Doxorubicin Influences the Expression of Glucosylceramide Synthase in Invasive Ductal Breast Cancer
et al. (2012) Doxorubicin Influences the Expression of Glucosylceramide Synthase in Invasive Ductal Breast
Cancer. PLoS ONE 7(11): e48492. doi:10.1371/journal.pone.0048492
Doxorubicin Influences the Expression of Glucosylceramide Synthase in Invasive Ductal Breast Cancer
Xiaofang Zhang 0
Xiaojuan Wu 0
Peng Su 0
Yongsheng Gao 0
Bin Meng 0
Yanlin Sun 0
Li Li 0
Zhiqiang Zhou 0
Gengyin Zhou 0
Ashraf B. Abdel-Naim, Faculty of Pharmacy, Ain Shams University, Egypt
0 1 Department of Pathology, Shandong University School of Medicine , Jinan, Shandong , P.R. China , 2 Department of Pathology, Academy of Tumor Treatment and Prevention , Jinan, Shandong , P.R.China
Introduction: Glucosylceramide synthase (GCS) is one enzyme that provides a major route for ceramide clearance. Recent evidence has indicated an important role for GCS in multidrug resistance (MDR) tumors. Doxorubicin (DOX)can modulate the expression of GCS in leukemia and ovary cell lines. However, few studies have investigated their relationship in breast cancer; Methods: We collected 84 excision biopsies from patients with invasive ductal breast cancer of whom 33 patients had undergone preoperative chemotherapy. Immunohistochemistry was used to analyze the expression of GCS protein and significantly showed that the expression of GCS was higher in the samples from patients treated with preoperative chemotherapy(p = 0.018). In order to investigate the underlying mechanism, breast cancer cell lines were cultured with different concentrations of DOX, and mRNA and protein levels of GCS were then detected; Results: DOX significantly upregulated the expression of GCS at both the mRNA and protein level in ERa-positive MCF-7 cells.We then block down the Sp1 site of GCS promoter, which inhibited the DOX-mediated increase in GCS expression; and after Era was inhibited in MCF-7 cells, the up-regulation of GCS by DOX also been inhibited. Conclusions: In conclusion, our data demonstrated the novel finding that DOX could modulate the expression of GCS through the Sp1 site of GCS promoter in ERa-positive breast cancer cells.
Breast cancer is one of the most common causes of death in
women due to cancer worldwide. Besides surgical methods,
chemotherapy and endocrine therapies are also used in the
treatment of breast cancer. The resistance of tumors to
chemotherapy occurs not only to single cytotoxic drugs, but also as
a cross-resistance to a range of drugs with different structures and
cellular targets. This phenomenon is termed multidrug resistance
(MDR), and is one of the contributing factors that prevents
survival rates for breast cancer improving further . Several
factors have been reported to be responsible for MDR, including
the overexpression of the adenosine triphosphate (ATP)-binding
cassette (ABC) membrane transporter family .
The accumulation of recent evidence has pointed towards an
important role for glucosylceramide synthase (GCS) in MDR.
Sphingolipids, which include ceramide and sphingosine, are
essential structural components of cell membranes. Furthermore,
they also play an important role in regulating the proliferation,
survival and apoptosis of cells. GCS is a pivotal enzyme that
transfers UDPglucose to ceramide to form glucosylceramide (GC)
. We and others have shown that MDR cancer cells have high
levels of GCS compared to drug-sensitive cells [4,5]. Transfection
with GCS could increase the level of MDR in breast cancer cell
lines , whereas its inhibition has proven to be useful in altering
responses to chemotherapy in numerous human tumor cell lines
Anthracycline-based chemotherapy (treatment regimens that
involve anthracyclines such as doxorubicin or epirubicin) has been
used clinically for over two decades. Several studies have
confirmed that doxorubicin (adriamycin) can modulate the
expression of GCS in the leukemia cell line, HL-60, and an ovary
cell line, NCI/ADR-RES [8,9]. Few studies have shown whether
doxorubicin influences the expression of GCS in breast cancer
tissue samples and breast cancer cells. This study aimed to rectify
this omission from the literature.
