RESUMO
Infection with Trichomonas vaginalis produces a malodorous seropurulent vaginal discharge due to several chemicals, including polyamines. The presence of 1,4-diamino-2-butanone (DAB) reduces the amount of intracellular putrescine by 90%, preventing the cotransport of exogenous spermine. DAB-treated parasites present morphological changes, which are restored by adding exogenous putrescine into the culture medium. However, the effect of polyamines over the trichomonad proteomic profile is unknown. In this study, we used a proteomic approach to analyze the polyamine-depletion and restoration effect by exogenous putrescine on T. vaginalis proteome. In the presence of inhibitor DAB, we obtained 369 spots in polyamine-depleted condition and observed 499 spots in the normal culture media. With DAB treatment, the intensity of 43 spots was increased but was found to be reduced in 39 spots, as compared to normal conditions. Interestingly, in DAB-treated parasites restored with a medium with added exogenous putrescine, 472 spots were found, of which 33 were upregulated and 63 were downregulated in protein intensity. Some of these downregulated proteins in DAB-treated parasites are involved in several cellular pathways such as glycolysis, glycolytic fermentation, arginine dihydrolase pathway, redox homeostasis, host cell binding mediated by carbohydrate, chaperone function, and cytoskeletal remodeling. Interestingly, the intensity of some of the proteins was restored by adding exogenous putrescine. In conclusion, the presence of DAB altered the proteomic profile of T. vaginalis, resulting in a decrease in the intensity of 130 proteins and an increase in the intensity of 43 proteins that was restored by the addition of putrescine.
Assuntos
Proteoma/efeitos dos fármacos , Putrescina/análogos & derivados , Putrescina/metabolismo , Espermina/metabolismo , Trichomonas vaginalis/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Meios de Cultura/metabolismo , Regulação para Baixo , Feminino , Proteômica/métodos , Putrescina/farmacologia , Vagina/química , Vagina/parasitologiaRESUMO
Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP) involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB) (an inhibitor of putrescine biosynthesis), diminished the amount and proteolytic activity of TvCP39 as compared with untreated parasites. Inhibition of putrescine biosynthesis also reduced â¼ 80% the tvcp39 mRNA levels according to RT-PCR and qRT-PCR assays. Additionally, actinomycin D-treatment showed that the tvcp39 mRNA half-life decreased in the absence of putrescine. However, this reduction was restored by exogenous putrescine addition, suggesting that putrescine is necessary for tvcp39 mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the tvcp39 mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization.
Assuntos
Cisteína Proteases/metabolismo , Proteínas de Protozoários/metabolismo , Putrescina/metabolismo , Trichomonas vaginalis/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Western Blotting , Divisão Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cisteína Proteases/genética , Expressão Gênica/efeitos dos fármacos , Microscopia Confocal , Proteólise/efeitos dos fármacos , Proteínas de Protozoários/genética , Putrescina/análogos & derivados , Putrescina/antagonistas & inibidores , Putrescina/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trichomonas vaginalis/citologia , Trichomonas vaginalis/genéticaRESUMO
α-Aminocarbonyl metabolites (e.g., 5-aminolevulinic acid and aminoacetone) and the wide spectrum microbicide 1,4-diamino-2-butanone (DAB) have been shown to exhibit pro-oxidant properties. In vitro, these compounds undergo phosphate-catalyzed enolization at physiological pH and subsequent superoxide radical-propagated aerobic oxidation, yielding a reactive α-oxoaldehyde and H2O2. DAB cytotoxicity to pathogenic microorganisms has been attributed to the inhibition of polyamine biosynthesis. However, the role played in cell death by reactive DAB oxidation products is still poorly understood. This work aims to clarify the mechanism of DAB-promoted pro-oxidant action on mammalian cells. DAB (0.05-10 mM) treatment of RKO cells derived from human colon carcinoma led to a decrease in cell viability (IC50 ca. 0.3 mM DAB, 24 h incubation). Pre-addition of either catalase (5 µM) or aminoguanidine (20 mM) was observed to partially inhibit the toxic effects of DAB to the cells, while N-acetyl-L-cysteine (NAC, 5 mM) or reduced glutathione (GSH, 5 mM) provided almost complete protection against DAB. Changes in redox balance and stress response pathways were indicated by the increased expression of HO-1, NQO1 and xCT. Moreover, the observation of caspase 3 and PARP cleavage products is consistent with DAB-triggered apoptosis in RKO cells, which was corroborated by the partial protection afforded by the pan-caspase inhibitor z-VAD-FMK. Finally, DAB treatment disrupted the cell cycle in response to increased p53 and activation of ATM. Altogether, these data support the hypothesis that DAB exerts cytotoxicity via a mechanism involving not only polyamine biosynthesis but also by DAB oxidation products.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Oxirredução , Putrescina/análogos & derivados , Espécies Reativas de Oxigênio/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Poliaminas/química , Poliaminas/metabolismo , Putrescina/metabolismo , Putrescina/farmacologia , Superóxidos/metabolismoRESUMO
