BACKGROUND: Tamoxifen is widely prescribed for the treatment of breast cancer. Its success has been attributed to the modulation of the estrogen receptor. I have previously proposed that the release of arachidonic acid from cells may also mediate cancer prevention. METHODS: Rat liver cells were radiolabelled with arachidonic acid. The release of [(3)H] arachidonic acid after various times of incubation of the cells with tamoxifen was measured. RESULTS: Tamoxifen, at micromolar concentrations, stimulates arachidonic acid release. The stimulation is rapid and is not affected by pre-incubation of the cells with actinomycin or the estrogen antagonist ICI-182,780. CONCLUSIONS: The stimulation of AA release by tamoxifen is not mediated by estrogen receptor occupancy and is non-genomic

ACB Cato

AE Wakeling

AR Brash

BS Katzenellenbogen

EC Dietze

F Peyrade

JD Croxtall

JI MacGregor

KP Whiting

L Levine

Lawrence Levine

M Obrero

ME Surette

MM Gottardis

S Mandlekar

SR Hammes

TA Chan

VC Jordan

W Zhang

WT Couldwell

English

PubMed

Springer - Publisher Connector

BioMed CentralBMC Cancer
ssOpen AcceResearch article
Tamoxifen stimulates arachidonic acid release from rat liver cells 
by an estrogen receptor-independent, non-genomic mechanism
Lawrence Levine*
Address: Department of Biochemistry, Brandeis University, Waltham, MA 02454, U.S.A
Email: Lawrence Levine* - llevine@brandeis.edu
* Corresponding author    
Abstract
Background: Tamoxifen is widely prescribed for the treatment of breast cancer. Its success has
been attributed to the modulation of the estrogen receptor. I have previously proposed that the
release of arachidonic acid from cells may also mediate cancer prevention.
Methods: Rat liver cells were radiolabelled with arachidonic acid. The release of [3H] arachidonic
acid after various times of incubation of the cells with tamoxifen was measured.
Results: Tamoxifen, at micromolar concentrations, stimulates arachidonic acid release. The
stimulation is rapid and is not affected by pre-incubation of the cells with actinomycin or the
estrogen antagonist ICI-182,780.
Conclusions: The stimulation of AA release by tamoxifen is not mediated by estrogen receptor
occupancy and is non-genomic.
Background
Clinically, breast cancer chemoprevention by tamoxifen
has been attributed to its antiestrogenic properties [1–3].
Tamoxifen also has been shown to prevent cancer in ani-
mal studies by a mechanism of action that is based, in
part, on its antiestrogenic activity [1,4]. However, at µM
levels, tamoxifen releases arachidonic acid (AA) from rat
liver cells [5]. This ability to release AA, a molecule whose
multiple bioactivities [6] include induction of apoptosis
[7], suggests a mechanism for cancer prevention that does
not require metabolism by cyclooxygenase [8]. I suggest
that AA release from cells may be a part of a mechanism
by which tamoxifen prevents cancer.
Tamoxifen has several biological effects, some of which
may be beneficial. It also has unfavorable effects, espe-
cially its estrogenic activity on the uterus [1,2]. One of the
many activities associated with perturbation of the plasma
membrane and/or release of AA may mediate some of
these biological effects.
Materials and Methods
The C-9 rat liver cell line was purchased from the Ameri-
can Type Culture Collection (Manassas, VA, USA). The
cells were maintained in Eagle's minimum essential
medium (MEM) supplemented with 10% fetal bovine
serum. [3H] AA (91.8 Ci/mmol) was obtained from NEN
Life Science Products, Inc. (Boston, MA, USA). ICI-
182,780 was purchased from Tocris Cookson, Inc. (Ball-
win, MO, USA). All other reagents were from Sigma
Chemical Co. (St. Louis, MO, USA).
Two days prior to experiments, the rat liver cells were
treated with 0.25% trypsin-EDTA and, after addition of
minimal essential media (MEM) containing 10% fetal calf
serum, the floating cells were seeded onto 35 mm culture
Published: 19 September 2003
BMC Cancer 2003, 3:24
Received: 09 June 2003
Accepted: 19 September 2003
This article is available from: http://www.biomedcentral.com/1471-2407/3/24
© 2003 Levine; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media 
for any purpose, provided this notice is preserved along with the article's original URL.Page 1 of 6
(page number not for citation purposes)
BMC Cancer 2003, 3 http://www.biomedcentral.com/1471-2407/3/24dishes. The plating densities varied from 0.1 to 0.5 × 105
cells/35 mm dish. The freshly seeded cultures were incu-
bated for 24-h to allow for cell attachment. After decanta-
tion of MEM containing the fetal bovine serum, 1.0 ml
fresh MEM containing 10% fetal bovine serum and [3H]
AA (0.2 µCi/ml) were added and the cells incubated for
another 24-h. The cells were washed 4 times with MEM
and incubated for various periods of time with 1.0 ml of
MEM containing 1.0 mg BSA/ml and different concentra-
tions of each compound. The culture fluids were then
decanted, centrifuged at 2000 × g for 10 min, and 200 µl
