Anxa2 Plays a Critical Role in Enhanced Invasiveness of the

Multidrug Resistant Human Breast Cancer Cells

Fei Zhang, Lin Zhang, Bin Zhang, Xiyin Wei, Yi Yang, Robert Z. Qi, Guoguang Ying,
Ning Zhang,*, and Ruifang Niu*,
Key Laboratory of Breast Cancer Prevention and Treatment, Ministry of Education, Tianjin Medical University
Cancer Institute and Hospital, Tianjin 300060, P.R. China, Department of Biochemistry, The Hong Kong
University of Science and Technology, Hong Kong, P.R. China, and Research Center of Basic Medical Science,
Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, P.R. China
Received May 24, 2009

Multidrug resistance (MDR) is the major cause of failure in cancer chemotherapy. Recent reports even
suggest that MDR is associated with elevated invasion and metastasis of tumor cells. In the current
study, we used a proteomic approach to identify genes that play an important role in MDR induced
cell migration. 2D-PAGE and MALDI-TOF/MS-based proteomics approach were used to separate and
identify differentially expressed proteins between MCF-7 and MCF-7/ADR, a p-glycoprotein-overexpressing adriamycin-resistance breast cancer cell line. Annexin a2 (Anxa2) was identified as highly
expressed in MCF-7/ADR cells, but not in MCF-7 cells. Small interference RNA-mediated gene
suppression demonstrated that Anxa2 was required for enhanced cell proliferation and invasion of the
MCF-7/ADR cells. Down-regulation of Anxa2 alone was not sufficient to revert the cell sensitivity to
adriamycin, suggesting that Anxa2 was not required for MDR phenotype. Taken together, our results
showed that expression of Anxa2 is enhanced when cancer cells, MCF-7, acquired drug resistance and
it plays an essential role in MDR-induced tumor invasion.
Keywords: breast cancer Anxa2 metastasis MDR 2-dimensional gel electrophoresis mass
spectrometry small interference RNA

1. Introduction
Breast cancer is one of the leading causes of death among
women. Chemotherapy plays an important role in the treatment of breast cancer at various stages. However, long-term
treatment often resulted in chemoresistance and even multidrug resistance (MDR), a phenomenon in which cross resistance of tumor cells to several structurally unrelated chemotherapeutic agents was developed after exposure to a single
cytotoxic drug.1,2 MDR is a major cause of treatment failure
and mortality for most cancer patients. Previous studies have
revealed several mechanisms contributing to drug resistance,
but it still remains as a formidable obstacle to reverse drug
resistance for effective treatment of human cancer.3-6
Recent studies showed that acquisition of MDR phenotype
is often associated with an elevated invasion/metastasis.7-10
Yang et al. observed increased motility, invasion, and metastasis
of certain P-glycoprotein-overexpressing MDR cancer cells
* To whom correspondence should be addressed. Ning Zhang, Tianjin
Medical University, Research Center of Basic Medical Sciences & Cancer
Institute and Hospital, Tianjin, 300060 China; e-mail, nzhangchina@yahoo.com.
Ruifang Niu, Tianjin Medical University Cancer Institute and Hospital,
Tianjin. 300060, China; e-mail, Niurf1982@yahoo.com.cn.

Key Laboratory of Breast Cancer Prevention and Treatment, Tianjin

Medical University Cancer Institute and Hospital.

The Hong Kong University of Science and Technology.

