HLECs used in this study were human dermal lymphatic endothelial cells (HDLECs). Primary HDLECs were purchased from ScienCell Research Laboratories, USA, and were identified by using immunofluorescent cyto-chemical technique via CD₃₁, podoplanin and Lyve1. Tissue culture flasks were peridiumed with fibronectin (1 mg/ml; Chemicon, USA). Cells were cultured in endothelial cell growth medium (ECGM) with endothelial cell growth factor (ScienCell Research Laboratories) in an incubator (Forma Series II HEPA Class 100, Thermo Co., USA) with 5% CO₂ at 37°C. The medium was changed every 2 days. When the cells became confluent, at 80% plating efficiency, they were treated with 5 ml phosphate-buffered saline (PBS) solution, digested with 1 ml 0.025% trypsin and 1 ml 0.02% ethylenediamine tetraacetic acid (EDTA) solution (ScienCell Research Laboratories), and observed by inversion microscope (Nikon TS100, Japan). Then the cells were retuned to culture at 37°C in 5% CO₂, and continued transfer of culture. The cells, which were at fifth generation, were used in the experiment.

The cultured HDLEC suspensions (5×10⁵ cells/ml) were used in this assay. The cultures were divided into NCTD groups and control groups. The tetrazolium-based colorimetric assay (MTT) was used to evaluate the inhibitory effect of NCTD on proliferation of HDLECs in vitro (tumor cytotoxicity test). After HDLECs were cultured in a 96-well plate peridiumed with fibronectin (5×10⁴ cells/ml × 100 μl/well) in culture medium (ECGM, 100 μl/well) overnight, they were without (negative control, equal ECGM solution) or treated with various concentrations (1.25–80 μg/ml, experimental groups; 6 wells per concentration) of NCTD (10 mg/2 ml, Jiangsu Kangxi Pharmaceutical Works, China) in fresh culture medium at 37°C in 5% CO₂ for 24 h. The tumor cell cytotoxicity was determined by MTT (methyltiazolyl tetrazolium, Sigma, MO, USA). The optical densities (A value) at 490 nm were measured with an ELISA reader (Elx800UV, Bio-Tek Co., USA). The A490 value of the experimental groups was divided by the A490 value of untreated controls and presented as a percentage of the cells. The inhibitory percent of NCTD on HDLECs (%) = (1-A490 value in the experimental group / A490 value of control group) × 100%. Three separate experiments were performed. The concentration of drug giving 50% growth inhibition (IC₅₀) was calculated from the formula IC₅₀= lg⁻¹{Xm-I [P-(3-Pm-Pn)/4]}. In equal experimental conditions, HDLECs were treated with 5 μg/ml concentration of NCTD at 6-h interval for 48 h, the optical densities (A value) and the inhibitory percent of NCTD on HDLECs were measured and calculated.

HDLECs (1×10⁵ cells/ml) were cultured on the fibronectin-peridium slides in a 96-well plate overnight. When slides were filled with 50–80% cells, they were treated without (negative control, equal ECGM solution) or treated with various concentrations (1.25–10 μg/ml, experimental groups) of NCTD, respectively. Then, the slides were fixed with 0.5 ml 4% formalin, washed with PBS solution, and stained with 0.5 ml fluorescence agent Hoechst 33258 (Sigma) and CY3 NHS ester (Lumiprobe, USA). Apoptosis of HDLECs was observed under a fluorescence microscope (Nikon Eclipse TE2000-U, Japan). Ten sample slides in each group were chosen by analysis. Visual fields (>10) were observed or >500 cells were counted per slide. The apoptotic percent of each group = apoptotic cells/cells in all × 100%.

HDLECs (1×10⁵ cells/ml) cultured in a 96-well plate peridiumed with fibronectin were without (negative control, equal ECGM solution) or treated with various concentrations (1.25–15.00 μg/ml, experimental groups; 6 wells per concentration) of NCTD at 37°C in 5% CO₂ for 24 h, then were made up into the cell suspension (5×10⁵ cells/ml), and suspended in 500 μl binding buffer. Tumor DNA was then stained for 15 min with 5 μl Annexin V-FITL and propidium iodine (PI, Sigma). DNA value and apoptotic rate of HDLECs in each group were determined by Cell Apoptotic Detection Kit (BioDev Co., China) and Fluorescent Activated Cell Sorter (420 type FACS flow cytometry, Becton-Dickinson, CA).

