J. Biomedical Science and Engineering, 2011, 4, 750-754
doi:10.4236/jbise.2011.412092 Published Online December 2011 (http://www.SciRP.org/journal/jbise/ JBiSE
Published Online December 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Embryoprotein TWIST distribution in breast cancer cells
MDA-MB-231 treated with 5-Fluorouracil and malnutrition
Jesica Escobar-Cabrera, Angelina Rodríguez-Torres, Laura Berumen-Segura,
Guadalupe García Alcocer*
Facultad de Química, Universidad Autónoma de Querétaro, Centro Universitario, Querétaro, México.
Received 16 August 2011; revised 19 October 2011; accepted 9 November 2011.
TWIST is a transcription factor that belongs to the
family of helix-loop-helix proteins involved in metas-
tasis with essential role regulating cell movement
during early development, as well as in the tumor
progression and metastasis of many cancers includ-
ing breast cancer. It will be interesting to study the
relation among cancer chemotherapy, malnutrition
and the transcription factors like TWIST in order to
explore the risk to metastasis. We used breast cancer
line MDA-MB-231. Cell cultures were treated with
5-Fluorouracil (5-Fu), as well as changes in serum
and nonessentials amino a cid (NEAA), to explore the
cell viability and the cellular distribution of TWIST
by immunocytochemistry. Our results indicate that
cell viability decreased significantly with 5-Fu treat-
ment whereas no changes were observed in malnutri-
tion treatment. On the other hand, TWIST protein
significantly increased its distribution in cytoplasm of
treated groups with malnutrition as well as in those
treated with 5-Fu compared with the control. These
results suggest that TWIST translocation was modi-
fied by the treatments and further studies are neces-
sary to suggest that TWIST could be a tag protein to
avoid metastasi s .
Keywords: TWIST; Breast Cancer; 5-Fu; 5-Fluorouracil;
NEAA; Nonessential Amino Acid
TWIST protein is a highly conserved transcription factor,
initially identified in Drosophila, with an essential role
in regulating cell movement during early development. It
belongs to the family of basic helix-loop-helix (bHLH)
proteins that can homodimerize or heterodimirize with
other proteins of the same family like E proteins, MASH1,
MASH2, SCL etc. [1-4]. TWIST has two homologues in
vertebrates that are expressed in a broad range of em-
bryonic and adult tissues: TWIST 1 and TWIST 2 .
The activation of the two TWIST gene could be associ-
ated with interleukin-6, which promotes STAT3 activa-
tion and their bound to the human TWIST promoter
starting their transcriptional activity . Besides the
important role during ontogeny, TWIST has been re-
ported over expressed in distinct types of cancer, in-
cluding breast cancer and metastasis .
Carcinogenesis involves initiation, progression and
metastasis . It is important to study the different
events involved in the cancer progress as TWIST over
expression, which has been related with the same bio-
logical activity during normal development in tumor
progression as cell migration [9,10]. The metastasis of
breast cancer remains essentially incurable . One of
the drugs used in breast cancer is 5-Fluorouracil (5-Fu)
which interferes with thymidylate synthase . It has
been reported by epidemiological studies the relation
between cancer and diet , as well as the relation of
chemotherapy treatment with metastasis remain unclear.
In this work, we study the viability and the cellular dis-
tribution of TWIST in cancer breast line cells MDA-
MB-231, treated with malnutrition and 5-Fu.
2. MATERIALS AND METHODS
2.1. Antibodies and Reagents
Antibodie against TWIST were purchased from Santa
Cruz Biotechnology, USA. Polyclonal anti-rabbit-HRP
were obtained from Invitrogen. 5-Fluorouracil drug was
obtained from Columbia Laboratories, Mx (50 µg/µl),
DAB (Sigma, Germany) Culture medium (DMEM),
amino acid glutamine, nonessentials amino acids and
antibiotics were obtained from Gibco (Zurich Switzer-
2.2. Cell line
The breast cancer cell line used was MDA-MB-231. The
J. Escobar-Cabrera et al. / J. Biomedical Science and Engineering 4 (2011) 750-754 751
cells were cultured in Dulbecco’s modified Eagle’s me-
dium (DMEM) with 10% and 2% fetal bovine serum
(FBS), 1% L-glutamine, 1% nonessential amino acids
(NEAA) 1% antibiotics mix (penicillin-streptomycin).
