RESUMEN
Methionine, in addition to its role in protein synthesis, participates in 3 important cellular functions: as AdoMet in transmethylation; as decarboxylated-AdoMet in aminopropylation; as homocysteine its demethylated form, in trans-sulphuration. Here we provide evidence from the literature and from our own work for a fourth role for its oxoacid: 4-methylthio-2-oxo-butanoate (MTOB) in apoptosis [28,29]. MTOB enters 2 pathways: (a) transamination by glutamine-transaminase K to methionine[13,14].(b)oxidative decarboxylation by the mitochondrial Branched-Chain-Oxo-Acid-Dehydrogenase-Complex to methional and finally to methylthiopropanoyl CoA (MTPCoA) [26,27]. Some of the methional formed after MTOB decarboxylation leaks into the cytoplasm as free methional [29]. Exogenous methional induces apoptosis in normal and cancer cells in culture [28, 29] but not in those overexpressing the antiapoptotic gene bcl2 [30]. In physiologically-induced apoptosis e.g; trophic factor (IL3) withdrawal, methional leakage is decreased [29] suggesting that MTPCoA is also involved in apoptosis. Both methional and MTPCoA give rise to metabolites that may act as cross-linking agents. In the case of methional, the CH3-S moiety is lost and malondialdehyde (MDA) is formed when methional is subjected to ( )OH attack [29]. MDA generated in situ from 1,3-propanediol, induces DNA-protein cross-linking [41].With regard to MTPCoA, it is metabolized to malonic semialdehyde CoA (MASACoA) with loss of the CH3-S moiety [48,49]. The capacity of MASACoA to form cross-links has not yet been established experimentally, but it could be a substrate for one of the histone acyl transferases [50, 51] and so form amides via the CoA at one end and imines by its CHO group at the other, with amino groups on proteins. Chromatin cross-linking/condensation is one of the hall-marks of apoptosis [40]. Methional, MDA and other apoptogenic aldehydes like 4-hydroxy-2-nonenal are oxidized by ALDHs to non-apoptogenic carboxylic acids [29,44, 45,68] but retain their apoptotic activity when the ALDHs are inhibited [98,110]. MASACoA would also lose its cross-linking capacity if its CoA moiety were putatively hydrolysed by ALDHs and/or acylCoA thioesterases [56,58,88,89]. ALDH inhibitors that control cellular MDA and possibly MASACoA homeostasis are cited as examples of targeted therapeutic approaches in chemoresistant cancers [62,84,97,98,110].
Asunto(s)
Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos , Metionina/análogos & derivados , Metionina/metabolismo , Aldehídos/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Liasas/metabolismo , Metionina/antagonistas & inhibidores , Metionina/farmacología , Mitocondrias/enzimología , Oxidación-Reducción , Antagonistas de Prostaglandina/farmacología , Transducción de Señal , Transaminasas/metabolismoRESUMEN
This study demonstrates that synthetic isopeptides formed on BSA can be quantitatively analyzed by a surface plasmon resonance-based biosensor method. A monoclonal IgM antibody 81D4, that reacts with the synthetic isopeptide and also with the natural isopeptide cross-link in D-dimer (but not with its non-cross-linked fibrin monomer), was covalently immobilized to a carboxymethylated dextran surface, a CM5 surface. Its immunocapturing efficiency was found to be good. The affinity of the interaction between the monoclonal 81D4 and the synthetic isopeptide was estimated to approximately 4x10(-7)M. Good reactivity was also observed when human plasma spiked with this isopeptide was used as test solution. Cross-linked D-dimer in the plasma of patients is a marker of disseminated intravascular coagulation (DIC) which occurs late in sepsis. This biosensor method has the potential to be developed into a rapid sensitive assay for measuring the level of natural isopeptide cross-links in proteins in the plasma of patients with a suspected diagnosis of sepsis.