Materials and Methods
Tissue samples from 84 patients with invasive ductal breast
carcinoma who underwent complete dissection of the breast and
axillary lymph nodes and 5 patients with accessory breast who
underwent complete dissection of the tissue were collected at the
Qilu Hospital Shandong University, China, between January and
September 2007. Thirty-three of the patients had also undergone
preoperative chemotherapy (CAF protocol: cyclophosphamide,
doxorubicin and 5-fluorouracil).
Tumor samples were paraffin-embedded and histopathological
variables, including tumor size, lymph node metastasis, histological
subtype, and histological grade were determined by reviewing
pathology reports and hematoxylin and eosin (H&E) stained
sections. Patient and tumor characteristics are summarized in
The use of these tissues was approved by the Research Ethics
Committee of Shandong Medical University, and we obtained
informed written consent for pathological evaluation from all
participants involved in our study.
Two drug-sensitive breast cancer cell lines, MCF-7
(ERpositive) and MDA-MB-231 (ER-negative), were obtained from
the American National Cancer Institute. The multidrug-resistance
breast cancer cell line, MCF-7/ADM, was selected from MCF-7
using doxorubicin treatment in stages . All the cells were
maintained in RPMI-1640 medium (Gibco, USA) containing 10%
FBS at 37uC in a humidified atmosphere containing 5% CO2.
Number of patients (%)
ERa -estrogen receptor a.
Immunocytochemical or Immunohistochemical Analyses
Immunohistochemical staining was carried out using the
DAKO Envision Detection Kit (Dako, Carpinteria, CA, USA).
In brief, tissue blocks were cut into 4 mm-thick sections, dried,
deparaffinized, and rehydrated. Antigen retrieval was performed
in a microwave oven for 15 min in 10 mM citrate buffer at
pH 6.0. For the cell culture experiments, after cells were
embedded in 4% neutral formaldehyde for 2 h, PBS with 0.5%
Tween-20 was added for 30 min at room temperature. For all
samples, endogenous peroxidase activity was blocked with a 3%
H2O2-methanol solution. The slides were blocked with 10%
normal goat serum for 10 min and were incubated with GCS
antibody (1:500) overnight at 4uC. The slides were then probed
with HRP-labeled polymer conjugated to a secondary antibody for
30 minutes. The antibody against GCS was a kind gift from Dr. D.
Marks (Mayo Clinical Center, Rochester, USA). After the
MCF7/ADM cell line was confirmed to overexpress GCS protein, it
was used as a positive control in this research. Otherwise, the
normal breast tissues were also used as control.
Staining results were interpreted by a breast pathologist who
was blinded to patient outcomes. Tumors with 1% or more
positively stained nuclei were considered positive for estrogen
receptor(ER) .A dual semi-quantitative scale that combined
staining intensity and the percentage of positive cells was used to
evaluate GCS protein staining. The staining intensity was scored
as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The
percentage of positive cells was scored as follows: 0, no staining
or staining in ,5% of the tumor cells; 1, staining in 5% to 25% of
the cells; 2, staining in 26% to 50% of the cells; 3, staining in 51%
to 75% of the cells; and 4, staining in .75% of the cells. As the
overexpression of GCS is related to MDR, cytoplasmic staining
was considered positive; an IHC score $4 was defined as high
expression and ,4 was considered to be low expression.
Treatment with Doxorubicin
Cells were treated with various concentrations of DOX
(0.1 mM, 0.2 mM and 0.7 mM) for 24 or 48 hours. The
concentrations of DOX were calculated according to the IC50
of the cells (IC50 = drug concentration (mmol/L) that results in the
50% inhibition of cell growth).
