ARTICLE
Auteur(s) : Gayle S Nobert, Martha M Kraak, Sarah Crawford
Department of Biology, Cancer Biology Laboratory, Southern
Connecticut State University, New Haven, CT 06515
Breast cancer is the most common form of cancer in women in the
United States and Europe. However it is well known that Asian women
are much less susceptible to developing this type of malignancy.
Asians consume 20-50 times more soy per capita than their Western
counterparts [1] and a high dietary intake of soy products has been
linked to a lowered incidence of breast cancer [2, 3]. Soy is a
rich source of the phytoestrogen genistein, which has a chemical
structure similar to steroidal estrogens, particularly
17β-estradiol (E2) which is capable of binding estrogen receptors
(ER). The main purpose of this study was to explore potential
differential responses of the ER+ MCF7 primary breast carcinoma
cell line cultured as microscopic solid tumors in vitro to the
growth inhibitory effects of genistein as opposed to that of
tamoxifen. Tamoxifen is an endocrine therapy that has been used for
many years in breast cancer patients. Several important clinical
trials have suggested that this anti-estrogen is effective in the
prevention or attenuation of continued malignant cell proliferation
following primary chemotherapy treatment [4]. Tamoxifen functions
as a competitive inhibitor that binds to the active site of the
estrogen receptor, thereby preventing estrogen from stimulating the
growth of cancer cells. Although tamoxifen has shown promising
results in the prevention/treatment of ER+ breast cancers, there
are some problems with its use, particularly associated with the
occurrence of resistance after prolonged use and in older patients
[5]. For these reasons, this experimental study was designed to
compare the cytotoxic effects of tamoxifen with the isoflavone
genistein used individually and in combination to treat solid
tumors derived from ER+ MCF7 breast carcinoma cells in vitro.This
research also investigated the effects of the vitamin A derivative
all-trans-retinoic acid (ATRA) on the MCF7 breast carcinoma cell
line. Studies on the relationship between the development of breast
cancer and vitamin A deficiency indicate that retinoid-dependent
cell signaling pathways may inhibit the process of carcinogenesis
and that dysregulated expression of vitamin A targets may promote
malignant transformation [6]. Retinoic acid receptors (RAR) are
members of the nuclear hormone receptor family, and, as such, share
similar protein domain structures to the ER. The cytotoxic effects
of ATRA used alone or in conjunction with genistein in experimental
tumors derived from the ER+ MCF7 cell line were analyzed in this
study.As an approach to the problem of acquired drug resistance
frequently encountered in the treatment of breast cancer, the
anti-inflammatory parthenolide was included in this research study.
Parthenolide, the active component of the medicinal plant Feverfew,
is known to exert inhibitory effects on the nuclear factor kappa B
(NFκB) survival pathway by blocking the phosphorylation and
degradation of the NFκB inhibitor IκB [7]. Since activation of the
NFκB survival pathway has been shown to limit the effectiveness of
conventional chemotherapy interventions, successful inhibition of
this pathway may result in the restoration of cell sensitivity to
chemotherapeutic agents, thereby thwarting mechanisms of acquired
drug resistance.The experimental tool for this study was breast
carcinoma cell line MCF7 cultured in the form of microscopic solid
tumors, termed multicellular tumor spheroids (MTS). Sutherland
proposed the MTS model in studies demonstrating that the tumor
spheroid microenvironment resembles the solid tumor environment in
vivo [8]. Most importantly, these studies suggested that MTS
display both intrinsic and acquired drug-resistant properties
similar to those observed in solid tumors in vivo [9, 10].
Methods and aims
Cell line and culture conditions
The ER+ MCF7 cell line was obtained from the American Type Culture
Collection (ATCC). Monolayer stock cultures were routinely
maintained in alpha MEM or EMEM (Gibco) with 10 % fetal bovine
serum (FBS) (Atlanta Biologicals), 1 % non-essential amino
acids (NEAA) (Sigma), 1 % penicillin/streptomycin mixture
(Sigma), and 1 % sodium pyruvate (Sigma). Low-serum media was
prepared using 1 % FBS in place of 10 % FBS. Media
supplemented with β-estradiol was prepared by adding 10 nM
β-estradiol (Sigma) to the low-serum or complete media preparation.