Asunto(s)
Técnicas Biosensibles , Inmunoensayo , Péptidos/metabolismo , Plasma/metabolismo , Albúmina Sérica Bovina/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Plasma/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
The pharmacomodulation of the N atom of alpha,beta-acetylenic aminothiolesters or the replacement of the thiolester moiety by more electrophilic groups did not permit any clear rationale to be established for improving the selective growth-inhibitory activity of this family of compounds over that of the previously synthesized alpha,beta-acetylenic aminothiolesters DIMATE and MATE [G. Quash, G. Fournet, J. Chantepie, J. Goré, C. Ardiet, D. Ardail, Y. Michal, U. Reichert, Biochem Pharmacol 64 (2002) 1279-92]. Hence DIMATE and MATE were investigated more thoroughly for selectivity and growth-inhibitory activity using human prostate epithelial normal cells (HPENC) on the one hand and human prostate epithelial cancer cells (DU145) on the other. Unequivocal evidence was obtained showing that both compounds were reversible growth inhibitors of HPENC but irreversible growth inhibitors of DU145. Growth-inhibition of DU145 was due to the induction of early apoptosis as revealed by the flow cytometric analytical profile of inhibitor-treated cells, of the decrease in the redox potential and increase in superoxide anion content of their mitochondria. Of the two intracellular enzymes: aldehyde dehydrogenases 1 and 3 (ALDH1 and ALDH3) targeted by DIMATE and MATE, ALDH3 was inhibited to the same extent by both compounds whereas ALDH1 was less susceptible to inhibition by MATE. As the induction of ALDH3 by xenobiotics is hormone-dependent, MATE, the more selective of the two inhibitors, is a useful tool not only for examining the role of the ALDH3 isoform in hormone-sensitive and resistant prostate cancer cells in culture but also for investigating if it can inhibit the growth of xenografts of prostate cancer in immunodeficient mice.
Asunto(s)
Aldehído Deshidrogenasa/antagonistas & inhibidores , Antineoplásicos/síntesis química , Apoptosis , Células Epiteliales/efectos de los fármacos , Compuestos de Sulfhidrilo/síntesis química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células Epiteliales/citología , Ésteres , Humanos , Isoenzimas/antagonistas & inhibidores , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción , Neoplasias de la Próstata , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacología , Superóxidos/metabolismo , Trasplante HeterólogoRESUMEN
6S,8S-Bis(3-methylthiopropanoyl) thiolesters of lipoic acid were synthesized with the carboxyl moiety of lipoate modified as methyl or water soluble choline esters. Evaluation on different cell lines in culture showed that they possessed modest antiproliferative activity. However, the 6-fold decrease in IC50 (from 270 to 45 microM) observed with the water soluble 6S,8S-bis(3-methylthiopropenoyl) thiolester dehydro derivative on a human epithelial prostate cancer cell line (DU145) argues in favor of 3-methylthiopropanoyl metabolites as endogenous growth regulatory (apoptogenic) compounds derived from methionine.
Asunto(s)
Aldehídos/química , Antineoplásicos/síntesis química , Ésteres/síntesis química , Propionatos/química , Ácido Tióctico/síntesis química , Aldehídos/metabolismo , Antineoplásicos/farmacología , Ésteres/farmacología , Humanos , Concentración 50 Inhibidora , Masculino , Metionina/química , Imitación Molecular , Neoplasias de la Próstata/patología , Compuestos de Sulfhidrilo/química , Ácido Tióctico/farmacología , Células Tumorales CultivadasRESUMEN
Plasma TG (transglutaminase) [FXIII (Factor XIII)] stabilizes fibrin and plays an essential role in haemostasis. In the present paper, we report a simple colorimetric assay for measuring FXIII activity. The advantage of this approach, compared with all the other solid-phase assays described so far for measuring TG activity, is that the first substrate, namely the synthetic dipeptide, N-benzyloxycarbonyl-L-Gln-L-Gly, is coupled covalently by its C-terminus with amine-substituted polystyrene plates. Covalent coupling eliminates the problem of desorption of proteins such as casein and N,N'-dimethylcasein (first substrates) when 'blocking' buffers containing proteins, e.g. BSA, are employed, or when samples of plasma or cell homogenates are used. The principle of the assay itself is based on the incorporation of the well-known second substrate, 5-(biotinamido)pentylamine, into the gamma-carboxamide of glutamine in the immobilized dipeptide. The amount of biotinylated amine bound to the plate, as measured by the phosphatase activity of Extravidin phosphatase attached to the biotin moiety, is directly proportional to the TG activity. The method shows strong correlation (r = 0.96) with the radiometric assay for FXIII activity. For plasma samples, a linear response was obtained in the range 0-1.33 i.u. (international unit)/ml, versus the Behring standard. In a preliminary clinical investigation, the method was applied to normal and pathological plasma samples from patients with liver failure and disseminated intravascular coagulation. In the isolation of TG from guinea-pig liver, it was used to measure enzyme activity after each purification step. This method is rapid, sensitive and can easily be applied to routine clinical analyses and to specific research problems.
