All cell culture reagents including fetal bovine serum (FBS), TrypLE™, DMEM medium and transfection reagent, Turbofect^TM were from Invitrogen (Carlsbad, CA, U.S.A.). All chemicals including toluidine blue O and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich (St Louis, MO, U.S.A.). Ribociclib was from MedChemExpress (Monmouth Junction, NJ, U.S.A.). Antibodies against PARP, caspase 3 active form, survivin, ERK1/2 and ERK1/2 phosphorylated at Thr 202/Tyr 204, p38MAPK, p38MAPK phosphorylated at Thr180/Tyr182, p53 phosphorylated at Ser15 and p53 acetylated at Lys379 were from Cell Signaling (Danvers, MA, U.S.A.). Antibodies against α-tubulin and GAPDH, as well as anti-rabbit and anti-mouse IgG conjugated horseradish peroxidase antibodies were from GeneTex Inc (Irvine, CA, U.S.A.). Antibody against Sp1 and normal IgG were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Immobilon Western Chemiluminescent HRP Substrate was from Millipore (Billerica, MA, USA). All materials for western analysis were from Bio-Rad (Hercules, CA, U.S.A.). Cell Proliferation ELISA, BrdU assay kit was from Roche (Indianapolis, IN, USA). PG13-luciferase construct (p53-luc) with p53 binding sites (Addgene plasmid #16642) and p21/WAF1 promoter luciferase construct (p21 pro-luc, Addgene plasmid #16451) as described previously (29) were kindly provided by Dr. Bert Vogelstein. A Dual-Glo luciferase assay system and renilla-luciferase construct were from Promega (Madison, WI, U.S.A.).

WMJ-J compounds were synthesized as described previously (28).

The murine LEC line SV-LEC was kindly provided by Dr. J.S. Alexander (Shreveport, LA). SV-LECs were cultured as previously described (30). Primary human lymphatic endothelial cells (HLEC, C-12217), MV2 basal and growth medium were purchased from PromoCell (Heidelberg, Germany). HLECs were maintained in MV2 growth medium in a humidified 37°C incubator. The MV2 growth medium contains growth supplements including 5% fetal calf serum, 5 ng/ml epidermal growth factor, 10 ng/ml basic fibroblast growth factor, 20 ng/ml insulin-like growth factor, 0.5 ng/ml vascular endothelial growth factor, 1 μg/ml ascorbic acid, 0.2 μg/ml hydrocortisone.

We used the colorimetric MTT assay to determine cell viability as described previously (28).

Human lymphatic endothelial cells (HLECs) (2 × 10⁴ per well) seeded in 48-well tissue culture plates were starved in MV2 basal medium in the absence of growth supplements for 24 h. After starvation, cells were incubated in MV2 growth medium containing growth supplements with or without WMJ-S-001 at indicated concentrations for another 24 h. A Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche) based on the colorimetric detection of the incorporation of BrdU was used to determine cell proliferation following the manufacturer's instructions.

SV-LCEs were treated with indicated concentrations of WMJ-S-001 for 24 h. Cells were harvested and fixed in 70% ethanol at 0°C for another 24 h. After fixation and washed with phosphate-citric acid buffer, cells were stained in the dark for 30 min with staining buffer [(0.1% Triton X-100, 100 μg/ml RNase A and 25 μg/ml propidium iodide (PI)]. The FACScan and Cellquest program (BD Biosciences, San Jose, CA, U.S.A.) were used to perform flow-cytometric analysis. The percentage of PI-stained cells in the G0/G1, S, G2/M, or subG1 (Apoptosis, Apo) region was analyzed using the FCS Express (De Novo Software, Glendale, CA, U.S.A) or ModFit (BD Biosciences, San Jose, CA, U.S.A.) program.

After treatment, cells were harvested in lysis buffer [0.5% NP-40, 140 mM NaCl, 10 mM Tris (pH 7.0), 0.05 mM pepstatin A, 2 mM PMSF and 0.2 mM leupeptin]. Cell lysate with equal amounts of protein were subjected to SDS-PAGE and transferred onto a NC membrane (Pall Corporation, Washington, NY, U.S.A.). After transfer, membrane was incubated with 5% non-fat milk-containing blocking buffer for 1 h. Specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies were used to recognize target proteins. Enhanced chemiluminescence was used to detect immunoreactivity according to manufacturer's instructions. To obtain the quantitative data, a computing densitometer with a scientific imaging system (Biospectrum AC System, UVP) was used.

