Wolf-Dieter Lehmann

Alterations of phospholipid concentration and species composition of the intestinal mucus barrier in ulcerative colitis: a clue to pathogenesis.

Background: Phospholipids are essential for the normal function of the intestinal mucus barrier. The objective of this study was to systematically investigate phospholipids in the intestinal mucus of humans suffering from inflammatory bowel diseases, where a barrier defect is strongly supposed to be pathogenetic. Methods: Optimal mucus recovery was first validated in healthy mice and the method was then transferred to the endoscopic acquisition of ileal and colonic mucus from 21 patients with ulcerative colitis (UC), 10 patients with Crohns disease (CD), and 29 healthy controls. Nano‐electrospray ionization tandem mass spectrometry (ESI‐MS/MS) was used to determine phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) in lipid extracts of mucus specimens. Results: Human and rodent mucus contained very similar phospholipid species. In the ileal and colonic mucus from patients suffering from UC, the concentration of PC was highly significantly lower (607 ± 147 pmol/100 &mgr;g protein and 745 ± 148 pmol/100 &mgr;g protein) compared to that of patients with CD (3223 ± 1519 pmol/100 &mgr;g protein and 2450 ± 431 pmol/100 &mgr;g protein) and to controls (3870 ± 760 pmol/100 &mgr;g protein and 2790 ± 354 pmol/100 &mgr;g protein); overall, P = 0.0002 for ileal specimens and P < 0.0001 for colonic specimens. Independent of disease activity, patients suffering from UC showed an increased saturation grade of PC fatty acid residues and a higher LPC‐to‐PC ratio. Conclusions: The intestinal mucus barrier of patients with UC is significantly altered concerning its phospholipid concentration and species composition. These alterations may be very important for the pathogenesis of this disease and underline new therapeutic strategies. Inflamm Bowel Dis 2009

Profiling assay for lipoxygenase products of linoleic and arachidonic acid by gas chromatography-mass spectrometry

A method for determination of the lipoxygenase products of linoleic acid (9- and 13-hydroxyoctadecadienoic acid; 9-HODE, 13-HODE) and of arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acid; 5-, 8-, 9-, 11-, 12-, and 15-HETE) is described. The method combines solid-phase extraction, derivatization to the corresponding fully hydrogenated methylester/trimethylsilylether derivatives and capillary gas chromatography coupled with electron impact mass spectrometry. Each regioisomeric HODE and HETE shows a unique pair of mass spectrometric fragment ions originating from fission of the fatty acid carbon chain at the hydroxylated position. The carboxyl-terminal fragment is used for quantification relative to a carboxyl-18O2-labeled analogue added as internal standard and the methyl-terminal fragment is monitored for confirmation. The assay can be extended for quantification of the complete hydroxylation profile of linoleic and arachidonic acid. Applications of this assay are demonstrated for the quantification of HODEs and HETEs in normal, hyperplastic, and neoplastic mouse epidermis. In mouse epidermis papilloma, the tissue levels of 8- and 12-HETE were found to be increased by one to two orders of magnitude compared to levels in normal epidermis.

Molecular interaction of artemisinin with translationally controlled tumor protein (TCTP) of Plasmodium falciparum

Malaria causes millions of death cases per year. Since Plasmodium falciparum rapidly develops drug resistance, it is of high importance to investigate potential drug targets which may lead to novel rational therapy approaches. Here we report on the interaction of translationally controlled tumor protein of P. falciparum (PfTCTP) with the anti-malarial drug artemisinin. Furthermore, we investigated the crystal structure of PfTCTP. Using mass spectrometry, bioinformatic approaches and surface plasmon resonance spectroscopy, we identified novel binding sites of artemisinin which are in direct neighborhood to amino acids 19-46, 108-134 and 140-163. The regions covered by these residues are known to be functionally important for TCTP function. We conclude that interaction of artemisinin with TCTP may be at least in part explain the antimalarial activity of artemisinin.

Significantly greater antioxidant anticancer activities of 2,3-dehydrosilybin than silybin.

Silybin or silymarin extract has been used to treat liver diseases, and has now been entered into clinical trials for cancer treatment. Here, we compared antioxidant and anticancer activities between silybin and its oxidized form 2,3-dehydrosilybin (DHS). With IC50 at three-fold lower concentrations than silybin, DHS inhibited reactive oxygen species generation in glucose-glucose oxidase system and HepG2 cells. Compared with silybin, DHS elicited greater protection against H2O2-induced HepG2 cell death and galactosamine-induced liver injury in vivo. It is known that oxidants induce releases of metalloproteinases (MMP)-2,-9 which are responsible for invasive and metastasis potentials of transformed cells. DHS at 10 microM markedly inhibited MMP-2,-9 releases as well as invasiveness, while silybin at 90 microM had marginal effects. DHS but not silybin at 30 microM induced apoptosis and loss of mitochondrial membrane potentials. LD50 of DHS was five-fold lower than that of silybin. Our data suggest that DHS may be more useful therapeutically than silybin.

