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  • 1. Abdallah, Qasem M. A.
    et al.
    Phillips, Roger M.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cosentino, Laura
    Abdel-Rahman, Hamdy
    Etzad, Jasarat
    Wheelhouse, Richard T.
    Kiakos, Konstantinos
    Bingham, John P.
    Hartley, John A.
    Patterson, Laurence H.
    Pors, Klaus
    Minor structural modifications to alchemix influence mechanism of action and pharmacological activity2012In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 83, no 11, p. 1514-1522Article in journal (Refereed)
    Abstract [en]

    Alchemix is an exemplar of a class of anthraquinone with efficacy against multidrug resistant tumours. We have explored further the mechanism of action of alchemix and investigated the effect of extending its side arm bearing the alkylating functionality with regard to DNA binding and activity against multidrug resistant cancer cells. Increasing the distance between the intercalating chromophore and the alkylating functionality of ICT2901 (propyl), ICT2902 (butyl) and ICT2903 (pentyl), led to a higher number of DNA alkylation sites, more potent topoisomerase II inhibition and generated more apoptotic and necrotic cells when analysed in p53-proficient HCT116 cells. Intriguingly, alchemix, the compound with the shortest distance between its intercalative chromophore and alkylating functionality (ethyl), did not conform to this SAR. A different toxicity pattern against DNA repair defective CHO cell lines as well as arrest of cells in Cl supports a somewhat distinct mode of action by alchemix compared with its analogues. Importantly, both alchemix and ICT2901 demonstrated greater cytotoxic activity against anthraquinone-resistant MCF-7/adr cells than wild-type MCF-7 cells. Subtle synthetic modification in this anthraquinone series has led to significant changes to the stability of DNA-compound complexes and cellular activity. Given that the failure of chemotherapy in the clinic is often associated with MDR, the results of both alchemix and ICT2901 represent important advances towards improved therapies.

  • 2.
    Artursson, Elisabet
    et al.
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Andersson, Per Ola
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Akfur, Christine
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Linusson, Anna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Börjegren, Susanne
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Ekström, Fredrik
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Catalytic-site conformational equilibrium in nerve-agent adducts of acetylcholinesterase: Possible implications for the HI-6 antidote substrate specificity2013In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 85, no 9, p. 1389-1397Article in journal (Refereed)
    Abstract [en]

    Nerve agents such as tabun, cyclosarin and Russian VX inhibit the essential enzyme acetylcholinesterase (AChE) by organophosphorylating the catalytic serine residue. Nucleophiles, such as oximes, are used as antidotes as they can reactivate and restore the function of the inhibited enzyme. The oxime HI-6 shows a notably low activity on tabun adducts but can effectively reactivate adducts of cyclosarin and Russian VX. To examine the structural basis for the pronounced substrate specificity of HI-6, we determined the binary crystal structures of Mus musculus AChE (mAChE) conjugated by cyclosarin and Russian VX and found a conformational mobility of the side chains of Phe338 and His447. The interaction between HI-6 and tabun-adducts of AChE were subsequently investigated using a combination of time resolved fluorescence spectroscopy and X-ray crystallography. Our findings show that HI-6 binds to tabun inhibited Homo sapiens AChE (hAChE) with an IC50 value of 300 μM and suggest that the reactive nucleophilic moiety of HI-6 is excluded from the phosphorus atom of tabun. We propose that a conformational mobility of the side-chains of Phe338 and His447 is a common feature in nerve-agent adducts of AChE. We also suggest that the conformational mobility allow HI-6 to reactivate conjugates of cyclosarin and Russian VX while a reduced mobility in tabun conjugated AChE results in steric hindrance that prevents efficient reactivation.

  • 3. Bellisola, Guiseppe
    et al.
    Fracasso, Giulio
    Ippoliti, Rodolfo
    Menestrina, Gianfranco
    Rosén, Anders
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Soldà, Silvia
    Udali, Silvia
    Tomazzolli, Rossella
    Tridente, Giuseppe
    Colombatti, Marco
    Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase2004In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 67, no 9, p. 1721-1731Article in journal (Refereed)
    Abstract [en]

    Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS-PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls (P<0.05). Conversely, auranofin failed to protect cells from the toxicity of pre-reduced ricin which does not require intracellular reduction of disulfide between the two ricin subunits. We conclude that TrxR, by activating disulfide reductase activity of PDI, can ultimately lead to reduction/activation of ricin and immunotoxin in the cell.

  • 4. Buss, Joan L
    et al.
    Neuzil, Jiri
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology.
    Gellert, Nina
    Weber, Christian
    Ponka, Prem
    Pyridoxal isonicotinoyl hydrazone analogs induce apoptosis in hematopoietic cells due to their iron-chelating properties2003In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 65, no 2, p. 161-172Article in journal (Refereed)
    Abstract [en]

    Analogs of pyridoxal isonicotinoyl hydrazone (PIH) are of interest as iron chelators for the treatment of secondary iron overload and cancer. PIH, salicylaldehyde isonicotinoyl hydrazone (SIH), and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (NIH), the toxicity of which vary over two orders of magnitude, were selected for a study of their mechanisms of toxicity. PIH analogs and their iron complexes caused concentration- and time-dependent apoptosis in Jurkat T lymphocytes and K562 cells. Bcl-2 overexpression was partially anti-apoptotic, suggesting mitochondrial mediation of apoptosis. Since the pan-caspase inhibitor zVAD-fmk did not reduce lysosomal and mitochondrial destabilization, these events occur upstream of caspase activation. In contrast, phosphatidylserine externalization and the development of apoptotic morphology were inhibited significantly, indicating the role of caspases in mediating these later events. Since overexpression of CrmA had no effect on apoptosis, caspase-8 is not likely involved. Fe3+ complexes of SIH and NIH, which accumulated in 59Fe-labeled mouse reticulocytes during incubation with the chelators, also caused apoptosis. BSA, which promotes release of the complexes from cells, reduced the toxicity of SIH and NIH, suggesting that the induction of apoptosis by PIH analogs involves toxic effects mediated by their Fe3+ complexes. Moreover, analogs of these agents lacking the iron-chelating moiety were non-toxic. ⌐ 2002 Published by Elsevier Science Inc.

