RESUMO
Amyloid-ß oligomers (Aßo) are the most pathologically relevant Aß species in Alzheimer's disease (AD), because they induce early synaptic dysfunction that leads to learning and memory impairments. In contrast, increasing VEGF (Vascular Endothelial Growth Factor) brain levels have been shown to improve learning and memory processes, and to alleviate Aß-mediated synapse dysfunction. Here, we designed a new peptide, the blocking peptide (BP), which is derived from an Aßo-targeted domain of the VEGF protein, and investigated its effect on Aß-associated toxicity. Using a combination of biochemical, 3D and ultrastructural imaging, and electrophysiological approaches, we demonstrated that BP strongly interacts with Aßo and blocks Aß fibrillar aggregation process, leading to the formation of Aß amorphous aggregates. BP further impedes the formation of structured Aßo and prevents their pathogenic binding to synapses. Importantly, acute BP treatment successfully rescues long-term potentiation (LTP) in the APP/PS1 mouse model of AD, at an age when LTP is highly impaired in hippocampal slices. Moreover, BP is also able to block the interaction between Aßo and VEGF, which suggests a dual mechanism aimed at both trapping Aßo and releasing VEGF to alleviate Aßo-induced synaptic damage. Our findings provide evidence for a neutralizing effect of the BP on Aß aggregation process and pathogenic action, highlighting a potential new therapeutic strategy.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Camundongos , Animais , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Doença de Alzheimer/metabolismo , Memória/fisiologia , Aprendizagem , Sinapses/metabolismo , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismoRESUMO
We report a spectroscopic and theoretical study of the interaction between double-stranded oligonucleotides containing either adenine-thymine or guanine-cytosine alternating sequences and N1-(Acridin-9-ylcarbonyl)-1,5,9,14,18-pentazaoctadecane, or ASC, which is formed by the covalent bonding of spermine and 9-amidoacridine moieties via a trimethylene chain. Solutions containing the oligonucleotides and the conjugate at different molar ratios were studied using complementary spectroscopic techniques, including electronic absorption, fluorescence emission, circular dichroism, and Raman spectroscopy. The spectroscopical properties of ASC at both the vibrational and the electronic levels were described by means of ab initio quantum-chemical calculations on 9-amidoacridine, used as a model compound. Molecular dynamics calculations, based on the QM/MM methodology, were also performed using previously docked structures of two oligonucleotide-ASC complexes containing the A-T and the G-C sequence. Our data, taken all together, allowed us to demonstrate that conjugation of spermine to acridine modulates and gives additional properties to the interaction of the latter with DNA. As the ASC molecule has a high affinity by the polyamine transport system, these results are promising for their application in the development of new anti-tumour drugs.
Assuntos
Acridinas/química , DNA/química , Espectrofotometria Ultravioleta/métodos , Análise Espectral Raman/métodos , Espermina/química , Dicroísmo CircularRESUMO
A series of polyamine conjugates were synthesized and evaluated for their ability to target the polyamine transporter (PAT) in two Chinese hamster ovary (CHO) cell lines (PAT-active CHO and PAT-inactive CHOMG). This systematic study identified salient features of the polyamine architecture required to target and enter cells via the PAT. Indeed, the separation of charges, the degree of N-alkylation, and the spacer unit connecting the N(1)-terminus to the appended cytotoxic component (anthracene) were found to be key contributors to optimal delivery via the PAT. Using the CHO screen, the homospermidine motif (e.g., 4,4-triamine) was identified as a polyamine vector, which could enable the selective import of large N(1)-substituents (i.e., naphthylmethyl, anthracenylmethyl and pyrenylmethyl), which were cytotoxic to cells. The cell selectivity of this approach was demonstrated in B-16 murine melanoma cells and normal melanocytes (Mel-A). Three polyamine areas (recognition and transport, vesicle sequestration and polyamine-target interactions) were identified for future research.
