Targeting pheochromocytoma/paraganglioma with polyamine inhibitors.

BACKGROUND: Pheochromocytomas (PCCs) and paragangliomas (PGLs) are neuroendocrine tumors that are mostly benign. Metastatic disease does occur in about 10% of cases of PCC and up to 25% of PGL, and for these patients no effective therapies are available. Patients with mutations in the succinate dehydrogenase subunit B (SDHB) gene tend to have metastatic disease. We hypothesized that a down-regulation in the active succinate dehydrogenase B subunit should result in notable changes in cellular metabolic profile and could present a vulnerability point for successful pharmacological targeting. METHODS: Metabolomic analysis was performed on human hPheo1 cells and shRNA SDHB knockdown hPheo1 (hPheo1 SDHB KD) cells. Additional analysis of 115 human fresh frozen samples was conducted. In vitro studies using N1,N11-diethylnorspermine (DENSPM) and N1,N12- diethylspermine (DESPM) treatments were carried out. DENSPM efficacy was assessed in human cell line derived mouse xenografts. RESULTS: Components of the polyamine pathway were elevated in hPheo1 SDHB KD cells compared to wild-type cells. A similar observation was noted in SDHx PCC/PGLs tissues compared to their non-mutated counterparts. Specifically, spermidine, and spermine were significantly elevated in SDHx-mutated PCC/PGLs, with a similar trend in hPheo1 SDHB KD cells. Polyamine pathway inhibitors DENSPM and DESPM effectively inhibited growth of hPheo1 cells in vitro as well in mouse xenografts. CONCLUSIONS: This study demonstrates overactive polyamine pathway in PCC/PGL with SDHB mutations. Treatment with polyamine pathway inhibitors significantly inhibited hPheo1 cell growth and led to growth suppression in xenograft mice treated with DENSPM. These studies strongly implicate the polyamine pathway in PCC/PGL pathophysiology and provide new foundation for exploring the role for polyamine analogue inhibitors in treating metastatic PCC/PGL. PRéCIS: Cell line metabolomics on hPheo1 cells and PCC/PGL tumor tissue indicate that the polyamine pathway is activated. Polyamine inhibitors in vitro and in vivo demonstrate that polyamine inhibitors are promising for malignant PCC/PGL treatment. However, further research is warranted.

[Leishmania donovani: structural insignt in the recognition of C-methylated analogues of spermidine as natural polyamine].

The ability of alpha-, beta-, gamma- and omega-methylated spermidine analogues to restore the growth of L. donovani promastigotes that were depleted of putrescine and spermidine was investigated. Only beta-methylated spermidine, like natural spermidine was capable of restoring the growth of L. donovani, while the remaining three analogues turned out to be inactive. Considering that alpha-methylated spermidine is a functionally active spermidine surrogate both in vivo and in vitro, this analogue can be considered as an antidote in the host-parasite system, especially in cases where inhibitors of polyamine biosynthesis are used for the therapy of leishmaniasis.

Targeting polyamines and inflammation for cancer prevention.

Increased polyamine synthesis and inflammation have long been associated with intraepithelial neoplasia, which are risk factors for cancer development in humans. Targeting polyamine metabolism (by use of polyamine synthesis inhibitors or polyamine catabolism activators) and inflammation (by use of nonsteroidal anti-inflammatory drugs) has been studied for many cancers, including colon, prostate, and skin. Genetic epidemiology results indicate that a genetic variant associated with the expression of a polyamine biosynthetic gene is associated with risk of colon and prostate cancers. A clinical trial of difluoromethylornithine (DFMO), a selective inhibitor of polyamine synthesis, showed that the 1 year treatment duration reduced prostate volume and serum prostate-specific antigen doubling time in men with a family history of prostate cancer. A second, clinical trial of DFMO in combination with sulindac, a NSAID in patients with prior colon polyps found that the 3-year treatment was associated with a 70% reduction of all, and over a 90% reduction of advanced and/or multiple metachronous colon adenomas. In this chapter, we discuss that similar combination prevention strategies of targeting polyamines and inflammation can be effective in reducing risk factors associated with the development of human cancers.

Role of polyamines and cAMP-dependent mechanisms on 5alpha-dihydrotestosterone-elicited functional effects in isolated right atria of rat.