  • 5.
    Chatzakos, Vicky
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Rundlöf, Anna-Klara
    Karolinska Institutet.
    Ahmed, Dilruba
    Karolinska Institutet.
    De Verdier, Petra J.
    Karolinska Institutet.
    Flygare, Jenny
    Karolinska Institutet.
    Inhibition of sphingosine kinase 1 enhances cytotoxicity, ceramide levels and ROS formation in liver cancer cells treated with selenite2012In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 84, no 5, p. 712-721Article in journal (Refereed)
    Abstract [en]

    High doses of selenite have been shown to induce cell death in acute myeloid leukemia and lung cancercells. In this study, we combined selenite treatment with modulators of sphingolipid metabolism in thehepatocellular carcinoma cell line Huh7. Treatment with 20 mM of selenite reduced the viability of Huh7cells by half and increased the levels of long chain C14-, C16-, C18- and C18:1- ceramides by two-fold.Inhibition of neutral sphingomyelinase with 3-O-methylsphingosine significantly reduced the cytotoxiceffect of selenite. In line with this result, selenite caused a 2.5-fold increase in the activity of neutralsphingomyelinase. The sphingosine kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophe-nyl)thiazole (SK1-II) sensitized the cells to the cytotoxic effects of selenite. Preincubation with 10 mM ofSK1-II prior to treatment with 10 mM of selenite caused induction of apoptosis and gave rise to a 2.5-foldincrease in C14-, C16-, C18- and C18:1- ceramides. Instead, 50 mM of SK1-II combined with 10 mM ofselenite caused accumulation of cells in G1/S phases, but less apoptosis and accumulation of ceramides.The formation of reactive oxygen species (ROS) after treatment with 10 mM of selenite was maximallyenhanced by 1 mM of SK1-II. Moreover, combined treatment with SK1-II and 10 mM of selenitesynergistically reduced the number of viable Huh7 cells, while the non-tumorigenic hepatocyte cell lineMIHA remained unaffected by the same treatment. These results raise the possibility that a combinationof selenite and SK1 inhibitors could be used to treat liver cancer cells, that are regarded as drug resistant,at doses that are non-toxic to normal liver cells.

  • 6.
    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.

  • 7. 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.

  • 8.
    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.

  • 9.
    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.

  • 10.
    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.

  • 11.
    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.

  • 12.
    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.

  • 13.
    Fotoohi, A K
    et al.
    KI, Stockholm.
    Wrabel, A
    KI Stockholm.
    Moshfegh, A
    KI, Stockholm.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Albertioni, F
    KI, Stockholm.
    Molecular mechanisms underlying the enhanced sensitivity of thiopurine-resistant T-lymphoblastic cell lines to methyl mercaptopurineriboside2006In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 72, no 7, p. 816-823Article in journal (Refereed)
    Abstract [en]

    Methylmercaptopurine riboside (meMPR), a cellular metabolite of 6-mercaptopurine (6-MP), is a potent inhibitor of de novo purine synthesis (DNPS). Human MOLT4 T-lymphoblastic leukaemia cells that have acquired resistance to 6-MP or 6-thioguanine (6-TG) as a consequence of defective transport exhibit enhanced sensitivity to meMPR. HPLC-based analysis of the transport of meMPR revealed normal uptake of this compound by our thiopurine-resistant cell sublines, suggesting a route of transport distinct from that for 6-MP and 6-TG. Studies on the wild-type parental leukemic cells showed that adenosine, dipyridamole and nitrobenzylthioinosine inhibit uptake of meMPR to a significant extent, whereas Na+ ions have no influence on this process. Transfection of these leukemic cells with small interference RNA molecules targeting the gene encoding the first member of the family of equiliberative nucleoside transporters (ENT1) strongly reduced the initial rate of meMPR transport. Our resistant cell lines exhibited 30-52% reductions (p < 0.005) in their levels of mRNA encoding several proteins involved in de novo purine synthesis, i.e., aminoimidazole carboxamide ribonucleotide formyltransferase, glycinamide ribonucleotide transformylase and guanine monophosphate synthetase. Consequently, the rate of de novo purine synthesis in these resistant sublines was decreased by 50%. Furthermore, the levels of ribonucleoside triphosphates in these cells were significantly lower than in the non-resistant parental cells. In combination, a reduced rate of de novo purine synthesis together with low levels of ribonucleoside triphosphates can explain the enhanced sensitivity of our thiopurine-resistant cell lines to meMPR. In this manner, meMPR bypasses the mechanisms of resistance to thiopurines and is even more cytotoxic towards resistant than towards wild-type cells. © 2006.

  • 14. 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.

  • 15.
    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.

  • 16.
    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.

  • 17.
    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.

  • 18.
    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.

  • 19. Imtiaz, Bushra
    et al.
    Tolppanen, Anna-Maija
    Kivipelto, Miia
    Stockholm University, Faculty of Social Sciences, Aging Research Center (ARC), (together with KI). University of Eastern Finland, Finland.
    Soininen, Hilkka
    Future directions in Alzheimer's disease from risk factors to prevention2014In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 88, no 4, p. 661-670Article, review/survey (Refereed)
    Abstract [en]

    The increase in life expectancy has resulted in a high occurrence of dementia and Alzheimer's disease (AD). Research on AD has undergone a paradigm shift from viewing it as a disease of old age to taking a life course perspective. Several vascular, lifestyle, psychological and genetic risk factors influencing this latent period have been recognized and they may act both independently and by potentiating each other. These risk factors have consequently been used to derive risk scores for predicting the likelihood of dementia. Despite population differences, age, low education and vascular risk factors were identified as key factors in all scoring systems. Risk scores can help to identify high-risk individuals who might benefit from different interventions. The European Dementia Prevention Initiative (EDPI), an international collaboration, encourages data sharing between different randomized controlled trials. At the moment, it includes three large ongoing European trials: Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER), Prevention of Dementia by Intensive Vascular Care (preDIVA), and Multidomain Alzheimer Prevention study (MAPT). Recently EDPI has developed a Healthy Aging through Internet Counseling in Elderly (HATICE) program, which intends to manage modifiable risk factors in an aged population through an easily accessible Internet platform. Thus, the focus of dementia research has shifted from identification of potential risk factors to using this information for developing interventions to prevent or delay the onset of dementia as well as identifying special high-risk populations who could be targeted in intervention trials.

