Inhibitory effects of prostaglandin E2 on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma

Abstract

Background: Several studies have described an increased cyclooxygenase-2 (COX-2) expression in pancreatic cancer, but the role of COX-2 in tumour development and progression is not clear. The aim of the present study was to examine expression of COX-2 in cancer cells and stromal cells in pancreatic cancer specimens, and to explore the role of PGE2 in pancreatic stellate cell proliferation and collagen synthesis.

Methods: Immunohistochemistry and immunofluorescence was performed on slides from whole sections of tissue blocks using antibodies against COX-2 and α-smooth muscle actin (αSMA). Pancreatic stellate cells (PSC) were isolated from surgically resected tumour tissue by the outgrowth method. Cells were used between passages 4 and 8. Collagen synthesis was determined by [(3)H]-proline incorporation, or by enzyme immunoassay measurement of collagen C-peptide. DNA synthesis was measured by incorporation of [(3)H]-thymidine in DNA. Cyclic AMP (cAMP) was determined by radioimmunoassay. Collagen 1A1 mRNA was determined by RT-qPCR.

Results: Immunohistochemistry staining showed COX-2 in pancreatic carcinoma cells, but not in stromal cells. All tumours showed positive staining for αSMA in the fibrotic stroma. Cultured PSC expressed COX-2, which could be further induced by interleukin-1β (IL-1β), epidermal growth factor (EGF), thrombin, and PGE2, but not by transforming growth factor-β1 (TGFβ). Indirect coculture with the adenocarcinoma cell line BxPC-3, but not HPAFII or Panc-1, induced COX-2 expression in PSC. Treatment of PSC with PGE2 strongly stimulated cAMP accumulation, mediated by EP2 receptors, and also stimulated phosphorylation of extracellular signal-regulated kinase (ERK). Treatment of PSC with PGE2 or forskolin suppressed both TGFβ-stimulated collagen synthesis and PDGF-stimulated DNA synthesis.

Conclusions: The present results show that COX-2 is mainly produced in carcinoma cells and suggest that the cancer cells are the main source of PGE2 in pancreatic tumours. PGE2 exerts a suppressive effect on proliferation and fibrogenesis in pancreatic stellate cells. These effects of PGE2 are mediated by the cAMP pathway and suggest a role of EP2 receptors.

Figure 1

COX-2 expression in formalin-fixed, paraffin-embedded tumour tissue from pancreatic cancer and isolated pancreatic stellate cells. A. Immunohistochemistry of COX-2 expression in tumour tissue from pancreatic cancer. COX-2 positive cells - red colour, fibrotic stroma αSMA positive - brown colour. B. Immunofluorescence of COX-2 expression in tumour tissue from pancreatic cancer. COX-2 positive cells - red colour, stroma αSMA positive - green colour. C. Immunofluorescence staining of cultured pancreatic stellate cells, passage five; COX-2 positive cells - red colour, αSMA positive cells - green colour, nucleus -blue colour. D. Expression of COX-2 and αSMA in different cell passage numbers. E. Expression of EP2 receptors, TGFβ receptors and PDGF receptors in two different cell passages F. Induction of COX-2 protein by EGF (10 nM), TGFβ (10 ng/ml), PGE₂ (10 uM), and PDGF (10 ng/ml) in two different cell passages. Cells in serum-free medium were stimulated with agonists for 24 hours before cells were harvested and lysates examined by Western blots as described in Methods. Results are from one representative experiment of four. G. Concentration dependent induction of COX-2 protein by IL-1β. Cells were stimulated in serum-free medium for 24 hours. Results are from one typical experiment of three.

Figure 2

Induction of COX-2 protein in pancreatic stellate cells by indirect coculture with pancreatic adenocarcinoma cell lines. A. Effect of coculture with Panc-1, HPAFII, and BxPC-3 cells. Cells were cocultured in serum-free medium for 48 hours, before harvesting and analysis by Western blots as described in Methods. Results are from one typical of three experiments. B. Inhibition of COX-2 induction by coculture with BxPC-3 cells when stellate cells were pretreated with IL-1 receptor antagonist (1 μg/ml) for 1 hour before coculture for 48 hours. Results are from one typical of four experiments.

