10Z-Hymenialdisine and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck KGaA. Hymenialdisine was dissolved in DMSO to a stock concentration of 10 mM and stored at −20°C.

The three human pancreatic adenocarcinoma cell lines MIA PaCa-2 (cat. no. CRL-1420), SW 1990 (cat. no. CRL-2172) and BxPC-3 (cat. no. CRL-1687) and the immortalized human endothelial cell line EA.hy926 (cat. no. CRL-2922) were purchased from the American Type Culture Collection. MIA PaCa-2 and SW-1990 cells were cultured in DMEM and BxPC-3 cells were cultured in RPMI medium (both from Sigma Aldrich; Merck KGaA) in a 37°C humidified incubator with 5% CO₂. These media were supplemented with 10% FBS, 10,000 U/ml penicillin, 25 µg/ml amphotericin B and 10 mg/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.).

Colony formation assays were performed using Diff-Quik solution (Sysmex Corporation). MIA PaCa-2, SW-1990 and BxPC-3 cells were seeded at 5×10² cells into each well of a six-well plate and cultured for 1 day at 37°C. The cells were then treated with various concentrations (0, 1, 2, 5 and 10 µM) of 10Z-Hymenialdisine. After 7 days of incubation at 37°C, the plates were gently washed with PBS and subsequently stained with Diff-Quik solution, which included Diff-Quik fixative reagent for 5 sec, Diff-Quik solution I (eosinophilic) for 5 sec and Diff-Quik solution II (basophilic) for 5 sec, as designated in the manufacturer's protocol at room temperature. Colonies were determined to be formed when there were ≥50 cancer cells.

Cell proliferation assays were performed using a Premix WST-1 Cell proliferation Assay System (Takara Bio, Inc.) according to the manufacturer's protocol. MIA PaCa-2, SW-1990 and BxPC-3 cells were seeded at 2×10⁵ cells into each well of a 96-well plate to a total volume of 100 µl and cultured for 1 day at 37°C. The cells were then treated with various concentrations (0, 1, 2, 5, 10 and 20 µM) of 10Z-Hymenialdisine and DMSO (equivalent to the concentration contained in 20 µM) at room temperature. After incubation for 72 h at 37°C, absorbance was measured at 450 nm using a Spectra Max 340 spectrophotometer (Molecular Devices, LLC).

MIA PaCa-2, SW-1990 and BxPC-3 cells were seeded at 1×10⁴ cells/chamber in a four-chamber glass slide and cultured for 1 day at 37°C. The cells were treated with 10Z-Hymenialdisine (5 µM) for 2 h at room temperature, followed by stimulation with TNF-α (1 ng/ml) for 20 min. 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 with PBS, permeabilized with 0.1% Triton-X for 3 min, and incubated with 3% bovine serum albumin (BSA; FUJIFILM Wako Pure Chemical Corporation) for 1 h at room temperature. Subsequently, slides were treated with rabbit antibodies against NF-κB p65 (Cell Signaling Technology, Inc.; cat. no. 8242; 1:400) overnight at 4°C and then treated with goat anti-rabbit IgG secondary antibodies H&L (Alexa Fluor^® 488) (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. Cell nuclei were subsequently stained with DAPI (50 ng/ml) at room temperature for 10 min. Images of the stained slides were captured using a BZ-X710 fluorescent microscope at ×400 magnification (Keyence Corporation).

MIA PaCa-2, SW-1990 and BxPC-3 cells were seeded at 2×10⁶ cells/dish in 100-mm dishes and cultured for 1 day at 37°C. The cells were treated with or without 10Z-Hymenialdisine (5 µM) for 2 h and then stimulated with or without TNF-α (1 ng/ml) for 20 min before the end of incubation. After the indicated treatment, nuclear protein was extracted using a Nuclear Extract Kit (Active Motif, Inc.) according to the manufacturer's protocol. The concentration of proteins in the nuclear extract was evaluated using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.) and stored at −80°C until use. The activity of NF-κB was measured using a Trans AM NF-κB p65 Active Transcription Assay Kit (Active Motif, Inc.) according to the manufacturer's protocol. A total of 5 micrograms of nuclear extract protein was used in the NF-κB activity assay.