SV-LECs (7 × 10⁴ cells/well) were transfected with PG13-luciferase (p53-luc) or p21 promoter-luciferase (p21 pro-luc) plus renilla-luciferase for reporter assay or transfected with negative control siRNA (NC) or survivin siRNA for MTT, flowcytometry, immunoblotting and invasion assay using Turbofect transfection reagent (Invitrogen, Carlsbad, CA, U.S.A.) per manufacturer's instructions.

After transfection, SV-LECs were treated with vehicle or WMJ-S-001 for another 24 h. A Dual-Glo luciferase assay system kit (Promega, Madison, WI, U.S.A.) was employed to determine the luciferase reporter activity per manufacturer's instructions. The reporter activity was normalized based on renilla-luciferase activity.

Target gene silencing in SV-LECs was performed as described previously (31). Negative control scramble siRNA and pre-designed siRNAs targeting the murine survivin (BIRC5) were purchased from Sigma-Aldrich (St Louis, MO, U.S.A). The siRNA oligonucleotides were as follows: survivin siRNA, 5′-cgauagaggagcauagaa-3′ and negative control scramble siRNA, 5′-gaucauacgugcgaucaga-3′.

SV-LECs were seeded in the 12-well tissue culture plates. After growing to confluence, SV-LECS were starved with serum-free DMEM medium for 24 h. Pipette tips were used to create scratch wounds in monolayers of SV-LECs. Cells were washed with PBS, followed by the treatment with WMJ-S-001 at different concentrations in the presence or absence of 10% FBS for another 24 h. Cells were fixed with cold 4% paraformaldehyde and stained with 0.5% toluidine blue. After staining, an OLYMPUS Biological Microscope digital camera (Yuan Li Instrument Co., Taipei, Taiwan) was used to take photographs at 40× magnification. Cell migration rate was determined by calculating the migrated cells in the wound area.

We performed cell invasion assays as described previously (20). 0.2% gelatin solution was used to coat the lower face of the filter in the transwell plate (Corning, NY, U.S.A.). The lower chambers were filled with containing 10% FBS-containing DMEM medium (SV-LECs) or growth supplements-containing MV2 medium (HLECs). SV-LECs or HLECs (2 × 10⁴ cells per chamber) were seeded in the upper chambers in the serum-free DMEM medium or MV2 basal medium with or without WMJ-S-001. After 18 h, the non-invaded cells in the upper chamber were removed by gently scraped with a cotton swab. The invaded cells in the lower face of the filter were fixed, stained with toluidine blue (0.5% in 4% paraformaldehyde) and photographed using an optical microscope (Nikon, Japan) at ×40. The number of stained cells that invaded through the filter were counted. We also quantified cell invasion by dissolving the stained cells in 33% acetic acid and measuring the absorbance at 570 nm.

After treatment as indicated, cells were harvested for isolation of total RNA and complementary DNA (cDNA) synthesis as previously described (31). We used GoTaq qPCR Master Mix (Promega, Madison, WI, U.S.A.) and StepOne Real-Time PCR systems (Applied Biosystems, Grand Island, NY U.S.A.) to perform RT- qPCR. The cycling conditions were as follows: hot-start activation for 2 min at 95°C, followed by 40 cycles of denaturation for 15 s at 95°C, annealing/extension for 60 s at 60°C. The primers used to transcribe survivin and GAPDH are as follows: human survivin forward, 5′-gcctttccttaaaggccatc-3′; human survivin reverse, 5′-aacccttcccagactcca ct-3′; human GAPDH forward, 5′-gtcagtggtggacctgacct-3′; human GAPDH reverse, 5′-aggggtctacatggcaactg-3′; mouse survivin forward, 5′-atcgccaccttcaagaactg-3′; mouse survivin reverse, 5′-tgactgacgggtagtctttgc-3′; mouse GAPDH forward, 5′-ccttcattgacctcaactac-3′; mouse GAPDH reverse, 5′-ggaaggccatgccagtgagc-3′.