The putrescine analogue 1,4-diamino-2-butanone (DAB) is highly toxic to various microorganisms, including Trypanosoma cruzi. Similar to other α-aminocarbonyl metabolites, DAB exhibits pro-oxidant properties. DAB undergoes metal-catalyzed oxidation yielding H(2)O(2), NH(4)(+) ion, and a highly toxic α-oxoaldehyde. In vitro, DAB decreases mammalian cell viability associated with changes in redox balance. Here, we aim to clarify the DAB pro-oxidant effects on trypomastigotes and on intracellular T. cruzi amastigotes. DAB (0.05-5 mM) exposure in trypomastigotes, the infective stage of T. cruzi, leads to a decline in parasite viability (IC(50)c.a. 0.2 mM DAB; 4 h incubation), changes in morphology, thiol redox imbalance, and increased TcSOD activity. Medium supplementation with catalase (2.5 µM) protects trypomastigotes against DAB toxicity, while host cell invasion by trypomastigotes is hampered by DAB. Additionally, intracellular amastigotes are susceptible to DAB toxicity. Furthermore, pre-treatment with 100-500 µM buthionine sulfoximine (BSO) of LLC-MK2 potentiates DAB cytotoxicity, whereas 5 mM N-acetyl-cysteine (NAC) protects cells from oxidative stress. Together, these data support the hypothesis that redox imbalance contributes to DAB cytotoxicity in both T. cruzi and mammalian host cells.
Assuntos
Oxidantes/farmacologia , Putrescina/análogos & derivados , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Animais , Linhagem Celular , Modelos Biológicos , Oxidantes/toxicidade , Oxirredução , Proteínas de Protozoários/metabolismo , Putrescina/farmacologia , Putrescina/toxicidade , Compostos de Sulfidrila/metabolismo , Superóxido Dismutase/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/patogenicidadeRESUMO
The α-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB's cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37°C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other α-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml-1) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(â¢), and those with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(â¢) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 µM) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Putrescina/análogos & derivados , Espécies Reativas de Oxigênio/química , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Linhagem Celular , Ferritinas/efeitos dos fármacos , Radicais Livres/análise , Radicais Livres/toxicidade , Haplorrinos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Radical Hidroxila/química , Radical Hidroxila/metabolismo , Metais/química , Consumo de Oxigênio/efeitos dos fármacos , Poliaminas/química , Putrescina/química , Putrescina/farmacologia , Superóxidos/química , Superóxidos/metabolismo , Transferrina/efeitos dos fármacosRESUMO
Polyamines are important regulators of growth and differentiation in a variety of cells, including parasitic protozoa. Promastigotes of Leishmania species have high levels of putrescine and spermidine and their growth can be inhibited by polyamine biosynthesis antagonists. The putrescine analogue 1,4-diamino-2-butanone (DAB) is microbicidal against Tritrichomonas foetus and Trypanosoma cruzi, so we tested its effects on Leishmania amazonensis proliferation, viability, organization, putrescine transport and synthesis as well as in vitro infectivity. DAB impaired promastigote proliferation dose-dependently (IC(50) 144 microM) and the parasite putrescine concentration was reduced by nearly 50 %. This analogue markedly inhibited both ornithine decarboxylase activity and [H(3)]putrescine uptake by promastigotes. Pre-treatment with DAB for 24 h led to compensatory enhancement of putrescine uptake, indicating an adaptive mechanism in DAB-treated parasites. Remarkably, DAB caused mitochondrial damage, assessed by transmission electron microscopy, and 3 h treatment with 1 mM DAB enhanced lipid peroxidation, whereas incubation with 10 mM DAB or for 24 h resulted in decreased peroxidation levels in the parasites. This effect was probably due to the loss of mitochondrial function, demonstrated by the diminished reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), not observed in macrophages. Murine macrophages infected with L. amazonensis amastigotes treated with DAB had parasite loads significantly (P<0.05) lower than controls, presumably due to interference with putrescine uptake and/or synthesis. These results suggest that putrescine may be involved in leishmanial survival, possibly by maintaining the parasite's mitochondrial function. The use of analogues to interfere with polyamine/diamine synthesis and transport may shed light on its function in intracellular parasite survival and lead to identification of new targets for leishmaniasis chemotherapy.
