Roswell Park Memorial Institute (RPMI)-1640 medium (C11875500BT), Dulbecco's modified Eagle's medium (DMEM) with high glucose (C11995500BT), fetal bovine serum (FBS; #10099-141), penicillin-streptomycin (SV30010), 0.25% trypsin-EDTA (#25200-072), Pierce RIPA buffer (#89901), Pierce BCA Protein Assay kit (#23227) and SuperSignal™ West Pico Chemiluminescent Substrate (#34080) were all purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay (MTS; G5430) was provided by Promega Corporation (Madison, WI, USA). The exogenous VEGF-C (CYT-527) was purchased from ProSpec-Tany TechnoGene Ltd. (East Brunswick, NJ, USA). Annexin V-FITC apoptosis detection kit (KGA108) was obtained from KeyGen Biotech Co., Ltd. (Jiangsu, China). Transwell chambers (8.0 µm; #3422) were obtained from Corning Life Sciences (Corning, NY, USA). The In Vitro Angiogenesis assay kit (ECM625) and nitrocellulose (NC) membrane (0.45 µm; HATF00010) were purchased from Millipore (Billerica, MA, USA). Rabbit polyclonal antibody against VEGF-C (#2445), VEGFR-3 (#2485) and β-actin (#4967) were obtained from Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal antibody against MMP-2 (ab37150) and MMP-9 (ab38898) were purchased from Abcam (Cambridge, MA, USA). HRP-conjugated goat anti-rabbit secondary antibody (E030120) was purchased from EarthOx Life Science (Millbrae, CA, USA). Culture flask and plates were purchase from NEST Biotechnology Co., Ltd. (Wuxi, Jiangsu, China). All the other chemicals used, unless otherwise stated, were obtained from Sigma Chemicals (St. Louis, MO, USA).

PZH was obtained from and authenticated by Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd. (Zhangzhou, China) (Chinese FDA approval no. Z35020242, lot no. 1009039). Stock solutions of PZH were prepared just before use by dissolving the PZH powder in phosphate-buffered saline (PBS) to a concentration of 20 mg/ml. The working concentrations of PZH were made by diluting the stock solution in the culture medium.

Human metastatic CRC cell lines HCT-8, HCT-116 and SW620 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The HLEC was purchased from the JENNIO Biological Technology Co., Ltd. (Guangzhou, China). HCT-8, HCT-116 cells and HLECs were grown in RPMI-1640; SW620 cells were grown in DMEM. All cell media were supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, and cultured at 37°C with 5% CO₂ in a humidified incubator (Forma 3110; Thermo Fisher Scientific, Inc.).

CRC cells were seeded into 6-well plates for 12 h, which was followed by PZH treatment for the indicated period of time. HLECs were first grown in complete RPMI-1640 (10% FBS) until ~60% confluency, and then continuously cultured in FBS-free medium overnight. The medium was replaced with RPMI-1640 with 2% FBS and cells were treated with 5 ng/ml VEGF-C and/or various concentrations of PZH for the indicated period of time.

Morphology of HLECs was observed using a phase-contrast microscope (FMIL/DFC295; Leica, Wetzlar, German) after PZH and/or exogenous VEGF-C treatment for 24 h. The images of five randomly selected views were captured at a magnification of ×200.

Cell viability was assessed using MTS assay. CRC cells and HLECs were seeded in 96-well plates. After PZH and/or exogenous VEGF-C treatment for 24 or 48 h, 10 µl MTS was added to each well after which the samples were incubated for 1 h at 37°C. The resulting absorbance was measured at 490 nm using an ELISA reader (Model ELx800; BioTek, Winooski, VT, USA).

After incubation with various concentrations of PZH and/or exogenous VEGF-C for 24 h, apoptosis of HLECs was determined by FACSCalibur and Annexin V-FITC apoptosis detection kit, as previously described (14). Staining was performed according to the manufacturer's instructions. In the present study, Annexin V/PI double-negative population (labeled as LL in the FACS diagram) indicates viable cells; Annexin V-positive/PI-negative or Annexin V/PI double-positive population (labeled as LR or UR in the FACS diagram) represents cells undergoing early or late apoptosis, respectively.

Migration ability of the CRC cell lines was evaluated by wound-healing, as previously described (15). Briefly, CRC cells were seeded into 6-well plates (10⁶ cells/well in 2 ml culture medium). After 12 h of incubation, cells were scraped away vertically in each well using a P200 pipette tip. Five randomly selected views along the scraped line were photographed from each well using a phase-contrast inverted microscope at a magnification of ×100. Cells were then treated with the indicated concentrations of PZH for 24 h and another set of images were captured in the same way. A reduction in the scraped area indicates migration of cells. For HLECs, the migration assay was performed using Transwell cell culture chambers as previously described (18). The inserts were placed within a 24-well chamber containing 0.7 ml RPMI-1640 with 10% FBS to serve as a chemoattractant. As before, HLECs were seeded into 6-well plates and treated with different concentrations of PZH and/or exogenous VEGF-C for 24 h. Cells (5×10⁴ cells) were seeded into the inserts suspended in 0.2 ml of serum-free RPMI-1640. The cells were incubated at 37°C with 5% CO₂ for 12 h before the upper surface of the filter was scraped to remove nonmigratory cells. Migrated cells were then fixed and stained with crystal violet. For quantification, the average number of migrating cells/field was assessed by counting five random fields under a phase-contrast microscope at a magnification of ×200.

