Polyamines and cancer: Minireview article

Summary. The naturally occurring polyamines, spermine, spermidine and the diamine putrescine are widespread in nature. They have been implicated in growth and differentiation processes. Polyamines accumulate in cancerous tissues and their concentration is elevated in body fluids of cancer patients. Assays of urinary and blood polyamines have been used to detect cancer and to determine the success of therapy. Drugs which inhibit the synthesis of polyamines can prevent cancer and may also be used for therapeutic purposes. Ornithine decarboxylase, which catalyzes the rate limiting step in polyamine synthesis, can serve as a marker of proliferation. Recently, a new in vitro chemosensitivity test, based on the disappearance of ornithine decarboxylase in drug-treated cancer cells has been developed. The increasing interest in polyamines and their physiological functions may lead to a more extensive application of these compounds or their derivatives in cancer diagnosis and treatment.     

Changes in free polyamine concentration induced by salt stress in seedlings of different species

Growth rate, mineral composition and changes in polyamine concentration induced in response to salinity were studied in six crop species: spinach, lettuce, bean, pepper, beetroot and tomato. Salinity decreased growth rate, but sensitivity differed amongst the species: pepper being the most sensitive, followed by bean, tomato, lettuce and spinach, with beetroot being the most tolerant. The increase of Na⁺ and total cation with salinity in shoots was the highest in spinach and beetroot, the most tolerant species, while in pepper it was the lowest. Changes in putrescine (Put) concentration in shoots were related to salinity tolerance (increased in the most sensitive), while changes in spermidine (Spd; decreases) and spermine (Spm; increases) were similar with most species, except for pepper in which salinity strongly increased Put, Spd and Spm. Therefore, total polyamine concentration increased in pepper shoot, while it decreased in the other species. Thus, results show that Put accumulation was a consequence of salt stress in the most sensitive species, while salt tolerant species (beetroot) showed little change in polyamine concentration, and higher concentration in both Na⁺ and total cations. The role of polyamines or cation increased concentration after saline treatment in species with different salt tolerance is discussed.     

Determination of polyamines in human prostate by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of polyamines in human prostate has been developed. This method is based on pre-column derivatization with dansyl chloride (Dns-Cl). The derivatives were separated on a μBondapak C₁₈ column (250×4.6 mm I.D.; 10 μm), and eluted with methanol and distilled water using a one-step linear gradient. The column eluate was monitored by fluorescence detection (excitation, 370 nm; emission, 506 nm). The within-assay precision of the study (C.V.) was as follows: putrescine (PUT) 2.88%, spermidine (SPD) 2.94% and spermine (SP) 1.17%. The between-assay precision (C.V.) was: PUT 2.66%, SPD 3.06%, SP 2.79%. The recovery was greater than 97%. The detection limit for PUT, SPD and SP were 0.05, 0.08 and 0.06 nmol/ml, respectively. In contrast to other studies, sample or polyamine derivatives did not require extraction with an organic solvent such as ethanol, evaporation under vacuum or other condensation procedures. This is a simple, rapid and sensitive method that can be applied to the determination of polyamines in nearly all biological tissues and body fluids, such as urine and serum.     

Polyamines as cancer markers: applicable separation methods

Spermine, spermidine, putrescine and cadaverine are aliphatic amines widely spread in the human body. Their concentrations together with their acetyl conjugates increase significantly in the biological fluids and the affected tissues of cancer patients. Their concentrations decrease with the improvement in the patient’s condition on multiple therapy. Various chromatographic techniques are frequently used in monitoring concentrations of di- and polyamines in cancer. Among these techniques, thin-layer chromatography and liquid chromatography using pre- or postcolumn derivatization, separating on a reversed-phase or an ion-exchange column are the most commonly used. Besides, high-resolution capillary column gas chromatography (GC) is increasingly used over packed column GC, and in recent years, capillary zone electrophoresis has also gained some importance in polyamine determinations. The review examines the prospects and the limitations of polyamines as cancer markers using chromatographic and electrophoretic techniques.     