HDLECs (1×10⁵ cells/ml) cultured in a 96-well plate peridiumed with fibronectin overnight were without (negative control, equal ECGM solution) or treated with various concentrations (1.25–5.00 μg/ml, experimental groups; 6 wells per concentration) of NCTD for 24 h, and then observed under an inverted microscope (I×50/I×70, Olympus, Japan). Simultaneously in the control group and 2.50 μg/ml NCTD group, they were digested with 0.025% trypsin and 0.002% EDTA solution, centrifuged (200 g for 10 min), washed with PBS solution, and fixed in 2.5% glutaraldehyde-1% osmium tetroxide buffered with PBS (pH 7.2). The specimens were dehydrated in a graded ethanol series, embedded in Spury Resin, cut into 50–70 nm sections, double-stained with uranyl acetate-lead citrate, and analyzed by standard procedures under a transmission electron microscope (JEM-1230, Jeol Co., Japan).

The cultured HDLECs were inoculated in 4 culture dishes peridiumed with fibronectin (1×10⁵ cells/ml × 100 μl/dish) in fresh culture medium (ECGM, 100 μl/dish). When HDLECs were fused into a single layer in dishes, a straight line on the surface of the growthing cells was scraped with sterile blades by scraping line method. Then, the cells at side of the line were scraped off completely using a plastic scraper, washed with D-Hanks. The cultures were treated without (negative control, equal ECGM solution) or treated with various concentrations (1.25–5 μg/ml, experimental groups, 4 dishes per concentration) of NCTD in fresh culture medium at 37°C in 5% CO₂ for 24 h. Lastly, cell migration distance onto the scraped areas and migrating cells were measured and counted in 4 independent microscopic visual fields (×100) under inversion microscope (Nikon TS100, Japan), and expressed as mean number per 1 field, so as to determine the inhibitory effect of NCTD on proliferation and migration of HDLECs.

Living HDLECs were trypsinized with 0.25% trypsin and washed with fresh culture medium, suspended in the culture medium with 10% bovine calf serum (1×10⁵ cells/ml). The cell suspension was transferred to the above layer of the Matrigel invasion chamber (0.3 ml/every chamber), while 0.8 ml of RPMI-1640 medium with 10% bovine calf serum was only added to the bottom layer of the Matrigel invasion chamber. Then the cells were cultured in 50 ml/l CO₂ at 37°C for 24 h. The cells were treated without (untreated control group) or with various concentrations of NCTD (the six-concentration groups, every concentration ×6) in fresh culture medium (0.3 ml/every chamber), were cultured in an incubator with 50 ml/l at 37°C for 72 h. The passing-membrane cells were collected from the above layer of the Matrigel invasion chamber, centrifuged (200 r/min, 10 min), dyed by trypan blue dye, and counted in a hemocytometer. Each experiment was performed thrice.

First, the mixed fibrinogen gel was prepared with fibrin gel [which was from human fibrinogen (3 mg/ml; Bite Bio Co., Beijing, China) and human thrombin (50 U/ml; Sigma) dissolved with PBS], solidified by adding in order 500 μl fibrinogen, 100 μl ECGM and 10 μl thrombin solution, and incubated at 37°C in 5% CO₂ for 30 min to promote gelling, according to the literature. Then, 6-well plates were coated with the fibrinogen gel (0.05 ml/well) and incubated at 37°C for 1 h to promote gelling. HDLECs (5th-generation cells) suspension (1×10⁵/ml, 300 μl/well) were seeded on the gel surface, cultured in ECGM (2 ml) in an incubator with 5% CO₂ at 37°C. The medium was changed every 2 days. After 1 week, the cells were treated without (equal ECGM solution; the control group) or with 2.5 μg/ml NCTD, 100 μl mF4-31C1 (a soluble VEGFR-3 antibody) and NCTD+mF4-31C1 (experimental groups, 6/each group), respectively, for 2–4 days. Inverted phase-contrast light microscope (Olympus) was used to observe and record the morphological changes between the groups and the tube formation by HUVECs, i.e., lymphangiogenesis in vitro. Tube formation ability was quantified by counting the total number of cell clusters and branching in 5 randomly chosen microscopic fields per well under magnification ×100. Results were expressed as the mean percentage of branching over total cell clusters and expressed as a ratio to the control.