Assuntos
Antracenos/metabolismo , Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Poliaminas/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Melanoma Experimental , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Bis(7-amino-4-azaheptyl)dimethylsilane (AzhepSi) and its bis(ethyl) derivative [bis(7-ethylamino-4-azaheptyl)dimethylsilane] (EtAzhepSi) are the first examples of a new type of aliphatic tetramine with a dimethylsilane group incorporated into the central carbon chain. AzhepSi shares certain properties with the natural polyamines, but in contrast with spermidine and spermine it inhibits the growth of L1210 leukemia cells in culture at micromolar concentrations. The bis(ethyl) derivative of AzhepSi was made, in analogy to bis(ethyl) spermine, a polyamine derivative, which gained much attention during the last decade as a potential anticancer drug. Chinese hamster ovary (CHO) cells accumulate the dimethylsilyl tetramines considerably more and are more sensitive to these drugs than are CHO-MG cells, a polyamine uptake-deficient mutant. This and related observations demonstrate that AzhepSi and EtAzhepSi are preferentially taken up by a polyamine transport system. Both tetramines inhibit the growth of a variety of tumor cells at micromolar concentrations. AzhepSi turned out to be either equipotent or more potent, but in no case less potent than EtAzhepSi. When given alone at daily doses of 25 micromol/kg, the compounds did not prolong the survival time of L1210 leukemia mice. However, in combination with 2-(difluoromethyl)ornithine and neomycin, AzhepSi had a significant effect on the life span of the animals. The growth rate of 3LL Lewis lung carcinoma was reduced by both compounds at daily doses of 25 micromol/kg. The observations presented in this work suggest that the dimethylsilyl tetramines are antiproliferative agents in vitro and in vivo. Due to enhanced general toxicity, the introduction of N-ethyl substituents was of no advantage in the case of these polyamine analogues.
Assuntos
Antineoplásicos/farmacologia , Silanos/farmacologia , Animais , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Divisão Celular/efeitos dos fármacos , Cricetinae , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Leucemia L1210/tratamento farmacológico , Camundongos , Camundongos Endogâmicos DBARESUMO
The structural basis of the binding of the polyamine spermine to the monoclonal antibody SPM8-2 was studied using computer modelling, ELISA methods and chemical modifications of the binding site residues. Paratope modelling showed that the antibody combining site forms a highly negatively charged cavity mainly shaped by aspartic acid and tyrosine residues which contact the tetra-positively charged spermine molecule by electrostatic interactions and hydrogen bondings. The importance of the electrostatic environment for spermine binding to SPM8-2 is emphasised by the strong dependency on pH and ionic strength. Specific chemical modifications of carboxylate groups and tyrosine residues of the antibody adsorbed to microtiter plates resulted in decreased binding of the N1-biotin-spermine conjugate used to monitor the activity of the antibody. These observations are consistent with a key role of aspartate and tyrosine residues in complex formation with spermine. These studies, important to our understanding of antibody-hapten specificity, may also shed light on important motifs responsible for protein-polyamine interactions.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Simulação por Computador , Modelos Moleculares , Espermina/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Camundongos , Modelos Imunológicos , Dados de Sequência Molecular , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
The uptake and release of the natural polyamines putrescine, spermidine and spermine by mammalian cells are integral parts of the systems that regulate the intracellular concentrations of these biogenic amines according to needs. Although a general feature of all tissues, polyamine uptake into intestinal mucosa cells is perhaps the most obvious polyamine transport pathway of physiological and pathophysiological importance. Mutant cell lines lacking the ability to take up polyamines from the environment are capable of releasing polyamines. This indicates that uptake and release are functions of two different transport systems. The isolation of a transporter gene from a mammalian cell line is still lacking. Overaccumulation of polyamines is controlled by release and by a feedback regulation system that involves de novo synthesis of antizyme, a well known protein that also regulates the activity of ornithine decarboxylase. Recent work has demonstrated that Ca(2+)-signalling pathways are also involved. Although there is consensus about the importance of polyamine uptake inhibitors in the treatment of neoplastic disorders, a practically useful uptake inhibitor is still missing. However, the attempts to target tumours, and to increase the selectivity of cytotoxic agents by combining them with the polyamine structure, are promising. New, less toxic and more selective anticancer drugs can be expected from this approach.
