Effects of 4-OH Tamoxifen on HEC 1B Endometrial Cancer Cells

1
Effects of 4-OH Tamoxifen on HEC 1B Endometrial
Cancer Cells Tracey Einem, Carolina Boet, Dr.
Maria Cuevas1, Dr. Maha Zewail-Foote2 1Biology
Department, 2 Chemistry Department, Southwestern
University, 1001 E. University Ave. Georgetown,
TX 78626 einemt_at_southwestern.edu,
boetc_at_southwestern.edu, cuevasm_at_southwestern.edu,
footezm_at_southwestern.edu
Western Blot Analysis Cell protein extracts were
prepared from HEC 1B cells treated with 0, 1µM,
and 10µM 4-OH TAM for 24 hours at the incubating
conditions previously described. Western blot
analysis was performed using standard procedure.






ABSTRACT
Figure 3 DNA Gel Electrophoresis of extracts
from ER positive HEC 1A (lanes 2-4) and ER
negative HEC 1B (lanes 9-11) endometrial
adenocarcinoma cells treated with 10µM 4-OH TAM
and estrogen. Characteristic DNA fragmentation
(180-200bp) was not seen following DNA gel
electrophoresis in all samples. This suggests
apoptosis did not occur in either cell line.
Tamoxifen, a well-known drug for breast cancer
treatment, has been found to be an estrogen
antagonist in the breast, but a partial estrogen
agonist in the endometrium. In addition,
tamoxifen metabolites have been shown to produce
DNA adducts in a variety of tissues. In this
study, we investigated the potential of 4-OH
tamoxifen (4-OH TAM) to exert proliferative
effects via an ER independent pathway. For this
purpose, we used the ER negative human
endometrial adenocarcinoma cell line, HEC 1B, and
compared results with the ER positive human
endometrial adenocarcinoma cell line, HEC 1A. We
treated ER negative HEC 1B cells with different
estrogen and 4-OH TAM concentrations ranging from
0-100?M and incubated at 37ºC, 5 CO2 atmosphere.
Using a dye-exclusion assay and colorimetric
method (MTS Assay) we observed that lower
concentrations of 4-OH TAM had little effect on
HEC 1B cell proliferation. However, at higher
concentrations (10,100?M), cell proliferation was
inhibited by almost 100 within 24 hours. When
HEC 1B cells were treated with different doses of
estrogen, we observed an initial proliferative
response after 24 hours at low doses, followed by
a partial inhibition of growth after 2 to 3 days.
However, at the highest dose (100?M), we
observed a complete inhibition of cell
proliferation. In contrast, ER positive HEC 1A
cells were refractory to low dose estrogen but,
like HEC1B cells, were completely growth
inhibited by 100?M estrogen. When ER positive
HEC 1A cells were treated with higher
concentrations of 4-OH TAM, no inhibition was
observed at 1?M whereas at 10?M, a steady decline
in cell growth was found. Similar to HEC 1B
cells, complete inhibition was observed at 100?M
within 24 hours. In order to determine if
apoptosis is the underlying mechanism of cell
death, we incubated HEC 1B cells with 10?M 4-OH
TAM for 24 hours and assayed for DNA laddering
and expression of the pro-apoptotic protein,
caspase 8. Preliminary data did not indicate
involvement of an apoptotic pathway. These
results suggest that 4-OH TAM is promoting cell
death via necrosis.
RESULTS
Figure 4 Western Blot analysis of caspase 8 from
4-OH TAM-treated HEC 1B (ER negative) endometrial
adenocarcinoma cells. Protein extracts and
western blot analysis were conducted as described
in Materials and Methods. 10µL of protein
extract from HEC 1B cells treated with 1µM or
10µM 4-OH TAM were fractioned on a 12
polyacrylamide gel, transferred to a PVDF
membrane, and immunoblotted with mouse anti-human
caspase 8 (BD Biosciences) followed by
chemiluminescence. Protein extracts from HEC 1B
cells grown in the absence of compound were used
as a negative control. The inactive form of
caspase 8 (55kDa) is cleaved into smaller
activated subunits of 40/36 kDa (doublet) and 23
kDa. A strong band of approximately 55 kDa was
detected in all cells, both treated and
untreated. A 23kDa band was observed in control
and in 1µM HEC 1B cells, whereas a faint band was
observed in HEC 1B cells treated with 10µM 4-OH
TAM. Thus, the active form of caspase 8 appeared
the least expressed at 10µM 4-OH TAM.
