The Masked Polar Group Incorporation (MPGI) Strategy in Drug Design: Effects of Nitrogen Substitutions on Combretastatin and Isocombretastatin Tubulin Inhibitors.

Colchicine site ligands suffer from low aqueous solubility due to the highly hydrophobic nature of the binding site. A new strategy for increasing molecular polarity without exposing polar groups-termed masked polar group incorporation (MPGI)-was devised and applied to nitrogenated combretastatin analogues. Bulky ortho substituents to the pyridine nitrogen hinder it from the hydrophobic pocket while increasing molecular polarity. The resulting analogues show improved aqueous solubilities and highly potent antiproliferative activity against several cancer cell lines of different origin. The more potent compounds showed moderate tubulin polymerization inhibitory activity, arrested the cell cycle of treated cells at the G2/M phase, and subsequently caused apoptotic cell death represented by the cells gathered at the subG0/G1 population after 48 h of treatment. Annexin V/Propidium Iodide (PI) double-positive cells observed after 72 h confirmed the induction of apoptosis. Docking studies suggest binding at the colchicine site of tubulin in a similar way as combretastatin A4, with the polar groups masked by the vicinal substituents. These results validate the proposed strategy for the design of colchicine site ligands and open a new road to increasing the aqueous solubility of ligands binding in apolar environments.

Oxidized phospholipids exert a dual effect on bile acid-induced CCL2 expression in pancreatic acini.

BACKGROUND: oxidized phospholipids (oxPLs) generated in inflammatory diseases could play a key role by inducing pro- and anti-inflammatory effects. OBJETIVES: we investigated the effect of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and oxidized POPC (oxPOPC) in the inflammatory response triggered in pancreatic acini. METHODS: control acini were incubated in the absence or presence of either POPC or oxPOPC (≤100 µM). In additional experiments, oxPOPC effects were evaluated in sodium taurocholate (NaTc)-treated acini. CCL2 and TLR4 mRNA expression was analyzed by RT-qPCR. By western blot, JNK-MAPK, JAK and IκBα in cytoplasm as well as p65-NF-kB and p-STAT3 in the nucleus were evaluated. The involvement of TLR4, JNK-MAPK, JAK as well as NF-kB, STAT3 and PPARγ was assessed using pharmacological inhibition. RESULTS: no effect was found in response to POPC. Conversely, in response to oxPOPC (10 µM), JNK-MAPK and JAK acted as TLR4-downstream signals, leading to CCL2 upregulation mainly through NF-kB activation. Moreover, TLR4 non-dependent mechanisms induced STAT3 activation in oxPOPC-treated acini. Mediated by PPARγ, oxPOPC (50 µM) inhibited the CCL2 overexpression found in NaTc-treated acini. CONCLUSIONS: oxPOPC exerts pro- and anti-inflammatory effects in pancreatic acinar cells mediated by TLR4 and PPARγ signals, respectively. This dual action proved to be dependent on the concentration. The molecular mechanisms involved in the oxPL response could be useful for new therapeutic approaches to the treatment of oxPLs-related inflammatory pathologies.

Dexamethasone down-regulates the inflammatory mediators but fails to reduce the tissue injury in the lung of acute pancreatitis rat models.

Pulmonary complications are frequent in the course of acute pancreatitis. We investigate the effects of dexamethasone on lung injury in mild and severe AP. Mild and severe acute pancreatitis was induced in rats by bile-pancreatic duct obstruction and infusion of 3.5% sodium taurocholate into the bile-pancreatic duct, respectively. Dexamethasone (1 mg/kg) was given by intramuscular injection 1 h after acute pancreatitis. Plasma amylase activity was measured to evaluate the pancreas damage. Lungs were harvested for analysing mRNA expression of monocyte chemoattractant protein-1 (MCP-1), cytokine-induced neutrophil chemoattractant (CINC), P-selectin and intercellular adhesion molecule-1 (ICAM-1), myeloperoxidase (MPO) activity (as index of neutrophil infiltration) and histological examination. Dexamethasone reduced the hyperamylasemia and hindered the pulmonary upregulation of MCP-1, CINC, P-selectin and ICAM-1, in both mild and severe acute pancreatitis. Despite this, dexamethasone treatment failed to reduce MPO activity and histological alterations developed in lungs during acute pancreatitis, either in bile-pancreatic duct obstruction or sodium taurocholate model. We conclude that pulmonary local factors different from inflammatory mediators contribute to leukocyte recruitment, so that although dexamethasone down-regulated the lung expression of chemokines and adhesion molecules during acute pancreatitis it was not able to prevent leukocyte infiltration, which could be responsible for maintaining the lung injury in either mild or severe acute pancreatitis.

