EMD for research use was from Sigma-Aldrich (cat. no. E7881, Beijing, China), with thepurity of ≥90%, as determined by high-performance liquid chromatography. A stock solution (1 mM) was prepared by dissolving EMD in dimethyl sulfoxide (DMSO). Recombinant human VEGF₁₆₅ (cat. no. 293-VE) is a product of R&D Systems (Minneapolis, MN, USA). A potent inhibitor of VEGFR-2, ZD6474 (Selleck Chemicals Inc., cat. no. S1046, Shanghai, China), and a broad spectrum MMP inhibitor, Batimastat (Bat; Santa Cruz Biotechnology Inc., cat. no. SC-203833, Santa Cruz, CA, USA) were used in this study and served as positive controls. In addition, L-sulforaphane (SFP, Sigma-Aldrich, cat. no. S6317, Beijing, China) was used as a positive control in tube formation assay.

The human umbilical vein cell line, EA.hy 926, was purchased from cell bank of Shanghai Institute for Biological Sciences, CAS. EA.hy 926 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Beijing, China) in a humidified incubator containing 5% CO₂ at 37°C. Prior to performing each assay, the ECs were serum starved for 4 h. MDA-MB-231 human breast cancer cells, purchased from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Inc.), 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Inc.) in a humidified incubator containing 5% CO₂ at 37°C.

Five- to-six-week-old female NOD/SCID mice were purchased from Vital River Laboratories (Beijing, China) to establish orthotopic and experimental lung metastatic xenograft model. Eight- to-nine-week-old male Sprague Dawley (SD) rats were also purchased from Vital River Laboratories to obtain thoracic aorta in the rat aortic ring angiogenesis assay. The animal experimental protocol complied with the Animal Management Rules of the Chinese Ministry of Health (document no. 55, 2001), and was approved by the Animal Ethics Committees of Jiangxi University of Traditional Chinese Medicine. All of the animal experiments were conducted in strict accordance with the requirements listed in this protocol. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

For the growth assay, MDA-MB-231 cell xenografts were established by injection of 1×10⁷ cells at the mammary fat pad in NOD/SCID mice. After 3 weeks of growth, the tumors were removed and 1×1×1 mm tumor pieces were then implanted at the mammary fat pad of mice. The mice bearing tumor chunks were randomly divided into three groups: control, EMD (40 mg/kg per day) treatment group, and EMD (80 mg/kg per day) treatment group. Forty-eight hours later, EMD was administered by gavage on a regimen of 6-day dosing per week for 5 weeks. Tumor growth was assessed by measuring volumes of tumors with electronic calipers every 3–4 days continuously. All of the mice were sacrificed 5 weeks after inoculation of the cancer cells and the tumors were collected and weighed.

To establish experimental lung metastasis xenograft model, 2×10⁶ MDA-MB-231 cells in 200 μl normal saline were injected through tail vein of NOD/SCID mice. The mice were divided into control, EMD (40 mg/kg per day) treatment group, and EMD (80 mg/kg per day) treatment group. EMD treatment was began 24 h after tumor injection. After 8-week treatment the formation of metastatic foci in lung tissues was measured by nest reverse-transcription polymerase chain reaction (RT-PCR). The mice were euthanized and dissected, and lungs were snap frozen in liquid nitrogen. Total RNA was isolated from each lung using the QIAshredder and RNeasy Protect Mini kit (Qiagen) for detection of human cytokeratin 19 (ck19) by nest PCR. For the survival assay, the experimental lung metastasis xenograft model was established using the same method as in vivo experimental metastasis assay, but the observation period was 120 days.