Research Center of Basic Medical Science, Tianjin Medical University

Cancer Institute and Hospital.
10.1021/pr900461c CCC: $40.75

2009 American Chemical Society

treated with P-gp transportable drugs;11 Li et al. reported that

up-regulation of CD147 (also knows as extracellular matrix
metalloproteinase inducer, EMMPRIN) and matrix metalloproteinase-2, -9 were induced by P-glycoprotein substrates in
multidrug resistant breast cancer cells, and expression of CD147
and MMPs correlated with the invasiveness of the tumor
cells.12,13 Stephen Hiscox showed that, following acquisition
of tamoxifen resistance, breast cancer cells displayed an altered
growth rate associated with increased aggressive behavior in
vitro,14-16 and elevated invasion and metastasis properties of
drug resistant cancer cell lines have also been demonstrated
in animal models.17-19 Taken together, development of MDR
not only prohibits effective chemotherapy, but also exacerbates
the metastatic symptom of cancer patients.
Invasion/metastasis is a multistep process, including detachment of tumor cells from the primary sites, intravasation into
circulation, spread of tumor cells through circulation, extravasation to the secondary sites, and growth into new tumors.20,21
Spectra of genes are up-regulated to orchestrate the invasion
and metastasis of tumor cells to the secondary sites. Among
them, several reports have revealed that the cytosolic level of
Annexin A2 (Anxa2), a calcium dependent phospholipid binding protein, is enhanced in metastatic cancers.22-26 Overexpression of Anxa2 has been proposed to be a potential marker
for cancer diagnosis and prognosis.27-32 However, the role of
Anxa2 in cancer metastasis remains unclear.
Journal of Proteome Research 2009, 8, 50415047 5041
Published on Web 09/18/2009

research articles

Zhang et al.

In this study, we investigate the molecular mechanism of

enhanced invasion/metastasis in MDR cancer cells. It has been
documented that MCF-7/ADR, a p-glycoprotein overexpressing
adriamycin-resistant breast cancer cell, shows elevated invasion/metastasis in comparison with its parental MCF-7 cell.33-36
A 2D-PAGE based proteomic approach was deployed to screen
for the proteins with altered level of expression upon cellular
acquisition of MDR, and small interference RNA technique was
used to further verify the functional relevance. Our study
identified Anxa2 as an essential factor which was responsible
for the enhanced invasiveness of the MCF-7/ADR cells.

2. Materials and Methods

2.1. Cell Culture. MCF-7 human breast cancer cells (adriamycin-sensitive) and their MCF-7/ADR multidrug-resistant
variant (adriamycin-resistance) were obtained from Dr. ZiZheng Hou of the Detroit Hospital, Detroit, MI. The cells were
cultured in RPMI 1640 supplemented with 10% (v/v) fetal
bovine serum (Hyclone, Logan, UT), 100 U/mL penicillin and
100 U/mL streptomycin, maintained in a humidified atmosphere at 37 C under 5% CO2, and were passaged every 2-3
days when digestion was made by a mixture of 0.025% trypsin
and 0.01% EDTA (Gibco BRL, Rockville, MD). The MCF-7/ADR
cells were continuously exposed to 0.5 M adriamycin for the
maintenance of the MDR phenotype, but cultured in drug-free
medium for at least 1 month before use in any experiment.
2.2. Two-Dimensional Gel Electrophoresis and Image
Analysis. For sample preparation, cells were harvested, washed
three times with ice-cold PBS without Ca2+ or Mg2+ to remove
serum proteins and stored as cell pellets in aliquots of 1 107
cells/tube at -80 C until use. The cell pellets were resuspended
in lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS,
0.5% IPG buffer (pH 3-10 NL), 20 mM tris, 40 mM DTT, 1 mM
PMSF and a mixture of protease inhibitors at room temperature
for 1 h. The lysates were centrifuged at 20 000g for 1 h at 4 C.
Protein concentrations were determined using the modified
Bio-Rad protein assay kit. Fifty micrograms or 500 g of protein
was loaded onto IPG strips for analytical or preparative gels,
respectively. The strips were rehydrated at 20 V for 14 h at 20
C and isoelectric focusing was performed sequentially at 500,
1000, 3000, and 5000 V, for 1 h each plus 8000 V for 5 h. The
strips were then equilibrated for 15 min in buffer I (50 mM
Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 100 mM DTT and 2%
SDS) and 15 min in buffer II (buffer I with 250 mg of