To facilitate MTS aggregation, tissue culture plates (Fisher
Scientific) were pre-coated with 1 % agarose (Sigma) prepared
in sterile deionized water. The growth of MTS was achieved by
transferring cells from the stock monolayer population at a fixed
density (2-3 × 105 cells per 3 mm agarose-treated
dish). Under these conditions, spheroids formed spontaneously
within 48 hours. All cultures were incubated at 37°C in 5 %
CO2.
Reagents
All drugs were obtained from Sigma Chemical Company. Genistein was
used at a dose range of 5-100 μM. Tamoxifen was used at a dose
range of 5-10 μM. ATRA was used at concentrations of 1 to 10 μM.
Parthenolide was used at a dose range of 1-5 μM. All stock
solutions were stored at 4° C.
Results
Dose/response assays
Experimental reagents were added either as single agents or in
combination 48 hours after cells were plated as either monolayer or
MTS cultures and incubated at 37°C for a period of 2-5 days. MTS
viability assays were carried out post drug-treatment by the
following method. Cell pellets were prepared by centrifugation,
re-suspended in phosphate buffered saline (PBS) and an aliquot of
cells was transferred to plates containing fresh media and
incubated for 24 hours to allow for re-attachment. Failure of MTS
re-attachment indicated loss of cell viability confirmed by trypan
blue staining. Each experiment was carried out at least two times.
Survival assays
Post-drug treatment survival was assessed by culturing aliquots of
treated cells in fresh media containing no drugs or additives and
incubated for 5-7 days to assay residual culture viability
utilizing trypan blue staining.
Cell imaging
To assess general morphology of MTS prior to, during, and
post-treatment, the cells were photographed using the Acquis
digital photomicroscopy system.
Data analysis
All data were analyzed using GraphPad Prism (GraphPad Software, San
Diego, CA).
Genistein’s cytotoxic effects on ER+ MCF-7multicellular tumor
spheroids are estrogen-independent
MTS cultured in complete media were treated with genistein at a
dose range of 5-100 μM for 5 days. Maximum sensitivity was observed
at doses of 25-50 μM with less than 1% survival post treatment in
this dose range, and complete toxicity was achieved at 50 μM
genistein ( (figure 1) ). The
addition of 10 nM β-estradiol to the media did not affect tumor
response to genistein ( (figure 2A) ). The
IC50 value for genistein was approximately 10 μM.
Antiproliferative effects of tamoxifen are
estrogen-dependent
MTS cultured in complete media to which 10 nMβ-estradiol was added
displayed a two-fold increase in growth inhibition by low doses of
tamoxifen (2.5 to 5 μM) as compared to MTS treated in the absence
of added estrogen ( (figure 2B) ).
Increasing the tamoxifen dose range from 7.5 to 10 μM produced a
greater differential response in MTS treated in the presence of
β-estradiol. To further assess the effects of β-estradiol on
tamoxifen sensitivity, MTS were cultured in low-serum containing
media to which 10 nM, 20 nM β-estradiol or no β-estradiol was
added. A potent estrogen-dependent response was observed under
these conditions. In the absence of added estrogen, 5 μM
tamoxifen produced only a 20 % reduction in MTS viability. In
contrast, low serum media containing 10 nM β-estradiol to
which 5 μM tamoxifen was added produced approximately 50%
growth inhibition. Likewise, when the tamoxifen concentration was
doubled to 10 μM, a 40% reduction in viability was observed in
the absence of estrogen whereas in the media containing estrogen,
the inhibitory effects of the same dose of tamoxifen approached
90%.
Further experiments to assess tamoxifen sensitivity to exogenous
estrogen levels involved cultivating MCF7 cells in media containing
no phenol red to ensure that no additional estrogenic growth
factors present in phenol red were affecting these results. These
experiments showed that the absence of phenol red had no
significant effect on the estrogen-dependent cytotoxic effects of
tamoxifen ( (figure 2C) ).
Tamoxifen and genistein used in combination produced enhanced
responses in MCF7 MTS in both the presence and absence of
supplemental estrogen
Treatment of MTS in media supplemented with 10 nM β-estradiol with
tamoxifen alone produced 70 % growth inhibition ( (figure 3) ). The
addition of 5 μM genistein increased the inhibitory effects to
almost 100 % 48 hours post treatment. In the absence of
supplemental β-estradiol, treatment with 10 μM tamoxifen as
the sole agent inhibited cell growth by only 20 %. However,
combining this dose of tamoxifen with 50 μM genistein produced
an 85 % reduction in viability 48 hours post treatment
ATRA is an effective cytotoxic agent on the MCF7 cell line
cultivated as multicellular tumor spheroids
MTS were treated at concentrations ranging from 1-10 μM ATRA.