Urinary polyamine evaluation for effective diagnosis of various cancers

With a newly modified analytical method, the concentrations of free and acetylated urinary polyamines were simultaneously determined in a control group (32 cases) and patients with various types of cancers (104 cases, 20 males and 84 females) by gas chromatography-nitrogen-phosphorus detection. Significant concentration differences between normal subjects and various cancer patients were found. The various types of cancers (advanced gastric carcinoma, ovarian cancer, acute myelocyte leukemia, non-Hodgkin's lymphoma) gave unique patterns of urinary polyamine profile as well as significant differences of concentration. To indirectly evaluate the possible involvement of enzymes, precursor-to-product concentration ratios were compared between controls and patients with various types of cancers.     

Determination of dansylated polyamines in red blood cells by liquid chromatography-tandem mass spectrometry

The concentration of polyamines in red blood cells (RBCs) is considered to be an index of cell proliferation. This index has been demonstrated to be of clinical importance for the follow-up and treatment of some cancer patients. The concentration of polyamines in RBCs is usually determined by high-performance liquid chromatography (HPLC) with fluorescence detection. In the current work, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of putrescine, spermidine, and spermine, the three major polyamines in RBCs. The polyamines were dansylated and analyzed by an LC gradient of 20-min duration on a C18 column on-line with a tandem mass spectrometer. An internal standard (1,8-diaminooctane) was used for quantification. This method exhibited excellent linearity for the three polyamines with regression coefficients higher than 0.99. The limits of detection for putrescine, spermidine, and spermine were 0.10, 0.75, and 0.50 pmol/ml, respectively. The intrarun precision values for putrescine, spermidine, and spermine all were better than 10%, and the interrun precision values were 13%, 9%, and 20%, respectively. The LC-MS/MS method is sufficiently simple and reliable enough to replace the currently used HPLC method with fluorescence detection in which putrescine is not always detectable.     

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.     

Arginine pathways and the inflammatory response: Interregulation of nitric oxide and polyamines: Review article

Summary. An early response to an acute inflammatory insult, such as wound healing or experimental glomerulonephritis, is the conversion of arginine to the cytostatic molecule nitric oxide (NO). This anti-bacterial phase is followed by the conversion of arginine to ornithine, which is the precursor for the pro-proliferative polyamines as well as proline for the production of extracellular matrix. This latter, pro-growth phase constitutes a repair phase response. The temporal switch of arginine as a substrate for the cytostatic iNOS/NO axis to the pro-growth arginase/ ornithine/polyamine and proline axis is subject to regulation by inflammatory cytokines as well as interregulation by the arginine metabolites themselves. Arginine is also the precursor for another biogenic amine, agmatine. Here we describe the capacity of these three arginine pathways to interregulate, and propose a model whereby agmatine has the potential to serve in the coordination of the early and repair phase pathways of arginine in the inflammatory response by acting as a gating mechanism at the transition from the iNOS/NO axis to the arginase/ODC/polyamine axis. Due to the pathophysiologic and therapeutic potential, we will further examine the antiproliferative effects of agmatine on the polyamine pathway.     

Metabolism and function of polyamines in plants: recent development (new approaches)

A review is presented of the recent developments in the metabolism andfunction of polyamines in plants. Polyamines appear to be involved in a widerange of plant processes so their exact role is not completely understood. Inthis review, the metabolic pathways involved in polyamine biosynthesis anddegradation are explained, along with the transport and conjugation of thesecompounds. The methodologies involved in the analysis of polyamine functionusing metabolic inhibitors and genetic and molecular approaches are described.The occurrence and distribution of polyamine-derived alkaloids are also dealtwith. The direction of future research in the study of plant polyamines isindicated.     