Bioinformatic and experimental fishing for artemisinin-interacting proteins from human nasopharyngeal cancer cells

Determining interacting cellular partners of drugs by chemical proteomic techniques is complex and tedious. Most approaches rely on activity-based probe profiling and compound-centric chemical proteomics. The anti-malarial artemisinin also exerts profound anti-cancer activity, but the mechanisms of action are incompletely understood. In the present investigation, we present a novel approach to identify artemisinin-interacting target proteins. Our approach overcomes usual problems in traditional fishing procedures, because the drug was attached to a surface without further chemical modification. The proteins identified effect among others, cell cycle arrest, apoptosis, inhibition of angiogenesis, disruption of cell migration, and modulation of nuclear receptor responsiveness. Furthermore, a bioinformatic approach confirmed experimentally identified proteins and suggested a large number of other interacting proteins. Theoretically predicted interaction partners may serve as a starting point to complete the whole set of proteins binding artemisinin.

Biliary phosphatidylcholine and lysophosphatidylcholine profiles in sclerosing cholangitis

AIM To analyze phospholipid profiles in intrahepatic bile from patients with primary sclerosing cholangitis (PSC) and secondary sclerosing cholangitis (SSC). METHODS Intrahepatic bile specimens collected via endoscopic retrograde cholangiography from 41 patients were analyzed. Fourteen of these patients were diagnosed with PSC, 10 with SSC, 11 with choledocholithiasis or no identifiable biliary disease, and 6 with cholangiocellular carcinoma (CCC). Bile acid, cholesterol, protein, and bilirubin contents as well as pancreas lipase activity in bile were determined by biochemical methods. Phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) species were quantified using nano-electrospray ionization tandem mass spectrometry. RESULTS Bile from all the examined patient groups showed a remarkably similar PC and LPC species composition, with only minor statistical differences. Total biliary PC concentrations were highest in controls (8030 ± 1843 μmol/L) and lowest in patients with CCC (1969 ± 981 μmol/L) (P = 0.005, controls vs SSC and CCC, respectively, P < 0.05). LPC contents in bile were overall low (4.2% ± 1.8%). Biliary LPC/PC ratios and ratios of biliary PC to bilirubin, PC to cholesterol, PC to protein, and PC to bile acids showed no intergroup differences. CONCLUSION PC and LPC profiles being similar in patients with or without sclerosing cholangitis, these phospholipids are likely not of major pathogenetic importance in this disease group.

Phosphorylation of multifunctional galectins by protein kinases CK1, CK2, and PKA

Phosphorylation is known to have a strong impact on protein functions. We analyzed members of the lectin family of multifunctional galectins as targets of the protein kinases CK1, CK2, and PKA. Galectins are potent growth regulators able to bind both glycan and peptide motifs at intra- and extracellular sites. Performing in vitro kinase assays, galectin phosphorylation was detected by phosphoprotein staining and autoradiography. The insertion of phosphoryl groups varied to a large extent depending on the type of kinase applied and the respective galectin substrate. Sites of phosphorylation observed in the recombinant galectins were determined by a strategic combination of phosphopeptide enrichment and nano-ultra-performance liquid chromatography tandem mass spectrometry (nanoUPLC-MS/MS). By in silico modeling, phosphorylation sites were visualized three-dimensionally. Our results reveal galectin-type-specific Ser-/Thr-dependent phosphorylation beyond the known example of galectin-3. These data are the basis for functional studies and also illustrate the analytical sensitivity of the applied methods for further work on human lectins.

Profiling of Monohydroxylated Fatty Acids in Normal, Hyperplastic and Neoplastic Mouse Epidermis by Gas Chromatography-Mass Spectrometry

Hydroxylated long-chain fatty acids in epidermal tissue originate mainly from enzyme-catalyzed lipid peroxidation or oxidation reactions involving lipoxygenase or cytochrome P-450 enzymes and free fatty acids as substrates. To quantify these products in small amounts of epidermal tissue, a profiling assay for monohydroxylated fatty acids based on gas chromatography-mass spectrometry (GC-MS) was set up and applied to normal, hyperplastic and neoplastic mouse epidermis. For detection of hydroxylated fatty acids by GC-MS, two methods have been established: formation of the corresponding pentafluorobenzyl- ester/trimethylsilylether (TMS) derivatives and their detection by negative chemical ionization MS (1) or formation of the corresponding methylester/TMS derivatives and their detection by positive ion electron impact (EI)-MS (2). In spite of its lower absolute sensitivity, we selected EI-MS since only with this approach can structure-specific mass spectrometric fragment ions be obtained.

