Spermine metabolism and radiation-derived reactive oxygen species for future therapeutic implications in cancer: an additive or adaptive response.

Destruction of cells by irradiation-induced radical formation is one of the most frequent interventions in cancer therapy. An alternative to irradiation-induced radical formation is in principle drug-induced formation of radicals, and the formation of toxic metabolites by enzyme catalyzed reactions. Thus, combination therapy targeting polyamine metabolism could represent a promising strategy to fight hyper-proliferative disease. The aim of this work is to discuss and evaluate whether the presence of a DNA damage provoked by enzymatic ROS overproduction may act as an additive or adaptive response upon radiation and combination of hyperthermia with lysosomotropic compounds may improve the cytocidal effect of polyamines oxidation metabolites. Low level of X-irradiations delivers challenging dose of damage and an additive or adaptive response with the chronic damage induced by spermine oxidase overexpression depending on the deficiency of the DNA repair mechanisms. Since reactive oxygen species lead to membrane destabilization and cell death, we discuss the effects of BSAO and spermine association in multidrug resistant cells that resulted more sensitive to spermine metabolites than their wild-type counterparts, due to an increased mitochondrial activity. Since mammal spermine oxidase is differentially activated in a tissue specific manner, and cancer cells can differ in term of DNA repair capability, it could be of interest to open a scientific debate to use combinatory treatments to alter spermine metabolism and deliver differential response.

Bidirectional fluxes of spermine across the mitochondrial membrane.

The polyamine spermine is transported into the mitochondrial matrix by an electrophoretic mechanism having as driving force the negative electrical membrane potential (ΔΨ). The presence of phosphate increases spermine uptake by reducing ΔpH and enhancing ΔΨ. The transport system is a specific uniporter constituted by a protein channel exhibiting two asymmetric energy barriers with the spermine binding site located in the energy well between the two barriers. Although spermine transport is electrophoretic in origin, its accumulation does not follow the Nernst equation for the presence of an efflux pathway. Spermine efflux may be induced by different agents, such as FCCP, antimycin A and mersalyl, able to completely or partially reduce the ΔΨ value and, consequently, suppress or weaken the force necessary to maintain spermine in the matrix. However this efflux may also take place in normal conditions when the electrophoretic accumulation of the polycationic polyamine induces a sufficient drop in ΔΨ able to trigger the efflux pathway. The release of the polyamine is most probably electroneutral in origin and can take place in exchange with protons or in symport with phosphate anion. The activity of both the uptake and efflux pathways induces a continuous cycling of spermine across the mitochondrial membrane, the rate of which may be prominent in imposing the concentrations of spermine in the inner and outer compartment. Thus, this event has a significant role on mitochondrial permeability transition modulation and consequently on the triggering of intrinsic apoptosis.

Polyamine catabolism: target for antiproliferative therapies in animals and stress tolerance strategies in plants.

Metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine, has been a target for antineoplastic therapy since these naturally occurring alkyl amines were found essential for normal mammalian cell growth. Intracellular polyamine concentrations are maintained at a cell type-specific set point through the coordinated and highly regulated interplay between biosynthesis, transport, and catabolism. A correlation between regulation of cell proliferation and polyamine metabolism is described. In particular, polyamine catabolism involves copper-containing amine oxidases and FAD-dependent polyamine oxidases. Several studies showed an important role of these enzymes in several developmental and disease-related processes in both animals and plants through a control on polyamine homeostasis in response to normal cellular signals, drug treatment, environmental and/or cellular stressors. The production of toxic aldehydes and reactive oxygen species, H(2)O(2) in particular, by these oxidases using extracellular and intracellular polyamines as substrates, suggests a mechanism by which the oxidases can be exploited as antineoplastic drug targets. This minireview summarizes recent advances on the physiological roles of polyamine catabolism in animals and plants in an attempt to highlight differences and similarities that may contribute to determine in detail the underlined mechanisms involved. This information could be useful in evaluating the possibility of this metabolic pathway as a target for new antiproliferative therapies in animals and stress tolerance strategies in plants.