Assuntos
Poliaminas , Animais , Transporte Biológico , Cálcio/metabolismo , Humanos , Poliaminas/metabolismo , Poliaminas/farmacologiaRESUMO
N1-Dansylspermine and related sulfonamides of the natural polyamines are very potent blockers of NMDA-type glutamate receptors. They exhibit pharmacological properties which were not predicted from the constituents of the conjugates. Cytotoxicity and calmodulin antagonism of N1-dansylspermine were especially impressive. Calmodulin antagonism implies that N1-dansylspermine prevents induction of ornithine decarboxylase and inhibits its own active uptake via the polyamine transport system. Structure-activity considerations demonstrated that an aromatic character of the substituent is not required; amide bond formation with an aliphatic sulfonic acid is sufficient to transform spermine into a highly toxic calmodulin antagonist. Cytotoxicity and calmodulin antagonism are properties which are intrinsic to spermine, but they are observed only at very high concentrations. Amide bond formation at N1 with a lipophilic residue appears to 'amplify' these normally latent properties. The use of polyamine conjugates structurally related to the amides described in this work for targeting tumours may be marred by their calmodulin antagonism.
Assuntos
Amina Oxidase (contendo Cobre) , Antineoplásicos/farmacologia , Calmodulina/antagonistas & inibidores , Compostos de Dansil/farmacologia , Poliaminas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Espermina/análogos & derivados , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Calmodulina/metabolismo , Bovinos , Compostos de Dansil/química , Compostos de Dansil/metabolismo , Técnicas In Vitro , Leucemia L1210 , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Poliaminas/química , Poliaminas/metabolismo , Putrescina/metabolismo , Espermina/química , Espermina/metabolismo , Espermina/farmacologia , Relação Estrutura-Atividade , Especificidade por Substrato , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologiaRESUMO
The transglutaminase-mediated insertion of putrescine into casein was inhibited competitively by alpha-difluoromethylornithine (alpha-DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase. Preincubation of the amine acceptor (casein) or the enzyme itself with the inhibitor did not affect enzyme activity. Alpha-DFMO is a poorer substrate for transglutaminase (Km = 2.10 mM) than putrescine (Km = 0.17 mM). The inhibitory effect was also found with fibronectin as amine acceptor.
Assuntos
Aciltransferases/antagonistas & inibidores , Ornitina/análogos & derivados , Animais , Caseínas/metabolismo , Eflornitina , Cobaias , Cinética , Fígado/enzimologia , Ornitina/metabolismo , Ornitina/farmacologia , Putrescina/metabolismo , TransglutaminasesRESUMO
An inverse correlation was found between cellular transglutaminase activity and metastatic potential of four cloned cell lines derived from a primary nickel-induced rat rhabdomyosarcoma. Cellular transglutaminase activity as assessed with endogenous cellular protein or exogenous methylated casein was greatest in the clone F9-4/8 which is the least metastasizing. When the putrescine-binding capacity of one cellular derived protein - fibronectin - was examined with exogenous transglutaminase, it was found that the fibronectin derived from the clone F9-4/8 showed the lowest binding capacity compared with those from the other clones. However, when the overall binding capacity of cellular proteins from each cell line was examined no differences could be detected. The results are discussed in the light of the well-known role of fibronectin in cellular adhesion.