Cytotoxic effects were observed for all ATRA doses tested with
maximum sensitivity at 10 μM with less than 1% cell survival in
recovery assays ( (figure 4) ). The IC50
value was 2 μM. Combined treatment of MCF7 cells cultured as MTS
with low doses of genistein and ATRA produced synergistic effects.
For example genistein 2.5 μM combined with ATRA 1 μM resulted in
the absence of cell survival in recovery assays (figures 5 and
6). This combined effect was dramatically greater than the effects
observed following a single dose of genistein or ATRA at
significantly higher concentrations.
Combination treatment of MCF-7 spheroids with parthenolide and
genistein produced additive effects that were independent of
estrogen
MCF7 MTS treated with parthenolide at a dose range of 1-5 μM for 48
hours showed that a complete cytotoxic response was achieved at 3
μM in the presence or absence of supplemental 10 nM
β–estradiol ( (figure 6) ).
Moreover, combination therapy using genistein and parthenolide
produced a synergistic effect independent of added estrogen ( (figure 7) ).
Combined treatment using 50 μM genistein and 2.5 μM parthenolide
produced almost complete cytotoxicity under conditions that
produced only 60 % growth inhibition with parthenolide alone
as compared to 80 % growth inhibition using genistein alone.
Similar results were obtained in the absence of added estrogen.
Combination treatment of MCF-7 spheroids with tamoxifen and
parthenolide yielded synergistic effects that were
estrogen-dependent
Combined treatment of MCF7 spheroids for 48 hours with 10 μM
tamoxifen and 2.5 μM parthenolide produced approximately 98 %
growth inhibition in the presence of estrogen ( (figure 8) ). In
contrast, tamoxifen alone produced only 30 % loss of viability
and parthenolide showed 40 % cell viability. When the assays
were repeated in the absence of supplemental estrogen, combined
treatment produced only 70 % inhibition.
Discussion
The results of this study suggest that the growth inhibitory
effects of genistein on the ER+ MCF7 cell line were independent of
β-estradiol levels, whereas sensitivity to tamoxifen was markedly
reduced in the absence of estrogen supplementation. In contrast,
treatment with both genistein and tamoxifen produced enhanced
combined effects independent of exogenous β-estradiol.
These pre-clinical findings suggest potential therapeutic
applications. First, they suggest that combined tamoxifen/genistein
treatment protocols might produce significantly greater therapeutic
effects than either of these drugs administered individually.
Secondly, the observed reduction in therapeutic effect of tamoxifen
under conditions of low estrogen suggest a potential explanation
for the decreased clinical effectiveness and increased resistance
to tamoxifen observed in post-menopausal women whose levels of
circulating estrogen are generally low. The molecular basis of the
estrogen-dependence of tamoxifen’s growth inhibitory effect on
MCF-solid tumors is not clear. We have proposed a model to explain
this observation ( (figure 9) )
suggesting that tamoxifen’s interference in ER+ signaling depends
on estrogen since the estrogen-ER complex is the functional complex
that may be competitively inhibited by tamoxifen. Under conditions
of low estrogen, the role of the estrogen receptor in sustaining
the malignant phenotype may be substantially reduced, reflected by
decreased sensitivity of the tumor to tamoxifen.
It is possible that genistein might be more effective than
tamoxifen in post-menopausal women as its effects were observed to
be independent of estrogen levels in this pre-clinical study. This
observation is consistent with its pleiotropic effects on cell
cycle control. For example, recent research suggests that growth
factor activated phosphotidylinositol-3 kinase PI3k/AKT signaling
may be down-regulated by genistein [11], which in turn may alter
expression of ERα [12].