Abstract 1993: Fishing for artemisinin-interacting proteins from human nasopharyngeal cancer cells

Determining cellular target molecules of drugs by chemical proteomic techniques is complex and tedious. Most approaches rely on activity-based probe profiling and compound-centric chemical proteomics. The antimalarial artemisinin also exerts profound anti-cancer activity, but the mechanisms of action are incompletely understood. In the present study, we have identified artemisinin-interacting target proteins from human nasopharyngeal carcinoma cell line CNE1. Thereby, our approach overcomes usual problems in traditional fishing procedures, because the drug was attached to a polystyrene surface without further chemical modification. Using mass spectrometry we have identified 20 proteins which effect cell cycle arrest, apoptosis, inhibition of angiogenesis, disruption of cell migration, and modulation of nuclear receptor responsiveness. One protein out of the twenty was further investigated in vitro for direct ligand binding. Furthermore, a blind-docking based approach confirmed experimentally identified proteins and suggested further interacting proteins. Theoretically predicted interaction partners may serve as a starting point to complete the whole set of proteins binding artemisinin. In our investigations, we have comprised artemisinins role in cancer treatment in a greater shape through a systematic biological strategy and thus, are able to show novel insights into its mode of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1993. doi:1538-7445.AM2012-1993

Abstract 3265A: GIST-associated c-Kit mutants exhibit differential affinities to hsp90 and its co-chaperone Cdc37

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Hsp90 inhibitors have been introduced to clinical treatment of imatinib-resistant GIST, but their efficacy is dependant on KIT mutations. We analysed the role of the chaperone Hsp90 as stabilizing factor for wild-type and oncogenic, misfolded c-Kit. Methods: the intracellular part of wild-type and mutated c-Kit (aa544-977) fused with an N-terminal TAP-tag was expressed in HEK293T cells. The clinically most abundant primary and imatinib-resistant secondary c-Kit mutations were selected: V560D; V559D; del557,558; L576P; K642E; V560D+D820A; del557,558+Y823D; D820A. A shortened TAP assay followed by LC-MS/MS was applied for identification of interaction partners. For verification a GST pulldown assay was used. Relative quantifications of mutation-specific interaction partners were performed by quantitative immunoblotting using an antibody raised against the C-terminus of human c-Kit. In addition, two GIST cell lines containing an imatinib-resistant and an imatinib-sensitive c-Kit mutation were investigated by quantitative immunoblotting. The activityof hsp90 inhibitor 17-AAG was studied in whole cell lysates of HEK293T cells expressing selected mutated c-Kit constructs. Results: Using the TAP-c-Kit constructs for a shortened TAP assay, Hsp90 and its cochaperone Cdc37 were identified as specific interaction partners for the GIST-associated c-Kit mutants. This interaction was confirmed using GST-Cdc37 pulldown analysis. Differential analysis of c Kit/Hsp90 interaction revealed mutation-specific affinities (up to a relative factor of about 8), with D820A showing the lowest, close-to wild-type affinity, and L576P showing the highest affinity. Among the two GIST cell lines studied, the imatinib-resistant mutant (c-Kit K642E) exhibited a 2.5 fold higher affinity to Hsp90 compared to the imatinib-sensitive mutant (c-Kit V560D+D820A). In both cell lines, the Cdc37 and Hsp90 affinities are observed in parallel. A closely related situation was observed for the interactions of the corresponding TAP-c-Kit constructs. In addition, the influence of the When analysing hsp90 inhibitor 17AAG (17-(allylamino)-17-demethoxygeldanamycin)) incells expressing selected mutated c-Kit constructs, western blot analyses showed significant degradation of total c-Kit relative to untreated controls in all mutants studied. Amongst these, mutant D820A was found to be less sensitive to 17AAG. Wild-type c-Kit showed no sensitivity against 17AAG. Conclusion: this study demonstrates mutation-specific affinities between Hsp90 and Cdc37 with c-Kit mutants. It also points to a conformity between this phenomenon and the 17AAG effect. These results allow a better understanding of the potential of hsp90 inhibition in GIST therapy and better selection of candidates for treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3265A. doi:10.1158/1538-7445.AM2011-3265A