RESUMO
The biogenic polyamines, spermine (Spm) and spermidine, are organic polycations present in millimolar concentrations in all eukaryotic cells participating in the regulation of vital cellular functions including proliferation and differentiation. The design and biochemical evaluation of polyamine analogues are cornerstones of polyamine research. Here we synthesized and studied novel C-methylated Spm analogues: 2,11-dimethylspermine (2,11-Me2Spm), 3,10-dimethylspermine (3,10-Me2Spm), 2-methylspermine, and 2,2-dimethylspermine. The tested analogues overcame growth arrest induced by a 72 h treatment with α-difluoromethylornithine, an ornithine decarboxylase (ODC) inhibitor, and entered into DU145 cells via the polyamine transporter. 3,10-Me2Spm was a poor substrate of spermine oxidase and spermidine/spermine-N1-acetyltransferase (SSAT) when compared with 2,11-Me2Spm, thus resembling 1,12-dimethylspermine, which lacks the substrate properties required for the SSAT reaction. The antizyme (OAZ1)-mediated downregulation of ODC and inhibition of polyamine transport are crucial in the maintenance of polyamine homeostasis. Interestingly, 3,10-Me2Spm was found to be the first Spm analogue that did not induce OAZ1 and, consequently, was a weak downregulator of ODC activity in DU145 cells.
Assuntos
Inibidores da Ornitina Descarboxilase/farmacologia , Ornitina Descarboxilase/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Poliaminas/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Espermina/análogos & derivados , Espermina/metabolismo , Transporte Biológico , Metilação de DNA , Humanos , Masculino , Ornitina Descarboxilase/metabolismo , Neoplasias da Próstata/metabolismo , Especificidade por Substrato , Células Tumorais Cultivadas , Poliamina OxidaseRESUMO
Tumorigenesis is accompanied by the metabolic adaptation of cells to support enhanced proliferation rates and to optimize tumor persistence and amplification within the local microenvironment. In particular, cancer cells exhibit elevated levels of biogenic polyamines. Inhibitors of polyamine biosynthesis and inducers of their catabolism have been evaluated as antitumor drugs, however, their efficacy and safety remain controversial. Our goal was to investigate if drug-induced modulation of polyamine metabolism plays a role in dedifferentiation using differentiated human hepatocyte-like HepaRG cell cultures. N¹,N11-diethylnorspermine (DENSpm), a potent inducer of polyamine catabolism, triggered an epithelial-mesenchymal transition (EMT)-like dedifferentiation in HepaRG cultures, as shown by down-regulation of mature hepatocytes markers and upregulation of classical EMT markers. Albeit the fact that polyamine catabolism produces H2O2, DENSpm-induced de-differentiation was not affected by antioxidants. Use of a metabolically stable spermidine analogue showed furthermore, that spermidine is a key regulator of hepatocyte differentiation. Comparative transcriptome analyses revealed, that the DENSpm-triggered dedifferentiation of HepaRG cells was accompanied by dramatic metabolic adaptations, exemplified by down-regulation of the genes of various metabolic pathways and up-regulation of the genes involved in signal transduction pathways. These results demonstrate that polyamine metabolism is tightly linked to EMT and differentiation of liver epithelial cells.
RESUMO
Replacing protium with deuterium is an efficient method to modulate drug metabolism. N-alkylated polyamine analogues are polyamine antimetabolites with proven anticancer efficacy. We have characterized earlier the preferred metabolic routes of N1,N12-diethylspermine (DESpm), N1-benzyl-N12-ethylspermine (BnEtSpm) and N1,N12-dibenzylspermine (DBSpm) by human recombinant spermine oxidase (SMOX) and acetylpolyamine oxidase (APAO). Here, we studied the above analogues, their variably deuterated counterparts and their metabolites as substrates and inhibitors of APAO, SMOX, semicarbazide-sensitive amine oxidase (SSAO), diamine oxidase (DAO) and monoamine oxidases. We found that targeted deuteration efficiently redirected the preferable cleavage site and suppressed reaction rate by APAO and SMOX in vitro We found a three- to six-fold decline in Vmax with moderate variable effect on Km when deuterium was located at the preferred hydrogen abstraction site of the analogue. We also found some of the metabolites to be potent inhibitors of DAO and SSAO. Surprisingly, analogue deuteration did not markedly alter the anti-proliferative efficacy of the drugs in DU145 prostate cancer cells, while in mouse embryonic fibroblasts, which had higher basal APAO and SMOX activities, moderate effect was observed. Interestingly, the anti-proliferative efficacy of the analogues did not correlate with their ability to suppress polyamine biosynthetic enzymes, induce spermidine/spermine-N1-acetyltransferase or deplete intracellular polyamine levels, but correlated with their ability to induce SMOX. Our data show that selective deuteration of N-alkyl polyamine analogues enables metabolic switching, offering the means for selective generation of bioactive metabolites inhibiting, e.g. SSAO and DAO, thus setting a novel basis for in vivo studies of this class of analogues.
