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Cystic fibrosis (CF) is a monogenic disease, characterized by massive chronic lung inflammation. The observed variability in clinical phenotypes in monozygotic CF twins is likely associated with the extent of inflammation. This study sought to investigate inflammation-related aberrant DNA methylation in CF twins and to determine to what extent acquired methylation changes may be associated with lung cancer.Blood-based genome-wide DNA methylation analysis was performed to compare the DNA methylomes of monozygotic twins, from the European CF Twin and Sibling Study with various degrees of disease severity. Putatively inflammation-related and differentially methylated positions were selected from a large lung cancer case-control study and investigated in blood by targeted bisulphite next-generation-sequencing. An inflammation-related locus located in the

For better lung cancer diagnosis and therapy, early detection markers of tumor dissemination are urgently needed, as most lung cancers do not show symptoms until extensive metastasis formation has already taken place. Our previous studies showed that in non-small cell lung cancer (NSCLC) early tumor dissemination is associated with a loss of chromosome 4q12-q32 and the presence of disseminated tumor cells (DTC) in the bone marrow. In order to identify the potential target gene in this region, a screen for methylation-dependent expression was performed. Lung cancer cell lines showing a loss of 4q as well as a normal bronchial epithelial cell line as control were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) followed by expression profiling. Seven genes within the 4q target region, which have been associated with a positive DTC status before were found to be regulated by hypermethylation. QRT-PCR in an independent sample set identified HERC5 as a potential target gene. Quantitative methylation analysis of these lung tissue samples revealed that HERC5 promoter hypermethylation was significantly associated with positive DTC status (p = 0.020) and occurrence of brain metastases (p = 0.015). In addition, hypermethylation of the HERC5 promoter in NSCLC was identified as a predictor for poor survival for Stage I adenocarcinoma patients (p = 0.022) and also for poor overall survival in metastatic lung cancer patients (p = 0.028). In conclusion, HERC5 may function as a prognostic marker and is associated with tumor dissemination in lung cancer.

Pancreatic ductal adenocarcinoma (PDAC) occurs mainly in people older than 50 years of age. Although great strides have been taken in treating PDAC over the past decades its incidence nearly equals its mortality rate and it was quoted as the 4th leading cause of cancer deaths in the U.S. in 2012. This review aims to focus on research models and scientific developments that help to explain the extraordinary resistance of PDAC towards current therapeutic regimens. Furthermore, it highlights the main features of drug resistance including mechanisms promoted by cancer cells or cancer stem cells (CSCs), as well as stromal cells, and the acellular components surrounding the tumor cells—known as peritumoral desmoplasia—that affects intra-tumoral drug delivery. Finally, therapeutic concepts and avenues for future research are suggested, based on the topics discussed.

Pancreatic cancer ranks among the tumors most resistant to chemotherapy. Such chemoresistance of tumors can be mediated by various cellular mechanisms including dysregulated apoptosis or ineffective drug concentration at the intracellular target sites. In this review, we highlight recent advances in experimental chemotherapy underlining the role of cellular transporters in drug resistance. Such contribution to the chemoresistant phenotype of tumor cells or tissues can be conferred both by uptake and export transporters, as demonstrated by in vivo and in vitro data. Our studies used human pancreatic carcinoma cells, cells stably transfected with human transporter cDNAs, or cells in which a specific transporter was knocked down by RNA interference. We have previously shown that 5-fluorouracil treatment affects the expression profile of relevant cellular transporters including multidrug resistance proteins (MRPs), and that MRP5 (ABCC5) influences chemoresistance of these tumor cells. Similarly, cell treatment with the nucleoside drug gemcitabine or a combination of chemotherapeutic drugs can variably influence the expression pattern and relative amount of uptake and export transporters in pancreatic carcinoma cells or select for pre-existing subpopulations. In addition, cytotoxicity studies with MRP5-overexpressing or MRP5-silenced cells demonstrate a contribution of MRP5 also to gemcitabine resistance. These data may lead to improved strategies of future chemotherapy regimens using gemcitabine and/or 5-fluorouracil.

