Oliver Bruhn

Down-Regulation of ATP-Binding Cassette C2 Protein Expression in HepG2 Cells after Rifampicin Treatment Is Mediated by MicroRNA-379

microRNAs (miRNAs), which contribute to the post-transcriptional processing through 3′-untranslated region-interference, have been shown to be involved in the regulation of ATP-binding 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 miRNA-mediated post-transcriptional gene regulation. We screened the expression of 377 human miRNAs in HepG2 cells after 48 h of treatment with 5 μM 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-of-function 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 ± 0.43 after 24 h. In addition, miR-379 increased (maximally 4.10 ± 1.33-fold after 48 h), whereas ABCC2 protein decreased with a maximal fold change of 0.47 ± 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.

MicroRNA profiling in K-562 cells under imatinib treatment: influence of miR-212 and miR-328 on ABCG2 expression.

Background Despite the enormous success of imatinib in chronic myeloid leukemia (CML), therapy resistance has emerged in a significant proportion of patients, partly because of the overexpression of ABC efflux transporters. Methods Using an array comprising 667 miRNAs, we investigated whether the expression of microRNAs (miRNAs) is altered in CML K-562 cells becoming resistant to increasing concentrations of imatinib. ABCB1 and ABCG2 mRNA (quantitative real-time PCR) and protein expression (western blot) were quantified under short-term and 4 months’ imatinib treatment. Interaction of miR-212 and miR-328 with ABCG2 was investigated by transfection experiments and reporter gene assays using respective miRNA precursors or miRNA inhibitors. Results Although ABCB1 protein was not expressed, ABCG2 protein was 7.2-fold elevated after long-term treatment with 0.3 µmol/l imatinib and decreased gradually at higher concentrations. miRNAs miR-212 and miR-328 were identified to correlate inversely with ABCG2 expression under these conditions. Short-term treatment also induced ABCG2 protein concentration dependently and caused a downregulation of miR-212, but not of miR-328 at all tested concentrations (P=0.050). Reporter gene assays confirmed miR-212 to target the 3′-UTR region of ABCG2. In contrast, transfection of anti-miR-212 revealed an upregulation of ABCG2 protein expression, whereas the effect of anti-miR-328 was weak. Conclusion Our study suggests an association of imatinib treatment, miRNA downregulation and ABCG2 overexpression, possibly contributing to the mechanisms involved in imatinib distribution and response in CML therapy.

Polymorphisms of the drug transporters ABCB1, ABCG2, ABCC2 and ABCC3 and their impact on drug bioavailability and clinical relevance

Introduction: Human ATP-binding cassette (ABC) transporters act as translocators of numerous substrates across extracellular and intracellular membranes, thereby contributing to bioavailability and consequently therapy response. Genetic polymorphisms are considered as critical determinants of expression level or activity and subsequently response to selected drugs. Areas covered: Here the influence of polymorphisms of the prominent ABC transporters P-glycoprotein (MDR1, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and the multidrug resistance-associated protein (MRP) 2 (ABCC2) as well as MRP3 (ABCC3) on the pharmacokinetic of drugs and associated consequences on therapy response and clinical outcome is discussed. Expert opinion: ABC transporter genetic variants were assumed to affect interindividual differences in pharmacokinetics and subsequently clinical response. However, decades of medical research have not yielded in distinct and unconfined reproducible outcomes. Despite some unique results, the majority were inconsistent and dependent on the analyzed cohort or study design. Therefore, variability of bioavailability and drug response may be attributed only by a small amount to polymorphisms in transporter genes, whereas transcriptional regulation or post-transcriptional modification seems to be more critical. In our opinion, currently identified genetic variants of ABC efflux transporters can give some hints on the role of transporters at interfaces but are less suitable as biomarkers to predict therapeutic outcome.

