Polyamines and Their Metabolites as Diagnostic Markers of Human Diseases

Polyamines, putrescine, spermidine and spermine, are ubiquitous in living cells and are essential for eukaryotic cell growth. These polycations interact with negatively charged molecules such as DNA, RNA, acidic proteins and phospholipids and modulate various cellular functions including macromolecular synthesis. Dysregulation of the polyamine pathway leads to pathological conditions including cancer, inflammation, stroke, renal failure and diabetes. Increase in polyamines and polyamine synthesis enzymes is often associated with tumor growth, and urinary and plasma contents of polyamines and their metabolites have been investigated as diagnostic markers for cancers. Of these, diacetylated derivatives of spermidine and spermine are elevated in the urine of cancer patients and present potential markers for early detection. Enhanced catabolism of cellular polyamines by polyamine oxidases (PAO), spermine oxidase (SMO) or acetylpolyamine oxidase (AcPAO), increases cellular oxidative stress and generates hydrogen peroxide and a reactive toxic metabolite, acrolein, which covalently incorporates into lysine residues of cellular proteins. Levels of protein-conjuagated acrolein (PC-Acro) and polyamine oxidizing enzymes were increased in the locus of brain infarction and in plasma in a mouse model of stroke and also in the plasma of stroke patients. When the combined measurements of PC-Acro, interleukin 6 (IL-6), and C-reactive protein (CRP) were evaluated, even silent brain infarction (SBI) was detected with high sensitivity and specificity. Considering that there are no reliable biochemical markers for early stage of stroke, PC-Acro and PAOs present promising markers. Thus the polyamine metabolites in plasma or urine provide useful tools in early diagnosis of cancer and stroke.     

Inhibition of mast cell secretion by oxidation products of natural polyamines.

Mast cells secrete many biologically active compounds upon stimulation by immunoglobulin E (IgE) and specific antigen (Ag), anaphylatoxins, as well as a number of cationic compounds which include drugs, kinins and neuropeptides. The effects of the two naturally occurring polyamines, spermine (SP) and spermidine (SPD), on mast cell secretion were studied because they have been implicated in the modulation of cellular processes, possibly through their cationic charge or the regulation of calcium ions. SP and SPD over the range of 10(-7) to 10(-4) M inhibited the release of 5-hydroxytryptamine (5-HT, serotonin) triggered by compound 48/80 (C48/80) in a time- and concentration-dependent manner, as long as at least 2% calf serum (CS) was present. SP also inhibited secretion of both histamine and serotonin stimulated immunologically by using IgE and anti-rat IgE. This inhibition was not accompanied by cytotoxicity. The major available polyamine metabolites tested, N1-acetyl spermine (N1-acSP) and N8-acetyl spermidine (N8-acSPD), also showed inhibition in the presence of CS, whereas putrescine, N8,N1-hexamethylene-bis-acetamide (HMBA) and benzylamine did not. Fetal bovine serum (FBS), as well as human and rat serum, which do not contain polyamine oxidase, did not result in any inhibition with the polyamines tested. Inhibitors of the polyamine oxidase blocked the polyamine effect, indicating that the inhibition of mast cell secretion must derive from aldehydes produced from these polyamines. Addition of the aldehyde inhibitor phenylhydrazine (phi-HDZ), simultaneously with, but not following the polyamines, blocked their inhibitory effect, further strengthening the involvement of aldehydes. These results indicate that naturally occurring polyamines may regulate mast cell secretion through metabolic products of polyamine oxidase, a similar enzyme of which is also present in human liver, placenta and pregnant serum.     

