Evidence is growing in favour of a relationship between cancer and chronic inflammation, and particularly of the role of a polyamine and histamine metabolic interplay involved in these physiopathological problems, which are indeed highly complex biological systems. Decodification of the complex inter- and intra-cellular signalling mechanisms that control these effects is not an easy task, which must be helped by systems biology technologies, including new tools for location and integration of database-stored information and predictive mathematical models, as well as functional genomics and other experimental molecular approaches necessary for hypothesis validation. We review the state of the art and present our latest efforts in this area, focused on the amine metabolism field.

The design and synthesis of SpmTrien (1,12-diamino-3,6,9-triazadodecane), an isosteric and charge-deficient analogue of spermine with excellent chelating properties towards Cu 2+ ions, as well as novel N 1 - and N 12 -Ac-SpmTriens and bis-Et-SpmTrien ( N 1 , N 12 -diethyl-1,12-diamino-3,6,9-triazadodecane) are described. Possible applications of SpmTrien and its derivatives to the investigation of the enzymes of polyamine metabolism and spermine cellular functions, including interaction with DNA, are discussed.

Colon cancer in humans is influenced by both genetic and dietary risk factors. The majority of colon cancers have somatic mutations in the APC (adenomatous polyposis coli) tumour-suppressor gene. Dietary arginine enhances the risk of APC-dependent colon carcinogenesis in mouse models by a mechanism involving NOS2 (nitric oxide synthase 2), as elimination of NOS2 alleles suppresses this phenotype. DFMO (difluoromethylornithine), a specific inhibitor of polyamine synthesis, also inhibits dietary arginine-induced colon carcinogenesis in C57BL/6J- Apc Min /J mice. The primary consequence of dietary arginine is to increase the adenoma grade in these mice. Either loss of NOS2 alleles or inhibition of polyamine synthesis suppresses the arginine-induced increase in adenoma grade. In addition to promoting intestinal carcinogenesis, polyamines can also reduce the efficacy of certain intestinal cancer chemopreventive agents. The NSAID (non-steroidal anti-inflammatory drug) sulindac is a potent inhibitor of intestinal carcinogenesis in the C57BL/6J- Apc Min /J mouse model and is used to treat humans with FAP (familial adenomatous polyposis). Dietary putrescine reduces the ability of sulindac to suppress intestinal tumorigenesis in the mouse model. These data suggest that reducing polyamine metabolism and dietary polyamine levels may enhance strategies for colon cancer chemoprevention.

Cytotoxic products of polyamines generated in situ by an enzyme-catalysed reaction may be useful as a new avenue in combating cancer. This study demonstrated that MDR (multidrug-resistant) cancer cells (colon adenocarcinoma and melanoma) are significantly more sensitive than the corresponding WT (wild-type) ones to H 2 O 2 and aldehydes, the products of BSAO (bovine serum amine oxidase)-catalysed oxidation of spermine. Moreover, cytotoxicity was considerably greater when the treatment was carried out at 42°C than at 37°C. TEM (transmission electron microscopy) observations showed major ultrastructural alterations of the mitochondria. These were more pronounced in MDR than in WT cells. After treatment with BSAO/spermine, a higher mitochondrial membrane depolarization and an increased mitochondrial activity in drug-resistant cells were observed.

