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  • 1.
    Chen, Xiaomei
    et al.
    Univ Michigan, Coll Pharm, Dept Pharmaceut Sci, 428 Church St, Ann Arbor, MI 48109 USA..
    Keep, Richard F.
    Univ Michigan Hlth Syst, Dept Neurosurg, Ann Arbor, MI USA..
    Liang, Yan
    Univ Michigan, Coll Pharm, Dept Clin Pharm, 428 Church St, Ann Arbor, MI 48109 USA..
    Zhu, Hao-Jie
    Univ Michigan, Coll Pharm, Dept Clin Pharm, 428 Church St, Ann Arbor, MI 48109 USA..
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hu, Yongjun
    Univ Michigan, Coll Pharm, Dept Pharmaceut Sci, 428 Church St, Ann Arbor, MI 48109 USA..
    Smith, David E.
    Univ Michigan, Coll Pharm, Dept Pharmaceut Sci, 428 Church St, Ann Arbor, MI 48109 USA..
    Influence of peptide transporter 2 (PEPT2) on the distribution of cefadroxil in mouse brain: A microdialysis study2017In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 131, p. 89-97Article in journal (Refereed)
    Abstract [en]

    Peptide transporter 2 (PEPT2) is a high-affinity low-capacity transporter belonging to the proton-coupled oligopeptide transporter family. Although many aspects of PEPT2 structure-function are known, including its localization in choroid plexus and neurons, its regional activity in brain, especially extracellular fluid (ECF), is uncertain. In this study, the pharmacokinetics and regional brain distribution of cefadroxil, a beta-lactam antibiotic and PEN 2 substrate, were investigated in wildtype and Pept2 null mice using in vivo intracerebral microdialysis. Cefadroxil was infused intravenously over 4 h at 0.15 mg/min/kg, and samples obtained from plasma, brain ECF, cerebrospinal fluid (CSF) and brain tissue. A permeability surface area experiment was also performed in which 0.15 mg/min/kg cefadroxil was infused intravenously for 10 min, and samples obtained from plasma and brain tissues. Our results showed that PEPT2 ablation significantly increased the brain ECF and CSF levels of cefadroxil (2- to 2.5-fold). In contrast, there were no significant differences between wildtype and Pept2 null mice in the amount of cefadroxil in brain cells. The unbound volume of distribution of cefadroxil in brain was 60% lower in Pept2 null mice indicating an uptake function for PEPT2 in brain cells. Finally, PEPT2 did not affect the influx clearance of cefadroxil, thereby, ruling out differences between the two genotypes in drug entry across the blood-brain barriers. These findings demonstrate, for the first time, the impact of PEPT2 on brain ECF as well as the known role of PEPT2 in removing peptide-like drugs, such as cefadroxil, from the CSF to blood.

  • 2. Del Tredici, Andria L.
    et al.
    Vanover, Kim E.
    Knapp, Anne E.
    Bertozzi, Sine M.
    Nash, Norman R.
    Burstein, Ethan S.
    Lameh, Jelveh
    Currier, Erika A.
    Davis, Robert E.
    Brann, Mark R.
    Mohell, Nina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Olsson, Roger
    Piu, Fabrice
    Identification of novel selective V-2 receptor non-peptide agonists2008In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 76, no 9, p. 1134-1141Article in journal (Refereed)
    Abstract [en]

    Peptides with agonist activity at the vasopressin V-2 receptor are used clinically to treat fluid homeostasis disorders such as polyuria and central diabetes insipidus. of these peptides, the most commonly used is desmopressin, which displays poor bioavailability as well as potent activity at the V-1b receptor, with possible stress-related adverse effects. Thus, there is a strong need for the development of small molecule chemistries with selective V-2 receptor agonist activity. Using the functional cell-based assay Receptor Selection and Amplification Technology (R-SAT (R)), a screening effort identified three small molecule chemotypes (AC-94544, AC-88324, and AC-110484) with selective agonist activity at the V-2 receptor. One of these compounds, AC-94544, displayed over 180-fold selectivity at the V-2 receptor compared to related vasopressin and oxytocin receptors and no activity at 28 other G protein-coupled receptors (GPCRs). All three compounds also showed partial agonist activity at the V-2 receptor in a cAMP accumulation assay. In addition, in a rat model of central diabetes insipidus, AC-94544 was able to significantly reduce urine output in a dose-dependent manner. Thus, AC-94544, AC-88324, and AC-110484 represent novel opportunities for the treatment of disorders associated with V-2 receptor agonist deficiency.

  • 3.
    Ekegren, Titti
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Aquilonius, Sten-Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Gomes-Trolin, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    A comparative study of methionine adenosyltransferase activity and regional distribution in mammalian spinal cord2000In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 60, no 3, p. 441-445Article in journal (Refereed)
    Abstract [en]

    To provide a background for future studies on neurodegenerative changes in the spinal cord, the present study analysed the distribution of the activity of methionine adenosyltransferase (ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6, MAT), an enzyme that catalyses the synthesis of the biological methyl group donor S-adenosylmethionine (AdoMet), in spinal cords from bovine and pig, and compared the results with those from human spinal cord. The enzyme activity was estimated by a radiochemical method measuring the rate of formation of [(3)H]AdoMet from L-[methyl-(3)H]methionine and ATP. The MAT activity (V(max)) was quite homogeneously distributed between spinal regions and species investigated (19-50 pmol [(3)H]AdoMet/mg protein/minute), with the highest level found in the male bovine group. The bovine group (both males and females) also presented a 20% higher enzymatic activity in the dorsal horn as compared to the ventral horn and white matter areas. In the pig spinal cord, the highest level of activity was found in the white matter. The lowest affinity for methionine (highest K(m)) was found in the human spinal cord. Whole spinal cords of one cat and one rhesus monkey were also analysed and the levels of MAT activity were similar to that of humans and bovine females, respectively. Studies of MAT stability in the rat spinal cord (post-mortem time 0-72 hr) showed a significant decrease in enzyme activity during the interval of 0-8 hr (23 degrees ). From this time point on and up to 72 hr (4 degrees ), the significant decrease in the activity remained at 60% of the initial value.