HDLEC suspension (3×10⁵ cells/ml, 400 μl/well) was seeded on the fibronectin-peridium slides in a 96-well plate overnight, cultured continually for 48 h. When cells were fused into a single layer, they were treated without (equal ECGM solution; the control group) or treated for 24 h with 2.5 μg/ml NCTD, 100 μl mF4-31C1 (a soluble VEGFR-3 antibody), and NCTD+mF4-31C1, respectively. Then, the slides were fixed with the solution of methanol and acetone (v/v=1: 1), were used in the experiment.

VEGF-C, VEGF-D, VEGFR-3 protein products from HDLECs of each group were determined by streparidinperoxidase (S-P) staining method, according to the kit brochure (Jinmei Biotechnology Co., Ltd., Shanghai). The slides from each group were washed with PBS solution, treated with 3% H₂O₂ and 1% TritonX-100 solution, were added in order with A reagents, i.e., primary antibody [rabbit anti-human monoclonal antibody VEGF-C (invitrogen, USA), VEGF-D (Abcam, USA), VEGFR-3 (Cell Signaling, USA)], B reagent (biotinylated anti-rabbit secondary), C reagent (HRP logo Streptavidin) and DAB solution, respectively. Then, slides were rinsed in distilled water, dehydrated through alcohol and xylene and mounted coverslip using a permanent mount medium and observed by optic microscope (Olympus). For negative control, the slides were treated with PBS in place of primary antibody. Six sample slides in each group were chosen by analysis. Visual fields (>10) were observed or >500 cells were counted per slide.

The drug experiment for HDLECs in vitro was the same as for immunocytochemistry. Expression of VEGF-C, VEGF-D, VEGFR3 proteins from HDLECs of each group were determined by western blot analysis (Lowry method; Lowry method protein kit, Puli Lai Co., Shanghai, China). Cells were lysed with 200 ml of cell lysis buffer (Promega) containing a cocktail of protease inhibitors (Nacalai Tesque) and the supernatant of the lysed cells was recovered. An aliquot of 20 mg of proteins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition, and were then transferred to a PVDF membrane. One hour after being blocked with PBS containing 5% non-fat milk, the membrane was incubated overnight, was then added in order with each primary antibody [(rabbit anti-human VEGF-C, VEGF-D, VEGFR-3 antibodys (1:500; Abcam), and rabbit anti-human β-actin antibody (1:5000; Cell Signaling, USA)] diluted with PBST containing 5% non-fat milk at 4°C, an appropriate anti-rabbit HRP-labeled secondary antibody (1:5000; Abcam), HistoFine (Dako, Glostrup, Denmark) at room temperature for 2 h. The target proteins were visualized by an enhanced chemiluminescent (ECL) reagent (GE Healthcare, USA), imaged on the Bio-Rad chemiluminescence imager. The gray value and gray coefficient ratio of each protein was analyzed and calculated.

The drug experiment for HDLECs in vitro was the same as immunocytochemistry. Expression of VEGF-C, VEGF-D, VEGFR3 mRNAs from HDLECs of each group were determined by quantitative real-time polymerase chain reaction (QRT-PCR) assay. QRT-PCR was performed as described by the manufacturer. Total RNA was extracted from HDLECs using the TRIzol reagent. Concentration of RNA was determined by the absorption at 260. The primers for amplification were designed and synthesized by Shanghai Sangon Co. The primers for VEGF-C, VEGF-D, VEGFR3 and GAPDH were as follows: VEGF-C (106 bp) 5′AGT CGC GAC AAA CAC CTT CT3′ (sense), 5′GTA GCT CGT GCT GGT GTT CA3′ (anti-sense); VEGF-D (194 bp) 5′CAG TGG GTT GAT CTG GGA CT3′ (sense), 5′CAT GCA GGG TAG GAT GGT CT3′ (anti-sense); VEGFR-3 (117 bp) 5′CGG CTT CAG CTG TAA AGG AC3′ (sense), 5′ TTG TAA AAC ACC TGG CCT CC3′ (anti-sense); GAPDH (128 bp) 5′GCC TCC AAG GAG TAA GAC CC3′ (sense), 5′TGT GAG GAG GGG AGA TTC AG3′ (anti-sense). Polymerse chain reactions were performed in a 20-μl reaction volume. RT-PCR reaction was run in the following conditions: at 94°C for 5 min; at 94°C for 30 sec, at 55°C for 30 sec, at 72°C for 30 sec, 35 circles; at 72°C for 10 min. The amplifying conditions were control group: at 50°C for 2 min, NCTD group: at 95°C for 5 min, mF4-31C1 group: at 95°C for 45 sec, and NCTD+mF4-31C1 group: at 60°C, 45 sec; 40 circles. PCR products (10 μl) were placed onto 15 g/l agarose gel and observed by EB (ethidium bromide) staining using the ABI PRISM 7300 SDS software.

