The murine mammary carcinoma cell stably expressing the firefly luciferin gene (4T1-luc) and non-labeled 4T1 cells were obtained from Dr Ganlin Zhang, Beijing Hospital of Traditional Chinese Medicine (Beijing, China). The cell line was cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) and supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 mg/ml streptomycin and 100 U/ml penicillin at 37°C in a humidified atmosphere with 5% CO₂.

SET was extracted by the Dalian Institute of Chemical Physics, Chinese Academy of Sciences (Dalian, China). ASA was purchased from the National Institutes for Food and Drug Control (Beijing, China).

Two kilograms of Caulis Spatholobi were decocted thrice with anhydrous ethanol. The liquids were merged and concentrated to constant weight on a rotary vacuum evaporator. Further purification of the extracts (20 g) was performed by HPD-100 macroporous adsorbent column chromatography eluted with different concentrations of alcohol. Respectively, the fractions of 20, 40, 60, 80 and 100% alcohol were concentrated to constant weight on a rotary vacuum evaporator and crushed into powder before experiments. The extracts were named SE1, SE2, SE3, SET, and SE4, respectively.

The screening of cytotoxicity was assessed by MTT (Amresco, USA) assay as previously described (11). Briefly, the 4T1 cells were plated into a 96-well cell culture cluster at 5×10³ per well. Then, the cells were treated with SE1, SE2, SE3, SET, and SE4 in different concentration, respectively, for 24, 48, 72 and 96 h. Another experiment was performed using SET as treatment, which was identified by the above preliminary test. The method was conducted as mentioned above, however, the time of drug treatment was adjusted to 3, 6, 12 and 24 h. At mentioned time points, MTT was added to each well till the final concentration of 0.5 mg/ml, and the cells were incubated at 37°C for 4 h. A total of 150 µl of dimethyl sulfoxide (DMSO) was replaced in each well instead of medium with MTT. With fully mixing, the absorbances of solution were determined in 570 nm performed a microplate reader (Molecular Devices, USA). The inhibitory rate of 4T1 cells was calculated as follows. Inhibitory rate = (1 - mean experimental absorbance/mean negative control absorbance) × 100%. Half maximal inhibitory concentration was calculated as previously described (12).

Fresh blood was obtained as previously described (13). In brief, blood was obtained by retro-orbital venous plexus sampling, which was collected into an Eppendorf tube mixed with acid citrate dextrose solution (38 mM citric acid/75 mM trisodium citrate/100 mM dextrose). The blood was centrifuged at 280 × g for 8 min at room temperature, supernatant containing plasma and buffy coat were transferred to a new tube, centrifuged at 280 × g for 4 min. Then, platelet-rich plasma (PRP) was collected into a fresh tube and prepared for study.

The different doses of SET were designed as low, medium, high (20, 40 and 80 µg/ml) groups and ASA (300 µM) as positive control group compared with negative control. The following study in vitro was based on identical methods. The platelet aggregation was measured by whole blood aggregometers (Chrono-Log, Havertown, PA, USA) (14). Briefly, 500 µl of whole blood with equivalent volume of normal saline (NS) was placed in aggregometer, incubated for 10 min at 37°C with stirring at 1,200 rpm. The samples were treated by SET as inhibitors. ADP (10 µM) was added to the mixture as activator for platelet aggregation. Finally, the rates of aggregation were recorded through a curve. To determine the optimal dose, the different concentrations of SET were used to inhibit the platelet aggregation induced by ADP.

As described by the previous method, PRP was used within 2 h after centrifugation. Next, carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE; Beyotime, Shanghai, China), a sort of cell fluorescent label, was incubated with PRP at a final concentration of 5 µM for 10 min. The labeled platelets were washed by Tyrode's solution twice, then, resuspended with fresh Tyrode's solution. The next section of test was performed after at least 1 h. The TCIPA was detected by confocal microscopy and flow cytometry assay as previously described (15). For the microscopic observation of TCIPA, briefly, 1×10⁵ cells/ml 4T1 cells, pre-treated by SET and ASA, were plateted in the confocal dish until the logarithmic phase. Fluorescently labeled platelets were placed into the dish and incubated with tumor cells for 30 min at 37°C. The non-aggregated platelets were removed. T-P cell aggregation was visualized by confocal microscopy (Olympus, Tokyo, Japan). For flow cytometry assay, 1×10⁵ pre-treated 4T1 cells were incubated with 200 µl of platelet preloaded with CFDA-SE for 10 min. A final concentration of 2% paraformaldehyde was added as a fixative stored at 4°C, prepared to flow cytometry assay.