  • 20. 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.

  • 21. Jostarndt, K
    et al.
    Rubic, T
    Kuhn, H
    Anthosen, M W
    Andera, L
    Gellert, N
    Trottman, M
    Weber, Christian
    Johansen, B
    Hrboticky, N
    Neuzil, Jiri
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology.
    Enzymatically modified low-density lipoprotein upregulates CD36 in low-differentiated monocytic cells in a peroxisome proliferator-activated receptor-γ-dependent way2004In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 67, no 5, p. 841-854Article in journal (Refereed)
    Abstract [en]

    Peroxisome proliferator-activated receptor-γ (PPARγ) has been suggested to upregulate CD36. Since free oxidized polyunsaturated fatty acids are PPARγ ligands, we studied the effects of LDL modified by the simultaneous action of sPLA2 and 15-lipoxygenase (15LO) on CD36 expression and PPARγ activation in monocytic cells. Exposure of MM6 cells, which do not express CD36 or other scavenger receptors, to such enzymatically modified LDL (enzLDL) resulted in upregulation of CD36 surface protein and mRNA expression. Similar effects were observed with free 13-hydroperoxyoctadecadienoic acid but not its esterified counterpart. Less pronounced effects were observed with LDL modified by 15LO alone. Upregulation of CD36 was inversely correlated to the state of cell differentiation, as showed by lower response to enzLDL of the scavenger receptor-expressing MM6-sr and THP1 cells. Importantly, LDL modified by sPLA2 and 15LO did not efficiently induce upregulation CD36 in PPARγ-deficient macrophage-differentiated embryonic stem cells confirming a role of PPARγ in CD36 expression in cells stimulated with enzLDL. Our data show that LDL modified with physiologically relevant enzymes stimulates CD36 expression in non-differentiated monocytes and that this process involves PPARγ activation. These effects of enzLDL can be considered pro-atherogenic in the context of early atherosclerosis.

  • 22.
    Lindgren, Maria
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Rosenthal-Aizman, Katri
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Saar, Külliki
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Eiríksdóttir, Emelía
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sassian, Meeri
    Östlund, Pernilla
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cell-penetrating peptide2006In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 71, no 4, p. 416-425Article in journal (Refereed)
    Abstract [en]

    Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Here, we present a new approach to overcome the setback of drug resistance by designing a conjugate of a cell-penetrating peptide and the cytostatic agent methotrexate (MTX). Two different peptides, YTA2 and YTA4, were designed and their intracellular delivery efficiency was characterized by fluorescence microscopy and quantified by fluorometry. MTX was conjugated to the transport peptides and the ability of the peptide–MTX conjugates to inhibit dihydrofolate reductase, the target enzyme of MTX, was found to be 15 and 20 times less potent than MTX. In addition, in vitro studies were performed in a drug resistant cell model using the 100-fold MTX resistant breast cancer cells MDA-MB-231. At a concentration of 1 mM, the peptide–MTX conjugates were shown to overcome MTX resistance and kill the cells more efficiently than MTX alone. Estimated EC50’s were determined for MTX, MTXYTA2 and YTA2 to be 18.5, 3.8 and 20 µM, respectively. In summary, cell-penetrating peptide conjugation of MTX is a new way of increasing delivery, and thereby, the potency of already well-characterized therapeutic molecules into drug resistant tumour cells.

  • 23. 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.

  • 24.
    Lotfi, Kourosh
    et al.
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pharmacology.
    Karlsson, Karin
    Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Haematology UHL.
    Fyrberg, Anna
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Juliusson, Gunnar
    Lund University Hospital.
    Jonsson, Viggo
    Oslo University.
    Peterson, Curt
    Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pharmacology.
    Eriksson, Staffan
    Swedish University of Agricultural Sciences, The Biomedical Center, Uppsala.
    Albertioni, Freidoun
    Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    The pattern of deoxycytidine- and deoxyguanosine kinase activity in relation to messenger RNA expression in blood cells from untreated patients with B-cell chronic lymphocytic leukemia2006In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 71, no 6, p. 882-890Article in journal (Refereed)
    Abstract [en]

    Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) catalyze the first step in the intracellular cascade of fludarabine (2-fluoroadenine-β- d-arabinofuranoside) and cladribine (2-chlorodeoxyadenosine) phosphorylation, which leads to activation of these prodrugs, commonly used for treatment of chronic lymphocytic leukemia (CLL). Thus, resistance to nucleoside analogues may primarily be due to low levels of deoxynucleoside kinase activity. The purpose of this study was to investigate the activity profiles of dCK and dGK and characterize the possible relationship between the levels of dCK enzymatic activities and mRNA levels in B-CLL cells from untreated patient samples in an attempt to determine the best approach for predicting sensitivity to nucleoside analogues and thereby optimizing treatment of CLL. For this purpose, dCK and dGK analyses were done in blood cells from 59 untreated symptomatic patients with CLL. The dGK activity towards 2-chlorodeoxyadenosine was significantly lower than of dCK (median 73 pmol/mg protein/min (85-121, 95% CI) versus 353 pmol/mg protein/min (331-421)). The median dCK mRNA level was 0.107 (0.096-0.120, 95% CI). There was a lack of correlation between the activities of dCK and dGK, which indicates that these proteins are regulated independently. We also found that the dCK and dGK activity measurement towards their endogenous substrates were comparable to the nucleoside analogues tested. Such variations in enzyme activities and mRNA levels may well explain differences in clinical responses to treatment. There was no correlation between the levels of dCK mRNAs and enzymatic activities using a quantitative real-time PCR procedure. Sequencing of dCK mRNA did not reveal alternate splicing or mutations in the coding region. The relation between activity and mRNA levels was studied by short interfering RNA (siRNA) method, which showed that in the siRNA treated cells the down-regulation of dCK expression, and activity followed each other. However, in control cells the mRNA levels remained stable but the protein activity markedly decreased. These data demonstrate that the dCK activity is not reflected by dCK mRNA expression that indicates a post-translational mechanism(s). © 2005 Elsevier Inc. All rights reserved.