  • 4.
    Eklund, Birgitta I.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gunnarsdottir, S.
    Elfarra, A.A.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Human glutathione transferases catalyzing the bioactivation of anticancer thiopurine prodrugs2007In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 73, no 11, p. 1829-1841Article in journal (Refereed)
    Abstract [en]

    cis-6-(2-Acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG) are thiopurine prodrugs provisionally inactivated by an α,β-unsaturated substituent on the sulfur of the parental thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). The active thiopurines are liberated intracellularly by glutathione (GSH) in reactions catalyzed by glutathione transferases (GSTs) (EC 2.5.1.18). Catalytic activities of 13 human GSTs representing seven distinct classes of soluble GSTs have been determined. The bioactivation of cAVTP and tAVTG occurs via a transient addition of GSH to the activated double bond of the S-substituent of the prodrug, followed by elimination of the thiopurine. The first of these consecutive reactions is rate-limiting for thiopurine release, but GST-activation of this first addition is shifting the rate limitation to the subsequent elimination. Highly active GSTs reveal the transient intermediate, which is detectable by UV spectroscopy and HPLC analysis. LC/MS analysis of the reaction products demonstrates that the primary GSH conjugate, 4-glutathionylbuten-2-one, can react with a second GSH molecule to form the 4-(bis-glutathionyl)butan-2-one. GST M1-1 and GST A4-4 were the most efficient enzymes with tAVTG, and GST M1-1 and GST M2-2 had highest activity with cAVTP. The highly efficient GST M1-1 is polymorphic and is absent in approximately half of the human population. GST P1-1, which is overexpressed in many cancer cells, had no detectable activity with cAVTP and only minor activity with tAVTG. Other GST-activated prodrugs have targeted GST P1-1-expressing cancer cells. Tumors expressing high levels of GST M1-1 or GST A4-4 can be predicted to be particularly vulnerable to chemotherapy with cAVTP or tAVTG.

  • 5.
    Elinder, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Selhorst, Philippe
    Vanham, Guido
    Öberg, Bo
    Vrang, Lotta
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Inhibition of HIV-1 by non-nucleoside reverse transcriptase inhibitors via an induced fit mechanism: Importance of slow dissociation and relaxation rates for antiviral efficacy2010In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 80, no 8, p. 1133-1140Article in journal (Refereed)
    Abstract [en]

    The importance of slow dissociation of non-nucleoside reverse transcriptase inhibitors (NNRTIs) for antiviral effect has been investigated. The kinetic characteristics of a series of NNRTIs interacting with wild type and drug resistant variants of HIV-1 RT (EC 2.7.7.49) were analyzed by SPR biosensor technology. The antiviral effect was determined in MT-4 and peripheral blood mononuclear cells. Due to extremely slow dissociation rates and a complex interaction mechanism, rate constants could not be quantified. Instead, interaction characteristics were qualitatively analyzed using simulated sensorgrams. The simplest model describing these interactions adequately was an induced fit mechanism, i.e. a mechanism involving the formation of an initial enzyme-inhibitor complex subsequently transformed into a more stable complex. Differences in rates of dissociation from the initial complex and rates of relaxation from the induced complex explained (1) the differences in the amounts of formed complex, (2) the stability of the complex and (3) the antiviral efficacies of the compounds. The effect of NNRTI binding site mutations also correlated with these kinetic characteristics. MIV-170 was the most effective inhibitor of wild type and mutant HIV-1 in cell culture, a property that was associated with the formation of the largest amount of complex and the slowest relaxation and dissociation rates. This study supports the hypothesis that the efficacy of anti-HIV drugs is dependent on slow dissociation from the target, thereby maximizing the duration of the inhibitory effect. It also illustrates the strength of simulating interaction data for qualitative analysis of tight-binding drugs and the importance of resolving the kinetic mechanism of drug-target interactions.

  • 6.
    Eriksson, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Höglund, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Lindhagen, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Åleskog, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Hassan, Sadia Bashir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Ekholm, Carina
    Fhölenhag, Karin
    Jensen, Annika Jenmalm
    Löthgren, Agneta
    Scobie, Martin
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Parrow, Vendela
    Identification of AKN-032, a novel 2-aminopyrazine tyrosine kinase inhibitor, with significant preclinical activity in acute myeloid leukemia2010In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 80, no 10, p. 1507-1516Article in journal (Refereed)
    Abstract [en]

    Aberrant signal transduction by mutant or overexpressed protein kinases has emerged as a promising target for treatment of acute myeloid leukemia (AML). We here present a novel low molecular weight kinase inhibitor, AKN-032, targeting the FMS-like tyrosine kinase 3 (FLT3) and discovered in a new type of screening funnel combining the target therapy approach with sequential cellular screens. AKN-032 was identified among 150 selected hits from three different high throughput kinase screens. Further characterization showed inhibitory activity on FLT3 enzyme with an IC50 of 70 nM. Western blot analysis revealed reduced autophosphorylation of the FLT3-receptor in AML cell line MV4-11 cells after exposure to AKN-032. Flow cytometry disclosed cytotoxic activity against MV4-11, but not against non-malignant 3T3-L1 fibroblast cells. Using a fluorometric microculture cytotoxicity assay, AKN-032 was tested against 15 cell lines and displayed a potent cytotoxic activity in AML cell lines MV4-11 (IC50 = 0.4 mu M) and Kasumi-1 (IC50 = 2.3 mu M). AKN-032 was also highly cytotoxic in tumor cells from AML patients in vitro. Furthermore, AKN-032 demonstrated significant antileukemic effect in a relatively resistant in vivo hollow fiber mouse model. No major toxicity was observed in the animals. In conclusion. AKN-032 is a promising new kinase inhibitor with significant in vivo and in vitro activity in AML Results from the hollow fiber mouse assay suggest a favorable toxicity profile. Future studies will focus on pharmacokinetic properties, toxicity as well as further clarifying the mechanisms of action of AKN-032 in AML.

