BackgroundEpidemiological studies have shown direct associations between type 2 diabetes and obesity, both conditions associated with hyperglycaemia and hyperinsulinemia, and the risk of pancreatic cancer. Up to 80% of pancreatic cancer patients present with either new-onset type 2 diabetes or impaired glucose tolerance at the time of diagnosis. Recent population studies indicate that the incidence of pancreatic cancer is reduced among diabetics taking metformin. In this study, the effects of exposure of pancreatic cancer cells to high glucose levels on their growth and response to metformin were investigated.MethodsThe human pancreatic cancer cell lines AsPC-1, BxPC-3, PANC-1 and MIAPaCa-2 were grown in normal (5 mM) or high (25 mM) glucose conditions, with or without metformin. The influence by metformin on proliferation, apoptosis and the AMPK and IGF-IR signalling pathways were evaluated in vitro.ResultsMetformin significantly reduced the proliferation of pancreatic cancer cells under normal glucose conditions. Hyperglycaemia however, protected against the metformin-induced growth inhibition. The anti-proliferative actions of metformin were associated with an activation of AMP-activated protein kinase AMPKThr172 together with an inhibition of the insulin/insulin-like growth factor-I (IGF-I) receptor activation and downstream signalling mediators IRS-1 and phosphorylated Akt. Furthermore, exposure to metformin during normal glucose conditions led to increased apoptosis as measured by poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, exposure to high glucose levels promoted a more robust IGF-I response and Akt activation which correlated to stimulated AMPKSer485 phosphorylation and impaired AMPKThr172 phosphorylation, resulting in reduced anti-proliferative and apoptotic effects by metformin.ConclusionOur results indicate that metformin has direct anti-tumour activities in pancreatic cancer cells involving AMPKThr172 activation and suppression of the insulin/IGF signalling pathways. However, hyperglycaemic conditions enhance the insulin/IGF-I responses resulting in an altered AMPK activation profile and prevent metformin from fully switching off the growth promoting signals in pancreatic cancer cells.

BackgroundDegraded extracellular matrix can stimulate the innate immune system via the Toll-Like Receptor-4 (TLR4). In the pancreas, syndecan-anchored heparan sulphate (HS) on the ductal epithelium can be cleaved off its protein cores by the proteases (trypsin and elastase) and potentially activate TLR4 signalling.MethodsTo investigate this signalling event, a low sulphated HS (500 μg/ml) was infused into the biliary-pancreatic duct of C57BL/6J wild-type mice. Phosphate buffered saline (PBS) and lipopolysaccharide (LPS) were used as negative and positive controls, respectively. Mice were sacrificed after 1, 3, 6, 9, and 48 hours and tissues were analysed for neutrophil and cytokine contents. In order to study the TLR4 signalling pathway of HS in the pancreas, genetically engineered mice lacking TLR4, Myeloid Differentiation primary response gene (88) (MyD88) or Interferon Regulatory Factor 3 (IRF3) were subjected to pancreatic infusion of HS.ResultsNeutrophil sequestration and corresponding myeloperoxidase (MPO) activity in the pancreas were increased 9 hours following HS challenge. In wild-type mice, the monocyte chemoattractant protein-1(MCP-1) increased at 3 hours after infusion, while RANTES increased after 9 hours.TLR4, MyD88, and IRF3 knockout mice showed an abrogated neutrophil recruitment and myeloperoxidase activity in the HS group, while the LPS response was only abolished in TLR4 and MyD88 knockouts.ConclusionsThe results of this study show that HS is capable of initiating a TLR4-dependent innate immune response in the pancreas which is distinctly different from that induced by LPS. This inflammatory response was mediated predominantly through IRF3- dependent pathway. Release of HS into the pancreatic duct may be one important mediator in the pancreatic ductal defence.

No abstract

BackgroundHeparan sulphate is known to have various functions in the animal body, including surveillance of tissue integrity. Administered intraperitoneally, it induces a systemic inflammatory response syndrome and when given locally in the pancreas it initiates a protective inflammatory response. The aim of the present study was to investigate the underlying mechanisms behind cell recruitment following intra-ductal infusion of heparan sulphate.MethodsRats were subjected to intraductal-infusion of heparan sulphate, lipopolysaccharide and phosphate buffered saline into the pancreas. Pancreatic tissue was harvested 1, 3, 6, 9 or 48 hours after infusion and stained immunohistochemically for myeloperoxidase, ED-1, CINC-1 and MCP-1, as well as using eosin hematoxylin staining. Furthermore, MPO activity and MCP-1 and CINC-1 concentrations of tissue homogenates were measured. All differences were analyzed statistically using the Mann-Whitney U-test.ResultsDuring HS infusion, a rapid influx of macrophages/monocytes, as visualized as ED-1 positive cells, was seen reaching a maximum at 6 hours. After 48 hours, the same levels of ED-1 positive cells were noted in the pancreatic tissue, but with different location and morphology. Increased neutrophil numbers of heparan sulphate treated animals compared to control could be detected only 9 hours after infusion. The number of neutrophils was lower than the number of ED-1 positive cells. On the contrary, LPS infusion caused increased neutrophil numbers to a larger extent than heparan sulphate. Furthermore, this accumulation of neutrophils preceded the infiltration of ED-1 positive cells. Chemokine expression correlates very well to the cell infiltrate. MCP-1 was evident in the ductal cells of both groups early on. MCP-1 preceded monocyte infiltration in both groups, while the CINC-1 increase was only noticeable in the LPS group.ConclusionsOur data suggest that heparan and LPS both induce host defense reactions, though by using different mechanisms of cell-recruitment. This implies that the etiology of pancreatic inflammation may influence how the subsequent events will develop.