  • 25. Lundgren, B
    et al.
    Andersson, K
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    DePierre, J W
    Effects of dietary treatment with 11 dicarboxylic acids, diethylcarboxylic esters and fatty acids on peroxisomal fatty acid beta-oxidation, epoxide hydrolases and lauric acid omega-hydroxylation in mouse liver.1992In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 43, no 4Article in journal (Refereed)
    Abstract [en]

    C57B1/6 male mice were exposed through their diet to 11 dicarboxylic acids, carboxylic acids and diethyldicarboxylesters for 10 days. For the diacids and diethylesters this treatment resulted in a chain length-dependent induction of lauryl-CoA oxidase and cyanide-insensitive palmitoyl-CoA oxidation activities. A chain length of 12 carbon atoms or more seemed to be necessary for induction of these two activities. In addition, the same chain length dependence was observed for induction of lauric acid omega + omega-1 hydroxylase activity and increase in the protein content of the mitochondrial fraction. Treatment with two "natural" fatty acids, i.e. lauric and palmitic acid gave no effect at all on these various parameters. In no case was induction of cytosolic and mitochondrial epoxide hydrolase activities observed. Instead, a slight decrease in these activities was observed after administration of diacids with a chain length of 4-8 carbon atoms, whereas microsomal epoxide hydrolase activity was concurrently induced.

  • 26.
    Lundgren, B
    et al.
    Stockholm University.
    Meijer, J
    DePierre, J W
    Induction of cytosolic and microsomal epoxide hydrolases and proliferation of peroxisomes and mitochondria in mouse liver after dietary exposure to p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid.1987In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 36, no 6Article in journal (Refereed)
    Abstract [en]

    The effects of dietary exposure to 0.125% (w/w) p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid on the content of peroxisomes and levels of certain xenobiotic-metabolizing enzymes in mouse liver have been investigated. In agreement with the literature on rat liver 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were found to cause extensive proliferation of peroxisomes (as judged by the total levels of "mitochondrial" protein, carnitine acetyltransferase, cyanide-insensitive palmitoyl-CoA oxidation and catalase) in mouse liver. On the other hand, exposure to p-chlorophenoxyacetic acid did not significantly affect any of these parameters. As with certain other peroxisome proliferators, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid increased total cytochrome oxidase activity as well. In addition, dietary exposure to 2,4-dichlorophenoxyacetic acid and to 2,4,5-trichlorophenoxyacetic acid resulted in increases in the activities of cytosolic and microsomal epoxide hydrolases in mouse liver and generally less pronounced increases in the total cytosolic glutathione transferase activity and microsomal content of cytochrome P-450. In the case of cytochrome P-450, this process can be said to be a true induction (i.e. the amount of enzyme protein is increased), because the assay procedure for cytochrome P-450 measures holoenzyme amount. Immunoquantitation demonstrated that this was also the case for the changes in cytosolic epoxide hydrolase. The dramatic differences in proliferation of peroxisomes and induction of xenobiotic-metabolizing enzymes seen here with compounds differing relatively little in structure may indicate that a receptor mechanism of some kind is involved.

  • 27.
    Madeja, Zbigniew
    et al.
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden / Jagiellonian University, Kraków, Poland.
    Sroka, Jolanta
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden / Jagiellonian University, Kraków, Poland.
    Nyström, Christina
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    Björkhem-Bergman, Linda
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    Nordman, Tomas
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    Damdimopoulos, Anastasios
    Karolinska Institutet, Huddinge, Sweden.
    Nalvarte, Ivan
    Karolinska Institutet, Huddinge, Sweden.
    Eriksson, Lennart C.
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    Spyrou, Giannis
    Foundation of Biomedical Research, Academy of Athens, Greece / Karolinska Institutet, Huddinge, Sweden.
    Olsson, Jerker M.
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    Björnstedt, Mikael
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    The role of thioredoxin reductase activity in selenium-induced cytotoxicity2005In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 69, no 12, p. 1765-1772Article in journal (Refereed)
    Abstract [en]

    The selenoprotein thioredoxin reductase is a key enzyme in selenium metabolism, reducing selenium compounds and thereby providing selenide to synthesis of all selenoproteins. We evaluated the importance of active TrxR1 in selenium-induced cytotoxicity using transfected TrxR1 over-expressing stable Human Embryo Kidney (HEK-293) cells and modulation of activity by pretreatment with low concentration of selenite. Treatment with sodium selenite induced cytotoxity in a dose-dependent manner in both TrxR1 over-expressing and control cells. However, TrxR1 over-expressing cells, which were preincubated for 72h with 0.1 microM selenite, were significantly more resistant to selenite cytotoxicity than control cells. To demonstrate the early effects of selenite on behaviour of HEK-293 cells, we also investigated the influence of this compound on cell motility. We observed inhibition of cell motility by 50 microM selenite immediately after administration. Moreover, TrxR1 over-expressing cells preincubated with a low concentration of selenite were more resistant to the inhibitory effect of 50 microM selenite than those not preincubated. It was also observed that the TrxR over-expressing cells showed higher TrxR1 activity than control cells and the preincubation of over-expressing cells with 0.1 microM selenite induced further significant increase in the activity of TrxR1. On the other hand, we demonstrated that TrxR1 over-expressing cells showed decreased glutathione peroxidase activity compared to control cells. These data strongly suggest that TrxR1 may be a crucial enzyme responsible for cell resistance against selenium cytotoxicity.