Figure 1. 2-D protein profiles from human breast cancer cell line
MCF-7 and its multidrug resistant subline MCF-7/ADR. Proteins
were run on IPG DryStrips (pH 3-11 NL) and further separated
using SDS-PAGE (12%) gels and visualized by silver staining. (A)
Representative images of 2-D gel for human breast cancer cell
line MCF-7 and MCF-7/ADR. (B) Representative images of enlarged view of the spots differentially expressed between the two
cell lines. (C) Anxa2 was overexpressed in MCF-7/ADR cells
compared with MCF-7 cells.

iodoacetamide instead of DTT). The second dimension was

performed on homogeneous 12% SDS-PAGE gels, and the
separated proteins were detected by silver staining for analytical
gels and coomassie blue staining for preparative gels. All
samples were pools of three independent cell preparations and
analyzed for at least three times. Image acquisition was
performed by UMAX image scanner at 300 dpi and the images
were analyzed using the ImageMaster 2D Elite software. Only
protein spots which consistently showed at least 3-fold difference were considered to be differentially expressed.
2.3. Trypsin Digestion, MALDI-TOF-MS and Protein
Identification. Coomassie blue-stained protein spots were
excised using a clean ophthalmic scalpel and transferred to an

Table 1. Proteins with Altered Expression in MCF-7 and MCF-7/ADR Cells

no.

accession
number

1
2

gi|1384068
gi|182311

gi|8923900

4
5
6
7
8
9
10

gi|45786109
gi|13623417
gi|386854
gi|35440
gi|265222
gi|4504893
gi|1335012

5042

protein name

MW (Da)

pI

Down-Regulated Proteins in MCF-7/ADR Cells

NADPH-flavin reductase
22105.4262
7.13
fructose-1,6-bisphosphatase
36804.8007
6.54
Up-Regulated Proteins
cytidine 5-monophosphate
N-acetylneuraminic
acid synthetase
Anxa2
Ubiquitin carboxyl-terminal esterase L1
type II keratin subunit protein
unnamed protein product
beta 2-microglobulin
kininogen 1
beta-gonadotropin

Protein scores greater than 66 are significant (p < 0.05).

Journal of Proteome Research Vol. 8, No. 11, 2009

in MCF-7/ADR Cells
48348.6472
8.02

38579.8085
24808.4609
52752.5195
23497.8203
11431.0336
47883.2079
15487.9648

7.57
5.53
5.31
5.43
5.86
6.62
8.43

Mascot
scorea

peptides
match/total

sequence
coverage (%)

110
125

11/46
14/55

55
44

68

7/25

24

216
100
83
73
79
68
74

17/26
8/31
9/36
6/19
8/28
6/25
7/21

53
37
21
29
28
22
26

Pivotal Role of Anxa2 in MDR Breast Cancer Cells

research articles
hydroxycinnamic acid (CHCA). Samples were spotted onto
stainless steel MALDI target plates and then analyzed using the
4700 Proteomics Discovery System MALDI-TOF mass spectrometer (Applied Biosystems, Framingham, MA) in the positive
ion reflector mode. Peptide masses were obtained for the range
700-4000 Da and the acquired peptide mass fingerprints were
used to search through the NCBI nonredundant (NCBI nr)
Protein Data Base with the Mascot software (www.matrixscience.com). After removal of known contamination peaks
such as keratin and autoproteolysis peaks, the following search
parameters were used in all mascot searches: human species,
monoisotopic peptide masses, tolerance of one missed cleavage, and a maximum error tolerance of 100 ppm, carbamidomethylation and oxidation of methionine as fixed and
variable modification. Protein scores greater than 66 were
considered as significant (p < 0.05).

Figure 2. Down-regulation of Anxa2 decreased cell proliferation.

(A) RT-PCR and Western blotting analysis of Anxa2 in MCF-7/
ADR cells, control cells and three stable clones of si-Anxa2/MCF7ADR cells. The Control cells were transfected with a siRNA
plasmid containing a scrambled sequence in MCF-7/ADR cells.
-Actin was used as an internal control. Anxa2 expression was
significantly decreased both at mRNA and protein levels in three
stable clones. (B) Cell proliferation activity was decreased after
knockdown of Anxa2 in MCF-7/ADR cells; each point and bar
shows the mean ( SD for triplicates. Statistical analysis was
carried out with one-way ANOVA, *P < 0.05 vs control cells. (C)
Down-regulation of Anxa2 induced a decrease in the proportion
of G2/M + S phase cells compared to the control. All the
experiments were repeated at least three times.