Assuntos
Antiprotozoários/farmacologia , Leishmania/metabolismo , Poliaminas/metabolismo , Putrescina/análogos & derivados , Animais , Antiprotozoários/metabolismo , Proliferação de Células , Células Cultivadas , Leishmania/efeitos dos fármacos , Leishmania/crescimento & desenvolvimento , Leishmania/ultraestrutura , Peroxidação de Lipídeos , Macrófagos/parasitologia , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ornitina Descarboxilase/metabolismo , Putrescina/metabolismo , Putrescina/farmacologia , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologiaRESUMO
The protozoan Giardia lamblia is the most frequent intestinal parasite of first-world countries and a major cause of waterborne disorder often referred to as traveler's diarrhea. We have previously noticed that the putrescine analog 1,4-diamino-2-butanone (DAB) remarkably inhibits the growth of anaerobic trichomonad and Trypanosoma cruzi parasites. Here, we examined the role of polyamines in Giardia cells using this putrescine analog. DAB impaired parasite proliferation dose-dependently. The analog induced increased flagella numbers and sometimes four ventral disks as well as asymmetrical division, indicating truncated or deregulated cytokinesis. Electron microscopy analysis revealed that DAB also triggered the encystment process. Oxidative stress was evaluated by measuring lipid peroxidation by thiobarbituric acid reactive substances (TBARS) detection. Trophozoites incubated either with 1 mM of DAB or putrescine for 18 h displayed increased lipoperoxide levels. Addition of 200 microM aminoguanidine, a polyamine/diamine oxidase inhibitor, partially reverted the DAB, but not the putrescine effects, indicating that the DAB effects are due, at least in part, to DAB oxidation end products. These data indicate that polyamines play a role in Giardia cell division, differentiation, and antioxidant defenses.
Assuntos
Giardia lamblia/efeitos dos fármacos , Giardia lamblia/crescimento & desenvolvimento , Putrescina/análogos & derivados , Animais , Giardia lamblia/ultraestrutura , Estresse Oxidativo , Poliaminas/metabolismo , Putrescina/metabolismo , Putrescina/farmacologia , Trofozoítos/efeitos dos fármacos , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/ultraestruturaRESUMO
Trypanosoma cruzi is the etiological agent of American trypanosomiasis. Most of the available data on trypanosomatid parasites were obtained from African trypanosomes. Parasitic protozoa polyamine metabolism and transport pathways comprise valuable targets for chemotherapy. T. cruzi cannot synthesize putrescine, but its uptake from the extracellular milieu can promote parasite survival. Nevertheless, little is known about the cell biology of this diamine in T. cruzi. Here we notice that the putrescine analogue 1,4-diamino-2-butanone (DAB) inhibited T. cruzi epimastigotes' in vitro proliferation and produced remarkable mitochondrial destruction and cell architecture disorganization, as assessed by transmission electron microscopy. Mitochondrial damage was confirmed by MTT reduction. We decided to analyze the oxidative stress undergone by DAB-treated parasites. Thiobarbituric-acid-reactive substances were measured to assess lipid peroxidation. Analogue effects were dose-dependent; 5 mM DAB only slightly enhanced peroxidation, whereas 10 mM DAB significantly (P < 0.05) diminished it. These data indicate that putrescine uptake by this diamine auxotrophic parasite may be important for epimastigote axenic growth and cellular organization.
Assuntos
Putrescina/análogos & derivados , Trypanosoma cruzi/efeitos dos fármacos , Animais , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo , Putrescina/metabolismo , Putrescina/toxicidade , Trypanosoma cruzi/fisiologia , Trypanosoma cruzi/ultraestruturaRESUMO
We utilized our modification of the amplified fragment length polymorphism technique for the determination of changes occurring in the DNA methylation patterns during the dimorphic transition of the fungi Mucor rouxii, Yarrowia lipolytica, and Ustilago maydis. To determine the specificity of differential methylation in regards to dimorphism, we obtained the yeast-like form of the three fungi under conditions that induced mycelial growth, by addition of 1,4-diaminobutanone (DAB), an inhibitor of ornithine decarboxylase in the case of M. rouxii and Y. lipolytica. In an odc null mutant of U. maydis, repression of the dimorphic transition was brought about by limitation in the amounts of exogenous putrescine. Yeasts from the three fungi thus obtained conserved a significant number of the differential DNA fragments with the methylation pattern displayed by normal yeasts, indicating their true correlation with dimorphism. Our results also confirm a role of polyamines in differential DNA methylation and fungal dimorphic transition.