Polyamines as biomarkers for plant regeneration capacity: improvement of regeneration by modulation of polyamine metabolism in different genotypes of indica rice

The importance of cellular polyamine (PA) levels and the ratio of putrescine (Put) to spermidine (Spd) for plant regeneration ability via somatic embryogenesis in several commercially grown indica rice varieties is reported here. The genotypes namely NDR-624, IR-20, IR-36, BJ-1 (having Put:Spd ratio2.3) showed superior plant regeneration while KL, PB-1 and TN-1 (having Put:Spd ratio3.8) showed moderate plant regeneration ability. The genotypes namely HS, Bindli, DV-85, ACB-72, IR-64 and IR-72 (having Put:Spd ratio5.0) showed poor plant regeneration ability. In contrast KH-7 (Put:Spd ratio10.0) showed no response at all. Favorable modification of cellular PA titers and their Put:Spd ratio by the addition of exogenous PAs (Put, Spd) or their biosynthesis inhibitor, difluoromethylarginine (DFMA) led to the induction/promotion of plant regeneration in poorly responding genotypes. These results showed a close relationship between cellular PA levels and their Put:Spd ratio with in vitro morphogenetic capacity in indica rice and suggest that the cellular PAs and Put:Spd ratios are important determinants (biomarkers) of plant regeneration ability in indica rice, and the improvement/induction of plant regeneration in morphogenetically poor and recalcitrant species could be achieved by modulating PA metabolism.     

Essential,deadly, enigmatic: Polyamine metabolism and roles in fungal cells

Polyamines are essential metabolites found in all organisms. Intracellular polyamine levels are tightly maintained by biosynthesis, degradation, uptake and excretion processes that involve regulatory mechanisms – such as the antizyme inhibitory protein – that are conserved across the kingdoms of life, indicating that polyamine levels are critical to cell function. Nonetheless, the biochemical roles of polyamines and their involvement in numerous fundamental cellular processes including aging, cell cycle progression and growth only become apparent when polyamine homeostasis is perturbed. Thus, while polyamines are present in most cells and essential for cell growth, their biochemical functions are largely enigmatic. Studies in fungi have contributed to our basic understanding of polyamines, and might continue to bridge knowledge gaps regarding polyamine metabolism and cell function. Moreover, when considering the impact of fungi – directly or indirectly, for good or for ill – on human society, closing gaps in our understanding of polyamine functions in fungal physiology is an important goal in itself that might lead to the discovery of new targets for enhancing beneficial fungal interactions and diminishing those detrimental to crop and human health. To facilitate progress towards this prospect, here we appraise what is known about polyamine metabolism in fungi, how prevalent polyamines impact fungal physiology and metabolism, and how the levels of each polyamine are maintained in the fungal cell – thus pointing to how they might be perturbed.     

Catabolism of polyamines

Summary. Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N¹-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects.Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N⁸-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.     

Biosynthesis, oxidation and conjugation of aliphatic polyamines in higher plants

Summary. This chapter will focus on polyamine biosynthesis, oxidation, conjugation processes, mainly to hydroxycinnamic acids, and compartmentation of enzymes, substrates and products, giving an overview about recent results especially in higher plants. New research advances regarding the cloning of the main cDNA encoding for polyamine biosynthetic and oxidative enzymes, will be taken into consideration. Received January 4, 2000 Accepted March 7, 2000     

Polyamine uptake in cultured astrocytes: characterization and modulation by protein kinases

The properties and regulation of the polyamine transport system in brain are still poorly understood. The present study shows, for the first time, the existence of a polyamine transport system in cerebellar astrocytes and suggests that polyamine uptake is mediated by a single and saturable high-affinity transport system for putrescine, spermine, and spermidine (K:(m) = 3.2, 1.2, and 1.8 microM:, respectively). Although substitution of NaCl by choline chloride produced a decrease in the putrescine, spermine, and spermidine uptake, it seems that polyamine transport in cerebellar astrocytes is not mediated by an Na(+) cotransport as in the presence of Na(+) and cholinium, polyamine uptake was much lower than when measured in a sucrose-based medium. On the other hand, ouabain, gramicidin (a Na(+) ionophore), and ionomycin (a Ca(2+) ionophore) produced a strong inhibition of polyamine uptake, suggesting that membrane potential could have an important role in the functioning of the astroglial polyamine uptake system. Moreover, protein kinase C inhibition produced an enhancement of polyamine uptake, whereas stimulation of protein kinase C with phorbol esters inhibited polyamine uptake. Alternatively, the tyrosine kinase inhibitor genistein caused a marked reduction in the uptake. No effects on polyamine uptake were observed with inhibitors and activators of cyclic AMP-dependent protein kinase or when Ca(2+)/calmodulin-dependent protein kinase II was inhibited with KN-62. These results suggest that the polyamine uptake system in cerebellar astrocytes could be modulated by protein kinase C and tyrosine kinase activities.     