  • 7.
    Eriksson, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Kalushkova, Antonia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Hilhorst, Riet
    Rickardson, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Göransson Kultima, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    de Wijn, Rik
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Öberg, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Parrow, Vendela
    Höglund, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    AKN-028 induces cell cycle arrest, downregulation of Myc associated genes and a dose dependent reduction of kinase activity in acute myeloid leukemia2014In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 87, no 2, p. 284-291Article in journal (Refereed)
    Abstract [en]

    AKN-028 is a novel tyrosine kinase inhibitor with preclinical activity in acute myeloid leukemia (AML), presently undergoing investigation in a phase I/II study. It is a potent inhibitor of the FMS-like kinase 3 (FLT3) but shows in vitro activity in a wide range of AML samples. In the present study, we have characterized the effects of AKN-028 on AML cells in more detail. AKN-028 induced a dose-dependent G(0)/arrest in AML cell line MV4-11. Treatment with AKN-028 caused significantly altered gene expression in all AML cell types tested (430 downregulated, 280 upregulated transcripts). Subsequent gene set enrichment analysis revealed enrichment of genes associated with the proto-oncogene and cell cycle regulator c-Myc among the downregulated genes in both AKN-028 and midostaurin treated cells. Kinase activity profiling in AML cell lines and primary AML samples showed that tyrosine kinase activity, but not serine/threonine kinase activity, was inhibited by AKN-028 in a dose dependent manner in all samples tested, reaching approximately the same level of kinase activity. Cells sensitive to AKN-028 showed a higher overall tyrosine kinase activity than more resistant ones, whereas serine/threonine kinase activity was similar for all primary AML samples. In summary, AKN-028 induces cell cycle arrest in AML cells, downregulates Myc-associated genes and affect several signaling pathways. AML cells with high global tyrosine kinase activity seem to be more sensitive to the cytotoxic effect of AKN-028 in vitro.

  • 8. Fuhrmann, A.
    et al.
    Lopes, P. C.
    Sereno, J.
    Pedro, J.
    Espinoza, D. O.
    Pereira, Maria J.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical diabetology and metabolism.
    Reis, F.
    Eriksson, J. W.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Carvalho, E.
    Molecular mechanisms underlying the effects of cyclosporin A and sirolimus on glucose and lipid metabolism in liver, skeletal muscle and adipose tissue in an in vivo rat model2014In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 88, no 2, p. 216-228Article in journal (Refereed)
    Abstract [en]

    Cyclosporin A (CsA) and sirolimus (SRL) are immunosuppressive agents (IAs) associated with dyslipidemia, insulin resistance and new onset diabetes after transplantation (NODAT). However, the molecular mechanisms involved are not fully understood. We investigated the effects of six-week treatment of either CsA or SRL on glucose and lipid metabolism in Wistar rats. The results show that, compared with vehicle-treated rats, SRL-treated rats were significantly lighter starting at week 5. CsA or SRL caused glucose intolerance, increased storage of lipids in the liver and skeletal muscle, and decreased the insulin-stimulated glucose uptake in isolated adipocytes. Furthermore, these agents significantly decreased genes involved in insulin action and glucose uptake, such as, IRS-1, Glut4 and Glut1, and increased genes and/or proteins involved in hepatic lipogenesis and gluconeogenesis, while decreasing them in adipose tissue. After either treatment PGC1 alpha gene expression was down regulated in skeletal muscle, an important player in fatty acid oxidation. Moreover, there was an increase in IL-6 gene expression in adipose tissue in the SRL-treated rats, suggesting stimulation of lipolysis. The results of the present study suggest that CsA and SRL lead to metabolic alterations in liver, muscle and adipose tissue, which may contribute to the development of dyslipidemia and insulin resistance associated with immunosuppressive therapy. (C) 2014 Elsevier Inc. All rights reserved.

  • 9.
    Gabl, Michael
    et al.
    Univ Gothenburg, Dept Rheumatol & Inflammat Res, Gothenburg, Sweden..
    Holdfeldt, Andre
    Univ Gothenburg, Dept Rheumatol & Inflammat Res, Gothenburg, Sweden..
    Sundqvist, Martina
    Univ Gothenburg, Dept Rheumatol & Inflammat Res, Gothenburg, Sweden..
    Lomei, Jalal
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Dahlgren, Claes
    Univ Gothenburg, Dept Rheumatol & Inflammat Res, Gothenburg, Sweden..
    Forsman, Huamei
    Univ Gothenburg, Dept Rheumatol & Inflammat Res, Gothenburg, Sweden..
    FPR2 signaling without beta-arrestin recruitment alters the functional repertoire of neutrophils2017In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 145, p. 114-122Article in journal (Refereed)
    Abstract [en]

    G-protein coupled receptor (GPCR) biased agonism or functional selectivity has become an essential concept in GPCR research over the last years. Receptor-specific biased agonists selectively trigger one signaling pathway over another and induce a restricted/directed functional response. In this study, we aimed to characterize the concept of biased agonism for FPR2, a member of the formyl peptide receptor (FPR) subfamily of GPCRs. We show that the earlier described FPR2-activating pepducin F2Pal(10) is a biased FPR2 agonist. The effects of F2Pal(10) on neutrophil function differed in several aspects compared to those mediated by WKYMVM, a conventional FPR2-specific peptide agonist. Upon interaction with FPR2 expressed by neutrophils both F2Pal(10) and WKYMVM activated the PLC-PIP2-Ca2+ signaling pathway and the superoxide-generating NADPH-oxidase, but only WKYMVM activated the receptor to recruit B-arrestin. The functional consequences linked to a lack of B-arrestin recruitment were further explored, and we demonstrate that FPR2 desensitization occurred independent of B-arrestin. Despite this, reactivation of desensitized receptors achieved through a disruption of the cytoskeleton and through a novel FPR2 cross-talk mechanism with P2Y(2)R (the ATP receptor) and PAFR (the receptor for PAF) differed between F2Pal(10)-desensitized and WKYMVM-desensitized neutrophils. Further, the inability to recruit beta-arrestin was found to be associated with a reduced rate of receptor internalization and impaired chemotaxis in neutrophils. In summary, we provide experimental evidence of biased agonism for FPR2 and our data disclose critical roles of beta-arrestin in neutrophil chemotaxis and reactivation of desensitized receptors. (C) 2017 Elsevier Inc. All rights reserved.