Quantitative Real-time PCR (qPCR) to Detect the mRNA
of GCS and ERa
Total RNA was isolated using Trizol (Invitrogen, Carlsbad,
USA) and quantitative real-time PCR (qPCR) was used to detect
GCS mRNA. qPCR was performed using SYBR Green Real-time
PCR MasterMix (TOYOBO, Japan). The primers for GCS were
as follows: sense: 59-CCT TTC CTC TCC CCA CCT TCC
TCT-39, antisense: 59-GGT TTC AGA AGA GAG ACA CCT
GGG-39 ; The primers of ERa were as follows: sense:
59-CACACGGCACAGTAGCGAG-39 . b-actin (sense: 59-ACC CCC ACT
GAA AAA GAT GA-39, antisense:59-ATC TTC AAA CCT CCA
TGA TG-39) was used as an internal control set. The final volume
was 20 ml, and an iCycler iQ Real-Time PCR Detection System
(Bio-Rad) was used for qPCR. The amplification data were
calculated using the DDCq method. The DDCq method was used
to calculate relative mRNA expression. The relative target gene
expression was calculated using 2-DDCq, where DDCq = target
Cq - control Cq, DDCq = DCq target -DCq calibrator.
Figure 1. Expression of GCS and ERa protein in invasive ductal breast carcinoma samples. The expression of GCS and ERa protein was
detected in all samples by immunohistochemical staining. For GCS, cytoplasmic staining was considered positive; and for ERa, nuclei staining was
considered positive. The figure shows two cases that were positive or negative respectively. A, B) An invasive ductal breast carcinoma sample that the
expression of ERa and GCS were both negative. C,D) An invasive ductal breast carcinoma sample that the expression of ERa and GCS were both
Western Blot to Analyze GCS Protein Expression
All the cells were cultured for another 48 h after treatment, the
confluent cells were then lysed in buffer containing 50 mM Tris-HCl
(pH 8.0), 150 mM NaCl, 0.5% Triton-X100, 2 mM EDTA
(PH 8.0), 5 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, and
10 mg/ml aprotinin for 20 min on ice. The complex was centrifuged
at 12,0006g for 10 min at 4uC. As described previously , equal
aliquots of protein (50 mg) were resolved using 412% gradient
PAGE. The transferred nitrocellulose blot was blocked with 5%
fatfree milk powder in TBS at room temperature for 2 h. The membrane
was immunoblotted with murine monoclonal antibody C219 against
human P-gp (0.7 mg/ml, Santa Cruz) or with GCS-1.2 antiserum
(diluted 1:1,000) in 5% fat-free milk in TBS-0.1% Tween-20. As
a control for equivalent protein loading, the filters were
simultaneously incubated with rabbit polyclonal antibody against human
bactin (diluted 1:1,000). Detection was performed using enhanced
chemiluminescence (Amersham Pharmacia Biotech, Piscataway,
Transfection with GC-rich/Sp1 Decoy
Phosphorothioated double-stranded ODNs (decoy ODNs)
containing the sequence of the GCS putative Sp1/GC-rich
binding site (59-ATTCCGGGGGCGGGGGCATG-39) and mock
ODNs (59-CATGCCATCGCTACCGGGGC-39) were
transfected (final concentration, 0.5 mmol/L) into breast cancer cells
suspended in RPMI 1640 using lipofectamine reagent (Invitrogen,
Carlsbad, USA) for 12 hours. After transfection, cells were
incubated with 10% FCS-RPMI 1640 for 12 hours and then
treated with DOX for 24 h . Then, qPCR or Western blotting
were used to assess the expression of GCS. Rates of apoptosis were
assessed with flow cytometry as described above.
Transfection with ERa Interference Plasmid
The RNA interference sequence targeted to ERa was selected
according to the previous studies .The RNAi sequences
targeted to MDR1 were forward, 59-UCAUCGCAUUCC
UUGCAAAdTdT-39, and reverse,
59-UUUGCAAGGAAUGCGAUGAdTdT-39. These oligonucleotides were annealed after
phosphorylation of their 59 terminate, and then subcloned into
pSUPER.neo+GFP to generate pSUPER-ERai.
