently transfected with either ERa or ERb
expression vectors along with a 3x-ERE-TATA-Luc plas-
mid. Both ERa (Fig. 2A, bar 4) and ERb (Fig. 2B, bar 4)
are able to effectively mediate Cd transcriptional activation.
Melatonin (100 nm) inhibited ERa-mediated transactiva-
tion by 45–60% (Fig. 2A, bar 5), whereas ERb-mediated
transcription was not affected by this concentration of
melatonin (Fig. 2A, bar 5). Therefore, we can conclude
from this set of experiments that melatonin is a specific
inhibitor of ERa-mediated transcription induced by Cd in
the same way that the pineal hormone inhibits ERa-medi-
ated transcription induced by estradiol. The mutant
ERa(K302G, K303G), which is unable to bind to CaM,
was stimulated by 1 lm Cd (Fig. 2C, lane 4). Melatonin
was unable to counteract Cd transcriptional activation
mediated by ERa (K302G, K303G) (Fig. 2C, bar 5).
To date, no data have been available indicating whether
or not Cd can activate ERa/AP1 and/or ERb/AP1
pathways. To address this question, HeLa cells were
transfected with either ERa or ERb along with the reporter
plasmid D coll 73-Luc (containing an AP1 binding site). We
found that Cd synergizes with EGF to significantly enhance
AP1 activity in ERa-transfected cells (Fig. 3A, bar 3)
proving that treatment with Cd can stimulate ERa/AP1
pathways. Our data show that in ERa/AP1 Cd (1 lm)
activation is significantly higher than that obtained with
1 nm E2(Fig. 3A, compare bar 3 and 5). Very importantly,
the synergistic effect of EGF and Cd in cells expressing ERa
was sensitive to melatonin, this inhibition being statistically
significant (Fig. 3A, lane 4). Interestingly, Cd does not
diminish AP1 activity in ERb-transfected cells (Fig. 3B, bar
3) as E2does (Fig. 3B, bar 5). Melatonin does not have any
inhibitory effect on AP1 activity mediated by Cd through
ERb (Fig. 3B, bar 4). From these experiments we can infer
that melatonin regulates Cd-ERa-mediated transcription
not only in ERE-dependent pathways but also in AP1
pathways, and importantly, the pineal hormone inhibits the
ERa/AP1 pathway independently of the ERa activator (E2
or Cd), whereas no effect was observed in cells expressing
Cadmium is a heavy metal that is dispersed throughout the
modern environment and that accumulates in the body.
Human exposure to Cd occurs primarily through dietary
sources, cigarette smoking and drinking water [25–28].
Prolonged exposure to Cd has been linked to multiple toxic
effects in both humans and animals and this heavy metal
has been classified as a human carcinogen by the Interna-
tional Agency for Research on Cancer.
Breast cancer is the leading cause of death in woman
between the ages of 35 and 45 . Because the ER is a
mediator of growth, molecules that bind and activate this
receptor can potentially increase the risk of breast cancer.
Thus, there is strong evidence that Cd is a potent
nonsteroidal estrogen in vivo (metalloestrogen). Cd has
been reported to mimic the effects of E2in the estrogen
responsive cell line MCF7 stimulating cell proliferation 
as a result of its ability to form a high affinity complex with
the hormone binding of the ER . As E2also stimulates
AP1 [30, 31], our aim was to test the effects of Cd in both
ERE- and AP1 pathways.
Melatonin is an indole hormone secreted by the pineal
gland only during the night or, more exactly, in darkness.
Among the properties of melatonin, we can underline its
role as an oncostaticagent on hormone-dependent
tumors [13, 18, 20]. It has also been described that
melatonin exerts anti-proliferative effects on MCF7 cells,
which has become a useful model to study the anti-
estrogenic effect of the pineal hormone [32–34]. In
synchronized MCF7 cells E2-induced proliferation is
inhibited by co-treatment with melatonin. As treatment
with Cd also stimulates growth of this cell line, we decide
to test the ability of melatonin to act as an anti-
proliferative agent; our results show that melatonin can
block the proliferation of MCF7 cells triggered by Cd
ControlCadmiumEstradiolMelatonin Cadmium +
Absorbance 570 nm (% of control)
Fig. 1. Cadmium (Cd) induced cell proliferation. Inhibitory effect
of melatonin. MCF7 cells were seeded in 96-multiwell plates at a
density of 6000 per well and incubated for 48 hr at 37?C in DMEM
with 5% sFCS. At 60–80% confluence, the media were replaced by
fresh ones containing 5% cFCS as well as CdCl2(1 lm), 17b-est-
radiol (10 nm), melatonin (1 nm) or Cd (1 lm) plus melatonin
(1 nm). Cells were cultured for 5 days and cell proliferation was
measured by the MTT method. a, P < 0.001 versus control; b,
P < 0.001 versus Cd.
Mel inhibits Cd-induced ERa activation
thus confirming, one more time, the anti-estrogenic
nature of melatonin actions .
Both ERa and ERb bind the same DNA sequence and
activate the transcription of genes regulated by ERE.