  • 28. Minami, S. Sakura
    et al.
    Shen, Vivian
    Le, David
    Krabbe, Grietje
    Asgarov, Rustam
    Stockholm University, Faculty of Science, Department of Neurochemistry. Gladstone Institute of Neurological Diseases, United States.
    Perez-Celajes, Liberty
    Lee, Chih-Hung
    Li, Jinhe
    Donnelly-Roberts, Diana
    Gan, Li
    Reducing inflammation and rescuing FTD-related behavioral deficits in progranulin-deficient mice with alpha 7 nicotinic acetylcholine receptor agonists2015In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 97, no 4, p. 454-462Article in journal (Refereed)
    Abstract [en]

    Mutations in the progranulin gene cause frontotemporal dementia (FTD), a debilitating neurodegenerative disease that involves atrophy of the frontal and temporal lobes and affects personality, behavior, and language. Progranulin-deficient mouse models of FTD exhibit deficits in compulsive and social behaviors reminiscent of patients with FTD, and develop excessive microgliosis and increased release of inflammatory cytokines. Activation of nicotinic acetylcholine receptors (nAChRs) by nicotine or specific alpha 7 nAChR agonists reduces neuroinflammation. Here, we investigated whether activation of nAChRs by nicotine or alpha 7 agonists improved the excessive inflammatory and behavioral phenotypes of a progranulin-deficient FTD mouse model. We found that treatment with selective alpha 7 agonists, PHA-568487 or ABT-107, strongly suppressed the activation of NF-kappa B in progranulin-deficient cells. Treatment with ABT-107 also reduced microgliosis, decreased TNF alpha levels, and reduced compulsive behavior in progranulin-deficient mice. Collectively, these data suggest that targeting activation of the alpha 7 nAChR pathway may be beneficial in decreasing neuroinflammation and reversing some of the behavioral deficits observed in progranulin-deficient FTD.

  • 29.
    Nguyen, Minh Vu Chuong
    et al.
    University of Grenoble 1, France .
    Lardy, Bernard
    University of Grenoble 1, France .
    Rousset, Francis
    University of Grenoble 1, France .
    Hazane-Puch, Florence
    University Hospital CHU Grenoble, France .
    Zhang, Leilei
    University of Grenoble 1, France .
    Trocme, Candice
    University Hospital CHU Grenoble, France .
    Serrander, Lena
    Östergötlands Läns Landsting, Heart and Medicine Center, Department of Infectious Diseases. Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Krause, Karl-Heinz
    Medical Faculty and University Hospital, Geneva, Switzerland.
    Morel, Francoise
    University of Grenoble 1, France .
    Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox42013In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 85, no 11, p. 1644-1654Article in journal (Refereed)
    Abstract [en]

    NADPH oxidase Nox4 is expressed in a wide range of tissues and plays a role in cellular signaling by providing reactive oxygen species (ROS) as intracellular messengers. Nox4 oxidase activity is thought to be constitutive and regulated at the transcriptional level; however, we challenge this point of view and suggest that specific quinone derivatives could modulate this activity. In fact, we demonstrated a significant stimulation of Nox4 activity by 4 quinone derivatives (AA-861, tBuBHQ tBuBQ and duroquinone) observed in 3 different cellular models, HEK293E, T-REx (TM), and chondrocyte cell lines. Our results indicate that the effect is specific toward Nox4 versus Nox2. Furthermore, we showed that NAD(P)H:quinone oxidoreductase (NQO1) may participate in this stimulation. Interestingly, Nox4 activity is also stimulated by reducing agents that possibly act by reducing the disulfide bridge (Cys226, Cys270) located in the extracellular E-loop of Nox4. Such model of Nox4 activity regulation could provide new insight into the understanding of the molecular mechanism of the electron transfer through the enzyme, i.e., its potential redox regulation, and could also define new therapeutic targets in diseases in which quinones and Nox4 are implicated.

  • 30.
    Nilsson, Kristofer F.
    et al.
    Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
    Lundgren, Michael
    nalyscentrum, Stockholm, Sweden.
    Agvald, Per
    Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
    Adding, Chistofer
    Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
    Linnarsson, Dag
    Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
    Gustafsson, Lars
    Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
    Formation of new bioactive organic nitrites and their identification with gas chromatography-mass spectrometry and liquid chromatography coupled to nitrite reduction2011In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 82, no 3, p. 248-259Article in journal (Refereed)
    Abstract [en]

    Nitric oxide (NO) donors, notably organic nitrates and nitrites are used therapeutically but tolerance develops rapidly, making the use of e.g. nitroglycerin difficult. NO donation in the pulmonary vascular bed might be useful in critically ill patients. Organic nitrites are not associated with tachyphylaxis but may induce methaemoglobinemia and systemic hypotension which might hamper their use. We hypothesised that new lung-selective NO donors can be identified by utilizing exhaled NO as measure for pulmonary NO donation and systemic arterial pressure to monitor hypotension and tolerance development. Solutions of alcohols and carbohydrates were reacted with NO gas and administered to ventilated rabbits for evaluation of in vivo NO donation. Chemical characterization was made by liquid chromatography with on-line nitrite reduction (LC-NO) and by gas chromatography-mass spectrometry (GC-MS). In vivo experiments showed that the hydroxyl-containing compounds treated with NO gas yielded potent NO donors, via nitrosylation to organic nitrites. Analyses by LC-NO showed that the reaction products were able to release NO in vitro. In GC-MS the reaction products were determined to be the organic nitrites, where some are new chemical entities. Non-polar donors preferentially increased exhaled NO with less effect on systemic blood pressure whereas more polar molecules had larger effects on systemic blood pressure and less on exhaled NO. We conclude that new organic nitrites suitable for intravenous administration are produced by reacting NO gas and certain hydroxyl-containing compounds in aqueous solutions. Selectivity of different organic nitrites towards the pulmonary and systemic circulation, respectively, may be determined by molecular polarity.

