Andrew Duffy

Sodium salicylate inhibits proliferation and induces G1 cell cycle arrest in human pancreatic cancer cell lines.

The mutations most common in pancreatic cancer decrease the ability to control Gl to S cell cycle progression and cellular proliferation. In colorectal cancer cells, nonsteroidal anti-inflammatory drugs inhibit proliferation and induce cell cycle arrest. We examined whether sodium salicylate, an aspirin metabolite, could inhibit proliferation in human pancreatic cancer cell lines (BxPC3 and Panc-1). Quiescent cells were treated with medium containing 10% fetal calf serum, with or without salicylate. Cellular proliferation was measured by MTT assay and bromodeoxyuridine incorporation. The fractions of cells in G0/G1, S, and G2/M phases of the cell cycle were quantitated by fluorescence-activated cell sorting. Results were compared between groups by two-tailed t test. Cydin Dl expression was determined by Western blot analysis and prostaglandin E2 expression by enzyme-linked immunosorbent assay. Serum-starved cells failed to proliferate, with most arrested in the Gl phase. Salicylate significantly inhibited serum-induced progression from Gl to S phase, cellular proliferation, and the expression of cyclin Dl. The concentrations at which 50% of serum-induced proliferation was inhibited were 1.2 mmol/L (Panc-1) and 1.7 mmol/L (BxPC3). The annproliferative effect of sodium salicylate was not explained by inhibition of prostaglandin E2 production. This study provides further evidence in a noncolorectal cancer model for the antineoplastic effects of nonsteroidal anti-inflammatory drugs.

Mitogen-activated protein kinases signal inhibition of apoptosis in lipopolysaccharide-stimulated neutrophils

BACKGROUND Neutrophil (PMN) apoptosis is critical to the resolution of infection and the limitation of inflammation. Bacterial endotoxin (lipopolysaccharide [LPS]) inhibits PMN apoptosis and activates the p38 mitogen-activated protein kinase (MAPK) signal cascade. The role of p38 and other MAPKs (ERK and SAPK/JNK) in regulating PMN apoptosis after LPS stimulation is unknown. We hypothesize that MAPK activation by LPS signals inhibition of PMN apoptosis. METHODS PMNs were isolated from the blood of healthy human volunteers and incubated with PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or 0.1% dimethyl sulfoxide (vehicle) for 1 hour before treatment with LPS (0, 10, or 1000 ng/mL). Neutrophil MAPK activation was determined by Western blot analysis for phosphorylated p38, ERK, and SAPK/JNK. Apoptosis was quantified by flow cytometry with use of propidium iodide and annexin V. RESULTS LPS inhibited PMN apoptosis and activated p38 and ERK in a dose- and time-dependent fashion. SAPK/JNK was not activated by LPS. Treatment of cells with ERK inhibitor before LPS stimulation abrogated LPS signaled inhibition of PMN apoptosis. Conversely, p38 inhibition with SB203580 augmented inhibition of apoptosis by LPS. CONCLUSIONS These data demonstrate opposing roles of MAPKs in mediating PMN apoptosis after LPS stimulation. We conclude that LPS signal transduction by ERK inhibits PMN apoptosis while activation of p38 promotes apoptosis.

Inhibited neutrophil apoptosis: proteasome dependent NF-kappaB translocation is required for TRAF-1 synthesis.

