Indomethacin induces apoptosis via a MRP1-dependent mechanism in doxorubicin-resistant small-cell lung cancer cells overexpressing MRP1

Small-cell lung cancers (SCLCs) initially respond to chemotherapy, but are often resistant at recurrence. The non-steroidal anti-inflammatory drug indomethacin is an inhibitor of multidrug resistance protein 1 (MRP1) function. The doxorubicin-resistant MRP1-overexpressing human SCLC cell line GLC4-Adr was highly sensitive for indomethacin compared with the parental doxorubicin-sensitive line GLC4. The purpose of this study was to analyse the relationship between hypersensitivity to indomethacin and MRP1 overexpression. The experimental design involved analysis of the effect of MRP1 downregulation on indomethacin-induced cell survival and apoptosis in GLC4-Adr and GLC4, using siRNA. In addition the effect of indomethacin on glutathione levels and mitochondrial membrane potential was investigated. Small interfering RNAs directed against MRP1 reduced MRP1 mRNA levels twofold and reduced efflux pump function of MRP1, which was reflected by a 1.8-fold higher accumulation of MRP1 substrate carboxyfluorescein, in si-MRP1 versus si-Luciferase-transfected GLC4-Adr cells. Multidrug resistance protein 1 downregulation decreased initial high apoptosis levels 2-fold in GLC4-Adr after indomethacin treatment for 24 h, and increased cell survival (IC50) from 22.8±2.6 to 30.4±5.1 μM following continuous indomethacin exposure. Multidrug resistance protein 1 downregulation had no effect on apoptosis in GLC4 or on glutathione levels in both lines. Although indomethacin (20 μM) for 2 h decreased glutathione levels by 31.5% in GLC4-Adr, complete depletion of cellular glutathione by L-buthionine (S,R)-sulphoximine only resulted in a small increase in indomethacin-induced apoptosis in GLC4-Adr, demonstrating that a reduced cellular glutathione level is not the primary cause of indomethacin-induced apoptosis. Indomethacin exposure decreased mitochondrial membrane potential in GLC4-Adr cells, suggesting activation of the mitochondrial apoptosis pathway. Indomethacin induces apoptosis in a doxorubicin-resistant SCLC cell line through an MRP1-dependent mechanism. This may have implications for the treatment of patients with MRP1-overexpressing tumours

The Interactions Between Rapeseed Lipoxygenase and Native Polyphenolic Compounds in a Model System

The focus of the present research was to study inhibition of lipoxygenase activity by rapeseed native polyphenols and the interactions between those compounds and the enzyme. The enzyme and polyphenolic compounds (polyphenols, phenolic acids) were extracted from rapeseed (Brassica napus) varieties Aviso and PR45DO3. The total phenolic compounds concentration in tested rapeseed was 1,485–1,691 mg/100 g d.m. (dry matter) and the free phenolic acids content in both rapeseed varieties was about 76 μg/100 g d.m. The isolated proteins showed lipoxygenase activity. Prooxidant properties of phenolic compounds in the presence of lipoxygenase and linoleic acid were observed rather in the case of extracts containing a relatively high concentration of miscellaneous polyphenols. Antioxidant properties were recorded in the case of phenolic acid extracts which contain only 1.4–1.9% of phenolics present in raw phenolic extracts. We propose that the prooxidant effect of phenolic compounds comes from quinone and oxidized polyphenols formation. The observed antioxidant activity of phenolic acid extracts is probably due to their ability to scavenge free radicals formed from linoleic acid. However, reduction of lipoxygenase ferric to ferrous ions, which prevent the activation of the enzyme and inhibited its activity, was also observed

Genotoxicity of Nicotiana tabacum leaves on Helix aspersa

Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant

PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity

The EphB4 receptor tyrosine kinase together with its preferred ligand, ephrin-B2, regulates a variety of physiological and pathological processes, including tumor progression, pathological forms of angiogenesis, cardiomyocyte differentiation and bone remodeling. We previously reported the identification of TNYL-RAW, a 15 amino acid-long peptide that binds to the ephrin-binding pocked of EphB4 with low nanomolar affinity and inhibits ephrin-B2 binding. Although ephrin-B2 interacts promiscuously with all the EphB receptors, the TNYL-RAW peptide is remarkably selective and only binds to EphB4. Therefore, this peptide is a useful tool for studying the biological functions of EphB4 and for imaging EphB4-expressing tumors. Furthermore, TNYL-RAW could be useful for treating pathologies involving EphB4-ephrin-B2 interaction. However, the peptide has a very short half-life in cell culture and in the mouse blood circulation due to proteolytic degradation and clearance by the kidneys and reticuloendothelial system. To overcome these limitations, we have modified TNYL-RAW by fusion with the Fc portion of human IgG1, complexation with streptavidin or covalent coupling to a 40 KDa branched polyethylene glycol (PEG) polymer. These modified forms of TNYL-RAW all have greatly increased stability in cell culture, while retaining high binding affinity for EphB4. Furthermore, PEGylation most effectively increases peptide half-life in vivo. Consistent with increased stability, submicromolar concentrations of PEGylated TNYL-RAW effectively impair EphB4 activation by ephrin-B2 in cultured B16 melanoma cells as well as capillary-like tube formation and capillary sprouting in co-cultures of endothelial and epicardial mesothelial cells. Therefore, PEGylated TNYL-RAW may be useful for inhibiting pathological forms of angiogenesis through a novel mechanism involving disruption of EphB4-ephrin-B2 interactions between endothelial cells and supporting perivascular mesenchymal cells. Furthermore, the PEGylated peptide is suitable for other cell culture and in vivo applications requiring prolonged EphB4 receptor targeting

The SHiP experiment at the proposed CERN SPS Beam Dump Facility

The Search for Hidden Particles (SHiP) Collaboration has proposed a general-purpose experimental facility operating in beam-dump mode at the CERN SPS accelerator to search for light, feebly interacting particles. In the baseline configuration, the SHiP experiment incorporates two complementary detectors. The upstream detector is designed for recoil signatures of light dark matter (LDM) scattering and for neutrino physics, in particular with tau neutrinos. It consists of a spectrometer magnet housing a layered detector system with high-density LDM/neutrino target plates, emulsion-film technology and electronic high-precision tracking. The total detector target mass amounts to about eight tonnes. The downstream detector system aims at measuring visible decays of feebly interacting particles to both fully reconstructed final states and to partially reconstructed final states with neutrinos, in a nearly background-free environment. The detector consists of a 50 m long decay volume under vacuum followed by a spectrometer and particle identification system with a rectangular acceptance of 5 m in width and 10 m in height. Using the high-intensity beam of 400 GeV protons, the experiment aims at profiting from the 4 x 10(19) protons per year that are currently unexploited at the SPS, over a period of 5-10 years. This allows probing dark photons, dark scalars and pseudo-scalars, and heavy neutral leptons with GeV-scale masses in the direct searches at sensitivities that largely exceed those of existing and projected experiments. The sensitivity to light dark matter through scattering reaches well below the dark matter relic density limits in the range from a few MeV/c(2) up to 100 MeV-scale masses, and it will be possible to study tau neutrino interactions with unprecedented statistics. This paper describes the SHiP experiment baseline setup and the detector systems, together with performance results from prototypes in test beams, as it was prepared for the 2020 Update of the European Strategy for Particle Physics. The expected detector performance from simulation is summarised at the end