Androgens produce acute vasodilation of systemic, pulmonary, and coronary arteries in several mammal preparations and increase cardiomyocyte contractility. A decrease of the spontaneous beating of sinoatrial cells has also been described. The aim of this study was to characterize the direct effect of 5alpha-dihydrotestosterone on the spontaneous chronotropism and inotropism in the same preparation as an approach to establish the effect on cardiac output and their mechanism of action. The effects were studied on isolated right atria of Wistar rats placed in an organ bath in Tyrode solution at 37 degrees C and bubbled with carbogen. In male rats, the acute administration of 5alpha-dihydrotestosterone, a nonaromatizable derivate of testosterone, elicited a positive inotropism, which was associated with a negative chronotropism. As reported in the left atria, polyamines and beta-adrenoceptors played a role in 5alpha-dihydrotestosterone-elicited positive inotropism because the effect was antagonized by alpha-difluoromethylornithine, an inhibitor of polyamine synthesis, and atenolol, a beta1-adrenoceptor blocker, but not on the negative effect on chronotropism. The androgen increased the sinoatrial node recovery time, suggesting an effect on the mechanisms of spontaneous diastolic depolarization involved in atria pacemaking. These effects of 5alpha-dihydrotestosterone are not hormonally regulated because they are similarly produced in estrogenized females and gonadectomized male and female rats. These results suggest that the androgen could acutely improve cardiac performance.

The polyamines regulate endothelial cell survival during hypoxic stress through PI3K/AKT and MCL-1.

Hypoxia-dependent angiogenesis is an inherent feature of solid tumors, and a better understanding of the molecular mechanisms of hypoxic cell-death should provide additional targets for cancer therapy. Here, we show a novel role of the polyamines in endothelial cell (EC) survival during hypoxia. Polyamine depletion by specific inhibition of ornithine decarboxylase was shown to protect ECs from hypoxia-induced apoptosis. Inhibition of the polyamines resulted in a significant induction of PI3K/AKT and its down-stream target MCL-1, i.e. an anti-apoptotic member of the BCL-2 family. Specific inhibitors of PI3K reversed the decrease of hypoxia-induced apoptosis as well as the induction of MCL-1 in polyamine-deprived cells. Moreover, siRNA-mediated down-regulation of MCL-1 was found to counter-act the protective effect of polyamine inhibition. We conclude that the polyamines regulate hypoxia-induced apoptosis in ECs through PI3K/AKT and MCL-1 dependent pathways. Our results may have important implications for the modulation of hypoxia-driven neovascularization.

Ornithine decarboxylase inhibition by alpha-difluoromethylornithine activates opposing signaling pathways via phosphorylation of both Akt/protein kinase B and p27Kip1 in neuroblastoma.

Ornithine decarboxylase (ODC) is a key enzyme in mammalian polyamine biosynthesis that is up-regulated in various types of cancer. We previously showed that treating human neuroblastoma (NB) cells with the ODC inhibitor alpha-difluoromethylornithine (DFMO) depleted polyamine pools and induced G1 cell cycle arrest without causing apoptosis. However, the precise mechanism by which DFMO provokes these changes in NB cells remained unknown. Therefore, we further examined the effects of DFMO, alone and in combination with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or Akt/protein kinase B (PKB) inhibitor IV, on the regulation of cell survival and cell cycle-associated pathways in LAN-1 NB cells. In the present study, we found that the inhibition of ODC by DFMO promotes cell survival by inducing the phosphorylation of Akt/PKB at residue Ser473 and glycogen synthase kinase-3beta at Ser9. Intriguingly, DFMO also induced the phosphorylation of p27Kip1 at residues Ser10 (nuclear export) and Thr198 (protein stabilization) but not Thr187 (proteasomal degradation). The combined results from this study provide evidence for a direct cross-talk between ODC-dependent metabolic processes and well-established cell signaling pathways that are activated during NB tumorigenesis. The data suggest that inhibition of ODC by DFMO induces two opposing pathways in NB: one promoting cell survival by activating Akt/PKB via the PI3K/Akt pathway and one inducing p27Kip1/retinoblastoma-coupled G1 cell cycle arrest via a mechanism that regulates the phosphorylation and stabilization of p27Kip1. This study presents new information that may explain the moderate efficacy of DFMO monotherapy in clinical trials and reveals potential new targets for DFMO-based combination therapies for NB treatment.

Mepacrine antagonises tumour cell growth induced by natural polyamines.

BACKGROUND: Mepacrine is an antiproliferative agent, characterised by an aliphatic chain similar to that of natural polyamines whose activation is closely associated with cell proliferation and may lead to malignant transformation and neurodegenerative diseases. This study aims to investigate a possible antagonism between mepacrine and polyamines in tumour proliferation. MATERIALS AND METHODS: MCF-7 and Vero cells were cultured in Eagle's minimum essential medium and then subjected to graded concentrations of putrescine, spermine and spermidine alone and in combination with mepacrine. Methyl thiazole tetrazolium test and Western-blotting were performed. RESULTS: Putrescine and spermidine at 0.5 mg/l significantly stimulated cell growth, whereas mepacrine treatment confirmed the enhanced p21 expression previously reported by a recent study and growth inhibition. When used in combination, mepacrine antagonized MCF-7 growth induced by polyamines. CONCLUSION: Our results suggest that mepacrine may represent a choice in the treatment of tumours induced by the modified concentration of polyamines.