The HLEC tube formation was assessed using the In Vitro Angiogenesis assay kit as previously described (15). Briefly, HLECs were treated with PZH and/or exogenous VEGF-C for 24 h, harvested and diluted 2×10⁵ cells/well in 200 µl medium individually, were seeded into 1:1 ECMatix gel (v/v) coated 48-well plate, and incubated for 8 h. The series of tube-like structures were photographed using a phase-contrast microscope at a magnification of ×40.

Cells were treated with PZH and/or exogenous VEGF-C for 24 h after which they were lysed using Pierce RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktails. The lysates were then centrifuged at 14,000 rpm for 20 min, and the resulting protein concentrations were determined using BCA protein assay reagent kit. A total of 50 µg of protein for each sample was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and resolved using 20 V for 10 min→ 80 V for 30 min→ 120 V for 1 h, then transferred onto nitrocellulose membranes. Following blocking with 5% non-fat dry milk, the membranes were incubated with antibodies against VEGF-C, VEGFR-3, MMP-2, MMP-9 and/or β-actin (1:1,000 dilution) overnight at 4°C and subsequently incubated with HRP-conjugated anti-rabbit secondary antibodies (1:5,000 dilution) for 1 h at room temperature. The membranes were then exposed with enhanced chemiluminescence (ECL) detection using SuperSignal™ West Pico Chemiluminescent Substrate. Image Lab™ Software (version 3.0) was used for densitometric analysis and quantification of western blot analyses.

All data were collected based on the mean of three experiments. Statistical analysis was performed using the SPSS software (version 17.0) for Windows (SPSS, Inc., Chicago, IL, USA) using one-way ANOVA. P<0.05 was considered to indicate a statistically significant result.

We first determined the effect of PZH on the viability of various CRC cell lines using MTS assay. PZH treatment at 0.25, 0.5 and 0.75 mg/ml for 24 or 48 h significantly reduced cell viability in the HCT-8, HCT-116 and SW620 cells (P<0.05) in a dose- and time-dependent manner (Fig. 1). We next evaluated the effect of PZH on migration of various CRC cell lines using a wound-healing assay. Twenty-four hours post-wounding, untreated HCT-8, HCT-116 and SW620 cells migrated into the wounded (clear) area of the cell monolayer, whereas PZH treatment dose-dependently inhibited migration of all these three cell lines (Fig. 2).

To explore a potential mechanism underlying the antimetastasis activity of PZH, we performed western blotting to examine the protein expression of VEGF-C in HCT-8, HCT-116 and SW620 cells. We found that PZH treatment profoundly and dose-dependently reduced the expression of VEGF-C (Fig. 3).

As seen from the cell morphology, VEGF-C stimulation increased the confluency of HLECs, while PZH treatment decreased cell confluency in a dose-dependent manner (Fig. 4A). Moreover, we found that cell viability of HLECs increased to 105.56% at 24 h and 113.56% by 48 h (P<0.05) after VEGF-C stimulation compared to control cells that did not receive VEGF-C, using an MTS assay. Treatment with 0.0625–0.5 mg/ml of PZH for 24 h decreased the viability of VEGF-C-stimulated cells from 97.94 to 35.07% and from 81.01 to 24.10% after 48 h (P<0.05 vs. PZH-untreated cells) (Fig. 4B).

The effect of PZH on apoptosis in the VEGF-C-stimulated HLECs was determined by Annexin V/PI staining followed by FACS analysis. The results indicated that the percentage of cells undergoing apoptosis after treatment with 0.0625, 0.125 or 0.25 mg/ml of PZH (including the early and late apoptotic cells) was not significantly different when compared with the control cells that did not receive PZH treatment (Fig. 5).

Transwell assays were performed to determine the effects of PZH on migration of HLECs. VEGF-C stimulated cells had enhanced migratory capacity by ~35.74% (P<0.0327). Treatment with 0.0625–0.25 mg/ml of PZH dose-dependently reduced the cell migratory ability by 104.81–20.49% (P<0.05) (Fig. 6). Similarly, exogenous VEGF-C stimulation led to a 33.69% (P<0.0218) increase in tube formation of HLECs, compared to control cells that did not receive VEGF-C, which in turn was decreased by PZH treatment by 114.81–10.49% (P<0.05) (Fig. 7).

VEGFR-3 is the cognate receptor of VEGF-C that is expressed in HLECs. Binding of VEGF-C to VEGFR-3 results in proliferation of HLECs as well as the formation of LVs. Moreover, activation of the VEGF-C/VEGFR-3 signaling pathway can lead to the upregulation of matrix metalloproteinases (MMPs). Expression of MMP-2 and MMP-9 has been associated with the migration ability of VEGF-C-stimulated cells and lymphangiogenesis (20). To further explore the anti-lymphangiogenesis properties of PZH, we used western blot analysis to determine the relative expression of these proteins after PZH treatment. We found that VEGFR-3, MMP-2 and MMP-9 were upregulated after VEGF-C stimulation and downregulated with PZH treatment (Fig. 8).