Assuntos
Deutério/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Poliaminas/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Inativação Metabólica/genética , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Poliaminas/química , Espermina/química , Espermina/metabolismo , Poliamina OxidaseRESUMO
Polyamine metabolism is an attractive anticancer drug target, since polyamines are absolutely required for cellular proliferation, and increased levels of polyamines and their biosynthetic enzyme ornithine decarboxylase (ODC) are associated with cancer. Triethylenetetramine (TETA) is a charge-deficient isosteric analogue of the polyamine spermidine (Spd) and a Cu(II)-chelating compound used for the treatment of Wilson's disease, and it has been implicated as a potential anticancer therapeutic drug. In the present study, we studied the effects of TETA in comparison with two other Cu(II)-chelators, D-penicillamine (PA) and tetrathiomolybdate (TTM), on polyamine metabolism in DU145 prostate carcinoma, MCF-7 breast carcinoma and JEG-3 choriocarcinoma cells. TETA induced antizyme, down-regulated ODC and inhibited [(14)C] Spd uptake. Moreover, it completely prevented α-difluoromethylornithine (DFMO)-induced increase in [(14)C] Spd uptake, and inhibited [(14)C] putrescine (Put) uptake and ODC activity in vivo Seven-day treatment of DU145 cells with TETA caused growth cessation by reducing intracellular polyamine levels and suppressing the formation of hypusinated eukaryotic translation initiation factor 5A (eIF5A). TETA or its N-acetylated metabolites also inhibited spermine (Spm), diamine and semicarbazide-sensitive amine oxidases and decreased the level of intracellular reactive oxygen species. Moreover, TETA inhibited the utilization of Put as energy source via the tricarboxylic acid (TCA) cycle, as indicated by decreased production of (14)CO2 from [(14)C] Put. These results indicate that TETA attacks multiple proven anticancer drug targets not attributed to copper chelation, which warrants further studies to reveal its potential in cancer chemoprevention and cure.
Assuntos
Proliferação de Células/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Poliaminas/metabolismo , Trientina/farmacologia , Amina Oxidase (contendo Cobre) , Linhagem Celular Tumoral , Eflornitina/metabolismo , Feminino , Humanos , Células MCF-7 , Masculino , Molibdênio/farmacologia , Penicilamina/metabolismo , Putrescina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espermina/metabolismoRESUMO
Eukaryotic translation initiation factor 5A (eIF5A) is essential for cell proliferation, becoming functionally active only after post-translational conversion of a specific Lys to hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Deoxyhypusine synthase (DHS) is the rate-limiting enzyme of this two-step process, and the polyamine spermidine is the only natural donor of the butylamine group for this reaction, which is very conserved-hypusine biosynthesis suffers last when the intracellular spermidine pool is depleted. DHS has a very strict substrate specificity, and only a few spermidine analogs are substrates of the enzyme and can support long-term growth of spermidine-depleted cells. Herein, we compared the biological properties of earlier unknown enantiomers of 3-methylspermidine (3-MeSpd) in deoxyhypusine synthesis, in supporting cell growth and in polyamine transport. Long-term treatment of DU145 cells with α-difluoromethylornithine (inhibitor of polyamine biosynthesis) and (R)-3-MeSpd did not cause depletion of hypusinated eIF5A, and the cells were still able to grow, whereas the combination of α-difluoromethylornithine with a racemate or (S)-3-MeSpd caused cessation of cell growth. Noticeably, DHS preferred the (R)- over the (S)-enantiomer as a substrate. (R)-3-MeSpd competed with [(14)C]-labeled spermidine for cellular uptake less efficiently than the (S)-3-MeSpd (Ki = 141 µM vs 19 µM, respectively). The cells treated with racemic 3-MeSpd accumulated intracellularly mainly (S)-3-MeSpd, but not DHS substrate (R)-3-MeSpd, explaining the inability of the racemate to support long-term growth. The distinct properties of 3-MeSpd enantiomers can be exploited in designing polyamine uptake inhibitors, facilitating drug delivery and modulating deoxyhypusine synthesis.