Eppendorf tube; stained gel slabs were cut into small pieces
and destained using washing buffer (25 mM ammonium
bicarbonate, 50% acetonitrile) for 15 min. Washing step was
repeated for at least three times until no color was visible. The
gel pieces were dehydrated with 100% acetonitrile and dried
in a Speedvac centrifuge. Digestion was performed with
sequencing grade trypsin. After tryptic digestion, the peptide
fragments were extracted three times with 20 L of 5% TFA in
50% acetonitrile, dried, and redissolved in 10 L of 0.5% TFA.
The samples were desalted with ZipTipC18 according to the
manufactures instructions and eluted with 2.5 L of 50%
acetonitrile containing 0.5% TFA and 3 mg/mL R-cyano-4-

2.4. Western Blotting and Immunodetection of Anxa2.

Cellular proteins were extracted with RIPA cell lysis buffer and
20 g of lysates was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. The membrane was incubated
in a blocking solution consisting of 5% milk in 10 mM TrisHCl (pH 8.0), 150 mM NaCl, and 0.1% Tween 20 at room
temperature for 1 h. The mouse monoclonal antibody against
Anxa2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) was
then added followed by incubation overnight at 4 C and
washing with TBST three times for 10 min. Detection was made
using horseradish peroxidase (HRP)-linked goat anti-mouse IgG
secondary antibody (1:5000, Santa Cruz Biotechnology, Santa
Cruz, CA) and ECL kit (Pierce Biotechnology, Rockford, IL)
according to the manufacturers protocol. Mouse monoclonal
antibody against -actin (1:2000, Santa Cruz Biotechnology,
Santa Cruz, CA) was used as a loading control.
2.5. SiRNA Design, Construction and Transfection. The
SiRNA expression vector pGCsilencer H1/hygro was obtained
from Genechem company (Genechem, Shanghai, China). The
targeting sequence 5-GGTCTGAATTCAAGAGAAA-3 against
Anxa2 was designed and verified to be specific by Blast search
against the human genome, and a sequence of similar GC
content which does not match any known human coding
sequence was used for negative control. Transfection was
mediated with Lipofectamine 2000 (Invitrogen, Carlsbad, CA)
according to the manufacturers instruction, and three separate
MCF-7/ADR stable transfectants were generated after 14 days
selection by 300 g/mL Hygromycin for both Anxa2 siRNA
(pGCsi-Anxa2) and the control (pGCsi-control). The transfectants were routinely maintained under selection but withdrawn
of the drug before performing drug-sensitivity assays.
2.6. RT-PCR Analysis of the Stable Transfectants. For RTPCR, total RNA was Trizol-extracted, reverse-transcribed using
the Reverse Transcription System (Invitrogen, Carlsbad, CA),
and PCR amplified using a Taq DNA polymerase kit (Takara
Bio., Dalian, China). The PCR primers 5-CTCCCGCAGTGAAGTGGA CAT-3 (forward) and 5-TTG AAA GCA GGG CCA
CAA AGT-3 (reverse) were synthesized by Augct Biotechnology
Co. (Augct Biotechnology Co., Beijing, China), which generated
a fragment of 466 bp in length. -Actin was amplified with the
primers 5-GGC CGG GAC CTG ACT GAC TAC-3 (forward) and
5- GCC GCC AGA CAG CAC TGT GTT-3 (reverse) with product
fragment of 363 bp. The PCR reaction conditions were as
follows: initial at 94 C for 5 min, followed by 35 cycles of 94
C (1 min), 60 C (30 s) and 72 C (30 s), and a final extension
at 72 C for 7 min. The amplified fragments were separated by
1% (w/v) agarose gel electrophoresis, stained by 0.3 mg/mL
Journal of Proteome Research Vol. 8, No. 11, 2009 5043

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Zhang et al.