After treatment as indicated, cells were cross-linked with formaldehyde (1%) for 10 min at 37°C. Cross-linking was quenched by adding 1.25 M glycine. After harvesting cells with ice-cold PBS, the cell pellet was resuspended in SDS lysis buffer. Samples were sonicated five times (for 15 s each) and centrifuged (10 min) to collect supernatants. An aliquot of each sample was used as “Input.” The remainder of the soluble chromatin was diluted in ChIP dilution buffer. Immunoprecipitation was performed by adding normal IgG, anti-p53, or anti-Sp1 antibodies plus protein A-magnetic beads (Millipore, Billerica, MA, U.S.A.) with a gentle rotation at 4°C for 18 h. The immune complexes were washed sequentially in the following buffers: low-salt, high-salt, LiCl immune complex washing buffer and Tris-EDTA buffer. After last wash, elution buffer (100 μl each) was added twice to elute the immune complex. The cross-linked chromatin complex was reversed by adding 0.2 M NaCl and heating for 4 h at 65°C. GP^TM DNA purification spin columns (Viogene, New Taipei City, Taiwan) were used to purify DNA. Purified DNA was used to perform PCR with PCR Master Mix (Promega, Madison, WI, U.S.A.). To amplify the survivin promoter fragment, the following primers were used: forward, 5′-accgcagcagaaggtacaac-3′ and reverse, 5′-agacgactcaaacgcaggat-3′. The cycling conditions were as follows: initial denaturation for 5 min at 95°C, followed by 30-cycles of 30 s at 95°C, 30 s at 56°C and 45 s at 72°C and final extension for another 10 min at 72°C. PCR products were analyzed by agarose gel electrophoresis (1.5%).

The basement membrane matrix, matrigel (Becton Dickinson, Mountain View, CA, USA) was used to perform the tube formation assay as previously described (32). Matrigel was polymerized for 30 min at 37°C. HLECs were seeded onto the matrigel in MV2 basal medium with or without WMJ-S-001 at indicated concentrations. After 24 h, cells were photographed using an optical microscope (Nikon, Japan) at ×40.

We have different people analyzing data (analyst) and conducting experiments (operator) for blinding. The same cell in every single experiment was used to evaluate the WMJ-S-001's effects vs. the related control. Therefore, formal randomization was not employed.

In this study, the data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (33). Results represented as mean ± standard error of mean (S.E.M) (n ≥ 3), where ‘n' refers to independent values, and not replicates. Normalization was performed to compare the differences after the treatment to control for unwanted sources of variation and to reveal relevant trends. To reduce the effect of variation from different exposure of immunoblotting, the protein expression levels or the status of protein modification were expressed by normalization that generates control values with no variance (SEM = 0). Such data are not subjected to parametric statistical analysis. SigmaPlot 10 (Build 10.0.0.54; Systat Software, San Jose, CA, U.S.A.) was used to perform statistical analysis. Statistical comparisons between two groups were evaluated by the Mann–Whitney test for non-parametric analysis or unpaired Student's t-test for parametric analysis. Kruskal-Wallis test followed by Dunn's multiple comparison for non-parametric analysis or one-way analysis of variance (ANOVA) with Tukey's post-hoc test for parametric analysis was used to evaluate the statistical comparisons between more than two groups. A P-value smaller than 0.05 was defined as statistically significant.