Novel agmatine analogue, gamma-guanidinooxypropylamine (GAPA) efficiently inhibits proliferation of Leishmania donovani by depletion of intracellular polyamine levels.

The efficacy of gamma-guanidinooxypropylamine (GAPA), a novel agmatine analogue against protozoan parasite, Leishmaniadonovani was evaluated. Wild-type and ornithine decarboxylase-overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. GAPA inhibited the growth of both promastigotes and amastigotes. Ornithine decarboxylase (ODC) activity and polyamine levels were markedly lower in cells treated with GAPA and proliferation was rescued by addition of putrescine or spermidine. GAPA inhibited L. donovani recombinant ODC with K(i) value of approximately 60microM. The ODC-overexpressors showed significant resistance to GAPA. GAPA has pK(a) 6.71 and at physiological pH the analogue can mimic protonated state of putrescine and can probably use putrescine transport system. Transport of putrescine in wild-type L. donovani promastigotes was inhibited by GAPA. We for the first time report that GAPA is a potential antileishmanial lead compound and it possibly inhibits L. donovani growth by depletion of intracellular polyamine levels.

Single chain fragment anti-heparan sulfate antibody targets the polyamine transport system and attenuates polyamine-dependent cell proliferation.

The growth-promoting polyamines are polybasic compounds that efficiently enter cancer cells by as yet incompletely defined mechanisms. Strategies to inhibit their internalization may have important implications in the management of tumor disease. Here, we show that cellular binding and uptake of polyamines are inhibited by a single chain variable fragment anti-heparan sulfate (HS) antibody. Polyamine uptake was inhibited in a dose-dependent manner, and was associated with compensatory up-regulation of ornithine decarboxylase (ODC), i.e. the key enzyme of the polyamine biosynthesis pathway. Conversely, depletion of intracellular polyamines by the specific ODC-inhibitor alpha-difluoromethylornithine (DFMO) resulted in increased cellular binding of polyamine and anti-HS antibody. Importantly, anti-HS antibody also efficiently targeted DFMO-induced polyamine uptake, and combined polyamine biosynthesis inhibition by DFMO, and uptake inhibition by anti-HS antibody attenuated tumor cell proliferation in vitro. In conclusion, cell-surface HS proteoglycan is a relevant target for antibody-mediated inhibition of the uptake of polyamines, and polyamine-dependent cell proliferation.

[Characteristics of polyamine biosynthesis regulation and tumor growth rate in hormone-dependant grafted breast tumors of mice and rats].

Effect of the inhibitors of polyamines biosynthesis on completely or partially hormone-dependant breast tumors (mouse Ca755 carcinoma and Walker W-256 carcinosarcoma) is essentially special: in contrary to hormone-dependant tumors, this effect may be not only breaking but stimulating as well. Change-over from one to another mode of reaction is conditioned, most probable, by hormonal status, which is determined by one or another estral cycle phase. Biochemical mechanisms of this change-over are closely connected with polyamines metabolism, namely the degree of polyamines (especially spermine) interconvertion and physiological reactivity level of the system controlling expression of ornithin-decarboxilase. At that, the first of these pathways is predominant for completely hormone-dependant Ca755 and the second one -for partially hormone-dependant W-256.

Polyamines in the brain: distribution, biological interactions, and their potential therapeutic role in brain ischaemia.

The endogenous polyamines (spermine, spermidine, and putrescine) are present at relatively high concentrations in the mammalian brain and play crucial roles in a variety of aspects of cell functioning. Stroke is the third most common cause of death and the leading cause of disability among adults in the western world. Brain polyamine levels change dramatically following cerebral ischaemia. Polyamines may be involved in the pathophysiological processes underlying brain ischaemia through several possible mechanisms. These include direct effects on ion channels and receptors modulating potassium, and most importantly calcium trafficking, or through the production of toxic metabolites. Considerable evidence shows that the non-competitive polyamine antagonists, ifenprodil and eliprodil, are neuroprotective. Interestingly, novel polyamine analogues, such as N(1)-dansylspermine, BU36b, and BU43b, have also recently been shown to have neuroprotective potential. The exact mechanisms of the neuroprotection afforded by the polyamine antagonists and their clinical applicability is worthy of further study.