Asunto(s)
Bioensayo/métodos , Colorimetría/métodos , Factor XIII/análisis , Hepatocitos/metabolismo , Fallo Hepático/sangre , Animales , Bioensayo/instrumentación , Colorimetría/instrumentación , Cobayas , Humanos , Miniaturización , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We have previously shown that the addition of 4-methylthio-2-oxobutanoate (MTOB) to cultures of methionine dependent neoplastic cells which lack endogenous MTOB restores their capacity to grow in the absence of exogenous methionine. Transition state inhibitors of the MTOB transaminase,responsible for the transamination of MTOB to methionine, had also been designed and selected for their capacity to inhibit the proliferation of methionine dependent neoplastic cells but not that of normal cells in culture. We now show that the transition state analogue : L-methionine ethyl esterpyridoxal(MEEP) with a structure corresponding to the oxo acid receptor covalently linked to pyridoxamine and the amine donor analogue: D-aspartate beta hydroxamate (D-AH) are efficient inhibitors of MTOB transaminase. [3H] MEEP uptake into transformed HeLa cells is similar to that in normal MRC5 cells, yet growth inhibition is seem in the transformed but not in the normal cells.MEEP irreversibly inhibits the activity of this enzyme when added to HeLa cells in culture but not that of the purified rat liver enzyme, probably due to pyridoxal phosphate already bound in the active site. On the contrary, D-AH is a noncompetitive reversible inhibitor of the purified rat liver enzyme in vitro and also inhibits intracellular HeLa MTOB transaminase. Furthermore, in HeLa cells both inhibitors induce DNA strand breaks typical of apoptotic cell death. These results provide evidence that MTOB transaminase is a potential target for antiproliferative agents which could selectively affect methionine-dependent neoplastic cells. The transition state intermediale : MEEP as an amine acceptor analogue was found to be 20 fold more effective than D-AH as the amine donor analogue in inducing apoptosis.