The induction of polyamine catabolism by specific anti-tumour polyamine analogues has increased interest in the roles polyamine catabolism play in cell growth, death and response to various anti-tumour agents. The relatively recent finding of an inducible mammalian spermine oxidase (SMO/PAOh1), in addition to the two-step spermidine/spermine N 1 -acetyltransferanse (SSAT)/ N 1 -acetylpolyamine oxidase (APAO) catabolic pathway, underscores the complexities of the regulation of polyamine catabolism by various stimuli. Furthermore, recent data indicate that infectious agents and mediators of inflammation can also up-regulate polyamine catabolism. Induction of SSAT by these agents can reduce intracellular polyamine concentrations and cell growth rate, thus providing a beneficial mechanism by which cells may adapt to inflammatory stress. However, increased polyamine catabolism can also result in substantial increases in intracellular reactive oxygen species (ROS) through the production of H 2 O 2 as a by-product of either APAO or SMO/PAOh1 activity. This increased generation of ROS can have different results, depending on the mechanism of induction and cell types involved. Targeted killing of tumour cells by agents that stimulate SSAT/APAO and/or SMO/PAOh1 is obviously a ‘good’ effect. However, induction of SMO/PAOh1 by inflammation or infectious agents has the potential to produce sufficient ROS in normal, non-tumour cells to lead to DNA damage, mutation and, potentially, carcinogenic transformation (‘bad’). The variation in the induction of these polyamine catabolic enzymes, as well as the level and timing of this induction will dictate the cellular outcome in the presence of both desirable and undesirable effects (‘ugly’). Here we discuss the relative role of each of the steps in polyamine catabolism in response to inflammatory stress.

The Myc oncogenes are dysregulated in 70% of human cancers. They encode transcription factors that bind to E-box sequences in DNA, driving the expression of a vast amount of target genes. The biological outcome is enhanced proliferation (which is counteracted by apoptosis), angiogenesis and cancer. Based on the biological effects of Myc overexpression it was originally assumed that the important Myc target genes are those encoding components of the cell cycle machinery. Recent work has challenged this notion and indicates that Myc target genes encoding metabolic enzymes deserve attention, as they may be critical arbiters of Myc in cancer. Thus targeting metabolic enzymes encoded by Myc-target genes may provide a new means to treat cancer that have arisen in response to deregulated Myc oncogenes.

Both polyamine metabolism and DNA methylation play an important role in normal and malignant growth. Specific enzyme inhibitors or drugs that interfere with these metabolic pathways have proven to be potential anticancer agents. Since DNA methylation and polyamine metabolism depend on a common substrate, i.e. S -adenosylmethionine, interaction between both pathways can be expected. Little is known about the relationship between these pathways but studies are available indicating that polyamines and DNA methylation are directly or indirectly interconnected, metabolically as well as physiologically with respect to the regulation of cell growth, differentiation and cancer development. These considerations give rise to the possibility that, by targeting both pathways, a more profound and effective inhibitory effect on the growth of malignant cells can be achieved. In previous studies we showed that 6-MP (6-mercaptopurine) as well as MTX (methotrexate), well-known drugs in the treatment of acute lymphoblastic leukaemia, inhibit DNA methylation and induce apoptosis in malignant blood cells. Our recent results show that combined treatment with 6-MP, MTX and drugs interfering with polyamine metabolism has additive/synergistic effects on the growth, cell viability and/or apoptotic death of leukaemic cells. Such a combination therapy could have great clinical value for patients in which therapy using inhibitors of thiopurines/purine metabolism has failed.

ODC (ornithine decarboxylase) is a central regulator of cellular polyamine synthesis. ODC is a highly regulated enzyme stimulated by a variety of growth-promoting stimuli. ODC overexpression leads to cellular transformation. Cellular ODC levels are determined at transcriptional and translational levels and by regulation of its degradation. Here we review the mechanism of ODC degradation with particular emphasis on AzI (antizyme inhibitor), an ODC homologous protein that appears as a central regulator of ODC stability, cellular polyamine homoeostasis and cellular proliferation.

While polyamine homoeostasis is clearly important in maintenance of normal cell function, the roles of these cations, as well as the enzymes that regulate their metabolism, in the neoplastic process are not clear. In particular, the polyamine catabolic enzyme SSAT (spermidine/spermine N 1 -acetyltransferase) seems to have different roles in tumorigenesis, depending upon the particular system being analysed. In attempts to clarify the function of SSAT in tumour development, we have utilized the Apc Min /+ mouse, which carries a mutant allele of the Apc (adenomatous polyposis coli) gene, rendering it susceptible to the formation of multiple adenomas in the small intestine and colon. Using genetically engineered animals (i.e. transgenic and knockout mice), we have shown that SSAT acts as a tumour promoter in the Apc Min /+ model. Modulation of tumorigenesis is not associated with changes in tissue levels of either spermidine or spermine. These findings, along with those made in other animal models of cancer, have prompted us to propose that metabolic flux through the polyamine biosynthetic and catabolic pathways, and the consequent changes in levels of various metabolites within the cell (i.e. the metabolome), is critical to tumour development. The metabolic flux model represents a novel way of thinking about the role of polyamines in cell physiology and the neoplastic process.

