NS-398 was obtained from Sigma-Aldrich (St. Louis, MO, USA). The NS-398 was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) at a stock concentration of 100 mM, and stored at −20°C. SB-203580 was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA) and dissolved in DMSO at a stock concentration of 10 mM. All stock solutions were wrapped in foil and maintained at 4°C or −20°C.

The PANC-1 pancreatic cancer cell line was purchased from the Shanghai Institutes for Biological Sciences (Shanghai, China). The cells were maintained in humidified room air containing 5% CO₂ at 37°C, and were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). Cells in the logarithmic phase of growth were used in all subsequent experiments.

The pancreatic cancer cells were seeded at a concentration of 5×10³ cells/200 µl/well in 96-well culture plates for the cell proliferation assay. Briefly, the cultured wells were treated with 20 µl/well of Cell Counting Kit-8 reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) during the final 2 h of a 24 h incubation period at 37°C, and the optical density (OD) of each well was measured at 450 nm using a Model 680 Bio-Rad microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The results of cell proliferation measurement were expressed as the absorbance at OD450.

The pancreatic cancer cells (3×10⁵) were seeded in each well of a 6-well plate (Corning Incorporated, Corning, NY, USA) with 1 ml complete medium. The cells were starved in serum-free DMEM for 24 h at 37°C prior to stimulation. Following stimulation with NS 398 for the durations indicated subsequently in the Figure legends and the Results section, the supernatant from each culture medium was collected by centrifugation (4,000 × g) at 4°C, and the prostaglandin E2 (PGE-2) released into the conditioned medium (100 µl of the cell supernatant) was quantified using an ELISA kit for PGE-2 (KGE004b; R&D Systems, Minneapolis, MN, USA), strictly following the manufacturer's protocol. The OD of each well was determined within 30 min using a microplate reader (Thermo Labsystems, Santa Rosa, CA, USA) at 450 nm.

The human CD147 siRNA and negative control siRNA were purchased from GenePharma (Shanghai, China). The siRNA sequence of CD147 was as follows: Sense 5′-GGUUCUUCGUGAGUUCCUCTT-3′ and antisense 5′-GAGGAACUCACGAAGAACCTG-3′. The negative control (NC siRNA sequence was as follows: Sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense 5′-ACGUGACACGUUCGGAGAATT-3′. DharmaFECT-4 transfection reagent were purchased from Dharmacon, Inc. (Lafayette, CO, USA). The PANC-1 cells were seeded in 6-well plates at a density of 2.5×10⁵ cells/well in MEM, and were grown for 16 h at 37°C. Transfection was performed according to the manufacturer's instructions, using 4 µl of DharmaFECT-4 reagent and siRNA, at a final concentration of 100 nM/well. The cells were cultured for a further 24 h at 37°C, following which the total protein extracts (see the 'western blot analysis' section below) were isolated and analyzed using rabbit anti-human CD147 (cat. no. 13287; 1:1,000) and rabbit anti-human β-actin (cat. no. 4970; 1:1,000) antibodies (both obtained from Cell Signaling Technology, Danvers, MA, USA) for immunoblotting. The cells were collected, washed twice with pre-cooled phosphate-buffered saline (PBS), and invasion was measured.

The cells were lysed in SDS lysis buffer on ice for 30 min. Cell debris was removed by centrifugation at 14,000 g at 4°C for 5 min, and the protein contents of the cell lysates were determined using a Quick Start™ Bio-Rad Protein Assay kit (cat. no. CA5000201; Bio-Rad Laboratories, Inc.). The cell lysates with equal protein content (20 or 40 µg) were then loaded and separated by 12% SDS-PAGE (cat. no. PG113; Promoton, Nantong, Jiangsu, China). The protein bands were then electro-transferred onto a polyvinylidene fluoride membrane (Merck Millipore, Billerica, MA, USA) and blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (pH 7.4) for 1 h at 37°C. Subsequently, the membrane was incubated with the following antibodies, as appropriate: anti-c-Jun N-terminal kinase (JNK; cat. no. 9252; 1:1,000; rabbit anti-human), anti-phosphorylated (p-)JNK (cat. no. 9251; 1:1,000; rabbit anti-human), anti-extracellular signal-regulated kinase (ERK; cat. no. 9102; 1:1,000; rabbit anti-human), anti-p-ERK (cat. no. 9101; 1:1,000; rabbit anti-human), anti-p38 (cat. no. 9212; 1:1,000; rabbit anti-human), anti-p-p38 mitogen-activated protein kinases (MAPKs; cat. no. 9211; 1:1,000; rabbit anti-human; all antibodies from Cell Signaling Technology, Inc.) and anti-MMP-2 (cat. no. sc-13594; 1:500; mouse anti-human; Santa Cruz Biotechnology, Dallas, TX, USA) for at least 4 h. After washing three times, the membranes were incubated for 1 h at room temperature with species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-mouse antibody, cat. no. 115-035-003; or goat anti-mouse antibody, cat. no. 111-035-003; 1:2,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Immunoreactive bands were visualized using Immobilon Western chemiluminescent HRP substrate (Merck Millipore). As an internal control, the contents of β-actin in the samples were also immunoblotted using polyclonal anti-actin antibody as a primary antibody. The quantitative analysis of the proteins was performed by normalizing against β-actin by using Image Pro Plus 6.0 software (Media Cybernetics, Inc., Silver Spring, MD, USA).