Before transfection, cells were seeded in 6-well plates at the density
of 16106 cells per well and incubated at 37uC in an atmosphere with
5% CO2 for 12 h. For each well, 10 ml (2 mg/ml) of lipofectamine
(Invitrogen, Carlsbad, USA) or 5 ml (1 mg/ml) of vector was diluted
into 250 ml of 1640 culture medium without serum. After incubated
for 10 min at room temperature, the diluted vector and lipofectamine
were mixed together and incubated for 20 min. Then the mixture was
adding to the cells washed three times by medium without serum. Six
hours later, the medium was replaced with 1 ml of complete 1640
culture medium (The final concentration of plasmid was 5 mg/ml).As
control, 10 ml of lipofectamine and 5 ml (1 mg/ml) of
pSUPER.neo+GFP were also transfected.
Assays were performed as described previously . Briefly,
cells were seeded in 96-well plates (1,000 cells/well) in 0.1 ml
RPMI 1640 medium containing 10% FBS and cultured at 37uC
for 24 h before the addition of adriamycin or mitoxantrone, both
of which were obtained from Sigma, USA. Drugs were added in
FBS-free medium (0.1 ml). After they were cultured for 72 h, cells
were stained with 150 ml sterile MTT dye (5 mg/ml,
SigmaAldrich, USA) for 4 h at 37uC; subsequently, the culture medium
was removed and 150 ml of dimethyl sulfoxide (DMSO,
SigmaAldrich, USA) was added and thoroughly mixed for 10 min.
Absorbance at 490 nm was recorded using an automatic multiwell
spectrophotometer (Bio-Rad-Coda, Richmond, CA). The MDR
reversal effect was evaluated as the alteration of IC50.
Apoptotic rates were assessed with flow cytometry using the
Annexin V-fluorescein isothiocyanate/propidium iodide (PI) kit
(Bipec Biopharma Corp, USA). Samples were washed with
icecold PBS twice and resuspended in binding buffer at a density of
16106 cells/ml. The cells were stained with Annexin V-FITC and
gently vortexed. After 15 min incubation at 48uC in the dark, PI
was added to the cells for another 5 min incubation at 48uC in
the dark. The results were analyzed by flow cytometry (FACScan,
BD Biosciences, USA).
The Chi-square test or the Fishers exact test was used to
analyze the relationship between the expression of GCS and
chemotherapy. Cellular data were presented as the mean 6
standard deviation, and one-way ANOVA and Dunnetts T3 was
used to determine the statistical significance. P-values ,0.05 were
considered statistically significant. All calculations were performed
using the SPSS16.0 for windows statistical software package
(SPSS, Chicago, IL, USA).
Figure 4. Effects of DOX on GCS mRNA and protein levels in Sp1 blocked MCF-7 cells. Cells were transfected with the Sp1 decoy ODNs or
mock ODNs for 24 h. Then, 0.2 mmol/L DOX was added to the cells for 24 h. The GCS mRNA was then analyzed by qPCR. The protein levels of GCS
was measured by western blotting. A, B) Effects of DOX on GCS mRNA levels in transfected MCF-7 cells. C) Effects of DOX on GCS protein levels in
transfected MCF-7 cells. An, cells transfected with Sp1 ODNs for 24 h; AnA, cells treated with 0.2 mmol/L DOX for 24 h after transfection with Sp1
ODNs; Mu, cells transfected with mock Sp1 ODNs for 24 h; MuA, cells treated with 0.2 mmol/L DOX for 24 h after transfection with mock Sp1 ODNs.
**p,0.01 compared with untreated cells.
Expression of GCS Protein in the Breast Tissue Samples
The expression of GCS protein was detected in all samples by
immunohistochemical staining (Figure 1). In the normal ductal
epitheliums, the staining often weak and was considered negative.
Overall, 33.3% of all the invasive carcinoma samples were positive
for GCS (28/84); in tumors resected from patients who were
treated with the CAF protocol, the expression of GCS was
significantly increased (p = 0.018). Further analysis showed that, in
the ER-positive tumor samples, the CAF protocol increased the
positive rate of GCS protein from 28.57% (10/35) to 60% (12/20)
(p = 0.022); however, in the ER-negative samples, CAF protocol
did not induce the expression of GCS.