Nest RT-PCR was used to detect expression of the human ck19 gene in lung tissues of tumor-bearing mice. Total RNA was isolated from each lung using the QIAshredder and RNeasy Protect Mini kit (Qiagen). Ck19 was reverse transcribed in the presence of 2 μl enzyme mix, 10 μl RNA and outer primer for 30 min at 50°C, according to the manufacturer’s instructions (Qiagen OneStep RT-PCR kit, Qiagen). After Taq polymerase activation for 15 min at 95°C, samples were amplified for 37 cycles at 94°C for 45 sec, 58°C for 45 sec and 72°C for 90 sec. Target primers for amplifying ck19 (outer primer) were designed using Primer Designer (Scientific & Educational Software Version 2.0). The forward primer for ck19 (GenBank no. BC010409) was 5′-cca cgt cgt cct tcg gag gcc-3′ (64–84 bp) and reverse primer was 5′-gttc cgt ctc aaa ctt ggt tc-3′ (529–549 bp). After a final extension for 10 min at 72°C, the RT-PCR product was further subjected to nest PCR using the Qiagen Multiplex PCR kit (Qiagen). The inner primers were forward 5′-tac agc cac tac tac acg acc atc c-3′ (432–456 bp) and reverse 5′-gga caa tcc tgg agt tct caa tg-3′ (488–510 bp). The nest PCR profile was as follows: 3 min at 94°C, followed by 35 three-step cycles of 45 sec at 94°C, 45 sec at 60°C and 1 min at 72°C. PCR reactions were subjected to final extension at 72°C for 10 min. Nest RT-PCR analysis was performed using the Mastercycler gradient (Eppendorf). The β-actin gene was used as an internal control for standardization and the primers, Tm, and cycles are the same as reported (23). The nest PCR product was separated by 4% agarose (UltraPure™ Agarose, Invitrogen) gel electrophoresis, and the gels were viewed by UV transillumination and photographed by the UVP EC3 gel imaging system.

MVD was determined by CD34 staining against ECs in tumor tissues. Firstly, 4-μm-thick sections from paraffin-embedded formalin-fixed MDA-MB-231 tumor tissues were made. After dewaxing, and hydration, the slides were incubated with Proteinase K at 37°C for 15 min to retrieve antigen, and then washed in PBS (0.01 mol/l, 5 min, three times). In order to block endogenous peroxidase activity, the sections were treated with 3% H₂O₂ in methanol for 10 min. Followed by blocking with 10% normal goat serum (Cell Signaling, Danvers, MA, USA) and washing in PBS, the slides were incubated with anti-CD34 antibody (1:50; cat. no. 3569; Cell Signaling) or PBS (0.01 mol/l) at 4°C overnight. The slices were incubated with second antibody (HRP-labeled goat anti-mouse antibody) at room temperature for 60 min, and then peroxidase activity was detected by SignalStain^® DAB substrate kit (Cell Signaling). All the slides were checked under light microscopy (Olympus, BX-63), and images were analyzed by Image Pro plus software 5.0 (Media Cybernetics Inc. Silver Spring, MD, USA).

The tube formation assay was used to investigate the effects of EMD on angiogenesis in vitro. Briefly, 80 μl of liquid growth factor-reduced Matrigel (BD Biosciences, San Jose, CA, USA) were added to each well of a 96-well plate. After 45-min incubation at 37°C, 3×10⁴ EA.hy 926 cells per well in 100 μl complete culture medium containing vehicle, ZD6474 or EMD was seeded in each well. Then, 100 μl serum-free medium containing VEGF₁₆₅ (final concentration 2 ng/ml) was added. After 24-h incubation at 37°C and 5% CO₂, the images of each well were recorded by an inverted microscope (Leica, DMI 3000B, Germany).

In the present study, an ex vivo tube formation system was used to evaluate the anti-angiogenic effect of EMD. Male SD rats (8–9-week-old) were euthanized and thoracic aortas were retrieved. After rinsing with 1% antibiotic/antimycotic cocktail in 1X PBS (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B; Invitrogen), the surrounding fibro-adipose tissue of thoracic aorta was completely removed with fine microdissection scissors and the thoracic aorta was cut into 1 mm thick rings with a scalpel blade. Then, individual ring was implanted on a Matrigel-coated 96-well microtiter plate. Matrigel was added again to embed and fix rings. After 30-min incubation in 5% CO₂ at 37°C, the aorta rings were incubated in human endothelial serum-free medium (Gibco, Carlsbad, CA, USA) supplemented with 2% FBS, 50 U/ml penicillin and 50 μg/ml streptomycin (Invitrogen) for 24 h and then treated with different doses of EMD for 13 days. Finally, 8 μg/ml Calcein AM (BD Biosciences) was added to stain microvessels. Photographs of the microvessels were obtained using an inverted fluorescence microscope (Leica, DMI 3000B, Germany).