Assuntos
Lisina/análogos & derivados , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Espermidina/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Eflornitina/farmacologia , Expressão Gênica , Humanos , Lisina/biossíntese , Lisina/metabolismo , Masculino , Inibidores da Ornitina Descarboxilase/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Espermidina/análogos & derivados , Espermidina/farmacologia , Estereoisomerismo , Especificidade por Substrato , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Spermidine/spermine N (1)-acetyltransferase (SSAT) is a catabolic regulator of polyamines, ubiquitous molecules essential for cell proliferation and differentiation. In pathological conditions, the increased polyamine catabolism has been shown to mediate its cellular functions not only by changed polyamine levels but also by the availability of metabolites shared with other metabolic pathways or by production of toxic compounds. Our previous results showed that mice overexpressing SSAT (SSAT mice) developed a myeloproliferative disease and the bone marrow microenvironment partly contributed to its development. In this study, the physiological role of SSAT and polyamines in bone remodeling was characterized. Skeletal development of the SSAT mice appeared outwardly similar to wild-type mice until maturity, after which the SSAT mice developed kyphosis. With aging, the SSAT overexpression elicited increased bone perimeter with strikingly thinned cortical bone, decreased trabecular thickness and increased trabecular number in mice. In vitro studies showed that the maturation of SSAT overexpressing osteoblasts was impaired and the expression of bone formation marker genes was dramatically decreased. The polyamine pattern in osteoblasts of SSAT mice was distorted in comparison with wild-type mice. However, treatment of osteoblasts with a SSAT-inducing functional polyamine analogue suggested that defective osteoblastogenesis resulted rather from other consequences of enhanced SSAT activity than lowered levels of the higher polyamines. In comparison to SSAT overexpressing mice, SSAT deficiency led to opposite changes in osteoblastogenesis and differences in bone phenotype in mice. In conclusion, the level of SSAT enzyme activity affected osteoblastogenesis and hence influenced bone remodeling and the bone phenotype in mice. Furthermore, our results suggest the contribution of the catabolic part of the polyamine cycle, other than polyamine depletion, in pathophysiological processes of bone remodeling.
Assuntos
Acetiltransferases/genética , Desenvolvimento Ósseo/genética , Remodelação Óssea/genética , Osteoblastos/metabolismo , Acetiltransferases/biossíntese , Animais , Animais Geneticamente Modificados , Cifose/genética , Cifose/patologia , Camundongos , Fenótipo , Poliaminas/metabolismoRESUMO
In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor. Nicotinamide is a precursor of NAD(+), an important cofactor linking cellular redox states with energy metabolism. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine-spermine N(1)-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD(+) levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD(+)-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes.
Assuntos
Dieta , Nicotinamida N-Metiltransferase/deficiência , Nicotinamida N-Metiltransferase/metabolismo , Obesidade/enzimologia , Obesidade/prevenção & controle , Acetiltransferases/metabolismo , Adipócitos/metabolismo , Tecido Adiposo/enzimologia , Tecido Adiposo/metabolismo , Tecido Adiposo Branco/enzimologia , Tecido Adiposo Branco/metabolismo , Animais , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético , Fígado Gorduroso , Técnicas de Silenciamento de Genes , Intolerância à Glucose , Transportador de Glucose Tipo 4/deficiência , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Resistência à Insulina , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , Niacinamida/metabolismo , Nicotinamida N-Metiltransferase/genética , Obesidade/etiologia , Obesidade/genética , Ornitina Descarboxilase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , S-Adenosilmetionina/metabolismo , Sirtuína 1/metabolismo , Espermina/análogos & derivados , Espermina/metabolismo , Magreza/enzimologia , Magreza/metabolismo , Poliamina OxidaseRESUMO
To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2(-/-) female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2(-/-) mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2(-/-)mammary fat pad showed a decrease in the content of myristic acid (C14), a principal substrate for protein myristoylation and a potential peroxisomal ß-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2(-/-) mice. The results point to disturbances of lipid metabolism in the mammary fat pad that in turn may result in abnormal epithelial growth. The work reveals impaired mammary gland development as a new category of peroxisomal disorders.