Assuntos
Metilação de DNA , DNA Fúngico/metabolismo , Fungos/crescimento & desenvolvimento , Fungos/metabolismo , Poliaminas/metabolismo , Putrescina/análogos & derivados , Impressões Digitais de DNA , DNA Fúngico/análise , DNA Fúngico/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Fungos/genética , Mucor/genética , Mucor/crescimento & desenvolvimento , Mucor/metabolismo , Micélio/crescimento & desenvolvimento , Técnicas de Amplificação de Ácido Nucleico , Ornitina Descarboxilase/genética , Inibidores da Ornitina Descarboxilase , Putrescina/metabolismo , Putrescina/farmacologia , Ustilago/genética , Ustilago/crescimento & desenvolvimento , Ustilago/metabolismo , Yarrowia/genética , Yarrowia/crescimento & desenvolvimento , Yarrowia/metabolismoRESUMO
The effects of the putrescine analogue 1-aminooxy-3-aminopropane on fungal polyamine metabolism were evaluated using Sclerotinia sclerotiorum as an experimental model. The compound inhibited ornithine decarboxylase, spermidine synthase, and S -adenosyl-methionine decarboxylase in mycelial extracts. Addition of 1-aminooxy-3-aminopropane at 1 mM to the culture medium did not reduce mycelial growth and caused a 29% decrease in free spermidine and a two-fold increase in free spermine. When added 4.5 h before the determination of ornithine decarboxylase, 1-aminooxy-3-aminopropane reduced in vivo activity of this enzyme by 40-50%. When added 48 h before the determination, 1-aminooxy-3-aminopropane at 0.01 and 0.1 mM caused a slight increase of in vivo ornithine decarboxylase activity, while it had no effect at 1 mM. Comparison of the action of 1-aminooxy-3-aminopropane with that of other inhibitors of polyamine biosynthesis suggested that its effects on in vivo ornithine decarboxylase activity resulted from a balance between direct inhibition of enzyme activity and indirect stimulation of enzyme synthesis and/or activity mediated by the decrease in spermidine levels, which in turn was due to inhibition of spermidine synthase and S -adenosyl-methionine decarboxylase. The potential of 1-aminooxy-3-aminopropane as a tool for studies on fungal polyamine metabolism and for the control of plant diseases of fungal origin is discussed.
Assuntos
Ascomicetos/efeitos dos fármacos , Poliaminas Biogênicas/biossíntese , Doenças das Plantas/microbiologia , Propilaminas/farmacologia , Adenosilmetionina Descarboxilase/efeitos dos fármacos , Adenosilmetionina Descarboxilase/metabolismo , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/metabolismo , Ornitina Descarboxilase/efeitos dos fármacos , Ornitina Descarboxilase/metabolismo , Propilaminas/metabolismo , Putrescina/análogos & derivados , Espermidina Sintase/efeitos dos fármacos , Espermidina Sintase/metabolismoRESUMO
This work describes the synthesis and characterization of six new dinuclear platinum complexes having N,N'-di-(2-aminoethyl)-1,3-diamino-2-propanol, aryl substituted N-benzyl-1,4-butanediamines and N-benzyl-1,6-hexanediamines as ligands. They were prepared by the reaction of cis-[PtCl(2)(DMSO)(2)] (DMSO=dimethyl sulfoxide) with the appropriate ligand in water, except for one of them, which was prepared from K(2)PtCl(4). We also report the cytotoxic activity and cellular accumulation of three of these complexes in a human small-cell lung carcinoma cell line and its resistant subline. Resistant cells exhibited a lesser degree of cross-resistance to these compounds when compared to cisplatin. The accumulation of platinum in both cell lines followed the same pattern, i.e. approximately the same intracellular platinum concentration yielded the same cytotoxic effect independent of the nature of the platinum complex used.