It is still difficult to isolate and propagate LECs from different organs (7, 34, 35). This limits the studies on lymphangiogenesis or signaling mechanisms in LECs. In this study, we selected a SV40 large T-expressed immortalized murine LEC line (SV-LEC), which retain their “lymphatic” endothelial characteristics after repeated passages (30, 36), to overcome these limitations. A MTT assay was used to examine whether WMJ-S-001, a novel aliphatic hydroxamate-based compound, affects SV-LEC viability. As shown in Figure 1A, WMJ-S-001 reduced SV-LEC viability in the concentration- and time-dependent manners. We next determined whether WMJ-S-001's inhibitory actions on the cell viability attributes to its anti-proliferative effects. After starvation with serum-free medium for 24 h, SV-LECs were stimulated with serum (10% FBS) for another 6 to 72 h in the presence or absence of WMJ-S-001 (10 μM). As shown in Figure 1B, WMJ-S-001 significantly reduced serum-induced SV-LEC proliferation. In addition, WMJ-S-001 concentration-dependently inhibited cell proliferation in SV-LECs after 48 h exposure to serum (Figure 1C). We next used flow cytometry with propidium iodide (PI) staining to examine whether WMJ-S-001 alters cell cycle progression or induces apoptosis. Treatment of WMJ-S-001 for 24 h significantly reduced the percentage of PI-stained cells in the S region as compared with the control group (Figure 1D). This effect was accompanied by a concomitant increase in the percentage of PI-stained cells in the G1 region after treatment of WMJ-S-001 (Figure 1D). In addition, WMJ-S-001 at concentrations of 10 μM or lower did not significantly cause apoptosis (sub-G1, apoptotic region). However, WMJ-S-001 at 30 μM significantly induced cell apoptosis in SV-LECs (Figure 1D). We next examined whether WMJ-S-001 activates caspase3, an apoptosis marker. WMJ-S-001 at concentrations higher than 10 μM (20 and 30 μM) markedly increased the cleaved (active) form of caspase 3 and its substrate, PARP (Figure 1E). It suggests that inhibiting LEC proliferation and causing apoptosis may contribute to WMJ-S-001's anti-lymphangiogenic effects.

We next examined whether WMJ-S-001 alters cell motility, a pivotal step in lymphangiogenesis, in SV-LECs after serum (10% FBS) exposure. WMJ-S-001 significantly inhibited serum-induced LEC migration as determined by wound-healing migration assay (Figure 2A). We also used transwell invasion assay to examine WMJ-S-001's effects on serum-induced cell invasion. As shown in Figure 2B, WMJ-S-001 at 10 and 30 μM significantly reduced the number of invading cell penetrating the gelatin-coated transwell filter barrier, using serum (10% FBS) as the chemoattractant. These observations indicate that WMJ-S-001 is capable of inhibiting LEC migration and invasion.

Growing evidence has demonstrated that suvivin not only regulates mitosis and apoptosis, but also plays a critical role in angiogenesis (37, 38). However, whether endothelial survivin participates in lymphangiogenesis remains unclear. We thus determined whether WMJ-S-001 alters survivin level in SV-LECs. Results from immunoblotting analysis demonstrated that survivin exhibits high expression levels in SV-LECs (Figure 3A). However, treatment of cells with WMJ-S-001 for 18 or 24 h concentration-dependently caused survivin reduction (Figure 3A). We also determined whether WMJ-S-001 affects survivin mRNA level in SV-LECs. As shown in Figure 3B, WMJ-S-001 at 10 or 30 μM significantly reduced survivin mRNA expression. It indicates that WMJ-S-001 may reduce survivin expression at the transcriptional level. A survivin siRNA oligonucleotide was employed to determine whether survivin reduction decreases cell viability in SV-LECs. Survivin siRNA significantly reduced the basal level of survivin (Figure 3C) and decreased cell viability (Figure 3D) in SV-LECs. Results from flow-cytometric analysis further demonstrated that survivin reduction mimics the enhancing effects of WMJ-S-001 in reducing the percentage of PI-stained cells in the S region. Survivin siRNA also caused G2/M cell cycle arrest and apoptosis in SV-LECs (Figure 3E). We examined whether survivin reduction affects SV-LEC motility. As shown in Figure 3F, survivin siRNA mimicked the WMJ-S-001's effects in reducing the number of invading cell penetrating the gelatin-coated transwell filter barrier, using serum as the chemoattractant. In contrast, negative control siRNA was without effects (Figure 3F). These observations suggest that survivin reduction may contribute to WMJ-S-001's inhibitory effects on SV-LEC motility and subsequent lymphangiogenesis.

