Transcript Presentation (powerpoint file)

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, Nobels vägen 16, KI Solna Campus, Karolinska Institutet,
Box 280, Department of Cell and Molecular Biology4, Karolinska Institutet, 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
Polyamine structures and functions
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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.
Polyamine metabolism
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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.
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.
PA analytical methods
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TLC, paper chromatography late 60’ and 70’, staining and radioactivity (C14) for
quantitification.
HPLC pre- and post column derivatization of analytes using reversed phase column
to separate analytes. Benzyl-Cl, Dansyl-Cl precolumn and OPA post column
derivatization
Detection is commonly based on either UV or fluorescence
Dansyl-Cl and OPA (orthophaldialdehyde) commonly existing analysis methods
nowadays.
During past ten year several LC-MS/MS based analytical methods have been
developed, and they allow accurate identification of the analyte and 10-100 fold
higher sensitivity comparing above described methods.
PA analytical methods OPA vrs Dansyl-Cl
Dansyl-method
OPA-method
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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
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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)
LC-MS/MS analysis of polyamines
and their analogues
Fig.1. A representative graph of LC-MS/MS
analyses of 14 different N-alkylated
polyamine analogues
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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.
Polyamine flux analysis
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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 OPApolyamine determination
method.
Smell of Cancer –project
 Instead of dogs we use
an electronic nose made
by a Finnish company
Environics Oy.
 Designed for detecting
chemical weapons
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.
Therapeutics
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Polyamine metabolism is a rational therapeutic target against inflammation (ROS)
mediated organ damage in Type 2 diabetes, cancer and parasitic diseases.
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Polyamine metabolism based therapeutics 1) PA-synthesis inhibitors 2) PAantimetabolites i.e. suppress biosynthesis, uptake and induce catabolism of natural
polyamines 3) Partly functional polyamine mimetics.
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Furthermore, we are studying drug ”secondary” targets of currently used polyaminebased drugs Trien (a drug for Wilson’s disease).
1) Biosynthesis inhibitor
DFMO (ODC)
2) PA-antimetabolites
3) PA-functional mimetics
TETA (Trien) metabolites; PAmetabolism
eIF5A post translational modification
Deoxyhypusine synthase (DHS) possesses stereospecificity for 1-methylspermidine (J Biol
Chem. 2007 Nov 30;282(48):34700-6. )
The (S) 1-MeSpd substitutes for Spd in supporting growth
and is a better substrate of DHS than the (R) isomer
Growth support in DFMO treated cells
Deoxyhypusine formed/dpm
DHS inhibition
7000
300 uM
1 mM
6000
5000
4000
3000
2000
1000
0
control R/S-MeSpd R-MeSpd S-MeSpd
Spd
DHS substrate for Homospermidine synthesis
Hspd formed (%)
5
D: DFMO, R: R-MeSpd S: S-MeSpd,
R,S: R,S-Me2Spm, R,R: R,R-Me2Spm,
25 uM
10 uM
4
3
2
1
0
R/S-MeSpd
R-MeSpd
S-MeSpd
Spd
S,S: S,S-Me2Spm
Hyvönen M et et al. 2007, JBC 282, 34700-34706
Spd or
1-MeSpd
+
14C-Ptc
DHS
14C-Hspd
Polyamines and viruses
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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
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.
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