Assuntos
Metabolismo dos Lipídeos , Glândulas Mamárias Animais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Peroxissomos/metabolismo , Tecido Adiposo/metabolismo , Animais , Ácidos e Sais Biliares/química , Células Epiteliais/citologia , Ácidos Graxos/química , Feminino , Homeostase , Lactação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Poliaminas/química , Frações Subcelulares , Fatores de TempoRESUMO
INTRODUCTION: Polyamines play a fundamental role during embryogenesis by regulating cell growth and proliferation and by interacting with RNA, DNA and protein. The polyamine pools are regulated by metabolism and uptake from exogenous sources. The use of certain inhibitors of polyamine synthesis causes similar defects to those seen in alcohol exposure e.g. retarded embryo growth and endothelial cell sprouting. METHODS: CD-1 mice received two intraperitoneal injections of 3 g/kg ethanol at 4 h intervals 8.75 days post coitum (dpc). The fetal head, trunk, yolk sac and placenta were collected at 9.5 and 12.5 dpc and polyamine concentrations were determined. RESULTS: No measurable quantity of polyamines could be detected in the embryo head at 9.5 dpc, 12 h after ethanol exposure. Putrescine was not detectable in the trunk of the embryo at that time, whereas polyamines in yolk sac and placenta were at control level. Polyamine deficiency was associated with slow cell growth, reduction in endothelial cell sprouting, an altered pattern of blood vessel network formation and consequently retarded migration of neural crest cells and growth restriction. DISCUSSION: Our results indicate that the polyamine pools in embryonic and extraembryonic tissues are developmentally regulated. Alcohol administration, at the critical stage, perturbs polyamine levels with various patterns, depending on the tissue and its developmental stage. The total absence of polyamines in the embryo head at 9.5 dpc may explain why this stage is so vulnerable to the development of neural tube defect, and growth restriction, the findings previously observed in fetal alcohol syndrome.
Assuntos
Poliaminas Biogênicas/metabolismo , Etanol/toxicidade , Transtornos do Espectro Alcoólico Fetal/etiologia , Retardo do Crescimento Fetal/etiologia , Defeitos do Tubo Neural/etiologia , Animais , Modelos Animais de Doenças , Desenvolvimento Embrionário/efeitos dos fármacos , Etanol/administração & dosagem , Feminino , Transtornos do Espectro Alcoólico Fetal/metabolismo , Transtornos do Espectro Alcoólico Fetal/patologia , Retardo do Crescimento Fetal/induzido quimicamente , Retardo do Crescimento Fetal/metabolismo , Idade Gestacional , Cabeça , Homeostase/efeitos dos fármacos , Camundongos , Defeitos do Tubo Neural/induzido quimicamente , Defeitos do Tubo Neural/metabolismo , Fenótipo , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Distribuição Tecidual , Saco Vitelino/efeitos dos fármacos , Saco Vitelino/metabolismoRESUMO
The metabolism of polyamines, the cationic small molecules essential for cell proliferation and differentiation, is altered in cancer cells and can be exploited in cancer diagnosis and therapy. Spermidine/spermine N(1)-acetyltransferase (SSAT), which regulates intracellular levels of polyamines by catabolizing spermidine and spermine, has a controversial role in the development of cancers. In this study, the polyamine metabolism and function of SSAT were characterized in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoid leukemia patient samples. Also, mice overexpressing SSAT and having a myeloproliferative phenotype were analyzed for their response to decitabine and histone deacetylase inhibitor trichostatin A. The presence of epigenetic factors in the bone marrow cells of SSAT mice was analyzed. Elevated levels of spermidine and spermine, as well as increased activity of SSAT, were detected in AML, CML, and acute lymphoid leukemia patients compared with the controls. However, we found SSAT activity to be associated with white blood cell count only in AML and CML patients. Decitabine treatment brought the peripheral blood and bone marrow cell counts of SSAT mice to the level of wild-type mice. Spermidine/spermine N(1)-acetyltransferase mice had increased histone methylation and an increased level of histone deacetylase 1 in their bone marrow cells. The study suggests that SSAT influences the development of myeloid malignancies, and epigenetic factors partly contribute to the SSAT overexpression-induced myeloproliferative disease in mice.
