Loss of TRAIL-Receptors Is a Recurrent Feature in Pancreatic Cancer and Determines the Prognosis of Patients with No Nodal Metastasis after Surgery

Introduction

Agonistic antibodies targeting TRAIL-receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are being developed as a novel therapeutic approach in cancer therapy including pancreatic cancer. However, the cellular distribution of these receptors in primary pancreatic cancer samples has not been sufficiently investigated and no study has yet addressed the issue of their prognostic significance in this tumor entity.

Aims and Methods

Applying tissue microarray (TMA) analysis, we performed an immunohistochemical assessment of TRAIL-receptors in surgical samples from 84 consecutive patients affected by pancreatic adenocarcinoma and in 26 additional selected specimens from patients with no lymph nodes metastasis at the time of surgery. The prognostic significance of membrane staining and staining intensity for TRAIL-receptors was evaluated.

Results

The fraction of pancreatic cancer samples with positive membrane staining for TRAIL-R1 and TRAIL-R2 was lower than that of cells from surrounding non-tumor tissues (TRAIL-R1: p<0.001, TRAIL-R2: p = 0.006). In addition, subgroup analyses showed that loss of membrane staining for TRAIL-R2 was associated with poorer prognosis in patients without nodal metastases (multivariate Cox regression analysis, Hazard Ratio: 0.44 [95% confidence interval: 0.22−0.87]; p = 0.019). In contrast, analysis of decoy receptors TRAIL-R3 and -R4 in tumor samples showed an exclusively cytoplasmatic staining pattern and no prognostic relevance.

Conclusion

This is a first report on the prognostic significance of TRAIL-receptors expression in pancreatic cancer showing that TRAIL-R2 might represent a prognostic marker for patients with early stage disease. In addition, our data suggest that loss of membrane-bound TRAIL-receptors could represent a molecular mechanism for therapeutic failure upon administration of TRAIL-receptors-targeting antibodies in pancreatic cancer. This hypothesis should be evaluated in future clinical trials.     

Alpha1-antitrypsin, old dog, new tricks. Alpha1-antitrypsin exerts in vitro anti-inflammatory activity in human monocytes by elevating cAMP

Regulation of serine protease activity is considered to be the sole mechanism for the function of alpha1-antitrypsin (AAT). However, recent reports of the anti-inflammatory effects of AAT are hard to reconcile with this classical mechanism. We discovered that two key activities of AAT in vitro, namely inhibition of endotoxin-stimulated tumor necrosis factor-alpha and enhancement of interleukin-10 in human monocytes, are mediated by an elevation of cAMP and activation of cAMP-dependent protein kinase A. As expected with this type of mechanism, the AAT-mediated rise in cAMP and the impact on endotoxin-stimulated tumor necrosis factor-alpha and interleukin-10 was enhanced when the catabolism of cAMP was blocked by the phosphodiesterase inhibitor rolipram. These effects were still observed with modified forms of AAT lacking protease inhibitor activity.     

Immunomodulatory effect of selenosemicarbazides and selenium inorganic compounds, distribution in organs after selenium supplementation

Antioxidant properties of selenium producing a protective barrier against free radicals play an important role in numerous metabolic and immunologic processes associated with oxidation- reduction reactions which take place during intracellular digestion of phagocyted bacteria. The aim of our study was to examine the properties of an organic compound of selenium, 4-(o-tolilo)- selenosemicarbazide of p-chlorobenzoic acid in terms of its retention in organs, effect on erythropoesis and phagocytic abilities of neutrophiles as well as antioxidant properties in neutrophiles tested with NBT test. This compound as well as inorganic sodium selenate was given to Swiss mice at the dose of 10^–3 g Se/kg for the period of 10 days. The concentrations of selenium in livers of mice treated with sodium selenate and selenosemicarbazide were found to be higher than in controls (18,7 g lg^–1 and 23.2 g lg^–1 vs. 12 g lg^–1, respectively). Analysis of blood cells count has shown a significant decrease in neutrophile levels in both groups treated with selenium. The influence of selenium compounds on phagocytosis and especially NBT test has been determined (3.8% of positive cells in the controls vs. 2.2% and 0.9% in the groups treated with sodium selenate and selenosemicarbazide, respectively). Our preliminary investigations suggest that selenosemicarbazides are biologically active compounds and can modify neutrophile functions.     