MIA PaCa-2, SW-1990 and BxPC-3 cells were seeded at 4×10⁵ cells/well in six-well plates containing medium (MIA PaCa-2 and SW-1990 cells were cultured in DMEM and BxPC-3 cells were cultured in RPMI medium) and cultured for 1 day at 37°C. Then, the medium was changed, and the cells were cultured for an additional 2 h with or without 10Z-Hymenialdisine (5 µM) at 37°C. Total RNA was extracted from cell pellets using an RNeasy Plus Mini Kit (Qiagen KK) according to the manufacturer's protocol and quantified using a Nano Drop 1000 spectrophotemer (Thermo Fisher Scientific, Inc.) using 260 nm wavelength. Total RNA (1 µg) was reverse transcribed using Super Script III First-Strand Synthesis Super Mix for RT-qPCR kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RT-qPCR was carried out using TaqMan Fast Advanced Master Mix (cat. no. 4444964; Thermo Fisher Scientific, Inc.) and TaqMan Gene Expression Assays for IL-8 (Hs01553824_g1; cat. no. 4331182; Thermo Fisher Scientific, Inc.), VEGF (Hs00900055_m1; cat. no. 4331182; Thermo Fisher Scientific, Inc.) and GAPDH (Hs99999905_m1; cat. no. 4331182; Thermo Fisher Scientific, Inc.) with an Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions for PCR were as follows: 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 relative expression levels of VEGF and IL-8 were normalized to the expression of GAPDH in each sample using the relative standard curve method (25).

MIA PaCa-2, SW-1990 and BxPC-3 cells were seeded at 4×10⁵ cells/well in six-well plates containing medium (MIA PaCa-2 and SW-1990 cells were cultured in DMEM and BxPC-3 cells were cultured in RPMI medium) and cultured for 1 day at 37°C. The culture medium was subsequently changed, and the cells were cultured for an additional 72 h at 37°C in the presence of different concentrations of 10Z-Hymenialdisine (0, 1, 2 and 5 µM). After incubation, the culture medium was collected and centrifuged at 400 × g for 5 min at 14°C to remove particles. The supernatants were frozen at −80°C until use. The concentrations of IL-8 and VEGF were determined using human IL-8 (R&D Systems, Inc.; cat. no. D8000C) and human VEGF (R&D Systems, Inc.; cat. no. DVE00) ELISA kits according to the manufacturer's protocol.

Tube formation assays were conducted using EA.hy926 cells and Matrigel matrix (Corning Inc.) in the presence of the supernatants of pancreatic cancer cells. Cell supernatants were collected from the cancer cell culture medium (MIA PaCa-2 and SW-1990 cells were cultured in DMEM and BxPC-3 cells were cultured in RPMI medium) via centrifugation at 400 × g for 5 min at 14°C. MIA PaCa-2, SW-1990 and BxPC-3 cells were seeded at 4×10⁵ cells/well in six-well plates containing medium (MIA PaCa-2 and SW-1990 cells were cultured in DMEM and BxPC-3 cells were cultured in RPMI medium) and cultured for 1 day at 37°C. The culture media were then changed, and the cells were incubated for an additional 48 h at 37°C in medium with or without 10Z-Hymenialdisine (5 µM), after which the supernatants were collected and centrifuged at 400 × g for 5 min at 14°C to remove particles. EA.hy926 cells (1.2×10⁴ cells/well) were seeded into each well of a 96-well plate coated with Matrigel. Matrigel was pre-coated for 30 min at room temperature. Then, the cells were cultured for 1 day at 37°C with 50 µl 10% FBS and 50 µl of the aforementioned supernatant. Tube formation was observed by a BZ-X710 fluorescent microscope at ×40 magnifications (Keyence Corporation) and evaluated by determining the number of the endotubes generated by EA.hy926 cells. A total of four random fields of view were analyzed per sample.

