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Polyamines as diagnostic tools and therapeutics

Polyamines as diagnostic tools and therapeutics. Tuomo A. Keinänen 1,2 , Merja Häkkinen 2 , Antti Roine 3 , Antti Tuokko 3 , Mervi T. Hyvönen 4 , Niku KJ Oksala 5 , and Jouko Vepsäläinen 2

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Polyamines as diagnostic tools and therapeutics

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  1. Polyamines as diagnostic tools and therapeutics Tuomo A. Keinänen1,2, Merja Häkkinen2, Antti Roine3, Antti Tuokko3, Mervi T. Hyvönen4, Niku KJ Oksala5, and Jouko Vepsäläinen2 Department of Microbiology, Tumor and Cell Biology1, Nobelsvägen 16, KI Solna Campus, KarolinskaInstitutet, Box 280, Department of Cell and Molecular Biology4, KarolinskaInstitutet, P.O. Box 285, SE-171 77 Stockholm, Sweden. School of Pharmacy2, Biocenter Kuopio, University of Eastern Finland, Kuopio Campus, P.O. Box 1627, FI-70211 Finland. Medical School3, University of Tampere, Department of Surgery5, Tampere University Hospital, Tampere, Finland. The 10-th Annual Conference of New Visby Network in Hepatitis C, Feb 10-13, 2013

  2. Polyamine structures and functions • Spermidine and spermine are organic cations present at millimolar concentrations in eukaryotic cells • In bacteria put and spd but not spermine exist; including ariginine decarboxylse mediated polyamine production • In bacteria and plants variety of polyamine based compounds have been characterized • Spider toxins and snake venoms contain also polyamine derivatives affecting to ion channel modulation • Polyamines are essential for cell growth and differentiation. • Act as ROS scavengers and participitate in immunoresponse.

  3. Polyamine metabolism • Polyamine metabolism is very tightly controlled under different physiolocial conditions, like in response to stimuli for growth or differentiation. • ODC and AdometDC (biosynthesis); spermidine/spermine-N1-acetyltransferase SAT and spermine oxidase SMO (interconversion) are inducible enzymes, rest of the PA metabolizing enzymes are constitutively expressed. • Myc mediated ODC dysregulation is associated with cancer • Dysregulated SMO is associated with cancer development in prostate, venticulus and colon.

  4. Polyamine therapeutic fields A polymorphism that delays fibrosis in hepatitis C promotes alternative splicing of AZIN1, reducing fibrogenesis. Paris AJ, Snapir Z, Christopherson CD, Kwok SY, Lee UE, Ghiassi-Nejad Z, Kocabayoglu P, Sninsky JJ, Llovet JM, Kahana C, Friedman SL. Hepatology. 2011 Dec;54(6):2198-207. Chemically induced oxidative stress increases polyamine levels by activating the transcription of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase in human hepatoma HUH7 cells. Smirnova OA, Isaguliants MG, Hyvonen MT, Keinanen TA, Tunitskaya VL, Vepsalainen J, Alhonen L, Kochetkov SN, Ivanov AV. Biochimie. 2012 Sep;94(9):1876-83. Induction of autophagy by spermidine promotes longevity. Eisenberg T, Knauer H, Schauer A, Büttner S, Ruckenstuhl C, et al.. Nat Cell Biol. 2009 Nov;11(11):1305-14. Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis-induced colon tumorigenesis. Goodwin AC, Destefano Shields CE, Wu S, Huso DL, Wu X, Murray-Stewart TR, Hacker-Prietz A, Rabizadeh S, Woster PM, Sears CL, Casero RA Jr. Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15354-9 Evaluating the utility of N1,N12-diacetylspermine and N1,N8-diacetylspermidine in urine as tumor markers for breast and colorectal cancers. Umemori Y, Ohe Y, Kuribayashi K, Tsuji N, Nishidate T, Kameshima H, Hirata K, Watanabe N. Clin Chim Acta. 2010 Dec 14;411(23-24):1894-9.

  5. PA analytical methods • TLC, paperchromatographylate 60’ and 70’, staining and radioactivity (C14) for quantitification. • HPLC pre- and postcolumnderivatization of analytesusingreversedphasecolumn to separateanalytes. Benzyl-Cl, Dansyl-Clprecolumn and OPA postcolumnderivatization • Detection is commonlybased on either UV orfluorescence • Dansyl-Cl and OPA (orthophaldialdehyde) commonlyexistinganalysismethodsnowadays. • Duringpasttenyearseveral LC-MS/MS basedanalyticalmethodshavebeendeveloped, and theyallowaccurateidentification of the analyte and 10-100 foldhighersensitivitycomparingabovedescribedmethods.

  6. PA analytical methods OPA vrs Dansyl-Cl Dansyl-method OPA-method • Time-consuming sample preparation • 60 min one sample analysis • Detection of primary and secondary amines • Signal/noise ratio excellent • N1N12-DiAc-Spm could be detected • N1/N8 Ac-Spd are co-eluting in same peak (N8-Ac-Spd is rarely detected) • 3 samples in 2 hours (40 min) • Separation of N1- and N8-AcSpd • Sensitive to any -NH3 impurities, increased background, lowering signal noise ratio • Analytes having only secondary amines remain undetectable

  7. LC-MS/MS analysis of polyamines and their analogues • Three different quantitative LC-MS/MS analysis methods for polyamines and their analogues from different sample matrixes were developed. • LC-MS/MS detection offers high sensitivity and reliable identification of each analyte but is sensitive to sample matrix effects and therefore require the use of internal standards for each analyte to achieve reliable results from distinct sample matrix. • Polyamine metabolism is disturbed in many metabolic disorders like cancer, and polyamine levels from urine could be used as disease diagnostic tool and for monitoring treatment response. • Currently available polyamine-based cancer diagnostics are based on analysis on N1N12DiAcSpm (and/or N1N8DiAcSpd) from urine samples. Fig.1. A representative graph of LC-MS/MS analyses of 14 different N-alkylated polyamine analogues

  8. Polyamine flux analysis • LC-MS/MS allows C13 or N15 enriched ornithine to be used as tracer to monitor polyamine flux • Practically all currently used methods require either Carbon-14 or tritium labeled ornithine and determination of radioactivity of each polyamine pool with simultaneous quantification of total PA-level in order to determine specific radioactivity. • One alternative is the use of 4-fluoroornithine and determination of fluorinated PA species by using OPA-polyamine determination method.