Multiple uracil-DNA glycosylase activities in Deinococcus radiodurans

The extremely radiation resistant bacterium, Deinococcus radiodurans, contains a spectrum of genes that encode for multiple activities that repair DNA damage. We have cloned and expressed the product of three predicted uracil-DNA glycosylases to determine their biochemical function. DR0689 is a homologue of the Escherichia coli uracil-DNA glycosylase, the product of the ung gene; this activity is able to remove uracil from a U : G and U : A base pair in double-stranded DNA and uracil from single-stranded DNA and is inhibited by the Ugi peptide. DR1751 is a member of the class 4 family of uracil-DNA glycosylases such as those found in the thermophiles Thermotoga maritima and Archaeoglobus fulgidus. DR1751 is also able to remove uracil from a U : G and U : A base pair; however, it is considerably more active on single-stranded DNA. Unlike its thermophilic relatives, the enzyme is not heat stable. Another putative enzyme, DR0022, did not demonstrate any appreciable uracil-DNA glycosylase activity. DR0689 appears to be the major activity in the organism based on inhibition studies with D. radiodurans crude cell extracts utilizing the Ugi peptide. The implications for D. radiodurans having multiple uracil-DNA glycosylase activities and other possible roles for these enzymes are discussed.     

Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, 1323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.     

alpha1-antitrypsin and its C-terminal fragment attenuate effects of degranulated neutrophil-conditioned medium on lung cancer HCC cells, in vitro

BACKGROUND: Tumor microenvironment, which is largely affected by inflammatory cells, is a crucial participant in the neoplastic process through promotion of cell proliferation, survival and migration. We measured the effects of polymorphonuclear neutrophil (PMN) conditioned medium alone, and supplemented with serine proteinase inhibitor alpha-1 antitrypsin (AAT) or its C-terminal fragment (C-36 peptide), on cultured lung cancer cells. METHODS: Lung cancer HCC cells were grown in a regular medium or in a PMN-conditioned medium in the presence or absence of AAT (0.5 mg/ml) or its C-36 peptide (0.06 mg/ml) for 24 h. Cell proliferation, invasiveness and release of IL-8 and VEGF were analyzed by [3H]-thymidine incorporation, Matrigel invasion and ELISA methods, respectively. RESULTS: Cells exposed to PMN-conditioned medium show decreased proliferation and IL-8 release by 3.9-fold, p < 0.001 and 1.3-fold, p < 0.05, respectively, and increased invasiveness by 2-fold (p < 0.001) compared to non-treated controls. In the presence of AAT, PMN-conditioned medium loses its effects on cell proliferation, invasiveness and IL-8 release, whereas VEGF is up-regulated by 3.7-fold (p < 0.001) compared to controls. Similarly, C-36 peptide abolishes the effects of PMN-conditioned medium on cell invasiveness, but does not alter its effects on cell proliferation, IL-8 and VEGF release. Direct HCC cell exposure to AAT enhances VEGF, but inhibits IL-8 release by 1.7-fold (p < 0.001) and 1.4-fold (p < 0.01) respectively, and reduces proliferation 2.5-fold (p < 0.01). In contrast, C-36 peptide alone did not affect these parameters, but inhibited cell invasiveness by 51.4% (p < 0.001), when compared with non-treated controls. CONCLUSIONS: Our data provide evidence that neutrophil derived factors decrease lung cancer HCC cell proliferation and IL-8 release, but increase cell invasiveness. These effects were found to be modulated by exogenously present serine proteinase inhibitor, AAT, and its C-terminal fragment, which points to a complexity of the relationships between tumor cell biological activities and local microenvironment.     

Rsp1p, a J domain protein required for disassembly and assembly of microtubule organizing centers during the fission yeast cell cycle

Regulation of microtubule organizing centers (MTOCs) orchestrates the reorganization of the microtubule (MT) cytoskeleton. In the fission yeast Schizosaccharomyces pombe, an equatorial MTOC (eMTOC) at the cell division site disassembles after cytokinesis, and multiple interphase MTOCs (iMTOCs) appear on the nucleus. Here, we show that, upon eMTOC disassembly, small satellites carrying MTOC components such as the gamma-tubulin complex travel in both directions along interphase MTs. We identify rsp1p, an MTOC protein required for eMTOC disassembly. In rsp1 loss-of-function mutants, the eMTOC persists and organizes an abnormal microtubule aster, while iMTOCs and satellites are greatly reduced. Conversely, rsp1p overexpression inhibits eMTOC formation. Rsp1p is a J domain protein that interacts with an hsp70. Thus, our findings suggest a model in which rsp1p is part of a chaperone-based mechanism that disassembles the eMTOC into satellites, contributing to the dynamic redistribution of MTOC components for organization of interphase microtubules.