Asunto(s)
Antineoplásicos/farmacología , Asparagina/análogos & derivados , Asparagina/farmacología , Metionina/análogos & derivados , Metionina/metabolismo , Metionina/farmacología , Piridoxal/análogos & derivados , Piridoxal/farmacología , Piridoxamina/análogos & derivados , Piridoxamina/farmacología , Transaminasas/antagonistas & inhibidores , Animales , Apoptosis/genética , Asparagina/metabolismo , Proliferación Celular/efectos de los fármacos , D-Aminoácido Oxidasa/análisis , D-Aminoácido Oxidasa/metabolismo , Fragmentación del ADN , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glutamina/metabolismo , Células HeLa/efectos de los fármacos , Células HeLa/enzimología , Humanos , Hígado/enzimología , Piridoxal/metabolismo , Ratas , Transaminasas/análisisRESUMEN
The 81D1C2 monoclonal antibody (Mab) directed against the Nepsilon-(gamma-L-glutamyl)-L-lysine isopeptide was found to cross-react on Enzyme Immuno Assay (EIA) with acylated lysines. Using a differential screening EIA procedure, a new Mab 81D4 was selected, which did not cross-react with acylated lysines but exhibited strong reactivity with Nepsilon-(gamma-L-glutamyl)-L-lysine formed by covalently coupling the gamma-carboxyl of NalphaCBZ OtBu glutamic acid to epsilon-NH2 derivatized microtiter plates. When Nepsilon-(gamma-L-glutamyl)-L-lysine isopeptides were generated on gamma-carboxyl derivatized plates, only lysine isopeptides with blocked alpha-amines were reactive, regardless of whether the bond formed by the amine blocking agent was a carbamate with carbobenzyloxychloride or an amide with acetic anhydride. The 81D4 Mab showed little or no affinity for free Nepsilon-(gamma-L-glutamyl)-L-lysine (IC50>5 mM), for N1 or N4 mono(gamma-Poly L-glutamyl)putrescine, and for N1 mono(gamma-Poly L-glutamyl)spermidine (IC50>5 mM). However, when these same isopeptides were synthesized as cross-links between two protein chains--Nepsilon-(gamma-L-glutamyl)-L-lysine between Poly L-glutamate and Poly L-lysine; N1N4 -bis(gamma-Poly L-glutamyl)putrescine, N1N8 -bis(gamma-Poly L-glutamyl)spermidine between Poly-L-glutamate chains--very good reactivity was observed (IC50 400 microM for lysine; 80 microM for putrescine and spermidine). In addition to the chemically synthesized isopeptide cross-links that were recognized by this Mab, the naturally occurring Nepsilon-(ã-L-glutamyl)-L-lysine isopeptide cross-links in D-dimer, which are formed by the action of plasma transglutaminase (Factor XIII) on fibrin, were also detected on immunoblots using 81D4 as the primary antibody.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Lisina/inmunología , Putrescina/inmunología , Espermidina/inmunología , Animales , Especificidad de Anticuerpos , Productos de Degradación de Fibrina-Fibrinógeno/inmunología , Humanos , Técnicas para Inmunoenzimas , RatonesRESUMEN
BACKGROUND/AIMS: Short-term efficacy of local gamma interferon delivered via a single injection of an adenovirus-gamma interferon vector has been reported in immunocompetent animals which develop spontaneous liver cancer. However the long-term outcome was not examined. The aim of this randomized trial was to assess in an immunodeficient mouse ectopic model the benefit, if any, of the long-term efficacy of intratumoral injections of gamma interferon itself. METHODOLOGY: 77 mice were randomly assigned to 4 groups. Gamma interferon treated groups received a dose of 5000, 10000 or 20000 IU per animal versus phosphate-buffered saline. The follow-up lasted 46 days. RESULTS: Significant differences were noted in mice receiving 20000 IU compared to controls: increase in survival (p=0.0485), slowing down of tumor growth in large tumors (p=0.009), increase in necrosis (p=0.004). The preferential staining in necrotic areas with anti-Class II antibody and the accumulation of nuclear debris indicated that neutrophils were involved. CONCLUSIONS: Gamma interferon could accentuate the migration of non-specific immune cells to necrotic areas which occur spontaneously in large tumors. These results in animals bearing large tumor suggest that it may be worthwhile to explore local gamma interferon delivery to patients with extensive hepatocarcinoma.
