PS VII, with a purity of >99%, was isolated from the root and rhizome of T. tschonoskii Maxim. which was obtained from the Qinba Mountains (Ankang, China) (13). The chemical structure of PS VII is shown in Fig. 1. PS VII was dissolved in dimethylsulfoxide (DMSO) at 1 M and stocked at −20°C in aliquots. Trypan blue, Triton X-100, pyruvate, penicillin G and streptomycin were obtained from Sigma (St. Louis, MO, USA). RPMI-1640 and fetal bovine serum (FBS) were purchased from Corning Inc. (Corning, NY, USA). Rabbit polyclonal anti-MMP-2 and MMP-9 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Materials and chemicals for electrophoresis were obtained from Bio-Rad Laboratories (Hercules, CA, USA). All other chemicals were of analytical reagent grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

SW620 and LoVo human colon carcinoma cell lines [American Type Culture Collection (ATCC), Rockville, MD, USA] were cultured in RPMI-1640 medium, and human umbilical vein endothelial cells (HUVECs; which were supplied by Prof. Zhou Siyuan, Department of Pharmacology) were cultured in Dulbecco’s modified Eagle’s medium. For cell maintenance, the basal medium was supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. The solvent control contained an equivalent amount of DMSO corresponding to the highest concentration of PS VII that was used.

The effects of PS VII on cell viability were determined using an MTT assay. Cells were seeded in a 96-well plate at a density 5,000 cells per well and incubated with or without PS VII for 24 h at 37°C. After incubation, 20 μl MTT solution (5 mg/ml) was added to each well and the plate was incubated for a further 4 h at 37°C. The supernatants were aspirated carefully and 200 μl DMSO was added, and then the plate was subjected to vibration for 20 sec. The optical density (OD) of the cell suspension was measured at a wavelength of 490 nm using a microplate reader (iMark model 680; Bio-Rad Laboratories). The inhibition rate was calculated using the following formula: Inhibition = (1-OD_{treatment group}/OD_{control group}) × 100. All experiments were performed in triplicate.

Cells contained in dishes were pretreated with or without PS VII (0.3 and 1 μM) for 24 h. Following pretreatment, the cells were detached from the dishes via incubation with 15 mM EDTA, washed twice with phosphate-buffered saline (PBS) and once with serum-free medium (SFM), counted and seeded into 96-well plates at a density of 5,000 cells per well. For cell-matrix attachment assays (13), the 96-well plates (Costar, Corning Inc., Corning, NY, USA) were precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) or bovine serum albumin as a background control, incubated for 1 h at room temperature (RT) and 1 h at 37°C, and washed with PBS, prior to the assay. Plates with CRC cells were incubated for 1 h at 37°C, cells were washed twice with PBS and attached cells were assessed using the MTT method. For cell-cell attachment assays (14) HUVECs were plated in growth medium at a density of 30,000 cells per well and incubated at 37°C in 5% CO₂ for 24 h prior to the assay. The plates containing the CRC cells were incubated in a humidified atmosphere for 1 h, washed four times with warm SFM, stained with a 0.25% rose bengal-PBS solution and incubated for 5 min at RT. Subsequently, cells were washed twice with SFM. Rose bengal was dissolved in a 95% ethanol-PBS solution (1:1) and detected using a microplate reader. Duplicate wells containing only HUVECs were included in each experiment as background controls. After subtracting the OD value of background wells, the rate of attachment was determined as the ratio of attaching cells to that of the non-washed group.

Cells were seeded in 6-well plates at a density of 5×10⁵ cells per well. Once the cells reached 90% confluence, a wound area was carefully created by scraping the cell monolayer with a sterile 200-μl pipette tip, from one end of the well to the other. The detached cells were removed by washing with PBS. Subsequently, the cells were incubated at different concentrations of PS VII (0.3 and 1 μM). Migration of cells into the wounded region was observed using an Olympus CK-2 inverted microscope (Olympus, Tokyo, Japan) and images were captured at 0 and 24 h at ×100 magnification. The wound area was measured using the image processing program ImageJ (NIH, Bethesda, Maryland, USA). The cell wound closure rate was calculated using the following equation: Wound closure = [1-(wound area at T_t/wound area at T₀) × 100, where T_t is the time passed since wounding and T₀ is the time the wound was created. The experiments were performed in triplicate.

Cell invasion was determined using Matrigel-coated Transwell cell culture chambers (8 μm pore size) (Millipore, Billerica, MA, USA) as described previously (15,16). Cells were maintained for 24 h in SFM, trypsinized and resuspended in serum-free RPMI-1640 medium. Following resuspension, cells were placed in the upper chamber of the Transwell insert (5×10⁴ cells per well) and incubated with different concentrations of PS VII (0.3 or 1 μM). RPMI-1640 medium containing 10% FBS was added to the lower chamber. The cells were incubated at 37°C in an incubator supplemented with 5% CO₂ for 24 h; non-invasive cells in the upper chamber were removed by wiping with a cotton swab and invasive cells were fixed with 4% formaldehyde in PBS and stained with 1% crystal violet in 2% ethanol. Images of cells on the lower surface of the filter were captured under a light microscope (TS100; Nikon Corporation, Tokyo, Japan; ×100 magnification). The inserts were washed with 33% acetic acid. The absorbance of the washing buffer at 570 nm was determined for each well using a microplate reader. Cell-free inserts which only contained medium were included in duplicate throughout each experiment as OD background controls. The reported OD data represent the mean background-corrected values ± standard deviation (SD) obtained from three independent experiments in duplicate.