Assuntos
Putrescina/metabolismo , Rabdomiossarcoma/metabolismo , Transglutaminases/metabolismo , Animais , Adesão Celular , Células Clonais/metabolismo , Fibronectinas/metabolismo , Níquel , Ligação Proteica , Ratos , Rabdomiossarcoma/induzido quimicamente , Rabdomiossarcoma/secundárioRESUMO
Protein-bound polyamines were isolated from the plasma of mice using antipolyamine antibodies covalently linked to magnetic latex spheres. Their subsequent separation by polyacrylamide gel electrophoresis (PAGE) showed that in plasma from normal mice, 3 proteins (27, 55 and 82 kDa) carrying polyamines could be visualized, whereas in mice bearing the Lewis lung carcinoma at least 8 other proteins of higher molecular mass (5 of 94, 110, 130, 145 and 160 kDa, and 3 of greater than 170 kDa) had bound polyamines. These protein-bound polyamines could be detected from the first week after tumour graft; they increased during the second and third week but decreased thereafter. These proteins were not bound by immunolatex spheres preincubated with spermine bound to a protein-carrier insulin. Moreover, the appearance of these protein-bound polyamines was not a consequence of the inflammatory process since in mice infected with heat-inactivated Brucella abortus, with the exception of a 65 kDa protein, polyamines were bound to the same proteins found in normal mice. In mice grafted with the Lewis lung carcinoma the concomitant decrease in transglutaminase-mediated polyamine (e.g. putrescine) binding capacity of plasma proteins provides additional evidence for the presence in vivo of polyamines already bound to plasma proteins.
Assuntos
Neoplasias Pulmonares/sangue , Poliaminas/sangue , Animais , Proteínas Sanguíneas/análise , Carcinoma/sangue , Feminino , Camundongos , Camundongos Endogâmicos , Transplante de Neoplasias , Ligação Proteica , Putrescina/sangueRESUMO
In cells, protein degradation is a key pathway for the destruction of abnormal or damaged proteins as well as for the elimination of proteins whose presence is no longer required. Among the various cell proteases, the proteasome, a multicatalytic macromolecular complex, is specifically required for the degradation of ubiquitinated proteins. In normal cells, the proteasome ensures the elimination of numerous proteins that play critical roles in cell functions throughout the cell cycle. Defects in the activity of this proteolytic machinery can lead to the disorders of cell function that is believed to be the root cause of certain diseases. Indeed, many proteins involved in the control of cell cycle transitions are readily destroyed by the proteasome once their tasks have been accomplished. Moreover, because proteasome inhibitors can provoke cell death, it has been suggested that proteasomes must be continually degrading certain apoptotic factors. For these reasons, proteasome inhibition has become a new and potentially significant strategy for the drug development in cancer treatment. The proteasome possesses three major peptidase activities that can individually be targeted by drugs. Different classes of proteasome inhibitors are reviewed here. In addition, we present new pseudopeptides with the enriched nitrogen backbones bearing a side chain and a modified C-terminal position that inhibit proteasome activity.
Assuntos
Antineoplásicos/uso terapêutico , Doenças Neurodegenerativas/tratamento farmacológico , Inibidores de Proteases/uso terapêutico , Animais , Cisteína Endopeptidases , Humanos , Complexos Multienzimáticos/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma , UbiquitinasRESUMO
A simple covalent enzyme-linked immunoassay procedure (CELIA) is described for the routine determination of free and immune complex-bound antibodies in sera. Assays for the latter could not have been performed by adsorption ELISA due to the high ionic strength of the reassociating buffer. For the measurement in human sera of free naturally occurring IgG and IgM antibody directed against the hapten spermine, polycarboxystyrene microtiter plates with covalently coupled spermine were used. For the determination of immune complex-bound antipolyamine IgG and IgM antibody titers, serum was first dissociated at pH 2.3 in tubes and then reassociated at pH 8.1 in the wells of a microtiter plate containing covalently bound spermine. The reactivity of anti-spermine antibodies was increased from 2- to 13-fold after dissociation and reassociation compared to that of non-dissociated area. The apparent reaction constant (Rapp.) of free IgG antibodies to spermine in the sera of 19 bronchopulmonary patients with cancer differed significantly from Rapp. values of IgG antibodies having this specificity in ten other patients with non-malignant disease.