Protein-bound γ-glutamylpolyamines have highlighted a new pathway in polyamine metabolism. Human foreskin keratinocytes offer a suitable model for this study. Indeed, they develop polymerized envelopes, as they differentiate, rich in ϵ-(γ-glutamyl)lysine and N 1 , N 8 -bis(γ-glutamyl)spermidine cross-links. We have found that the selective oxidation of N 1 -(γ-glutamyl)spermidine and N -(γ-glutamyl)spermine by FAD-dependent polyamine oxidase (PAO) may be one of the cellular mechanisms regulating the preferential formation of a sterically defined bis(γ-glutamyl)spermidine cross-link. The significance of this finding is unknown, but it suggests that the target of this PAO-modulation is to achieve the biochemical prerequisite for production of a normal epidermal stratum corneum.

The production of polyamines has been shown to be an effective target for a drug against the West African form of sleeping sickness caused by Trypanosoma brucei gambiense . T. brucei belongs to the group of protozoan parasites classed as trypanosomatids. Parasitic species of this group are the causative agents of various tropical diseases besides African sleeping sickness, e.g. Chagas' disease ( Trypanosoma cruzi ), cutaneous ( Lesihmania spp.) and visceral ( Leishmania donovani ) leishmaniasis. The metabolism of polyamines in the parasites is a potential target for the development of new drugs for treatment of these diseases. The key steps in polyamine synthesis are catalysed by ODC (ornithine decarboxylase) and AdoMetDC (S-adenosylmethionine decarboxylase). In the present paper, some of the available information on ODC and AdoMetDC in trypanosomatids will be described and discussed.

Colorectal cancer is one of a number of cancers that may be amenable to prevention. The NSAIDs (non-steroidal anti-inflammatory drugs) have been shown to be effective chemopreventative agents in humans, but their mechanism of action is not clear. The polyamines are cellular polycations that are essential for cell growth and are overproduced in cancer cells. It is our hypothesis that inhibition of polyamine metabolism is an integral part of the mechanism of cancer prevention mediated by NSAIDs.

To sustain growth and support metabolic requirements, mammals assimilate energy-producing molecules and nutrients from food. These molecules are distributed throughout the body in order to meet the requirements of the internal organs. The various demands of the different organs are to a large extent met by regulatory processes consisting of a complex interaction between hormones, growth factors and cytokines. Normal metabolic activity and partitioning of nutrients between individual organs is affected by a number of events such as stress, a limited supply of nutrients, infection or tumour growth. Since the intestine has the highest metabolic activity of all the internal organs, a tumour will initially compete with the gut for nutrients and energy-providing molecules. The polyamines represent a class of molecules where the demand in the body increases during tumour growth. A tumour can partly obtain the polyamines required to support its growth by up-regulating its own biosynthetic capacity and partly by increasing uptake from the body pool. Rather than limiting the exogenous supply of dietary polyamines we have used another approach to manipulate polyamine pools in mice. When the lectin phytohaemagglutinin is included in the diet, a fully reversible dose-dependent growth of the small intestine occurs leading to an extensive accumulation of polyamines in the intestinal epithelia. This approach of reducing the availability of exogenous polyamines to a growing tumour will be discussed.