Neutrophil (PMN) apoptosis regulates local and systemic inflammation during sepsis. Tumor necrosis factor receptor-associated factors (TRAFs) have been implicated as mediators of apoptosis; however, the signaling pathways for their production in stimulated PMN are unclear. We hypothesize that NF-kappaB translocation is necessary for the induction of TRAF-1 in PMNs with prolonged survival. Neutrophils were isolated from the blood of healthy volunteers by Ficoll gradient centrifugation and red blood cell sedimentation. Neutrophil NF-kappaB was inhibited with a proteasome inhibitor, PSI-I. Cells were treated with PSI-I (30 microM) or vehicle (DMSO 0.2%) for 50 min then incubated over an 18-h time course with LPS (10 to 1000 ng/mL), tumor necrosis factor alpha (TNFalpha) (2 to 20 ng/mL) or control media. In vitro apoptosis was quantified by propidium iodide FACS analysis. Total cellular TRAF-1 was detected by Western blot analysis of cell lysates. Steady state TRAF-1 mRNA was detected by RPA. NF-kappaB activity was determined by Western blot analysis for nuclear p65. Means and standard errors were calculated; data were analyzed by ANOVA. Lipopolysaccharide (LPS) and TNFalpha increased PMN nuclear p65 and steady state TRAF-1 mRNA. Apoptosis was inhibited by TNFalpha and LPS at 12 and 18 h (P < 0.01). Incubation of cells in the NF-kappaB inhibitor PSI-I blocked LPS and TNFalpha-induced inhibition of apoptosis (P < 0.05) and the induction of both nuclear p65 and TRAF-1 mRNA. These data demonstrate that inhibition of PMN apoptosis and TRAF-1 induction by LPS and TNFalpha is NF-kappaB dependent.

Inhibition of Neutrophil Apoptosis after Severe Trauma Is NF???? Dependent

Background: Systemic inflammation may inhibit neutrophil (PMN) apoptosis and promote multiple organ dysfunction syndrome. We hypothesize that severe trauma causes dysregulation of PMN apoptosis. Methods: Neutrophils were isolated from trauma patients (24-72 hours after injury; n = 16) and controls (healthy volunteers) and incubated for 18 hours. In separate experiments, control cells were treated ± the nuclear factor kappa beta (NFκβ) inhibitor pyrrolidinithiocarbamate then incubated with 25% patient or control plasma. Apoptosis was quantified by enzyme-linked immunosorbent assay for histone-associated DNA and annexin V fluorescence-activated cell sorter. NFκβ activation was determined by Western blot for phosphorylated Iκβ. Results: Apoptosis was inhibited in trauma patient PMN. Neutrophil apoptosis correlated with multiple organ dysfunction syndrome score, Acute Physiology and Chronic Health Evaluation II, and platelet count. Patient plasma inhibited apoptosis and induced phosphorylation of Iκβ in control cells. Inhibition of PMN apoptosis by patient plasma was blocked by pretreatment with pyrrolidinithiocarbamate. Conclusion: NFκβ-dependent inhibition of neutrophil apoptosis occurs after trauma. Early inhibition of PMN apoptosis is dependent on the magnitude of injury.

Activated neutrophils induce nitric oxide production in Kupffer cells.

Neutrophils (PMN) are proposed to contribute to hepatic dysfunction during sepsis. Transmigrating PMN have been demonstrated to adhere to and injure parenchymal cells (hepatocytes); however, the effect of sepsis-activated PMN on hepatic macrophages or Kupffer cells (KC) is poorly characterized. We hypothesize that PMN influence KC inflammatory mediator production, including nitric oxide. Rodent KC were co-cultured with PMN obtained from controls (Norm-PMN) or endotoxemic rats [lipopolysaccharide (LPS)-PMN] for 18 h. After an 18-h incubation, supernatants and cell lysates of the KC were analyzed for nitric oxide (NO) production. Co-cultures with LPS-PMN/KC demonstrated significantly increased production of nitrite and up-regulation of inducible nitric oxide synthase (iNOS) protein compared to KC alone or Norm-PMN/KC co-cultures. Immunohistochemistry revealed preferential iNOS protein staining in the cytoplasm of KC cultured with LPS-PMN compared to controls. Nitrite production in co-cultures of KC and LPS-PMN where cell contact was inhibited by a cell impermeable but diffusable membrane was significantly reduced. These data provide evidence that KC can be stimulated directly by activated PMN for production of NO. Further, they suggest another mechanism by which PMN modulate hepatic function during sepsis.