The cultures were maintained in a humidified incubator
at 37˚C with 5% CO2. To measure viability, cell were
seeded at 3 × 104 per well in 12-well dish for 48 hours,
for each treatment. The cells from three dishes were
trypsinized and stained with trypan blue. Each experi-
ment was rep roduced 3 times.
2.3. Immunocytochemical Analysis
The cells were washed three times with PBS (phosphate
buffer saline, pH:7.4) and then peroxidase activity was
blocked for 30 minutes in a PBS solution, containing 1%
hydrogen peroxide for 1 hour. The cells were washed
and blocked with milk 3% (BIO-RAD) in PBT for two
hours. For specific immunoreactivity detection, the cells
were washed three times with PBS and incubated with
the rabbit polyclonal anti-TWIST antibody in PBT
(phosphate buffer saline with triton 0.5% X-100, 1:200)
at 4˚C overnight. The next day the cells were washed
three times with PBS, and then incubated with the sec-
ondary anti rabbit- HRP antibody (1:300) in PBT and
finally the stain was developed with DAB. Negative
control was stained by omitting the primary antibody.
We analyzed 3 different experiments with 250 cells
2.4. Statistical Analysis
The cell viability was calculated as the percentage over
control. Differences in mean values were evaluated us-
ing either one-way ANOVA with Tukey test to deter-
mine differences among treatments groups. P < 0.05 was
regarded as statistically significant. All data are given
mean ± standard error.
To understand the role of FBS on proliferation and vi-
ability of MDA-MB-231 cells, we cultured cells with
10%, 2% FBS and 0.5% albumin for 48 hours. The cell
viability with 2% FBS was 82.66% ± 10.74% ee and
16.5% ± 5.32% ee with albumin, both in relation to the
control group (10% of FBS). The albumin treatment
showed statistical difference compared to control. The
2% of FBS concentration was elected to the following
experiments (F i gure 1).
To evaluated the effect of nonessential amino acids
we cultured cells with 1%, 0.5% and without NEAA.
The viability obtain ed did not have statistical differences
compared to the control with NEAA 1% (Data not
shown). The condition with NEAA depletion was used
in the following experiments on viability with 5-Fu
added and the cellular distributio n of TWIST.
To know the 5-Fu effect on cell viability, the cultures
were subjected to different drug concentrations or with-
out 5-Fu (control group), the cells growth allowed 33.1%
± 13.8% ee in 5 µM concentration; 39.01% ± 8.75% ee
in 50 µM, 34.54% ± 6.08% ee in 100 µM and 19.6% ±
9.58% ee in 200 µM. All concentrations showed sign ifi-
cant difference compared with the control but not among
the treatments (Figure 2).
Figure 1. Proliferation and viability in malnutrition cells. The
MDA-MB-231 viability cells significantly decreases in albu-
min treatment (P < 0.05). The results are the mean values of
three independent experiments ± standard error.
Figure 2. 5-Fluorouracil effects in MDA-MB-231 cells. All
5-Fu different concentrations caused significant proliferation
and viability decrease compared with the control (with no drug)
and non statistical differences were observed among treatments
(P < 0.05). The groups were cultured with 2% FBS and opti-
mal nonessential amino acids concentration (1%). The results
are given as mean values of 3 independent experiments ±
opyright © 2011 SciRes. JBiSE
J. Escobar-Cabrera et al. / J. Biomedical Science and Engineering 4 (2011) 750-754
In the control group the staining was observed in
96.3% ± 1.3% ee of total cells analyzed (250), the deple-
tion of nonessential amino acids showed staining cells in
88.6% ± 5.3% ee, the 5-Fu (50 uM) in the 80.6% ±
13.3% ee, the combination of depletion of NEAA and
5-Fu in 87% ± 3.8% ee. The immunocytochemical re-
sults indicated different TWIST staining zones: nucleus,
cytoplasm and both (nucleus-cytoplasm) (Figure 3). The
treatments did not cause significant difference immu-
nostain decrease compared to the control in the total
cells. On the other hand, in the specific zone the deple-
tion of NEAA, the 5-Fu and both factors added together
showed a significant decrease in nucleus-cytoplasm stain
compared to the control (Figure 4).
