MDA-MB-231 and MDA-MB-157 cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics (100 μM penicillin and 100 μM streptomycin). Human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Institute of Biochemistry (Shanghai, China) and maintained in RPMI-1640 medium supplemented with 10% FCS and antibiotics (100 μM penicillin and 100 μM streptomycin). Cells were maintained in a humidified cell incubator with 5% CO₂ at 37°C.

Viability of cells was determined using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, Carlsbad, CA, USA). MDA-MB-231 cells, MDA-MB-157 cells or HUVECs were plated at a density of 5×10³ cells/cm², cultured until 60% confluency was reached and treated with various concentrations of KP-10 (Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe) (ChinaPeptides Co., Ltd., Biomart.cn, Shanghai, China) (0, 20, 40, 60, 80 and 100 ng) for 48 h. The cells were treated with 0.5 mg/ml MTT for 4 h and lysed with dimethyl sulfoxide (DMSO). Absorbance rates were measured at 550–560 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

Apoptosis was determined using an apoptosis detection kit (KeyGen, Nanjing, China). Briefly, cells were collected, washed twice in ice-cold phosphate-buffered saline (PBS), and then resuspended in binding buffer at a density of 1×10⁶ cells/ml. Cells were simultaneously incubated with fluorescein-labeled Annexin V and propidium iodide (PI) for 20 min. The mixture was then analyzed on a FACSCalibur (BD Biosciences, Baltimore, MD, USA). Annexin V-FITC generated signals were detected on FL1 at a wavelength of 525 nm, and PI signals were monitored using a detector reserved for phycoerythrin emission (FL2, 575 nm). Data were analyzed using CellQuest software from BD.

The migration assay was performed using a Boyden chamber (8-μm pore size polycarbonate membrane; Cell Biolabs, San Diego, CA, USA). Cells were resuspended in FBS-free DMEM to a concentration of 3×10⁵ cells/ml. The upper chamber was loaded with 200 μl of cell suspension and the lower chamber was loaded with 600 μl of DMEM containing 10% FBS. The filter was fixed in 4% paraformaldehyde (Sigma) and stained with crystal violet (CV; Beyotime, Shanghai, China). The cells on the upper side of the filter were wiped off using a cotton swab. The cells that migrated to the undersurface of the membrane were counted using a light microscope. Ten microscopic fields (x400) were randomly selected to count the cells.

Cells were grown in a 6-well dish. A confluent monolayer of cells was scratched with a 200-μl pipette tip to simulate a wound. Cells were washed twice with PBS and then supplemented with medium and incubated for 4 h at 37°C. Cell migration into the wounded area was monitored microscopically. Images were captured at the interface of the unwounded and wounded areas.

Cell monolayers were plated in black, clear-bottomed 96-well plates overnight. Cells were then incubated in serum-free media at 37°C for 1 h prior to stimulation with FLIPR Calcium 4 reagent (Molecular Devices, Sunnyvale, CA, USA). Cells were then incubated for 1 h at 37°C while ligands were prepared in a separate flat-bottomed 96-well plates. Cells were then tested for intensity of fluorescence on the NOVOstar (BMG Labtech, UK) over 60 sec with ligands being added 1:10 at 11 sec.

Fifty micrograms of protein was applied to 10% polyacrylamide gels with 1% gelatin incorporated as a substrate for gelatinolytic proteases. After running the gel the SDS was removed by washing twice in 2.5% Triton X-100 for 30 min. The gels were incubated overnight in zymography development buffer containing 50 mM Tris-HCl (pH 7.4), 2 mM NaN₃ and 5 mM CaCl₂. After development, the gels were stained for 3 h in 45% methanol/10% glacial acetic acid containing 1% (w/v) Coomassie blue R-250 and subsequently partially destained with the same solution without dye. The gelatinolytic activity of each MMP was evaluated as a clear band against the blue-stained gelatin background.

The present study was conducted using protocols approved by the Animal Care and Use Committee of China Medical University. NOD/SCID mice were allowed a standard rat diet and water ad libitum, and maintained on a 10-/14-h light/dark cycle. MDA-MB-231 or MDA-MB-157 cells (1×10⁸) were injected into NOD/SCID mice by subcutaneous injection. After the tumor diameters reached 3–5 mm, the mice were divided randomly into 6 groups (MDA-MB-231, MDA-MB-231 + PBS, MDA-MB-231 + KP-10, MDA-MB-157, MDA-MB-157 + PBS and MDA-MB-157 + KP-10). Tumor growth was monitored every 5 days after the first week of inoculation. All mice were sacrificed at day 60. All of the tumor tissues were collected, fixed in 4% formaldehyde and embedded in paraffin for immunostaining.