Assuntos
Acetiltransferases/metabolismo , Leucemia Mieloide/patologia , Contagem de Leucócitos , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/análogos & derivados , Azacitidina/uso terapêutico , Decitabina , Humanos , Leucemia Mieloide/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poliaminas/metabolismoRESUMO
Spermidine/spermine N(1)-acetyltransferase (SSAT) regulates intracellular polyamine levels by catabolizing spermidine and spermine which are essential for cell proliferation and differentiation. Hematological characterization of SSAT overexpressing mice (SSAT mice) revealed enhanced myelopoiesis and thrombocytopoiesis leading to increased amounts of myeloid cells in bone marrow, peripheral blood, and spleen compared to wild-type animals. The level of SSAT activity in the bone marrow cells was associated with the bone marrow cellularity and spleen weight which both were significantly increased in SSAT mice. The result of bone marrow transplantations indicated that both the intrinsic SSAT overexpression of bone marrow cells and bone marrow microenvironment had an impact on the observed hematopoietic phenotype. The Lineage-negative Sca-1(+) c-Kit(+) hematopoietic stem cell (HSC) compartment in SSAT mice, showed enhanced proliferation, increased proportion of long-term HSCs and affected expression of transcription factors associated with lineage priming and myeloid differentiation. The proportions of common myeloid and megakaryocytic/erythroid progenitors were decreased and the proportion of granulocyte-macrophage progenitors was increased in SSAT bone marrow. The data suggest that SSAT overexpression and the concomitantly accelerated polyamine metabolism in hematopoietic cells and bone marrow microenvironment affect lineage commitment and lead to the development of a mouse myeloproliferative disease in SSAT mice.
Assuntos
Acetiltransferases/genética , Hematopoese , Transtornos Mieloproliferativos/metabolismo , Poliaminas/metabolismo , Acetiltransferases/metabolismo , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/patologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Polyamines, spermidine, spermine and their precursor putrescine, are ubiquitous cell components essential for normal cell growth. Increased polyamine levels and enhanced biosynthesis have been associated with malignant transformation and tumor formation, and thus, the polyamines have been considered to be a meaningful target to cancer therapies. However, clinical cancer treatment trials using inhibitors of polyamine synthesis have been unsuccessful probably due to compensatory uptake of polyamines from extracellular sources. The antizyme proteins regulate both polyamine biosynthesis and transport, and thus, the antizymes could provide an efficient approach to control cellular proliferation compared to the mere inhibition of biosynthesis. To define the role of antizymes in proliferative processes associated with the whole animal, we have generated transgenic mice overexpressing mouse antizyme 1 gene under its own regulatory sequences. Antizyme 1 protein was abundantly expressed in various organs and the expressed antizyme protein was functional as ornithine decarboxylase activity was significantly reduced in all tissues analyzed. However, antizyme 1 overexpression caused only minor changes in tissue polyamine levels demonstrating the challenges in using the "antizyme approach" to deplete polyamines in a living animal. Neither were there any changes in cellular proliferation in the proliferative tissues of transgenic animals. Interestingly though, there was occurrence of abnormally high level of apoptosis in the non-proliferating part of the colon epithelia. Otherwise, the transgenic founder mice appeared healthy and out of seven founders six were fertile. However, none of the founders could transmit the transgene suggesting that the antizyme 1 overexpression may be deleterious to transgenic gametes.