Additional studies reported here suggest that combined treatment
of MCF7 cells with tamoxifen/genistein produced enhanced cytotoxic
effects. The effects of genistein on AKT pathway activation may
affect downstream targets such as the transcriptional regulator
NFκB. Moreover, recent studies have suggested that NFκB inhibition
markedly enhances the sensitivity of resistant breast cancer tumor
cells to tamoxifen [13]. The studies reported here show that this
effect is estrogen-dependent, whereas combined therapy using
genistein and the NFκB inhibitor parthenolide produced enhanced
cytotoxic responses in MCF7 solid tumors independent of estrogen
levels ( (figure 10) ).
The results of this research showed that ATRA produced important
cytotoxic responses in MCF7 cells that were synergistic with
genistein in combined treatment. Although the anti-cancer effects
of retinoic acid have traditionally been attributed to the
induction of differentiation, research studies suggest that
PI3K/AKT signaling may also be a target of this agent [6]. With
respect to the combined treatment effects of ATRA/genistein, these
two agents may interact with this pathway and NFκB signaling to
produce a greater inhibitory effect on tumor survival.
In conclusion, these pre-clinical research findings suggest
possible clinical applications relevant to the use of tamoxifen in
women with low levels of circulating estrogen and suggest that
genistein might be a useful clinical alternative, particularly in
post-menopausal women in whom breast cancer occurs more frequently.
Moreover, this research suggests that combined treatment approaches
involving the use of tamoxifen in conjunction with agents that
inhibit NFκB pathway signaling, such as parthenolide and genistein,
warrant further study.
Acknowledgments
The research was supported by Connecticut State University research
grants awarded to Dr. S. Crawford.
References
1 Dampier K, Hudson EA, Howells LM, Manson MM,
Walker RA, Gescher A. Difference between human breast
cell lines in susceptibility towards growth inhibition by
genistein. Br J Cancer 2001; 85: 618-24.
2 Hall M, Couse J, Korach KS. The multifaceted
mechanisms of estradiol and estrogen receptor signaling. J Biol
Chem 2001; 276: 36869-72.
3 McMichael-Philips D, Harding C, Morton M,
et al. Effects of soy protein supplementation on epithelial
proliferation in the histologically normal breast. Am J Clin Nutr
1998; 68: 1431S.
4 Ryden L, Jonsson PE, Chebil G, et al. Two
years of adjuvant tamoxifen in premenopausal patients with breast
cancer: a randomised, controlled trial with long-term follow-up.
Eur J Cancer 2005; 41: 256-64.
5 Goss PE. Changing clinical practice: extending the
benefits of adjuvant endocrine therapy in breast cancer. Semin
Oncol 2004; 31(6 Suppl 12): 15-22.
6 Seewaldt VL, Kim JH, Parker MB, Dietze EC,
Srinivasan KV, Caldwell LE. Dysregulated expression of
cyclin D1 in normal human mammary epithelial cells inhibits
all-trans-retinoic acid-mediated G0/G1 phase arrest and
differentiation in vitro. Exp Cell Res 1999; 249: 70-85.
7 Zhang S, Lin ZN, Yang CF, Shi X,
Ong CN, Shen HM. Suppressed NF-kappaB and sustained JNK
activation contribute to the sensitization effect of parthenolide
to TNF-alpha-induced apoptosis in human cancer cells.
Carcinogenesis 2004; 25: 2191-9.
8 Sutherland RM. Cell and environmental interactions in
tumor microregions: the multicell spheroid model. Science 1988;
240: 177-84.
9 Desoize B, Jardlier J. Multicellular resistance: a
paradigm for clinical resistance? Crit Rev Oncol Hematol 2000; 36:
193-207.
10 Fracasso G, Colombatti M. Effect of therapeutic
macromolecules in spheroids. Crit Rev Oncol Hematol 2000; 36:
159-78.
11 Gong L, Li Y, Nedeljkovic-Kurepa A,
Sarkar FH. Inactivation of NF-κB by genistein is mediated via
Akt signaling pathway in breast cancer cells. Oncogene 2003; 22:
4702-9.
12 Stoica GE, Franke TF, Moroni M, et al.
Effect of estradiol on estrogen receptor-α gene expression and
activity can be modulated by the ErB2/ PI3-K/Akt pathway. Oncogene
2003; 22: 7998-8011.
13 de Graffenried L, Chandrasekar W,
Friedrichs WE, et al. NFkB inhibition markedly enhances
sensitivity of resistant breast cancer tumor cells to tamoxifen.
Ann Oncol 2004; 15: 885-90.
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