Assuntos
Anticorpos/análise , Especificidade de Anticorpos , Técnicas Imunoenzimáticas , Poliaminas/imunologia , Animais , Complexo Antígeno-Anticorpo/sangue , Cabras , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/análise , Imunoglobulina M/análise , Pneumopatias/imunologia , Neoplasias Pulmonares/imunologia , Reprodutibilidade dos Testes , Espermina/imunologiaRESUMO
The reactivity of an anti-spermine MAb (SPM8-2) toward polyamines either free or bound to a solid surface was investigated using equilibrium dialysis and ELISA methods. When polyamines were covalently linked to hydrophilized microtiter plates using carbodiimide, the MAb SPM8-2 reacted both with spermine and spermidine, with a higher affinity for the latter, but did not show any reactivity towards bound putrescine. In contrast, the MAb SPM8-2 reacted with all three polyamines bound to the microtiter plates with glutaraldehyde, with an affinity in the order: putrescine > spermidine > spermine. Equilibrium dialysis and competitive ELISA tests showed that the MAb SPM8-2 exhibited high affinity for free spermine and 50% and 5% cross-reactivity with free spermidine and putrescine respectively. The affinity of the MAb SPM8-2 for putrescine, spermidine and spermine appears to depend on whether the polyamine is free or bound. The antigenicity of the polyamines differs according to the nature of their link to the solid phase. These observations are discussed in the light of the structural modification produced by covalent binding of the polyamines. It is also concluded that when antibodies are used, due care has to be exercised in choosing the appropriate immunoassay for determining the specificity of antibodies directed against small haptens such as the polyamines.
Assuntos
Anticorpos Monoclonais/metabolismo , Poliaminas/imunologia , Espermina/imunologia , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Poliaminas/químicaRESUMO
Cancer cells have high-affinity polyamine uptake systems with a low stringency for structural features. Putrescine, spermidine, and spermine have, therefore, been considered as potential vectors for the selective accumulation in tumors of therapeutically or diagnostically useful structures and elements. We envisaged N-benzyl derivatives of the polyamines as vectors of (10)B and (18)F for boron neutron capture therapy (BNCT) and tumor imaging by positron emission tomography (PET), respectively. In the present work, the synthesis, transport characteristics, DNA-binding properties, and cytotoxicity of several N-benzyl derivatives of putrescine and spermidine are described. The fluorinated spermidine derivative N-(3-[(4-aminobutyl)amino]propyl)[(4-fluorophenyl)methyl]amine (N(1)-4-Fbz-spd) may be useful for PET because of its high accumulation in cancer cells via the polyamine transport system. Among the boron-containing benzyl polyamines, N-(4-aminobutyl)([4-(dihydroxyboryl)phenyl]methyl)amine (4-Bbz-put) and N-(3-[(4-aminobutyl)amino]propyl)([4-(dihydroxyboryl)phenyl]methyl)amine (N(1)-4-Bbz-spd) should be suitable for BNCT, because their accumulation in B16 melanoma cells was more efficient than that of borocaptate and borophenylalanine, two reference compounds used in BNCT.
Assuntos
Compostos de Boro/síntese química , Putrescina/análogos & derivados , Putrescina/síntese química , Espermidina/análogos & derivados , Espermidina/síntese química , Animais , Transporte Biológico Ativo , Compostos de Boro/metabolismo , Terapia por Captura de Nêutron de Boro , Linhagem Celular , DNA/química , Putrescina/metabolismo , Espermidina/metabolismo , Espermina/análogos & derivados , Relação Estrutura-Atividade , Tomografia Computadorizada de Emissão , Células Tumorais CultivadasRESUMO
Although the exact functions of polyamines in the nervous system remain still unclear, they are thought to have a physiological role in intracellular signal processing and neurotransmission. Polyamine deprivation which consists in the reduction of both the endogenous and exogenous sources of polyamines is a promising treatment for cancer. In a previous study we have shown that this treatment provokes an analgesic effect in rats submitted to brief phasic nociceptive tests. The present study examined the effect of polyamine deprivation on pain-related behaviors and spinal c-fos expression evoked in the formalin test presumed to better reflect clinical pain, using morphine as analgesia control. Polyamine deprivation per se altered the characteristic pain-related behaviors, reducing the interphase depression of pain, without inducing changes in the spinal Fos staining. In addition this treatment prevented the antinociceptive effect of morphine both on behavioral responses and on spinal c-fos expression. In polyamine-deprived rats, despite morphine injection, nociceptive scores remained dramatically high during the intermediate and the late phases of the response and the number of Fos immunoreactive neurons remained largely higher in deeper layers than in morphine control rats. Altogether these data support a modulatory role of polyamines both on the neuronal circuitry mediating sensory information, and on mechanisms underlying morphine analgesia.