Assuntos
Transformação Celular Neoplásica/genética , Ornitina Descarboxilase/biossíntese , Poliaminas/metabolismo , Proteínas/genética , Animais , Transporte Biológico/genética , Regulação da Expressão Gênica , Homeostase , Camundongos , Camundongos Transgênicos , Ornitina Descarboxilase/genética , Proteínas/metabolismo , Distribuição TecidualRESUMO
Polyamine metabolism is intimately linked to the physiological state of the cell. Low polyamines levels promote growth cessation, while increased concentrations are often associated with rapid proliferation or cancer. Delicately balanced biosynthesis, catabolism, uptake and excretion are very important for maintaining the intracellular polyamine homeostasis, and deregulated polyamine metabolism is associated with imbalanced metabolic red/ox state. Although many cellular targets of polyamines have been described, the precise molecular mechanisms in these interactions are largely unknown. Polyamines are readily interconvertible which complicate studies on the functions of the individual polyamines. Thus, non-metabolizable polyamine analogues, like carbon-methylated analogues, are needed to circumvent that problem. This review focuses on methylated putrescine, spermidine and spermine analogues in which at least one hydrogen atom attached to polyamine carbon backbone has been replaced by a methyl group. These analogues allow the regulation of both metabolic and catabolic fates of the parent molecule. Substituting the natural polyamines with methylated analogue(s) offers means to study either the functions of an individual polyamine or the effects of altered polyamine metabolism on cell physiology. In general, gem-dimethylated analogues are considered to be non-metabolizable by polyamine catabolizing enzymes spermidine/spermine-N¹-acetyltransferase and acetylpolyamine oxidase and they support short-term cellular proliferation in many experimental models. Monomethylation renders the analogues chiral, offering some advantage over gem-dimethylated analogues in the specific regulation of polyamine metabolism. Thus, methylated polyamine analogues are practical tools to meet existing biological challenges in solving the physiological functions of polyamines.
Assuntos
Poliaminas/metabolismo , Animais , Humanos , MetilaçãoRESUMO
We have shown previously that the polyamine spermidine is indispensable for differentiation of 3T3-L1 preadipocytes. In the present study, we examined the mechanism of spermidine function by using the polyamine biosynthesis inhibitor α-difluoromethylornithine in combination with the metabolically stable polyamine analogues γ-methylspermidine or (R,R)-α,ω-bismethylspermine. At the early phase of differentiation, spermidine-depleted 3T3-L1 cells showed decreased translation of the transcription factor C/EBPß (CCAAT/enhancer-binding protein ß), decreased PP2A (protein phosphatase 2A) activity and increased cytoplasmic localization of the RNA-binding protein HuR (human antigen R). The amount of HuR bound to C/EBPß mRNA was reduced, whereas the amount of bound CUGBP2, an inhibitor of C/EBPß translation, was increased. ANP32 (acidic nuclear phosphoprotein 32) proteins, which are known PP2A inhibitors and HuR ligands, bound more PP2A and HuR in spermidine-depleted than in control cells, whereas immunodepletion of ANP32 proteins from the lysate of spermidine-depleted cells restored PP2A activity. Taken together, our data shows that spermidine promotes C/EBPß translation in differentiating 3T3-L1 cells, and that this process is controlled by the interaction of ANP32 with HuR and PP2A.
Assuntos
Adipogenia/efeitos dos fármacos , Proteínas ELAV/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatase 2/metabolismo , Espermidina/farmacologia , Células 3T3-L1 , Adipogenia/genética , Animais , Proteínas ELAV/genética , Eflornitina/farmacologia , Feminino , Masculino , Camundongos , Proteínas Nucleares/genética , Poliaminas/química , Poliaminas/farmacologia , Proteína Fosfatase 2/genética , Ratos , Ratos WistarRESUMO
Biogenic polyamines spermine and spermidine participate in numerous cellular processes including transcription, RNA processing and translation. Specifically, they counteract oxidative stress, an alteration of cell redox balance involved in generation and progression of various pathological states including cancer. Here, we investigated how chemically induced oxidative stress affects polyamine metabolism, specifically the expression and activities of enzymes catalyzing polyamine synthesis (ornithine decarboxylase; ODC) and degradation (spermidine/spermine-N(1)-acetyltransferase; SSAT), in human hepatoma cells. Oxidative stress induced the up-regulation of ODC and SSAT gene transcription mediated by Nrf2, and in case of SSAT, also by NF-κB transcription factors. Activation of transcription led to the elevated intracellular activities of both enzymes. The balance in antagonistic activities of ODC and SSAT in the stressed hepatoma cells was shifted towards polyamine biosynthesis, which resulted in increased intracellular levels of putrescine, spermidine, and spermine. Accumulation of putrescine is indicating for accelerated degradation of polyamines by SSAT - acetylpolyamine oxidase (APAO) pathway generating toxic products that promote carcinogenesis, whereas accelerated polyamine synthesis via activation of ODC is favorable for proliferation of cells including those sub-lethally damaged by oxidative stress.