Effect of peroxides on spermine transport in rat brain and liver mitochondria.

The polyamine spermine is transported into the matrix of various types of mitochondria by a specific uniporter system identified as a protein channel. This mechanism is regulated by the membrane potential; other regulatory effectors are unknown. This study analyzes the transport of spermine in the presence of peroxides in both isolated rat liver and brain mitochondria, in order to evaluate the involvement of the redox state in this mechanism, and to compare its effect in both types of mitochondria. In liver mitochondria peroxides are able to inhibit spermine transport. This effect is indicative of redox regulation by the transporter, probably due to the presence of critical thiol groups along the transport pathway, or in close association with it, with different accessibility for the peroxides and performing different functions. In brain mitochondria, peroxides have several effects, supporting the hypothesis of a different regulation of spermine transport. The fact that peroxovanadate can inhibit tyrosine phosphatases in brain mitochondria suggests that mitochondrial spermine transport is regulated by tyrosine phosphorylation in this organ. In this regard, the evaluation of spermine transport in the presence of Src inhibitors suggests the involvement of Src family kinases in this process. It is possible that phosphorylation sites for Src kinases are present in the channel pathway and have an inhibitory effect on spermine transport under regulation by Src kinases. The results of this study suggest that the activity of the spermine transporter probably depends on the redox and/or tyrosine phosphorylation state of mitochondria, and that its regulation may be different in distinct organs.

Cytotoxicity of spermine oxidation products to multidrug resistant melanoma M14 ADR2 cells: sensitization by the MDL 72527 lysosomotropic compound.

It has been confirmed that multidrug resistant (MDR) human melanoma cells are more sensitive than their wild-type counterparts to H2O2 and aldehydes, the products of bovine serum amine oxidase (BSAO)-catalyzed oxidation of spermine. The metabolites formed by BSAO and spermine are more toxic than exogenous H2O2 and acrolein, even though their concentration is lower during the initial phase of incubation due to their more gradual release than the exogenous products. Both wild-type and MDR cells, after pre-treatment with MDL 72527, an inactivator of polyamine oxidase and a lysosomotropic compound, show to be sensitized to subsequent exposure to BSAO/spermine. Evidence of ultrastructural aberrations and acridine orange release from lysosomes is presented in this work that is in favor of the permeabilization of the lysosomal membrane as the major cause of sensitization by MDL 72527. Owing to its lysosomotropic effect, pre-treatment with MDL 72527 amplifies the ability of the metabolites formed from spermine by oxidative deamination to induce cell death. Since it is conceivable that combined treatment with a lysosomotropic compound and BSAO/spermine would be effective against tumor cells, it is of interest to search for such novel compounds, which might be promising for application in a therapeutic setting.

The combined treatment with chloroquine and the enzymatic oxidation products of spermine overcomes multidrug resistance of melanoma M14 ADR2 cells: a new therapeutic approach.

It has been confirmed that multidrug resistant (MDR) melanoma cells (M14 ADR2) are more sensitive than their wild-type counterparts (M14 WT) to H2O2 and aldehydes, the products of bovine serum amine oxidase (BSAO)-catalyzed oxidation of spermine. The metabolites formed by BSAO and spermine are more toxic, in M14 cells, than exogenous H2O2 and acrolein, even though their concentration is lower during the initial phase of incubation due to their more gradual release than the exogenous products. Binding of BSAO to the cell membrane and release of the reaction products of spermine into the immediate vicinity of the cells, or directly into the cells, may explain the apparently paradoxical phenomenon. Both WT and MDR cells, after pre-treatment for 24 h, or longer, with the lysosomotropic compound chloroquine (CQ), show to be sensitized to subsequent exposure to BSAO/spermine enzymatic system. Evidence of ultrastructural aberrations and acridine orange release from lysosomes is presented in this study that is in favor of the permeabilization of the lysosomal membrane as the major cause of sensitization by CQ. Pre-treatment with CQ amplifies the ability of the metabolites formed from spermine by oxidative deamination to induce cell death. Melanocytes, differently from melanoma cells, were unaffected by the enzymatic system, even when preceded by CQ treatment. Since it is conceivable that combined treatment with a lysosomotropic compound and BSAO/spermine would be effective against tumour cells, it is of interest to search for such novel compounds, which might be promising for application in a therapeutic setting.