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.

Suppression of TNF-alpha production by S-adenosylmethionine in human mononuclear leukocytes is not mediated by polyamines.

Endotoxin-induced cytokine production is an important mechanism in the development of several types of liver damage. Methionine, some of its precursors and metabolites were reported to have protective effects against such injury. The aim of this study was to investigate whether methionine, its precursors or metabolites [phosphatidylcholine, choline, betaine, S-adenosylmethionine (SAM)] have a modulating effect on tumor necrosis factor alpha (TNF-alpha) production by endotoxin-stimulated human mononuclear leukocytes and whether SAM-dependent polyamines (spermidine, spermine) are mediators of SAM-induced inhibition of TNF-alpha synthesis. Methionine and betaine had a moderate stimulatory effect on TNF-alpha production, whereas phosphatidylcholine (ID(50) 5.4 mM), SAM (ID(50) 131 microM), spermidine (ID(50) 4.5 microM) and spermine (ID(50) 3.9 microM) had a predominantly inhibitory effect. Putrescine did not alter TNF-alpha release. Inhibitors of polyamine synthesis that blocked either putrescine (difluoromethylornithine) or spermine (CGP48664A) production did not affect TNF-alpha synthesis. Endotoxin stimulation of leukocytes did not alter the intracellular levels of polyamines. In addition, supplementation with SAM did not change the intracellular concentration of either polyamine measured. We conclude that phosphatidylcholine-induced immunosuppression is not caused by methionine and polyamines are not involved in SAM-induced inhibition of TNF-alpha production. The limitation of TNF-alpha release by spermidine is specific and is not due to its conversion into spermine.

Polyamine-mediated reduction in human airway epithelial migration in response to wounding is PGE2 dependent through decreases in COX-2 and cPLA2 protein levels.

Both ornithine decarboxylase inhibition to deplete polyamines and cyclooxygenase inhibition diminish the migration response to injury of human airway epithelial cells in tissue culture monolayers by approximately 75%. Restoration of normal migration responses is achieved in the polyamine depleted system either by exogenous reconstitution of polyamines or the addition of prostaglandin E(2) (PGE(2)). However, only PGE(2) was able to restore migration in the cyclooxygenase-inhibited systems. Western blot for cyclooxygenase-2 and cytosolic phospholipase A(2) protein levels and ELISAs for PGE(2) secretion demonstrate dramatic increases over 24-48 h after monolayer wounding. These increases are completely abolished by polyamine depletion or cyclooxygenase inhibition. We conclude that polyamine inhibition decreases cellular migration in response to injury in airway epithelial cells at least in part through inhibiting normal PGE(2) production in response to injury. This may be brought about by decreases in cytosolic phospholipase A(2) and cyclooxygenase-2 protein levels.

The use of anticancer drugs in antiparasitic chemotherapy.

Many similarities exist between cancer cells and parasites. A potentially lucrative starting point for the discovery of novel drugs to combat parasites is to examine available compounds developed against cancer for antiparasitic properties. Here, we review the use of current and promising anticancer agents for treating major human parasitic diseases.

[Effect of alpha-difluoromethylornithine and polyhexamethyleneguanidine (PMG), polyamine biosynthesis inhibitors on leucosis L1210 growth kinetics and life expectancy of animals with cancer].

The article deals with the study of polyamines content and y-glutamiltranspeptidase (gamma-GTP) activity in leucosis L1210 cells under the influence of inhibitors of polyamines synthesis such as alpha-difluoromethylornithine (alpha-DFMO) and polyhexamethylenguanidine (PMG). Injections of alpha-DFMO and PMG to animals essentially reduce putrescine and spermidine concentrations, and the levels of spermine and gamma-GTP activity increase under this influence. These modulation were associated with L1210 leucosis growth retardation. Antiblastic effect was dependent on inhibitors' doses and mode of injections' course. Under the optimum conditions the retardation index was 90-98%. The animals with retarded tumor growth had essentially longer survival time frame than blank tumor-bearing animals (index was 37.2 for a-DFMO and 67.5 for PMG).

Significance of targeting polyamine metabolism as an antineoplastic strategy: unique targets for polyamine analogues.