Physiological functions of polyamines and regulation of polyamine content in cells

Polyamines (putrescine, spermidine, and spermine) are essential for normal cell growth. The polyamine level in cells is regulated by biosynthesis, degradation, and transport. The role of antizyme on polyamine biosynthesis and transport in mammalian cells and characteristics of polyamine transport in Escherichia coli and yeast are described briefly in this review. In addition, the effects of polyamines on protein synthesis and the NMDA receptor are outlined. Finally, the correlation between acrolein produced from polyamines by polyamine oxidase and chronic renal failure and brain stroke is summarized. Increased levels of polyamine oxidase and acrolein are good markers of chronic renal failure and brain stroke.     

N-Benzylpolyamines as vectors of boron and fluorine for cancer therapy and imaging: synthesis and biological evaluation

Cancer cells have high-affinity polyamine uptake systems with a low stringency for structural features. Putrescine, spermidine, and spermine have, therefore, been considered as potential vectors for the selective accumulation in tumors of therapeutically or diagnostically useful structures and elements. We envisaged N-benzyl derivatives of the polyamines as vectors of (10)B and (18)F for boron neutron capture therapy (BNCT) and tumor imaging by positron emission tomography (PET), respectively. In the present work, the synthesis, transport characteristics, DNA-binding properties, and cytotoxicity of several N-benzyl derivatives of putrescine and spermidine are described. The fluorinated spermidine derivative N-(3-[(4-aminobutyl)amino]propyl)[(4-fluorophenyl)methyl]amine (N(1)-4-Fbz-spd) may be useful for PET because of its high accumulation in cancer cells via the polyamine transport system. Among the boron-containing benzyl polyamines, N-(4-aminobutyl)([4-(dihydroxyboryl)phenyl]methyl)amine (4-Bbz-put) and N-(3-[(4-aminobutyl)amino]propyl)([4-(dihydroxyboryl)phenyl]methyl)amine (N(1)-4-Bbz-spd) should be suitable for BNCT, because their accumulation in B16 melanoma cells was more efficient than that of borocaptate and borophenylalanine, two reference compounds used in BNCT.     

Alpha-methyl polyamines: efficient synthesis and tolerance studies in vivo and in vitro. First evidence for dormant stereospecificity of polyamine oxidase

Efficient syntheses of metabolically stable alpha-methylspermidine 1, alpha-methylspermine 2, and bis-alpha,alpha'-methylated spermine 3 starting from ethyl 3-aminobutyrate are described. The biological tolerance for these compounds was tested in wild-type mice and transgenic mice carrying the metallothionein promoter-driven spermidine/spermine N(1)-acetyltransferase gene (MT-SSAT). The efficient substitution of natural polyamines by their derivatives was confirmed in vivo with the rats harboring the same MT-SSAT transgene and in vitro with the immortalized fibroblasts derived from these animals. Enantiomers of previously unknown 1-amino-8-acetamido-5-azanonane dihydrochloride 4 were synthesized starting from enantiomerically pure (R)- and (S)-alaninols. The studies with recombinant human polyamine oxidase (PAO) showed that PAO (usually splits achiral substrates) strongly favors the (R)-isomer of 4 that demonstrates for the first time that the enzyme has hidden potency for stereospecificity.     

Synthesis and antitumor activity of spergualin analogues. II. Chemical modification of the spermidine moiety

Chemical modifications of the spermidine moiety of an antitumor antibiotic, spergualin (Ia), and the structure-activity relationship are described. Replacement of spermidine with other polyamines decreased the antitumor activity against mouse leukemia L1210. Analogues containing an oxidized spermidine moiety that probably formed during oxidation with amine oxidase were inactive. Spermidine is indispensable for the antitumor activity. A facile method for the synthesis of glyoxyloyl polyamine, a key intermediate of spergualin-related compounds, is also reported.     

alpha-Methyl polyamines: metabolically stable spermidine and spermine mimics capable of supporting growth in cells depleted of polyamines.