Reactive oxygen species spermine metabolites generated from amine oxidases and radiation represent a therapeutic gain in cancer treatments.

The most frequent interventions in cancer therapy are currently the destruction of cells by irradiation or administration of drugs both able to induce radical formation and toxic metabolites by enzyme-catalyzed reactions. The aim of this study was to determine the cell viability of cells undergoing a DNA damage threshold accomplished by ROS overproduction via both ectopic expression of murine spermine oxidase (mSMOX) and bovine serum amine oxidase (BSAO) enzymes. Low dose of X-irradiation delivers a challenging dose of damage as evaluated in proficient Chinese hamster AA8 cell line and both deficient transcription-coupled nucleotide excision repair (NER) UV61 cells and deficient base excision repair (BER) EM9 cells, at 6 and 24 h after exposure. The priming dose of ROS overexposure by mSMOX provokes an adaptive response in N18TG2, AA8 and EM9 cell lines at 24 h. Interestingly, in the UV61 cells, ROS overexposure by mSMOX delivers an earlier adaptive response to radiation. The enzymatic formation of toxic metabolites has mainly been investigated on wild-type (WT) and multidrug-resistant (MDR) cancer cell lines, using and spermine as substrate of the BSAO enzyme. MDR cells are more sensitive to the toxic polyamine metabolites than WT cells, thus indicating a new therapeutic strategy to overcome MDR tumors. Since SMOX in mammals is differentially activated in a tissue-specific manner and cancer cells can differ in terms of DNA repair and MDR capabilities, it could be of interest to simultaneously treat with very low dose of X-rays and/or to alter SMOX metabolism to generate a differential response in healthy and cancer tissues.

Novel reversible monoamine oxidase A inhibitors: highly potent and selective 3-(1H-pyrrol-3-yl)-2-oxazolidinones.

Monoamine oxidases (MAOs) are involved in various psychiatric and neurodegenerative disorders; hence, MAO inhibitors are useful agents in the therapy of Parkinson's disease, Alzheimer's dementia, and depression syndrome. Herein we report a novel series of 3-(1H-pyrrol-3-yl)-2-oxazolidinones 3-7 as reversible, highly potent and selective anti-MAO-A agents. In particular, 4b, 5b, and 4c showed a K(i-MAO-A) of 0.6, 0.8, and 1 nM, respectively, 4c being 200000-fold selective for MAO-A with respect to MAO-B.

Interaction of bovine serum amine oxidase with the polyamine oxidase inactivator MDL 72527.

MDL 72527 was considered a selective inhibitor of FAD-dependent polyamine oxidases. In the present communication, we demonstrate that MDL 72527 inactivates bovine serum amine oxidase, a copper-containing, TPQ-enzyme, time-dependently at 25 degrees C. In striking contrast, the enzyme remained active after incubation with excessive MDL 72527 at 37 degrees C, even after 70 h of incubation. Inactivation of BSAO with MDL 72527 at 25 degrees C did not involve the cofactor, as was shown by spectroscopy and by reaction with phenylhydrazine. Docking of MDL 72527 is difficult, owing to its size and two lipophilic moieties, and it has been shown that minor changes in reaction rate of substrates cause major changes in K(m) and k(cat)/K(m). We hypothesise that subtle conformational changes between 25 and 37 degrees C impair MDL 72527 from productive binding and prevent the nucleophilic group from reacting with the double bond system.