Upregulation of GCS mRNA in MCF-7 Cells, but not in
qPCR was adopted to detect the expression of GCS mRNA,
which was approximately 10.2-fold higher in MCF-7 cells than in
MDA-MB-231 cells. As shown in Fig. 2A, DOX at 0.1 mM,
0.2 mM, and 0.7 mM for 24 h increased GCS mRNA expression
in MCF-7 cells by 6.51-fold, 56.7-fold and 11.4-fold, respectively,
compared with untreated MCF-7 cells. In addition, the expression
of GCS after DOX treatment increased in a time-dependent
manner. MCF-7 cells treated with 0.2 mmol/L DOX for 24 h had
an approximate increase in GCS mRNA of 56.7-fold relative to
control levels, while treatment with the same concentration of
DOX for 48 h upregulated GCS mRNA levels by approximately
370-fold compared to controls.
In MDA-MB-231 cells, the low concentration of DOX (0.1 mM)
decreased the level of GCS mRNA by 35.47% (p,0.01) and
0.7 mM DOX increased GCS mRNA levels by only
approximately 1.34-fold relative to the controls (p,0.01) (Fig. 3A).
Compared with MCF-7 cells, the effect of DOX on the expression
of GCS mRNA in MDA-MB-231 cells was much smaller, and was
Alteration of GCS Protein in the Treated Cells
GCS protein expression was analyzed by Western blotting, as
shown in Fig. 2B and Fig. 3B. After MCF-7 cells were treated with
DOX, there was a significant increase in GCS protein expression,
especially after treatment with 0.2 mM DOX for 48 h (Fig. 2B).
GCS protein levels did not significantly change in MDA-MB-231
cells after DOX treatment (Fig. 3B).
Changes in GCS Expression after Sp1 was Blocked
After cells were transfected with the Sp1 decoy ODNs, GCS
mRNA levels were significantly decreased by approximately
99.97% in MCF-7 cells (Figure 4A) and 99.92% in
MDA-MB231 cells (Figure 5A) compared with the controls. Then, DOX was
added to both cell lines for 24 hours. Real-time PCR and Western
blotting showed that transfection with Sp1 decoy ODNs
significantly inhibited the DOX-induced elevation of GCS mRNA
(Figure 4) and protein levels (Figure 5) in both MCF-7 and
Alteration of GCS mRNA and Protein in the ERa
Interference MCF-7 Cells
After cells were transfected with the ERa interference plasmid
pSUPER-ERai, GCS mRNA levels were significantly decreased
by approximately 84.75% in MCF-7 cells (Figure 6A) compared
with the controls. Then, DOX was added to both cell lines for 24
hours. Real-time PCR and Western blotting showed that after
transfected with pSUPER-ERai, the DOX-induced elevation of
both GCS mRNA (Figure 6A) and protein levels (Figure 6B) was
significantly inhibited in MCF-7 cell lines.
Figure 6. Effects of DOX on GCS mRNA and protein in the ERa blocked MCF-7 cells. Cells were transfected with the plasmid pSUPER-ERai
or pSUPER(control) for 246h. Then, 0.2 mmol/L DOX was added to the cells for 24 h. The GCS mRNA was then analyzed by qPCR. The protein levels of
GCS was measured by western blotting. A) The effects of RNA interference on ERa mRNA in transfected MCF-7 cells. B,C) The effects of DOX on GCS
mRNA levels in transfected MCF-7 cells. D) Effects of DOX on GCS protein levels in transfected MCF-7 cells.
Figure 7. Effects of DOX on IC50 values of DOX in each group. Cytotoxicity assays were performed as described in the Materials and
Methods. The IC50 is the drug concentration (mmol/L) that results in a 50% inhibition of cell growth. A, B) The survival curve in MCF-7 cells and
MDAMB-231 cells, respectively.C, D) The IC50 value of each group. A21, cells treated with 0.2 mmol/L DOX for 24 h; An, cells transfected with Sp1 ODNs for
24 h; AnA, cells treated with 0.2 mmol/L DOX for 24 h after their transfection with Sp1 ODNs; Mu, cells transfected with mock Sp1 ODNs for 24 h;
MuA, cells treated with 0.2 mmol/L DOX for 24 h after they were transfected with mock Sp1 ODNs. **p,0.01 compared with MCF-7 cells. # p,0.05
compared with MDA-MB-231 cells.