Female BALB/c nu-nu 12 mice (4 weeks old; weight range, 13–15 g) were purchased from Charles River Laboratories, Inc. The animals were housed in standard Plexiglas cages (maximum of 5 mice per cage) in a room maintained at constant temperature (20–26°C) and humidity (40–60%) with a 12-h light/dark cycle. Mice were fed regular autoclaved chow and were provided water ad libitum. Anesthesia was administered by inhalation of isoflurane. Induction was conducted at 4–5% concentration and maintenance at 2–3%. Euthanasia was performed by cervical dislocation after adequate anesthesia. All experiments were conducted according to the Guidelines for Animal Experiments of Nagoya City University Graduate School of Medicine Sciences and were approved (approval no. Medical Animal 20–020) by the Animal Care and Use Committee of the Nagoya City University Graduate School of Medical Sciences (Nagoya, Japan).

BxPC-3 human pancreatic cells (5×10⁶ cells in 100 µl PBS) were injected subcutaneously into the left flank of each mouse. Tumor volume was measured once a week using calipers. When the tumors reached a volume of ~50 mm³, the mice were randomly divided into two groups (6 mice per group).

Mice in group I were administered DMSO (the same concentration as group II) diluted with PBS as a control, whereas mice in group II received intraperitoneal injections of 10Z-Hymenialdisine (10 mg/kg/week) diluted with PBS. The tumor volume was calculated according to the following formula: Tumor volume (mm³)=(AxB²)/2, where A and B represented the longest and shortest diameters in millimeters, respectively. Mice were injected intraperitoneally with DMSO or 10Z-Hymenialdisine once a week for 5 weeks and then sacrificed 1 week later. Finally, the tumors were excised and fixed in 10% formaldehyde at 4°C for 24 h for further analysis.

Subcutaneous xenograft tumors were fixed with 4% paraformaldehyde at 4°C for 6 h and embedded in paraffin. The sections (3 µm) were mounted on 3-amino-propyltriethoxysilane-coated slides. Dewaxed paraffin sections were placed in a microwave (10 min, 600 watts) to recover antigens before staining. The following antibodies (obtained from Cell Signaling Technology, Inc.) were used at 4°C for 24 h: Rabbit monoclonal anti-Ki67 antibodies (cat. no. 9027; 1:500), mouse monoclonal anti-CD31 antibodies (cat. no. 3528; 1:1,000), rabbit monoclonal anti-NF-κB p65 antibodies (cat. no. 8242; 1:100). Biotin-conjugated secondary antibodies (all, Dako; Agilent Technologies, Inc) were also used at room temperature for 45 min: Anti-rabbit secondary antibodies (cat. no. K4003; 1:500) and anti-mouse secondary antibodies at room temperature for 45 min (cat. no. K4001; 1:1,500). Liqid DAB + Substrate Chromogen System (cat. no. K3467) were used as the chromogen for detection at room temperature for 10 min. Hematoxylin was used for nuclear counterstaining at room temperature for 30 sec.

For the quantification of NF-κB p65 activation, the number of nucleus-positive cells was counted. Nuclear translocation of p65 was considered to be a marker of NF-κB activation. Proliferation was quantified by determining the percentage of Ki67-positive cells. Results were expressed as the mean number of Ki67-positive cells ± SD per high-power field. A total of three fields were examined from each tumor for each of the two treatment groups. For the quantification of microvessel density in sections stained for CD31, any distinct area of positive staining for CD31 was counted as a single vessel. Results were expressed as the mean number of vessels ± the SD per high-power field. A total of three random fields were examined and counted from each tumor for each of the two treatment groups. Images of the stained slides were captured using a BZ-X710 fluorescent microscope at ×200 magnification (Keyence Corporation).

All experiments, except for in vivo procedures, were performed in triplicate. All experimental data are presented as the mean ± SD. Multiple group comparisons were performed using one-way ANOVA with Dunnett's post-hoc tests for subsequent individual group comparisons. Comparisons between two groups were performed using unpaired Student's t-tests. Comparisons between multiple groups with >1 reference group were analysed using Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed with EZR Version 1.54 (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a modified version of R commander designed to add statistical functions frequently used in biostatistics (26).

