Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were obtained from Gibco-BRL (Grand Island, NY, USA). Zileuton was obtained from GaoMeng Chemicals (Beijing, China). The multiclonal antibody against 5-LOX was purchased from Cayman Chemicals Co. (Ann Arbor, MI, USA). Primers were synthesized by Shanghai Biotech (Shanghai, China). The reverse transcription system was purchased from Promega Biotechnology (Madison, WI, USA). Total RNA isolation kit was obtained from Invitrogen Biotechnology (Shanghai, China).

Tumor tissue specimens were obtained from 48 pancreatic cancer patients who received surgery at the Affiliated Hospital of Nantong University from 2004 to 2006. All 48 cases of pancreatic samples were fixed in 10% buffered formalin, embedded in paraffin and cut into sections with a 4-μm thickness. One section each was stained with hematoxylin and eosin for classification. Additionally, fresh pancreatic cancer tissues were partly sufficient for storage at −80°C for RT-PCR. The patients included 20 women and 28 men. The mean age was 57.2 years, and ranged from 30 to 72 years. All patients had not been treated with NSAIDs or radiotherapy and chemotherapy before surgery. We obtained the approval of the Medical Ethics Committees of Affiliated Hospital of Nantong University for conduction of this study and we complied with the Helsinki declaration.

Human pancreatic cancer cell strain SW1990 was purchased from the American Type Culture Collection (Rockville, MD, USA) and cultivated in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO₂. A stock solution of zileuton was made in DMEM and the final concentration of DMEM for all treatments including the negative control was maintained at 0.1%.

SW1990 cells were treated with 40, 20 and 10 μg/ml of zileuton for 24 h. For analysis, cells were fixed with 4% paraformaldehyde at room temperature for 1 h. Immunohistochemical and immunocytochemical staining of 5-LOX were performed using the streptavidin-peroxidase method using an anti-5-LOX antibody at a dilution of 1:50. Negative control sections were processed in the same manner, replacing the primary antibody with buffered saline. The stained sections were reviewed and scored using an Olympus microscope. The sections were then scored as having positive or negative staining. Positive staining was defined as 5% or more of the epithelial cells staining positively (14).

Cell viability was measured using the MTT assay. Exponentially growing cells were plated onto 96-well plates containing 4,000 cells/well in 200 μl medium for 24 h. The medium was then replaced with either control medium or medium containing zileuton at 40, 20, 10, 5 and 1 μg/ml for 24, 48 and 72 h, respectively. Twenty microliters of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide stock solution (5 mg/ml; Sigma-Aldrich) was added into each well, and the cells were further incubated at 37°C for 4 h. The supernatant was replaced with 150 μl of DMSO to dissolve the formazan product. The optical density (OD) was measured at a wavelength of 570 nm. The percentage of viability was calculated using the equation: Viability (%) = (1 - ODt/ODc) × 100, where ODt and ODc are the optical densities of the treated and control cultures, respectively.

Cell apoptosis was measured using the TUNEL assay (Roche Diagnostics, Germany). SW1990 cells were seeded onto 6-well plates that contained coverslips and were then incubated for 24 h. The medium was then replaced with either control medium or medium containing zileuton at 40, 20 or 10 μg/ml, incubated for 24 h and then fixed with 4% paraformaldehyde. Cells were then washed with PBS, chilled in an ice bath for 2 min with permeabilization solution, washed again with PBS and incubated with TUNEL mixture of terminal deoxynucleotidyl transferase and dUTP in DNA-labeling solution for 1 h at 37°C. Cells were then rinsed twice with PBS, incubated with 50 μl of enzyme-labeled anti-fluorescein antibody solution in the dark for another 30 min. After the cells were rinsed with PBS, 3,3-diaminobenzidine was added for color development and hematoxylin was used for counterstaining. For each experimental group, a total of 1,000 cells from 5 high-power field images were examined under a microscope.

Cells (1×10⁷) were seeded into 50-ml dishes and incubated for 24 h at 37°C. Then zileuton at 40, 20, and 10 μg/ml was directly added to the dishes and incubated for an additional 24 h. Cells were collected, washed with PBS and resuspended in PBS. Apoptotic cell death was identified by double supravital staining with recombinant FITC (fluorescein isothiocyanate)-conjugated Annexin V and propidium iodide (PI), using the Annexin V-FITC Apoptosis Detection kit (Becton-Dickinson, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Flow cytometric analysis was performed immediately after supravital staining. Data acquisition and analysis were performed in a Becton-Dickinson FACSCalibur flow cytometer using CellQuest software. The distribution of cells in the cell-cycle phases was determined using flow cytometric analysis of DNA content. Briefly, after treatment with zileuton at 20 μg/ml for 24 h, cells were fixed with ice-cold 70% ethanol and stored at −20°C. Prior to flow cytometry, the cells were washed and resuspended at 1×10⁷ cells/ml in PBS and incubated with 100 μg/ml RNase and 50 μg/ml PI at 37°C for 30 min. Samples were analyzed using a flow cytometer (FACSCalibur type; BD Biosciences, San Diego, CA, USA). The apoptotic cells were detected on a DNA content histogram as a sub-diploid or pre-G1 peak.