For lung seeding and metastasis analysis, the mouse model of pulmonary metastasis was established by vein tail injection as previously described (16). Briefly, 2×10⁴ 4T1-luc cells in 100 µl of NS were injected into 8-week-old female Balb/c mice by vein tail. Simultaneously, mice were administrated with different doses of SET (2.2, 4.4 and 8.8 mg/kg). ASA (22.5 µmol/kg) was used as positive control drug compared with model group (tumor only). Imaging assay was conducted after injection of tumor cells and daily therapy. Mice were anesthetized by isoflurane gas, then visualized by using small animal in vivo imaging system (Kodak, USA) 10 min after injection of luciferin. Until the mice of model group died, mice were sacrificed and the lungs were collected into 4% paraformaldehyde for observation and hematoxylin and eosin (H&E) staining (17) using a microscope (Olympus). Survival curve was employed to evaluate overall survival. The study was approved by the Ethics Committee of Beijing Administration Rule of Laboratory Animal, Beijing, China.

The method of cell plating and drug treatment was measured as mentioned above. In the logarithmic phase, the 4T1 cells cultured in FBS-free medium were incubated with 200 µl PRP, or not, for 10 min. Then, the reaction was terminated and samples were collected by centrifugation at 900 × g for 10 min. Supernatant was stored at −80°C until the activities of Matrix metalloproteinase (MMP)-2 and −9 were detected by zymography.

Gelatin zymography was used to detect the activity of MMP-2 and −9 in the platelet supernatants as previously described (18). In brief, samples of TCIPA were separated by 10% SDS-PAGE with copolymerized gelatin (0.2%; Sigma Chemical Co., St. Louis, MO, USA) with a substrate for gelatinolytic proteases. After electrophoresis, the gels were washed with 2.5% Triton X-100 for 20 min repeated three times, and then incubated for 48 h at 37°C in incubating buffer (25 mM Tris HCl, 0.9% NaCl, 5 mM CaCl₂ and 0.05% Na₃N, pH 7.5). The conditioned medium of 4T1 cells was used as control. After 72 h reaction, gels were fixed and stained in 0.1% Coomassie blue R-250 (Sigma Chemical Co.), 40% methanol and 10% acetic acid for 1 h and then discolored in 8% acetic acid with 4% methanol. The gelatinolytic activities were presented by separate bands.

The methods of cell plating and drug treatment were measured as indicated above. Total RNA was isolated by TRIzol reagent (Life Technologies, Grand Island, NY, USA). The detailed procedure of RT-PCR was measured by RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) (19). According to the kit instruction, 1 µg total RNA was performed to the following reverse transcriptase in a final volume of 20 µl with AMV reverse transcriptase. Subsequently, several specific primers were used to amplify cDNAs of selectin P (SELP) forward, 5′-CGAGCCCAACAACAAGAA-3′ and reverse, 5′-GGGTAGCAGGAGCAGGTAT-3′; selectin P ligand (SELPLG) forward, 5′-CTGTCACTGAGGCAGAGTCG-3′ and reverse, 5′-TGAGCAGCCACGGTGTTG-3′; GPVI forward, 5′-CCTTCCATCTTACCCACA-3′ and reverse, 5′-CTGCTGAAAGCCAAGTTAT-3′; integrin α2b (ITGA2B) forward, 5′-TGACAGGGAGCAGGAAGA-3′ and reverse, 5′-GCTGGGCGTGGATAGTTT-3′. GAPDH (20) forward, 5′-GTGGATATTGTTGCCATCA-3′ and reverse, 5′-ACTCATACAGCACCTCAG-3′ was compared with each experiment as internal control. The PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining.

The values of data were presented as means ± standard deviation. Statistical significance was determined by one-way analysis of variance followed by LSD post hoc test using SPSS Statistics 17.0. P<0.05 was considered to be statistically significant, *P<0.05, **P<0.01, ***P<0.001.

Taking breast cancer as cell model, we screened the proliferation inhibitory effect of SET in 4T1 cells by MTT assay. The proliferation of 4T1 cells was inhibited by SE1, SE2, SE3, SET, SE4 in a time- and dose-dependent manner, respectively. Taking the 50% inhibition rate as the cytotoxic criteria, to avoid the non-selective cytotoxicity, various doses of tested drugs and prolonged treatment times were designed and the cell viability was evaluated (Fig. 1A-E). The result showed little cytotoxicity for the tested extracts with low doses when they are treated for <48 h. In contrast, in long-term treatment (>48 h), the non-selective cytotoxicity can be clearly detected in all of the high dose extracts (>400 µg/ml). Based on preliminary analysis, in order to determine a drug that interacts with 4T1 cells with low non-selective cytotoxicity, SET was selected for more detailed testing. The morphological analysis revealed that the SET treatment was time- and dose-dependent (Fig. 1F). Such influence of SET was further quantified by MTT assay. As shown in Fig. 1G after 6-h treatment, cell inhibition rate approached or exceeded 50%, which implied that <6 h of treatment is optimal for efficacy study. Moreover, IC₅₀ values calculated at 3, 6, 12 and 24 h were 2929.153, 1309.211, 132.26 and 26.458 µg/ml (Fig. 1H), respectively. Thus, the safe and effective concentrate interval (<400 µg/ml) and treatment time (6 h) of SET were determined for further research.