The high viability in 10% and 2% of FBS cultures com-
pared to those cultures with 0.5% BSA-medium with
lower viability were consistent with the viability in os-
teoblasts experiments, as well as in stem cells added
with different FBS concentrations [14,15]. On the other
hand, our results were different from those experiments
in Chinese hamster ovary cells because they reported a
decrease in the viability with FBS . They also dif-
fered from the experiments in human mesenchymal
steam cells that reported an increased viability with the
FBS reduction and observed a higher level of viability
when used 1% human serum albumin instead of FBS
. One possibility in the viability d ifferences with the
FBS could be associated with the distinct cell lines re-
quirements and their specific growth nutrients sensibili-
ties . In human cells, the growth factors also partici-
pate in the capacity to synthesize the necessary proteins
for their survivor and proliferation, even in conditions of
nutrients dep rivation  .
Our results indicate that the viability was not affected
by nonessentials amino acids as has been reported before
with mice feds deficient tryptophan tumoral cells [20-
22]. On the other hand, a decrease in the viability has
been reported in rat brain postmitotic neurons depleted
in tryptophan, isoleucin, metionin and cholin . The
differences could be associated with the use of essentials
amino acids instead nonessentials. The essentials amino
acids like leucine, isoleucine and valine increase the
protein synthethize, and decreases proteolisis. They are
also important for cell survival . Another possibility
could be associated to the serum grow factors activity,
that can improve the cell development inducing the use
of peptides, like source of amino acids in nutrients dep-
rivation conditions .
The viability cells decrease by the citotoxic effects of
Fluorouracil was consistent with those reported in
linfoblastic cell cancer, thyroid cell cancer, and mouse
Figure 3. TWIST cell distribution in MDA-MB-231 cells.
Representative cells of TWIST protein immunocytochemistal
staining, (A) Stained cell in nucleus; (B) Stained cell in cyto-
plasmic zone; (C) Stain of TWIST in nucleus and cytoplasm.
40X magnifications. Bar 10 µm.
Figure 4. TWIST distribution in treated cells with 5-Fu and
malnutrition. Control cells with optimal nonessentials amino
acids concentrations (1) showed no statistical difference among
cytoplasm (C) and nucleus-cytoplasm (NC) zones. The stain-
ing was higher in cytoplasm than nucleus-cytoplasm in all
treated groups, compared to the control. The lines indicated a
mean ± ee. The treatments were: control with 1% NEAA (1),
without NEAA (2), 1% NEAA and 5-Fu (3) without NEAA
and 5-Fu (4).
tumor cell [25-27]. The 5-Fu damage may be associated
with a fail on repair system , and could be possible
that higher doses induce total cell death. The Fluorouracil
toxicity mechanism could be associated with the sin-
tetase timidilate inh ibition vía Fas .
The depletion on NEAA, the treatment with 5-Fu and
both factors added together were not enough to eliminate
TWIST immunosignal from cells MDA-MB-231. It was
interesting to observe a cytoplasm immunosignal in-
crease in all treated groups; those results may be related
to changes in protein conformation or mutations in the
TWIST gen induced by the treatment . The increase
of TWIST presence in cytoplasm affected its function as
The authors thank to Dr. Teresa García Gasca and Dr. Aracely Anaya
opyright © 2011 SciRes. JBiSE
J. Escobar-Cabrera et al. / J. Biomedical Science and Engineering 4 (2011) 750-754 753
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