Evaluation of Chemosensitivity in Treated Cells
After treatment, we assessed the influence of DOX on the
cellular response to anti-neoplastic drugs. The results showed that
the IC50 of MCF-7 cells increased from 0.315560.0179 mmol/L
to 2.16460.0899 mmol/L after they were treated with 0.2 mM
DOX for 24 hours (p,0.01). After they were transfected with Sp1
decoy ODNs, the IC50 for DOX decreased to
0.0111360.00007 mmol/L, but did not change when cells were
treated with DOX for 24 h (p.0.05) (Fig. 7A, Fig. 7C).
In MDA-MB-231 cells, the IC50 for DOX was
0.124160.0179 mmol/L, which increased to
0.136660.0116 mmol/L after they were treated with 0.2 mM
DOX for 24 hours (p.0.05) (Fig. 7). After transfection with Sp1
decoy ODNs, the IC50 for DOX was reduced to
0.011460.0059 mmol/L and DOX could not induce the
upregulation of cellular drug resistance (Fig. 7B, 7D). These combined
results suggested that the Sp1/GC-rich element may play an
important role in the regulation of GCS expression in the presence
of DOX in MCF-7 cells, but not in MDA-MB-231 cells.
Alteration of Apoptosis Rate in the Treated Cells
The apoptosis rate was detected by flow cytometry using the
Annexin V-fluorescein isothiocyanate/propidium iodide (PI) kit.
Annexin V-positive, PI-negative cells were scored as early
apoptotic cells. The apoptosis rates estimated in the present study
only included early apoptotic cells, which were marked as LR in
Figures 8 and Figure 9. After MCF-7 cells were treated with
0.2 mM DOX for 24 hours, the rate of apoptosis increased from
10.1761.92% to 20.060.87%. After transfection with Sp1 decoy
ODNs, the apoptosis rate increased to 35.664.86%, and was
unaffected by DOX (Figure 8A and Figure 9A).
In MDA-MB-231 cells, DOX increased the apoptosis rate from
7.0362.61% to 12.461.67%. After transfection with Sp1 decoy
ODNs, the apoptosis rate increased and the rise of apoptosis rate
by DOX disappeared (Figure 8B and Figure 9B).
Sphingolipids, which include ceramides and sphingosine, were
first isolated and characterized in the late 1800s. However, they
have been long regarded as structural and insert components of
cell membranes. In recent years, many studies have shown that
they are still associated with a myriad of cell processes, including
proliferation, cell survival and death. Ceramide, an important
member of sphingolipid metabolism, has been proven to be
a second messenger in the process of apoptosis [13,14]. Cellular
stress is known to increase intracellular ceramide levels. Therefore,
it is easy to understand that increased ceramide levels are observed
in response to many anti-cancer drugs, including doxorubicin,
vincristine, paclitaxel, etoposide, PSC 833 and fenretinide.
The regulation of ceramide levels involves many enzymes, such
as ceramide synthase and sphingomyelinase, which are responsible
for the generation of ceramide, sphingomyelin synthase and
ceramidase . GCS is one enzyme that provides a major route
for ceramide clearance. As an enzyme that catalyzes the first step
in glycosphingolipid synthesis, GCS transfers UDPglucose to
ceramide to form glucosylceramide (GC). Increased intracellular
ceramide can induce the upregulation of GCS , whereas the
overexpression of GCS is association with decreased rates of
apoptosis in many cancer types .