The biological functions of polyamine oxidation products by amine oxidases: Perspectives of clinical applications

Summary. The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H₂O₂ and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H₂O₂ generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.     

Protein-polyamine conjugation by transglutaminase in cancer cell differentiation: Review article

Summary. Considerable and intense progress has been made in the understanding of the chemistry, molecular biology and cell biology of transglutaminases (TGases: EC 2.3.2.13). The knowledge that very different processes such as cell growth, reproduction and death are dependent on the presence of adequate levels of these enzymes and that the amount of both free and protein-conjugated polyamines, formed by the enzyme, are capable of modulating the differentiation and proliferative capability of several cell types, has prompted a multitude of researchers to study the role of these fascinating molecules in cancer cell differentiation.     

Tissue transglutaminase in normal and abnormal wound healing: Review article

Summary. A complex series of events involving inflammation, cell migration and proliferation, ECM stabilisation and remodelling, neovascularisation and apoptosis are crucial to the tissue response to injury. Wound healing involves the dynamic interactions of multiple cells types with components of the extracellular matrix (ECM) and growth factors. Impaired wound healing as a consequence of aging, injury or disease may lead to serious disabilities and poor quality of life. Abnormal wound healing may also lead to inflammatory and fibrotic conditions (such as renal and pulmonary fibrosis). Therefore identification of the molecular events underlying wound repair is essential to develop new effective treatments in support to patients and the wound care sector.Recent advances in the understating of the physiological functions of tissue transglutaminase a multi functional protein cross-linking enzyme which stabilises tissues have demonstrated that its biological activities interrelate with wound healing phases at multiple levels. This review describes our view of the function of tissue transglutaminase in wound repair under normal and pathological situations and highlights its potential as a strategic therapeutic target in the development of new treatments to improve wound healing and prevent scarring.     

Leishmania tropica major: effect of paromomycin and pentamidine on polyamine levels in the skin of normal and infected mice

The polyamine content of the skin of BALB/c and C3H mice was determined at intervals, after injecting Leishmania tropica major. In BALB/c mice, putrescine and spermidine levels increased three- to seven-fold; in C3H mice, spontaneous recovery occurred after 3 weeks, accompanied by a reduction in putrescine and spermidine levels. Ornithine decarboxylase activity was negligible in normal, uninfected skin of both BALB/c and C3H mice, but increased steadily during infection. Treatment with drugs that inhibit the growth of leishmanial amastigotes in the skin of mice also reduced polyamine levels and ornithine decarboxylase activity of previously infected skin. There was a close correlation between the therapeutic activity of the drugs and their effect on polyamine content and synthesis. The aminoglycoside paromomycin, which was chemotherapeutically more effective than pentamidine, also had a greater effect on polyamine levels. S-adenosyl-L-Methionine decarboxylase activity in the skin of BALB/c and C3H mice was only slightly affected by the parasites. Polyamine levels and ornithine decarboxylase activity could possibly serve as means for measuring the growth of leishmanial parasites in skin and other tissues and as a measure of the efficacy of anti-leishmanial chemotherapeutics.     

Biogenic amines and apoptosis: Minireview article

Summary. The programmed cell death is a very complex mechanism involving many factors, among them the intracellular concentration of biogenic amines (BA) appears to be important for apoptosis triggering. The mitochondrial damage is imputable to hydrogen peroxide and aldehydes, produced by amine oxidases (AO)-mediated oxidation of BA. On the other hands, the apoptosis protection observed by high BA concentration appears to be related to their scavenger effect of ROS and/or their interaction with membrane pores. Also monoamine oxidase (MAO) inhibitors, like propargylamines, preserve the mitochondria integrity by inhibiting MAO and therefore the production of H₂O₂ and aldehydes and, as cations, by regulating membrane pores, like BA.As general conclusion, apoptosis is protected by high concentration of BA and/or other cations while it is favoured by ROS produced by AOs or other mechanisms.