Transcript Document

Effects of 4-OH Tamoxifen on HEC 1B Endometrial Cancer Cells
Tracey Einem, Carolina Boet, Dr. Maria
1Biology
1
Cuevas ,
Dr. Maha
2
Zewail-Foote
Department, 2 Chemistry Department, Southwestern University,
1001 E. University Ave. Georgetown, TX 78626
[email protected], [email protected], [email protected], [email protected]
ABSTRACT
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 proapoptotic 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.
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.
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.
RESULTS
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 4OH 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).
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).
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).
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.
REFERENCES
Cell Lines & Tissue Culture Conditions
MTS Assay
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.
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.
MATERIALS & METHODS
4-OH Tamoxifen and Estrogen (Sigma) were dissolved in ethanol, stored, and protected from light in stock
solutions of 1mM at -20˚C.
DISCUSSION
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.
Compounds
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 4OH TAM.
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.
OBJECTIVES
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 McCoy’s 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 37˚C in a 5% CO2 atmosphere.
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.
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.
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).
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