Anxa2 Plays a Critical Role in Enhanced Invasiveness of the Multidrug

Sep 18, 2009 - Multidrug resistance (MDR) is the major cause of failure in cancer chemotherapy. Recent reports even suggest that ... Small interferenc...
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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, [email protected]. Ruifang Niu, Tianjin Medical University Cancer Institute and Hospital, Tianjin. 300060, China; e-mail, [email protected]. † 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, 5041–5047 5041 Published on Web 09/18/2009

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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 µM 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 µg 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

3

gi|8923900

4 5 6 7 8 9 10

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

a

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).

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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. β-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 µL of 5% TFA in 50% acetonitrile, dried, and redissolved in 10 µL of 0.5% TFA. The samples were desalted with ZipTipC18 according to the manufacture’s instructions and eluted with 2.5 µL 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 µg 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 manufacturer’s protocol. Mouse monoclonal antibody against β-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 manufacturer’s instruction, and three separate MCF-7/ADR stable transfectants were generated after 14 days selection by 300 µg/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. β-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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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 µg/mL) and RNase (1 µg/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 µL 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 µL 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

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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 µm 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 µL of 0.1% bovine serum albumin (BSA)-DMEM was added into each of the upper chamber, and the lower chamber was filled with 500 µL 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 manufacturer’s 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 (µM)

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 β-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). References (1) Beck, W. T. Mechanisms of multidrug resistance in human tumor cells. The roles of P-glycoprotein, DNA topoisomerase II, and other factors. Cancer Treat. Rev. 1990, 17, 11–20. (2) Beck, W. T.; Cirtain, M. C.; Danks, M. K.; Felsted, R. L.; Safa, A. R.; Wolverton, J. S.; Suttle, D. P.; Trent, J. M. Pharmacological, molecular, and cytogenetic analysis of “atypical” multidrugresistant human leukemic cells. Cancer Res. 1987, 47 (20), 5455– 60. (3) Fojo, T.; Bates, S. Strategies for reversing drug resistance. Oncogene 2003, 22 (47), 7512–23.

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