Gemcitabine is widely used as first-line chemotherapeutic drug in the treatment of pancreatic cancer. Our previous experimental chemotherapy studies have shown that treatment of human pancreatic carcinoma cells with 5-fluorouracil (5-FU) alters the cellular transporter expression profile and that modulation of the expression of multidrug resistance protein 5 (MRP5; ABCC5) influences the chemoresistance of these tumor cells. Here, we studied the influence of acute and chronic gemcitabine treatment on the expression of relevant uptake and export transporters in pancreatic carcinoma cells by reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR, and immunoblot analyses. The specific role of MRP5 in cellular gemcitabine sensitivity was studied by cytotoxicity assays using MRP5-overexpressing and MRP5-silenced cells. Exposure to gemcitabine (12 nM for 3 days) did not alter the messenger RNA (mRNA) expression of MRP1, MRP3, MRP5, and equilibrative nucleoside transporter 1 (ENT1), whereas high dosages of the drug (20 µM for 1 hour) elicited up-regulation of these transporters in most cell lines studied. In cells with acquired gemcitabine resistance (up to 160 nM gemcitabine), the mRNA or protein expression of the gemcitabine transporters MRP5 and ENT1 was upregulated in several cell lines. Combined treatment with 5-FU and gemcitabine caused a 5- to 40-fold increase in MRP5 and ENT1 expressions. Cytotoxicity assays using either MRP5-overexpressing (HEK and PANC-1) or MRP5-silenced (PANC1/shMRP5) cells indicated that MRP5 contributes to gemcitabine resistance. Thus, our novel data not only on drug-induced alterations of transporter expression relevant for gemcitabine uptake and export but also on the link between gemcitabine sensitivity and MRP5 expression may lead to improved strategies of future chemotherapy regimens using gemcitabine in pancreatic carcinoma patients.

The multidrug resistance protein MRP2 (ABCC2) acts as an ATP-dependent conjugate export pump in apical membranes of polarized cells and confers multidrug resistance. Purified MRP2 is essential for the detailed functional characterization of this member of the family of ATP-binding cassette (ABC) transporter proteins. In human embryonic kidney cells (HEK293), we have permanently expressed MRP2 containing an additional C-terminal (His)6-tag. Immunoblot and immunofluorescence analyses detected the MRP2-(His)6 overexpressing clones. Isolated membrane vesicles from the MRP2-(His)6-expressing cells were active in ATP-dependent transport of the glutathione S-conjugate leukotriene C4 and were photoaffinity-labelled with 8-azido-[alpha-32P]ATP. MRP2-(His)6 was solubilized from membranes of MRP2-(His)6-cells and purified to homogeneity in a three-step procedure using immobilized metal affinity chromatography, desalting, and immunoaffinity chromatography. The identity of the pure MRP2-(His)6 was verified by MS analysis of tryptic peptides. The purified MRP2-(His)6 glycoprotein was reconstituted into proteoliposomes and showed functional activity as ATPase in a protein-dependent manner with a Km for ATP of 2.1 mM and a Vmax of 25 nmol ADP x mg MRP2-1 x min-1. This ATPase activity was substrate-stimulated by oxidized and reduced glutathione and by S-decyl-glutathione. Future studies using pure MRP2 reconstituted in proteoliposomes should allow further insight into the molecular parameters contributing to MRP2 transport function and to define its intracellular partners for transport and multidrug resistance.

Tumor cell proliferation and metastasis proceed via a network of interdependent molecular events with a vast array of molecular players and signal transduction mechanisms differing in various types of human tumors. In the sequence of events necessary for carcinogenesis, arachidonate metabolites have been documented to play a significant role at several steps. Arachidonate metabolism in human cells occurs via several enzymatic pathways, including enzymes such as cyclo-oxygenases and lipoxygenases. This review pays particular attention to one member of the lipoxygenase family of enzymes, namely 12-lipoxygenase, since an arachidonate metabolite generated via 12-lipoxygenase action, 12(S)-HETE, has been shown to elicit various prometastatic effects of tumor cells in vivo and in vitro. We focus especially on mechanisms of activation and modulation of 12-lipoxygenase expression in human tumor cells, since various tumor cells express 12-lipoxygenase or are responsive to metabolites derived from 12-lipoxygenase action, thus offering a potential for successful therapeutic intervention against such tumors