Statistical analyses were performed using SPSS 13.0. The data are presented as mean ± SD. Statistical differences were evaluated using Student’s t-test or the χ² test. P<0.05 was considered statistically significant.

As shown in Fig. 1, the cultured HDLECs began to grow at 6 h, maturated at 24 h, which were predominantly of shuttle-shape, or accumulation, with abundant cytoplasm, clear nuclei (control group, Fig. 1Aa1); after NCTD treatment, the morphology of HDLECs showed visible cell aggregation, floating, nuclear condensation or fragmentation, cataclysm, apoptotic bodies, or even death (Fig. 1Aa2–a4). Furthermore, NCTD inhibited markedly the HDLEC proliferation in a dose- and time-dependent manner with the IC₅₀ value 6.8 μg/ml (Fig. 1B). Thus, NCTD inhibits significantly the proliferation and growth of HLECs.

In this study, apoptosis of HLECs was evaluated by flow cytometry (FCM), Hoechst immunofluorescence dye and transmission electron microscopy. As shown in Fig. 2, after treatment with NCTD for 24 h, NCTD induced significantly HDLEC apoptosis in vitro when compared with control group (1.280±0.010 vs. 16.380±1.400%, P<0.05), with a dose- and time-dependent manner, i.e., apoptosis percent of HDLECs (total cells under right quadrant of cells) increased (FCM analysis, Fig. 2A); viable HDLECs decreased, apoptotic cells which presented bright blue or orange dyes, even with fine or fragmented nuclei increased (fluorescence microscopy observation, Fig. 2B), along with increase of NCTD concentration. In addition, Fig. 2C shows under electron microscope irregular cells with abundant microvilli, clear cell organelles, larger nucleus/cytoplast ratio, irregular nuclei and chromatin enrichment compared to the control group (Fig. 2Cc1); and decreased microvilli, golgiosome atrophy, mitochondria swelling, cytoplast vacuoles, nuclear shrinkage, chromosome condensation, chromatin aggregation and typical apoptosis bodies in NCTD (2.5 μg/ml) group (Fig. 2Cc2–c4). It is thus clear that NCTD induces significantly apoptosis of HLECs.

The effects of NCTD on migration of the cultured HDLECs, i.e., cell migration distance onto the scraped areas and migrated cells were measured by scraping line method under an inversion microscope. As shown in Fig. 3, after treatment with NCTD for 24 h and along with increase of NCTD concentration, migrated cells (11.5±3.4, 17.7±2.8 or 32.5±4.3 vs. 56.3±5.2 n, P=0.000; Fig. 3Bb1) onto the scraped areas were decreased, cell migration distance (75.6±9.7, 163.4±15.3 or 258.7±28.7 vs. 405.7±20.5 μm, P=0.000; Fig. 3Bb2) was shortened significantly, when compared with control group. It was shown that NCTD inhibited significantly migration of HLECs in vitro, in a dose-dependent manner.

The effect of NCTD on invasion of the cultured HDLECs was measured by Matrigel invasion experiment. As shown in Fig. 4, HDLECs in control group passed more of the membrane and had more invasive capability in vitro; NCTD began to inhibit the invasion of HDLECs at the concentration of 5 μg/ml and as its concentration increased, their passing membrane cells markedly decreased, the trypan blue dyed cells, namely the dead passing-membrane cells obviously increased (P<0.01). At 50 μg/ml of NCTD, the invasive action of HDLECs was inhibited almost completely. Thus, NCTD inhibited significantly invasion of HLECs in a dose-dependent manner.