Figure 1A Percent Survival for ER negative HEC
1B human endometrial cancer cells treated with
different concentrations of 4-OH TAM. HEC 1B
cells treated with lower concentrations of 4-OH
TAM (1-3 days) resulted in no significant
difference in percent survival when compared to
untreated cells (red line).
Figure 1B Percent Survival for ER negative HEC
1B human endometrial cancer cells treated with
different concentrations of estrogen. HEC 1B
cells treated with lower concentrations of
estrogen (1-3 days) resulted in no significant
difference in percent survival when compared to
untreated cells (red line).
INTRODUCTION
Tamoxifen has been researched for over twenty
years as an anti-estrogenic drug used to combat
breast cancer. By competing for estrogen
receptors, tamoxifen has been proven to reduce
the proliferation of breast cancer by 49 through
its antagonistic properties (1). On the other
hand, in the endometrium, tamoxifen has been
shown to be a partial agonist. In addition, the
agonistic effect of tamoxifen has been shown to
depend on cell type, promoter context, and
estrogen receptor subtype (? or ?) (2). It has
also been shown that tamoxifen and its
metabolite, 4-OH TAM, produce DNA adducts in the
human, rat, and mouse endometrium as well as in
the mouse and rat liver supporting the existence
of an alternative ER independent pathway (3,4).
By covalently binding to nucleic acids, DNA
adducts may disrupt the duplex structure
resulting in mutations. If repair mechanisms are
not able to remove the mutations upon
replication, mutations located within significant
genes (such as proto-oncogenes) may initiate an
unregulated cell cycle (5). In the end, this
uncontrollable cell growth lends way to
cancer. In the breast, MCF-7 human
adenocarcinoma cells treated with tamoxifen or
4-OH TAM have been demonstrated to undergo a
cessation of cell proliferation (6). However, in
the endometrium, studies conducted using ER
positive HEC 1A human adenocarcinoma cells
treated with tamoxifen have exhibited both
agonistic and antagonistic tendencies with
respect to cell proliferation (7). Furthermore,
the effects of tamoxifen and its metabolites on
the cell growth of ER negative human endometrial
cancer cells (such as HEC 1B) have not been
extensively explored. Therefore, this study
intended to investigate the effects of 4-OH TAM
and estrogen on the proliferation of HEC 1B
endometrial cancer cells lacking an ER while
using ER positive HEC 1A endometrial cancer cells
as a means of comparison. By varying the
concentration of 4-OH TAM and estrogen, the
antagonistic or agonistic growth effects were
analyzed using MTS assay. Tamoxifen-induced DNA
adduct formation can result in an agonistic
effect due to the mechanisms explained above.
However, DNA damage (caused by DNA adducts) can
also lead to apoptosis (8). In general,
apoptosis is a pathway leading to programmed cell
death with visible characteristics such as
morphological change, chromatin condensation, and
DNA fragmentation (9). If the apoptotic pathway
is not utilized, necrosis occurs through the
unintentional lysing of the cell. Overall,
analyzing the effects of 4-OH TAM and estrogen on
ER negative HEC 1B endometrial cancer cells
permits a better understanding of the ER
independent pathway.
DISCUSSION
In the present study, we attempted to determine
the effects of 4-OH TAM on ER negative HEC 1B
endometrial cancer cells while using ER positive
HEC 1A endometrial cancer cells as a means of
comparison. Through MTS Assay analysis, we
discovered that lower concentrations of 4-OH TAM
and estrogen had no effect on HEC 1B cell
proliferation. However, higher concentrations of
4-OH TAM produced a significant inhibitory effect
on the growth of HEC 1A and HEC 1B cell lines
when compared to non-treated cells. In addition,
HEC 1B and HEC 1A cells were inhibited by higher
concentrations of estrogen when compared to
controls. Despite the presence of the estrogen
receptor within the HEC 1A cells, 100µM estrogen
led to a complete inhibition of growth.