Assuntos
Acetiltransferases/genética , Carcinoma Hepatocelular/patologia , Ornitina Descarboxilase/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Poliaminas/metabolismo , Ativação Transcricional/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Regulação para Cima/efeitos dos fármacosRESUMO
In macrophages, basal polyamine (putrescine, spermidine, and spermine) levels are relatively low but are increased upon IL-4 stimulation. This Th2 cytokine induces Arg1 activity, which converts arginine into ornithine, and ornithine can be decarboxylated by ODC to produce putrescine, which is further converted into spermidine and spermine. Recently, we proposed polyamines as novel agents in IL-4-dependent E-cadherin regulation in AAMs. Here, we demonstrate for the first time that several, but not all, AAM markers depend on polyamines for their IL-4-induced gene and protein expression and that polyamine dependency of genes relies on the macrophage type. Remarkably, Arg1-deficient macrophages display rather enhanced IL-4-induced polyamine production, suggesting that an Arg1-independent polyamine synthesis pathway may operate in macrophages. On the other side of the macrophage activation spectrum, LPS-induced expression of several proinflammatory genes was increased significantly in polyamine-depleted CAMs. Overall, we propose Arg1 independently produced polyamines as novel regulators of the inflammatory status of the macrophage. Indeed, whereas polyamines are needed for IL-4-induced expression of several AAM mediators, they inhibit the LPS-mediated expression of proinflammatory genes in CAMs.
Assuntos
Arginase/fisiologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Poliaminas/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Citometria de Fluxo , Perfilação da Expressão Gênica , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Óxido Nítrico/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Receptores Proteína Tirosina Quinases/fisiologia , Receptor TIE-2RESUMO
BE-3-3-3-3 (1,15-(ethylamino)4,8,12-triazapentadecane) is a bis(ethyl)polyamine analogue under investigation as a therapeutic agent for breast cancer. Since estradiol (E(2)) is a critical regulatory molecule in the growth of breast cancer, we examined the effect of BE-3-3-3-3 on estrogen receptor α (ERα) positive MCF-7 cells in the presence and absence of E(2). In the presence of E(2), a concentration-dependent decrease in DNA synthesis was observed using [(3)H]-thymidine incorporation assay. In the absence of E(2), low concentrations (2.5-10 µM) of BE-3-3-3-3 increased [(3)H]-thymidine incorporation at 24 and 48 h. BE-3-3-3-3 induced the expression of early response genes, c-myc and c-fos, in the absence of E(2), but not in its presence, as determined by real-time quantitative polymerase chain reaction (qPCR). BE-3-3-3-3 had no significant effect on these genes in an ERα-negative cell line, MDA-MB-231. Chromatin immunoprecipitation assay demonstrated enhanced promoter occupation by either E(2) or BE-3-3-3-3 of an estrogen-responsive gene pS2/Tff1 by ERα and its co-activator, steroid receptor co-activator 3 (SRC-3). Confocal microscopy of BE-3-3-3-3-treated cells revealed membrane localization of ERα, similar to that induced by E(2). The failure of BE-3-3-3-3 to inhibit cell proliferation was associated with autophagic vacuole formation, and the induction of Beclin 1 and MAP LC3 II. These results indicate a differential effect of BE-3-3-3-3 on MCF-7 cells in the absence and presence of E(2), and suggest that pre-clinical and clinical development of polyamine analogues might require special precautions and selection of sensitive subpopulation of patients.