Figure 3. Down-regulation of Anxa2 decreased MCF-7/ADR cell invasion. (A) Same amounts of cell suspension with serum-free media
were loaded into the upper, matrigel coated surface of the transwell inserts, and FBS-containing media were loaded in the lower well
of the assay chamber. Then, the number of cells invaded and attached to the bottom of the membrane was counted. (B) The number
of cells invaded was evaluated in three fields for each experimental group; each column and bar shows the mean ( SD for triplicates.
The experiments were repeated at least three times, and statistical analysis was carried out with one-way ANOVA, *P < 0.05 vs control
cells.

ethidium bromide and analyzed using a Kodak 2D imageanalysis software.

2.7. Cell Proliferation Assay. Both flow cytometer and MTT
methods were used to determine the cell proliferation. For flow
cytometer analysis, cells were harvested, washed three times
with ice-cold PBS and fixed in ice-cold 70% ethanol overnight.
The cells were then washed with PBS and incubated with
propidium iodide (50 g/mL) and RNase (1 g/mL) at 37 C
for 30 min. Flow cytometric analysis was performed on a
Beckman Coulter EPICS analyzer. The cell cycle phase distribution was calculated from the resultant DNA histogram using
Multicycle AV software. The Proliferation Index (PI) was
calculated using the following equation: PI ) (S + G2/M)/[(G0/
G1) + S + (G2/M)] 100%. The results were the averages of 3
experiments. For MTT, 2 103 cells were plated in 96-well
microtiter plates per well. Upon analysis, 20 L of 5 mg/mL
MTT in PBS was added to each well and the cells were
incubated for another 4 h at 37 C. The supernatant was then
removed, and 200 L of DMSO was added to each well. The
absorbency of each sample was measured with a micro ELISA
reader using test and reference wavelength of 570 and 630 nm.
The surviving cells were measured every day for 5 consecutive
days.
2.8. Adriamycin Sensitivity Assay. MTT-based cytotoxicity
assay was used to determine the cell sensitivity to adriamycin.
Five 103 cells were seeded in 96-well plates and different
5044

Journal of Proteome Research Vol. 8, No. 11, 2009

concentrations of adriamycin were added accordingly 24 h

later. Three replicates were prepared for each treatment and
three blank wells containing only media without drug were also
included as control in all experiments. After 72-h incubation,
cell viability was examined by the ability of viable cells to
reduce MTT dye to formazan. The relative drug resistance was
determined by comparing the IC50 values of experiment and
control groups. The IC50 was defined as the concentration of
the drug causing 50% inhibition of cell growth, as compared
to the untreated control and the IC50 values were calculated
by linear regression of percent survival versus drug concentration.
2.9. Cell Invasion Assay. Invasion assays of MCF-7/ADR or
transfectants were performed in 24-well Boyden chamber plates
with polycarbonate membrane filter inserts with 8 m pores
(Corning Costar Corporation, Cambridge, MA). The above
surface of the porous membrane of transwell inserts were
coated with matrigel (BD Biosciences, San Jose, CA). Cell
suspension (1 104 to 1 105) in 200 L of 0.1% bovine serum
albumin (BSA)-DMEM was added into each of the upper
chamber, and the lower chamber was filled with 500 L of 10%
FBS-DMEM. After incubation for 6-36 h, noninvaded cells in
the inserts were removed with cotton swabs. Invaded cells on
the bottom side of the membrane were fixed and stained with
the 3 Step Stain Set kit (Richard-Allen Scientific, Kalamazoo,
MI) according to the manufacturers instructions. The stained
membranes were cut and placed on a glass slide, and the