Metabolism of leukotrienes via omega-oxidation represents a major degradative and inactivating pathway of these biologically active icosanoids. Isonicotinic acid hydrazide (isoniazid) inhibited this process in rats in vivo, in the isolated perfused rat liver, and in hepatic microsomes. The in vivo catabolism of leukotriene E4 via N-acetyl-leukotriene E4 to its omega-oxidized metabolites was inhibited by 50% or 71% using single intravenous isoniazid doses of 0.6 mmol or 1.0 mmol/kg body mass, respectively. Isoniazid interfered with leukotriene catabolism at the initial omega-oxidation step, resulting in an accumulation of N-acetyl-leukotriene E4. Analogous although weaker inhibition of leukotriene omega-oxidation in vivo was observed by pretreatment with isonicotinic acid 2-isopropylhydrazide and monoacetyl hydrazine. In the isolated perfused liver, isoniazid at concentrations varying over 0.2-10 mM decreased the omega-oxidation of cysteinyl leukotrienes dose-dependently by up to 94%. omega-Oxidation of both leukotriene E4 and leukotriene B4 by rat liver microsomes was inhibited by isoniazid, isonicotinic acid 2-isopropylhydrazide, and monoacetyl hydrazine with half-maximal concentrations in the range of 5-15 mM. Our measurements indicate that the impairment of leukotriene omega-oxidation by isoniazid involves both cytochrome-P450-dependent enzyme systems responsible for omega-oxidation of leukotriene E4 and leukotriene B4. In effect, under isoniazid treatment one can expect a prolongation of the proinflammatory actions of endogenously produced leukotrienes.

Tumor cell proliferation and metastasis proceed via a network of interdependent molecular events with a vast array of molecular players and signal transduction mechanisms differing in various types of human tumors. In the sequence of events necessary for carcinogenesis, arachidonate metabolites have been documented to play a significant role at several steps. Arachidonate metabolism in human cells occurs via several enzymatic pathways, including enzymes such as cyclo-oxygenases and lipoxygenases. This review pays particular attention to one member of the lipoxygenase family of enzymes, namely 12-lipoxygenase, since an arachidonate metabolite generated via 12-lipoxygenase action, 12(S)-HETE, has been shown to elicit various prometastatic effects of tumor cells in vivo and in vitro. We focus especially on mechanisms of activation and modulation of 12-lipoxygenase expression in human tumor cells, since various tumor cells express 12-lipoxygenase or are responsive to metabolites derived from 12-lipoxygenase action, thus offering a potential for successful therapeutic intervention against such tumors.

Arachidonate metabolites are potent mediators generated in endotoxin shock. Following endotoxin administration (15 mgkg) into unanesthetized rats, we found a rapid biliary secretion of peptide leukotrienes. Analysis of bile for peptide leukotrienes included organic solvent extractions, reversed phase-HPLC, radioimmunoassay (RIA), and spectrophotometry. The major immunoreactive endogenous leukotriene (LT) from bile was eluted between LTC4 and LTD4 in three chromatographic systems. It corresponded thereby to a biliary metabolite of injected LTC4 and LTD4 which in turn showed the ultraviolet spectrum of a peptide leukotriene. This demonstration of endotoxin-induced generation of peptide LTs in vivo was possible by sequential HPLC and RIA analyses in bile into which peptide LTs are eliminated from blood.

A significant increase in the production of cysteinyl leukotrienes was observed after mechanical or thermal trauma in the anesthesized rat. The amount of biliary N-acetyl-leukotriene E4, which represents a suitable indicator for blood plasma leukotrienes, was used as a measure of leukotriene generation. Cysteinyl leukotrienes were rapidly eliminated from blood plasma into bile where N-acetyl-leukotriene E4 was the major metabolite. Leukotrienes were at a much lower concentration in blood plasma than in bile and differed in the pattern of metabolites. The detected amounts of leukotrienes were sufficient to induce known phenomena associated with trauma, such as tissue edema and circulatory and respiratory dysfunction. Increased leukotriene generation appears to play an important role in the pathophysiology of tissue trauma.

The cysteinyl leukotrienes LTC4, LTD4, and LTE4 play an important role within the network of mediators of endotoxin shock since they are capable of eliciting the generation of other lipid mediators of shock. Three lines of evidence support the conclusion that the cysteinyl leukotrienes are involved in the lethal shock-inducing action of endotoxin. (1) Endotoxin elicits the systemic production of cysteinyl leukotrienes in vivo. (2) Injection of these leukotrienes into sensitive species causes shock-like reactions, myocardial depression, and extravasation of plasma. (3) Cysteinyl leukotriene receptor antagonists and inhibitors of leukotriene synthesis prevent experimental endotoxin shock. Moreover, endotoxin induces a strong inhibition of the hepatobiliary elimination of cysteinyl leukotrienes, which normally represents the major pathway for deactivation of these mediators. This dual action of endotoxin, comprising enhanced synthesis and simultaneously impaired deactivation of potent mediators, points to a novel type of toxin action. The role of cysteinyl leukotrienes in endotoxin action does not exclude the involvement of additional shock-inducing factors released by endotoxin and their possible contribution to the formation of lipid mediators, including leukotrienes.