  • 10.
    Garcia-Bennett, Alfonso
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Nees, Matthias
    VTT Technical Research Center of Finland, Department of Medical Biotechnology, Turku, Finland.
    Fadeel, Bengt
    Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm and Childhood Cancer Research Unit, Department of Women's and Children's Health, Astrid Lindgren's Children's Hospital, Karolinska University Hospital, Stockholm.
    In search of the Holy Grail: Folate-targeted nanoparticles for cancer therapy2011In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 81, no 8, p. 976-984Article in journal (Refereed)
    Abstract [en]

    Targeted drug therapy or "smart" drug delivery, potentially combined with simultaneous imaging modalities to monitor the delivery of drugs to specific tissues, is arguably the "holy grail" of pharmacology. Therapeutic approaches that exploit nanoparticles to deliver drugs selectively to cancer cells are currently considered one of the most promising avenues in the area of cancer therapeutics and imaging. The potential to deliver active chemotherapeutic drugs in the vicinity or directly within specific tumors via receptor mediated pathways, and to image tumors through the use of nanoparticles has been conceptually and experimentally shown for several classes of nanoparticles. Nanoparticles functionalized with the vitamin folic acid are of particular interest as a variety of malignant tumors are known to overexpress the folate receptor(s). Indeed, several nanoparticle architectures with improved retention time, administration route, biocompatibility, absorption, and clearance are being proposed and are in late stage clinical development. This commentary highlights some of the most important concepts related to nanoparticles and folate-mediated drug delivery and imaging in cancer research.

  • 11.
    Glisovic, Tina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Söderberg, Malin
    Christian, Kyle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lang, Matti A.
    Raffalli-Mathieu, Francoise
    Interplay between transcriptional and post-transcriptional regulation of Cyp2a5 expression2003In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 65, no 10, p. 1653-1661Article in journal (Refereed)
    Abstract [en]

    The cytochrome P450 (Cyp) 2a5 gene can be upregulated transcriptionally or by mRNA stabilization. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 interacting with the CYP2A5 mRNA has been shown to be a key post-transcriptional regulator of the Cyp2a5 gene. The aim of this study was to investigate if the transcriptional and post-transcriptional steps of Cyp2a5 expression are linked. This was done by modifying the transcription rate with transcriptional inducers (phenobarbital and cyclic AMP) and inhibitors (actinomycin D and 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole) and analyzing the effects upon post-transcriptional events. We found that inhibition of transcription led to relocalization of hnRNP A1 from the nucleus to the cytoplasm, to its strongly increased binding to the cytoplasmic CYP2A5 mRNA and to CYP2A5 mRNA stabilization. In contrast, stimulated transcription resulted in increased binding of nuclear hnRNP A1 to the Cyp2a5 promoter, and overexpression of hnRNP A1 led to stimulated transcription of a Cyp2a5 promoter-driven luciferase recombinant. This strongly suggests that the transcriptional and post-transcriptional stages of Cyp2a5 expression are interrelated and that the nucleocytoplasmic shuttling hnRNP A1 may coordinate these different steps.

  • 12.
    Gullbo, Joachim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Rickardson, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Darcy, Padraig
    Hägg, Maria
    Wickström, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Hassan, Sadia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Westman, Gunnar
    Brnjic, Slavica
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Linder, Stig
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Phenotype-based drug screening in primary ovarian carcinoma cultures identifies intracellular iron depletion as a promising strategy for cancer treatment2011In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 82, no 2, p. 139-147Article in journal (Refereed)
    Abstract [en]

    Primary cultures of patient tumor cells (PCPTC) have been used for prediction of diagnosis-specific activity and individual patient response to anticancer drugs, but have not been utilized as a model for identification of novel drugs in high throughput screening. In the present study, ovarian carcinoma cells from three patients were tested in response to a library of 3000 chemically diverse compounds. Eight hits were retrieved after counter screening using normal epithelial cells, and one of the two structurally related hit compounds was selected for further preclinical evaluation. This compound, designated VLX 50, demonstrated a broad spectrum of activity when tested in a panel of PCPTCs representing different forms of leukemia and solid tumors and displayed a high tumor to normal cell activity. VLX 50 induced delayed cell death with some features of classical apoptosis. Significant in vivo activity was confirmed on primary cultures of human ovarian carcinoma cells in mice using the hollow fiber model. Mechanistic exploration was performed using gene expression analysis of drug exposed tumor cells to generate a drug-specific signature. This query signature was analyzed using the Gene Set Enrichment Analysis and the Connectivity Map database. Strong connections to hypoxia inducible factor 1 and iron chelators were retrieved. The mechanistic hypothesis of intracellular iron depletion leading to hypoxia signaling was confirmed by a series of experiments. The results indicate the feasibility of using PCPTC for cancer drug screening and that intracellular iron depletion could be a potentially important strategy for cancer therapy.