Pivotal Role of Anxa2 in MDR Breast Cancer Cells

research articles
Most of the spots correlated well between MCF-7 and MCF7/ADR cells, showing equivalent intensity between the two sets
of gels. Quantitative analysis by ImageMaster 2D Platinum
software revealed that 67 spots showed differentiated intensity,
with p-values less than 0.05. Among them, 10 spots were
selected, cut out, and subjected to in-gel digestion. Eight spots
showed increased intensity and two with decreased intensity
in MCF-7/ADR cells. MALDI-TOF MS analysis followed by a
database search revealed the identity of these proteins as listed
in Table 1. Several proteins have been previously reported to
be differentially expressed in drug resistance cell lines, indicating that our method is suitable for this study.37-39 Within these
10 proteins, Anxa2 was significantly up-regulated in MCF-7/
ADR cells (Figure 1B). Western blotting analysis further confirmed that Anxa2 level was significantly up-regulated in MCF7/ADRcells(Figure1C).Anxa2,acalcium-dependentphospholipid
binding protein, has been implicated in a number of biological
responses, such as cell proliferation, angiogenesis, ion channel
activation, and cell-cell communication.40,41 We speculate that
Anxa2 may also play a role in an elevated invasion of MDR
cancer cells.

Figure 4. Down-regulation of Anxa2 did not reverse the drug

resistance phenotype of MCF-7/ADR cells. (A) MCF-7 cells were
significantly more sensitive to the treatment compared with MCF7/ADR cells. (B) All of the three Anxa2 knockdown clones retained
adriamycin-resistant property compared with control and MCF7/ADR cells; each point and bar shows the mean ( SD for
triplicates. All the experiments were repeated at least three times.
Table 2. IC50 of Adriamycin in MCF-7, MCF-7/ADR, Anxa2
Knockdown Clones and Their Negative Controla
cell type

IC50 (M)

MCF-7
MCF-7/ADR
Control
Control 1
Control 2
Control 3

3.72 ( 0.38*
39.11 ( 4.19
35.12 ( 6.49
38.90 ( 8.83
36.14 ( 5.14
41.48 ( 3.97

a
IC50 values are presented as mean ( SD from at least three
experiments performed in triplicate, *P < 0.05 vs. control cells.

number of invaded cells on the bottom surface of the membrane was counted using a bright field light microscope. All
assays were performed in triplicates. Statistical analysis was
done using one-way ANOVA, and P-value was calculated based
on two-tailed test.
2.10. Statistics. Statistics were conducted using SPSS software. All experiments were carried out at least 3 times, the
results are presented as mean ( standard errors (SEM), and
one-way ANOVA was used for data analysis. A value of P < 0.05
was considered to be statistically significant.

3. Results
3.1. MCF-7/ADR Cells Exhibits a Different 2D-PAGE
Pattern from MCF-7 Cells. Reproducible protein expression
patterns were obtained with a majority of proteins visible
between pH 5-8 in our triplicate analysis. As shown in Figure
1A, a panel of representative gel images selected from three
replicates, more than 900 protein spots are visible in each gel.

3.2. Down-Regulation
of
Anxa2
Decreased
Cell
Proliferation. A small RNA interference technique was used
to down-regulate the expression of Anxa2 in MCF-7/ADR cells
(Figure 2A). Semiquantitative PCR results showed that siRNA,
not control vector, significantly reduced the mRNA levels of
Anxa2 in MCF-7/ADR cell. Western blotting analysis of Anxa2
showed a more than 10-fold decrease at protein level, further
confirming the efficacy of siRNA techniques (Figure 2A).
Suppression of Anxa2 expression exhibited a marked inhibition
in cell proliferation (Figure 2B). The cell growth curves indicated that the three Anax2 suppression clones grew significantly
slower than MCF-7/ADR and the control siRNA cells (p < 0.05).
Cell cycle analysis showed that down-regulation of Anxa2
induced a decrease in the proportion of G2/M + S phase cells
compared to the control (Figure 2C).
3.3. Down-Regulation of Anxa2 Decreased MCF-7/ADR
Cell Invasion. A previous study demonstrated that Anxa2 was
overexpressed in invasive breast cancer and contributed to
tumor invasion and progression;25 we investigated the role of
Anxa2 in MDR induced invasiveness by using a Boyden
Chamber based assay. As shown in Figure 3A,B, MCF-7/ADR
showed a more than 4-fold increase in invasiveness in comparison with its parental MCF-7 cells, consistent with previous
report.33,34,36 In three Anxa2 knockdown clones, the invasion
properties were significantly blocked up to 70%, suggesting that
Anxa2 played an essential role in invasion of MCF-7/ADR cells
(p < 0.05).
3.4. Down-Regulation of Anxa2 Did Not Reverse the
Drug Resistance Phenotype of MCF-7/ADR Cells. To examine
whether Anxa2 is required to convey drug resistance phenotype
of MCF-7/ADR cells, MTT assay was used to check the
adriamycin cytotoxic effect on MCF-7/ADR cells depleted of
Anxa2. As shown in Figure 4A and Table 2, MCF-7/ADR is 10fold less sensitive to the treatment with adrianmycin, consistent
with previous report.12 All three Anxa2 knockdown clones, as
well as vector control transfected cells, did not show detectable
difference at IC50 of adriamycin in comparison with MCF-7/
ADR cells (p > 0.05) (Figure 4B, Table 2). Taken together, our
results suggest that elevated expression of Anxa2 is associated
with acquisition of multidrug resistance phenotype, but not
required for multidrug resistance.
Journal of Proteome Research Vol. 8, No. 11, 2009 5045