In order to assess the tolerance of the target enzyme spermine synthase for alpha-substituents on the aminopropyl moiety of the substrate spermidine, 1-methylspermidine (MeSpd, 2) was synthesized. It was determined that MeSpd is a poor substrate for spermine synthase and is not a substrate for spermidine N1-acetyltransferase, suggesting that alpha-methylated polyamines might be metabolically stable and therefore useful tools for studying polyamine effects in intact cells. On the basis of initial cellular results with 2, 1-methylspermine (MeSpm, 3) and 1,12-dimethylspermine (Me2Spm, 4) were also synthesized. When added to cells (L1210, SV-3T3, or HT29) depleted of both putrescine and spermidine by prior treatment with alpha-(difluoromethyl)ornithine (DFMO), these alpha-methylated polyamines were able to restore cell growth to that observed in the absence of DFMO. In accord with the enzyme data noted above, metabolic studies indicated a slow conversion of 2 to 3, but no metabolism of 4 in these cells. It was concluded from these results that the alpha-methylated polyamines are able to substitute for the natural polyamines spermidine and spermine in critical biochemical processes which involve polyamines for continued cell growth. In accord with the hypothesis, preliminary data indicate that MeSpd and Me2Spm are as effective as spermidine and spermine, respectively, in promoting the conversion of B-DNA to Z-DNA.     

The role of amine oxidases in xenobiotic metabolism

The amine oxidases of mammalian tissues are a heterogeneous family of enzymes that metabolise various monoamines, diamines and polyamines produced endogenously, or being absorbed as dietary or xenobiotic substances. The heterogeneous class of amine oxidases can be divided on an arbitrary basis of the chemical nature of their cofactors into two types. Monoamine oxidase (MAO) and an intracellular form of polyamine oxidase (PAO) contain flavin adenine dinucleotide (FAD) as their cofactor, whereas a second group of amine oxidases without FAD contain a cofactor possessing one or more carbonyl groups, making them sensitive to inhibition by carbonyl reagents such as semicarbazide; this group includes semicarbazide-sensitive amine oxidase (SSAO) and the connective tissue enzyme, lysyl oxidase. This article focuses on the general aspects of MAO's contribution to the metabolism of foreign toxic substances including toxins and illegal drugs. Another main objective of this review is to discuss the properties of PAO and SSAO and their involvement in the metabolism of xenobiotics.     

Alpha-methylated polyamines as potential drugs and experimental tools in enzymology

We describe synthesis of alpha-methylated analogues of the natural polyamines and their use as tools in unraveling polyamine functions. Experiments with alpha-methylated spermidine and spermine revealed that the polyamines are exchangeable in supporting cellular growth. Degradation of the analogues by polyamine oxidase disclosed hidden, aldehyde-guided stereospecificity of the enzyme.     

The use of novel C-methylated spermidine derivatives to investigate the regulation of polyamine metabolism

The polyamines are organic polycations present at millimolar concentrations in eukaryotic cells where they participate in the regulation of vital cellular functions including proliferation and differentiation. Biological evaluation of rationally designed polyamine analogs is one of the cornerstones of polyamine research. Here we have synthesized and characterized novel C-methylated spermidine analogs, that is, 2-methylspermidine, 3-methylspermidine, and 8-methylspermidine. 3-Methylspermidine was found to be metabolically stable in DU145 cells, while 8-methylspermidine was a substrate for spermidine/spermine N(1)-acetyltransferase (SSAT) and 2-methylspermidine was a substrate for both SSAT and acetylpolyamine oxidase. All the analogs induced the splicing of the productive mRNA splice variant of SSAT, overcame growth arrest induced by 72-h treatment with ornithine decarboxylase (ODC) inhibitor α-difluoromethylornithine, and were transported via the polyamine transporter. Surprisingly, 2-methylspermidine was a weak downregulator of ODC activity in DU145 cells. Our data demonstrates that it is possible to radically alter the biochemical properties of a polyamine analog by changing the position of the methyl group.     