  • 31.
    Palm-Espling, Maria E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Reaction of platinum anticancer drugs and drug derivatives with a copper transporting protein, Atox12012In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 83, no 7, p. 874-881Article in journal (Refereed)
    Abstract [en]

    Platinum (Pt) containing anticancer drugs have been used in cancer treatment for several decades as they trigger cell death upon DNA binding. Pt-containing anticancer drugs and drug derivates with a variety of ligands around the Pt center (with Cisplatin being most well known) exist today in clinics and in clinical trials. However, a major drawback with these drugs is limited efficacy due to side reactions resulting in cell resistance. The cellular copper (Cu) transport pathway is proposed to be responsible for part of these side reactions through interactions with the Pt-containing drugs and possibly cellular export of Pt. The cytoplasmic Cu chaperone, Atox1, was recently found to bind Cisplatin in vitro and, when over-expressed in Escherichia coli, in vivo. Here we investigate how the chemical properties of six Pt-substances differentially affect binding, unfolding, and aggregation of Atox1 in vitro using near- and far-UV circular dichroism (CD) spectroscopy and SDS-PAGE. The results show that both ligand type and orientation dictate the interactions with Atox1. Only substances with two good leaving groups in cis-configuration result in near-UV CD changes that report on Cu–Pt interactions. The different substances promote Atox1 unfolding in a pattern that can be explained by ligand chemistry and geometry. Our work emphasize that ligands around the Pt-center have decisive roles in tuning protein interactions (prior to DNA binding) and therefore they also dictate the level of drug side effects and cellular resistance.

  • 32. Permadi, H
    et al.
    Lundgren, B
    Andersson, K
    DePierre, J W
    Effects of perfluoro fatty acids on xenobiotic-metabolizing enzymes, enzymes which detoxify reactive forms of oxygen and lipid peroxidation in mouse liver.1992In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 44, no 6Article in journal (Refereed)
    Abstract [en]

    Male mice were exposed via their diet to perfluoro fatty acids of various chain-lengths (2-10 carbon atoms) at different doses (0.02 and 0.1% weight) and for different periods of time (2-10 days). Thereafter, we monitored effects on liver and body weights and a number of hepatic parameters, including mitochondrial protein content, microsomal contents of cytochromes P450 and b5, NADPH-cytochrome P450 reductase activity [measured as NADPH-cytochrome c reductase (EC 1.6.2.3)], microsomal and cytosolic epoxide hydrolase (EC 3.3.2.3) activities, cytosolic DT-diaphorase (EC 1.6.99.2), glutathione transferase (EC 2.5.1.18), glutathione peroxidase (EC 1.11.1.9) and superoxide dismutase (EC 1.15.1.1) activities, and levels of thiobarbituric acid-reactive material (as an indicator of lipid peroxidation) in the mitochondrial subfraction. The most dramatic changes observed were a 5-9-fold increase in mitochondrial protein, a 3-6-fold increase in the microsomal content of cytochrome P450, a 3-10-fold increase in cytosolic DT-diaphorase activity, an approximately 2-fold increase in cytosolic epoxide hydrolase activity and as much as a 60% decrease in the level of thiobarbituric acid-reactive compounds in the mitochondrial fraction. Smaller increases in microsomal epoxide hydrolase activity and decreases in cytosolic glutathione peroxidase activity were also observed. Of the perfluoro fatty acids tested, perfluorooctanoic acid caused the largest changes in the parameters examined here. Dietary exposure of mice to a 0.02% dose of this substance for 10 days results in a maximal or near-maximal effect in most cases.

  • 33.
    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.

  • 34.
    Sandler, Stellan
    et al.
    Institutionen för medicinsk cellbiologi, Uppsala universitet.
    Andersson, Annika K.
    Institutionen för medicinsk cellbiologi, Uppsala universitet.
    Larsson, Jenny
    Makeeva, Natalia
    Institutionen för medicinsk cellbiologi, Uppsala universitet.
    Olsen, Therese
    Institutionen för medicinsk cellbiologi, Uppsala universitet.
    Arkhammar, Per O.G.
    Novo Nordisk A/S.
    Bondo Hansen, John
    Novo Nordisk A/S.
    Karlssson, F. Anders
    Institutionen för medicinska vetenskaper, Uppsala universitet.
    Welsh, Nils
    Institutionen för medicinsk cellbiologi, Uppsala universitet.
    Possible role of an ischemic preconditioning-like response mechanism in KATP 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)
  • 35.
    Sato, Masaaki
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Monash University, Australia.
    Evans, Bronwyn A.
    Sandström, Anna L.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Chia, Ling Yeong
    Mukaida, Saori
    Bui, San
    Anh, Nguyen
    Lim, Linzi
    Tan, Christina Y. R.
    Baltos, Jo-Anne
    White, Paul J.
    May, Lauren T.
    Hutchinson, Dana S.
    Summers, Roger J.
    Bengtsson, Tore
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    alpha(1A)-Adrenoceptors activate mTOR signalling and glucose uptake in cardiomyocytes2018In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 148, p. 27-40Article in journal (Refereed)
    Abstract [en]

    The capacity of G protein-coupled receptors to modulate mechanistic target of rapamycin (mTOR) activity is a newly emerging paradigm with the potential to link cell surface receptors with cell survival. Cardiomyocyte viability is linked to signalling pathways involving Akt and mTOR, as well as increased glucose uptake and utilization. Our aim was to determine whether the am-adrenoceptor (AR) couples to these protective pathways, and increased glucose uptake. We characterised alpha(1A)-AR signalling in CHO-K1 cells co-expressing the human alpha(1A)-AR and GLUT4 (CHO alpha(1A)GLUT4myc) and in neonatal rat ventricular cardiomyocytes (NRVM), and measured glucose uptake, intracellular Ga2* mobilization, and phosphorylation of mTOR, Akt, 5' adenosine monophosphate-activated kinase (AMPK) and S6 ribosomal protein (S6rp). In both systems, noradrenaline and the alpha(1A)-AR selective agonist A61603 stimulated glucose uptake by parallel pathways involving mTOR and AMPK, whereas another alpha(1A)-AR agonist oxymetazoline increased glucose uptake predominantly by mTOR. All agonists promoted phosphorylation of mTOR at Ser2448 and Ser2481, indicating activation of both mTORC1 and mTORC2, but did not increase Akt phosphorylation. In CHO alpha(1A)GLUT4myc cells, siRNA directed against rictor but not raptor suppressed alpha(1A)-AR mediated glucose uptake. We have thus identified mTORC2 as a key component in glucose uptake stimulated by alpha(1A)-AR agonists. Our findings identify a novel link between the alpha(1A)-AR, mTORC2 and glucose uptake, that have been implicated separately in cardiomyocyte survival. Our studies provide an improved framework for examining the utility of alpha(1A)-AR selective agonists as tools in the treatment of cardiac dysfunction.