  • 13. Johansson, Stina M.
    et al.
    Salehi, A.
    Sandström, Marie E.
    Westerblad, H.
    Lundquist, I.
    Carlsson, Per-Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Fredholm, B.B.
    Katz, A.
    A(1) receptor deficiency causes increased insulin and glucagon secretion in mice2007In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 74, no 11, p. 1628-1635Article in journal (Refereed)
    Abstract [en]

    Adenosine influences metabolism and the adenosine receptor antagonist caffeine decreases the risk of type 2 diabetes. In this study the metabolic role of one adenosine receptor subtype, the adenosine A(1)R, was evaluated in mice lacking this receptor [A(1)R (-/-)]. The HbA1c levels and body weight were not significantly different between wild type [A(1)R (+/+)] and A(1)R (-/-) mice (3-4 months) fed normal lab chow. At rest, plasma levels of glucose, insulin and glucagon were similar in both genotypes. Following glucose injection, glucose tolerance was not appreciably altered in A(1)R (-/-) mice. Glucose injection induced sustained increases in plasma insulin and glucagon levels in A(1)R (-/-) mice, whereas A(1)R (+/+) control mice reacted with the expected transient increase in insulin and decrease in glucagon levels. Pancreas perfusion experiments showed that A(1)R (-/-) mice had a slightly higher basal insulin secretion than A(1)R (+/+) mice. The first phase insulin secretion (initiated with 16.7 mM glucose) was of the same magnitude in both genotypes, but the second phase was significantly enhanced in the A(1)R (-/-) pancreata compared with A(1)R (+/+). Insulin- and contraction-mediated glucose uptake in skeletal muscle were not significantly different between in A(1)R (-/-) and A(1)R (+/+) mice. All adenosine receptors were expressed at mRNA level in skeletal muscle in A(1)R (+/+) mice and the mRNA A(2A)R, A(2B)R and A(3)R levels were similar in A(1)R (-/-) and A(1)R (+/+) mice. In conclusion, the A(1)R minimally affects muscle glucose uptake, but is important in regulating pancreatic islet function.

  • 14. Lopes, P. C.
    et al.
    Fuhrmann, A.
    Carvalho, F.
    Sereno, J.
    Santos, M. R.
    Pereira, Maria João
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical diabetology and metabolism.
    Eriksson, Jan W.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical diabetology and metabolism.
    Reis, F.
    Carvalho, E.
    Cyclosporine A enhances gluconeogenesis while sirolimus impairs insulin signaling in peripheral tissues after 3 weeks of treatment2014In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 91, no 1, p. 61-73Article in journal (Refereed)
    Abstract [en]

    Cyclosporine A (CsA) and sirolimus (SRL) are immunosuppressive agents (IA) associated with new-onset diabetes after transplantation (NODAT). This study aims to evaluate the effects of 3-weeks of treatment with either CsA (5 mg/kg BW/day) or SRL (1 mg/kg BW/day) on insulin signaling and expression of markers involved in glucose metabolism in insulin-sensitive tissues, in Wistar rats. Although no differences were observed in fasting glucose, insulin or C-peptide levels, both treated groups displayed an impaired glucose excursion during both glucose and insulin tolerance tests. These results suggest glucose intolerance and insulin resistance. An increase in glucose-6-phosphatase protein levels (68%, p<0.05) and in protein-tyrosine phosphatase 1B (163%,p<0.05), a negative regulator of insulin was observed in the CsA-treated group in the liver, indicating enhanced gluconeogenesis and increased insulin resistance. On the other hand, glucokinase protein levels were decreased in the SRL group (35%, p < 0.05) compared to vehicle, suggesting a decrease in glucose disposal. SRI treatment also reduced peroxisome proliferator-activated receptor gamma coactivator 1 alpha protein expression in muscle (similar to 50%, p<0.05), while no further protein alterations were observed in muscle and perirenal adipose tissue nor with the CsA treatment. Moreover, the phosphorylation of key proteins of the insulin signaling cascade was suppressed in the SRL group, but was unchanged by the CsA treatment. Taken together, these data suggest that CsA treatment enhances gluconeogenic factors in liver, while SRL treatment impairs insulin signaling in peripheral tissues, which can contribute to the development of insulin resistance and NODAT associated with immunosuppressive therapy.

  • 15.
    Sandler, Stellan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Andersson, Annika K.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Larsson, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Makeeva, Natalia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Olsen, Therese
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Arkhammar, Per O. G.
    Hansen, John Bondo
    Karlsson, F. Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Welsh, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Possible role of an ischemic preconditioning-like response mechanism in K(ATP) channel opener-mediated protection against streptozotocin-induced suppression of rat pancreatic islet function2008In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 76, no 12, p. 1748-1756Article in journal (Refereed)
    Abstract [en]

    Potassium channel openers (KCOs) decrease insulin secretion from beta-cells. Some KCOs also protect against damage to beta-cell function and type 1 diabetes in animal models. Previously we have found that the KCO NNC 55-0118 counteracted islet cell dysfunction, and this was associated with a lowering of the mitochondrial membrane potential (Deltapsi). Presently we aimed to explore whether inhibition of insulin secretion per se or rather inhibition of mitochondrial function correlates to counteraction of beta-cell suppression. For this we used two novel KCOs (NNC 55-0321 and NNC 55-0462), which at certain concentrations have different actions regarding insulin secretion and the Deltapsi, with NNC 55-0321 being a potent inhibitor of Deltapsi and NNC 55-0462 being a potent inhibitor of insulin secretion. At 10 microM NNC 55-0321, but not with NNC 55-0462, the islet ATP content and ATP/ADP ratio was acutely decreased. This was accompanied by a complete protection against streptozotocin-induced suppression of islet insulin secretion using the former KCO. In cardiac research KCOs have been used to induce an ischemic preconditioning (IPC) response. In line with an IPC-like mechanism we found that NNC 55-0321 induced an initial free oxygen radical formation, PKC-epsilon isoform activation and a subsequent phosphorylation of the survival promoting factor Akt. Thus, KCOs may elicit mitochondrial events that resemble classical IPC seen in cardiomyocytes, and this could explain the enhanced islet cell function observed. KCOs with this property may be particularly interesting compounds to study as a rescue therapy during acute episodes of beta-cell suppression/destruction.

