Pancreatic Stellate Cells (PSCs) express Cyclooxygenase-2 (COX-2) and pancreatic cancer stimulates COX-2 in PSCs

In early primary PSCs, cytoplasmic COX-2 staining was detected (Figure 1). However, early primary cultured PSCs (quiescent cells) were α-SMA negative (Figure 1). After passage, PSCs flattened and developed long cytoplasmic extensions (activated PSCs), and showed positive immunostaining for COX-2 and α-SMA (Figure 2).

On days one and four in primary culture, PSCs expressed low levels of α-SMA (Figure 3). Between day 7 and day 20, α-SMA expression increased substantially (Figure 3). In. contrast, the COX-2 protein was detected in primary cultured PSC from day 1 through day 20 (Figure 3).

PSCs were treated with PANC-1 CM for 0.5, 1, 3, 6, 12, 24, 48, and 72 hours. PANC-1 CM caused sustained up-regulation of the COX-2 protein, which was maximally increased after 12 hours and remained elevated for at least 24 hours (Figure 4).

PSCs were treated with PANC-1 CM and control medium and PANC-1 CM with the mitogen-activated protein kinase kinases (MEK) inhibitor U0126 (10 μM) for 0.5, 1, 3, 6, 12, 24, 48, 72 hours. U0126 significantly inhibited PANC-1-induced expression of COX-2 (Figure 5).

Since COX-2 is increased by PANC-1 CM, the role of COX-2 in PANC-1 CM-induced PSC proliferation was investigated using a specific COX-2 inhibitor, NS398. PANC-1 CM increased PSC thymidine incorporation as well as cell number compared to control medium (Figure 6). Inhibition of COX-2 with NS398 resulted in a concentration-dependent decrease in thymidine incorporation and cell number.

Iscove's modified Dulbecco's medium (IMDM), Dulbecco's modified Eagle's medium (DMEM), albumin, and pronase were purchased from Sigma Chemical (St Louis, MO). Fetal bovine serum (FBS), glutamine, and antibiotics were purchased from Mediatech, Inc. (Herndon, VA). Collagenase P was purchased from the Roche Diagnostics Corporation (Indianapolis, IN), and deoxyribonuclease from Amersham Biosciences (Piscataway, NJ). Nycodenz was obtained from Nycomed Pharma AS (Oslo, Norway). U0126, a specific inhibitor of extracellular signal-regulated kinase (ERK) activation, was obtained from Calbiochem (San Diego, CA). NS398, COX-2 inhibitor was obtained from Cayman Chemicals (Ann Arbor, MI). ³H-methyl thymidine was purchased from ICN Pharmaceuticals (Costa Mesa, CA).

Male Sprague-Dawley rats weighing 200–250 g were used in accordance with standard institutional animal welfare guidelines, and protocols were approved by the Institutional Animal Care and Use Committee, Northwestern University School of Medicine.

Rat PSCs were isolated as previously described [3]. Briefly, the pancreas was digested with a mixture of collagenase P and pronase and deoxyribonuclease in Gey's balanced salt solution. The resulting suspension of cells was centrifuged in a 28.7% Nycodenz gradient at 1400 g for 23 minutes. Stellate cells then separated into a hazy band just above the interface of the Nycodenz solution and the aqueous buffer. Cells were harvested, washed, and resuspended in IMDM containing 10% FBS, 4 mmol/l glutamine, and antibiotics. PSCs were all used within two passages following isolation.