Assuntos
Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/toxicidade , Platina/metabolismo , Platina/toxicidade , Putrescina/análogos & derivados , Putrescina/química , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Resistencia a Medicamentos Antineoplásicos , Ligantes , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Compostos Organoplatínicos/química , Compostos Organoplatínicos/metabolismo , Platina/administração & dosagem , Platina/químicaRESUMO
NMR spectroscopy was used to characterize the binding properties of polyamines to Escherichia coli tRNA. The (15)N NMR spectra of three (15)N-enriched N-substituted putrescine derivatives (DMP, DEP and DBP) were recorded in the presence of tRNA, and the spin relaxation times of the nitrogen nuclei were measured. From these data, the activation parameters for the rotational correlation times of the (15)N nuclei were determined. The present data indicate that the nature of the amino substituents does play a relevant role in controlling the polyamine-tRNA interaction. This study also provides a rationale for the in vivo antiproliferative effect of DBP against tumoral cells.
Assuntos
Putrescina/análogos & derivados , RNA de Transferência/química , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Putrescina/química , RNA Bacteriano/químicaRESUMO
Trichomonad parasites such as Tritrichomonas foetus produce large amounts of putrescine (1,4-diaminobutane), which is transported out of the cell via an antiport mechanism which results in the uptake of a molecule of spermine. The importance of putrescine to the survival of the parasite and its role in the biology of T. foetus was investigated by use of the putrescine analogue 1, 4-diamino-2-butanone (DAB). Growth of T. foetus in vitro was significantly inhibited by 20 mM DAB, which was reversed by the addition of exogenous 40 mM putrescine. High-performance liquid chromatography analysis of 20 mM DAB-treated T. foetus revealed that putrescine, spermidine, and spermine levels were reduced by 89, 52, and 43%, respectively, compared to those in control cells. The DAB treatment induced several ultrastructural alterations, which were primarily observed in the redox organelles termed hydrogenosomes. These organelles were progressively degraded, giving rise to large vesicles that displayed material immunoreactive with an antibody to beta-succinyl-coenzyme A synthetase, a hydrogenosomal enzyme. A protective role for polyamines as stabilizing agents in the trichomonad hydrogenosomal membrane is proposed.
Assuntos
Poliaminas Biogênicas/biossíntese , Organelas/efeitos dos fármacos , Putrescina/análogos & derivados , Tritrichomonas foetus/efeitos dos fármacos , Tritrichomonas foetus/crescimento & desenvolvimento , Animais , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Microscopia Eletrônica , Movimento/efeitos dos fármacos , Putrescina/biossíntese , Putrescina/farmacologia , Espermidina/biossíntese , Espermina/biossíntese , Tritrichomonas foetus/metabolismo , Tritrichomonas foetus/ultraestruturaRESUMO
Polyamines (putrescine, spermidine and spermine) increase in proliferating tissues and are essential for cellular growth and cell division processes. We had previously shown that alkyl substituted putrescines can inhibit cell proliferation. We now tested the effects of the (N(alpha),N(omega)-dibenzyl derivatives of the simple diamines putrescine, cadaverine and 1,3-diaminopropane on the growth of three human squamous cell carcinoma (SCC) lines and a rat hepatoma (H-4-II-E) cell line. Survival assays were measured by treating exponentially-growing SCC cultures with N1,N4-dibenzylputrescine (DBP) (270 microM) or a rat hepatoma cell culture with DBP (100 microM) for 48 hrs. Inhibition of cell growth was measured either by the colony forming assay or by cell counting. DBP inhibited proliferation of the rat hepatoma (H-4-II-E) cell line and induced cytotoxicity when used at a concentration of 100 microM for >48 hrs. N1,N5-dibenzylcadaverine (DBC) also induced cytotoxicity at a similar concentration, while N1,N3-dibenzyl-1,3-diaminopropane (DBPr) was a much weaker inhibitor of cell growth. Inhibition of cell growth by DBP resulted in marked modifications of cell morphology, such as vacuole formation, decrease in size, pycnosis, change in staining behavior toward trypan blue and lack of adherence. DBP was also growth inhibitory in the three human SCC cell lines tested. The concentration of DBP required to achieve growth inhibition of SCC cells could be dramatically decreased in the presence of N1,N4-bis(buta-2,3-dienyl)butanediamine, a specific inhibitor of polyamine oxidase (PAOI). Moreover, although the presence of PAOI only prevented the oxidation (debenzylation) of approximately 20% of intracellular DBP over a 5-day period, it produced a 5-fold increase in the inhibition of cell proliferation by DBP. DBP (and DBC) inhibited putrescine uptake by rat hepatoma (H-4-II-E) cells in what appears to be a competitive reaction. A tenfold excess of putrescine over DBP did not inhibit the antiproliferative or cytotoxic effects of the latter. DBP administered for 72 hrs. depleted intracellular levels of putrescine, spermidine and spermine in the SCC lines by 50-100% of control values. It was found that DBP inhibited nucleic acid and protein synthesis at an early stage of cell proliferation, hence its growth inhibitory effect may be related to inhibition of the synthesis of macromolecules.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Putrescina/análogos & derivados , Células 3T3 , Animais , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Carcinoma de Células Escamosas/metabolismo , Divisão Celular/efeitos dos fármacos , Diaminas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Hepáticas Experimentais/metabolismo , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/efeitos dos fármacos , Ácidos Nucleicos/biossíntese , Ornitina Descarboxilase/efeitos dos fármacos , Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismo , Putrescina/farmacocinética , Putrescina/farmacologia , Ratos , Células Tumorais CultivadasRESUMO
Putrescine and spermidine were the only polyamines found in Paracoccidioides brasiliensis, a dimorphic fungus pathogenic for humans. Free polyamines (putrescine > spermidine) increased during the first 24 h of yeast growth, with a second peak at 42 h, and also during the first 12 h of mycelium-to-yeast transition (spermidine > putrescine). Conjugated and bound polyamines were also quantified. 1, 4-Diamino-2-butanone decreased free putrescine and spermidine accumulation by inhibiting the activity of ornithine decarboxylase. The increase in free polyamines corresponds to bud emergence in yeast growth and to the mycelium-to-yeast transition of P. brasiliensis.
Assuntos
Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/metabolismo , Putrescina/biossíntese , Espermidina/biossíntese , Inibidores da Ornitina Descarboxilase , Putrescina/análogos & derivados , Putrescina/farmacologiaRESUMO
Ornithine decarboxylase in Paracoccidioides brasiliensis, a dimorphic human pathogenic fungus, was more active at 37 degrees C in the yeast phase and at 30 degrees C in the mycelial phase. In contrast to other fungal systems, yeast growth and mycelium-to-yeast transition in P. brasiliensis were accompanied by a high activity of ornithine decarboxylase at the onset of the budding process, the activity of which was inhibited by 1,4-diamino-2-butanone. The activity of ornithine decarboxylase remained at a basal level during vegetative growth of both the mycelial phase and the late stage of yeast phase, and also through the yeast-to-mycelium transition.
Assuntos
Ornitina Descarboxilase/metabolismo , Paracoccidioides/enzimologia , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , América Latina , Micoses/microbiologia , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/crescimento & desenvolvimento , Putrescina/análogos & derivados , Putrescina/farmacologiaRESUMO
2-(Aminomethyl)-4-aminobutyric acid (isoornithine), 3-methylisoornithine, and 2,3-dimethylisoornithine were not decarboxylated by liver ornithine decarboxylase (ODC, EC 4.1.1.17) of thioacetamide-treated rats but were good competitive inhibitors of the enzyme (Ki ranged from 0.72 to 1.79 mM). When assayed in vivo in the treated rats, the above mentioned isoornithines were also found to inhibit liver ODC when administered 1 h before sacrifice. When the methylputrescines formally derived from the decarboxylation of several isoornithines were assayed on rat liver ODC, it was found that only 2,3-dimethylputrescine decreased the enzymatic activity. When assayed in vivo, it was found to decrease ODC activity by 60%, when the latter was measured 1 h after administration. The effect was reverted 4 h after administration of the drug. Isoornithines were not taken up by H-35 hepatoma cells; hence they did not affect their ODC activity. 2,3-Dimethylputrescine however, was transported into the cells and significantly decreased its ODC activity.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores da Ornitina Descarboxilase , Ornitina/análogos & derivados , Ornitina/química , Putrescina/análogos & derivados , Putrescina/química , Animais , Ligação Competitiva , Carcinoma Hepatocelular , Fígado/efeitos dos fármacos , Fígado/enzimologia , Neoplasias Hepáticas , Ornitina/metabolismo , Putrescina/metabolismo , Ratos , Relação Estrutura-Atividade , Tioacetamida/farmacologia , Células Tumorais CultivadasRESUMO