  • 16.
    Seeger, Christian
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Christopeit, Tony
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Fuchs, K.
    Grote, K.
    Sieghart, W.
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Corrigendum to "Histaminergic pharmacology of homo-oligomeric β3 γ-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology" (Biochemical Pharmacology (2012) 84 (341-351))2012In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 84, no 11, p. 1541-Article in journal (Refereed)
  • 17.
    Seeger, Christian
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Christopeit, Tony
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Fuchs, Karoline
    Grote, Katharina
    Sieghart, Werner
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Histaminergic pharmacology of homo-oligomeric beta 3 gamma-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology2012In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 84, no 3, p. 341-351Article in journal (Refereed)
    Abstract [en]

    A surface plasmon resonance biosensor assay was established for studying the interactions of 51 histaminergic and 15 GABAergic ligands with homo-oligomeric beta 3 GABA(A) receptors. Detergent solubilized receptors were successfully immobilized via affinity-capture on biosensor surfaces. The interaction kinetics of both histaminergic and GABAergic ligands were very rapid but affinities could be determined by steady-state analysis. Binding of several GABAergic ligands was observed, in agreement with previous data. Histamine and 16 histaminergic ligands were detected to directly bind to beta 3 GABA(A) receptors with micromolar affinity (K-D <300 mu M), thus extending previous evidence that beta 3 GABA(A) receptors can interact with histaminergic ligands. Histamine exhibited an affinity for these receptors comparable to that for human histamine type 1 (H1) or type 2 (H2) receptors. Furthermore, 13 of these histaminergic ligands appeared to compete with histamine. The discovery that H2, H3 and H4 receptor ligands interact with beta 3 receptors indicates a unique histaminergic pharmacology of these receptors. Due to their low affinity for the homo-pentameric beta 3 receptors these histaminergic drugs are not expected to modulate these receptors at clinically relevant concentrations. The results support the use of the new biosensor assay for the identification of drugs interacting with full length receptors and for fragment-based drug discovery of high affinity ligands for beta 3 receptors. Drugs with high affinity and selectivity for these receptors can be used to clarify the question whether beta 3 receptors do exist in the brain, and provide new avenues for the development of therapeutically active compounds targeting this novel histamine binding site. 

  • 18. Semaan, Walid
    et al.
    Desbiens, Louisane
    Houde, Martin
    Labonte, Julie
    Gagnon, Hugo
    Yamamoto, Daisuke
    Takai, Shinji
    Laidlaw, Tanya
    Bkaily, Ghassan
    Schwertani, Fadel
    Pejler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Levesque, Christine
    Desjardins, Roxane
    Day, Robert
    D'Orleans-Juste, Pedro
    Chymase inhibitor-sensitive synthesis of endothelin-1 (1-31) by recombinant mouse mast cell protease 4 and human chymase2015In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 94, no 2, p. 91-100Article in journal (Refereed)
    Abstract [en]

    Important structural differences imply that human and mouse mast cell chymases may differ with respect to their enzymatic properties. We compared in this study the catalytic efficiencies of recombinant human chymase (rCMA1) and its functional murine homologue recombinant mouse mast cell protease-4 (rmMCP-4) toward a fluorogenic chymase substrate (Suc-Ala-Ala-Pro-Phe-7-amino-4-methylcoumarin (AMC) and by their ability to convert Big-endothelin (ET)-1 into ET-1 (1-31) using a LC/MS/MS system. Activities toward a fluorogenic substrate (Suc-Leu-Leu-Val-Tyr-AMC) and Big ET-1 were also measured in extracts from mouse peritoneal mast cells, LUVA human mast cell-like cells and human aortas. The specificity of these activities was assessed with the chymase inhibitor TY-51469 (2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonyl-phenyl]thiazole-4-carboxylic acid). For similar affinities, rmMCP-4 showed a higher activity toward the fluorogenic substrate and a higher ability to process Big ET-1 as compared to recombinant CMA1 (chymase activity (k(cat)/K-M in mu M-1 s(-1)): 2.29 x 10(-4) vs. 6.41 x 10(-6); ET-1 (1-31) production: 2.19 x 10(-3) vs. 6.57 x 10(-5)), and both of these activities of mouse and human chymase were sensitive to TY-51469. Furthermore, extracts from mouse peritoneal mast cells, LUVA cells and human aorta homogenates contained processing activities toward the fluorogenic chymase substrate as well as Big ET-1, all of which were sensitive to TY-51469. Finally, the pressor responses to Big ET-1 but not to ET-1 were significantly reduced in conscious and free moving mMCP-4 KO mice when compared to wild type congeners. Our results suggest that both mouse and human chymases have potent ET-1 (1-31)-producing abilities, with the murine isoform being more efficient.

  • 19. Siedle, Bettina
    et al.
    Gustavsson, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Johansson, Senia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Murillo, Renato
    Castro, Victor
    Bohlin, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Merfort, Irmgard
    The effect of sesquiterpene lactones on the release of human neutrophil elastase2003In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 65, no 5, p. 897-903Article in journal (Refereed)
    Abstract [en]

    Sesquiterpene lactones (SLs) are natural products responsible for the anti-inflammatory activity of a variety of medicinal plants, mainly from the Asteraceae family. Here, we investigated whether they also influence the process of exocytosis of pro-inflammatory enzymes, such as the human neutrophil elastase (HNE). Altogether, eight structurally different SLs from the eudesmanolide, guaianolide, pseudoguaianolide, and germacranolide type were studied. Neutrophils were isolated from fresh human blood. After pre-incubation with different concentrations of the respective SL and cytochalasin B, the exocytosis of elastase was initiated either by platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine. Inhibition of HNE release was measured by p-nitroaniline formation. The SLs exhibited an inhibitory effect on elastase release from neutrophils challenged either by platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine. Concentration-response curves were recorded and the IC(50) values ranged from 2 to 30 microM. Studies on isolated HNE showed that a selective direct inhibition on HNE can be excluded. Interestingly, the inhibitory activity did not correlate with the number of alpha,beta-unsaturated carbonyl functions. The structure-activity relationship and the molecular mechanism are discussed.