  • 36.
    Schulze-Osthoff, Klaus
    et al.
    Institute of Biochemistry, Albert-Ludwigs-University, D-79104 Freiburg, Germany.
    Los, Marek Jan
    Division of Molecular Oncology, German Cancer Research Center, D-69120 Heidelberg, Germany.
    Baeuerle, Pa
    Institute of Biochemistry, Albert-Ludwigs-University, D-79104 Freiburg, Germany.
    Redox Signaling by Transcription Factors Nf-Kappa-B and Ap-1 in Lymphocytes1995In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 50, no 6, p. 735-741Article in journal (Refereed)
  • 37.
    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)
  • 38.
    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. 

  • 39. 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.

  • 40. 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.

  • 41.
    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. 

  • 42.
    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.

  • 43.
    Ulvestad, Maria
    et al.
    AstraZeneca, Sweden / Cellectis AB, Sweden / University of Oslo, Norway.
    Nordell, Pär
    AstraZeneca, Sweden.
    Asplund, Annika
    Cellectis AB, Sweden.
    Rehnström, Marie
    Cellectis AB, Sweden.
    Jacobsson, Susanna
    Cellectis AB, Sweden.
    Holmgren, Gustav
    University of Skövde, School of Life Sciences. University of Skövde, The Systems Biology Research Centre. Cellectis AB, Sweden / Sahlgrenska University Hospital, Sweden.
    Davidson, Lindsay
    University of Dundee, Scotland.
    Brolén, Gabriella
    AstraZeneca, Sweden.
    Edsbagge, Josefina
    Cellectis AB, Sweden.
    Björquist, Petter
    Cellectis AB, Sweden.
    Küppers-Munther, Barbara
    University of Skövde, School of Life Sciences. University of Skövde, The Systems Biology Research Centre. Cellectis AB, Sweden.
    Andersson, Tommy B.
    AstraZeneca, Sweden / Karolinska Institute, Sweden.
    Drug metabolizing enzyme and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells2013In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 86, no 5, p. 691-702Article in journal (Refereed)
    Abstract [en]

    Human embryonic and induced pluripotent stem cell-derived hepatocytes (hESC-Hep and hiPSC-Hep) have the potential to provide relevant human in vitro model systems for toxicity testing and drug discovery studies. In this study, the expression and function of important drug metabolizing cytochrome P450 (CYP) enzymes and transporter proteins in hESC-Hep and hiPSC-Hep were compared to cryopreserved human primary hepatocytes (hphep) and HepG2 cells. Overall, CYP activities in hESC-Hep and hiPSC-Hep were much lower than in hphep cultured for 4 h, but CYP1A and 3A activities were comparable to levels in hphep cultured for 48 h (CYP1A: 35% and 26% of 48 h hphep, respectively; CYP3A: 80% and 440% of 48 h hphep, respectively). Importantly, in hESC-Hep and hiPSC-Hep, CYP activities were stable or increasing for at least one week in culture which was in contrast to the rapid loss of CYP activities in cultured hphep between 4 and 48 h after plating. With regard to transporters, in hESC-Hep and hiPSC-Hep, pronounced NTCP activity (17% and 29% of 4 h hphep, respectively) and moderate BSEP activity (6% and 8% of 4 h hphep, respectively) were observed. Analyses of mRNA expression and immunocytochemistry supported the observed CYP and transporter activities and showed expression of additional CYPs and transporters. In conclusion, the stable expression and function of CYPs and transporters in hESC-Hep and hiPSC-Hep for at least one week opens up the possibility to reproducibly perform long term and extensive studies, e.g. chronic toxicity testing, in a stem cell-derived hepatic system. (C) 2013 Elsevier Inc. All rights reserved.

  • 44. Weber, Jonasz Jeremiasz
    et al.
    Clemensson, Laura Emily
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Schiöth: Functional Pharmacology.
    Schiöth, Helgi B.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Schiöth: Functional Pharmacology.
    Nguyen, Huu Phuc
    Olesoxime in neurodegenerative diseases: Scrutinising a promising drug candidate2019In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 168, p. 305-318Article, review/survey (Refereed)
    Abstract [en]

    Over the last years, the experimental compound olesoxime, a mitochondria-targeting cholesterol derivative, has emerged as a promising drug candidate for neurodegenerative diseases. Numerous preclinical studies have successfully proved olesoxime's neuroprotective properties in cell and animal models of clinical conditions such as amyotrophic lateral sclerosis, Huntington disease, Parkinson disease, peripheral neuropathy and spinal muscular atrophy. The beneficial effects were attributed to olesoxime's potential impact on oxidative stress, mitochondrial permeability transition or cholesterol homoeostasis. Although no significant benefits have been demonstrated in patients of amyotrophic lateral sclerosis, and only the first 12 months of a phase II/III clinical trial showed an improvement in motor symptoms of spinal muscular atrophy, this orphan drug may still offer undiscovered potential in the treatment of neurological diseases. In our earlier preclinical studies, we demonstrated that administration of olesoxime in mouse and rat models of Huntington disease improved psychiatric and molecular phenotypes. Aside from stabilising mitochondrial function, the drug reduced the overactivation of calpains, a class of calcium-dependent proteases entangled in neurodegenerative conditions. This observation may be credited to olesoxime's action on calcium dyshomeostasis, a further hallmark in neurodegeneration, and linked to its targets TSPO and VDAC, two proteins of the outer mitochondrial membrane associated with mitochondrial calcium handling. Further research into the mode of action of olesoxime under pathological conditions, including its effect on neuronal calcium homeostasis, may strengthen the untapped potential of olesoxime or other similar compounds as a therapeutic for neurodegenerative diseases.