Similarly, growth of HEC 1B (ER negative) cells
was inhibited when exposed to 100µM estrogen.
However, a greater inhibition within a shorter
amount of time was observed in ER negative HEC 1B
cells when compared to ER positive HEC 1A
cells. Due to the inhibitory action of these
compounds, we investigated the molecular
mechanism behind the observed cell death. DNA
Gel Electrophoresis and Western blot analysis of
the apoptotic protein, caspase 8, did not
demonstrate that apoptosis had occurred. From
the Western blot analysis, we saw a basal level
of expression of caspase 8 in controls and HEC 1B
cells treated with 1µM 4-OH TAM (10). On the
contrary, an under-expression of active caspase 8
was observed in HEC 1B cells treated with 10µM
4-OH TAM. These results lead us to conclude that
necrosis, instead of apoptosis, was the
underlying mechanism of cell death. Overall,
both estrogen and 4-OH TAM exposure resulted in
necrosis in both ER negative HEC 1B and ER
positive HEC 1A cells. These results differ from
previous studies in which 4-OH TAM was found to
be an estrogen agonist in HEC 1A cells using 1µM
concentrations (2). However, the fact that HEC
1B cells showed inhibition when exposed to 4-OH
TAM and estrogen supports the existence of an ER
independent pathway.
Figure 1C Percent Survival for ER negative HEC
1B human endometrial cancer cells treated with
higher concentrations of 4-OH TAM. HEC 1B cells
treated with higher concentrations of 4-OH TAM
(1-3 days) resulted in a significant inhibitory
effect (p 0.0001) at 10µM and 100µM when
compared to untreated cells (red line). No
significant difference was observed between 1µM
and control.
Figure 1D Percent Survival for ER negative HEC
1B human endometrial cancer cells treated with
higher concentrations of estrogen. When HEC 1B
cells were treated with higher concentrations of
estrogen (1-3 days), we observed an initial
proliferative response after 24 hours at 1µM and
10µM , followed by a partial inhibition of growth
after 2 to 3 days. A significant inhibitory
effect (p 0.0001) was seen at 100µM when
compared to untreated cells (red line).
OBJECTIVES
ACKNOWLEDGEMENTS
The objective of this study is to determine the
effects of 4-OH TAM on ER negative HEC 1B human
endometrial cells in comparison with the effects
observed with ER positive HEC 1A human
endometrial cells. Specifically, this study
aims to 1. Determine if 4-OH TAM has an
agonistic or antagonistic effect on the growth of
ER negative HEC 1B endometrial cancer
cells. 2. Investigate the molecular mechanism
of the agonistic or antagonistic effect on
proliferation.
We would like to thank Dr. Cheryl Walker for her
generous donation of the HEC 1A and HEC 1B
endometrial cancer cell lines and her technical
advice. Also, we would like to thank Dr. Martín
Gonzalez and Dr. Maria Todd for their technical
expertise. Finally, a special thanks to Dr. Romi
Burks for her statistical guidance.
REFERENCES
MATERIALS METHODS
1. Liu, X., Pisha, E., Tonetti, D.A., Yao, D.,
Yan, L., Yao, J., Burdette, J.E., Bolton, J.L.
(2003) Antiestrogenic and DNA Damaging Effects
Induced by Tamoxifen and Toremifene Metabolites.
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Castro-Rivera, E. and Safe, S. (2003)
17ß-Estradiol- and 4-hydroxytamoxifen-induced
transactivation in breast, endometrial and liver
cancer cells is dependent on ER-subtype, cell and
promoter context. J. Steroid Biochem. Molec.