Background: Two differently charged polypeptides, α-poly-L-lysine and poly-L-glutamate, have previously been shown to effectively reduce postoperative intraabdominal adhesions. Though α-poly-L-lysine showed toxicity in doses too close to the lowest therapeutic dose, the aim in the present study was to investigate the possible antiadhesive effect of another four cationic polypeptides. Materials/Methods: 125 mice were studied with a standardized and reproducible adhesion model and given epsilon poly-Llysine, lactoferrin, lysozyme and polyarginine respectively in a combination with poly-L-glutamate. Epsilon poly-L-lysine was also tested in different concentrations and as single treatment. Results: All four cationic polypeptides above showed a significantly better anti-adhesive effect than the controls receiving saline (p < 0.05). Epsilon poly-L-lysine had the best antiadhesive effect of the new substances tested in the experiment. Single treatment with the epsilon poly-Llysine showed toxic side effects. Discussion: We have shown that epsilon poly-L-lysine, polyarginine, lysozyme and lactoferrin, in descending order, all can reduce postoperative intraabdominal adhesions in mice when combined with poly-L-glutamate. There were side effects of epsilon poly-L-lysine resembling those of α-poly-L-lysine, although less toxic. The antiadhesive effect of epsilon poly-L-lysine did not reach the level of α-poly-L-lysine. Further studies will concentrate on additional investigation, trying to modify the α-poly-L-lysine to lower its toxicity. The less toxic epsilon poly-L-lysine also needs further attention in our research of antiadhesive bioactive polypeptides.

Epidemiological studies have shown direct associations between type 2 diabetes (T2D) and obesity, both conditions associated with hyperglycemia and hyperinsulinemia, and the risk of pancreatic cancer. Up to 80% of pancreatic cancer patients are diagnosed with either impaired glucose tolerance or new-onset T2D one or two years prior to their cancer diagnosis. Recent population studies indicate that the incidence of pancreatic cancer is reduced among diabetics taking metformin, an insulin-sensitizing and glucose lowering drug. We investigated the effects of exposure of pancreatic cancer cells to high glucose levels on their growth and response to metformin. We found that metformin significantly reduced the proliferation of pancreatic cancer cells under normal glucose conditions, but that hyperglycemia protected against the metformin-induced growth inhibition. The antiproliferative actions of metformin were associated with an activation of AMP-activated protein kinase AMPK(T172) together with a dose-dependent suppression of the insulin/insulin-like growth factor (IGF) signaling mediators IRS-1 and phosphorylated AKT. Furthermore, exposure to metformin during normal glucose led to increased apoptosis as measured by poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, exposure to high glucose promoted a more robust IGF-I response and AKT activation, that stimulated AMPK(S485) phosphorylation and suppressed AMPK(T172) phosphorylation resulting in reduced antiproliferative and apoptotic effects by metformin. In conclusion, our results indicate that metformin has direct antitumor activities in pancreatic cancer cells involving AMPK(T172) activation and modulation of the insulin/IGF signaling pathways. However, hyperglycemic conditions enhance the insulin/IGF-I responses resulting in an altered AMPK activation profile that prevent metformin from fully switching of the growth promoting signals in pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5156. doi:1538-7445.AM2012-5156

Purpose: The aim of this study was to evaluate the potential for combining the multikinase inhibitor sorafenib and the specific cyclo-oxygenase 2 (COX-2) inhibitor celecoxib as therapy in pancreatic adenocarcinoma cells and to test the hypothesis that a synergistic or additive effect on the Ras/MAPK/ERK and related pathways might be obtained. Experimental design: COX-2 positive (BxPC-1) and low/negative (MIAPaCa-2, PANC-1 and AsPC-1) human pancreatic adenocarcinoma cells were exposed to sorafenib and celecoxib combined treatment in vitro, after which cell viability and various growth promoting and survival signaling pathways were monitored by MTT, flow cytometry and Western blotting. Results: Combined treatment with sorafenib and celecoxib resulted in synergistic inhibition of pancreatic adenocarcinoma cell proliferation through COX-2 independent mechanisms. This regimen produced combination index (CI) values between 0.67 and 0.92 for the various cell lines, suggesting significant synergistic interactions between sorafenib and celecoxib, which also markedly inhibited the migratory capacity. The growth inhibition was associated with a reduction of basal ERK1/2 activity, accumulation of cells in the G0/G1 phase of the cell cycle, induction of apoptosis and poly(ADP-ribose) polymerase cleavage. These changes were accompanied by a significant reduction of p21WAF1/Cip1 levels, where celecoxib sensitized the cells to sorafenib-mediated p21WAF1/Cip1 suppression. Conclusion: p21WAF1/Cip1 levels are elevated frequently in human pancreatic adenocarcinoma and increases with disease progression. In this study we demonstrate that combined treatment with sorafenib and celecoxib synergistically induce growth inhibition and apoptosis through a process involving phospho-ERK1/2 and p21WAF1/Cip1 suppression. These data suggest the combined treatment of sorafenib and celecoxib as potential therapy for clinical testing in patients with pancreatic adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-226. doi:10.1158/1538-7445.AM2011-LB-226