The antiproliferative action of deoxyspergualin is different from that induced by amine oxidase

The amine oxidase activities contained in calf serum and human serum were detected at levels of 90.8 and less than 0.1 nmol O2/minute/ml serum, respectively, by measuring oxygen consumption coupled with spermidine oxidation. Deoxyspergualin (NKT-01) and spergualin (SGL) containing spermidine in their structure were also oxidized in calf serum at the rate of 3.6 and 11.6 nmol O2/minute/ml serum, respectively. To investigate whether amine oxidase is essential for NKT-01 and SGL to exhibit their antiproliferative activities or not, the in vitro activities of NKT-01, SGL and polyamines against L1210 cells were examined in the presence of calf or human serum. Polyamines exhibited antiproliferative activity only in the presence of calf serum, while NKT-01 and SGL inhibited cell growth in the presence of both calf and human serum. In the presence of calf serum the activity of NKT-01 was inhibited by aminoguanidine, an amine oxidase inhibitor. Aminoguanidine did not inhibit the activity of NKT-01 in the presence of human serum. The activity of NKT-01 was shown at much lower concentrations in the presence of human serum than that in the presence of calf serum, and was strongly dependent on incubation time. The in vivo activities of NKT-01, SGL and SGL derivatives correlated with their in vitro activities in the presence of human serum. These results suggest that the in vivo antitumor activities of NKT-01, SGL and SGL derivatives may be attributed to a mechanism different from those of amine oxidase-oxidized product and represent a novel growth inhibitory action.     

Inhibition of enzymes of polyamine back-conversion by pentamidine and berenil.

Pentamidine and berenil, clinical antiparasitic amidines, have been found to be potent competitive inhibitors of human spermidine/spermine acetyltransferase (SSAT). Ki values were found to be 2.4 and 2 microM, respectively, with spermidine as substrate. A second enzyme of polyamine back-conversion, murine polyamine oxidase (PAO), was found to be competitively inhibited by pentamidine, with a Ki of 7.6 microM when N-acetylspermine was the substrate. Berenil, on the other hand, was an extremely weak inhibitor (Ki = 120 microM). The implication of the effect of inhibition of polyamine back-conversion on the growth of mammalian parasites is discussed.     

Serum effect on cellular uptake of spermidine, spergualin, 15-deoxyspergualin, and their metabolites by L5178Y cells

Spergualin (SG) and 15-deoxyspergualin (DSG) were more slowly incorporated into L5178Y cells than spermidine. SG and DSG inhibited carrier-mediated transport of [3H]spermidine competitively with inhibition constants of 0.67 mM and 0.45 mM, respectively. Addition of calf serum stimulated uptake of [3H]spermidine into the cells in a serum concentration-dependent manner. The effect was not observed when horse serum was used in place of calf serum. Preincubation of spermidine in calf serum for 1 hour before addition to cells remarkably decreased cellular incorporation of tritium. Three amine oxidase inhibitors, aminoguanidine, 3-hydroxybenzyloxyamine, and semicarbazide, inhibited stimulation of uptake of [3H]spermidine by calf serum and the decrease of it by preincubation in calf serum. So we propose that cellular incorporation or binding of products generated by oxidation of spermidine by amine oxidase in calf serum was much faster than that of spermidine itself and they were unstable and transformed quickly to unincorporable or non-binding substances if cellular targets were not present. Effect of amine oxidase inhibitors on cytotoxic activity of SG and DSG were determined in low and high concentrations of calf serum. In the presence of 10% calf serum in the basal medium, cytotoxicity to L5178Y cells by SG and DSG was suppressed at high drug concentrations (above 10 micrograms/ml) and enhanced at low drug concentrations (below 2.5 micrograms/ml) by amine oxidase inhibitors. In the presence of 0.5% calf serum suppression of cytotoxicity at high drug concentrations by amine oxidase inhibitors was also observed, but enhancement at low drug concentrations was obscure. These data may suggest the existence of two kinds of cytotoxic mechanism of SG and DSG, one dependent on and one independent of amine oxidase in serum.     