Figure 3

PGE₂-stimulated cAMP accumulation in pancreatic stellate cells. A. Dose-dependent effect of PGE₂ in the absence and presence of 0.5 mM isobutylmethylxanthine (IBMX). Cells were cultured as described in Methods, and were stimulated for 5 minutes. Results are presented as mean + S.E.M. of three replicates from one representative of three experiment. B. Effect of EP4 receptor antagonist (L161982, 10 μM) and EP2 receptor antagonist (AH6809, 30 μM) on PGE₂-stimulated cAMP accumulation (1 μM PGE₂). Cells were preincubated with antagonists for 30 minutes before stimulation with PGE₂ for 15 minutes in the presence of 0.5 mM IBMX. Results are presented as mean ± S.E.M. of five experiments.

Figure 4

Effect of different agonists on DNA synthesis and phosphorylation of ERK and Akt in pancreatic stellate cells. A. Effect of PDGF (10 ng/ml), EGF (10 nM), and TGFβ (10 ng/ml) on DNA synthesis. Cells in serum-free medium were stimulated for 24 hours, with [³H] thymidine added at 18 hours. DNA synthesis was assessed as described in Methods. Results are presented as mean +/−SEM of six experiments. B. Effect of pretreatment with indomethacin (10 μM) for one hour before stimulation of cells with PDGF for 24 hours. Results are presented as mean +/−SEM of three experiments. C. Effect of PDGF (10 ng/ml), EGF (10 nM), and TGFβ (10 ng/ml) on phosphorylation of Akt and ERK. Cells in serum-free medium were stimulated for 5 minutes before harvesting and analysis of cell lysates on Western blots. Blots are from one typical of four experiments. Histograms represent mean +/−SEM of four experiments. D. Effect of thrombin (1 U/ml), PGE₂ (10 μM), fluprostenol (10 μM), and forskolin (10 μM) on ERK phosphorylation. Cells were stimulated for 5 minutes before harvesting. Blots from one typical of four experiments are shown. Histograms represent mean +/−SEM of four experiments. * Sign. different from control.

Figure 5

Effect of PGE₂ and forskolin on DNA synthesis and cAMP accumulation. A. Effect of increasing concentrations of PGE₂ on PDGF-stimulated DNA synthesis. Results are presented as mean ± S.E.M. of four experiments. B. Effect of 1 μM forskolin on PDGF- stimulated DNA synthesis. Results are presented as mean ± S.E.M. of four experiments. C. Time-dependent effect of 1 μM PGE₂ on cAMP accumulation in the absence of IBMX. Results are from one typical of four experiments and are presented as mean ± S.E.M. of triplicates. D. Time-dependent effect of 5 μM forskolin on cAMP accumulation in the absence of IBMX. Results are from one typical of four experiments and are presented as mean ± S.E.M. of triplicates. * Sign. different from control. ** Sign. different from PDGF alone.

Figure 6

Effects of different agonists on collagen synthesis. A. Effect of TGFβ (10 ng/ml), PDGF (10 ng/ml) and EGF (10 nM) on collagen synthesis. Cells were cultured and stimulated with agonists for 48 hours, as described in Methods. [³H] proline was present for the last 24 hours of stimulation. Collagen was precipitated and radioactivity in collagen was determined as described in Methods. Results are presented as mean ± S.E.M. of five experiments. B. Effect of pretreatment with indomethacin (10 μM) for one hour before stimulation of cells with TGFβ for 48 hours. Results are presented as mean ± S.E.M. of three experiments. * Significantly different from control.

Figure 7

Effect of PGE2 and forskolin on TGFβ-stimulated collagen synthesis. A, B. Effects of different concentrations of PGE₂ on TGFβ-stimulated collagen synthesis. Cells were stimulated for 48 hours. Results are presented as mean ± S.E.M. of six experiments. C. Effect of forskolin on TGFβ stimulated collagen synthesis. Cells were stimulated for 48 hours. Results are presented as mean ± S.E.M of five experiments. D. Effect of TGFβ (10 ng/ml) alone and in combination with 100 nM PGE₂ on collagen 1A1 mRNA expression. Cells were stimulated for 24 hours before RNA was isolated and real time quantitative RT-qPCR was performed. Results are presented as mean ± S.E.M. of eight experiments. E. Effect of TGFβ (10 ng/ml) alone and in combination with 1 μM PGE2 on collagen 1A1 mRNA expression. Cells were stimulated for 24 hours before RNA was isolated and real time quantitative RT-qPCR was performed. Results are presented as mean + S.E.M. of three experiments. * Sign. different from control ** Sign. different from TGFβ alone. F. Effect of the EP4 receptor antagonist ( L161982 1 μM) on PGE₂ influence on TGFβ-stimulated collagen synthesis. Cells were stimulated for 48 hours. Results are presented as mean ± S.E.M. of three experiments. *Sign. different from control ** Sign. different from TGFβ alone.