Recently, accumulating evidence has pointed towards an
important role for GCS in MDR. Many drug-resistant cell lines
have been found to overexpress GCS, including breast cancer cell
lines, while the inhibition of GCS by antisense oligonucleotides or
by specific inhibitors could restore chemosensitivity in many
cancer cell lines . The breast cancer resistance cell line,
MCF-7/AdrR, is derived from the human ovarian carcinoma cell
line, OVCAR-8, and has been re-designated NCI/ADR-RES
. As this cell line has been an important and widely used
research tool over the last two decades, many data regarding
MDR in breast cancer now have to be re-evaluated. Therefore,
the function of GCS in breast cancer remains enigmatic. Our
research in 2009 showed that the suppression of GCS reversed
MDR in the breast cancer cell line MCF-7/ADM .
A GC-rich/Sp1 promoter binding region in important in the
regulation of GCS expression; furthermore, doxorubicin can
induce the activation of Sp1 and the upregulation of GCS and
apoptosis in the leukemia drug-resistant cell line HL-60/ADR in
addition to an ovarian cancer cell line . Numerous data have
shown that preoperative chemotherapy in breast cancer may
induce the expression of many MDR-related proteins, including
pglycoprotein (p-gp), and multidrug resistance-related protein
(MRP), amongst others . However, there is little evidence
regarding whether chemotherapeutic agents can modulate the
expression of GCS in vivo. Although our study only used
a relatively small number of samples, our research showed the
novel result that GCS expression was significantly increased after
patients were treated with the CAF protocol (p,0.05) (Figure 1
and Table 2).
In order to confirm whether doxorubicin induces the expression
of GCS, different concentrations of doxorubicin were added to
sensitive breast cancer cell lines, MCF-7 and MDA-MB-231. The
results demonstrated that doxorubicin upregulated the expression
of GCS at both the mRNA and protein levels in the ERa-positive
cell line, MCF-7 (Figure 2), while there was no change in GCS
expression in the ERa-negative cell line, MDA-MB-231 (Figure 3).
We then investigated the mechanism by which doxorubicin
influences GCS expression in MCF-7 cells. After blocking the Sp1
site of the GCS promoter, we found that DOX could not increase
GCS mRNA and protein expression levels (Figure 4). A cellular
resistance experiment (MTT assay) displayed that the drug
resistance of DOX-treated MCF-7 cells significantly increased
Histologic rade Grade I
compared with untreated MCF-7 cells, and the blockade of the
Sp1 site inhibited this phenomenon (Figure 7A and Figure7B).
This change was consistent with the mRNA and protein results.
In 2009, Ruckhaberle et al. analyzed microarray data regarding
GCS mRNA expression in 1,681 breast tumors and found that
GCS expression was associated with positive estrogen receptor
(ER) status, lower histological grading, low Ki67 levels and ErbB2
negativity (p,0.001 for all) . This study revealed the
expression profile of GCS in breast cancer at the mRNA level.
In 2011, Liu et al. detected the levels of GCS expression in normal
tissue and cancer tissue samples. Their results showed that breast
and other hormone-dependent organs (testis, cervix, ovary and
prostate) displayed the lowest levels of GCS mRNA, whereas the
liver, kidney, bladder and stomach displayed the highest expressed
levels of GCS. In breast cancer specimens, GCS overexpression is
highly associated with ERa- and HER2-positivity in breast cancers
that have metastasized . In our study, we found that DOX
induced the expression of GCS in ERa-positive MCF-7 cells
significantly, but the effection on ERa-negative MDA-MB-231
cells was much smaller. This finding suggests that Era may be
related to the expression of GCS. Then we inhibited the ERa of
MCF-7 cells via RNA interference, and the results displayed that
the DOX-induced upregulation of GCS mRNA and protein were
also been inhibited (Figure 6).
In conclusion, our data demonstrated the novel findings that
DOX could modulate the expression of GCS through the Sp1 site
of the GCS promoter in ERa-positive breast cancer cells.
We greatly appreciate the gift of GCS antiserum from Dr. R. Pagano and
Dr. D. Marks (Mayo Clinical and Foundation, Rochester, MN, USA).
Conceived and designed the experiments: GZ. Performed the experiments:
XZ XW PS YG BM ZZ. Analyzed the data: LL. Contributed reagents/
materials/analysis tools: YS. Wrote the paper: XZ.
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