Evaluation of N-acetylcysteine treatment in acute pancreatitis-induced lung injury.

OBJECTIVE: Pulmonary complications are frequent during acute pancreatitis (AP). We investigate the effects of N-acetylcysteine (NAC) on lung injury in mild and severe AP. ANIMALS AND TREATMENT: Mild and severe AP was induced in rats by bile-pancreatic duct obstruction (BPDO) and infusion of 3.5 % sodium taurocholate (NaTc) into the bile-pancreatic duct, respectively. NAC (50 mg/kg) was given 1 h before and 1 h after AP. METHODS: Amylase activity was measured in plasma. Lungs were harvested for mRNA expression analysis of monocyte chemoattractant protein-1 (MCP-1), cytokine-induced neutrophil chemoattractant (CINC), P-selectin and intercellular adhesion molecule-1 (ICAM-1), myeloperoxidase (MPO) activity and histological examination. RESULTS: Hyperamylasemia was reduced by NAC in both AP models. NAC down-regulated MCP-1, CINC and P-selectin in BPDO- but not in NaTc-induced AP. Pulmonary insults did not vary in mild AP and were exacerbated in severe AP by NAC treatment. NAC reduced lung MPO activity in mild but not in severe AP. CONCLUSIONS: Although NAC treatment down-regulated inflammatory mediators in lungs during AP it did not prevent leukocyte infiltration, which could be responsible for maintaining the lung injury. As a result, NAC aggravated the lung damage in severe AP and failed to exert beneficial effects in the mild disease model.

Effects of dexamethasone on intercellular adhesion molecule 1 expression and inflammatory response in necrotizing acute pancreatitis in rats.

OBJECTIVES: Adhesion molecules are involved in the inflammatory response during acute pancreatitis (AP). We investigated the effect of dexamethasone (Dx) on intercellular adhesion molecule 1 (ICAM-1) expression during AP and its consequences on leukocyte recruitment and pancreatic damage. METHODS: Acute pancreatitis was induced in rats by 3.5% sodium taurocholate for 3 hours and 6 hours. Dexamethasone (1 mg/kg) was administered either 30 minutes before or 1 hour after inducing AP. Messenger RNA ICAM-1 expression in pancreas and lung, membrane-bound ICAM-1 in acinar cells, and ICAM-1 plasma levels were analyzed. Histological examination of the pancreas and neutrophil infiltration in pancreas and lung were also measured. RESULTS: Prophylactic and therapeutic administration of Dx down-regulated ICAM-1 expression in pancreas and lung from early AP. Dexamethasone given before AP reduced the pancreatic damage, but lung inflammation was not prevented. Therapeutic Dx treatment was ineffective in avoiding leukocyte recruitment into the pancreas and lung in rats with AP. High ICAM-1 concentration was found in plasma during AP, which was not reduced by Dx treatments. CONCLUSIONS: Dexamethasone down-regulates ICAM-1 expression, but it does not completely prevent leukocyte recruitment during sodium taurocholate-induced AP.

Effect of dexamethasone on peripheral blood leukocyte immune response in bile-pancreatic duct obstruction-induced acute pancreatitis.