However, only ERa, but not ERb, which has a CaM
binding site, interacts with CaM and is specifically inhibited
by both CaM antagonists and melatonin [23, 24]. There-
fore, one of the aims of this work was first to test whether
or not both ERa and ERb are stimulated by Cd, and
secondly, whether melatonin specifically inhibited ERa but
showed no inhibitory effect over ERb-mediated transcrip-
tion. We found that both ERa and ERb are significantly
activated by Cd and that Cd-induced transcription medi-
ated by ERb reaches lower levels than that of ERa, as has
been previously described for E2. Cd-induced transcription
through ERa is inhibited by melatonin at ERE-driven
promoters, as previously shown for E2, whereas ERb
activation is not affected by treatment with the pineal
hormone. Importantly, the ERa (K302G, K303G) mutant,
which does not interact with CaM, is stimulated by Cd but
is not inhibited by melatonin. Therefore, melatonin acts as
an ERa specific inhibitor with independence of the agent
used for cell growth stimulation, either as previously shown
for E2or Cd.
We also addressed the effect of Cd in AP1 sites. It has
been reported that exposure of cells to Cd-induced signi-
ficant activation of AP1 and all three members of the MAP
kinase family in mouse epidermal JB6 cells. The induction
of AP1 activity by Cd appears to involve activation of Erks,
since the induction of AP1 activity by Cd was blocked by
pretreatment of cells with PD98058 . In primary rat
hepatocytes, it has been observed that Cd, through the
E2 (10 nM)
Mel (100 nM)
ERα (k302, 303G) ERβ
Fig. 2. Differential effect of melatonin on the transactivation properties of (A) ERa, (B) ERb and (C) ERa (K302G, K303G). HeLa cells
were transfected with 0.1 lg of ERa, ERb or ERa (K302G, K303G) expression vectors, 50 ng of the internal control plasmid pRL-TK and
0.75 lg of 3xERELuc (ERE-driven reporter plasmid). After 18–24 hr, medium was renewed and cells were stimulated for 24 hr with 10 nm
E2, 1 lm cadmium (Cd) and 100 nm melatonin as indicated. Luciferase activities were normalized to the Renilla luciferase activities. The
data are reported as fold induction relative to untreated cells, which were arbitrarily assigned as 1. The bars represent mean ± S.D. of three
independent experiments performed in duplicates. a, P < 0.001 versus control; b, P < 0.001 versus E2; c, P < 0.001 versus control;
d, P < 0.001 versus Cd; e, P < 0.001 versus control; f, P < 0.001 versus control.
E2 (10 nM)
Cd (1 mM)
Mel (100 nM)
Fig. 3. Effect of melatonin on cadmium (Cd)-dependent transactivation at an AP1 element. (A) ERa and (B) ERb HeLa cells were
transfected with 0.1 lg of the ERa expression vector, 50 ng of internal control plasmid pRL-TK and 0.75 lg of the AP1-containing reporter
plasmid (D coll. 73-Luc). Cultures were stimulated for 48 hr with 1 lg/mL EGF, 10 nm E2, 1 lm Cl2Cd and 100 nm of melatonin as
indicated. The data are reported as fold-induction relative to untreated cells, which were arbitrarily assigned as 1. The bars represent
mean ± S.D. of three independent experiments run in duplicates; a, P < 0.001 versus EGF; b, P < 0.001 versus EGF plus Cd;
c, P < 0.001 versus EGF plus Cd; d, P < 0.001 versus EGF plus estradiol; e P < 0.001 versus estradiol plus EGF.
Martı´nez-Campa et al.
generation of reactive oxygen species and prior to signifi-
cant cellular damage, activates the stress activated signal
protein JNK, regulates c-jun expression, and promotes the
binding of a redox sensitive transcription factor AP1 .
It has been documented that ERa and ERb signal in
opposite ways when complexed with the natural hormone
E2 from an AP1 site: with ERa, 17b-estradiol activates
transcription, whereas with ERb, 17b-estradiol inhibits
transcription . The effect of many environmental
estrogenic chemicals on AP1 has been tested with either
ERa or ERb in NIH 3T3 cells. Compounds such as
bisphenol A or t-methylbutylphenol activate only ERa but
not ERb-dependent AP1 transcriptional activity .
For all the reasons mentioned above, we tested the ability
of Cd to modulate both ERa- and ERb-dependent AP1
transactivation. We found that Cd significantly stimulates
AP1 activity in ERa-transfected cells. Interestingly, whereas
neither stimulates nor inhibits transcription on this system.
Because it has been previously described that CaM
antagonists and melatonin also inhibit ERa-dependent AP1
transcriptional activity [23, 24] we tested the ability of
melatonin to inhibit ERa-mediated transcription in AP1-
driven promoters. We found that melatonin significantly
inhibited ERa-mediated transactivation in AP1 sites;
therefore, melatonin also acts as a regulator of the ERa-
CaM/AP1 pathway when this pathway is stimulated by Cd.
In summary, as chemicals such as Cd mimic the effect of
estrogens, the high incidence of breast cancer among
women working in the chemical industry could be explained
because of the estrogenic properties of this heavy metal.
The protective effects of melatonin on Cd-induced breast
cancer cell proliferation, now demonstrated, point to a
possible role of this indolamine as a preventive agent for Cd
environmental or occupational contamination.
This work has been supported by grants (PI042603) from
the Health Institute ?Carlos III? and (BFI2003-06305) from
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