Chronic inflammation has been implicated in the pathogenesis of many severe autoimmune disorders, as well as in diabetes, pulmonary diseases, and cancer. Inflammation accompanies most solid cancers including pancreatic ductal adenocarcinoma (PDAC), one of the most fatal cancers with surgery being the only curative therapeutic approach currently available. In the present work, we investigated the role of the major proinflammatory cytokine tumor necrosis factor A (TNFA) in the malignancy of PDAC cells in vitro and in vivo. In vitro, TNFA strongly increased invasiveness of Colo357, BxPc3, and PancTuI cells and showed only moderate antiproliferative effect. TNFA treatment of mice bearing orthotopically growing PDAC tumors led to dramatically enhanced tumor growth and metastasis. Notably, we found that PDAC cells themselves secrete TNFA. Although inhibition of TNFA with infliximab or etanercept only marginally affected proliferation and invasiveness of PDAC cells in vitro, both reagents exerted strong antitumoral effects in vivo. In severe combined immunodeficient mice with orthotopically growing Colo357, BxPc3, or PancTuI tumors, human-specific anti-TNF antibody infliximab reduced tumor growth and metastasis by about 30% and 50%, respectively. Importantly, in a PDAC resection model performed with PancTuI cells, we found an even stronger therapeutic effect for both anti-TNF compounds. Infliximab and etanercept reduced the number of liver metastases by 69% and 42%, respectively, as well as volumes of recurrent tumors by 73% and 51%. Thus, tumor cell-derived TNFA plays a profound role in malignancy of PDAC, and inhibition of TNFA represents a promising therapeutic option particularly in adjuvant therapy after subtotal pancreatectomy.

No abstract

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and agonistic anti-DR4/TRAIL-R1 and anti-DR5/TRAIL-R2 antibodies are currently under clinical investigation for treatment of different malignancies. TRAIL activates DR4 and DR5 and thereby triggers apoptotic and non-apoptotic signaling pathways, but possible different roles of DR4 or DR5 in these responses has poorly been addressed so far. In the present work, we analyzed cell viability, DISC formation as well as IL-8 and NF-kappaB activation side by side in responses to TRAIL and agonistic antibodies against DR4 (mapatumumab) and against DR5 (lexatumumab) in pancreatic ductal adenocarcinoma cells. We found that all three reagents are able to activate cell death and pro-inflammatory signaling. Death-inducing signaling complex (DISC) analysis revealed that mapatumumab and lexatumumab induce formation of homocomplexes of either DR4 or DR5, whereas TRAIL additionally stimulated the formation of heterocomplexes of both receptors. Notably, blocking of receptors using DR4- and DR5-specific Fab fragments indicated that TRAIL exerted its function predominantly via DR4. Interestingly, inhibition of PKC by Goe6983 enabled DR5 to trigger apoptotic signaling in response to TRAIL and also strongly enhanced lexatumumab-mediated cell death. Our results suggest the existence of mechanisms that silence DR5 for TRAIL- but not for agonistic-antibody treatment.

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.

SignificanceLocated at the apical (blood-facing) site of brain capillary endothelial cells that form the blood–brain barrier (BBB), the efflux transporter P-glycoprotein (Pgp) restricts the brain entry of various lipophilic xenobiotics, which contributes to BBB function. Pgp may become saturated if exposed to too-high drug concentrations. Here, we demonstrate a second-line defense mechanism in human brain capillary endothelial cells—that is, Pgp-mediated intracellular lysosomal drug trapping. Furthermore, we describe a mechanism of drug disposal at the BBB, which is shedding of lysosomal Pgp/substrate complexes at the apical membrane of human and porcine BBB endothelial cells and subsequent phagocytosis by neutrophils. Thus, we have discovered a fascinating mechanism of how Pgp might contribute to brain protection.

microRNAs (miRNAs), which contribute to the post-transcriptional processing through 3Ј-untranslated region-interference, have been shown to be involved in the regulation of ATPbinding cassette (ABC) membrane transporters. The aim of this study was to investigate whether ABCC2, an important efflux transporter for various endogenous and exogenous compounds at several compartment barriers, is subject to miRNAmediated post-transcriptional gene regulation. We screened the expression of 377 human miRNAs in HepG2 cells after 48 h of treatment with 5 M rifampicin [a pregnane X receptor (PXR) ligand] or vehicle using reverse transcription-polymerase chain reaction-based low-density arrays. Specific miRNA, ABCC2 mRNA, and protein expression were monitored in HepG2 cells undergoing rifampicin treatment for 72 h. Loss-and gain-offunction experiments and reporter gene assays were performed for further confirmation. Highly deregulated miRNAs compared with in silico data revealed miRNA (miR) 379 as candidate miRNA targeting ABCC2 mRNA. Under rifampicin treatment, ABCC2 mRNA increased significantly, with a maximal fold change of 1.56 Ϯ 0.43 after 24 h. In addition, miR-379 increased (maximally 4.10 Ϯ 1.33-fold after 48 h), whereas ABCC2 protein decreased with a maximal fold change of 0.47 Ϯ 0.08 after 72 h. In contrast, transfection of miR-379 inhibitor led to an elevation of ABCC2 protein expression after rifampicin incubation for 48 h. We identify a miRNA negatively regulating ABCC2 on the post-transcriptional level and provide evidence that this miRNA impedes overexpression of ABCC2 protein after a PXR-mediated external transcriptional stimulus in HepG2 cells.

No abstract

Intestinal homeostasis disturbance through intestinal barrier disruption presumably plays a key role in inflammatory bowel disease (IBD) development. Genetic and candidate gene analyses in an Il10-deficient IBD mouse model system identified Cd14 as a potentially protective candidate gene. The role of Cd14 in colitis development was determined using dextran sulfate sodium (DSS)-induced acute and an Il10-deficiency-induced chronic model of intestinal inflammation. Intestinal permeability was investigated by fluorescein isothiocyanate-dextran uptake assay, quantitative RT-PCR analysis of tight junction proteins, myosin light chain kinase, and proinflammatory cytokine expression. Immunohistological staining of occludin, Ki-67, NF-κB-p65, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was performed, and intestinal inflammation severity was evaluated histologically. Untreated B6-Cd14 mice and wild-type controls did not differ in intestinal barrier function. However, DSS-treated Cd14-deficient and B6-Il10Cd14 mice exhibited more severe intestinal barrier disruption, with increased histological scores and proinflammatory cytokine expression, compared to controls. Therefore, Cd14 deficiency did not influence epithelial integrity under steady-state conditions but caused intestinal barrier dysfunction under inflammation. As expected, CD14 overexpression increased barrier integrity. No difference in intestinal epithelial NF-κB translocation was observed between the investigated groups. Intestinal myosin light chain kinase expression decreased in Cd14-deficient mice under steady-state conditions and in the chronic model, whereas no difference was detected in the DSS models. Thus, CD14 plays a protective role in IBD development by enhancing intestinal barrier function.