Accumulating evidence suggests an important role for cyclooxygenase-2 (COX-2) in the pathogenesis of a wide range of malignancies. The protumorigenic properties of COX-2 are generally thought to be mediated by its product, PGE2, which is shown to promote tumor spread and growth by multiple mechanisms but most importantly through modulation of the local immune response in the tumor. Pancreatic tumor cells produce various amounts of PGE2, some of them being even deficient in COX enzymes or other PGE2 synthases. Here we describe that, beside pancreatic tumor cells or stromal fibroblasts, human peripheral blood mononuclear cells can also produce PGE2 upon coculture with pancreatic cancer cells. Stimulating of cellular cPLA2 within PBMCs by secreted factors, presumably sPLA2, from tumor cells appeared crucial, while the direct contact between PBMCs and PDACs seemed to be dispensable for this effect. Our data is emphasizing the complex interactions participating in the formation of the tolerogenic immune milieu within pancreatic tumors.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal gastrointestinal malignancies. It is the fourth most frequent cause of cancer-related deaths in North America, the sixth in Europe, and the fifth in the UK [
Beside surgery and chemotherapy, cyclooxygenases (COX)—the constitutive (COX-1) and the inducible (COX-2)—have been investigated as targets for treatment and prevention of pancreatic cancer since their expression seems to correlate with poor prognosis in PDAC patients [
Several works have focused on the production of PGE2 and the activity of enzymes involved in the earlier steps of prostaglandin production in pancreatic cancer [
In our work, we describe that the culture of human pancreatic cancer cells (or their conditioned media) with peripheral blood mononuclear cells induces the release of PGE2 irrespective of the COX status and PGE2 producing capacity of the PDAC cell line used in the assay.
Human pancreatic carcinoma cell lines were obtained from ATCC (Manassas, VA) and grown in RPMI 1640 (BxPC-3, T3M-4, AsPC-1) or DMEM (MiaPaCa-2) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/mL), and streptomycin (100
Buffy coats of randomly selected healthy donors were obtained from the blood bank of the Heidelberg University, and peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over Histopaque 1077 gradient (Sigma, Hamburg, Germany), at 400 g, no brake, for 45 min at room temperature. Cells were cultured in RPMI supplemented with 10% FCS and penicillin (100 U/mL)/streptomycin (100
The supernatants for either PDAC cells or PBMCs were obtained after centrifugation at 10000 rpm for 5 minutes before application onto the cells or use for PGE2 ELISA experiments.
The PLA2 inhibitor N-{(2S,4R)-4-(Biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5 ylidenemethyl)-phenyl]acrylamide (Pyrrolidin 1) was obtained from Merck Biosciences, Germany and used at a final concentration of 1
The PGE2 measurement in culture media was performed using a PGE2 ELISA kit obtained from Enzo Life Sciences GmbH (Lörrach, Germany) following manufacturer’s instructions. Experiments were repeated in triplicate with at least three different blood donors, and statistical significance was calculated using paired Student’s
In our study we used several human pancreatic cancer cell lines from primary (MiaPaCa-2, BxPC-3) and metastatic origin (AsPC-1, T3M-4) that have been previously characterized to have various expression of the enzymes involved in PGE2 production. BxPC-3 have been formerly reported to intrinsically produce PGE2, since they are endowed with several active elements of the PGE2 enzymatic cascade, namely, cPLA2, COX-1, and COX-2 [
We tested the expression of PGE2 from these cells and found that AsPC-1 and MiaPaCa-2 did not produce any PGE2 after 48 hours of cultivation (Figure
Release of PGE2 from pancreatic carcinoma cells and peripheral blood mononuclear cells. (a) PGE2 production from PDAC monocultures of cells. Cells (AsPC-1, MiaPaCa-2, T3M-4, BxPC-3) were plated in 48-well plates at a density of
In our next step we aimed at establishing whether PGE2 production may be induced by a factor secreted by tumor cells or whether it requires direct cell to cell contact. Supernatants from cultured pancreatic cancer cells were applied onto preseeded PBMCs. We selected to use conditioned media from AsPC-1 and MiaPaCa-2 cells since, as already determined above, these cells were unable to produce PGE2 on their own. As shown in Figure
Release of PGE2 from peripheral blood mononuclear cells treated with PDAC supernatants. Supernatants (SN) from AsPC-1 and MiaPaCa-2 cells, derived 24 hrs after plating of the cells, were treated (SN + cPLA2 Inh.) or not (SN) with Pyrolidin 1 and applied onto cultured PBMCs (
The first enzymatic activity required for prostaglandin production is phospholipase A2, which among other enzymes catalyzes the release of arachidonic acid from cell membrane lipids [
PGE2 production from PBMCs in presence of supernatants from PDAC cell lines and specific inhibitors.
Treatment | Inhibitors | ||||
No inh. | cPLA2 | sPLA2 | COX-1 | COX-2 | |
PBMC + AsPC-1 SN1 | |||||
PBMC + MiaPaCa-2 SN1 |
PGE2 production was measured in the coculture supernatants 48 hours posttreatment. Data are represented as percentages of PGE2 production, compared to that induced by PDAC supernatants on PBMCs (taken as 100%) plus. The names and concentrations of respective inhibitors are indicated in Materials and Methods. The experiment was repeated with four independent donors. Standard error was calculated using SigmaPlot 10.1 program, and the statistical significance (*) of differences was assessed by a two-tailed Student’s paired
1SN-supernatant.
Very recent study by Omura et al. [
Omura et al. [
The levels of PGE2 induced by supernatants of AsPC-1 and MiaPaCa-2 cells applied on the PBMCs were lower than the ones achieved in the corresponding cocultures, but one has to take into account that the concentrations of the probable secreted stimulators should be much higher in the immediate proximity of the cells than in the overlaying medium that we used for the stimulation.
In our work we describe a previously unknown, feature of pancreatic cancer stromal interaction, pointing that pancreatic cancer cells can induce PGE2 production from immune cells and further modulate host cell functions in their favor through a mechanism that may also be characteristic of other tumor entities.
Pancreatic ductal adenocarcinoma
Cyclooxygenase
Peripheral blood mononuclear cells
Pancreatic stellate cells
Phospholipase A2.