Role of polyamines derived from arginine in differentiation and proliferation of human blood cells

L-Arginine is a precursor of polyamine, nitric oxide (NO), creatine, and agmatine and is essential for the differentiation and proliferation of blood cells, although the precise biological role of L-arginine is unclear. We have recently reported that the depletion of L-arginine in cultured medium prevented both proliferation and differentiation of blood cells (Shima et al., Blood First Edition Paper, October 6, 2005; DOI 10.1182). Since one of metabolic products of L-arginine in the cells is polyamine that associates with cell differentiation and proliferation, the effects of L-arginine on the human K562 cell line and human cord blood-derived CD34 positive cells were investigated by focusing on polyamines such as putrescine, spermidine, and spermine in the present study. When polyamines were added to the culture medium in the absence of L-arginine, the cells did not grow or differentiate well. However, when intracellular polyamines were depleted using ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), the proliferation and differentiation of K562 cells to erythrocytes were reduced even in the presence of L-arginine. Moreover, in the presence of DFMO, cell differentiation and proliferation were recovered by the addition of putrescine or spermidine in the presence of L-arginine. Accordingly, it was demonstrated that polyamines are essential for the proliferation and differentiation of the blood cells as the metabolites of L-arginine and the externally added polyamines are also effective by being taken up through polyamine transporter.     

Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases

The polyamines spermidine and spermine and their diamine precursor putrescine are naturally occurring, polycationic alkylamines that are essential for eukaryotic cell growth. The requirement for and the metabolism of polyamines are frequently dysregulated in cancer and other hyperproliferative diseases, thus making polyamine function and metabolism attractive targets for therapeutic intervention. Recent advances in our understanding of polyamine function, metabolic regulation, and differences between normal cells and tumour cells with respect to polyamine biology, have reinforced the interest in this target-rich pathway for drug development.     

Polyamine-iron chelator conjugate

The current study demonstrates unequivocally that polyamines can serve as vectors for the intracellular delivery of the bidentate chelator 1,2-dimethyl-3-hydroxypyridin-4-one (L1). The polyamine-hydroxypyridinone conjugate 1-(12-amino-4,9-diazadodecyl)-2-methyl-3-hydroxy-4(1H)-pyridinone is assembled from spermine and 3-O-benzylmaltol. The conjugate is shown to form a 3:1 complex with Fe(III) and to be taken up by the polyamine transporter 1900-fold against a concentration gradient. The K(i) of the conjugate is 3.7 microM vs spermidine for the polyamine transporter. The conjugate is also at least 230 times more active in suppressing the growth of L1210 murine leukemia cells than is the parent ligand, decreases the activities of the polyamine biosynthetic enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase, and upregulates spermidine-spermine N (1)-acetyltransferase. However, the effect on native polyamine pools is a moderate one. These findings are in keeping with the idea that polyamines can also serve as efficient vectors for the intracellular delivery of other iron chelators.     

Interactions between agmatine and polyamine uptake pathways in rat pulmonary artery endothelial cells

Agmatine, a product of arginine metabolism in vascular endothelial cells, is structurally similar to the natural polyamines, putrescine, spermidine and spermine. To test the hypothesis that agmatine and polyamines interacted at the level of the polyamine transporter, we determined if polyamines competed with agmatine for import and whether interventions modulating polyamine import exerted coordinate effects on agmatine uptake. Multiple lines of evidence were obtained to suggest that agmatine enters pulmonary artery endothelial cells (PAECs) via the polyamine transporter, though its intracellular disposition after uptake appears different from the natural polyamines.     