Total RNA was extracted from 22 cases of pancreatic cancer tissues, their corresponding non-tumor tissues and SW1990 cells treated with 20 μg/ml of zileuton for 24 and 48 h. After being reversely transcribed into cDNA, 1 μl of the RT product was used as a template for PCR. The primer sequences used to amplify the 5-LOX gene were: forward, 5′-TCA-TCG-TGG-ACT-TTG-AGC-TG-3′ and reverse, 5′-AGA-AGG-TGG-GTG-ATG-GTC-TG-3′. The primers for amplifying the β-actin gene were: forward, 5′-AAG-TAC-TCC-GTG-TGG-ATC-GG-3′ and reverse, 5′-ATG-CAT-TCA-CCT-CCC-CTG-TG-3′. The expected amplification fragment lengths of 5-LOX and β-actin were 262 and 486 bp, respectively. PCR was performed at 94°C for 5 min, 36 amplification cycles at 94°C for 40 sec, 54°C for 55 sec and 72°C for 1 min and a final extension at 72°C for 7 min. Amplification was performed in a Perkin-Elmer 2400 thermocycler (Applied Biosystems, Foster City, CA, USA). The PCR products were resolved by electrophoresis on 1.5% agarose gel and visualized after ethidium bromide staining and ultraviolet irradiation. The relative level of 5-LOX mRNA expression was analyzed by normalizing the band intensity of 5-LOX to that of β-actin. The detection was performed 6 times.

All data are expressed as mean ± SE. The significance of the difference between 2 groups was assessed by one-way ANOVA and the frequency of computing was performed by Chi-square or Fisher’s exact probability test using STATA software package. P<0.05 was considered to indicate a statistically significant result.

By RT-PCR, 5-LOX mRNA expression was detected in 6/48 cases (12.5%) of the non-tumor tissues and in 39/48 cases (81.3%) of the pancreatic cancer tissues (representative data shown in Fig. 1A). Additionally, 5-LOX protein expression was detected in 7/48 cases (14.6%) of the non-tumor tissues and in 39/48 cases (81.3%) of the pancreatic cancer tissues (Fig. 1B and C). The difference in 5-LOX expression between the non-tumor and tumor tissues was statistically significant (P<0.01). Furthermore, we correlated the tumor expression of 5-LOX with clinicopathological data and observed that 5-LOX mRNA (P<0.05, Table I) and protein (P<0.05, Table II) expression was statistically significantly associated with lymph node metastasis and TNM stage.

SW1990 cells were treated with 40, 20, 10, 5 and 1 μg/ml zileuton for 24, 48, and 72 h and cell viability was assessed by MTT assay. We observed that zileuton suppressed SW1990 cell proliferation in a concentration- and time-dependent manner (Fig. 2). Approximately 79% of the SW1990 cells were still viable after treatment with 20 μg/ml of zileuton for 72 h, while the viability decreased to 40% when the concentration of zileuton was increased to 40 μg/ml.

TUNEL assay results showed that zileuton induced apoptosis in the SW1990 cells (Fig. 3). Cells exhibited cytoplasmic shrinkage and nuclei were stained brown and the cytoplasm was stained blue which indicated apoptosis. After a 24-h exposure to zileuton at concentrations of 40, 20 or 10 μg/ml, the percentages of apoptotic cells estimated by TUNEL assay were 45.1, 31.3 and 24.7% respectively, which were significantly higher than that of the control (9.6%, P<0.05, Fig. 6A).

We analyzed the DNA content of zileuton-treated SW1990 cells by flow cytometric analysis. After a 24-h exposure to 20 μg/ml of zileuton, apoptotic cells in the sub-diploid population before the G0/G1 phase were observed by flow cytometry (Fig. 4).

During early apoptosis, phosphatidylserine, a phospholipid usually located on the inner surface of the plasma membrane, translocates to the outer plasma membrane due to the loss of membrane phospholipid symmetry. Annexin V preferentially binds to the negatively charged phosphatidylserine. Annexin V conjugated to fluorescein allows for detection of early apoptosis by flow cytometry or fluorescence microscopy. Early apoptotic cells bind Annexin V but do not exhibit intracellular staining with PI. As cells progress through apoptosis, the integrity of the plasma membrane is lost, allowing PI to penetrate and label the cells with a strong yellow-red fluorescence. The results in Fig. 5 demonstrate strong Annexin V staining in the SW1990 pancreatic cancer cells after 20 μg/ml zileuton treatment for 24 h, but no staining or only very weak staining was observed in the control cells.

The apoptotic cell levels estimated by flow cytometry were induced by zileuton in a concentration-dependent manner. The percentage of apoptotic cells estimated by flow cytometry and Annexin V/PI assay were 43.7, 29.8 and 23.3% respectively, which were significantly higher than that of the control (7.3%, P<0.05, Fig. 6B).

Our data indicated that zileuton induced apoptosis in SW1990 cells. Interestingly, the cellular levels of 5-LOX mRNA decreased with zileuton treatment (Fig. 7A and Table III). Furthermore, immunocytochemical staining revealed that the level of 5-LOX protein expression in the SW1990 cells was significantly decreased following zileuton treatment (Fig. 7B–D). Using image analysis software to quantify the 5-LOX mRNA levels, a significant difference between the zileuton-treated group and the control group was noted (Table III).