Assuntos
Analgésicos Opioides/farmacologia , Poliaminas Biogênicas/fisiologia , Hiperalgesia/fisiopatologia , Morfina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Imuno-Histoquímica , Masculino , Medição da Dor , Proteínas Proto-Oncogênicas c-fos/biossíntese , Ratos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
It is well established that inhibition of putrescine formation using D,L-2-(difluoromethyl)ornithine and feeding a polyamine-deficient diet together with non-absorbable antibiotics (neomycin and metronidazole), prevent almost completely the growth of tumors in rats. A similar regimen given to patients with prostate cancer not only reduced the titer of prostate specific antigen in serum, but surprisingly provoked at the same time an antalgic effect. This observation led us to study the potentiation effect of polyamine deprivation on pain threshold in healthy rats. Animals were fed for 2 weeks with an artificial diet of known polyamine content, in combination with antibiotics and 2-(difluoromethyl)ornithine, and were then submitted to pain stimuli using two models, the Randall-Selitto test and the Tail-Flick test. Polyamine deprivation produced in these models an increase in the latency of the response, even under conditions which did not produce significant changes of the polyamine concentrations in blood and brain. From these observations, we may conclude that the polyamines play a role in the perception of nociceptive stimuli under physiological conditions.
Assuntos
Analgesia , Poliaminas/antagonistas & inibidores , Animais , Dieta , Eflornitina/farmacologia , Poliaminas/administração & dosagem , Poliaminas/metabolismo , Ratos , Ratos WistarRESUMO
High red blood cell (RBC) spermidine levels have been observed in patients harboring various histological types of cancer. In an attempt to explain the mechanism of RBC spermidine uptake in the malignant cell growth process, erythrocytes from normal and (3LL) Lewis lung carcinoma grafted mice were incubated with [14C] spermidine in the presence of PBS or plasma from normal or 3LL grafted mice. Though RBC of cancer mice harbor abnormally elevated spermidine concentrations as compared with those of controls, when incubated with PBS, 3LL grafted mice exhibit [14C] spermidine uptake three times higher than that of normal mice erythrocytes in the same incubating conditions. Moreover, if plasma incubations always increase spermidine uptake in RBC from cancer mice compared with erythrocytes only incubated with PBS, plasma incubations are responsible for two opposite effects in RBC of normal mice: plasma increases spermidine uptake in PBS unwashed erythrocytes and lowers it in PBS washed RBC. One possible explanation for the observed plasma stimulating effect in RBC spermidine uptake might be the presence of a plasma component common to plasma from normal and cancer mice, able to interact with RBC membrane proteins. In erythrocytes from cancer mice, the high spermidine concentration would be responsible for the observed compactness in band 3 proteins involved in ionic transport through RBC stroma. In normal mice erythrocytes which exhibit low polyamine levels, the observed dissociation of band 3 proteins might explain the spermidine uptake in PBS washed normal RBC incubated with plasma. In vivo, this plasma component would participate in the stabilization of normal mice membrane proteins involved in RBC spermidine uptake.