For immunohistochemical staining of CD31, endogenous peroxidase activity was blocked in 4-μm tumor sections with 3% hydrogen peroxide for 30 min. Antigen retrieval was performed in citrate buffer (10 mM, pH 6.0) for 30 min at 95°C in a pressure cooker. The CD31 antibody (Sigma) was incubated with the sections at 1:500 overnight at 4°C. The sections were then incubated with a biotinylated secondary antibody for 1 h at room temperature, followed by incubation with a streptavidin-horseradish peroxidase (HRP) complex (Beyotime, Beijing, China) for 60 min at room temperature. The bound antibody was visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Beyotime). Sections were also counterstained with hematoxylin (Beyotime).

According to the method of Hong et al (13), the fertilized eggs were incubated at 37°C for 8 days and then divided into 2 groups (PBS and KP-10). After incubation for 3 more days, the CAM of each live egg was harvested and placed individually in a 6-well plate. Images of the blood vessels of each CAM were captured using Motic Images Plus^® (MediaCybernetics^®, Bethesda, MD, USA). The blood vessel area as a percentage of the total CAM area, blood vessel length and number of branch points were quantified using Image Pro^® analysis software (MediaCybernetics^®).

Proteins were resolved by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and detected using the following antibodies: E-cadherin (sc-7870), vimentin (sc-6260), N-cadherin (sc-7939) and β-actin (sc-47778) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunostaining was detected using an enhanced chemiluminescence (ECL) system (Amersham Biosciences, Westborough, MA, USA).

Data are presented as the means ± standard deviation (SD). Differences between groups were analyzed using the Student’s t-test for continuous variables. The Kaplan-Meier estimator was used to compare different patient groups, and P-values were calculated using the log-rank (Mantel-Cox) test. Statistical analysis was performed using GraphPad 5.0 software (GraphPad Software, La Jolla, CA, USA) and significance was established at P<0.05.

The inhibitory growth effects of KP-10 on breast cancer MDA-MB-231 and MDA-MB-157 cells were determined by MTT assay. This inhibition was dependent on the concentration of KP-10. The IC₅₀ values of KP-10 for MDA-MB-231 and MDA-MB-157 cells were 62.5 and 63.3 nM, respectively (P<0.05; Fig. 1A). The apoptotic ratio of KP-10-treated MDA-MB-231 or MDA-MB-157 cells was 5–15 times higher compared with the ratio of the respective control cells (P<0.05; Fig. 1B). The mobility of breast cancer cells was also inhibited by KP-10. The Transwell assay showed that less cells after KP-10 treatment migrated to the lower side of the membrane than the number of cells in the untreated ones (P<0.05; Fig. 1C). The wound-healing assay showed that the migration of MDA-MB-231 or MDA-MB-157 cells after KP-10 treatment was significant reduced when compared with that in the untreated cells (P<0.05; Fig. 1D). We also found that the activity of MMP-2 and MMP-9 was inhibited by KP-10 in the MDA-MB-231 and MDA-MB-157 cells (Fig. 1E). Furthermore, both MDA-MB-231 and MDA-MB-157 cells exhibited a marked increase in [Ca²⁺] in response to treatment with KP-10 (Fig. 1F). Notably, KP-10 had similar effects on HUVECs (Fig. 1).

The antitumor properties of KP-10 were evaluated using mouse breast cancer models. Both tumor volume and the weight of the KP-10-treated tumors were lower compared to the untreated ones (P<0.05; Fig. 2A and B). KP-10 also increased the survival rate of the tumor-bearing mice (P<0.05; Fig. 2C). In addition, the blood vessel density of the tumors from the mice in the KP-10-treated group was significantly reduced compared to that in the untreated group using immunostaining for CD31 expression (P<0.05; Fig. 2D).

We evaluated the inhibitory effect of KP-10 on in vivo angiogenesis by using a chick embryo chorioallantoic membrane angiogenesis assay. The results showed that KP-10 inhibited neovascularization in the chick CAM model (P<0.05; Fig. 2E).

Based on the antitumor roles of KP-10 in breast cancer cells, we carried out western blotting to identify the mechanism of KP-10. Compared with the untreated cells, KP-10-treated MDA-MB-231 and MDA-MB-157 cells showed a higher expression level of epithelial marker E-cadherin, while lower expression levels of mesenchymal markers vimentin and N-cadherin were noted (Fig. 3).