  • 20.
    Strese, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Wickström, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Fuchs, Peder Fredlund
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dale, Tim
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    The novel alkylating prodrug melflufen (J1) inhibits angiogenesis in vitro and in vivo2013In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 86, no 7, p. 888-895Article in journal (Refereed)
    Abstract [en]

    Aminopeptidase N (APN) has been reported to have a functional role in tumor angiogenesis and repeatedly reported to be over-expressed in human tumors. The melphalan-derived prodrug melphalan-flufenamide (melflufen, previously designated J1) can be activated by APN. This suggests that this alkylating prodrug may exert anti-angiogenic properties, which will possibly contribute to the anti-tumoral activity in vivo. This work presents a series of experiments designed to investigate this effect of melflufen. In a cytotoxicity assay we show that bovine endothelial cells were more than 200 times more sensitive to melflufen than to melphalan, in HUVEC cells the difference was more than 30-fold and accompanied by aminopetidase-mediated accumulation of intracellular melphalan. In the chicken embryo chorioallantoic membrane (CAM) assay it is indicated that both melflufen and melphalan inhibit vessel ingrowth. Two commercially available assays with human endothelial cells co-cultured with fibroblasts (TCS Cellworks AngioKit, and Essen GFP-AngioKit) also illustrate the superior anti-angiogenic effect of melflufen compared to melphalan. Finally, in a commercially available in vivo assay in mice (Cultrex DIVAA angio-reactor assay) melflufen displayed an anti-angiogenic effect, comparable to bevacizumab. In conclusion, this study demonstrates through all methods used, that melphalan-flufenamide besides being an alkylating agent also reveals anti-angiogenic effects in different preclinical models in vitro and in vivo. 

  • 21.
    Turpaev, Kyril
    et al.
    Institut de Chimie des Substances Naturelles, Gif-sur-Yvette, France.
    Ermolenko, Mikhail
    Cresteil, Thierry
    Drapier, Jean Claude
    Benzylidenemalononitrile compounds as activators of cell resistance to oxidative stress and modulators of multiple signaling pathways: A structure-activity relationship study2011In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 82, no 5, p. 535-547Article in journal (Refereed)
    Abstract [en]

    Benzylidenemalononitrile (BMN) tyrphostins are well known as potent tyrosine kinase inhibitors. Moreover, in recent years it has been recognized that members of the tyrphostin family possess additional biological activities independent of their ability to inhibit protein tyrosine kinases. In this study, we examined the relationship between the structure of 49 BMNs and related compounds, and their capacity to induce heme oxygenase 1 (HO-1) gene expression in U937 human monocytic cells, to activate upstream signaling pathways and to protect cells against menadione-induced oxidative stress. It was found that the electron-withdrawing (NO(2), CN, halogen) groups in BMN molecules and double meta-MeO substituents increased the HO-1 gene induction, while the electron-donating groups in ortho/para position (OH, MeO and N-morpholino) significantly decreased it. The magnitude of activation of c-Jun, Nrf2, p38 MAPK, and p70S6K correlated with specific substitution patterns in the BMN structure. BMN-dependent maximal up-regulation of HO-1 required parallel increase in Nrf2 and phospho-c-Jun cellular levels. Liquid chromatography mass spectrometry (LC-MS) analysis revealed that BMNs can generate conjugates with one or two glutathione equivalent(s). This study supports the hypothesis that BMNs induce the expression of protective genes by alkylating sensitive cysteine residues of regulatory factors.

  • 22.
    Wickström, Malin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Danielsson, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Rickardson, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Isaksson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Lövborg, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Pharmacological profiling of disulfiram using human tumor cell lines and human tumor cells from patients2007In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 73, no 1, p. 25-33Article in journal (Refereed)
    Abstract [en]

    The thiocarbamate drug disulfiram has been used for decades in the treatment of alcohol abuse. Disulfiram induces apoptosis in a number of tumor cell lines and was recently by us proposed to act as a 26S proteasome inhibitor. In this work we characterized disulfiram in vitro with regard to tumor-type specificity, possible mechanisms of action and drug resistance and cell death in human tumor cell lines and in 78 samples of tumor cells from patients using the fluorometric microculture cytotoxicity assay and the automated fluorescence-imaging microscope ArrayScan®. Disulfiram induced cytotoxicity in a biphasic pattern in both cell lines and patient tumor cells. Disulfiram induced apoptosis as measured by cell membrane permeability, nuclear fragmentation/condensation and caspase-3/7 activation using high content screening assays. For many of the cell lines tested disulfiram was active in sub-micromolar concentrations. When comparing the log IC50 patterns with other cytotoxic agents, disulfiram showed low correlation (R < 0.5) with all drugs except lactacystin (R = 0.69), a known proteasome inhibitor, indicating that the two substances may share mechanistic pathways. Disulfiram was more active in hematological than in solid tumor samples, but substantial activity was observed in carcinomas of the ovary and the breast and in non-small cell lung cancer. Disulfiram also displayed higher cytotoxic effect in cells from chronic lymphocytic leukemia than in normal lymphocytes (p < 0.05), which may indicate some tumor selectivity. These results together with large clinical experience and relatively mild side effects encourage clinical studies of disulfiram as an anti-cancer agent.