Our aim was to analyze the effects of dexamethasone (Dx) (1mg/kg), prophylactically or therapeutically administered, on the inflammatory response triggered by peripheral blood leukocytes during acute pancreatitis (AP) induced in rats by bile-pancreatic duct obstruction (BPDO) and their consequences in the progress of the disease. Flow cytometry was used to analyze the distribution of the major leukocyte populations, the CD45 expression and the activated state of monocytes as reflected by the membrane-bound intercellular adhesion molecule-1 (ICAM-1) and the production of tumor necrosis factor-alpha (TNF-alpha) and monocyte chemoattract protein-1 (MCP-1) in response to lipopolysaccaride (LPS). Interleukin-6 (IL-6) plasma levels, pancreatic fluid content and histology of pancreas sections were also evaluated. Dx, given either before or after AP, blunted the monocyte increase induced by BPDO-induced AP, but did not change lymphocyte and neutrophil counts. Membrane-bound ICAM-1 expression did not vary in circulating monocytes during BPDO, either in Dx-treated or non-treated rats. Both Dx treatments inhibited TNF-alpha and MCP-1 production in non-stimulated and LPS-stimulated monocytes, whose response was found to be higher than in controls from early AP. Leukocyte CD45 expression was found to be reduced in rats with AP and shifted to control values in Dx-post-treated rats. Cytokinemia as well as pancreatic edema and leukocyte infiltration found in BPDO rats were reduced by Dx given either before or after AP. We conclude that prophylactic and therapeutic Dx treatments inhibited the inflammatory response triggered by circulating leukocytes in rats with BPDO-induced AP, thus contributing to reducing the severity of the disease.

N-acetylcysteine in acute pancreatitis.

Premature trypsinogen activation and production of oxygen free radicals (OFR) are early pathogenic events which occur within acinar cells and trigger acute pancreatitis (AP). OFR exert their harmful effects on various cell components causing lipid peroxidation, disturbances in calcium homeostasis and DNA damage, which lead to increased cell injury and eventually cell death. This review presents the most recent data concerning the effects of N-Acetylcysteine (NAC), in the treatment of AP. NAC is an antioxidant capable of restoring the levels of Glutathione, the most important cellular antioxidant. Studies show the beneficial effects of NAC treatment in preventing OFR production and therefore attenuating oxidative damage. Additionally, NAC treatment has been shown to prevent the increase in cytosolic Ca(2+) concentration and reduce the accumulation of enzymes in acinar cells during AP. The prevention, by NAC, of these pathological events occurring within acinar would contribute to reducing the severity of AP. NAC is also capable of reducing the activation of transcription factors especially sensitive to the cellular redox state, such as Nuclear factor-κB, signal transducer and activator of transcription-3 and mitogen-activated protein kinase. This leads to a down-regulation of cytokines, adhesion molecules and chemokine expression in various cell types during AP. These findings point to NAC as a powerful therapeutic treatment, attenuating oxidative-stress-induced cell injury and other pathological events at early stages of AP, and potentially contributing to reducion in the severity of disease.

Signal transduction of MCP-1 expression induced by pancreatitis-associated ascitic fluid in pancreatic acinar cells.

Pancreatitis-associated ascitic fluid (PAAF) is known to contribute to the progression of acute pancreatitis (AP). We have investigated the capability of PAAF to activate the expression of MCP-1 in pancreatic acinar cells and the involvement of MAPK, NF-kappaB and STAT3 as downstream signalling transduction pathways. The actions of dexamethasone (Dx) and N-acetylcysteine (NAC) on the PAAF's acinar effects have also been evaluated. Acinar cells were incubated for 1 hr with PAAF collected from rats with severe AP induced by sodium taurocholate in the absence or presence of Dx (10(-7) M) or NAC (30 mM). MCP-1 mRNA expression, phospho-p38-MAPK, IkappaB alpha, nuclear p65 levels and nuclear translocation of STAT3 were analysed. In response to PAAF, overexpression of MCP-1, phosphorylation of p38-MAPK, degradation of IkappaB alpha and increases in p65 nuclear levels and STAT3 activity were found in acinar cells. PAAF-mediated MCP-1 up-regulation was completely suppressed by Dx and NAC. MAPK activation was only inhibited by NAC, NF-kappaB activation was repressed by Dx and NAC, and STAT3 pathway was strongly blocked by Dx and significantly reduced by NAC. In conclusion, acinar cells were activated by PAAF to produce MCP-1, mainly via NF-kappaB and STAT3 pathways. Both downstream pathways were targeted by Dx and NAC to repress the PAAF-mediated acinar MCP-1 up-regulation.

Targeting peripheral immune response reduces the severity of necrotizing acute pancreatitis.