Antimicrobial peptides and proteins of the horse - insights into a well-armed organism

Antimicrobial peptides play a pivotal role as key effectors of the innate immune system in plants and animals and act as endogenous antibiotics. The molecules exhibit an antimicrobial activity against bacteria, viruses, and eukaryotic pathogens with different specificities and potencies depending on the structure and amino-acid composition of the peptides. Several antimicrobial peptides were comprehensively investigated in the last three decades and some molecules with remarkable antimicrobial properties have reached the third phase of clinical studies. Next to the peptides themselves, numerous organisms were examined and analyzed regarding their repertoire of antimicrobial peptides revealing a huge number of candidates with potencies and properties for future medical applications. One of these organisms is the horse, which possesses numerous peptides that are interesting candidates for therapeutical applications in veterinary medicine. Here we summarize investigations and knowledge on equine antimicrobial peptides, point to interesting candidates, and discuss prospects for therapeutical applications.

Cellular uptake of imatinib into leukemic cells is independent of human organic cation transporter 1 (OCT1)

Purpose: In addition to mutated BCR-ABL1 kinase, the organic cation transporter 1 (OCT1, encoded by SLC22A1) has been considered to contribute to imatinib resistance in patients with chronic myeloid leukemia (CML). As data are conflicting as to whether OCT1 transports imatinib and may serve as a clinical biomarker, we used a combination of different approaches including animal experiments to elucidate comprehensively the impact of OCT1 on cellular imatinib uptake. Experimental Design: Transport of imatinib was studied using OCT1-expressing Xenopus oocytes, mammalian cell lines (HEK293, MDCK, V79) stably expressing OCT1, human leukemic cells, and Oct1-knockout mice. OCT1 mRNA and protein expression were analyzed in leukemic cells from patients with imatinib-naïve CML as well as in cell lines. Results: Transport and inhibition studies showed that overexpression of functional OCT1 protein in Xenopus oocytes or mammalian cell lines did not lead to an increased cellular accumulation of imatinib. The CML cell lines (K562, Meg-01, LAMA84) and leukemic cells from patients expressed neither OCT1 mRNA nor protein as demonstrated by immunoblotting and immunofluorescence microscopy, yet they showed a considerable imatinib uptake. Oct1 deficiency in mice had no influence on plasma and hepatic imatinib concentrations. Conclusions: These data clearly demonstrate that cellular uptake of imatinib is independent of OCT1, and therefore OCT1 is apparently not a valid biomarker for imatinib resistance. Clin Cancer Res; 20(4); 985–94. ©2013 AACR.

A novel horse α-defensin: gene transcription, recombinant expression and characterization of the structure and function

Defensins are a predominant class of antimicrobial peptides, which act as endogenous antibiotics. Defensins are classified into three distinct sub-families: theta-, beta-, and alpha-defensins. Synthesis of alpha-defensin has been confirmed only in primates and glires to date and is presumably unique for a few tissues, including neutrophils and Paneth cells of the small intestine. Antimicrobial activities of these peptides were shown against a wide variety of microbes including bacteria, fungi, viruses and protozoan parasites. In the present study, we report the characterization of the equine alpha-defensin DEFA (defensin alpha) 1. Transcription analysis revealed that the transcript of the gene is present in the small intestine only. An alignment with known alpha-defensins from primates and glires displayed a homology with Paneth-cell-specific alpha-defensins. DEFA1 was recombinantly expressed in Escherichia coli and subsequently analysed structurally by CD and molecular modelling. To examine the antimicrobial properties, a radial diffusion assay was performed with 12 different micro-organisms and the LD90 (lethal dose killing > or =90% of target organism) and MBC (minimal bactericidal concentration) values were examined. DEFA1 showed an antimicrobial activity against different Gram-positive and Gram-negative bacteria and against the yeast Candida albicans. Using viable bacteria in combination with a membrane-impermeable fluorescent dye, as well as depolarization of liposomes as a minimalistic system, it became evident that membrane permeabilization is at least an essential part of the peptides mode of action.

