Escin (C₅₅H₈₆O₂₄; CID 6476031) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Merck KGaA). Escin solution (50 mM) was prepared in DMSO, stored as small amounts at −20°C, then thawed and diluted in cell culture medium as required. Recombinant human tumor necrosis factor (TNF)-α was purchased from R&D Systems Inc.

The human pancreatic adenocarcinoma cell lines, BxPC-3, AsPC-1 and SW1990, and the immortalized human endothelial cell line, EA.hy 926, were purchased from the American Type Culture Collection. The BxPC-3 and AsPC-1 cell lines were maintained in RPMI-1640 medium, while the SW1990 and EA.hy 926 cell lines were maintained in DMEM (both from Sigma-Aldrich; Merck KGaA). Each medium was replenished with 10% fetal bovine serum (FBS), 10 mg/ml streptomycin, 10,000 U/ml penicillin, and 25 µg amphotericin B (all from Gibco; Thermo Fisher Scientific, Inc.). All the cell lines were cultured at 37°C in a humidified incubator with 5% CO₂.

The cytotoxicity of escin was assessed with a Premix WST-1 Cell Proliferation Assay System (Takara Bio, Inc.) according to the manufacturer's protocols. Briefly, BxPC-3, AsPC-1 and SW1990 cell lines were seeded at 2×10³ cells/100 µl/well in 96-well plates and cultured for 1 day. Then, various concentrations of escin (0–30 µM) and DMSO (equivalent to the concentration contained in 30 µM escin) were added to the cells. After incubation for 72 h, the absorbance at 450 nm was measured using a SpectraMax 340 spectrophotometer (Molecular Devices, LLC).

The BxPC-3, AsPC-1 and SW1990 cell lines were initially seeded at 1×10⁴ cells/chamber in a 4-chamber slide glass and cultured overnight. The cells were then treated with escin (10 µM) for 2 h and stimulated with TNF-α (1 ng/ml) for 15 min before the end of the incubation. The cells that had not been treated were used as controls. The cells were then washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Next, the cells were washed and permeabilized with 0.1% Triton-X for 3 min and incubated with blocking buffer (3% BSA, FUJIFILM Wako Pure Chemical Corporation) for 1 h at room temperature. The cells were probed with anti-NF-κB p65 antibody (cat. no. 8242T; Cell Signaling Technology, Inc.) overnight at 4°C. Subsequently, the cells were washed and incubated with Alexa Fluor^® 488 goat anti-rabbit IgG (cat. no. ab150077; Abcam) for 1 h at room temperature. Primary and secondary antibodies were used at 1:400 and 1:500 dilution with 3% BSA, respectively. The nuclei were visualized with DAPI staining at room temperature for 10 min. Images of the stained slides were captured using a BZ-X710 fluorescent microscope at ×100 (Keyence Corporation).

The BxPC-3, AsPC-1 and SW1990 cell lines were seeded at 2×10⁶ in 100-mm dishes with 10% FBS and cultured to ~80% confluence. The cells were then treated with escin (10 µM) in 5% FBS for 2 h and stimulated with or without TNF-α (5 ng/ml) for 30 min before the end of the incubation. Nuclear extracts were obtained from the cells using a Nuclear Extraction kit (Active Motif, Inc.). The concentrations of intranuclear proteins were measured with a Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.). Nuclear extracts were stored at −80°C until further use. The NF-κB activity was determined using a Trans AM NF-κB p65/p50 Transcription Factor Assay kit (cat. no. 40096; Active Motif, Inc.) according to the manufacturer's protocols. A total of 4 µg nuclear extract was used for the NF-κB activity assays.