The lymphatic capillary-like structures (i.e., lymphangiogenesis) formed from the three-dimensional culture of HDLECs in vitro was observed by inverted phase-contrast light microscopy. As shown in Fig. 5, when seeded on the mixed fibrinogen gel for 24 h, HDLECs started to paste the well walls, grew, spread out, formed cell groups composed of multi-angular or pseudopod cells (Fig. 5Aa1); formed tubular rudiment structure at the third day (Fig. 5Aa2); had some typical lymphatic capillary-like tubes, with obvious pipe walls, the lumen and progressive branches after 1 week (Fig. 5Aa3). At the second day in the process of lymphatic tube formation, HDLECs started to grow in the form of pasting the wall of the well, to spread out and form tubular rudimental structures in control group (Fig. 5Bb1); HDLECs lost the capacity of the above lymphatic tube formation, with visible cell aggregation, floating, nuclear fragmentation, apoptosis and necrosis, after using NCTD (Fig. 5Bb2), mF4-31C1 (Fig. 5Bb3) or NCTD+mF4-31C1 (Fig. 5Bb4) for 2 days. By counting the total number of cell clusters and branching of lymphatic tube formation, it was found that the lymphatic capillary-tube number in NCTD, mF4-31C1 or NCTD+mF4-31C1 group was less than that in the control group (10.9±2.3, 32.8±5.4 or 9.8±1.5, vs. 68.4±5.2, all P<0.000), while the lymphatic number in NCTD or NCTD+mF4-31C1 group was less than that in mF4-31C1 group (10.9±2.3 or 9.8±1.5, vs. 32.8±5.4, all P<0.01). One week after lymphatic tube formation, there are some typical lymphatic tube structures in the control group (Fig. 5Cc1); the formed structure of lymphatic tubes was destroyed, with visible cell aggregation, floating, nuclear fragmentation, apoptosis and necrosis, after using NCTD (Fig. 5Cc2), mF4-31C1 (Fig. 5Cc3) or NCTD+mF4-31C1 (Fig. 5C44) for 4 days. It was shown that NCTD or mF4-31C1 obviously inhibited and destroyed the forming of lymphangiogenesis and formed lymphangiogenesis from the 3-D culture of HDLECs in vitro, while this effect of NCTD or NCTD+mF4-31C1 was stronger. Thus, NCTD or mF4-31C1 effectively inhibits lymphatic tube formation of HLECs.

VEGF-C, VEGF-D, VEGFR-3 at protein and mRNA levels in lymphangiogenesis on 3-D culture of HDLECs were measured by immunohistochemistry (streparidin-peroxidase staining method), western blotting and quantitative real-time polymerase chain reaction (QRT-PCR). As shown in Figs. 6 and 7, at protein level, the expression of VEGF-C, VEGF-D and VEGFR-3 in NCTD group or NCTD+mF4-31C1 group was decreased significantly when compared with control group (P<0.001); but the expression of VEGF-C in NCTD or NCTD+mF4-31C1 group was lower than that of mF4-31C1 group (P<0.05); and there was no statistical difference in VEGF-D expression between control group and mF4-31C1 group (Figs. 6 and 7A–C). At the mRNA level, the expression of VEGF-C, VEGF-D and VEGFR3 mRNAs was decreased significantly in all experimental groups when compared with control group; but the expression of VEGF-C mRNA in NCTD or NCTD+mF4-31C1 group was also lower than that of mF4-31C1 group (P<0.05); and the expression of VEGF-C, VEGF-D or VEGFR3 mRNAs in NCTD+mF4-31C1 group was significantly lower than those of mF4-31C1 (P<0.005) or NCTD (P=0.01) group (Fig. 7C and D). It was shown that NCTD downregulated the expression of VEGF-C, VEGF-D and VEGFR-3 at protein and mRNA levels in the process of lymphatic tube-formation of HLECs; mF4-31C1 did not effect VEGF-D expression; the downregulation of VEGF-C, VEGF-D/VEGFR3 by the combined application of NCTD and mF4-31C1 can be enhanced. Thus, we believe that NCTD inhibit lymphangiogenesis in HLECs by simultaneously blocking VEGF-C, VEGF-D and VEGFR-3.