The repertoire of equine intestinal α-defensins

BackgroundDefensins represent an important class of antimicrobial peptides. These effector molecules of the innate immune system act as endogenous antibiotics to protect the organism against infections with pathogenic microorganisms. Mammalian defensins are classified into three distinct sub-families (α-, β- and θ-defensins) according to their specific intramolecular disulfide-bond pattern. The peptides exhibit an antimicrobial activity against a broad spectrum of microorganisms including bacteria and fungi. Alpha-Defensins are primarily synthesised in neutrophils and intestinal Paneth cells. They play a role in the pathogenesis of intestinal diseases and may regulate the flora of the intestinal tract. An equine intestinal α-defensin (DEFA1), the first characterised in the Laurasiatheria, shows a broad antimicrobial spectrum against human and equine pathogens. Here we report a first investigation of the repertoire of equine intestinal α-defensins. The equine genome was screened for putative α-defensin genes by using known α-defensin sequences as matrices. Based on the obtained sequence information, a set of oligonucleotides specific to the α-defensin gene-family was designed. The products generated by reverse-transcriptase PCR with cDNA from the small intestine as template were sub-cloned and numerous clones were sequenced.ResultsThirty-eight equine intestinal α-defensin transcripts were determined. After translation it became evident that at least 20 of them may code for functional peptides. Ten transcripts lacked matching genomic sequences and for 14 α-defensin genes apparently present in the genome no appropriate transcript could be verified. In other cases the same genomic exons were found in different transcripts.ConclusionsThe large repertoire of equine α-defensins found in this study points to a particular importance of these peptides regarding animal health and protection from infectious diseases. Moreover, these findings make the horse an excellent species to study biological properties of α-defensins. Interestingly, the peptides were not found in other species of the Laurasiatheria to date. Comparison of the obtained transcripts with the genomic sequences in the current assembly of the horse (EquCab2.0) indicates that it is yet not complete and/or to some extent falsely assembled.

Challenges in pharmacogenetics

The attempt to optimize drug treatment of patients by using evidenced-based medicine considering individual physiological and disease-related conditions is standard of modern medicine. Pharmacogenetics (PGx) has contributed to individualization considering hereditary genetic information; however, increasingly, pharmacogenomics is becoming essential, particularly in relation to modern oncology. New technologies such as next-generation sequencing and rapid development of computational and information sciences will help to better elucidate the consequences of genetic variation, considering also epigenetics and gene–environmental interactions and their translation into clinically relevant individual phenotypes. This review highlights the current challenging and most promising examples of PGx.

In vitro potential of equine DEFA1 and eCATH1 as alternative antimicrobial drugs in rhodococcosis treatment.

ABSTRACT Rhodococcus equi, the causal agent of rhodococcosis, is a severe pathogen of foals but also of immunodeficient humans, causing bronchopneumonia. The pathogen is often found together with Klebsiella pneumoniae or Streptococcus zooepidemicus in foals. Of great concern is the fact that some R. equi strains are already resistant to commonly used antibiotics. In the present study, we evaluated the in vitro potential of two equine antimicrobial peptides (AMPs), eCATH1 and DEFA1, as new drugs against R. equi and its associated pathogens. The peptides led to growth inhibition and death of R. equi and S. zooepidemicus at low micromolar concentrations. Moreover, eCATH1 was able to inhibit growth of K. pneumoniae. Both peptides caused rapid disruption of the R. equi membrane, leading to cell lysis. Interestingly, eCATH1 had a synergic effect together with rifampin. Furthermore, eCATH1 was not cytotoxic against mammalian cells at bacteriolytic concentrations and maintained its high killing activity even at physiological salt concentrations. Our data suggest that equine AMPs, especially eCATH1, may be promising candidates for alternative drugs to control R. equi in mono- and coinfections.

Antimicrobial properties of the equine α-defensin DEFA1 against bacterial horse pathogens

Defensins are small effector molecules of the innate immune system, synthesised by various organisms including plants and animals. The peptides act as endogenous antibiotics with an antimicrobial activity against a broad spectrum of microbes including bacteria, fungi and viruses. alpha-Defensins are a subgroup of the defensin family, their synthesis is limited to some tissues and furthermore to some mammalian species including the horse. Equine DEFA1 is an enteric alpha-defensin exclusively produced in Paneth cells. The peptide showed an activity against a broad spectrum of microbes, but typical pathogens of the horse were not included in the previous antimicrobial studies. Here, we report the antibacterial properties of DEFA1 against clinical isolates of typical horse pathogens including Rhodococcus equi, various streptococci strains, Salmonella choleraesuis, and Pasteurella multocida. The recombinantly expressed DEFA1 peptide exerted potent activity against these pathogenic bacteria. The highest susceptibility showed R. equi. Three genetically different strains of R. equi were killed at low micromolar concentrations, comparable with conventionally used antibiotics.