Colony formation assays revealed that the growth of pancreatic cancer cell lines (MIA PaCa-2, SW-1990 and BxPC-3) was suppressed by 10Z-Hymenialdisine in a concentration-dependent manner (Fig. 1A). Consistent with this, WST-1 assays revealed that 10Z-Hymenialdisine inhibited the proliferation of all three pancreatic cancer cell lines in a concentration-dependent manner, with significant inhibition observed at 10 µM (Fig. 1B). In subsequent experiments, to avoid cytotoxicity of 10Z-Hymenialdisine, the concentration of 10Z-Hymenialdisine was set at 5 µM.

Immunocytochemical analysis revealed that p65 translocated into the nucleus of cells treated with TNF-α alone, whereas in cells treated with TNF-α and 10Z-Hymenialdisine (5 µM), p65 remained in the cytoplasm. The translocation of p65 into the nucleus was then examined using ELISA (Fig. 2A and B). The results demonstrated that 10Z-Hymenialdisine significantly decreased the translocation of p65 into the nucleus. This effect was confirmed with TNF-α. TNF-α alone significantly increased p65 translocation; however, the addition of 10Z-Hymenialdisine significantly decreased this effect (Fig. 2B).

RT-qPCR revealed that 10Z-Hymenialdisine (5 µM) significantly decreased the mRNA expression levels of IL-8 and VEGF in pancreatic cell lines (Fig. 3A and B). In addition, ELISA revealed that the protein secretion levels of IL-8 and VEGF were reduced by 10Z-Hymenialdisine (1–5 µM; Fig. 4A and B). In the present experiment, all pancreatic cancer cell lines exhibited reduced levels of IL-8 and VEGF proteins in the presence of different concentrations of 10Z-Hymenialdisine (1–5 µM). For cells treated with 5 µM 10Z-Hymenialdisine, all pancreatic cancer cell lines demonstrated significant reductions in both IL-8 and VEGF. It was confirmed that the concentration of 5 µM was the most appropriate to inhibit angiogenic factors without causing cytotoxicity.

Subsequently, tube formation assays were performed using human endothelial cells. Tube formation was enhanced when the cells were incubated with supernatants from untreated pancreatic cancer cell lines, but was significantly decreased when the cells were incubated with supernatants from 10Z-Hymenialdisine-treated pancreatic cancer cell lines (Fig. 5A). Tube formation was then evaluated by counting the number of endotubes. Endotube numbers were significantly increased when the cells were incubated with supernatants from cancer cell lines and significantly decreased when the cells were incubated with supernatants from 10Z-Hymenialdisine-treated pancreatic cancer cell lines (Fig. 5B).

BxPC-3 cells were subcutaneously injected into mice, after which tumors were allowed to form. When the tumors had grown, mice were injected intraperitoneally with 10Z-Hymenialdisine (10 mg/kg) or DMSO for 5 weeks. One mouse in the control group succumbed in the middle of the treatment course owing to the maintenance 2–3% inhalation of isoflurane anesthesia. 10Z-Hymenialdisine significantly inhibited tumor growth 14 days after treatment (Fig. 6A-C). Body, liver and kidney weights were not decreased compared with those in the control group (Fig. 6D-F).

Resected pancreatic cancer tumors were evaluated using immunocytochemical analysis. It was revealed that p65 translocation into the nucleus was decreased in groups treated with 10Z-Hymenialdisine compared with the control group. To determine the effects of 10Z-Hymenialdisine on proliferation and angiogenesis, the expression of Ki-67, a cell proliferation marker, and CD31, a microvessel density marker, were examined in tumor tissues. As demonstrated in Fig. 7, 10Z-Hymenialdisine significantly decreased the activation of NF-κB (p65) and inhibited the expression of Ki-67 and CD31 (Fig. 7A-D).

The expression levels of targeted mRNAs in resected pancreatic cancer tumors were then evaluated using RT-qPCR. The results revealed that 10Z-Hymenialdisine significantly decreased the mRNA expression levels of IL-8 and VEGF in pancreatic cancer tumors compared with that in control tumors (Fig. 8A and B).