Assuntos
Eritrócitos/metabolismo , Neoplasias Pulmonares/sangue , Espermidina/sangue , Animais , Transporte Biológico , Radioisótopos de Carbono , Membrana Eritrocítica/metabolismo , Feminino , Técnicas In Vitro , Cinética , Proteínas de Membrana/sangue , Camundongos , Camundongos Endogâmicos , Ligação Proteica , Valores de ReferênciaRESUMO
It has previously been demonstrated that during Lewis Lung carcinoma growth, red blood cell spermidine levels increase concomitantly with tumor volume. If [14C] putrescine or 2-methylputrescine are administered, [14C] spermidine and methylspermidine, respectively, accumulate in red blood cells in proportion with the tumor volume. In the present work the metabolic transformation of 2-hydroxyputrescine, a natural derivative of putrescine, to hydroxyspermidine, was studied in tumor bearing mice. After a single i.p. injection of 2-hydroxyputrescine, higher concentrations of hydroxyspermidine were found in the tumor than in liver. In the red blood cells of Lewis lung carcinoma-bearing mice, hydroxyspermidine was detected between 24 hours and 96 hours after i.p. injection of 2-hydroxyputrescine. The concentration of hydroxyspermidine found in red blood cells was proportional to the tumor volume. Hydroxyspermidine has potential as a marker of malignant cell proliferation in human oncology.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Espermidina/análogos & derivados , Espermidina/metabolismo , Animais , Carcinoma Pulmonar de Lewis/sangue , Carcinoma Pulmonar de Lewis/patologia , Divisão Celular , Fígado/metabolismo , CamundongosRESUMO
Dimethylsilane tetramines are structural analogues of spermine with a (CH3)2 Si-group incorporated into the central carbon chain. They have potential as anticancer drugs. Their cytotoxic effect was considered to rely mainly on their polyamine antagonist property. In order to obtain new ideas about cellular mechanisms, which are potential targets of the dimethylsilane polyamines, the effects of these compounds on some basic cell functions, such as protein and DNA synthesis, and calmodulin antagonism were studied. In addition, their mode of accumulation in cells was investigated. It became evident that the intracellular accumulation of dimethylsilane polyamines is almost exclusively achieved via the polyamine transport system. However, the exchange of a part of the intracellular natural polyamines against dimethylsilane polyamines has only a small effect on polyamine uptake. Binding to the endoplasmic reticulum and inhibition of protein synthesis are presumably important for the cytotoxic action of bis(11-amino-4,8-diazaundecyl)dimethylsilane, a hexamine, but seem of no importance for the tetramines. Calmodulin antagonism, however, is likely to contribute to their cytotoxic effect.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Calmodulina/análogos & derivados , Poliaminas/farmacologia , Poliaminas/farmacocinética , Silanos/farmacologia , Silanos/farmacocinética , Animais , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Calmodulina/metabolismo , Agregação Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Cricetinae , DNA de Neoplasias/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Leucina/antagonistas & inibidores , Leucina/metabolismo , Leucemia L1210/tratamento farmacológico , Leucemia L1210/metabolismo , Metenamina/farmacocinética , Metenamina/farmacologia , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Ratos , Espermidina/antagonistas & inibidores , Espermidina/farmacocinética , Timidina/antagonistas & inibidores , Timidina/metabolismo , Poliamina OxidaseRESUMO
The monoclonal antispermine antibody Spm8-2 was obtained by immunizing mice with a thyroglobulin-spermine conjugate. The molecular requirements for polyamines binding to this antibody were investigated by ELISA binding and inhibition tests, using a variety of natural polyamines and synthetic polyamine analogs. Four major structural determinants are important for the binding of polyamines by the antibody: (1) terminal amino groups: N-alkylation of both terminal amino groups of the polyamines leads to an important drop in the affinity for the antibody; (2) number of methylene groups spacing the amino groups: the four carbon chains appear to present the optimum length since the antibody binds polyamines with repeats of the aminobutyl moiety more actively than their homologues with shorter or longer carbon chains; (3) number of amino groups: the affinity of Spm8-2 for free homologous polyamines varied in the following order: pentamines > tetramines > triamines > diamines, showing the importance of the number of positive charges of the polyamines in the antibody-antigen reaction; the importance of charges is further emphasized by the dependence of antibody binding on the ionic strength of the medium; (4) N-acylation of one terminal amino group: the antibody binds more actively N1-acetylspermidine than spermidine or spermine. The binding properties of Spm8-2 suggest the presence of two recognition sequences, one selective for N-acylaminopropyl moieties, the second for the aminobutyl moiety.