Biol. 8423-31. 3. Carthew, P., Edwards, R.E.,
Nolan, B.M., Tucker, M.J., Smith, L.L. (1999)
Compartmentalized Uterotrophic Effects of
Tamoxifen, Toremifene, and Estradiol in the
Ovariectomized Wister (Han) Rat. Toxicological
Sciences. 48197-205. 4. Lyman, S.D. and Jordan,
V.C. (1985) Metabolism of Tamoxifen and Its
Uterotrophic Activity. Biochemical Pharmacology.
342787-2794. 5. Martin, E.A., Brown, K.,
Gaskell, M., et al., (2003) Tamoxifen DNA Damage
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Mass Spectrometry. Cancer Research. 63
8461-8465. 6. Wang, S., Zhang, B., and Faller,
D.V. (2004) BRG1/BRM and prohibitin are required
for growth suppression by estrogen antagonists.
The EMBO Journal. 232293-2303. 7. Castro-Rivera,
E. and Safe, S. (1998) Estrogen-and
Antiestrogen-responsiveness of HEC 1A Endometrial
Adenocarcinoma Cells in Culture. J. Steroid
Biochem. Molec. Biol. 64287-295. 8. Wyllie,
A.H. (1995) The genetic regulation of apoptosis.
Current Opinions in Genetics and Development.
597-104. 9. Dietze, E.C., Caldwell, L.E.,
Grupin, S.L., Mancini, M., Seewaldt, V.L. (2001)
Tamoxifen but Not 4-OH tamoxifen Initiates
Apoptosis in p53(-) Normal Human Mammary
Epithelial Cells by Inducing Mitochondrial
Depolarization. J. Biol. Chem. 276
5384-5394. 10. Esposti, M.D., Ferry, G.,
Masdehors, P., Boutin, J.A., Hickman, J.A., Dive,
C. (2003) Post-translational Modification of Bid
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Cell Lines Tissue Culture Conditions HEC 1B and
HEC 1A human endometrial cancer cells were a
generous gift from Dr. Cheryl Walker at M.D.
Anderson in Smithville. HEC 1A cells were
maintained in McCoys 5A medium with 1
L-glutamine (Gibco) and supplemented with 10
Fetal Bovine Serum. HEC 1B cells were maintained
in Minimum Essential Medium (MEM) (Gibco)
supplemented with 10 Fetal Bovine Serum, 2
L-glutamine, and 1 sodium pyruvate. Cells were
grown in 75cm2 culture flasks at 37C in a 5 CO2
atmosphere. Compounds 4-OH Tamoxifen and
Estrogen (Sigma) were dissolved in ethanol,
stored, and protected from light in stock
solutions of 1mM at -20C. MTS Assay HEC 1B (ER
negative) and HEC 1A (ER positive) cells were
seeded in triplicates into 96-well plates
(10,000cells/well). Media was replaced using 5
charcoal stripped bovine serum and cells were
allowed to recover for 24 hours. Cells were then
incubated with 200µL of various 4-OH TAM and
estrogen concentrations (nM to µM range) for 1-3
days. MTS assay was performed using CellTiter
96?AQueous One Solution Cell Proliferation Assay
(Promega). Absorbance was read at 490nm using a
lysis plate reader (BioRad). DNA Gel
Electrophoresis Cell DNA extracts were prepared
from HEC 1A and HEC 1B cells treated with 10µM
4-OH TAM and estrogen for 24 hours at the
incubating conditions previously described. DNA
laddering analysis was performed using a DNA
Laddering Assay kit (Cayman).
Figure 2B Percent Survival for ER positive HEC
1A human endometrial cancer cells treated with
higher concentrations of estrogen. Complete
inhibition (p 0.0001) was observed with HEC 1A
cells treated with 100µM estrogen (1-3 days). In
contrast, HEC 1A cells treated with 1µM and 10µM
estrogen were refractory to low dose estrogen
when compared to control (red line).
Figure 2A Percent Survival for ER positive HEC
1A human endometrial cancer cells treated with
higher concentrations of 4-OH TAM. A significant
inhibition (p 0.0001) was seen with HEC 1A
cells treated with 10µM and 100µM 4-OH TAM (1-3
days) when compared to untreated cells (red
line). No inhibition was observed at 1µM.