OBJECTIVE: A complex cascade of immunologic events leads to the development of systemic inflammatory response in acute pancreatitis (AP). Our aim was to evaluate the effects of two different immunomodulating treatments: Dexamethasone (Dx) and N-acetylcysteine (NAC), on the progression of necrotizing AP. DESIGN: Prospective, random, and control study. Laboratory animals. SETTING: University-based research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Retrograde infusion of 3.5% of sodium taurocholate into pancreatic-biliary duct was used to induce AP in rats. Dx (1 mg/kg) was administered 30 mins before or 1 hr after AP, and NAC (50 mg/kg) was given 1 hr before and 1 hr after inducing AP. MEASUREMENTS AND MAIN RESULTS: Dx and NAC treatments reduced the severity of AP in terms of amylasemia, pancreatic edema, and pancreatic and liver necrosis. Dx, administered before or after AP, and NAC reduced the leukocytosis induced by AP and blocked the ability of circulating monocytes to produce tumor necrosis factor-alpha and monocyte chemoattractant protein-1; however none of them significantly reduced the overexpression of intercellular cell adhesion molecule-1 found in monocytes 6 hrs after inducing AP. Leukocyte infiltration in the pancreas was attenuated in Dx-pretreated rats and significantly reduced 6 hrs after inducing AP in rats treated with NAC. However, neither Dx nor NAC were able to significantly reduce interleukin-6 in plasma or mitigate leukocyte infiltration in the lung. CONCLUSIONS: Our data demonstrated that treatments targeting the peripheral immune response reduced the severity of sodium taurocholate -induced AP attenuating pancreatic and liver injury, but they were not effective for limiting the spread of the inflammatory damage to the lung.

Extrapancreatic organ impairment during acute pancreatitis induced by bile-pancreatic duct obstruction. Effect of N-acetylcysteine.

Multiple organ failure is frequently associated with acute pancreatitis (AP). Our aim was to study pulmonary, hepatic and renal complications developed in the course of AP experimentally induced in rats by bile-pancreatic duct obstruction (BPDO), differentiating the complications caused by AP itself, from those directly caused by bile duct obstruction (BDO), after ligating the choledocus. N-acetylcysteine (NAC) was administered as a therapeutic approach. Myeloperoxidase activity revealed neutrophil infiltration in lungs from 12 h after BDO, even if AP was not triggered. Lactate dehydrogenase (LDH) activity indicated hepatocyte death from 48 h after BDO, and from 24 h following BPDO-induced AP onwards, an effect delayed until 48 h by NAC treatment. Rats with single cholestasis (BDO) and rats with BPDO-induced AP showed a significant increase in plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT) and bilirubin concentration from 12 h onwards, whose values were reduced by NAC treatment at early BPDO. No renal failure was found during 120 h of bile-pancreatic obstruction. Our results showed lung and liver impairment as a result of BDO, even if AP does not develop. Pancreatic damage and extrapancreatic complications during AP induced by BPDO were palliated by NAC treatment.

ICAM-1 and CD11b/CD18 expression during acute pancreatitis induced by bile-pancreatic duct obstruction: effect of N-acetylcysteine.

Different molecules are involved in the recruitment of leukocytes during inflammation. The aim was to investigate (i) the contribution of acinar cells to the overall production of ICAM-1 and (ii) the kinetics of leukocyte CD11b/CD18 expression during acute pancreatitis (AP) induced by bile-pancreatic duct obstruction (BPDO) to evaluate the contribution of both molecules to leukocyte homing. The role of reactive oxygen species (ROS) as mediators in the expression of ICAM-1 and CD11b/CD18 was examined by using N-acetylcysteine (NAC) as an antioxidant treatment. By mechanisms resistant to NAC treatment, acinar cells were able to produce ICAM-1 at first onset of AP; other cell sources contribute to maintaining increased ICAM-1 plasma levels during AP. By contrast, CD11b/CD18 was overexpressed in leukocytes in the course of AP by oxidant-dependent mechanisms. Since NAC treatment reduced neutrophil infiltration in the pancreas, we conclude that CD11b/CD18 over-expression is required for leukocyte recruitment; however, other adhesion molecules in addition to ICAM-1 seem to contribute to leukocyte homing during BPDO-induced AP.