Effects of EMD on invasion were measured by a 48-well microchemotaxis system (AP 48, Neuro Probe; Gaithersburg, MD, USA). Briefly, 5 μg of fibronectin in a volume of 50 μl was applied on the rough (lower) surface of the polycarbonate membrane and 5 μg/filter Matrigel was plated to the smooth (upper) surface. The lower chambers of the plates were then filled with 30 μl medium containing 0.1% BSA. Log-phase cells were harvested and re-suspended in culture medium with 0.1% BSA. Cell suspensions (100 μl containing 1×10⁵ cells) were added to the upper compartment and incubated for 16 h at 37°C in a 5% CO₂ atmosphere. After incubation, the filters were fixed with methanol and stained with 0.5% crystal violet for 60 min. The cells on the upper surface of the filters were removed by wiping with cotton swabs. The cells invading to the lower surface of the filter through Matrigel and filter were quantified with the Image Pro plus software 5.0 (Media Cybernetics Inc.), and representative results are illustrated in the figures. Each assay was performed in triplicate.

In vitro migration of MDA-MB-231 cells was measured using the AP 48 chamber (Neuro Probe), similar to the in vitro invasive assay, but without Matrigel pre-coating on the smooth (upper) surface of filters. Briefly, the underside of the polycarbonate membrane was coated with 10 μg/ml fibronectin overnight at 4°C. Thirty microliters of DMEM (with 10% FBS and 10 μg/ml Collagen I) was added to the lower chamber, and the chamber was covered by the filter. MDA-MB-231 cells were trypsinized and washed with FBS-free DMEM, and 100 μl of cell suspensions (in FBS-free DMEM, containing 2×10⁵ cells) with or without EMD were added to the upper chambers and incubated for 14 h in an incubator containing 5% CO₂ at 37°C. Determination of migrated cells was conducted as described for the in vitro invasive assay.

Log-phase cells EA.hy 926 cells were harvested, re-suspended and seeded on the 8 wells Nunc™ Lab-Tek™ II Chamber Slide™ system. The cells were grown at 37°C in a humidified CO₂ incubator until they were 50–70% confluent. The culture medium was aspirated from each well and the cells gently rinse twice in PBS at room temperature. Then, the cells were exposed to EMD and/or vehicle for 24 h. After exposure, the chambers of the Chamber Slide system were removed gently and the slides were rinsed twice in PBS. Then cells were fixed by incubation in 4% (v/v) paraformaldehyde in PBS for 20 min at room temperature. The slides were heated in antigen retrieval buffer [100 mM Tris, 5% (w/v) urea, pH 9.5] at 95°C for 10 min. After rinsed in PBS, cells were blocked by incubating the cells in 0.1% Triton X-100 in PBS for 15 min at room temperature. Then, cells were incubated with the primary antibodies at 4°C overnight. After rinsing in PBS, the slices were incubated with second antibody (HRP-labeled goat anti-mouse antibody) at room temperature for 60 min, and then peroxidase activity was detected by SignalStain^® DAB substrate kit (Cell Signaling). All the slides were checked under a light microscope (Olympus, BX-63), and images were analyzed by Image Pro plus software 5.0 (Media Cybernetics Inc.).

The activity of MMPs was measured by the SensoLyte^® 570 Generic MMP assay kit (AnaSpec, Fremont, CA, USA). This kit provides a FRET-based method to detect the activity of a variety of MMPs including MMP-1, 2, 3, 7, 8, 9, 10, 11, 12, 13 and 14. It uses 5-FAM (fluorophore) and QXL520™ (quencher) labeled FRET peptide substrates for continuous measurement of MMP activity. In an intact FRET peptide, the fluorescence of 5-FAM is quenched by SensoLyte. Upon the cleavage of FRET peptide by MMPs, the fluorescence of 5-FAM is recovered. Analyses were performed according to the manufacturer’s instructions. Briefly, supernatants of MDA-MB-231 cells were collected after incubation with or without EMD for 12 h. MMPs in the supernatants were activated by incubation with 4-aminophenylmercuric acetate for 1 h at 37°C. Fifty micro-liter MMPs-containing samples and 50 μl MMP substrate solution were added into a 96-well plate. The reagents were mixed by shaking the plate gently for 30 sec. After a 50-min incubation period at 37°C, the counter Victor3™, Perkin-Elmer (Waltham, MA, USA) was applied at Ex/Em=540 nm/575 and then the action was stopped by adding stop solution, and fluorescence intensity was measured.