  • 45.
    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.

  • 46.
    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.

  • 47.
    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.

  • 48.
    Zhang, Xiaonan
    et al.
    Karolinska Inst, Sweden.
    Pellegrini, Paola
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Saei, Amir Ata
    Karolinska Inst, Sweden.
    Hillert, Ellin-Kristina
    Karolinska Inst, Sweden.
    Mazurkiewicz, Magdalena
    Karolinska Inst, Sweden.
    Olofsson, Maria Hagg
    Karolinska Inst, Sweden.
    Zubarev, Roman A.
    Karolinska Inst, Sweden.
    D´arcy, Padraig
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Linder, Stig
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Karolinska Inst, Sweden.
    The deubiquitinase inhibitor b-AP15 induces strong proteotoxic stress and Check for mitochondrial damage2018In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 156, p. 291-301Article in journal (Refereed)
    Abstract [en]

    Human cancers are characterized by intrinsic or acquired resistance to apoptosis and evasion of apoptosis has been proposed to contribute to treatment resistance. Bis-benzylidine piperidone compounds, containing alpha,beta-unsaturated carbonyl functionalities, have been extensively documented as being effective in killing apoptosis-resistant cells and to display promising antineoplastic activities in a number of tumor models. We here explored the phenotypic response of colon cancer cells to b-AP15, a bis-benzylidine piperidone previously shown to inhibit the proteasome deubiquitinases (DUBs) USP14 and UCHL5. Whereas similar overall mRNA and protein expression profiles were induced by b-AP15 and the clinically available proteasome inhibitor bortezomib, b-APIS induced stronger increases of chaperone expression. b-AP15 also induced a stronger accumulation of polyubiquitinated proteins in exposed cells. These proteins were found to partially colocalize with organelle structures, including mitochondria. Mitochondrial oxidative phosphorylation decreased in cells exposed to b-APIS, a phenomenon enhanced under conditions of severe proteotoxic stress caused by inhibition of the VCP/p97 ATPase and inhibition of protein translocation over the ER. We propose that mitochondrial damage caused by the association of misfolded proteins with mitochondrial membranes may contribute to the atypical cell death mode induced by b-AP15 and related compounds. The robust mode of cell death induction by this class of drugs holds promise for treatment of tumor cells characterized by apoptosis resistance.

  • 49.
    Zhenchuk, Anna
    et al.
    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden.
    Lotfi, Koroush
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Juliusson, Gunnar
    Lund Strategic Center for Stem Cell Biology and Therapy, Department of Hematology, Lund University Hospital, Lund, Sweden.
    Albertioni, Freidoun
    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden.
    Commentary: Mechanisms of anti-cancer action and pharmacology of clofarabine: in BIOCHEMICAL PHARMACOLOGY, ISSN 0006-2952, vol 78, issue 11, pp 1351-13592009In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 78, no 11, p. 1351-1359Article in journal (Other academic)
    Abstract [en]

    Clofarabine, a next-generation deoxyadenosine analogue, was developed on the basis of experience with cladribine and fludarabine in order to achieve higher efficacy and avoid extramedullary toxicity. During the past decade this is the only drug granted approval for treatment of pediatric acute leukemia. Recent clinical studies have established the efficacy of clofarabine in treating malignancies with a poor prognosis, such as adult, elderly, and relapsed pediatric leukemia. The mechanisms of its anti-cancer activity involve a combination of direct inhibition of DNA synthesis and ribonucleotide reductase and induction of apoptosis. Due to this broad cytotoxicity, this drug is effective against various subtypes of leukemia and is currently being tested as an oral formulation and for combination therapy of both leukemias and solid tumors. In this review we summarize current knowledge pertaining to the molecular mechanisms of action and pharmacological properties of clofarabine, as well as clinical experiences with this drug with the purpose of facilitating the evaluation of its efficacy and the development of future therapies.

  • 50.
    Zimdahl Kahlin, Anna
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Helander, Sara
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Chemistry. Linköping University, Faculty of Medicine and Health Sciences.
    Skoglund, Karin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Söderkvist, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Comprehensive study of thiopurine methyltransferase genotype, phenotype, and genotype-phenotype discrepancies in Sweden2019In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 164, p. 263-272Article in journal (Refereed)
    Abstract [en]

    Thiopurines are widely used in the treatment of leukemia and inflammatory bowel diseases. Thiopurine metabolism varies among individuals because of differences in the polymorphic enzyme thiopurine methyltransferase (TPMT, EC 2.1.1.67), and to avoid severe adverse reactions caused by incorrect dosing it is recommended that the patients TPMT status be determined before the start of thiopurine treatment. This study describes the concordance between genotyping for common TPMT alleles and phenotyping in a Swedish cohort of 12,663 patients sampled before or during thiopurine treatment. The concordance between TPMT genotype and enzyme activity was 94.5%. Compared to the genotype, the first measurement of TPMT enzyme activity was lower than expected for 4.6% of the patients. Sequencing of all coding regions of the TPMT gene in genotype/phenotype discrepant individuals led to the identification of rare and novel TPMT alleles. Fifteen individuals (0.1%) with rare or novel genotypes were identified, and three TPMT alleles (TPMT*42, *43, and *44) are characterized here for the first time. These 15 patients would not have been detected as carrying a deviating TPMT genotype if only genotyping of the most common TPMT variants had been performed. This study highlights the benefit of combining TPMT genotype and phenotype determination in routine testing. More accurate dose recommendations can be made, which might decrease the number of adverse reactions and treatment failures during thiopurine treatment.

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