The charge of the agmatine analogues AO-Agm [ N -(3-aminooxypropyl)guanidine], GAPA [ N -(3-aminopropoxy)guanidine] and NGPG [ N -(3-guanidinopropoxy)guanidine] is deficient as compared with that of agmatine and they are thus able to inhibit agmatine transport in liver mitochondria. The presence of the guanidine group is essential for an optimal effect, since AO-Agm and NGPG display competitive inhibition, whereas that of GAPA is non-competitive. NGPG is the most effective inhibitor ( K i =0.86 mM). The sequence in the inhibitory efficacy is not directly dependent on the degree of protonation of the molecules; in fact NGPG has almost the same charge as GAPA. When the importance of the guanidine group for agmatine uptake is taken into account, this observation suggests that the agmatine transporter is a single-binding, centre-gated pore rather than a channel.

SAM ( S -adenosylmethionine, also known as AdoMet) is well known as the methyl donor for the majority of methyltransferases that modify DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modelling, epigenetic modifications and imprinting, which are all topics of great interest and importance in cancer research and aging. In total, 15 superfamilies of SAM-binding proteins have been identified, with many additional functions varying from methylation of phospholipids and small molecules such as arsenic to synthesis of polyamines or radical formation. SAM is regenerated from demethylated SAM via the methionine cycle, which involves folate. Imbalance of this cycle in humans, e.g. through folate shortage via dietary insufficiency, alcohol abuse, arsenic poisoning or hereditary factors, leads to depletion of SAM and human disease. In addition to its role as a methyl donor to modification enzymes that protect bacterial DNA against cognate restriction, SAM also serves as a co-factor for nucleases such as the type I restriction enzyme EcoKI, which is unable to restrict DNA in the absence of SAM. Finally, on a completely different tack, SAM can bind to certain RNA structures called riboswitches that control transcription or translation. In this way, expression of multiple genes can be regulated in a SAM-dependent manner, an unexpected finding that opens up new avenues into gene control. This minireview discusses some of these diverse and amazing roles of this small metabolite.

Polyamines are downstream mediators of genetic risk factors in human intestinal cancers. The adenomatous polyposis coli (APC) tumour-suppressor gene, which is mutated in essentially all human colon cancers, regulates the expression of several e-box transcription factors. These factors, in turn, regulate the transcription of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis. The Kirsten ras ( K-ras ) oncogene regulates the expression of several genes, including suppressing the expression of peroxisomal proliferator-activated receptor γ (PPARγ). This PPAR, in turn, activates the expression of the spermidine/spermine-N 1 -acetyltransferase (SSAT), the first enzyme in polyamine catabolism. The non-steroidal anti-inflammatory drug (NSAID) sulindac induces the transcription of SSAT via activation of PPARγ. Inactivation of the APC tumour-suppressor gene, and the activation of K-ras , have a combined effect on increasing tissue polyamine contents due to increased synthesis and decreased catabolism of the polyamines. Pharmacological strategies for suppressing ODC (e.g. the enzyme-activated inhibitor α-difluoromethylornithine) and activating SSAT (e.g. NSAIDs) are potent inhibitors of intestinal carcinogenesis in rodent models. Clinical trials combining these classes of agent in humans with risk factors for colon cancer are in progress.

Interestingly, there is a major difference in turnover rate between ornithine decarboxylases (ODCs) from various trypanosomatids. ODCs from Trypanosoma brucei and Leishmania donovani are both stable proteins, whereas ODC from Crithidia fasciculata is a metabolically unstable protein in the parasite. C. fasciculata ODC is also rapidly degraded in mammalian systems, whereas the closely related L. donovani ODC is not. The degradation of C. fasciculata ODC in the mammalian systems is shown to be dependent on a functional 26 S proteasome. However, in contrast to the degradation of mammalian ODC, the degradation of C. fasciculata ODC does not involve antizyme. Instead, it appears the degradation of C. fasciculata ODC may be associated with poly-ubiquitination of the enzyme.