Runx2 activity in MDA-MB-231 and EA.hy 926 nuclear extracts was detected using the TransAM™ AMl-3/Runx2 kit (Active Motif North America, Carlsbad, CA, USA) following the manufacturer’s instructions. Cell extracts were prepared using the nuclear extract kit (Active Motif). Then, 20 μl of extracts diluted in complete lysis buffer and containing 15 μg nuclear extract were added into a 96-well plate. This plate immobilizes oligonucleotides containing Runx2 consensus binding sites. Saos-2 nuclear extract served as a positive control for Runx2 activation and 20 μl complete lysis buffer served as the blank. The wild-type consensus oligonucleotide was provided as a competitor for Runx2 binding to monitor the specificity of the assay. After 1-h incubation at room temperature, the plate was washed three times with washing buffer. Diluted primary antibody (100 μl) was added into wells and incubated for 1 h at room temperature without agitation. After three washes, HRP-labeled secondary antibody was added and incubated for 1 h at room temperature. Then, 100 μl developing solution was added to initiate the color reaction. After 100 μl stop solution was added, the absorbance was measured within 5 min at 450 nm with a reference wavelength of 655 nm using an ELx800 microplate reader (BioTek, Winooski, VT, USA).

For western blot analysis, ECs were washed twice with PBS and then lysed by the addition of 1 ml lysis buffer (10 mmol/l Tris, pH 7.6, 150 mmol/l NaCl, 5 mmol/l EDTA, pH 8.0, 10 ml/l Triton X-100, 1 mmol/l DTT) containing 0.1 mmol/l PMSF. After 30 min on ice, lysates were collected and clarified by centrifugation at 15,000 g for 10 min at 4°C. Aliquots of whole cell lysates were subjected to 10% SDS-PAGE and then transferred to Hybond nitro-blotting membranes. The membranes were blocked with 3% bovine serum albumin in Tris-buffered saline containing 0.5 ml/l Tween-20 (TTBS) and then incubated with a 1:500–1,000 dilution of the indicated primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Immunoreactive proteins were detected using an enhanced chemiluminescence kit (Millipore). β-actin (Santa Cruz, SC-130301) served as an internal control.

The mean values were obtained from at least three independent tests. The data are presented as mean ± SD and analyzed with the GraphPad Prism 5.0 software program (La Jolla, CA, USA). Comparison among different groups was carried out by analysis of variance (one-way ANOVA). Differences between means were considered statistically significant at p<0.05.

Initially the study was to determine the inhibitory effects of EMD on breast cancer in vivo. The growth inhibition in MDA-MB-231 cell line-xenografts was determined first. Tumor-bearing mice were treated with various doses of EMD for 5 weeks. A significant suppression on tumor weight was found when the mice were treated with 80 mg/kg b.w. of EMD (Fig. 2A). These results indicated that EMD may contribute to breast cancer. Considering the clinical importance of metastasis in breast cancer patients, the role of EMD in inhibition of metastasis was evaluated in vivo. Experimental lung metastasis models were established by injecting human breast cancer MDA-MB-231 cells through tail vein of NOD/SCID mice and the tumor cells invaded into lung tissues were detected by amplification of human ck19 gene with nest RT-PCR assay. As shown in Fig. 2B, EMD at 80 mg/kg b.w. daily for 8 weeks reduced >70% ck19 expression compared to the vehicle control. To determine whether EMD influence survival of the tumor cell injected mice, overall survival assay was performed. Our data showed that EMD lengthen survival time of NOD/SCID mice in a dose-dependent manner (Fig. 2C).

Invasion and migration are the critical steps for the spread of tumor cells to distant organs. To determine whether inhibition of lung tumor formation by EMD in vivo was due to the ability of EMD to influence tumor cell migration and/or invasion, we next determined the effects of EMD on cell invasion and migration in vitro. As shown in Fig. 3A, EMD blocked trans-membrane invasion of MDA-MB-231 cells significantly, when MDA-MB-231 cells were incubated with 10, 20 and 40 μM EMD for 14 h. We next evaluated the effects of EMD on cell migration with similar methods. Here, we also found that migration of MDA-MB-231 cells was significantly blocked by EMD (Fig. 3B) in a dose-dependent manner. The data suggested that the inhibitory effects of EMD on metastasis may be associated with its significant inhibition on cell invasion and migration.