  • 23.
    Wickström, Malin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Viktorsson, Kristina
    Lundholm, Lovisa
    Aesoy, Reidun
    Nygren, Helen
    Sooman, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Vogel, Lotte Katrine
    Lewensohn, Rolf
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    The alkylating prodrug J1 can be activated by aminopeptidase N, leading to a possible target directed release of melphalan2010In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 79, no 9, p. 1281-1290Article in journal (Refereed)
    Abstract [en]

    The alkylating prodrug of melphalan, J1 (melphalanyl-l-p-fluorophenylalanyl ethyl ester) is currently in early clinical trials. Preclinical studies have shown that J1-mediated cytotoxicity is dependent on hydrolytic activity of tumor cells. In this report we have analyzed potential peptidases and esterases of importance for release of free melphalan from J1. Exposure of tumor cell lines to J1 resulted in a significant increased level of free intracellular melphalan, at least tenfold at Cmax, compared to exposure to melphalan at the same molar concentration. This efficient intracellular delivery could be inhibited in both magnitude and in time by bestatin, a broad spectrum inhibitor of the aminopeptidases, including the metalloproteinase aminopeptidase N (APN, EC 3.4.11.2.), and ebelactone A, an esterase inhibitor. These effects resulted, as expected, in decreased cytotoxic effects of J1. A specific role of APN in hydrolyzing J1 releasing free melphalan was demonstrated in vitro with pure APN enzyme. By using plasmid-based overexpression of APN or down regulation of endogenous APN with siRNA in different tumor cell lines we here confirm the involvement of APN in J1-mediated cytotoxic and apoptotic signaling. In conclusion, this study demonstrates a role of APN in the activation of the melphalan prodrug J1 and subsequently, its cytotoxicity. Given that APN is shown to be overexpressed in several solid tumors our data suggest that J1 may be activated in a tumor selective manner.

  • 24.
    Wincent, Emma
    et al.
    Swetox.
    Kubota, Akira
    Woods Hole Oceanographic Institution.
    Timme-Laragy, Alicia
    Woods Hole Oceanographic Institution.
    Jönsson, Maria E.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
    Hahn, Mark E
    Woods Hole Oceanographic Institution.
    Stegeman, John J
    Woods Hole Oceanographic Institution.
    Biological effects of 6-formylindolo[3,2-b]carbazole (FICZ) in vivo are enhanced by loss of CYP1A function in an Ahr2-dependent manner2016In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 110, p. 117-129Article in journal (Refereed)
    Abstract [en]

    6-Formylindolo[3,2-b]carbazole (FICZ) is a potent aryl hydrocarbon receptor (AHR) agonist that is efficiently metabolized by AHR-regulated cytochrome P4501 enzymes. FICZ is a proposed physiological AHR ligand that induces its own degradation as part of a regulatory negative feedback loop. In vitro studies in cells show that CYP1 inhibition in the presence of FICZ results in enhanced AHR activation, suggesting that FICZ accumulates in the cell when its metabolism is blocked. We used zebrafish (Danio rerio) embryos to investigate the in vivo effects of FICZ when CYP1A is knocked down or inhibited. Embryos were injected with morpholino antisense oligonucleotides targeting CYP1A (CYP1A-MO), Ahr2, or a combination of both. FICZ exposure of non-injected embryos or embryos injected with control morpholino had little effect. In CYP1A-MO-injected embryos, however, FICZ dramatically increased mortality, incidence and severity of pericardial edema and circulation failure, reduced hatching frequency, blocked swim bladder inflation, and strongly potentiated expression of Ahr2-regulated genes. These effects were substantially reduced in embryos with a combined knockdown of Ahr2 and CYP1A, indicating that the toxicity was mediated at least partly by Ahr2. Co-exposure to the CYP1 inhibitor alpha-naphthoflavone (αNF) and FICZ had similar effects as the combination of CYP1A-MO and FICZ. HPLC analysis of FICZ-exposed embryos showed increased levels of FICZ after concomitant CYP1A-MO injection or αNF co-exposure. Together, these results show that a functioning CYP1/AHR feedback loop is crucial for regulation of AHR signaling by a potential physiological ligand in vivo and further highlights the role of CYP1 enzymes in regulating biological effects of FICZ.

  • 25.
    Österroos, Albin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Kashif, Muhammad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Haglund, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Blom, Kristin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Höglund, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Andersson, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Eriksson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Larsson, R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Combination screening in vitro identifies synergistically acting KP372-1 and cytarabine against acute myeloid leukemia2016In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 118, p. 40-49Article in journal (Refereed)
    Abstract [en]

    Cytogenetic lesions often alter kinase signaling in acute myeloid leukemia (AML) and the addition of kinase inhibitors to the treatment arsenal is of interest. We have screened a kinase inhibitor library and performed combination testing to find promising drug-combinations for synergistic killing of AML cells. Cytotoxicity of 160 compounds in the library InhibitorSelect (TM) 384-Well Protein Kinase Inhibitor I was measured using the fluorometric microculture cytotoxicity assay (FMCA) in three AML cell lines. The 15 most potent substances were evaluated for dose-response. The 6 most cytotoxic compounds underwent combination synergy analysis based on the FMCA readouts after either simultaneous or sequential drug addition in AML cell lines. The 4 combinations showing the highest level of synergy were evaluated in 5 primary AML samples. Synergistic calculations were performed using the combination interaction analysis package COMBIA, written in R, using the Bliss independence model. Based on obtained results, an iterative combination search was performed using the therapeutic algorithmic combinatorial screen (TACS) algorithm. Of 160 substances, cell survival was <= 50% at <0.5 mu M for Cdk/Crk inhibitor, KP372-1, synthetic fascaplysin, herbimycin A, PDGF receptor tyrosine kinase inhibitor IV and reference-drug cytarabine. KP372-1, synthetic fascaplysin or herbimycin A obtained synergy when combined with cytarabine in AML cell lines MV4-11 and HL-60. KP372-1 added 24 h before cytarabine gave similar results in patient cells. The iterative search gave further improved synergy between cytarabine and KP372-1. In conclusion, our in vitro studies suggest that combining KP372-1 and cytarabine is a potent and synergistic drug combination in AML.

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Output format
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  • asciidoc
  • rtf