Inspired by the traditional anticoagulation efficacy of Caulis Spatholobi, by using the active extract (Fig. 1), we further questioned whether our newly prepared extract (SET) affects the inhibition of platelet aggregation induced by ADP. The platelet aggregation assay results clearly showed that, in the model group, the platelet aggregation could be obviously induced in response to ADP stimulation. In contrast, the SET treatment, with 4, 40 and 400 µg/ml doses, could largely reverse such changes, as represented by the decrease of resistance values (Ohm). This result suggested that the coagulation cascade was significantly blocked by SET. Notably, the anticoagulation activity of SET exhibits in a typical dose-dependent manner. SET at 40 µg/ml exerted the strongest efficacy (Fig. 2A and B). Taking these data into account, the 40 µg/ml dose interval was chosen for further efficacy study. A more detailed dose-dependent efficacy study was also performed. The treatment of SET, with 20, 40 and 80 µg/ml doses, significantly inhibited platelet aggregation dose-dependently. Compared with the model group, the SET treated groups showed an obviously decrease of platelets aggregation (Fig. 2C and D). Consequently, SET was able to inhibit the activation induced by ADP. However, the role of SET in the interaction between tumor and platelet and the inhibitory effects on the TCIPA are still unknown.

To further reveal the efficacy of SET in vitro, we focused on TCIPA, during which SET exerted the effectiveness on the adhesion of platelets to 4T1 cells. In this study, 4T1 cells were incubated with platelets stained with fluorescent probe CFDA-SE. In the presence or absences of SET, non-aggregated platelets were totally washed and removed. Then, the stained platelets were observed in the vicinity of tumor cells by using a confocal microscope (Fig. 3A). The result showed that the quantity and intensity of green fluorescence in model group was much clearer. In drug-treated groups, fluorescence intensity was markedly decreased, which represents the reduced level of TCIPA. Additionally, such effect was further quantified via flow cytometry analysis. In the absence of SET, TCIPA could be clearly detected as measured by the elevated positive fluorescent ratio. On the contrary, in the presence of SET, the fluorescent positive rate was decreased obviously (Fig. 3B). Therefore, SET was able to efficiently inhibit the platelet aggregation induced by tumor cells. Anti-TCIPA therapy may be a potential drug target for SET that regulates the process of tumor metastasis in the blood circulation.

To confirm the efficacy of SET in vivo, excluding the effect of SET on the tumor in situ, we selected the tail vein injection model for detecting and analyzing the intensity of lung metastasis of breast cancer. Briefly, for experiments, drug administration was initiated 2 days before modeling with low-, medium- and high-dose of SET, once daily. The ASA was also used for positive control. Metastatic process was measured by small animal in vivo imaging system. The bioluminescence imaging on the lung was first observed on the 21st day after tumor cell injection in the modeling group. As shown in Fig. 4A, mice treated with SET were visualized and showed less metastases compared to modeling mice. For quantitative analysis, the photon statistics measurements of coherent fields are shown in Fig. 4B. They are negatively influenced in a significant dose-dependent manner. To further analyze the intensity of metastasis in the lung, we performed general observation and H&E staining assay (Fig. 5A and B). Consistently, both of the results confirmed the obvious inhibition of lung metastasis in SET-treated mice. Moreover, the mice in the modeling group showned a poor survival rate compared with SET-treated mice (Fig. 4C). Thus, an obvious anti-metastasis efficacy of SET was evaluated. Consequently, SET has the possibility to be an anti-metastatic drug by targeting TCIPA in breast cancer.

In recent studies, MMP-2 and −9 were proven to play an important role in the association with platelet and tumor cell (21). Leading by this, zymography assay was measured to detect whether MMP-2 and −9 were released or not during TCIPA. As shown in Fig. 5C, we found that MMP-2 and −9 were released during TCIPA. However, the gray scale value of zymographic bands in each group had no statistical difference which indicated that SET failed to regulate the secretion of MMP-2 or −9 during T-P interaction. Consequently, the efficacy of SET on the inhibition of TCIPA was MMP-independent.

To investigate the exact mechanism of SET for blocking the association between platelets and tumor cells, we focused the efficacy of SET in the regulation of adhesive interaction between platelets and tumor cells. As commonly recognized, the key adhesive molecules such as SELP, SELPLG, GPVI, ITGA2B involved in tumor metastasis played a crucial role in TCIPA (22). Therefore, we detected the expression of above molecules as measured by RT-PCR assay. As expected, SET significantly inhibited SELP, GPVI, and ITGA2B expression compared with model group (Fig. 5D). While through analysis of RNA bands and measurement of the intensity of gray scale value, the expression of SELPLG could not be regulated by SET efficiently. We then identified that the expression of SELP, GPVI, and ITGA2B were suppressed by SET during TCIPA. Consequently, the mechanism of SET to inhibit TCIPA and reduce metastasis may relate with regulating the cell adhesion molecules and then suppressing adhesion of tumor cells and platelets.