Breakdown of the extracellular matrix by MMPs in surrounding tissues is a fundamental step of invasion and migration in tumor cell metastasis. Therefore, we examined the effects of EMD on MMPs in MDA-MB-231 cells. Fig. 4 shows the effects of EMD on MMPs in MDA-MB-231 cells. Using a fluorescence resonance energy transfer (FRET)-based analysis, we found that EMD exhibited significant suppression of FRET substrate cleavage of MMPs in a dose-dependent manner (Fig. 4A). To further determine whether EMD inhibited the functional activity or expression of MMPs, we analyzed the expression of MMP-9 and MMP-13 in MDA-MB-231 cells treated with EMD. As shown in Fig. 4B, EMD significantly decreased MMP-9 and MMP-13 expression of MDA-MB-231 in a dose-dependent manner. These data suggested that the anti-metastatic properties of EMD may be due to downregulation of expression of MMP-9 and MMP-13 in MDA-MB-231 cells.

To determine whether tumor growth and metastasis inhibition by EMD was associated with inhibition of tumor vessel formation, we next measured effects of EMD on microvessel density (MVD) in MDA-MB-231 tumor blocks by IHC assay. Representative results are illustrated in Fig. 5 and semi-quantitative analysis of CD34 in these tumor blocks was shown in Table I. A 42.49% (p<0.01 compared with CTRL) and 78.91% (p<0.01 compared with CTRL) reduction of positive area of CD34 staining were observed when the nude mice were treated with 40 and 80 mg/kg/day EMD, respectively.

To determine whether inhibition of tumor induced angiogenesis in vivo by EMD was due to a direct inhibition targeting ECs, we used the tube formation assay to evaluate the effects of EMD on angiogenesis. As shown in Fig. 6A, when EA.hy 926 cells were seeded on Matrigel, capillary-like structures with a lumen were formed. After exposure to EMD solution at various concentrations for 24 h, these structures were destroyed in a dose-dependent manner. In addition, rat aortic ring angiogenesis assay was used in this study to verify the anti-angiogenic effects of EMD. Similarly, new blood vessels, triggered by the injury of the dissection procedure and mediated by growth factors produced from aortic ring were demolished by EMD in a dose-dependent manner (Fig. 6B).

VEGFR-2 is a crucial regulator in all aspects of normal and pathological vascular endothelial cell biology. In order to understand whether VEGFR-2 is involved in the inhibition of angiogenesis induced by EMD, we next evaluated the effects of EMD on VEGFR-2 phosphorylation in EA.hy 926 cells. To this end, immunocytochemistry assay was employed and the results are shown in Fig. 7. From these data, it was found that EMD produced a dose-dependent decrease in phospho-VEGFR-2 expression. Collectively, these findings indicated that EMD inhibited angiogenesis by directly inhibiting activation of VEGFR-2 in human ECs.

Runx2 is a transcriptional factor of metastatic growth of breast cancer cells. Several genes required for the formation of metastatic foci, including MMP-9, MMP-13, bone sialoprotein, osteopontin, VEGF, are targets of this transcriptional factor. Therefore, next we examined Runx2 activities in ECs and MDA-MB-231 cells. As shown in Fig. 8A and C, EMD had no effect on total Runx2 expression, but caused a significant decrease of phospho-Runx2 expression. To confirm the western blot results, Runx2 transcription factor assay was employed. Here, an ELISA-based kit for the Runx2 transcription factor was used to analyze the effects of EMD on Runx2. Nuclear extracts incubated with EMD or vehicle were prepared and the binding activity between Runx2 with its target sequence was determined. The results showed that EMD significant decreased the binding activity of Runx2 to its target sequences in EA.hy 926 and MDA-MB-231 cells (Fig. 8B and D). Together, these findings suggested that the inhibitory effects of EMD on angiogenesis and metastasis were caused by inhibition of MMPs and VEGFR-2, which may be associated with suppression of Runx2 phosphorylation.