Polyamine modulation of iron uptake in CHO cells

Polyamines are ubiquitous molecules, which, like iron, are essential for cell growth. All eukaryotic cells are equipped with a specific polyamine transport system (PTS). Polyamines have primary and secondary amino groups which chelate bivalent metal cations such as Fe and Cu. In the present study, we investigated the potential contribution of naturally occurring polyamines and their active transport system to iron uptake. In presence of subtoxic Fe(III) (10microM), treatment of CHO cells with spermine, and to a lesser extent with spermidine (10-100microM), resulted in a marked cytotoxic effect. This cytotoxicity was prevented by the addition of an iron-chelator, deferioxamine, and was not observed in CHO-MG cells, a mutant cell line devoid of polyamine transport activity. Experiments using 14C-polyamines and 55Fe(III) revealed that these toxic effects were related to polyamine-modulation of iron uptake, and were dependent on the presence of the active PTS. These results demonstrated active uptake of polyamine-iron complexes via the PTS. The number of amino groups affected the efficacy of the studied natural polyamines to transport iron via the PTS. Spermine, a tetramine, was more efficient than the triamine spermidine. Co-transport of iron by the diamine putrescine was not observed. These results demonstrate that the cell polyamine transport system is a potential cell entry pathway for iron. The studied polyamines, spermine and spermidine, may be components of the pool of transferrin-independent iron-chelating vectors, which have recently attracted the attention of many investigators.     

Regulation of polyamine and cancer

This review describes my work in the field of polyamine research for the last 35 years. My research started with developing the improved synthesis of decarboxylated S-adenosylmethionine and then moved to the purification of spermidine synthase from rat prostate. I also took considerable efforts to find the synthetic procedure for various polyamines with high yield in order to prepare (15)N-labeled polyamines. On the basis of these methodological work, I searched for the inhibitor of spermidine synthase and found trans-4-methylcyclohexylamine (MCHA), the most effective one at the present time. I also developed a new analytical method for polyamines using stable isotope and ionspray ionization mass spectrometry (IS-MS). Based on these studies I examined the role of polyamines in liver regeneration and found that oral administration of MCHA effectively changed the concentration of polyamines and inhibited the hepatic growth. I also found the close relationship between the concentration ratio of spermidine to spermine and the extent of liver regeneration. These results may shed new light on the control of cell growth by polyamine in vivo.     

Selective cellular depletion of mitochondrial DNA by the polyamine analog N1,N12-bis(ethyl)spermine and its relationship to polyamine structure and function

N1,N8-Bis(ethyl)spermidine (BESPD) and N1,N12-bis(ethyl)spermine (BESPM) are minimally modified analogs of spermidine and spermine that deplete cellular polyamine pools by suppressing key polyamine biosynthetic enzymes. The consequences of polyamine depletion and the concomitant analog replacement of these pools were compared on two cellular DNA targets, mitochondrial DNA (mtDNA) and a defined nuclear DNA episome present in 935.1 mouse fibroblasts. The spermidine analog, BESPD, depleted cellular putrescine and spermidine pools, but not spermine pools, and had no effect on either DNA target. Treatment with the corresponding analog of spermine, BESPM, resulted in a near-total depletion of all three polyamine pools and a greater than 80% reduction in the cellular content of mtDNA, without affecting the levels of the nuclear episome. Topological forms analysis by Southern blotting of mtDNA and episomal DNA from BESPM-treated cells failed to reveal any forms interconversion, indicating the absence of analog-induced single- or double-strand break damage to either DNA target. The growth-dependent loss of mtDNA is consistent with a rapid cessation of mtDNA replication and subsequent dilution of existing mtDNA copies by cell division. Similar decreases in polyamine pools and mtDNA were also observed in L1210 cells treated with BESPM. When a comparable level of polyamine depletion was produced in L1210 cells by specific enzyme inhibitors, there was no effect on the cellular content of mtDNA, and BESPD was not rendered capable of decreasing mtDNA levels. Because the analogs are structurally similar to the naturally occurring polyamines and would be expected to have similar binding properties, the loss in mtDNA may reflect dysfunctional replacement by BESPM at spermine-specific binding sites in the mitochondrion.