research articles
4. Discussion
Recent studies demonstrated that there might be a functional
link between MDR and tumor invasion/metastasis. One representative example was the fact that enhanced properties of
invasion and metastasis were found in a drug resistant strain
of MCF-7 cells, suggesting that drug resistance and invasion/
metastasis might be inseparable cellular events during the
progression of malignant tumors.10,12-16,34 We speculate that
acquisition of drug resistance and metastasis is a complex
progress, involving changes of multiple cellular components.
Thus, in order to possess a relatively full spectrum of molecules
affected upon the acquisition of MDR, we employed a powerful
2D-PAGE coupled with MALDI-TOF/MS technique to study the
differential protein expression patterns between the MCF-7 and
MCF-7/ADR cells. Our results showed that Anxa2 was upregulated in the drug resistant MCF-7/ADR cells, in comparison
to MCF-7 cells. When the expression of Anxa2 in MCF-7/ADR
was reduced to a low level, the drug resistant MCF-7/ADR cells
lost their invasiveness, indicating that Anxa2 played a critical
role in the invasion/metastasis of MCF-7/ADR cells.
Among the identified proteins, there were metabolic proteins
(fructose-1, 6-bisphosphatase and NADPH-flavin reductase),
structure proteins (type II keratin subunit protein), and signaling intermediates (Anxa2 and -2-microglobulin). These proteins may play an important role in acquisition of drug
resistance or in the new more malignant phenotype of the drug
resistant strains. Interestingly, we also identified ubiquitin
carboxy-terminal hydrolase l (UCHL1), whose overexpression
was closely correlated with advanced progression and invasion
of various cancers including breast cancer,42-45 as expressed
at a much higher level in the MCF-7/ADR cells. Its role in
invasion/metastasis is still under investigation.
Down-regulation of Anxa2 by siRNA did not reverse adriamycin resistance in MCF-7/ADR cells. One plausible explanation is that Anxa2, up-regulated along with the acquisition of
MDR phenotype, was not required for drug resistance. The
other possibility is that a number of proteins played redundant
roles in MDR; therefore, knockdown of Anxa2 alone was
insufficient to reverse the drug resistance. Taken together, our
results suggest that Anxa2 is not required and is not sufficient
in the acquisition of MDR phenotype.
In conclusion, our results confirm the fact that MDR is
closely associated with enhanced invasion/metastasis of tumor
cells. Furthermore, our study revealed Anxa2 as a critical
component in MCF-7/ADR cells that mediated the invasion/
metastasis. We speculate that Anxa2 may be used as a biomarker for MDR and may be developed into an antimetastasis
drug target.

Acknowledgment. This research was supported by

grants from NFSC (30772529), Tianjin Commission of
Science and Technology (06TXTJJC14502) and 973 Project
(2010CB933900).
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