  9. Smell of Cancer –project • Instead of dogs we use an electronic nose made by a Finnish company Environics Oy. • Designed for detecting chemical weapons

  10. Detection of smell print differences between nonmalignant and malignant prostate cells with an electronic nose. Future oncology, 2012, 8(9), pp1157-1165 Roine et al.

  11. Therapeutics • Polyamine metabolism is a rational therapeutic target against inflammation (ROS) mediated organ damage in Type 2 diabetes, cancer and parasitic diseases. • Polyamine metabolism based therapeutics 1) PA-synthesis inhibitors 2) PA-antimetabolites i.e. suppress biosynthesis, uptake and induce catabolism of natural polyamines 3) Partly functional polyamine mimetics. • Furthermore, we are studying drug ”secondary” targets of currently used polyamine-based drugs Trien (a drug for Wilson’s disease). 1) Biosynthesis inhibitor DFMO (ODC) 2) PA-antimetabolites 3) PA-functional mimetics

  12. TETA (Trien) metabolites; PA-metabolism

  13. eIF5A post translational modification Deoxyhypusine synthase (DHS) possesses stereospecificity for 1-methylspermidine (J Biol Chem. 2007 Nov 30;282(48):34700-6. )

  14. 1 mM 300 uM 25 uM 10 uM The (S) 1-MeSpd substitutes for Spd in supporting growth and is a better substrate of DHS than the (R) isomer DHS inhibition 7000 6000 5000 4000 Deoxyhypusine formed/dpm 3000 2000 Growth support in DFMO treated cells 1000 0 Spd control R/S-MeSpd R-MeSpd S-MeSpd DHS substrate for Homospermidine synthesis 5 4 3 Hspd formed (%) 2 1 0 D: DFMO, R: R-MeSpd S: S-MeSpd, R,S: R,S-Me2Spm, R,R: R,R-Me2Spm, S,S: S,S-Me2Spm Spd R/S-MeSpd R-MeSpd S-MeSpd Spd or 1-MeSpd Hyvönen M et et al. 2007, JBC 282, 34700-34706 + 14C-Ptc 14C-Hspd DHS

  15. Polyamines and viruses • Adjuvant in vaccines, increase immunogenity. • Direct acting effects; capsid formation RNA/DNA packaging • eIF5A-hyp inhibition (eIF5A-hyp is vital for HIV REV-function) • Virus infection (stress) affects to PA-metabolism throught several mechanism some associated to host defence system to generate ROS. What is the role of disturbed polyamine metabolism in chronic infection secondary effects fibrosis, cirhosis and development of cancer. Metabolomic Profile of Hepatitis C Virus-Infected Hepatocytes Barbara Roe, Elizabeth Kensicki, Robert Mohney, William W. Hall PLoS One. 2011; 6(8): e23641. PA-metabolism affected Inhibition of multidrug-resistant HIV-1 by interference with cellular S-adenosylmethionine decarboxylase activity. Schäfer B, Hauber I, Bunk A, Heukeshoven J, Düsedau A, Bevec D, Hauber J. J Infect Dis. 2006 Sep 15;194(6):740-50 Antiviral activity of oxidized polyamines. Bachrach U. Amino Acids. 2007 Aug;33(2):267-72. Terminal deamination catabolism products are active Multivalent binding oligomers inhibit HIV Tat-TAR interaction critical for viral replication. Wang D, Iera J, Baker H, Hogan P, Ptak R, Yang L, Hartman T, Buckheit RW Jr, Desjardins A, Yang A, Legault P, Yedavalli V, Jeang KT, Appella DH. Bioorg Med Chem Lett. 2009 Dec 15;19(24):6893-7

  16. Summary • Both the currently used polyamine quantification methods are on routine use at UEF. • Three different LC-MS/MS methods have been developed for quantitative determination of polyamines and their analoques from different sample matrixes. • We have necessary ”know-how” in synthetic chemistry to prepare required standards and chemical tools for biological studies. • In collaboration with Tampere University Hospital we are searching novel methods for polyamine based diagnostic and therapies, like eNose project which may offer portable low cost solution for cancer diagnostics. • Anticancer (antiproliferative) drug development project using Trien as lead compounds are on progress and data obtained with Trien suggests interference of polyamine metabolism as potential Trien drug target.

  17. Acknowledgments Professor Britta Wahren and Adjunct professor Maria Issagulliantis, KI, Solna Sweden Professor Jouko Vepsäläinen and Professor Seppo Auriola, UEF, Kuopio Campus Professor Leena Alhonen, UEF, Kuopio Campus Professor Alex Khomutov, Professor S.N. Kochetkov, Alexander Ivanov, Olga Smirnova, Russian Academy of Sciences, Moscow. Merja Häkkinen, Mervi T. Hyvönen, Janne Weisel, UEF, Kuopio Campus Collabators at the Tampere University Hospital, Niku KJ Oksala, Antti Roine, Antti Tuokko Thank you

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