of the supernate counted for radioactivity. Radioactivity
recovered in the washes before the 6-h incubation was
compared to input radioactivity to calculate the % radio-
activity incorporated into the cells [9]. For PGI2 produc-
tion, 1.0 ml of MEM supplemented with 10% fetal bovine
serum, void of [3H]AA, was added after the first 24-h incu-
bation. The cells were incubated for another 24-h, washed
three times with MEM, then incubated with lactacystin
plus TPA and the compounds in MEM/BSA for various
periods of time. The culture fluids were decanted and ana-
lyzed for 6-keto-PGF1α,the stable hydrolytic product of
PGI2, by radioimmunoassay [10].
The [3H] AA release is presented as a percentage of the
radioactivity incorporated by the cells. Except for the time-
course experiments which used duplicate dishes. (Fig. 2),
three to six culture dishes were used for each experimental
point. The data are expressed as mean values ± SEM
(number of dishes). The data were evaluated statistically
by the unpaired Student's t-test. A P value < 0.05 was con-
sidered significant.
Results
The release of AA from rat liver cells after a 6-h incubation
with tamoxifen is dependent on the concentration of the
drug (Fig. 1). Even at 8 µM, the stimulation of AA release
is significant statistically. Tamoxifen (8 µM) also stimu-
lates significantly AA release from rat glial cells (data not
shown). After 30 min. incubation with 12 µM tamoxifen,
AA release from rat liver cells is stimulated (Fig. 2). Even
after a 5 min incubation, the AA release by 16 µM
tamoxifen is stimulated significantly, 1.4 ± 0.06 (7) vs 1.6
± 0.06 (7) % released in MEM/BSA and in the MEM/BSA
containing the 16 µM tamoxifen respectively (P < 0.02).
Tamoxifen (16 µM) also stimulates prostacyclin produc-
tion. After a 6-h incubation, the stimulation of 6-keto-
PGF1∝ production by 16 µM tamoxifen was 3.1 ± 0.18 fold
when tested with cells ranging from the 19th to the 50th
passage. The AA release by 8 µM tamoxifen after a 6-h
incubation is not affected by pre-incubation of the cells
for 2-h with 1 µM actinomycin (Fig. 3). Under these con-
ditions, AA release and prostaglandin production induced
by treatment of cells with lactacystin plus 12-0-tetrade-
canoyl-13-acetate or 15-deoxy-∆12,14-PGJ2 are inhibited
[11]. Pre-incubation of the cells with the estrogen antago-
nist ICI-182,780 (50 µM) [12]for 2-h does not signifi-
cantly affect the stimulation of AA release by 16 µM
tamoxifen (Fig. 4). ICI-182,780 (50 µM), however, did
affect AA release stimulated by 17β-estradiol, 22(R)cho-
lesterol, indomethacin, all-trans-retinoic acid and the
tyrosine analog of thiazolidinedione, GW7845 [11].
Discussion
Tamoxifen, in addition to its actions mediated by the
estrogen receptor (ER), inhibits protein kinase C [13] and
induces apoptosis in normal human mammary epithelial
cells [14]. Submicromolar concentrations of tamoxifen or
4-hydroxytamoxifen induce apoptosis in ER-positive
HeLa cells. However, both of these compounds, as well as
estrogen, at concentrations of 10–20 µM, induce apopto-
sis in ER-negative HeLa cells [15]. The induction of apop-
tosis probably contributes to the effectiveness of
tamoxifen in cancer prevention. AA, also induces apopto-
sis [8]. It has been suggested that cancer prevention by
nonsteroidal antiinflammatory drugs (NSAIDs) is medi-
ated by release of AA [8]. Several agents that prevent
The dependence of AA release on tamoxifen concentrationFigur  1
The dependence of AA release on tamoxifen concentration. 
The cells were incubated for 6-h. The analyses were per-
formed with triplicate dishes. Each bar gives the mean value 
and the brackets give the SEM. * = statistically different vs 
control. These data are representative of several independ-
ent experiments with the same results.Page 2 of 6
(page number not for citation purposes)
BMC Cancer 2003, 3 http://www.biomedcentral.com/1471-2407/3/24cancer, including retinoids, NSAIDs, vitamin D3 and some
anti-oxidants, release AA from rat liver cells [5].
The AA release by tamoxifen and other reagents studied in
my laboratory occurs with µM concentrations [5,9,11].
These experiments were carried out in the presence of BSA
(1.0 mg/ml), and therefore do not differentiate between
the protein-bound and free reagent. Thus, they are likely
overestimated values. Nevertheless, the possibility that
general necrotic cell death may cause AA release, must be
considered. Lactacystin, (5.4 µM) phenylmethylsulpho-
nyl flouride, (1 mM) carbobenzoxyleucylleucyleucinal,
(1.0 µM) and carbobenzoxyleucylleucylnorvalinal (0.5
µM) were tested for rat liver cell viability by a tetrazolium-
based assay. They were not toxic at these concentrations
[16]. Proteosome inhibitors are not toxic to several other
cells in culture [16]. No toxicity of tamoxifen, at concen-
trations of 10 to 20 µM for A549 human lung adenocarci-
noma (ER-negative) cells was reported [17]; nor was 10
µM tamoxifen toxic when tested on rat glial cells and
breast cancer MCF-7 cells [18]. Even when cell viability of