The invasive abilities of the pancreatic cancer cells were measured using a BD BioCoat™ BD Matrigel™ Invasion Chamber (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. Briefly, the PANC-1 cells were treated with inhibitor or siRNA for 24 h, as described above. The cells were then seeded onto the membrane of the upper chamber of the Transwell, at a concentration of 3–5×10⁵/ml in 2 ml DMEM. The medium in the upper chamber was serum-free, and the medium in the lower chamber contained 5% fetal calf serum as a source of chemoattractants. The number of cells that passed through the Matrigel-coated membrane were then counted on the undersides of the filters using a Nikon Eclipse Ti light microscope (Nikon Corporation, Tokyo, Japan).

Each sample was analyzed in triplicate, and experiments were repeated three times. All data are presented as the mean ± standard deviation. All statistical analyses were performed using Microsoft Office Excel (Microsoft, Albuquerque, New Mexico, USA) and STASTISCA (StatSoft, Tulsa, OK, USA). Differences between mean values were evaluated using a paired t-test. P<0.05 was considered to indicate a statistically significant difference.

The expression of PGE-2, a product of COX-2, is downregulated by NS-398 (17). In the present study, treatment of the PANC-1 cells with 5 µM NS-398 significantly reduced the levels of PGE-2, and 50 µM NS-398 decreased PGE-2 to the lowest levels observed. However, the CCK-8 kit, which was used to determine the effect of NS-398 on the proliferation of the PANC-1 pancreatic cancer cells, found that NS-398 had no significant inhibitory effect at either low or high concentrations (between 5 and 100 µM). To investigate why NS-398 was able to inhibit COX-2 activity, but did not lead to the inhibition of pancreatic cancer cell proliferation, 50 µM NS-398 was used to treat the pancreatic cancer cells over a specific period of time, and the expression levels of MAPK signaling molecules were analyzed. The results showed that the p-P38 protein was significantly upregulated, however, the p-JNK and p-ERK signaling molecules were unaffected. The increase in p-P38 was observed in the pancreatic cancer cells treated with NS-398 for 1 h, following which the levels of p-ERK gradually increased, peaked at 12 h and were maintained to 24 h (Fig. 1A–D).

Pretreatment of the cells with SB-203580, a P38 signaling molecule inhibitor (18), was observed to inhibit NS-398-induced P38 activation. Accordingly, treatment with SB-203580 led to NS-398 inhibiting the proliferation activity of the pancreatic cancer cells. Invasive in vitro experiments were also performed, which demonstrated that treatment with 50 µM NS-398 increased the invasiveness of the pancreatic cancer cells, whereas the P38 inhibitor, SB-203580, significantly reduced pancreatic cancer cell invasion, compared with the NS-398-treated cells. SB-203580 was observed to completely inhibit the NS-398-induced increase in pancreatic cancer cell invasiveness (Fig. 2A and B).

The activation of the P38 signaling pathway is closely associated with the activation of the CD147-MMP-2 signaling pathway (19). In order to determine the role of P38 signaling molecules in the induction of CD147 and downstream MMP-2 by NS-398, the PANC-1 cells were treated with 50 µM NS-398 for the indicated durations. The results revealed that the levels of CD147 and MMP-2 were upregulated following activation of the P38 signaling molecules. SB-203580 inhibited NS-398-induced P38 activation, and also inhibited the expression levels of CD147 and downstream MMP-2 (Fig. 3A and B).

To further confirm the hypothesis that NS-398 mediates the CD147-MMP-2 signaling pathway, involved in pancreatic cancer cell invasiveness, through the activation of P38 signaling molecules, CD147 siRNAs were transfected into PANC-1 cells for 24 h. The cells were then treated with or without 50 µM NS-398, and western blot analysis of the protein expression levels was performed. The results indicated that the NS-398-induced expression of MMP-2 was inhibited by the CD147 siRNAs, however, the activation induced by NS-398 remained. Invasive in vitro experiments were also performed, which revealed that the CD147 siRNAs significantly inhibited the NS-398-induced increase in pancreatic cancer cell invasiveness. CD147 siRNA treatment also restored the anti-proliferative effect of NS-398 in the pancreatic cancer cells (Fig. 4A–C).