R. Sharif

Heat Shock Protein 70 Increases Tumorigenicity and Inhibits Apoptosis in Pancreatic Adenocarcinoma

Pancreatic carcinoma is a malignant disease that responds poorly to chemotherapy because of its resistance to apoptosis. Heat shock proteins (Hsp) are not only cytoprotective but also interfere with the apoptotic cascade. Here, we investigated the role of Hsp70 in regulating apoptosis in pancreatic cancer cells. Hsp70 expression was increased in pancreatic cancer cells compared with normal pancreatic ductal cells. This was confirmed by increased mRNA levels for Hsp70 in human pancreatic cancer tissue compared with neighboring normal tissue from the same patient. Depletion of Hsp70 by quercetin decreased cell viability and induced apoptosis in cancer cells but not in normal pancreatic ductal cells. To show that this is a specific effect of Hsp70 on apoptosis, levels of Hsp70 were knocked down by short interfering RNA treatment, which also induced apoptosis in cancer cells as indicated by Annexin V staining and caspase activation. Daily administration of quercetin to nude mice decreased tumor size as well as Hsp70 levels in tumor tissue. These findings indicate that Hsp70 plays an important role in apoptosis and that selective Hsp70 knockdown can be used to induce apoptosis in pancreatic cancer cells.

Impact of Toll-like Receptor 4 on the Severity of Acute Pancreatitis and Pancreatitis-Associated Lung Injury in Mice

Background and Aims: Acute pancreatitis is an inflammatory disease involving acinar cell injury, and the rapid production and release of inflammatory cytokines, which play a dominant role in local pancreatic inflammation and systemic complications. Toll-like receptor 4 (TLR4) initiates a complex signalling pathway when it interacts with lipopolysaccharide (LPS), which ultimately results in a proinflammatory response. We hypothesised that TLR4 is important in the pathophysiology of acute pancreatitis, independently of LPS. Using two different models of acute pancreatitis, we investigated how genetic deletion of TLR4 or its co-receptor CD14 effects its progression and severity. Methods: We induced acute pancreatitis by administering either caerulein or l-arginine to wild-type, TLR4−/−, and CD14−/− mice. Control mice received normal saline injections. The severity of acute pancreatitis was determined by measuring serum amylase activity, quantifying myeloperoxidase (MPO) activity in the pancreatic tissue, and histologically assessing acinar cell injury. Results: It was found that administering caerulein and l-arginine to wild-type mice resulted in acute pancreatitis (as assessed by hyperamylasaemia, oedema, increased pancreatic MPO activity, and pancreatic necrosis) and associated lung injury. The same treatment to TLR4−/− or CD14−/− mice resulted in significantly less severe acute pancreatitis, and reduced lung injury. We found no evidence of either bacteria or LPS in the blood or in pancreatic tissue. Conclusions: The severity of acute pancreatitis is ameliorated in mice that lack either TLR4 or CD14 receptors. Furthermore, these results indicate that TLR4 plays a significant pro-inflammatory role independently of LPS in the progression of acute pancreatitis.

Protease-activated receptor-2 protects against pancreatitis by stimulating exocrine secretion

Background: Protease-activated receptor-2 (PAR-2) is present in the pancreas, where it has been shown to play a protective role during pancreatitis. However, the mechanism by which it protects against pancreatitis still remains to be elucidated. Acute pancreatitis is associated with premature zymogen activation and a blockage in digestive enzyme secretion. Aim: To examine the effects of PAR-2 activation on the severity of pancreatitis, and to determine whether its protective effects are mediated by affecting either premature activation or secretory blockage, or both. Results: The results confirmed that PAR-2 −/− mice have more severe pancreatitis than wild-type mice. Interestingly, intrapancreatic trypsin levels in the PAR-2 knockouts remained high after 6 h of pancreatitis, whereas they reverted to normal in the wild types. During pancreatitis, PAR-2 mRNA levels were upregulated in wild-type mice in response to supramaximal caerulein administration. Further, after a single injection of supramaximal caerulein, PAR-2 mRNA levels were also elevated, reaching a peak at 3 h. Stimulating PAR-2 with trypsin or the PAR-2-activating peptide, serine-leucine-isoleucine-glycine-arginine-leucine (SLIGRL), induced significantly more secretion from the acini of these caerulein-sensitised mice compared with the controls. PAR-2 activation also reversed the inhibition of secretion observed in both the caerulein and arginine models. Conclusions: Trypsin released during the early stages of pancreatitis activates PAR-2 receptors on the acinar cells and stimulates secretion from these cells. Thus, PAR-2 activation may decrease pancreatic injury and limit the severity of pancreatitis by allowing extracellular trypsin to act as a secretagogue.

An improved method for extracting myeloperoxidase and determining its activity in the pancreas and lungs during pancreatitis.

Objectives: This study was undertaken to examine the cause of variation in determined values of myeloperoxidase activity from sequestered neutrophils in pancreas and lungs during pancreatitis and to develop a reproducible method for the extraction and measurement of myeloperoxidase in these tissues. Methods: We measured myeloperoxidase in pancreatic and lung homogenates at different steps and evaluated the extent of inhibitory activity by measuring enzyme activity in the presence of homogenates from normal lungs and pancreata. To remove inhibitory activity from the homogenates, different methods like heat inactivation, inclusion of catalase inhibitor, and membranous pellet washing were evaluated. Results: Significant myeloperoxidase inhibitory activity was observed in pancreatic and lung homogenates, which could be effectively removed by the newly developed protocol. In extracts, myeloperoxidase activity can be determined by a spectrophotometric method, which is not only reproducible but is also adoptable for use in a plate reader or an autoanalyzer. Using this method, we studied the pattern of neutrophil sequestration over time in both pancreatic and lung tissue during caerulein-induced pancreatitis. Conclusions: Myeloperoxidase inhibition in the pancreas and lungs contributes to the variation observed in measurement of the enzyme.