Assuntos
Neoplasias da Mama/patologia , Mimetismo Molecular , Poliaminas/farmacologia , Receptores de Estrogênio/metabolismo , Sequência de Bases , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Primers do DNA , Replicação do DNA/efeitos dos fármacos , Feminino , Humanos , Poliaminas/química , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The study of polyamine flux, i.e. the circulating flow of polyamines through the interconnected biosynthetic and catabolic pathways, is of considerable interest because of the established links between the polyamine metabolism and many diseases, such as cancer and diabetes. To study polyamine flux in detail, a novel method based on following the label incorporation from the (13)C, (15)N-labeled polyamine precursors, arginine, methionine and ornithine, into polyamines by LC-MS/MS was implemented. This methodology was tested on three distinct cell lines with different spermidine/spermine-N (1)-acetyltransferase (SSAT) expression levels, i.e. non-transgenic, transgenic and knockout. These trials allowed the identification of the critical conditions for the successful polyamine flux measurement, such as the functional time frame of label incorporation, until plateau phase with the selected precursor is reached. The novel LC-MS/MS-based method for polyamine flux overcame the limitations of previous existing methodologies, with baseline separation of the different polyamine species and the exact quantification of the incorporated label. Moreover, the obtained results clearly show that the increased SSAT expression is associated with accelerated polyamine flux.
Assuntos
Aminoácidos/metabolismo , Poliaminas Biogênicas/metabolismo , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Calibragem , Isótopos de Carbono , Meios de Cultura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Isótopos de NitrogênioRESUMO
The polyamines, putrescine, spermidine, and spermine, are ubiquitous multifunctional cations essential for cellular proliferation. One specific function of spermidine in cell growth is its role as a butylamine donor for hypusine synthesis in the eukaryotic initiation factor 5A (eIF5A). Here, we report the ability of novel mono-methylated spermidine analogs (α-MeSpd, ß-MeSpd, γ-MeSpd, and ω-MeSpd) to function in the hypusination of eIF5A and in supporting the growth of DFMO-treated DU145 cells. We also tested them as substrates and inhibitors for deoxyhypusine synthase (DHS) in vitro. Of these compounds, α-MeSpd, ß-MeSpd, and γ-MeSpd (but not ω-MeSpd) were substrates for DHS in vitro, while they all inhibited the enzyme reaction. As racemic mixtures, only α-MeSpd and ß-MeSpd supported long-term growth (9-18 days) of spermidine-depleted DU145 cells, whereas γ-MeSpd and ω-MeSpd did not. The S-enantiomer of α-MeSpd, which supported long-term growth, was a good substrate for DHS in vitro, whereas the R-isomer was not. The long-term growth of DFMO-treated cells correlated with the hypusine modification of eIF5A by intracellular methylated spermidine analogs. These results underscore the critical requirement for hypusine modification in mammalian cell proliferation and provide new insights into the specificity of the deoxyhypusine synthase reaction.
Assuntos
Divisão Celular/efeitos dos fármacos , Lisina/análogos & derivados , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espermidina/farmacologia , Western Blotting , Linhagem Celular Tumoral , Humanos , Lisina/metabolismo , Masculino , Metilação , Espermidina/análogos & derivados , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
The prostate has the highest level of polyamines among all tissues, and it is the only tissue in which polyamines are purposely synthesized for export. It has been suggested that the high local polyamine concentrations suppress cell growth of primary prostatic carcinomas and that this growth control is lost when cancer cells metastasize. It has also been shown that the sensitivity to polyamine-induced growth arrest correlates with antizyme induction in prostate carcinoma cell lines. In this study, we evaluated the sensitivity of poorly metastatic (LNCaP) and highly metastatic (DU145) prostate cancer cell lines to conditional antizyme 1 over-expression. Antizyme 1 induction resulted in a marked loss of ODC activity and polyamine uptake in both cell lines. However, the proliferation of LNCaP cells was repressed by antizyme 1 induction, whereas the proliferation of DU 145 cells was not affected. Neither cell line showed any reduction in polyamine pools after manipulation nor did polyamine addition into the medium save the LNCaP cells from the growth retardation. The growth inhibition of LNCaP cells was accompanied by accumulation of cells in the G1 phase and depletion of cyclin E1 protein. These results confirm that different prostate cancer cell lines show diverse sensitivities to antizyme 1 which may not be directly polyamine related. The high gene transfer capacity of the used lentiviral vector makes the present approach a useful tool to study the therapeutic potential of antizyme 1 both in cell cultures and experimental animals.