The effect of several methylputrescines on the activity of insulin-induced ornithine decarboxylase (ODC) was examined in H-35 hepatoma cells. The induction involved both protein and m-RNA synthesis. Actinomycin D inhibited ODC activity when given up to 1 h after insulin treatment. When added to the medium 2 h or 3 h after the insulin, the activity was increased 100% and 80% respectively. Insulin-induced ODC from H-35 cells had a biphasic half-life, a shorter one of 46 min and a longer one of 90 min. 1-Methylputrescine and 2-methylputrescine were found to be competitive inhibitors of the ODC from H-35 cells with Ki values of 2.8 and 0.1 mM respectively. Putrescine itself was found to have a Ki = 2.4 mM. N-Methylputrescine was a very poor inhibitor of the cell free ODC while 1,4-dimethylputrescine did not show any inhibitory effect. When cellular ODC activity was measured, the four methylputrescines assayed as well as putrescine entirely abolished its activity in the H-35 cells when given at a 1 mM concentration together with insulin. 1-Methylputrescine and 1,4-dimethylputrescine abolished 60% of the activity at a 0.1 microM concentration. All the methylputrescines given at 0.1 mM concentrations decreased the putrescine content of the stimulated cells to the levels found in quiescent cells, but only 1-methyl and 2-methylputrescines decreased spermidine and spermine content. 1,4-Dimethyl and 1-methylputrescines showed a strong inhibition of ODC synthesis, while the other diamines were less inhibitory. At concentrations that abolished ODC activity, 1,4-dimethylputrescine decreased 70% of the total immunoreactive ODC bands, while 1-methyl and 2-methylputrescine decreased them by 50%, and N-methylputrescine and putrescine decreased them by 20%. The lack of decrease in immuno-reactive ODC with the latter two compounds was mainly due to the appearance of immunoreactive degradation products of ODC of low molecular weight. Putrescine and N-methylputrescine affected protein synthesis to a small extent in stimulated cells, while 1-methylputrescine decreased it to the level of non-stimulated cells. Insulin (1 microM concentration) stimulated DNA synthesis in the cells, and this stimulation was doubled in the presence of 2-methylputrescine or putrescine. It can be concluded that, among the methylputrescines assayed, 2-methylputrescine was the best inhibitor of cell-free ODC activity, while 1,4-dimethylputrescine and 1-methylputrescine were the best inhibitors of cellular ODC activity.
Assuntos
Insulina/farmacologia , Fígado/enzimologia , Ornitina Descarboxilase/metabolismo , Putrescina/análogos & derivados , Putrescina/farmacologia , Animais , DNA/biossíntese , Dactinomicina/farmacologia , Ativação Enzimática , Cinética , Fígado/efeitos dos fármacos , Neoplasias Hepáticas Experimentais , Ornitina Descarboxilase/biossíntese , Biossíntese de Proteínas , Ratos , Células Tumorais CultivadasRESUMO
1,4-Dimethylputrescine (2,5-hexanediamine) was separated into its racemic and meso isomers by fractional crystallization of its dibenzoyl derivative. The racemic form was resolved into its (+)- and (-)-isomers with (+)- and (-)-dibenzoyltartaric acids. None of the three isomers (meso, +, and -) inhibited ornithine decarboxylase (ODC) activity in vitro, while all the three were strongly inhibitory of ODC when assayed in vivo in rats or in H-35 hepatoma cells. In rat liver the three isomers also decreased the putrescine pool while only the (+)-isomer decreased spermidine content. In the H-35 cells the (-)- and (+)-isomers decreased the spermidine and spermine content. When ODC was induced in the latter by insulin it was found that the (-)-isomer strongly inhibited protein and ODC synthesis, while the (+)-isomer and the meso isomer were less inhibitory. The meso isomer was a good inducer of ODC antizyme in rat liver, while the (+)- and (-)-isomers were poor inducers of the former.
Assuntos
Inibidores da Ornitina Descarboxilase , Putrescina/análogos & derivados , Putrescina/farmacologia , Animais , Linhagem Celular , Feminino , Isomerismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Neoplasias Hepáticas Experimentais , Proteínas de Neoplasias/biossíntese , Rotação Ocular , Poliaminas/metabolismo , Putrescina/síntese química , Putrescina/isolamento & purificação , Ratos , Ratos Endogâmicos , Relação Estrutura-Atividade , Tioacetamida/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.