The importance of polyamines in prostatic growth and differentiation has prompted studies to evaluate the clinical relevance of the ornithine decarboxylase/polyamine system in prostatic cancer. These studies show that differences in biological behaviour of prostatic (cancer) cells are associated with changes in polyamine levels and/or the activity of their metabolic enzymes. Faulty antizyme regulation of polyamine homoeostasis may play an important role in the growth and progression of prostatic carcinoma. Treatment of human prostate carcinoma cells with inhibitors of polyamine metabolic enzymes or polyamine analogues induces cell growth arrest or (apoptotic) cell death. Our recent in vitro studies using conformationally restricted polyamine analogues show that these compounds inhibit cell growth, probably by inducing antizyme-mediated degradation of ornithine decarboxylase. Sensitivity of human prostate cancer cells for these compounds was increased in the absence of androgens. These results suggest that these analogues might have chemotherapeutic potential in case prostatic cancer has become androgen-independent. Pilot data in an in vivo model show that these analogues have effects on tumour cell proliferation, vascularity, blood perfusion and tissue hypoxia. Overall, these studies show that polyamines may serve as important biomarkers of prostatic malignancy and provide a promising target for chemotherapy of prostatic cancer.

The naturally occurring polyamines are found in all living cells, where they fulfil a number of critical functions in relation to cell growth. The quest to identify these functions has been the subject of five independent colloquia hosted by the Biochemical Society and today still occupies several hundred scientists across Europe, the U.S.A. and Japan.

Molecular, biochemical and genetic characterization of ornithine decarboxylase, S -adenosylmethionine decarboxylase and spermidine synthase establishes that these polyamine-biosynthetic enzymes are essential for growth and survival of the agents that cause African sleeping sickness, Chagas' disease, leishmaniasis and malaria. These enzymes exhibit features that differ significantly between the parasites and the human host. Therefore it is conceivable that exploitation of such differences can lead to the design of new inhibitors that will selectively kill the parasites while exerting minimal, or at least tolerable, effects on the parasite-infected patient.

In colon cancer, the activities of polyamine-synthesizing enzymes and polyamine content are increased 3–4-fold over that found in the equivalent normal colonic mucosa, and polyamines have even been attributed as markers of neoplastic proliferation in the colon. Furthermore, and in contrast with all other cell systems in the body, normal and neoplastic cells in the colon are exposed to high concentrations of putrescine from the lumen, synthesized by colonic microflora. While such a high polyamine supply may be of benefit in non-neoplastic colonic mucosal growth, the role of luminal polyamines in colon cancer is a clear concern. Luminal polyamines are readily taken up by neoplastic colonocytes, they are utilized in full to support neoplastic growth, and their uptake is strongly up-regulated by the mitogens known to play an important role in colonic carcinogenesis. Inhibition of polyamine synthesis and their uptake, impaired utilization of exogenous polyamines, and enhanced catabolism of polyamines in neoplastic colonocytes are therefore logical approaches in the chemoprevention of colorectal cancer.

Interest in polyamine catabolism has increased since it has been directly associated with the cytotoxic response of multiple tumour types to exposure to specific anti-tumour polyamine analogues. Human polyamine catabolism was considered to be a two-step pathway regulated by the rate-limiting enzyme spermidine/spermine N 1 -acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (APAO). Further, the super-induction of SSAT by several anti-tumour polyamine analogues has been implicated in the cytotoxic response of specific solid-tumour phenotypes to these agents. This high induction of SSAT has been correlated with cellular response to the anti-tumour polyamine analogues in several systems and considerable progress has been made in understanding the molecular mechanisms that regulate the analogue-induced expression of SSAT. A polyamine response element has been identified and the transacting transcription factors that bind and stimulate transcription of SSAT have been cloned and characterized. The link between SSAT activity and cellular toxicity is thought to be based on the production of H 2 O 2 by the activity of the constitutive APAO that uses the SSAT-produced acetylated polyamines. The high induction of SSAT and the subsequent activity of APAO are linked to the cytotoxic response of some tumour cell types to specific polyamine analogues. However, we have recently cloned a variably spliced human polyamine oxidase (PAOh1) that is inducible by specific polyamine analogues, efficiently uses unacetylated spermine as a substrate, and also produces toxic H 2 O 2 as a product. The results of studies with PAOh1 suggest that it is an additional enzyme in polyamine catabolism that has the potential to significantly contribute to polyamine homoeostasis and drug response. Most importantly, PAOh1 is induced by specific polyamine analogues in a tumour-phenotype-specific manner in cell lines representative of the major forms of solid tumours, including lung, breast, colon and prostate. The sensitivity to these anti-tumour polyamine analogues can be significantly reduced if the tumour cells are co-treated with 250 μM of the polyamine oxidase inhibitor N 1 , N 4 -bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527), suggesting that the H 2 O 2 produced by PAOh1 does in fact play a direct role in the observed cytotoxicity. These results strongly implicate PAOh1 as a new target that, in combination with SSAT, may be exploited for therapeutic advantage. The current understanding of the role and regulation of these two important polyamine catabolic enzymes are discussed.