CD45 expression on rat acinar cells: involvement in pro-inflammatory cytokine production.

CD45 transduces activation signals in inflammatory cells. We investigate CD45 expression on pancreatic acinar cells and examine its role in the inflammatory response which these cells have also shown under certain circumstances. Similar CD45 mRNA levels were found in acinar cells and leukocytes (positive control). Flow cytometric and immunohistochemical analysis showed a heterogeneous CD45 distribution on acinar cells. Activation of acinar cells by incubation with pancreatitis-associated ascitic fluid as evidencied by TNF-alpha production resulted in a decreased CD45 expression, suggesting that CD45 acts as a negative regulator of cytokine production. As a validation of this finding in vivo, a decrease in the acinar CD45 expression in parallel with an increased ability to produce TNF-alpha was found in rats with acute pancreatitis. Our data show that CD45 is constitutively expressed in acinar cells and suggest that it plays an important role in negatively regulating cytokine production.

Pro- and anti-inflammatory response of acinar cells during acute pancreatitis. Effect of N-acetyl cysteine.

We investigate the ability of acinar cells to produce tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) at different stages of acute pancreatitis (AP). Since oxidative stress is involved in the inflammatory response, the effect of N-acetyl cysteine (NAC) has also been evaluated. AP was induced in rats by bile-pancreatic duct obstruction (BPDO). NAC (50 mg/kg) was administered 1h before and 1h after BPDO. Acinar cells were incubated for 4 h at 37 degrees C in 5% CO2 atmosphere in absence and presence of 24-h BPDO-PAAF (20%, v/v) as stimulant agent. Acinar production of TNF-alpha and IL-10 was analysed by flow cytometry. Plasma amylase activity and histological studies of the pancreas indicated the severity of AP. PAAF significantly stimulated the acinar production of TNF-alpha and IL-10 in control rats. TNF-alpha production was also significantly stimulated in acinar cells of rats with AP, although a decrease in the pro-inflammatory response was found from 6 h after BPDO onwards. However, acinar cells failed to produce IL-10 from 3 h after BPDO. The protective effect of NAC treatment against oxidative cell damage reduced the pancreatic injury and maintained and enhanced the ability of acinar cells to produce IL-10 at early AP stages. As long as acinar cells were not severely damaged in the course of AP, greater ability to produce cytokines in response to PAAF was found in those with higher forward scatter (R2 cells). We suggest that the capability of acinar cells to maintain an appropriate balance between the production of pro- and anti-inflammatory mediators could contribute to determine the degree of severity of AP.

Kinetic study of TNF-alpha production and its regulatory mechanisms in acinar cells during acute pancreatitis induced by bile-pancreatic duct obstruction.

Cytokines play a critical role in acute pancreatitis (AP) but the contribution of different cell sources to cytokine production is unclear. Unfortunately, there are no data concerning the molecular mechanisms involved in the inflammatory response in humans during AP. For this reason, the aim of this study was to analyse the ability of acinar cells, in comparison with leukocytes, to produce TNF-alpha at different stages of AP induced in rats by bile-pancreatic duct obstruction (BPDO) and to investigate the time course of oxidant-sensitive mechanisms involved in cytokine production. The role of oxygen free radicals as messengers of the mechanisms underlying acinar cell TNF-alpha production was assessed in BPDO rats treated with N-acetylcysteine (NAC). While monocytes were not able to produce TNF-alpha until 12 h after inducing AP, acinar cells triggered TNF-alpha production from 6 h after BPDO, at which time the pancreas develops maximal oxidative stress. Phosphorylated p38-MAPK and activated NF-kappaB were detected in acinar cells from 6 h after BPDO. NAC treatment reduced pancreatic glutathione depletion during the early stages of AP and attenuated the activation of p38-MAPK and NF-kappaB for 48 h following BPDO. As a result, acinar cells in NAC-treated rats failed to produce TNF-alpha during AP. In addition, NAC delayed monocyte TNF-alpha production, thereby maintaining low TNF-alpha levels in plasma during BPDO. In conclusion, acinar cells contribute directly to the inflammatory response during BPDO-induced AP by producing TNF-alpha even before inflammatory cells in the peripheral blood. The blockade of oxidant-mediated signal transduction pathways induced by NAC treatment prevented acinar cell TNF-alpha production.