The zymographic analysis was adapted from Surgucheva et al (17). Cells were seeded in a 24-well plate at a density of 5×10⁵ cells per well and incubated with PS VII (0.3 or 1 μM) for 24 h at 37°C. Following incubation, conditional medium was harvested and then electrophoresed on 15% denaturing sodium dodecyl sulphate (SDS) polyacrylamide gels containing 1 mg/ml gelatin (Sangon, Shanghai, China). Gels were washed twice in rinsing buffer (50 mM Tris-HCl, 5 mM CaCl₂, 2.5% Triton-X 100, 1 μM ZnCl₂ and 0.05% NaN₃) for 1 h and then incubated for 24 h at 37°C in the rinsing buffer without Triton-X 100, so that renaturation of the enzyme could occur. Gels were stained with Coomassie blue R-250 (Bio-Rad Laboratories) and destained with 5% acetic acid containing 10% methanol. Gelatinolytic activities were visualized as clear bands against a blue background.

Following treatment with PS VII (0.3 and 1 μM) for 24 and 48 h, cells were washed twice with PBS and treated with extraction buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% deoxycholic acid). The cell extractions were collected, centrifuged at 10,000 × g for 15 min at 4°C and the cell lysates collected as supernatants. The cell lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% (w/v) non-fat milk in PBS containing 0.1% Tween-20, and then blotted with primary antibody. Subsequently, the membranes were incubated with an appropriate secondary antibody (horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG). The immuno-detected proteins were then revealed using enhanced chemiluminescence (ChemiScope5000; Clinx Science Instruments, CO., Ltd., Shanghai, China).

Results are expressed as the mean ± SD. Two group comparisons were evaluated using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

The viability of SW620 and LoVo cells treated with PS VII at different concentrations (0.1, 0.3, 1, 3, 10 or 30 μM) for 24 h was determined using an MTT reduction assay. As shown in Fig. 2, PS VII reduced the cell viability rate in a concentration-dependent manner. Higher concentrations of PS VII inhibited the growth of CRC cells directly. Hence, in order to observe the effect of PS VII on the attachment, migration and invasion of CRC cells more accurately and clearly, concentrations for the following experiments were selected which demonstrated no obvious cytotoxicity to the proliferation of the cells (0.3 and 1 μM).

To determine whether PS VII affects the attachment ability of CRC cells, the attachment of the cells to Matrigel and vascular endothelial cells was assessed. Matrigel is an extracellular matrix (ECM) which is composed of laminin, collagen type IV, nidogen and heparan sulfate glycoprotein. As shown in Fig. 3A, pretreatment with 0.3 and 1 μM PS VII for 24 h reduced the attachment of SW620 and LoVo cells to Matrigel following incubation for 1 h, compared with that of the control cells. The potential effect of PS VII on the attachment of CRC cells to vascular endothelial cells was investigated via incubation with HUVECs. As shown in Fig. 3B, SW620 and LoVo cells that were pretreated with 0.3 and 1 μM PS VII for 24 h demonstrated an impaired ability to attach to HUVECs after incubation for 1 h, compared with that of the control cells.

One characteristic of tumor metastasis is the increased migratory ability of tumor cells. The inhibition of migration of SW620 and LoVo cells by PS VII was investigated using wound-healing assays. Cells were incubated with different concentrations of PS VII for 24 h. Higher concentrations of PS VII (1 μM) were observed to significantly increase the inhibition of cell migration in the two cell lines compared with that of the control cells (Fig. 4).

In order to determine the inhibitory effect of PS VII on the invasion of SW620 and LoVo cells across the ECM, the cells that invaded through the Matrigel-coated polycarbonate filter in the Transwell chamber were analyzed. The results are presented in Fig. 5. The majority of SW620 and LoVo cells invaded from the upper to the lower chamber in the control group; however, the presence of PS VII inhibited the penetration of the Matrigel-coated filter by SW620 and LoVo cells (Fig. 5A). This inhibitory effect was greatest at a concentration of 1 μM. The quantification of cells in the lower chamber from Fig. 5B indicated that PS VII significantly inhibited SW620 and LoVo cell invasion and that this inhibitory effect was concentration-dependent.

The expression of MMPs is crucial to ECM degradation, which is required for cell invasion. Hence, the effect of PS VII on the expression of MMPs was investigated by western blot analysis. The results demonstrated that PS VII suppresses the expression levels of MMP-2 and MMP-9 proteins in a concentration-dependent manner (Fig. 6). These effects may lead to the inhibition of the invasive ability of SW620 and LoVo cells following exposure to PS VII.

Although the protein expression of MMP-2 and MMP-9 was downregulated by PS VII, the activity of these two enzymes during treatment with PS VII required further investigation. Gelatinolytic activity at molecular masses of 72 and 92 kDa (which correspond to the molecular mass of MMP-2 and MMP-9, respectively) was detected in the conditioned medium of cells treated with PS VII. The activity of MMP-2 and MMP-9 in the two cell lines was markedly repressed by PS VII in a concentration-dependent manner (Fig. 7).