We investigated the underlying mechanisms of WMJ-S-001 in repressing survivin expression in SV-LECs. Transcription factor p53 participates in various cellular processes such as cell cycle arrest, apoptosis or senescence by regulating diverse target genes (39). It is reported that p53 may suppress survivin expression by counteracting Sp1 binding to the survivin promoter region (40). Therefore, we explored whether WMJ-S-001 induces p53 activation in SV-LECs. Post-translational modifications such as phosphorylation or acetylation play important roles in regulating p53's activity (41). We thus examined WMJ-S-001's effects on p53 phosphorylation and acetylation in SV-LECs. As shown in Figure 4A, 6 h exposure to WMJ-S-001 led to concentration-dependent increases in p53 phosphorylation and acetylation. Results from reporter assay showed that treatment of cells with WMJ-S-001 for 24 h significantly increased PG13-luciferase (p53-luciferase) activity. WMJ-S-001 also increased the promoter-luciferase activity of p21, a p53 target gene, in SV-LECs (Figure 4B). Moreover, we performed a ChIP analysis to examine whether Sp1 or p53 is recruited to the endogenous survivin promoter region (−236 to −26) containing putative Sp1 and p53 binding sites, in WMJ-S-001-stimulated SV-LECs. As shown in Figure 4C, 6 h WMJ-S-001 exposure increased p53 binding to the survivin promoter region (−236/−26). This effect was accompanied by a concomitant decrease in Sp1 binding to the region.

p38-mediated signaling cascade has been shown previously to cause p53 phosphorylation and survivin reduction in cancer cells (23). We thus elucidated whether WMJ-S-001-induced p53 activation involves p38MAPK signaling in SV-LECs. As shown in Figure 4D, WMJ-S-001 time-dependently induced p38MAPK phosphorylation. A pharmacological p38MAPK inhibitor, p38 inhibitor III, significantly suppressed WMJ-S-001's effects in inducing p53 phosphorylation (Figure 4E) and survivin reduction (Figure 4F) in SV-LECs. It appears that WMJ-S-001 induces p38MAPK activation, resulting in p53 activation and survivin reduction in SV-LECs.

We next examined whether WMJ-S-001 reduces survivin expression and exhibits anti-lymphangiogenic activities in primary human LECs. As shown in Figure 5A, WMJ-S-001 at concentrations ranging from 10 to 30 μM significantly reduced survivin mRNA levels in human LECs (Figure 5A). WMJ-S-001's effects on cell proliferation in human LECs were examined using a BrdU incorporation assay. After starvation with MV2 basal medium for 24 h, human LECs were stimulated by MV2 growth medium in the absence or presence of WMJ-S-001 for another 24 h. The percentage of BrdU-labeled cells increased significantly after a 24 h treatment with MV2 growth medium. However, WMJ-S-001 reduced this increase in a concentration-dependent manner (Figure 5B). The effects of WMJ-S-001 on human LEC invasion were also determined using transwell invasion assay. As shown in Figure 5C, WMJ-S-001 significantly reduced the number of invading cells penetrating the gelatin-coated transwell filter barrier using MV2 growth medium as the chemoattractant (Figure 5C). We also examined whether ribociclib, a potent proliferation inhibitor targeting cyclin-dependent kinases (CDKs) 4/6 (42), affects human LEC invasion. As shown in Figure 5D, ribociclib, similar to WMJ-S-001, suppressed human LEC invasion. It is likely that WMJ-S-001's inhibitory effects on LEC invasion may also attribute to its anti-proliferative properties. Another key step of lymphangiogenesis is the tubular formation of LECs. We thus examined WMJ-S-001's effect on this step. Human LECs were seeded on the matrigel surface in complete MV2 growth medium in the presence of vehicle as control or WMJ-S-001. As shown in Figure 5E, cells incubated with MV2 growth medium became elongated, and formed capillary-like structure within 16 h. WMJ-S-001, however, concentration-dependently reduced the formation of capillary-like network (Figure 5E). Similarly, WMJ-S-001 also significantly reduced VEGF-C-induced tubular formation of human LECs (Figure 5E). Furthermore, WMJ-S-001 suppressed VEGF-C-induced ERK1/2 phosphorylation in human LECS (Figure 5F). Together these observations suggests that WMJ-S-001 exhibits anti-lymphangiogenic properties through suppressing cell proliferation, invasion and tubular formation of LECs. WMJ-S-001's actions in LECs may also attribute to survivin reduction.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.