Biliary pancreatitis-associated ascitic fluid activates the production of tumor necrosis factor-alpha in acinar cells.

OBJECTIVE: Acute pancreatitis is associated with increased cytokine release from different cell sources. We have investigated the ability of acinar cells, in comparison with inflammatory peripheral blood cells, to produce tumor necrosis factor (TNF)-alpha in response to pancreatitis-associated ascitic fluid (PAAF). DESIGN: Controlled, randomized animal study. SETTING: University research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Flow cytometry using phycoerythrin-labeled monoclonal anti-TNF-alpha antiserum. MEASUREMENTS AND MAIN RESULTS: PAAF (20%, v:v) obtained from rats with acute pancreatitis induced by bile-pancreatic duct obstruction significantly increased TNF-alpha production in acinar cells, as measured by flow cytometry using phycoerythrin-labeled monoclonal anti-TNF-alpha antiserum. Neither heating of PAAF nor the addition of soybean trypsin inhibitor or neutralizing amounts of anti-TNF-alpha monoclonal antiserum reduced the acinar cell TNF-alpha production. Monocytes and lymphocytes did not produce TNF-alpha in response to PAAF. Likewise, the typical monocyte and lymphocyte stimulating factors-lipopolysaccharide (10 microg/microL) and phorbol 12-myristate 13-acetate (250 ng/mL) plus ionomycin (1 microg/mL), respectively-were not able to produce TNF-alpha in acinar cells. By comparison of the two acinar cell populations differentiated by flow cytometry, R2 cells (with higher forward scatter values) showed a greater ability to produce TNF-alpha in response to PAAF than R1 cells. Acinar cell nuclear factor-kappaB was activated, but TNF-alpha production was not totally inhibited in presence of N-acetyl cysteine (30, 100 mM). CONCLUSIONS: The production of TNF-alpha from different cell sources is selectively activated. PAAF may be involved in the pathophysiology of acute pancreatitis by TNF-alpha production in acinar cells through mechanisms partially mediated by nuclear factor-kappaB activation. PAAF components, such as TNF-alpha or trypsin, are not responsible for acinar cell activation. TNF-alpha was induced by heat-resistant PAAF factors, displaying acinar cells with higher forward scatter (R2) a greater ability to increase the TNF-alpha production than R1 cells.

Contribution of circulating leukocytes to cytokine production in pancreatic duct obstruction-induced acute pancreatitis in rats.

Little information is available regarding the role of circulating leukocytes in the pathogenesis of acute pancreatitis (AP). Our aim was to explore the time-course of the potential role of inflammatory peripheral blood (PB) cells during AP induced in rats by pancreatic duct obstruction (PDO). Flow cytometry immunophenotyping was used to analyse the distribution of the major circulating leukocyte subsets, the activation state of circulating monocytes as reflected by both CD11b expression and TNF-alpha production and the relative contribution of T-cell derived pro- (TNF-alpha) and anti- (IL-10) inflammatory mediators at different stages of PDO-induced AP. A progressive increase in PB neutrophils and monocytes was observed up to 6h after PDO whereas lymphocytes, as well as CD4(+) and CD8(+) T-cell subsets, rose as early as 1.5 h after PDO and decreased thereafter. Monocytes were activated in PB from 6 h after inducing AP as reflected by increases in both CD11b expression and spontaneous TNF-alpha production; nevertheless, they showed the capability of producing TNF-alpha at earlier AP stages by lipopolysaccharide (LPS) stimulation. In contrast, T-cells were unable to produce TNF-alpha during AP neither spontaneously nor after stimulation with PMA/Ionomycin. Therefore, only PB monocytes contribute to increase TNF-alpha levels in plasma as observed from 12 h onwards after inducing AP. Interleukin-10 was produced by T-cells 6 h after PDO only after PMA/Ionomycin stimulation. We conclude that systemic inflammatory events are triggered off at early stages of PDO-induced AP, with the activation of circulating monocytes, though not T-cells, playing a central role.