A number of polyamine derivatives have demonstrated potential as therapeutic agents. For example, 1,12-bisethylspermine and bisnaphthalimide (elinafide) are currently in phase I clinical trials for the treatment of certain cancers. Here, the biological activities of two new groups of polyamine derivative, namely the oxa-polyamines and the bisnaphthalimides, are presented. The most active compounds in the oxa-polyamine and bisnaphthalimido series possessed IC 50 values of 2.93 and 1.38 μM, respectively, against MCF7 cells after 48 h of exposure. The structure–relationship activities of each group of compounds are discussed. Bisnaphthalimido compounds are DNA-binding agents. Addition of the bisnaphthalimides PK3, PK4, PK5, PK6 and PK7, at a concentration of 10 μM, to the calf thymus DNA duplex increased the T m of DNA by 11.55±0.56, 14.545±1.59, 6.23±2.45, 12.56±1.84 and 16.45±0.39°C respectively. With the exception of PK5, all compounds bind to DNA by intercalation as judged by effect of compounds on DNA mobility. Ethidium bromide displacement assay showed that all the compounds have significant affinity for calf thymus DNA (the drug concentration required to reduce the fluorescence of initially DNA-bound ethidium bromide by 50%, C 50 , was 1.21–17.33 μM). The order of DNA-binding strength was PK4 > PK3 > PK7 > PK6 > PK5. In HL-60 promyelocytic leukaemia cells, oxa-polyamine and bisnaphthalimido treatment resulted in a decline in cell proliferation and viability. The assays performed suggested that apoptosis was not the principal cell death mechanism involved in oxa-polyamine cytotoxicity. In contrast, HL-60 cell death induced by the bisnaphthalimido series was characterized by early exposure of phosphatidylserine exclusive from membrane damage, elevated caspase-3 activity, increased DNA instability and, ultimately, DNA fragmentation. Thus the principal cytotoxic members of the bisnaphthalimido series appear to induce apoptosis.

It is becoming apparent that control of protein synthesis by metabolites is more common than previously thought. Much of that control is exerted at the level of initiation of mRNA translation, orchestrated by upstream open reading frames (uORFs) and RNA secondary structure. S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis and both mammalian and plant AdoMetDCs are translationally regulated by uORFs in response to polyamine levels by distinct mechanisms.

The driving force of the cell cycle is the activities of cyclin-dependent kinases (CDKs). Key steps in the regulation of the cell cycle therefore must impinge upon the activities of the CDKs. CDKs exert their functions when bound to cyclins that are expressed cyclically during the cell cycle. Polyamine biosynthesis varies bicyclically during the cell cycle with peaks in enzyme activities at the G 1 /S and S/G 2 transitions. The enzyme activities are regulated at transcriptional, translational and post-translational levels. When cells are seeded in the presence of drugs that interfere with polyamine biosynthesis, cell cycle progression is affected within one cell cycle after seeding. The cell cycle phase that is most sensitive to polyamine biosynthesis inhibition is the S phase, while effects on the G 1 and G 2 /M phases occur at later time points. The elongation step of DNA replication is negatively affected when polyamine pools are not allowed to increase normally during cell proliferation. Cyclin A is expressed during the S phase and cyclin A/CDK2 is important for a normal rate of DNA elongation. Cyclin A expression is lowered in cells treated with polyamine biosynthesis inhibitors. Thus, polyamines may affect S phase progression by participating in the regulation of cyclin A expression.