three different breast cell lines (ER-positive MCF-7; ER-
negative MDA-MB-239 and ER-negative BT-20 cells) was
measured after incubation with 25 µM tamoxifen for 24-
h, the loss in viability was due to apoptosis [19] and was
not the result of necrotic cell death. Concentrations of
tamoxifen used in this report are comparable to those
found to induce apoptosis, not necrotic cell death. The
median concentration of tamoxifen and its metabolites
for clinical effectiveness in the treatment of breast cancer
varies from 0.8 µM to 2.4 µM, depending on the age of the
woman [20].
The stimulation of AA release by tamoxifen is non-tran-
scriptional as indicated by the lack of inhibition by actin-
omycin (Fig. 3). The stimulation of AA release by
tamoxifen also is not ER mediated as indicated by the lack
of inhibition by the estrogen antagonist ICI-182,780 (Fig.
Time course of release of AA during incubation with 12 µM tamoxifen (*) and MEM/BSA control ()F gure 2
Time course of release of AA during incubation with 12 µM tamoxifen (*) and MEM/BSA control (). Analyses were per-
formed on duplicate or triplicate dishes. The average value is recorded.Page 3 of 6
(page number not for citation purposes)
BMC Cancer 2003, 3 http://www.biomedcentral.com/1471-2407/3/244). Tamoxifen at micromolar concentration may be inter-
calating into the lipid bilayer of the plasma membranes
and affecting the fluidity and biochemical properties of
the cell [21]. Another possibility is that tamoxifen is acting
via G protein-coupled receptors [22,23].
Conclusions
Tamoxifen stimulates AA release from rat liver cells by a
non-genomic, ER-independent pathway. In view of induc-
tion of apoptosis by AA, its release per se could, in addition
to its effects on the ER, mediate cancer prevention.
Effect of pre-incubation of cells with 1 µM actinomycin on the stimulation of AA release by tamoxifenFigure 3
Effect of pre-incubation of cells with 1 µM actinomycin on the stimulation of AA release by tamoxifen. The cells were pre-incu-
bated for 2-h in the presence of 1 µM actinomycin. During the 2-h pre-incubation, the % AA released by incubation with the 
control MEM/BSA and MEM/BSA containing actinomycin was 4.2 ± 0.21(4) and 4.3 ± 0.11(4) respectively. Each bar gives the 
mean value and the brackets give the SEM. () = MEM/BSA; (■) = MEM/BSA containing 8 µM tamoxifen. The data recorded 
are the sum of the AA released during the 2-h pre-incubation plus the average of the subsequent 6-h incubation. They are rep-
resentative of three separate experiments with similar results.Page 4 of 6
(page number not for citation purposes)
BMC Cancer 2003, 3 http://www.biomedcentral.com/1471-2407/3/24Effect of pre-incubation of cells with 50 µM of estrogen antagonist ICI-182,780 for 2-h on the stimulation of AA release by tamoxifenFigure 4
Effect of pre-incubation of cells with 50 µM of estrogen antagonist ICI-182,780 for 2-h on the stimulation of AA release by 
tamoxifen. The cells were pre-incubated for 2-h with 50 µM ICI-182,780. The data recorded are the sum of the AA released 
during the 2-h pre-incubation plus the average of the subsequent 6-h incubation. During the 2-h pre-incubation, 3.1 ± 0.02(4) 
and 2.6 ± 0.26(4) % AA was released by the control MEM/BSA and MEM/BSA containing ICI-182,780 respectively. () = MEM/
BSA; (■) = MEM/BSA containing 16 µM tamoxifen. Each bar gives the mean value and the brackets give the SEM.Page 5 of 6
(page number not for citation purposes)
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Competing interests
None declared.
Acknowledgements
I thank Dr. Armen Tashjian Jr., Department of Cancer Cell Biology, Har-
vard School of Public Health, for the continued interest in these studies and 
Hilda B. Gjika for preparation of the manuscript.
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Tamoxifen stimulates arachidonic acid release from rat liver cells by an estrogen receptor-independent, non-genomic mechanism

Crossref

Abstract Background Tamoxifen is widely prescribed for the treatment of breast cancer. Its success has been attributed to the modulation of the estrogen receptor. I have previously proposed that the release of arachidonic acid from cells may also mediate cancer prevention. Methods Rat liver cells were radiolabelled with arachidonic acid. The release of [3H] arachidonic acid after various times of incubation of the cells with tamoxifen was measured. Results Tamoxifen, at micromolar concentrations, stimulates arachidonic acid release. The stimulation is rapid and is not affected by pre-incubation of the cells with actinomycin or the estrogen antagonist ICI-182,780. Conclusions The stimulation of AA release by tamoxifen is not mediated by estrogen receptor occupancy and is non-genomic.</p

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Tamoxifen stimulates arachidonic acid release from rat liver cells by an estrogen receptor-independent, non-genomic mechanism

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