The BxPC-3 cells were seeded at 2×10⁶ in 100-mm dishes with 10% FBS and cultured to ~80% confluence. The cells were then treated with escin (10 µM) in 5% FBS for 2 h and stimulated with or without TNF-α (5 ng/ml) for 30 min before the end of the incubation. Nuclear and cytoplasmic extracts were obtained from the cells using a Nuclear Extraction kit (Active Motif, Inc.). The concentrations of each protein were measured with a Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.). A total of 20 µg each protein extract was denatured at 90°C for 5 min and separated on 10% Mini-PROTEAN TGX Precast gels (Bio-Rad Laboratories, Inc.). The protein bands were transferred to nitrocellulose membranes and blocked in iBind Flex Solution (iBind Flex Buffer, iBind Flex Additive and distilled water; Thermo Fisher Scientific, Inc.) for 15 min at room temperature. The primary and secondary antibody reactions were performed using the iBind Flex Western System (Thermo Fisher Scientific, Inc.) for 2.5 h at room temperature according to the manufacturer's instructions. The membranes were incubated with anti-p65 (1:1,000; cat. no. 8242S; Cell Signaling Technology, Inc.), -p-p65 (1:1,000; cat. no. 3033S; Cell Signaling Technology, Inc.), -GAPDH (1:2,000; cat. no. SC-47724; Santa Cruz Biotechnology Inc.) and -TATA-box binding protein (TBP; 1:1,000; cat. no. 22006-1-AP; ProteinTech Group, Inc.) primary antibodies, then HRP-conjugated goat anti-rabbit polyclonal secondary antibody (1:2,000; cat. no. P0448; Agilent Technologies, Inc.). The protein-antibody complexes were visualized with a SuperSignal West Pico Chemiluminescent Substrate, SuperSignal West Femto Chemiluminescent Substrate, or Pierce ECL Western Blotting Substrate (all from Thermo Fisher Scientific, Inc.). The immunoreactive protein bands were detected using an Amersham Imager 600 (Cytiva).

The BxPC-3, AsPC-1 and SW1990 cell lines were seeded at 1×10⁵ in 6-well plates with 10% FBS and cultured to ~80% confluence. Then, the cells were treated with or without 10 µM escin and 5% FBS for 1 h and stimulated with TNF-α (1 ng/ml) for 15 min before the end of the incubation. Total RNA was extracted from the cell pellets using a RNeasy Plus Mini kit (Qiagen GmbH) according to the manufacturer's protocols and quantified using a NanoDrop^® 1000 (Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was reverse transcribed using a Super Script III First-Strand Synthesis Super Mix for RT-qPCR (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocols. RT-qPCR was performed using a Taqman Fast Advanced Master Mix and Taqman Gene Expression Assays for VEGF (Hs00900055_m1), IL-8 (Hs01553824_g1) and GAPDH (Hs99999905_m1) on a 7900HT Fast Real-Time PCR System (all from Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used: Initial denaturation at 95°C for 20 sec, followed by 40 cycles at 95°C for 1 sec and 60°C for 20 sec. The expression levels of VEGF and IL-8 were standardized to those of GAPDH in each sample, using the relative standard curve method (27).

The BxPC-3, AsPC-1 and SW1990 cell lines were seeded at 1×10⁵ cells/well in a 6-well plate containing cell specific medium as aforementioned, supplemented with 10% FBS and incubated overnight at 37°C. The culture media were then changed, and cells were incubated and stimulated with TNF-α (1 ng/ml) for 48 h in the presence of escin (10 µM). In addition, the BxPC-3 and SW1990 cells were also incubated for an additional 48 h in the presence of different doses of escin (0–10 µM) with 5% FBS after culturing overnight as aforementioned. The culture media were then collected and centrifuged at 400 × g for 5 min at 4°C to discard particulates and stored at −80°C until further use. The concentrations of IL-8 and VEGF were then measured using appropriate ELISA kits (R&D Systems, Inc.) following the manufacturer's protocols.

Tube formation was determined using the EA.hy 926 cell line and angiogenesis assays using Matrigel (Corning, Inc.). The BxPC-3 and SW1990 cell lines were seeded at 1×10⁵ cells/well in 6-well plates containing medium (RPMI-1640 and DMEM, respectively) supplemented with 10% FBS and incubated overnight at 37°C. The culture media were then changed, and the cells were incubated for an additional 48 h with or without escin (10 µM) in 2% FBS. The cell supernatants were then collected and centrifuged at 400 × g for 5 min at 4°C to discard particulates. Matrigel was added to a 96-well plate (50 µl/well) at 4°C and incubated for 30 min at 37°C for the Matrigel to solidify. The EA.hy 926 cells (1.2×10⁴ cells/well) were added on top of the Matrigel. The cells were then incubated with mixed medium (50 µl of the RPMI-1640 medium with 2% FBS and 50 µl of the aforementioned supernatant per well) for 16 h to form capillary-like structures. The cells incubated with RPMI medium with 2% FBS only were used as the control. The EA.hy 926 cells (1.2×10⁴ cells/well) were also added on top of the Matrigel and incubated with RPMI medium (2% FBS) containing 100 ng/ml recombinant IL-8 and VEGF (both from R&D Systems Inc.), respectively. The number of endotubes were counted under a confocal microscope (×40). A total of 4 fields of view were analyzed per sample.