Asunto(s)
Antineoplásicos/administración & dosificación , Carcinoma Hepatocelular/tratamiento farmacológico , Interferón gamma/administración & dosificación , Neoplasias Hepáticas/tratamiento farmacológico , Animales , Anticuerpos/análisis , Carcinoma Hepatocelular/patología , División Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/patología , Modelos Animales de Enfermedad , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inyecciones Intralesiones , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Necrosis , Neutrófilos , Análisis de SupervivenciaRESUMEN
Our aim in this commentary is to provide evidence that certain oxoacids formed in anaplerotic reactions control cell proliferation/apoptosis. In tumour cells with impaired Krebs cycle enzymes, some anaplerotic reactions do compensate for the deficit in oxoacids. One of these, oxaloacetate, derived from the transamination of asparagine but not of aspartate, is decarboxylated 4-fold more efficiently in polyoma-virus transformed cells than in their non-transformed counterparts. The deamidation of asparagine, in the cell culture medium, to aspartate by asparaginase decreases asparagine transamination and inhibits concomitantly the growth of asparaginase-sensitive lymphoma cells, suggesting a causal relationship between asparagine transamination and growth. Another oxoacid that can provide ATP when metabolised in mitochondria, but by the branched-chain oxoacid dehydrogenase complex (BCOADC), is 2-oxobutanoate. It has two origins: (a) deamination of threonine, and (b) cleavage of cystathionine, a metabolite derived from methionine. 2-Oxobutanoate in the presence of insulin promotes growth in G1/S arrested cells. But methionine also gives rise to another substrate of BCOADC, 4-methylthio-2-oxobutanoate (MTOB), which is synthesised exclusively from methylthioadenosine (MTA) by the action of MTA phosphorylase. In Met-dependent tumour cells with defective MTA phosphorylase, 2-oxobutanoate production would exceed that of MTOB. Further, BCOADC also has 3-fold greater affinity for 2-oxobutanoate than for MTOB; hence, the deficiency in 3-methylthio propionyl CoA, the final product of MTOB decarboxylation, would be exacerbated. Methional, the transient metabolic precursor in 3-methylthio propionyl CoA biosynthesis, is apoptogenic for both normal and bcl(2)-negative transformed cells in culture. Investigations of other causal relationships between the genes/enzymes mediating the homeostasis of anaplerotic oxoacids and cell growth/death may be worthwhile.
Asunto(s)
Apoptosis/fisiología , Ácidos Cetoglutáricos/metabolismo , Ácido Oxaloacético/metabolismo , Ácido Pirúvico/metabolismo , Aciltransferasas/metabolismo , Aciltransferasas/farmacología , Aminoácidos/metabolismo , Ciclo Celular , División Celular/fisiología , Línea Celular Transformada , Cisteína/fisiología , Sustancias de Crecimiento/metabolismo , Humanos , Metionina/fisiología , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
4-Amino-4-methyl-pent-2-ynthioc acid S-methyl ester (ampal thiolester: ATE) was used as a lead compound to synthesise new amino-substituted derivatives of alpha, beta acetylenic thiolester compounds as inhibitors of aldehyde dehydrogenase 1, (ALDH1). Of these compounds, the dimethyl derivative (DIMATE) was a competitive irreversible inhibitor (K(i) approximately 280 microM) of baker's yeast ALDH1 in vitro showing 80% inhibition at 400 microM when preincubated with the enzyme for 30min, whereas the trimethyl ammonium and the morpholine derivatives showed only 15% inhibition at 600 microM even after 60min preincubation. ATE inhibited ALDH1 activity in ALDH1-transfected L1210 T cells resistant to hydroperoxycyclophosphamide (HCPA) and inhibited growth synergistically in the presence of HCPA. In non-transfected L1210 counterparts ATE did not potentiate growth inhibition by HCPA. DIMATE was a 30-100-fold more effective growth inhibitor than ATE. Endogenous ALDH1 activities of BAF(3) cells over-expressing different levels of bcl(2) (0-100%) were similar (16-20mU/mg protein) and were all inhibited by DIMATE, reaching 20-30% at 4 microM. Up to 4 microM no apoptosis, as measured by DNA-fragmentation was observed, but at 8 and 10 microM DIMATE, DNA-fragmentation increased concomitantly with ALDH1 inhibition. No DNA-fragmentation was observed with ALDH1 irreversible inhibitors devoid of a thiolester group or with thiolesters which were not inhibitors of ALDH1. It was seen only with competitive irreversible inhibitors having the methanethiol and enzyme-inhibitory moieties. The methanethiol putatively released from DIMATE by ALDH1 esterase activity plays a role, albeit undefined, in lowering intramitochondrial glutathione levels which decreased by 47% as DNA-fragmentation increased.