The polyamines, putrescine, spermidine, and spermine, are naturally occurring polycationic alkylamines that are absolutely required for eukaryotic cell growth. Importantly, the polyamine metabolic pathway, as well as the requirement of polyamines for cell growth, is frequently dysregulated in cancer cells, thus providing a unique set of targets for therapeutic intervention. Ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis, is frequently up-regulated in preneoplastic cells, and has been implicated as an oncogene in multiple tumor types. Several model systems have demonstrated that inhibition of ODC's enzymatic activity and down-regulation of its expression are rational strategies for both chemotherapy and chemoprevention. Specific inhibitors of ODC, most notably 2-difluoromethylornithine (DFMO), have been used experimentally to validate polyamine metabolism as an antineoplastic strategy. However, multiple biochemical and clinical limitations to these ODC-targeting strategies minimize their value as therapeutic tools. Included among these limitations are poor bioavailability of the inhibitor, and the compensatory up-regulation of polyamine metabolism and transport that allow tumor cells to escape the growth inhibitory effects of blockers specifically targeting ODC. As a strategy to overcome the limitations of direct enzyme inhibition, several groups have pursued the design of polyamine analogues that specifically target the dysregulated polyamine metabolism found in tumors. These analogues have been developed specifically to target the specific polyamine transporter, thus competing with circulating natural polyamines. Additionally, most of the analogues examined thus far maintain the regulatory function of the natural polyamines, but are unable to functionally substitute for them in promoting growth. Specifically, individual analogues have demonstrated the ability to down-regulate each of the biosynthetic enzymes without causing compensatory increases in parallel systems or increases in polyamine uptake. Additionally, specific analogues produce tumor specific up regulation of the rate-limiting enzymes in polyamine catabolism. These results are particularly significant in that the products of polyamine catabolism, including H2O2, have been demonstrated to participate in the tumoricidal activity of specific analogues.

Drugs affecting the cell cycle via actions on the polyamine metabolic pathway.

Polyamines (putrescine, spermidine, and spermine) are ubiquitous cellular components that have multiple functions, including actions affecting the cell cycle. Polyamine biosynthesis and content is altered during the course of cell cycling via changes in two key biosynthetic enzymes, ornithine decarboxylase and S-adenosyl-methionine decarboxylase. Decreases in polyamine content and/or alterations in the relative amounts of polyamines can be achieved by treatment with inhibitors of these enzymes or by application of polyamine analogues, which subvert mechanisms for polyamine homeostasis and may interfere directly with polyamine-dependent processes. Such changes cause G1 and G2-M cell cycle blocks that can be brought about via induction of p21WAF1/CIP1.

Visually driven regulation of intrinsic neuronal excitability improves stimulus detection in vivo.

Neurons adapt their electrophysiological properties to maintain stable levels of electrical excitability when faced with a constantly changing environment. We find that exposing freely swimming Xenopus tadpoles to 4-5 hr of persistent visual stimulation increases the intrinsic excitability of optic tectal neurons. This increase is correlated with enhanced voltage-gated Na+ currents. The same visual stimulation protocol also induces a polyamine synthesis-dependent reduction in Ca2+-permeable AMPAR-mediated synaptic drive, suggesting that the increased excitability may compensate for this reduction. Accordingly, the change in excitability was prevented by blocking polyamine synthesis during visual stimulation and was rescued when Ca2+-permeable AMPAR-mediated transmission was selectively reduced. The changes in excitability also rendered tectal cells more responsive to synaptic burst stimuli, improving visual stimulus detection. The synaptic and intrinsic adaptations function together to keep tectal neurons within a constant operating range, while making the intact visual system less responsive to background activity yet more sensitive to burst stimuli.

Biomarkers and their use in cervical cancer chemoprevention.

Cervical cancer chemoprevention agents under study include diet and micronutrients (particularly beta-carotene, folate, and vitamins A, C, and E); medications such as retinoids (retinyl acetate gel, all-trans-retinoic acid, and 4-hydroxyphenylretinamide) that are chemically related to micronutrients; and other chemopreventives meant to affect the carcinogenic process at the cellular level, including such polyamine synthesis inhibitors as alpha-difluoromethylornithine. Agents become reasonable candidates for study when they have a biologic rationale, they are of low toxicity, and they can be taken for a long period of time. Since the human papillomavirus (HPV) is the major etiologic agent, the medication should show activity against HPV-positive preinvasive and invasive cell lines. The medication needs to be of low toxicity because it may be taken for long periods of time and less toxicity is tolerated in the precancerous setting. Until 1995, none of the studies used surrogate end point biomarkers (SEBs), relying instead on histologic and colposcopic regression as end points. All studies typically included subjects with cervical intraepithelial neoplasia. Conclusions to be drawn from these studies include the following: Though micronutrients are logical candidates for chemoprevention, they haven't worked consistently, and the reasons remain unclear. Furthermore, SEBs need to be validated in phase I trials. Finally, a better understanding of the role of HPV needs elucidation, including an understanding of the relationship of the medication to HPV status and of the immunobiology of HPV throughout the trial.