All experiments were performed in triplicate. All the experimental data are represented as the mean ± SD. Comparisons between two groups were assessed using unpaired t-tests, while comparisons between multiple groups were determined using one-way analysis of variance with Bonferroni's post hoc test for subsequent comparison of individual groups. P<0.05 was considered to indicate a statistically significant difference.

The short-term effects of escin on the proliferation of the PaCa cells (BxPC-3, AsPC-1 and SW1990) were examined using a WST-1 assay after incubating the cells with various concentrations of escin for 72 h. The proliferation of all the PaCa cell lines was significantly inhibited by >10 µM escin (Fig. 1). The half-maximal inhibitory concentration (IC₅₀) values were calculated from the results of the WST-1 assays and were 17.2, 15.9 and 14.1 µM for the BxPC-3, AsPC-1 and SW1990 cells, respectively. To avoid the effect of cytotoxicity induced by escin, the concentration of escin was set at less than the IC₅₀ value in the subsequent experiments.

Immunocytochemical analysis was subsequently performed to examine whether escin affected TNF-α-induced translocation of p65 to the nucleus in the PaCa cells (Fig. 2). In cells treated with TNF-α alone, p65 translocated into the nucleus, while in cells treated with escin, p65 remained in the cytoplasm. The activity of p65 translocated into the nucleus was then determined using ELISA (Fig. 3). The results demonstrated that escin significantly reduced the TNF-α-induced activity of p65 in the nucleus. The activity of p65 was higher in cells treated with both escin and TNF-α compared with that in cells that were untreated; however, it is considered that the activation of p65 by TNF-α stimulation was beyond the range of suppression by escin. These results supported the hypothesis that escin inhibited the translocation of p65. To further support the results that escin inactivated NF-κB, western blot analysis was also performed to determine the protein expression levels of p65 and p-p65 in the cytoplasm and nucleus from the BxPC-3 cells, which were found to be the most sensitive to escin in immunocytotochemical staining. Escin notably reduced TNF-α-induced activation of both total p65 and p-p65 in the cytoplasm and the nucleus (Fig. S1).

RT-qPCR revealed that escin (10 µM) significantly decreased TNF-α-induced mRNA expression levels of IL-8 and VEGF in PaCa cells (Fig. 4).

Next, ELISA was performed to evaluate the protein secretion levels of IL-8 and VEGF in PaCa cells. Escin (10 µM) significantly suppressed TNF-α-induced protein secretion of IL-8 and VEGF in the PaCa cells (Fig. 5A). With respect to the AsPC-1 cell line, secretion levels of both VEGF and IL-8 were significantly enhanced by the treatment with TNF, and the enhancement was significantly inhibited by escin (Fig. S2); however, the basal secretion levels of both the angiogenic cytokines were lower compared with that in the other cell lines. Therefore, the AsPC-1 cell line was excluded from the next experiment. In addition, escin also suppressed the secretion of these proteins in a dose-dependent manner in the BxPC-3 and SW1990 cells (Fig. 5B). With respect to the inhibition of IL-8 production, there were statistically significant differences; however, the inhibition of TNF-α-induced IL-8 production in the SW1990 cell line by escin was slightly lower compared with the inhibition of VEGF production.

Lastly, the effects of escin on tube formation in human endothelial cells were determined. Tube formation was enhanced when the cells were incubated with recombinant IL-8 and VEGF (Fig. S3). Tube formation was also enhanced when incubated with the supernatants from the untreated PaCa cells. However, tube formation was significantly decreased when the cells were incubated with supernatants from escin-treated PaCa cells (Fig. 6A and B).