The poorly differentiated pancreatic adenocarcinoma cell line PANC-1 (American Type Tissue Culture, Rockville, MD) was grown in DMEM in 75 cm² flasks. When the cells reached confluence, the serum-containing medium was removed and the cells were cultured in 20 ml of serum-free medium. After 24 hours, the medium was collected and the peptide containing fraction obtained by semi-purifying on a Sep-Pak Plus C18 Cartridge (Waters, Milford, MA). After washing, Sep-Paks were eluted with 50% acetonitrile (EM Science, Gibbstown, NJ) with 0.1% trifluoroacetic acid (J. T. Baker, Phillipsburg, NJ). The eluates were lyophilized and reconstituted in fresh IMDM with 1% FBS, forming what we refer to as PANC-1 conditioned medium (CM). In total extracts of 100 ml pf PANC-1 conditioned media were purified and reconstituted in 20 ml of media for PSC culture. Control medium consisted of only serum-free medium without PANC-1 cells which underwent are same Sep-Paking procedure. The eluates were also lyophilized, and then reconstituted in fresh IMDM with 1% FBS.

COX-2 and α-SMA expression in PSCs was evaluated by immunohistochemical staining. Cultured PSCs were grown directly on glass coverslips in six-well plates, and immunostained for COX-2 using peroxidase-labeled streptavidin for immunohistochemistry (KPL, Inc., Maryland) according to the manufacturer's instructions. Cells were fixed for 30 minutes in acetone at -20°C. Thereafter, glass coverslips were air-dried and stored at 4°C until the cells were stained. Endogenous peroxidase activity was blocked by incubation in methanol with 0.3% hydrogen peroxidase for 30 minutes. After immersion in normal goat serum for 30 minutes, the slides were incubated with COX-2 (murine) polyclonal antibody (Cell Signaling Technology, Inc., Beverly, MA) diluted 1:200 in tris-buffered saline (TBS) 1× bovine serum albumin and stored in a humid chamber overnight at 4°C. The slides were incubated with anti-mouse immunoglobulins for 10 minutes at 37°C, followed by peroxidase conjugated streptavidin for 30 minutes at room temperature. Finally, color was developed incubating the slides for 8 minutes, with diaminobenzine (DAB Reagent Set; KPL, Inc., Maryland). Expression of α-SMA was examined in a similar manner by using monoclonal anti-α-SMA antibody (Sigma, St Louis, MI).

Protein concentrations in the cell lysates were measured by the method of Lowry et al. [28] using bovine serum albumin as the standard.

Following the treatment of cells with PANC-1 CM for 48 hours, DNA synthesis was measured by adding to each well 0.5 μCi of ³H-methyl thymidine and incubating these plates for the final 24 hours. The cell protein was precipitated with 10% trichloroacetic acid overnight, washed twice with phosphate buffered saline (PBS) and then dissolved by adding 0.25 ml of 0.5 mol/l NAOH to each well. Incorporation of ³H-methyl thymidine into DNA was measured by adding 1 ml of scintillation cocktail (ScintiSafe Plus 50%, S × 25-5, Fischer Scientific, Pittsburgh, PA) followed by count measurements using the Wallac WinSpectral liquid scintillation counter (Wallac Turku, Finland).

Cells were washed with PBS, harvested by trypsinization using 0.5% trypsin-0.2% EDTA, resuspended in 100 μl culture medium, and counted using the Guava Personal Cytometer (Guava Technologies, Inc., Burlingame, CA).

Expression of COX-2 and α-SMA were detected by Western blotting. Protein extracts (5 μg) from each sample were separated by gel electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Known molecular weight protein standards were run alongside the samples. Separated proteins were then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA), which was incubated for one hour at room temperature in blocking buffer (TBS and 0.1% Tween 20 with 5% nonfat dry milk). A murine COX-2 polyclonal antibody was diluted 1:2000 buffer (TBS and 0.1% Tween 20 with 5% nonfat dry milk). After incubation with the primary antibody overnight at 4°C, the membrane was exposed to the secondary antibody with gentle agitation for one hour at room temperature. Western Blots were visualized using Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology, Inc., CA). α-SMA expression was examined in a similar manner by using monoclonal anti-α-SMA antibodies. Bands from individual western blots were quantified densitometrically and the mean ± SEM for each time point or concentration calculated for presentation.

All experiments were repeated at least twice. Data are expressed as mean ± SEM. Statistical analysis was performed using ANOVA with the Prism software package (GraphPad, San Diego, CA).